Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

Jonathan Van Blerkom; Patrick Davis; Verena Thalhammer

doi:10.1093/molehr/gan037

Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

Van Blerkom, Jonathan;Davis, Patrick;Thalhammer, Verena 2008-06-30 00:00:00 Abstract Whether exogenous factors influenced the level of mitochondrial polarity (ΔΨm) in the subplasmalemmal cytoplasm of the oocyte was investigated with denuded and cumulus-enclosed human and mouse oocytes between the germinal vesicle and metaphase II stage. Co-culture of denuded oocytes with cumulus masses or primary cumulus cell cultures demonstrated a ‘proximity’ effect with respect to the detectable level of ΔΨm in the oocyte. The specificity and reversibility of this effect on subplasmalemmal mitochondria were shown by repeated repositioning between cellular and acellular regions, which sequentially down- or up-regulated ΔΨm. Experimental studies with a nitric oxide (NO) donor and inhibitor of NO synthase indicate that NO produced by cumulus cells has a regulatory influence on ΔΨm in the subplasmalemmal cytoplasm of the corresponding oocyte. Culture of denuded and cumulus-enclosed (intact) oocytes in low and high oxygen atmospheres suggests that competition between oxygen and NO at the mitochondrial level may regulate the level of ΔΨm and maintain mitochondria homeostasis in the pre-ovulatory oocyte, with a shift to higher polarity occurring after ovulation. The role of exogenous influences on oocyte ΔΨm is discussed with respect to the regulation of developmental processes in the oocyte and early embryo. mitochondria, mitochondrial polarity, nitric oxide, cumulus cells, regulation of mitochondrial activity Introduction Mitochondria in the mature mouse and human oocyte and early embryo are structurally underdeveloped (Motta et al., 2000), but as the principal source of ATP, have a central role in the establishment of developmental competence (Van Blerkom, 2004, 2008; Harvey et al., 2007; Shoubridge and Wai, 2007). While the most abundant organelles in the oocyte, little is known about how their different functions (Van Blerkom 2004, 2008, for review), including respiratory activity, may be regulated during early development. In this regard, it has been proposed that levels of ATP generation are locally up- or down-regulated by endogenous factors, such as transient changes in free calcium or pH (redox potential; Dumollard et al., 2003, 2007; Van Blerkom et al., 2002, 2003; Van Blerkom, 2008). Here, a principal aim was to investigate whether oocyte mitochondrial activity is influenced by exogenous factors that may normally exist in vivo. We have previously reported that the largely morphologically homogenous population of mitochondria in the mouse and human oocyte can be distinguished on the basis of the potential difference across the inner mitochondrial membrane, commonly termed mitochondrial polarity (ΔΨm) (Van Blerkom et al., 2002). The magnitude of ΔΨm is a determinant of several mitochondrial functions, such as ATP generation, calcium release and sequestration (Van Blerkom and Davis, 2007; Van Blerkom, 2008, for review), and is the driving force for other activities, including protein translocation and modification within these organelles (Huang et al., 2002). Staining with the mitochondrial-specific potentiometric probe JC-1, whose fluorescent emission wavelength is ΔΨm-dependent (see below), detects a subplasmalemmal domain of high-polarized mitochondria in denuded oocytes (i.e. free of cells forming the corona radiata and cumulus oophorus) that contains <5% of the total organelle complement and is spatially stable in the oocyte and inherited by the early embryo (Van Blerkom and Davis, 2006). The presence and relative level of polarity in this mitochondrial domain (or microzone, Van Blerkom, 2008) have been associated with the normality of fertilization and early embryonic development (Van Blerkom and Davis, 2006, 2007). A second aim concerned the origin of high ΔΨm in the subplasmalemmal cytoplasm, and asked whether its occurrence coincided with the loss of direct communication between the oocyte and corona radiata/cumulus oophorus at the outset of pre-ovulatory maturation, as previously suggested (Van Blerkom et al., 2002). The results indicate that high polarity arises in the mature oocyte, well after the termination of direct communication between the somatic and germ cell compartments. The findings also indicate that nitric oxide (NO) produced by cumulus/coronal cells may regulate ΔΨm in the subplasmalemmal cytoplasm by competing with oxygen at the level of electron transport. The implications of exogenous regulation of ΔΨm are discussed with respect to developmental processes in the oocyte and early embryo. Materials and Methods Oocyte and embryo collection and culture Mouse Intact cumulus–oocyte complexes (COCs) were recovered from the ampullary region of the oviduct between 12 and 14 h after the administration of an ovulatory dose of human chorionic gonadotrophin (5 IU) to 40–60-day-old CD-1 mice that had been primed with 5 IU of equine gonadotrophin 48 h earlier. Hormonally primed animals were housed overnight with males and mating was confirmed by the detection of a vaginal plug at the times indicated above. Newly ovulated metaphase II (MII) oocytes and pronuclear stage mouse oocytes denuded of cumulus and coronal cells by a brief (3–5 min) exposure to hyaluronidase followed by repeated passages through a narrow bore (glass) micropipette. Preimplantation stage embryos were recovered from the reproductive tract on Days 1–4.5 post coitum. Oocytes and embryos were stained with JC-1 in 50 µl microdroplets of either M2 or KSOM for 30 min at a standard concentration of 1 µM/1 (InVitrogen, USA), as previously described (Van Blerkom et al., 2002, 2003). For coincident mitochondrial and DNA staining, DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich) was included at a concentration of 20 µg/ml. Intact COCs containing germinal vesicle (GV) stage oocytes were recovered from the antral follicles of naturally cycling mice cultured (Van Blerkom et al., 2002) and stained as above. Human The meiotic status of intact oocytes was determined within 45 min of follicular aspiration (Van Blerkom and Henry, 1988) from women (27–38 years) undergoing ovarian stimulation and ovulation induction for in vitro fertilization (IVF). COCs were considered appropriate for analysis if the corresponding oocytes were clearly at the GV stage or showed no evidence of first polar body abstriction. For the latter, our previous studies demonstrated that at the time of aspiration, such oocytes were between the GV breakdown (GVB) and metaphase I (MI) stages (see below). COCs with MII oocytes were donated by patients who requested that a specific number be inseminated. According to protocol, denuded oocytes found to be immature at ICSI (intracytoplasmic sperm insemination), or MII but unfertilized after conventional IVF, were available for research. Oocytes were characterized as unfertilized if they showed no evidence of pronuclear formation or second polar body abstriction at 22 h post-insemination (Van Blerkom et al., 2002). Cleavage and morula stage embryos were donated to research under the following conditions, listed in order of occurrence: (i) normally fertilized but multinucleated in one or both blastomeres at the 2-cell stage, (ii) dispermic penetration indicated by the presence of 3PN and (iii) normally fertilized but cyropreservation was not selected. The human oocytes examined in this study were morphologically normal and the embryos were free of fragments and showed uniform, stage-appropriate cell divisions. Mitochondrial staining of intact COCs Newly retrieved mouse and human COCs were stained with JC-1 or a combination of JC-1 and /DAPI at hourly intervals for up to 6 h (see below). After staining, they were individually placed in microdroplets of HEPES-buffered HTF (human) or KSOM (mouse) that had been previously deposited on glass coverslips, and gently compressed between a second coverslip until the flattened oocyte could be clearly visualized, and examined for mitochondrial and chromsomal fluorescence in the fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC) or DAPI channels, respectively (Van Blerkom et al., 2002, 2003). Slight compression of COCs allowed J-aggregate fluorescence throughout the subplasmalemmal cytoplasm to be detected with long working distance fluorescent objectives and minimal optical sectioning at ×20 magnification. Cumulus oophorus cultures Co-culture of denuded oocytes and preimplantation stage embryos with intact masses Fragments of human cumulus oophorus were individually extracted at ovum retrieval from clear follicular aspirates, washed through several changes of medium (HEPES-buffered HTF), and then pooled and cultured under oil in 100 µl microdroplets of pre-equilibrated HTF supplemented with 0.4% (w/v) bovine serum albumin (Fraction V, BSA) in either ΔT dishes (Bioptics Inc., Butler, PA, see below) or poly-D-lysine-coated glass bottom culture dishes (MatTek Inc., Ashland, MA) to which a layer of gelatin (0.1%) had been previously applied for 30 min. Newly ovulated mouse COCs were washed, pooled (2–4/culture) and transferred to 50–100 µl droplets of KSOM in ΔT or poly-D-lysine/gelatin-coated dishes. Four hours prior to oocyte/embryo co-culture, the microdroplets were reformed with fresh medium containing JC-1. Cultures were maintained in an atmosphere of 90% N2, 4.5% O2, 5.5% CO2 (low oxygen atmosphere, LOA) or 95% air–5% CO2 (high oxygen atmosphere, HOA), as noted below. Denuded oocytes or preimplantation stage embryos, or both, were deposited in a location-specific manner with respect to the cumulus masses according to the following scheme: (i) directly on the surface, (ii) within invaginations, such that the oocytes were largely surrounded, but not enclosed by the cumulus oophorus and (iii) at varying positions, from the immediate margin of the mass to ∼8 mm distant, as estimated by a micrometer incorporated into one eyepiece of a dissecting microscope. MII oocytes were initially oriented in ΔT dishes such that the first polar body faced the cumulus mass. However, it was found that the stability of this orientation was more reliably maintained on the poly-D-lysine/gelatin-coated surface described above, or on the ΔT glass surface, when the BSA content was reduced to ≥0.2% (w/v). After a 30–60 min staining, oocytes or embryos were sequentially removed according to location and transferred to 50 µl microdroplets of HEPES-buffered HTF in ΔT dishes for fluorescent microscopic examination. During examination, the microdroplet temperature was maintained at precisely 37°C by means of ΔT controller, which continuously regulated the electric current passing through the thermo-optically treated glass coverslip integrated into bottom of the ΔT culture dish (Van Blerkom et al., 1995; Bioptics Inc.). Oocytes and embryos were returned to culture and repositioned with respect to the cumulus masses such that those formerly on the surface were deposited several mm away, while those originally distant from the mass were deposited on the surface of the cumulus mass, or at intervals of ∼1–2 mm from the mass (see figures). After 30–60 min, the oocytes/embryos were extracted from JC-1 containing medium according to position and re-examined by fluorescence microscopy. Cycles of repositioning followed by fluorescence microscopy were repeated three times for the human, and twice for the mouse, using the same oocytes/embryos and cumulus containing culture droplets. In both instances, the only experimental variable was change in location with respect to cumulus mass. Co-culture with primary cumulus cell cultures Primary cultures of human cumulus cells were established from intact COCs disassociated with hyaluronidase for ICSI, or from spontaneous cumulus cell outgrowths that were gently displaced from the plastic culture dish surface with a glass micropipette (fire-polished to produce a small spherical bead) at 16–20 h after conventional IVF. Cumulus cells were washed in HTF, pooled and transferred to either ΔT dishes containing 250–500 µl droplets (under oil) of DMEM (Sigma-Aldrich) supplemented with 1 mM sodium pyruvate, 20 mM glucose and 10% fetal bovine serum (InVitrogen), or to similar microdroplets formed on the glass surface of gelatin/poly-D-lysine-coated culture dishes described above. Cultures were maintained in modular incubators (see below) under LOA or HOA conditions for at least 21 days with medium changes at intervals of 2 (250 µl cultures) or 3 days (500 µl cultures). For the mouse, primary cumulus cell cultures were derived from COCs disassociated with hyaluronidase shortly after ovulation, followed by two centrifugations, pellet re-suspensions, and seeding in DMEM. Four hours prior to the addition of oocytes or embryos, the medium was equilibrated with freshly prepared JC-1, or JC-1 and DAPI, at the above concentrations. Similar to co-culture with intact cumulus masses, the placement of denuded human and mouse oocytes, or preimplantation stage embryos, involved deposition on the surface of confluent human of mouse cumulus cells, or at intervals of ∼1–2 mm, from the edge of the monolayer (see figures), up to ∼6 mm. After staining, oocytes and embryos were sequentially removed with respect to location and observed by fluorescence microscopy. After examination, they were returned to culture, repositioned with respect to their former location, and re-examined by fluorescence microscopy. Similar to studies with intact cumulus masses, two or three cycles of JC-1 staining and repositioning were performed for mouse and human oocytes, respectively, using 1–21-day-old cumulus cultures. All MII human oocytes characterized as unfertilized were examined by DAPI fluorescence to determine whether sperm penetration had occurred, despite the absence of overt signs of fertilization. Oocytes that were initially stained with JC-1 alone were fixed in a phosphate-buffered saline (PBS) solution containing 3.7% formaldehyde, washed in PBS and stained with DAPI. None of the oocytes used in the present study had been penetrated. JC-1 staining of partially denuded COCs Fully enclosed mouse GV stage oocytes (Van Blerkom et al., 2002) were focally denuded of cumulus and coronal cells by the repeated application of gentle suction with a mouth-operated glass micropipette prepared with an internal tip diameter of ∼20 µm. A similar protocol was used for intact human GV to MI stage oocytes (Van Blerkom et al., 2002). The manipulated COCs were stained in JC-1, washed and examined by fluorescence microscopy in medium pre-equilibrated in LOA conditions. After examination, oocytes were completely denuded and either re-examined immediately, or individually transferred to fresh microdroplets of KSOM or HTF containing JC-1, and re-examined at 5–10 min intervals for 60 min. Rhodamine 123 staining of intact COCs and denuded oocytes The specificity and patterns of mitochondrial staining of intact and denuded mouse and human oocytes COC detected with JC-1 was confirmed using another mitochondria-specific fluorescent probe, rhodamine 123 (r123, Sigma-Aldrich). COCs and denuded oocytes placed on and near cumulus masses or cumulus cell cultures were exposed to r123 at a concentration of 10 µg/ml for 30–60 min (Van Blerkom, 2008) in either LOA or HOA. After washing in normal medium, they were transferred to ΔT dishes maintained at 37°C and sequentially imaged in the FITC and RITC channels. Incubation of COCs and denuded oocytes in the presence of SNP or L-NAME in atmospheres containing different oxygen concentrations An optimal concentration and exposure time for experiments that used SNP (sodium nitroprusside (Sigma-Aldrich), a NO donor, or L-NAME (Nw-Nitro-L-arginine methyl ester, Sigma-Aldrich), an inhibitor of inducible and endothelial nitric oxide synthase (NOS), were determined in preliminary studies. Denuded MII human and mouse oocytes were exposed to SNP at concentrations between 1 and 300 µM/l for up 3 h in the presence of JC-1, with oocytes examined for J-aggregate fluorescence at 30 min intervals under LOA conditions. For L-NAME, mouse and human COCs were exposed this inhibitor at concentrations between 1 µM and 1 mM/l, and the oocytes examined for J-aggregate fluorescence at hourly intervals for as many as 8 h. In the present study, a standard exposure to SNP for 30 min at a concentration of 150 µM/l in LOA was used, as these conditions abolished detectable J-aggregate fluorescence, which was restored during culture in inhibitor-free medium. L-NAME was used at a concentration of 200 µM/l for similar reasons. To determine whether the inhibition of NOS activity was reversible or had toxic effects on the suppressive influence of cumulus cells (on ΔΨm, see below), human and mouse cumulus cell cultures and intact masses were treated with L-NAME for 8 h, followed by replacement with inhibitor-free medium containing JC-1. Four hours later, denuded oocytes were deposited on the surface of cumulus cell cultures or masses for 30 min, examined by fluorescence microscopy, and then returned to culture, but positioned at a distance from the masses or cumulus cell monolayers. Oocytes were examined by fluorescence microscopy 30–45 min later and the restoration of J-aggregate fluorescence in a low oxygen environment indicated that NOS inhibition was reversible. Denuded oocyte and COC culture under LOA or HOA conditions with normal medium, or medium containing SNP or L-NAME, was performed in modular incubators (Billups Rothenberg, Del Mar, CA) using defined (±0.1%), pre-mixed atmospheres. Results JC-1 staining of intact and denuded human oocytes Fluorescent microscopic analysis of denuded oocytes stained with JC-1 and examined as whole mounts (i.e. uncompressed) confirmed earlier findings (see below) by demonstrating that all GV (n = 26), GVB to MI (n = 87) and MII (n = 49) human oocytes (e.g. MII, Fig. 1A) exhibited a distinct subplasmalemmal domain of J-aggregate fluorescence (arrows, Fig. 1B). J-aggregates develop in high-polarized mitochondria (≥−140 mV), and fluoresce bright orange in the FITC channel (arrows, Fig. 1B, Van Blerkom et al. 2006) and red in the RITC channel (arrows, Fig. 1C). To investigate whether a similar pattern of J-aggregate fluorescence occurred in intact (i.e. cumulus-enclosed) oocytes, newly aspirated COCs containing GV (n = 7; Fig. 2A), GVB (n = 11, Fig. 1D and E), MI (Fig. 2C–F; n = 29) or MII oocytes (Fig. 1F–H; n = 9) were stained with JC-1 for up to 6 h (e.g. 1 h, Fig. 1E–H; 2 h Fig. 2E and F; 4 h Fig. 2C and D; 6 h, Fig. 2A) under LOA conditions. As discussed below, most JC-1 staining was performed in a low oxygen environment because it closely approximates intrafollicular conditions. Fluorescent microscopic analysis of COCs involved compression between two glass coverslips, which facilitated the detection of J-aggregate fluorescence (e.g. Figs 1D and E and 2C–F) and enabled the meiotic status of the oocyte to be confirmed by DAPI chromosomal fluorescence (e.g. Fig. 1H). Figure 1: View largeDownload slide (A–C) The typical subplasmalemmal location of high-polarized mitochondria (arrows, B and C) in MII human oocytes (first polar body, PB1, A) stained with JC-1 after denudation of cumulus and coronal cells and imaging in the living state by fluorescence microscopy in the FITC (B) and RITC (C) channels. (D and E (GVB) and F–H (MII)) are intact human COCs (D, light microscopic images) stained for mitochondria with JC-1 (FITC channel, F; RITC channel, G) and for DNA with DAPI (arrow indicates MII chromosomes). J-aggregate fluorescence that reports high-polarized mitochondria was detected in the cumulus oophorus (CO) and transzonal processes (arrows, E), but not in the ooplasm (O). Cytoplasmic JC-1 monomeric green fluorescence showed the typical distribution of mitochondria in normal MII human oocytes, including a subplasmalemmal domain of organelles (arrow, F). Figure 1: View largeDownload slide (A–C) The typical subplasmalemmal location of high-polarized mitochondria (arrows, B and C) in MII human oocytes (first polar body, PB1, A) stained with JC-1 after denudation of cumulus and coronal cells and imaging in the living state by fluorescence microscopy in the FITC (B) and RITC (C) channels. (D and E (GVB) and F–H (MII)) are intact human COCs (D, light microscopic images) stained for mitochondria with JC-1 (FITC channel, F; RITC channel, G) and for DNA with DAPI (arrow indicates MII chromosomes). J-aggregate fluorescence that reports high-polarized mitochondria was detected in the cumulus oophorus (CO) and transzonal processes (arrows, E), but not in the ooplasm (O). Cytoplasmic JC-1 monomeric green fluorescence showed the typical distribution of mitochondria in normal MII human oocytes, including a subplasmalemmal domain of organelles (arrow, F). Figure 2: View largeDownload slide In intact human COCs containing immature oocytes (A, germinal vesicle stage, GV) high-polarized mitochondria are observed in the somatic cells that surround the oocyte but not in the ooplasm. (B) The normal pattern of mitochondrial fluorescence in the cumulus and oocyte of a GV stage human oocyte stained with the mitochondria-specific fluorescent probe rhodamine 123 and imaged in the FITC channel. (C–F) are JC-1 stained human COCs with different degrees of cumulus expansion and imaged by light (GVB, C; MI, E) and fluorescent microscopy in the FITC channel (D and F) while slightly compressed between two glass coverslips to maximize detection of the oocyte. J-aggregate fluorescence indicative of high-polarized mitochondria was detected in the cells surrounding the oocyte, but not in the MI oocyte. However, partial mechanical removal of portions of corona and cumulus cells from the zona pellucida resulted in the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, H). This domain was detectable after complete denudation of the somatic cumulus and coronal cells (arrows, I) and shortly thereafter, extended throughout the subplasmalemmal cytoplasm (J, FITC channel; K, RITC channel). Figure 2: View largeDownload slide In intact human COCs containing immature oocytes (A, germinal vesicle stage, GV) high-polarized mitochondria are observed in the somatic cells that surround the oocyte but not in the ooplasm. (B) The normal pattern of mitochondrial fluorescence in the cumulus and oocyte of a GV stage human oocyte stained with the mitochondria-specific fluorescent probe rhodamine 123 and imaged in the FITC channel. (C–F) are JC-1 stained human COCs with different degrees of cumulus expansion and imaged by light (GVB, C; MI, E) and fluorescent microscopy in the FITC channel (D and F) while slightly compressed between two glass coverslips to maximize detection of the oocyte. J-aggregate fluorescence indicative of high-polarized mitochondria was detected in the cells surrounding the oocyte, but not in the MI oocyte. However, partial mechanical removal of portions of corona and cumulus cells from the zona pellucida resulted in the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, H). This domain was detectable after complete denudation of the somatic cumulus and coronal cells (arrows, I) and shortly thereafter, extended throughout the subplasmalemmal cytoplasm (J, FITC channel; K, RITC channel). While J-aggregate-positive cells were detected throughout the cumulus oophorus and corona radiata (FITC channel, Figs 1E and F and 2A, D and F), a similar signal was not observed in the corresponding oocyte(s) (e.g. Fig. 1G, RITC channel). However, the occurrence of green cytoplasmic fluorescence at intensities similar to levels observed in the denuded oocytes (Fig. 1B) indicates JC-1 uptake by mitochondria whose ΔΨm was below the threshold required to promote the potential-driven multimerization of JC-1 monomers into J-aggregates (≥−140 mV; see below), and that the presence of somatic cells did not impede uptake. The latter possibility was also suggested by (i) the finding that JC-1 detected a domain of subplasmalemmal mitochondria that are normally high polarized in denuded oocytes, but occur at a lower polarity in the intact oocytes (arrows in Fig. 1F) and (ii) patterns and intensities of mitochondrial fluorescence in intact oocytes stained with r123 (Fig. 2B) that were similar to their denuded counterparts (Van Blerkom, 2008). Proximity effect of cumulus masses on Δψm in the human oocyte Whether the presence of cumulus/coronal cells, rather than transzonal process (TZP)-mediated intercellular contact between these cells and the oolemma, could be regulatory with respect to the level of ΔΨm in the subplasmalemmal cytoplasm was examined in human COCs (n = 15) stained with JC-1 after varying portions of surrounding cells were removed mechanically. Figure 2G is an example of partial cellular denudation in which J-aggregate fluorescence was detected in the corresponding subplasmalemmal cytoplasm of a MI stage human oocyte after a 20 min staining (arrows, Fig. 2H). The same oocyte is shown in Fig. 2I immediately following the removal of all cumulus and coronal cells. Within 15 min of complete denudation, J-aggregate fluorescence extended throughout the circumference of the oocyte (Fig. 2J; arrows, RITC channel, Fig. 2K). Similar results were obtained with COCs in which oocytes were at the GV (n = 6), GVB stages (n = 3), MI (n = 6) and MII (n = 3) stages. These findings indicate that the presence of cumulus cells, rather than their direct physical association with the oolemma, may influence the magnitude of ΔΨm in the subplasmalemmal cytoplasm. This notion was further investigated by co-culturing denuded immature oocytes (GV to MI, n = 48) and normal appearing, unfertilized MII oocytes (n = 43, e.g. Fig. 3B) with newly aspirated cumulus masses (Fig. 3A). As noted above, all denuded GV to MII human oocytes exhibited circumferential J-aggregate fluorescence after JC-1 staining. Prior to oocyte placement, cumulus masses were preloaded with JC-1 for 1 h, followed by location-specific deposition of oocytes as follows: (i) on the surface of the cumulus mass (arrows, Fig. 3A), (ii) into indentations in these structures or (iii) at intervals of ∼1–2 mm from the edge of the mass to cell-free locations (arrows, upper right, Fig. 3A). After 1 h, oocytes were extracted, pooled with respect to location and examined individually by fluorescence microscopy. Each co-culture study was repeated four times with cumulus cells and oocytes derived from different patients with a minimum of 10 oocytes/experiment. Figure 3: View largeDownload slide Co-culture of denuded human MI and MII human oocytes (B) with masses of cumulus oophorus retrieved from follicular aspirates showed a location-dependent influence on mitochondrial polarity (Δψm) in the subplasmalemmal cytoplasm. Oocytes placed on the surface or within indentations of cumulus masses (white arrows, A) were uniformly J-aggregate-negative (C and D), while those at a distance from the mass (black arrows, A), or within a few mm, were J-aggregate-positive throughout the subplasmalemmal cytoplasm (arrows, H). Oocytes in close proximity to, but not in direct contact with cumulus mass, showed asymmetric J-aggregate fluorescence (E–G). Where the initial orientation of the oocyte at the time of placement could be confirmed after staining, the J-aggregate-negative region in the subplasmalemmal cytoplasm had faced the cumulus during exposure to JC-1 (e.g. arrow, E). (I) The normal distribution of J-aggregate fluorescence imaged in the RITC channel in oocytes stained at a distance from cumulus masses. (J) The absence of detectable J-aggregate fluorescence in oocytes imaged in the RITC channel after JC-1 staining (see Fig. 1C) while on the surface or in close proximity to the edge to the mass. Normal subplasmalemmal J-aggregate fluorescence returned when J-aggregate-negative oocytes (e.g. C and D) were repositioned away from the cumulus mass (arrows, K–M). Culture of mouse (M) and human (H) oocytes in the presence of primary cultures of human cumulus cells (N, O1 and 2) maintained in vitro for as many as 21 days showed the same location-dependent response in ΔΨm in subplasmalemmal mitochondria as observed with intact masses. Human oocytes cultured on the surface of cumulus cell cultures were uniformly J-aggregate-negative (e.g. P) while those cultured at varying distances from the edge of the outgrowths exhibited asymmetric J-aggregate fluorescence (arrows, R–V), reduced intensity circumferential fluorescence (Q), or a normal subplasmalemmal signal (W). In all cases, placement of J-aggregate-negative oocytes in cell-free regions of the same culture was accompanied by the relatively rapid up-regulation of ΔΨm in the subplasmalemmal domain (X–Z1). Figure 3: View largeDownload slide Co-culture of denuded human MI and MII human oocytes (B) with masses of cumulus oophorus retrieved from follicular aspirates showed a location-dependent influence on mitochondrial polarity (Δψm) in the subplasmalemmal cytoplasm. Oocytes placed on the surface or within indentations of cumulus masses (white arrows, A) were uniformly J-aggregate-negative (C and D), while those at a distance from the mass (black arrows, A), or within a few mm, were J-aggregate-positive throughout the subplasmalemmal cytoplasm (arrows, H). Oocytes in close proximity to, but not in direct contact with cumulus mass, showed asymmetric J-aggregate fluorescence (E–G). Where the initial orientation of the oocyte at the time of placement could be confirmed after staining, the J-aggregate-negative region in the subplasmalemmal cytoplasm had faced the cumulus during exposure to JC-1 (e.g. arrow, E). (I) The normal distribution of J-aggregate fluorescence imaged in the RITC channel in oocytes stained at a distance from cumulus masses. (J) The absence of detectable J-aggregate fluorescence in oocytes imaged in the RITC channel after JC-1 staining (see Fig. 1C) while on the surface or in close proximity to the edge to the mass. Normal subplasmalemmal J-aggregate fluorescence returned when J-aggregate-negative oocytes (e.g. C and D) were repositioned away from the cumulus mass (arrows, K–M). Culture of mouse (M) and human (H) oocytes in the presence of primary cultures of human cumulus cells (N, O1 and 2) maintained in vitro for as many as 21 days showed the same location-dependent response in ΔΨm in subplasmalemmal mitochondria as observed with intact masses. Human oocytes cultured on the surface of cumulus cell cultures were uniformly J-aggregate-negative (e.g. P) while those cultured at varying distances from the edge of the outgrowths exhibited asymmetric J-aggregate fluorescence (arrows, R–V), reduced intensity circumferential fluorescence (Q), or a normal subplasmalemmal signal (W). In all cases, placement of J-aggregate-negative oocytes in cell-free regions of the same culture was accompanied by the relatively rapid up-regulation of ΔΨm in the subplasmalemmal domain (X–Z1). The principal finding was that proximity to cumulus masses had a pronounced effect on the relative magnitude of ΔΨm in the subplasmalemmal cytoplasm. The same results were obtained with JC-1 staining for 30 min (n = 23), 60 min (n = 38), 4 h (n = 25) or 6 h (n = 12). No J-aggregate fluorescence was observed in any oocyte placed within (Fig. 3C) or on the surface of the cumulus mass (Fig. 3D). For proximity studies, MII oocytes were initially positioned on the treated culture dish surface (see above) such that the first polar body stably faced the cumulus mass and the culture dishes were carefully handled to minimize potential disruption of this orientation. The findings demonstrate that the occurrence of J-aggregate fluorescence was distance dependant: oocytes directly adjacent to (≤∼1 mm), but not in direct contact with the mass, were all J-aggregate-negative (n = 35, similar to Fig. 3C and D), whereas those at a distance of ∼2 mm showed a partial domain of J-aggregate fluorescence (n = 27, e.g. arrow, Fig. 3E). This asymmetry diminished with distance such that at −3 mm, smaller regions of the subplasmalemmal cytoplasm were negatively affected (n = 16, e.g. Fig. 3F and G), and at ∼4 mm, the distribution and intensity of J-aggregate fluorescence was normal throughout the circumference of the subplasmalemmal cytoplasm and similar to oocytes in cell-free culture (n = 19; arrows, FITC channel, Fig. 3H: RITC channel, Fig. 3I). Figure 3J is a typical image of a J-aggregate-negative oocyte (RITC channel) that had been stained for 60 min with JC-1 while on the surface of a cumulus mass. The proximity effect of cumulus masses on Δψm is reversible Repositioning oocytes known to be J-aggregate-negative (e.g. Fig. 3J) away from the mass, or placing oocytes known to be J-aggregate-positive on the cumulus mass surface, demonstrated the second major finding of this study, that the apparent proximity effect was reversible. In all instances (n = 44), J-aggregate-positive oocytes became J-aggregate-negative within 30 min of placement on the surface of a cumulus mass. All J-aggregate-negative oocytes (n = 28, similar to Fig. 3C and D) showed a normal circumferential J-aggregate signal (arrows, Fig. 3K–M) when restained at a distance of ∼5 mm from the mass (e.g. upper arrows, Fig. 3A). The extent to which ΔΨm could be repeatedly up- or down-regulated in the subplasmalemmal domain was determined by sequentially repositioning oocyte(s) with respect to cumulus masses in the same JC-1 containing culture, with fluorescent examinations of short duration (∼2–3 s) made at each transition. Shifts in the magnitude of ΔΨm reported by J-aggregate fluorescence could be documented for the same oocytes (n = 61) during three cycles of repositioning. For example, Figures 4A–E show a typical cycle of proximity-related transitions in ΔΨm, from high (Fig. 4A) to low (Fig. 4B) to high (FITC channel, Fig. 4C; RITC channel, Fig. 4D), and back to low (Fig. 4E), that was specifically related to position with respect to a cumulus mass, as described above. These results indicate that the level of ΔΨm in the subplasmalemmal domain is not regulated by direct intercellular contact between the oocyte and somatic cell compartment but can be directly influenced by proximity to cumulus cells. Figure 4: View largeDownload slide The influence of cumulus masses or cumulus cell cultures on ΔΨm in the subplasmalemmal cytoplasm was demonstrated by several cycles of culture of the same oocyte(s) at areas distant from these cells (A and C, FITC channel; D, RITC channel), on their surface (B), or in close proximity (E). Human oocytes co-cultured with mouse cumulus masses or on the surface of primary cultures of cumulus cells (arrows, F and G) were uniformly J-aggregate-negative (H) but a normal subplasmalemmal signal returned when these oocytes were repositioned to cell-free areas in the same culture (I). The arrow in (I) denotes normal J-aggregate fluorescence in a human 2-cell embryo that was J-aggregate-negative when co-cultured with mouse cumulus cells. Newly ovulated MII mouse oocytes (O, J) stained with JC-1 showed J-aggregate fluorescence in the cumulus oophorus and corona radiata (CR, J), but not in the oocyte (O) when viewed in a slightly compressed state (K) in the FITC (L) or RITC channel (O, M). That uptake of JC-1 was not impeded by the enveloping cumulus and coronal cells was indicated by normal cytoplasmic monomeric green fluorescence, which occurred at higher intensity around the MII spindle (arrow, L), and by normal mitochondrial and chromosomal fluorescence after staining with r123 (N) and DAPI (O). (P) The normal subplasmalemmal distribution of J-aggregate fluorescence in denuded mouse oocytes detected between the GV and MII stages. Partial removal of the cumulus oophorus (CO) and corona radiata from the surface of the zona pellucida in GV stage oocytes was accompanied by the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, Q), which increased in intensity as more of this cellular layer was extracted (R). The focal domain of positive J-aggregate fluorescence remained detectable after most of these cells were removed (S, FITC channel; T, RITC channel), and involved the entire subplasmalemmal cytoplasm shortly after complete denudation (U). The differential effects on ΔΨm of culturing denuded MII mouse oocytes at different locations with respect to human or mouse cumulus masses or primary cell cultures (V) is shown in figures W–X. Oocytes at a distance from mouse cells were J-aggregate-positive (FITC, channel. W); those in close proximity showed asymmetric J-aggregate fluorescence (RITC channel, Y); those on the surface of cell cultures were uniformly J-aggregate-negative (X). Figure 4: View largeDownload slide The influence of cumulus masses or cumulus cell cultures on ΔΨm in the subplasmalemmal cytoplasm was demonstrated by several cycles of culture of the same oocyte(s) at areas distant from these cells (A and C, FITC channel; D, RITC channel), on their surface (B), or in close proximity (E). Human oocytes co-cultured with mouse cumulus masses or on the surface of primary cultures of cumulus cells (arrows, F and G) were uniformly J-aggregate-negative (H) but a normal subplasmalemmal signal returned when these oocytes were repositioned to cell-free areas in the same culture (I). The arrow in (I) denotes normal J-aggregate fluorescence in a human 2-cell embryo that was J-aggregate-negative when co-cultured with mouse cumulus cells. Newly ovulated MII mouse oocytes (O, J) stained with JC-1 showed J-aggregate fluorescence in the cumulus oophorus and corona radiata (CR, J), but not in the oocyte (O) when viewed in a slightly compressed state (K) in the FITC (L) or RITC channel (O, M). That uptake of JC-1 was not impeded by the enveloping cumulus and coronal cells was indicated by normal cytoplasmic monomeric green fluorescence, which occurred at higher intensity around the MII spindle (arrow, L), and by normal mitochondrial and chromosomal fluorescence after staining with r123 (N) and DAPI (O). (P) The normal subplasmalemmal distribution of J-aggregate fluorescence in denuded mouse oocytes detected between the GV and MII stages. Partial removal of the cumulus oophorus (CO) and corona radiata from the surface of the zona pellucida in GV stage oocytes was accompanied by the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, Q), which increased in intensity as more of this cellular layer was extracted (R). The focal domain of positive J-aggregate fluorescence remained detectable after most of these cells were removed (S, FITC channel; T, RITC channel), and involved the entire subplasmalemmal cytoplasm shortly after complete denudation (U). The differential effects on ΔΨm of culturing denuded MII mouse oocytes at different locations with respect to human or mouse cumulus masses or primary cell cultures (V) is shown in figures W–X. Oocytes at a distance from mouse cells were J-aggregate-positive (FITC, channel. W); those in close proximity showed asymmetric J-aggregate fluorescence (RITC channel, Y); those on the surface of cell cultures were uniformly J-aggregate-negative (X). The proximity effect of cumulus cells is not species-specific Human oocytes (n = 17) placed on the surface of mouse cumulus masses were all J-aggregate-negative (similar to Fig. 3C), but high polarity was re-established when repositioned away from the mass (similar to Fig. 3H). MII stage mouse oocytes (n = 60) placed on/in close proximity to human cumulus oophorus were all J-aggregate-negative (Fig. 4H), but all returned to a positive state when repositioned ≥4 mm distant (Fig. 4I). Whether cumulus cells directly influence ΔΨm was examined by simultaneously culturing mouse (M) or human oocytes (H) in primary cultures of human (Fig. 3O1 and 2) or mouse cumulus cells (Fig. 3N). Human oocytes stained with JC-1 while on the surface of the human cumulus cell layer (n = 10, Fig. 3O1) or at the immediate margin (n = 14; Fig. 3O2) were all J-aggregate-negative (Fig. 3P). In contrast, those located at a distance from the cumulus cells were all J-aggregate-positive (similar to Fig. 3W). Human oocytes cultured on the surface of mouse cumulus cells were J-aggregate-negative (n = 11, similar to Fig. 3P), but all returned to a positive state (Fig. 3Q) when repositioned to acellular regions of the same culture (H, Fig. 3N). Human oocytes placed at varying distances from the margins of mouse or human cumulus cells showed asymmetries in J-aggregate fluorescence that were proximity related (n = 27; arrows, Fig. 3R–V). Figure 3W is a typical image of subplasmalemmal J-aggregate fluorescence in MII oocytes that were the furthest from the cumulus cells. In all instances, repositioning J-aggregate-negative oocytes to regions distant from the cumulus cell layer resulted in the appearance of normal J-aggregate fluorescence (arrows, Fig. 3X–Z1). The suppressive effect of cumulus cells did not diminish for mouse or human cultures maintained for 21 days. For example, the arrows in Fig. 4F and G show different regions of a 5-day-old mouse cumulus cell culture in which human and mouse oocytes were placed in regions of comparatively high or low cell density, respectively. Similar to cultures up to Day 21, no detectable J-aggregate signal occurred in human or mouse oocytes stained with JC-1 while on the cellular surface (e.g. mouse oocytes, Fig. 4H). When repositioned to cell-free areas, all oocytes showed normal subplasmalemmal J-aggregate fluorescence (Fig. 4I). A multinucleated 2-cell human embryo (arrow, Fig. 4I) was included in this culture and showed normal J-aggregate fluorescence when removed from the immediate vicinity of mouse cumulus cells (see below). JC-1 staining of denuded mouse oocytes and intact COCs Intact mouse COCs retrieved from the ampullary region of the oviduct were stained with JC-1 for up to 4 h (n = 65, e.g. Fig. 4J, shown uncompressed). While high ΔΨm in the cumulus and coronal cells (Fig. 4K, compressed) was indicated by yellow-orange fluorescence in the FITC channel (Fig. 4L), the corresponding oocyte cytoplasm fluoresced green, indicating a ΔΨm below the threshold required to form J-aggregates. A pronounced accumulation of green fluorescence surrounding the MII spindle (arrow, Fig. 4L) demonstrated uptake of the JC-1 monomer and confirmed previous findings that mitochondria in this location were low polarized (Van Blerkom et al., 2002). When compressed COCs were viewed in the RITC channel, no detectable J-aggregate fluorescence was observed in the ooplasm (O, Fig. 4M). Similar to results from human studies, mouse COCs (n = 25) stained with r123 showed green mitochondrial fluorescence for both ooplasm and granulosa cells (FITC channel, Fig. 4N). After staining with DAPI, fluorescent nuclei in the cumulus oophorus and corona radiata, and MII chromosomes in the oocyte (arrow, Fig. 4O) were clearly evident. Staining with JC-1, r123 and DAPI in the examples shown was for 30 min, indicating that similar to human COCs, the presence of cumulus and coronal cells in the mouse did not impede the uptake of these stains by the oocyte. The typical pattern of subplasmalemmal J-aggregate fluorescence detected in all denuded mouse oocytes between the GV and MII stages (n = 155) is shown in Fig. 4P (see also, Van Blerkom et al., 2002). Similar to findings from manipulated human COCs, partial removal of the surrounding cumulus and coronal cells was spatially associated with J-aggregate formation. The arrows in Fig. 4Q denote J-aggregate fluorescence that developed in the exposed region of the subplasmalemmal cytoplasm in all GV stage mouse oocytes (n = 40) within 15 min after partial denudation of the cumulus oophorus (CO) and corona radiata. As more of the cellular envelope was removed, the corresponding exposed cell margins showed J-aggregate fluorescence (arrow, Fig. 4R, 10 min after denudation, n = 30). After complete denudation, the entire circumference of the subplasmalemmal cytoplasmic showed a normal J-aggregate signal (Fig. 4U). Similar findings were obtained for all oocytes at the MI (n = 30, Fig. 4S and T) and MII stages (n = 50; similar to Fig. 4W). These results indicate that the capacity of subplasmalemmal mitochondria in the mouse oocyte to assume a higher state of polarization is influenced by cumulus and coronal cells in a manner similar to their human counterparts. This conclusion was tested by co-culture of denuded MII mouse oocytes with intact mouse or human cumulus masses and primary cumulus cell cultures. Similar to findings with human oocytes, all MII oocytes (n = 70) stained with JC-1 while on the surface of, or deposited within fresh mouse (n = 25) or human cumulus masses (n = 45) were J-aggregate-negative (Fig. 4X). In both mouse (Fig. 3N) and human cumulus cell cultures (Fig. 4V), all mouse oocytes (n = 85) placed at a distance from the monolayer were J-aggregate-positive throughout the subplasmalemmal cytoplasm (similar to Fig. 4U), and J-aggregate-negative when deposited on the surface of the cumulus cells (similar to Fig. 4X). Mouse oocytes that remained fixed in position ∼1–2-mm from cumulus masses (n = 110) or cumulus monolayers (n = 70) showed asymmetries in the spatial distribution of J-aggregate fluorescence (Fig. 4Y, RITC channel) that were similar to the proximity-dependent patterns described above for human oocytes cultured under similar conditions. These findings indicate for two species that cumulus cells can influence the magnitude of ΔΨm in the subplasmalemmal cytoplasm of each other’s oocyte, regardless of stage of meiotic maturity. The suppression of high Δψm in the oocyte involves NO of cumulus-cell origin Media from 1–5-day-old cultures of mouse and human cumulus masses, or from primary cumulus cell cultures maintained for up to 1 week, and which were shown to suppress ΔΨm in fresh oocytes, were used for oocyte culture and JC-1 staining of human (n = 9) and mouse oocytes (n = 65). No effect on the normal intensity and distribution of J-aggregate fluorescence (similar to Figs 3H and 4I) was found under LOA or HOA conditions. This result suggested that putative regulators of ΔΨm were either short-lived, labile or did not accumulate in culture medium at levels that could detectably suppress ΔΨm under the conditions used. The observed rapidity with which polarity in the subplasmalemmal domain was up- or down-regulated, and the proximity effects described above, suggested that putative regulatory factors were short-lived, low molecular weight molecule(s) that could rapidly and freely diffuse through the zona pellucida and oolemma. For reasons described below, the gaseous lipophilic free radical NO was considered a likely candidate. To determine whether NO could influence levels of ΔΨm in a manner similar to the one observed in the cumulus co-culture studies described above, denuded MI and MII human oocytes were exposed to the NO donor SNP for 30 min under LOA (n = 38) or HOA (n = 19) conditions. The typical pattern of JC-1, J-aggregate fluorescence in human oocytes cultured in the absence of this NO donor is shown in Fig. 5A (LOA) and D (HOA), respectively, and as presented, closely reflect the actual intensity of fluorescence observed. Exposure to SNP at concentrations up to 1 mM/l in HOA conditions had no detectable effect on the relative intensity or distribution of subplasmalemmal J-aggregate fluorescence (1 mM/l, Fig. 5B; 500 µM/l, Fig. 5C). In contrast, culture in LOA conditions completely abolished detectable J-aggregate fluorescence in all oocytes at 150 µM/ml (Fig. 5E). Comparatively high SNP levels that had no evident effect on the intensity of JC-1 monomer or J-aggregate fluorescence in a HOA (e.g. 300 µM/l) showed a clear reduction in the relatively intensity of JC-1 monomer (green) fluorescence in LOA culture (Fig. 5G), indicating that uptake of JC-1 by mitochondria distant from the oolemma and their relative ΔΨm were likely being affected. Figure 5: View largeDownload slide (A and D) J-aggregate fluorescence in denuded MI and MII human oocytes cultured in an atmosphere containing ~20 or 4.5% O2, respectively. (B and C) Images of J-aggregate fluorescence in oocytes cultured in the presence of a nitric oxide (NO) donor at ~20% O2. J-aggregate fluorescence was absent in oocytes cultured in the presence of a NO donor (SNP) at 4.5% O2 (E) but returned to normal (F) during culture in SNP-free medium. At higher SNP concentrations, levels of cytoplasm JC-1 fluorescence were reduced (G), but returned to normal in the absence of the donor (H). Exposure of mouse COCs to L-NAME, an inhibitor of nitric oxide synthase for 1–4 h (I,K and N) showed a progressive increase in subplasmalemmal J-aggregate fluorescence (L and M, 2 h; FITC and RITC channels, respectively; O, 3 h). At 4 h, the intensity of the fluorescent signal in oocytes observed under compression (P, FITC channel; Q, RITC channel), was similar to levels observed in denuded oocytes (see Fig. 4U) examined as whole mounts after a 30 min staining with JC-1. (R) The intensity and distribution of J-aggregate fluorescence at 4 h in untreated mouse COCs in LOA conditions. (S–Z) The differential effects on ΔΨm of culturing cleavage stage embryos (human, S–V) and blastocyst (mouse, W–Z) in the presence or absence of cumulus cells (masses or cell cultures). Normal subplasmalemmal J-aggregate fluorescence (T) is undetectable in the presence of cumulus cells (U) and restored when embryos were relocated to cell-free areas in the same culture (V). The typical high-intensity J-aggregate fluorescence detected in the mural trophectoderm (mTR, W and X), and to a lesser extent in polar trophectoderm (pTR, W and X) of the expanded mouse blastocyst was abolished during co-culture with mouse or human cumulus masses or primary cumulus cultures (Y), but reappeared within minutes when embryos were cultured in cell-free areas (Z). BN, binucleated blastomeres. Figure 5: View largeDownload slide (A and D) J-aggregate fluorescence in denuded MI and MII human oocytes cultured in an atmosphere containing ~20 or 4.5% O2, respectively. (B and C) Images of J-aggregate fluorescence in oocytes cultured in the presence of a nitric oxide (NO) donor at ~20% O2. J-aggregate fluorescence was absent in oocytes cultured in the presence of a NO donor (SNP) at 4.5% O2 (E) but returned to normal (F) during culture in SNP-free medium. At higher SNP concentrations, levels of cytoplasm JC-1 fluorescence were reduced (G), but returned to normal in the absence of the donor (H). Exposure of mouse COCs to L-NAME, an inhibitor of nitric oxide synthase for 1–4 h (I,K and N) showed a progressive increase in subplasmalemmal J-aggregate fluorescence (L and M, 2 h; FITC and RITC channels, respectively; O, 3 h). At 4 h, the intensity of the fluorescent signal in oocytes observed under compression (P, FITC channel; Q, RITC channel), was similar to levels observed in denuded oocytes (see Fig. 4U) examined as whole mounts after a 30 min staining with JC-1. (R) The intensity and distribution of J-aggregate fluorescence at 4 h in untreated mouse COCs in LOA conditions. (S–Z) The differential effects on ΔΨm of culturing cleavage stage embryos (human, S–V) and blastocyst (mouse, W–Z) in the presence or absence of cumulus cells (masses or cell cultures). Normal subplasmalemmal J-aggregate fluorescence (T) is undetectable in the presence of cumulus cells (U) and restored when embryos were relocated to cell-free areas in the same culture (V). The typical high-intensity J-aggregate fluorescence detected in the mural trophectoderm (mTR, W and X), and to a lesser extent in polar trophectoderm (pTR, W and X) of the expanded mouse blastocyst was abolished during co-culture with mouse or human cumulus masses or primary cumulus cultures (Y), but reappeared within minutes when embryos were cultured in cell-free areas (Z). BN, binucleated blastomeres. Whereas J-aggregate fluorescence was undetectable after exposure to SNP in an LOA, the typical pattern and intensity of J-aggregate fluorescence was completely restored after transfer to normal JC-1-containing medium (in the same atmosphere), including all oocytes that had been exposed to SNP at high concentration (15 min, Fig. 5F). For example, the oocyte in Fig. 5H is the same as in Fig. 5G, and shows the restoration of subplasmalemmal J-aggregate fluorescence after a 30 min culture in normal medium. Similar to the oocyte transfer/repositioning studies with cumulus masses and primary cumulus cell cultures described above, the magnitude of ΔΨm in subplasmalemmal cytoplasm of human oocytes could be reversibly down- (similar to Fig. 5E) and up-regulated (similar to Fig. 5H) over several cycles with sequential culture in the presence or absence of SNP under conditions of LOA. These findings indicate that NO may have a significant influence on ΔΨm and that the magnitude of the effect may be related to O2 concentration, as indicated by the differential response to SNP under LOA and HOA conditions. Effects of NOS inhibition on Δψm in subplasmalemmal mitochondria Figure 5I, K and N are light microscopic images of newly ovulated mouse COCs containing MII oocytes and observed under compression after 1 (n = 15), 2 (n = 16) and 4 h (n = 12) of culture in an LOA, respectively, in the presence of the nitric oxide synthase (NOS) inhibitor, L-NAME; Figure 5J, L and O are the corresponding JC-1 fluorescent patterns seen in the FITC channel. All images closely approximate the actual intensity of JC-1 (green) and J-aggregate (orange) fluorescence observed under compression, which as described above, detects punctate J-aggregate fluorescence throughout the subplasmalemmal cytoplasm in the same approximate plane of focus. J-aggregate fluorescence was undetectable in all oocytes (n = 40) during the first hour of treatment with L-NAME. However, within 2 h, J-aggregate fluorescence was evident in the subplasmalemmal cytoplasm (n = 40; arrows, Fig. 5L, FITC channel; Fig. 5M, RITC channel). At 4 h (n = 25), levels of J-aggregate fluorescence in the subplasmalemmal cytoplasm of intact oocytes were similar to those observed in denuded oocytes examined under compression (FITC channel, Fig. 5P; RITC channel, Fig. 5Q). Under HOA conditions, J-aggregate fluorescence was detectable in intact mouse oocytes (n = 20) within the first hour of culture in the presence of L-NAME (similar to Fig. 5M) and by 2 h, was similar to patterns observed at 4 h under LOA conditions (i.e. similar to Fig. 5P and Q). This finding indicates that (i) suppression of NOS activity in COCs is associated with the development of high polarity in the subplasmalemmal mitochondria of the corresponding oocyte, and (ii) the apparent rate at which high ΔΨm occurs is influenced by the concentration of ambient oxygen. The specific effect of L-NAME-treated cumulus cells on oocyte ΔΨm was confirmed by culturing denuded MII human (n = 12) and mouse (n = 60) oocytes in the presence of L-NAME for 4–6 h, under LOA and HOA conditions. As expected, the intensity and distribution of J-aggregate fluorescence in the subplasmalemmal cytoplasm was the same as observed in their untreated (denuded) siblings (similar to Figs 1B and 4U). After exposure to L-NAME (LOA culture), mouse and human cumulus masses were rinsed through several changes of inhibitor-free medium and cultured in the same medium for 5 h, at which time the medium was exchanged with freshly equilibrated medium containing JC-1. After 1 h of culture, mouse MII (n = 30) and human MI (n = 18) and MII human oocytes (n = 12) were deposited on the surface of the masses, as described above, and stained for 60 min. None of the oocytes exhibited J-aggregate fluorescence (similar to Figs 3C and 4X). When repositioned away from the mass, a normal subplasmalemmal signal was evident in all oocytes within 30 min (similar to Figs 1B for human and 4P for mouse). This finding indicates that the suppression of NOS activity by L-NAME in human and mouse cumulus oophorus masses is reversible. The timing and extent of spontaneous J-aggregate formation in untreated mouse COCs was examined at 1–2 h intervals during a 16-h culture under LOA (n = 8 COCs/time point) and HOA conditions (n = 5 COCs/time point). Under HOA conditions, the pattern of subplasmalemmal J-aggregate fluorescence at 3 h was similar to (i) sibling COCs exposed to L-NAME for 1 h (similar to Fig. 5P and Q) and (ii) denuded oocytes stained with JC-1 for 30 min. Under LOA conditions, subplasmalemmal J-aggregate fluorescence remained relatively scant for as long as the cumulus oophorus remained intact (e.g. asterisk, Fig. 5R, 6 h). The spontaneous detachment of portions of the cumulus oophorus began at ∼8 h of culture and was accompanied by an increased density of J-aggregate fluorescence that reached levels similar to those shown in Fig. 5M and Q. This finding suggests that that the suppression of high polarity is associated with the degree to which an intact corona radiata and cumulus oophorus persists. The possibility that NO produced by cultured cumulus cells directly influences ΔΨm in the oocyte was examined by treating primary mouse and human cumulus cell cultures with L-NAME (for 8 h under LOA conditions), followed by the placement of denuded MII mouse (n = 30) and human (n = 12) oocytes on the surface a confluent monolayer. As described above, oocyte co-culture under conditions of LOA (but not HOA conditions) is associated with the absence of J-aggregate fluorescence. The occurrence of circumferential J-aggregate fluorescence in all oocytes (similar to Figs 3H and 4U) in L-NAME-treated cultures supports the suppression of NOS activity and NO production. After JC-1 staining, cumulus cultures were washed through several changes of medium, and then cultured for 12 h in the same atmosphere in the absence of L-NAME. The medium was exchanged for fresh medium containing JC-1 and denuded mouse (n = 40) and human (n = 7) oocytes were deposited on the surface of the cumulus cell monolayers. Similar to findings obtained with washed cumulus masses described above, all mouse (similar to Fig. 4H) and human oocytes (similar to Fig. 3C) were J-aggregate-negative, indicating that the effect of L-NAME on NOS activity in primary cumulus cell cultures was reversible. Influence of cumulus cells on Δψm during the preimplantation stages Whether the apparent depressive influence of NO on ΔΨm in the subplasmalemmal cytoplasm of the oocyte extended to mitochondria in preimplantation stage embryos, especially at the blastocyst stage, where mitochondrial development is well advanced, was examined by culturing human and mouse cleavage (2—4-cell; human, n = 11; mouse, n = 90), morula (human, 8—16-cell, n = l5; mouse, n = 70) and expanded blastocyst stage embryos (mouse, n = 35) in the presence of mouse or human cumulus masses, or cumulus cell cultures, as described above. The normal subplasmalemmal J-aggregate fluorescence observed in cleavage and morula stage blastomeres (Van Blerkom et al., 2002; human, Fig. 5S and T) was abolished when embryos were cultured on the surface of, or in close proximity to, intact cumulus masses or primary cell cultures (Fig. 5U), but returned when the same embryos were relocated to cell-free areas in the same culture dish (Fig. 5V). Binucleated (BN) human blastomeres are indicated by arrows in Fig. 5T and V. A similar depressive effect was observed for mouse blastocysts (Fig. 5W), where high intensity J-aggregate fluorescence typical of the trophectoderm (mural trophectoderm, mTR; polar trophectoderm, pTR, FITC channel, Fig. 5X) was reduced or abolished (Fig. 5Y). Restoration of subplasmalemmal J-aggregate fluorescence in trophectodermal cells occurred within 15 min of repositioning embryos away from the cumulus masses or high-density regions of cumulus cell cultures (Fig. 5Z). The positional manipulations had no effect on the inner cell mass (ICM, Fig. 5Y), whose mitochondria are normally low polarized (Fig. 5X, see below). These findings indicate that the influence of cumulus cells on ΔΨm is not stage-specific, species-specific or related to state of mitochondrial development. Discussion The association between mitochondria and developmental competence in the oocyte and early embryo has focused primarily on the relationship between bioenergetic capacity and the normality of stage-specific developmental processes (Muller-Hocker et al., 1996; Dumollard et al., 2007; Harvey et al., 2007; Van Blerkom, 2004, 2008, for reviews). The adverse developmental consequences of a bioenergetic deficit that may be associated with a subnormal mitochondrial complement in the oocyte (Santos et al., 2006; Zeng et al., 2007) or between blastomeres during early cleavage (resulting from disproportionate mitochondrial inheritance: Van Blerkom et al., 2000; Katayama et al., 2006; Shourbagy et al., 2006), indicate apparent numerical thresholds for these organelles that may be central to the normality of meiotic maturation, fertilization and preimplantation embryogenesis (May-Panloup et al., 2007; Shoubridge and Wai, 2007). While mitochondria in the oocyte and early embryo are viewed as largely homogenous with respect to morphology, function and activity, a spatially stable subplasmalemmal domain of organelles characterized by a high ΔΨm has been suggested to be competence-related (Van Blerkom, 2004, 2008; Van Blerkom and Davis, 2007). The bioenergetic contribution of mitochondria in this domain to the total cytoplasmic ATP appears to be marginal (Van Blerkom and Davis, 2007). However, their unique spatial position and the known influences of ΔΨm on mitochondrial functions suggest specialized or local regulatory functions during early development (Van Blerkom, 2008). For example, if they are spontaneously eliminated by minor fragmentation or subject to disproportionate segregation between blastomeres, the domain is not reconstituted (Van Blerkom and Davis, 2006), and in the human, affected blastomeres (with negligible loss of cytoplasm and mitochondria to fragments) cease to divide. A role for high-polarized mitochondria in fertilization was suggested by the failure of sperm penetration and cortical granule exocytosis (but not attachment to the oolemma) to occur in zona-free mouse oocytes when the magnitude of ΔΨm was experimentally reduced; these early events in the fertilization process took place when ΔΨm returned to normal levels (Van Blerkom and Davis, 2007). High-polarized mitochondria are detected with potentiometric probes that report differences in inner mitochondrial transmembrane potential (ΔΨm, polarity), and in this regard, the fluorescent stain JC-1 has been especially useful (Salvioli et al., 1997; Van Blerkom et al., 2002, 2003). After incorporation into the mitochondrial matrix and excitation at appropriate frequencies, mitochondria whose ΔΨm −100 mV or less fluorescence green, whereas those ≥140 mV fluoresce red (Smiley et al., 1991; Cossarizza et al., 1996). A shift to longer wavelengths (green to red) results from the potential-dependent multimerization of the JC-1 monomer into so-called J-aggregates (Reers et al., 1995). Is the level of Δψm in the oocyte, suppressed by communication with the corona radiata and cumulus oophorus? Our previous studies suggested that gap-junction-mediated communication between the oocyte and TZPs extending from the corona radiata and cumulus oophorus (Albertini, 2004) may be associated with a constitutive down-regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte (Van Blerkom et al., 2002; Van Blerkom, 2004). We proposed that the depressive influence was relieved after the detachment of TZPs at the outset of the pre-ovulatory maturation (i.e. resumption of at the GV stage) and may focally increase mitochondrial activities, such as ATP generation, regulation of free calcium levels or their ability to participate in certain signal transduction pathways (Van Blerkom et al., 2002, 2003). The notion of a specialized or local regulatory role for high-polarized mitochondria was also suggested by stage-specific mitochondrial translocations during mouse oocyte maturation (Van Blerkom and Runner, 1985) that did not seem to include high-polarized organelles localized to the subplasmalemmal domain (Van Blerkom et al., 2002). Here, the finding that J-aggregate fluorescence occurred in the subplasmalemmal cytoplasm GV stage oocytes where the overlying corona radiata and cumulus oophorus had been mechanically removed supports an earlier contention that direct intercellular communication between the oocyte and somatic cell compartments in COCs suppresses a shift to higher ΔΨm (Van Blerkom et al., 2002). Indeed, the present findings showed that the partial domain of J-aggregate fluorescence remained evident after complete cellular denudation, but within ∼15 min, high polarity was detectable throughout the subplasmalemmal cytoplasm. The possibility that a focal up-regulation of ΔΨm could be associated with the detachment of TZPs is an attractive one because prior to the resumption of meiosis, these cellular extensions provide a continuous conduit for the bidirectional flow of regulatory and signaling factors between the somatic and germ cell compartments, including secondary messages such as cyclic AMP, that maintain meiosis in an arrested state. If increased ΔΨm in the subplasmalemmal cytoplasm is an early aspect of pre-ovulatory maturation, exogenous regulation of ΔΨm may involve a similar mechanism of intercellular communication. The present studies confirm previous findings that described a distinct domain of subplasmalemmal high-polarized mitochondria, reported by coincident J-aggregate fluorescence, in denuded mouse and human oocytes between the GV and MII stages (Van Blerkom et al., 2002). In contrast, while J-aggregate fluorescent mitochondria were clearly detectable in the cumulus oophorus, corona radiata and within TZPs (at the GV stage), indicating that JC-1 was reporting high ΔΨm in the somatic cell compartment, high polarity in the subplasmalemmal domain of the corresponding oocyte was not evident until MII, well after TZPs had detached from the oolemma. This finding is inconsistent with the model of ΔΨm regulation by cell contact. Studies with r123 and DAPI, and prolonged staining with JC-1 at higher concentrations, support an earlier conclusion that the absence of J-aggregate fluorescent in the oocyte was not uptake related (Van Blerkom et al., 2002, 2003). Δψm in the subplasmalemmal cytoplasm of the oocyte is regulated by a freely diffusible factor originating from cumulus cells We report that the presence of cumulus cells alone had a pronounced suppressive effect on ΔΨm in the subplasmalemmal cytoplasm of denuded oocytes, and that the magnitude of the effect was proximity-dependent. Transitions in ΔΨm from high and low, and low and high, could be repeated several times for the same oocyte(s) under identical culture conditions, with the only variable being proximity to intact cumulus masses or confluent cumulus cell cultures. Exposure of denuded oocytes to medium extracted from micro-cultures of cumulus cell masses or primary cultures showed no detectable effect on ΔΨm, suggesting that a putative factor was likely of relatively low molecular weight, freely diffusible across the zona pellucida and oolemma, and either short-lived or unstable. The apparent influence of cumulus cells on ΔΨm is not species-specific, as human and mouse oocytes responded in a similar manner when exposed to the others cumulus masses or cultured cells. The possibility that a cumulus-derived factor capable of depressing ΔΨm in the subplasmalemmal cytoplasm is gaseous in nature was suggested by the rapidity with which levels of ΔΨm could be repeatedly up- or down-regulated, and the ‘proximity effect’ described above. NO, a ubiquitous gaseous lipophilic free radical that results from the conversion of L-arginine to NO and L-citrulline by NOS, was considered a likely candidate involved in the regulation of ΔΨm for the reasons set out in Table I. Table I. Properties of NO compatible with a role in the regulation of mitochondrial polarity. (i) NO effects mitochondrial respiration by competing with O2 binding to the ‘oxygen sensor,’ the heme moiety of the terminal acceptor in the electron transport chain, cytochrome C oxidase. (ii) Cytochrome C oxidase is located on the inner mitochondrial membrane and catalyzes the oxidation of cytochrome C and the reduction of O2 to water, a process coupled with the outward pumping of protons from the mitochondrial matrix (Erusalimsky and Moncada, 2007). Outward proton pumping across the inner mitochondrial membrane creates a proton gradient that has two components, a ΔΨm and a pH gradient, and the energy stored in either component drives the conversion of ADP to ATP by respiratory chain enzymes. (iii) NO has stimulatory or depressive effects on ΔΨm related to metabolic state and the local redox environment of the cytoplasm, and competition with oxygen at the level of the inner mitochondrial membrane is regulative with respect to respiration (Erusalimsky and Moncada, 2007). (iv) NO has a half-life measured in seconds and is rapidly and freely diffusible across cell membranes (Fostermann et al., 1984; Huet-Hudson, 2007). (i) NO effects mitochondrial respiration by competing with O2 binding to the ‘oxygen sensor,’ the heme moiety of the terminal acceptor in the electron transport chain, cytochrome C oxidase. (ii) Cytochrome C oxidase is located on the inner mitochondrial membrane and catalyzes the oxidation of cytochrome C and the reduction of O2 to water, a process coupled with the outward pumping of protons from the mitochondrial matrix (Erusalimsky and Moncada, 2007). Outward proton pumping across the inner mitochondrial membrane creates a proton gradient that has two components, a ΔΨm and a pH gradient, and the energy stored in either component drives the conversion of ADP to ATP by respiratory chain enzymes. (iii) NO has stimulatory or depressive effects on ΔΨm related to metabolic state and the local redox environment of the cytoplasm, and competition with oxygen at the level of the inner mitochondrial membrane is regulative with respect to respiration (Erusalimsky and Moncada, 2007). (iv) NO has a half-life measured in seconds and is rapidly and freely diffusible across cell membranes (Fostermann et al., 1984; Huet-Hudson, 2007). View Large NO is an important intracellular and intercellular signaling molecule that regulates gene expression (Bogdan, 2001) and redox signal transduction pathways (Huang et al., 1999; Kozhukhar et al., 2006). NO is produced in the ovarian follicle by cell-type-specific NOS isoforms (Zackisson et al., 1996; Jablonka-Shariff and Olson, 1997, 1998; Yamagata et al., 2002; Bu et al., 2003; Mitchell et al., 2004), and has pleiotropic influences on follicular biology that include the regulation of steroidogenesis, survival of pre-ovulatory granulosa cells and ovulation (Olson et al., 1996; Jablonka-Shariff and Olson, 1998; Nakamura et al., 2002; Maul, 2003; Mitchell et al., 2004). NO may have a direct effect on the normality of oocyte maturation via cGMP, cAMP and mitogen-activated protein kinase signaling pathways (Lander et al., 1996; Jablonka-Shariff and Olson, 1998; Sengoku et al., 2001; Nakamura et al., 2002; Bu et al., 2003; 2004; Tao et al., 2004; Viana et al., 2007). NO, O2 and Δψm Here, the main finding is that cumulus cells can modulate the level of ΔΨm in the oocyte, and results with SNP and L-NAME suggest that the putative regulatory agent is NO (summarized in Fig. 6). Because NO modulation of respiration occurs in an oxygen-dependent manner associated with competition between these gases at the mitochondrial level, interpretations of the effects of NO on mitochondrial activity can be problematic when studies are performed under normal atmospheric conditions. In vivo, cells normally experience O2 tensions ≤6% (Palacios-Callender et al., 2004; Erusalimsky and Moncada, 2007), and this level is especially relevant here because the pre-ovulatory intrafollicular milieu in women is hypoxic, with O2 concentrations between ∼1 and ≤4% typical of gonadotrophin-stimulated follicles (Van Blerkom et al., 1997; Huey et al., 1999). The importance of O2 concentration was indicated in the present study. When oocytes were treated with SNP in 95% air, there was little, if any, detectable effects on J-aggregate fluorescence, even at relatively high concentration. In contrast, culture in a 4.5% O2 atmosphere abolished detectable J-aggregate fluorescence, which was restored when the NO donor was removed. For intact COCs exposed to L-NAME, the rapidity with which high ΔΨm was detected in the subplasmalemmal ooplasm was related to atmospheric O2 concentration. These findings suggest that the level of ambient O2 at the surface of the oolemma may mediate the suppressive influence of NO on ΔΨm and possibly, corresponding levels of organelle activity. Figure 6: View largeDownload slide Diagrammatic summary of experimental evidence for the role of nitric oxide (NO) in the regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte. Cumulus-enclosed oocytes (top centre) showed no J-aggregate staining for polarized mitochondria while a band of polarized mitochondria (red) appeared in areas which had been partially denuded (top right). Cumulus-enclosed oocytes cultured in the presence of the nitric oxide synthase (NOS) inhibitor L-NAME developed J-aggregate staining (red) similar to fully denuded oocytes (centre) implicating a role for nitric oxide. Denuded oocytes cultured in close proximity to cumulus cells lost J-aggregrate staining (middle left) and this property was abolished by treating the cumulus cells with L-NAME (bottom left). Further evidence comes from treatment of denuded oocytes with the nitric oxide donor SNP in low oxygen resulting in no J- aggregate staining (middle right) although this effect was not seen in high oxygen (bottom right). Figure 6: View largeDownload slide Diagrammatic summary of experimental evidence for the role of nitric oxide (NO) in the regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte. Cumulus-enclosed oocytes (top centre) showed no J-aggregate staining for polarized mitochondria while a band of polarized mitochondria (red) appeared in areas which had been partially denuded (top right). Cumulus-enclosed oocytes cultured in the presence of the nitric oxide synthase (NOS) inhibitor L-NAME developed J-aggregate staining (red) similar to fully denuded oocytes (centre) implicating a role for nitric oxide. Denuded oocytes cultured in close proximity to cumulus cells lost J-aggregrate staining (middle left) and this property was abolished by treating the cumulus cells with L-NAME (bottom left). Further evidence comes from treatment of denuded oocytes with the nitric oxide donor SNP in low oxygen resulting in no J- aggregate staining (middle right) although this effect was not seen in high oxygen (bottom right). Intrafollicular hypoxia may enhance the suppressive effect of cumulus-derived NO owing to the competition between these gases, as described above. This may explain the relatively rapid appearance of high polarity in denuded oocytes cultured in an LOA and the absence of a SNP effect on high ΔΨm in near normal atmospheric conditions, which may reflect reduced competition with NO at a non-physiological O2 concentration. The findings also indicate that the concentration of NO at the surface of intact cumulus masses or confluent cumulus cell cultures is sufficient to suppress ΔΨm to levels below the threshold (≤140 mV) required for J-aggregate formation (Smiley et al., 1991; Reers et al., 1995) in a low oxygen environment. The relatively short half-life of NO may confine the reduction of ΔΨm to the subplasmalemmal domain if this region of the ooplasm is the site of maximal intracellular competition. In this regard, recent mathematical modeling of O2 distribution within COCs suggests that levels at surface of the zona pellucida are likely the same as in the follicular fluid (Clark et al., 2006). The dynamic balance between NO and O2 at the surface of the oolemma may change during the pre-ovulatory period if higher O2 concentrations occur in follicular fluid as a result of increased rates of diffusion from the perifollicular microvasculature (Van Blerkom and Trout, 2007, for review). Whether the diffusion of exogenous NO extends deeper into the cytoplasm and occurs internally at levels sufficient to influence the polarity of mitochondria distant from the oolemma could not be determined under the conditions used. However, as noted above, the ΔΨm of these mitochondria is normally below the level required for J-aggregate formation. We are currently investigating other methods to assess whether the ratio between NO and O2 influences polarity in mitochondria located in the interior of the ooplasm (Lemasters and Ramshesh, 2007). Possible developmental relevance of exogenous regulation of Δψm by NO It has been suggested that high-polarized mitochondria may be involved in the focal generation of elevated levels of ATP or may be more active in calcium release and sequestration (homeostasis) than their lower polarized counterparts and as such, could have spatially related regulatory roles in the oocyte and early embryo (Van Blerkom and Davis, 2007; Van Blerkom, 2008). Based on the evidence described above, the detectable influence of cumulus cells on ΔΨm in the subplasmalemmal domain appears to be directly mediated by NO. In the intact state, NO may enter the perivitelline space or the immature oocyte through TZPs. After detachment, these cellular extensions may still provide a conduit for NO as they persist at high density on the inner surface of the zona pellucida and remain associated with functional coronal and cumulus cells (Makabe and Van Blerkom, 2006). If mitochondrial activity in the subplasmalemmal cytoplasm is ΔΨm related, it could be under exogenous control prior to ovulation and progressively relaxed during the peri-ovulatory and early post-ovulatory periods. Because NO can de-energize mitochondria at the low O2 concentrations normally experienced in cells and tissues (Schweizer and Richter, 1994), the suppression of high polarity observed in vitro (under low O2 conditions) may be experienced in vivo and associated with a ‘metabolic homeostasis’ that depresses ATP generation and other energy requiring processes localized to the subplasmalemmal domain of the follicular oocyte. In this respect, the ‘quiet rules’ for metabolism that Leese et al. (2007) relate to viability for the preimplantation embryo may extend to the follicular oocyte. The up-regulation of ΔΨm in the ovulated MII oocyte may temporally coincide with, or facilitate, early events in fertilization (Van Blerkom and Davis, 2007). Two isoforms of NOS, endothelial (eNOS) and inducible (iNOS), have been identified by immunofluorescence in MII oocytes and preimplantation stage mouse embryos (Nishikimi et al., 2001). However, if the very weak staining detected by these investigators in the oocyte is indicative of low NOS of activity, NO regulation of ΔΨm may be largely, if not entirely, exogenous in origin. In contrast, up-regulation of NOS activity after fertilization suggests an endogenous source of NO in the early embryo that could influence ΔΨm in a manner similar to the depression observed here with cumulus masses or cumulus cell cultures. If modulation of mitochondrial activity in vivo involves endogenously produced NO, the stage-specific timing of increased NOS activity (Manser et al., 2004) may coincide with the natural loss of an exogenous source. Such a transition could influence patterns of gene expression in the early embryo, and may become particularly relevant at the blastocyst stage, where NOS activity is localized to the ICM (Nishikimi et al., 2001), whose structurally developed mitochondria normally occur in a low-polarized state (Van Blerkom et al., 2006). The present findings demonstrate that similar to their counterparts in the oocyte, high polarity in the developing mitochondria of the preimplantation stage embryo is also sensitive to suppression by cumulus cells that we propose involves exogenous NO. Whether spatially related changes in ΔΨm have specific coincident (local) effects on developmentally significant processes in the oocyte requires further study. Confirmation of such an association may also provide new insights into the origins of differential oocyte/embryo competence and its relationship to specific intrafollicular conditions (Van Blerkom and Trout, 2007, for review). In the same respect, the extent to which early events in embryonic development are associated with an elevation of ΔΨm in the subplasmalemmal cytoplasm needs to be investigated in the context of the present findings, because penetration, fertilization and early cleavage occur while the oocyte is still enclosed by or associated with residual cumulus and coronal cells, which as shown here, retain the capacity to depress ΔΨm. However, studies of fertilization in zona-free mouse oocytes showed that penetration and cortical granule exocytosis occurred in regions of the oolemma where the subjacent mitochondria were high polarized (Van Blerkom and Davis, 2007) and as reported here, this state develops in the MII oocyte after ovulation. References Albertini D. Van Blerkom J, Gregory L. Oocyte-granulosa cell interactions, Essential IVF: Basic Research and Clinical Applications. , 2004 Boston Kluwer Academic Publishers(pg. 43- 58) Bogdan C. 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Publisher: Oxford University Press
Copyright: © The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Subject: New Research Horizons
ISSN: 1360-9947
eISSN: 1460-2407
DOI: 10.1093/molehr/gan037
pmid: 18591214
Publisher site: See Article on Publisher Site

Abstract

Abstract Whether exogenous factors influenced the level of mitochondrial polarity (ΔΨm) in the subplasmalemmal cytoplasm of the oocyte was investigated with denuded and cumulus-enclosed human and mouse oocytes between the germinal vesicle and metaphase II stage. Co-culture of denuded oocytes with cumulus masses or primary cumulus cell cultures demonstrated a ‘proximity’ effect with respect to the detectable level of ΔΨm in the oocyte. The specificity and reversibility of this effect on subplasmalemmal mitochondria were shown by repeated repositioning between cellular and acellular regions, which sequentially down- or up-regulated ΔΨm. Experimental studies with a nitric oxide (NO) donor and inhibitor of NO synthase indicate that NO produced by cumulus cells has a regulatory influence on ΔΨm in the subplasmalemmal cytoplasm of the corresponding oocyte. Culture of denuded and cumulus-enclosed (intact) oocytes in low and high oxygen atmospheres suggests that competition between oxygen and NO at the mitochondrial level may regulate the level of ΔΨm and maintain mitochondria homeostasis in the pre-ovulatory oocyte, with a shift to higher polarity occurring after ovulation. The role of exogenous influences on oocyte ΔΨm is discussed with respect to the regulation of developmental processes in the oocyte and early embryo. mitochondria, mitochondrial polarity, nitric oxide, cumulus cells, regulation of mitochondrial activity Introduction Mitochondria in the mature mouse and human oocyte and early embryo are structurally underdeveloped (Motta et al., 2000), but as the principal source of ATP, have a central role in the establishment of developmental competence (Van Blerkom, 2004, 2008; Harvey et al., 2007; Shoubridge and Wai, 2007). While the most abundant organelles in the oocyte, little is known about how their different functions (Van Blerkom 2004, 2008, for review), including respiratory activity, may be regulated during early development. In this regard, it has been proposed that levels of ATP generation are locally up- or down-regulated by endogenous factors, such as transient changes in free calcium or pH (redox potential; Dumollard et al., 2003, 2007; Van Blerkom et al., 2002, 2003; Van Blerkom, 2008). Here, a principal aim was to investigate whether oocyte mitochondrial activity is influenced by exogenous factors that may normally exist in vivo. We have previously reported that the largely morphologically homogenous population of mitochondria in the mouse and human oocyte can be distinguished on the basis of the potential difference across the inner mitochondrial membrane, commonly termed mitochondrial polarity (ΔΨm) (Van Blerkom et al., 2002). The magnitude of ΔΨm is a determinant of several mitochondrial functions, such as ATP generation, calcium release and sequestration (Van Blerkom and Davis, 2007; Van Blerkom, 2008, for review), and is the driving force for other activities, including protein translocation and modification within these organelles (Huang et al., 2002). Staining with the mitochondrial-specific potentiometric probe JC-1, whose fluorescent emission wavelength is ΔΨm-dependent (see below), detects a subplasmalemmal domain of high-polarized mitochondria in denuded oocytes (i.e. free of cells forming the corona radiata and cumulus oophorus) that contains <5% of the total organelle complement and is spatially stable in the oocyte and inherited by the early embryo (Van Blerkom and Davis, 2006). The presence and relative level of polarity in this mitochondrial domain (or microzone, Van Blerkom, 2008) have been associated with the normality of fertilization and early embryonic development (Van Blerkom and Davis, 2006, 2007). A second aim concerned the origin of high ΔΨm in the subplasmalemmal cytoplasm, and asked whether its occurrence coincided with the loss of direct communication between the oocyte and corona radiata/cumulus oophorus at the outset of pre-ovulatory maturation, as previously suggested (Van Blerkom et al., 2002). The results indicate that high polarity arises in the mature oocyte, well after the termination of direct communication between the somatic and germ cell compartments. The findings also indicate that nitric oxide (NO) produced by cumulus/coronal cells may regulate ΔΨm in the subplasmalemmal cytoplasm by competing with oxygen at the level of electron transport. The implications of exogenous regulation of ΔΨm are discussed with respect to developmental processes in the oocyte and early embryo. Materials and Methods Oocyte and embryo collection and culture Mouse Intact cumulus–oocyte complexes (COCs) were recovered from the ampullary region of the oviduct between 12 and 14 h after the administration of an ovulatory dose of human chorionic gonadotrophin (5 IU) to 40–60-day-old CD-1 mice that had been primed with 5 IU of equine gonadotrophin 48 h earlier. Hormonally primed animals were housed overnight with males and mating was confirmed by the detection of a vaginal plug at the times indicated above. Newly ovulated metaphase II (MII) oocytes and pronuclear stage mouse oocytes denuded of cumulus and coronal cells by a brief (3–5 min) exposure to hyaluronidase followed by repeated passages through a narrow bore (glass) micropipette. Preimplantation stage embryos were recovered from the reproductive tract on Days 1–4.5 post coitum. Oocytes and embryos were stained with JC-1 in 50 µl microdroplets of either M2 or KSOM for 30 min at a standard concentration of 1 µM/1 (InVitrogen, USA), as previously described (Van Blerkom et al., 2002, 2003). For coincident mitochondrial and DNA staining, DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich) was included at a concentration of 20 µg/ml. Intact COCs containing germinal vesicle (GV) stage oocytes were recovered from the antral follicles of naturally cycling mice cultured (Van Blerkom et al., 2002) and stained as above. Human The meiotic status of intact oocytes was determined within 45 min of follicular aspiration (Van Blerkom and Henry, 1988) from women (27–38 years) undergoing ovarian stimulation and ovulation induction for in vitro fertilization (IVF). COCs were considered appropriate for analysis if the corresponding oocytes were clearly at the GV stage or showed no evidence of first polar body abstriction. For the latter, our previous studies demonstrated that at the time of aspiration, such oocytes were between the GV breakdown (GVB) and metaphase I (MI) stages (see below). COCs with MII oocytes were donated by patients who requested that a specific number be inseminated. According to protocol, denuded oocytes found to be immature at ICSI (intracytoplasmic sperm insemination), or MII but unfertilized after conventional IVF, were available for research. Oocytes were characterized as unfertilized if they showed no evidence of pronuclear formation or second polar body abstriction at 22 h post-insemination (Van Blerkom et al., 2002). Cleavage and morula stage embryos were donated to research under the following conditions, listed in order of occurrence: (i) normally fertilized but multinucleated in one or both blastomeres at the 2-cell stage, (ii) dispermic penetration indicated by the presence of 3PN and (iii) normally fertilized but cyropreservation was not selected. The human oocytes examined in this study were morphologically normal and the embryos were free of fragments and showed uniform, stage-appropriate cell divisions. Mitochondrial staining of intact COCs Newly retrieved mouse and human COCs were stained with JC-1 or a combination of JC-1 and /DAPI at hourly intervals for up to 6 h (see below). After staining, they were individually placed in microdroplets of HEPES-buffered HTF (human) or KSOM (mouse) that had been previously deposited on glass coverslips, and gently compressed between a second coverslip until the flattened oocyte could be clearly visualized, and examined for mitochondrial and chromsomal fluorescence in the fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC) or DAPI channels, respectively (Van Blerkom et al., 2002, 2003). Slight compression of COCs allowed J-aggregate fluorescence throughout the subplasmalemmal cytoplasm to be detected with long working distance fluorescent objectives and minimal optical sectioning at ×20 magnification. Cumulus oophorus cultures Co-culture of denuded oocytes and preimplantation stage embryos with intact masses Fragments of human cumulus oophorus were individually extracted at ovum retrieval from clear follicular aspirates, washed through several changes of medium (HEPES-buffered HTF), and then pooled and cultured under oil in 100 µl microdroplets of pre-equilibrated HTF supplemented with 0.4% (w/v) bovine serum albumin (Fraction V, BSA) in either ΔT dishes (Bioptics Inc., Butler, PA, see below) or poly-D-lysine-coated glass bottom culture dishes (MatTek Inc., Ashland, MA) to which a layer of gelatin (0.1%) had been previously applied for 30 min. Newly ovulated mouse COCs were washed, pooled (2–4/culture) and transferred to 50–100 µl droplets of KSOM in ΔT or poly-D-lysine/gelatin-coated dishes. Four hours prior to oocyte/embryo co-culture, the microdroplets were reformed with fresh medium containing JC-1. Cultures were maintained in an atmosphere of 90% N2, 4.5% O2, 5.5% CO2 (low oxygen atmosphere, LOA) or 95% air–5% CO2 (high oxygen atmosphere, HOA), as noted below. Denuded oocytes or preimplantation stage embryos, or both, were deposited in a location-specific manner with respect to the cumulus masses according to the following scheme: (i) directly on the surface, (ii) within invaginations, such that the oocytes were largely surrounded, but not enclosed by the cumulus oophorus and (iii) at varying positions, from the immediate margin of the mass to ∼8 mm distant, as estimated by a micrometer incorporated into one eyepiece of a dissecting microscope. MII oocytes were initially oriented in ΔT dishes such that the first polar body faced the cumulus mass. However, it was found that the stability of this orientation was more reliably maintained on the poly-D-lysine/gelatin-coated surface described above, or on the ΔT glass surface, when the BSA content was reduced to ≥0.2% (w/v). After a 30–60 min staining, oocytes or embryos were sequentially removed according to location and transferred to 50 µl microdroplets of HEPES-buffered HTF in ΔT dishes for fluorescent microscopic examination. During examination, the microdroplet temperature was maintained at precisely 37°C by means of ΔT controller, which continuously regulated the electric current passing through the thermo-optically treated glass coverslip integrated into bottom of the ΔT culture dish (Van Blerkom et al., 1995; Bioptics Inc.). Oocytes and embryos were returned to culture and repositioned with respect to the cumulus masses such that those formerly on the surface were deposited several mm away, while those originally distant from the mass were deposited on the surface of the cumulus mass, or at intervals of ∼1–2 mm from the mass (see figures). After 30–60 min, the oocytes/embryos were extracted from JC-1 containing medium according to position and re-examined by fluorescence microscopy. Cycles of repositioning followed by fluorescence microscopy were repeated three times for the human, and twice for the mouse, using the same oocytes/embryos and cumulus containing culture droplets. In both instances, the only experimental variable was change in location with respect to cumulus mass. Co-culture with primary cumulus cell cultures Primary cultures of human cumulus cells were established from intact COCs disassociated with hyaluronidase for ICSI, or from spontaneous cumulus cell outgrowths that were gently displaced from the plastic culture dish surface with a glass micropipette (fire-polished to produce a small spherical bead) at 16–20 h after conventional IVF. Cumulus cells were washed in HTF, pooled and transferred to either ΔT dishes containing 250–500 µl droplets (under oil) of DMEM (Sigma-Aldrich) supplemented with 1 mM sodium pyruvate, 20 mM glucose and 10% fetal bovine serum (InVitrogen), or to similar microdroplets formed on the glass surface of gelatin/poly-D-lysine-coated culture dishes described above. Cultures were maintained in modular incubators (see below) under LOA or HOA conditions for at least 21 days with medium changes at intervals of 2 (250 µl cultures) or 3 days (500 µl cultures). For the mouse, primary cumulus cell cultures were derived from COCs disassociated with hyaluronidase shortly after ovulation, followed by two centrifugations, pellet re-suspensions, and seeding in DMEM. Four hours prior to the addition of oocytes or embryos, the medium was equilibrated with freshly prepared JC-1, or JC-1 and DAPI, at the above concentrations. Similar to co-culture with intact cumulus masses, the placement of denuded human and mouse oocytes, or preimplantation stage embryos, involved deposition on the surface of confluent human of mouse cumulus cells, or at intervals of ∼1–2 mm, from the edge of the monolayer (see figures), up to ∼6 mm. After staining, oocytes and embryos were sequentially removed with respect to location and observed by fluorescence microscopy. After examination, they were returned to culture, repositioned with respect to their former location, and re-examined by fluorescence microscopy. Similar to studies with intact cumulus masses, two or three cycles of JC-1 staining and repositioning were performed for mouse and human oocytes, respectively, using 1–21-day-old cumulus cultures. All MII human oocytes characterized as unfertilized were examined by DAPI fluorescence to determine whether sperm penetration had occurred, despite the absence of overt signs of fertilization. Oocytes that were initially stained with JC-1 alone were fixed in a phosphate-buffered saline (PBS) solution containing 3.7% formaldehyde, washed in PBS and stained with DAPI. None of the oocytes used in the present study had been penetrated. JC-1 staining of partially denuded COCs Fully enclosed mouse GV stage oocytes (Van Blerkom et al., 2002) were focally denuded of cumulus and coronal cells by the repeated application of gentle suction with a mouth-operated glass micropipette prepared with an internal tip diameter of ∼20 µm. A similar protocol was used for intact human GV to MI stage oocytes (Van Blerkom et al., 2002). The manipulated COCs were stained in JC-1, washed and examined by fluorescence microscopy in medium pre-equilibrated in LOA conditions. After examination, oocytes were completely denuded and either re-examined immediately, or individually transferred to fresh microdroplets of KSOM or HTF containing JC-1, and re-examined at 5–10 min intervals for 60 min. Rhodamine 123 staining of intact COCs and denuded oocytes The specificity and patterns of mitochondrial staining of intact and denuded mouse and human oocytes COC detected with JC-1 was confirmed using another mitochondria-specific fluorescent probe, rhodamine 123 (r123, Sigma-Aldrich). COCs and denuded oocytes placed on and near cumulus masses or cumulus cell cultures were exposed to r123 at a concentration of 10 µg/ml for 30–60 min (Van Blerkom, 2008) in either LOA or HOA. After washing in normal medium, they were transferred to ΔT dishes maintained at 37°C and sequentially imaged in the FITC and RITC channels. Incubation of COCs and denuded oocytes in the presence of SNP or L-NAME in atmospheres containing different oxygen concentrations An optimal concentration and exposure time for experiments that used SNP (sodium nitroprusside (Sigma-Aldrich), a NO donor, or L-NAME (Nw-Nitro-L-arginine methyl ester, Sigma-Aldrich), an inhibitor of inducible and endothelial nitric oxide synthase (NOS), were determined in preliminary studies. Denuded MII human and mouse oocytes were exposed to SNP at concentrations between 1 and 300 µM/l for up 3 h in the presence of JC-1, with oocytes examined for J-aggregate fluorescence at 30 min intervals under LOA conditions. For L-NAME, mouse and human COCs were exposed this inhibitor at concentrations between 1 µM and 1 mM/l, and the oocytes examined for J-aggregate fluorescence at hourly intervals for as many as 8 h. In the present study, a standard exposure to SNP for 30 min at a concentration of 150 µM/l in LOA was used, as these conditions abolished detectable J-aggregate fluorescence, which was restored during culture in inhibitor-free medium. L-NAME was used at a concentration of 200 µM/l for similar reasons. To determine whether the inhibition of NOS activity was reversible or had toxic effects on the suppressive influence of cumulus cells (on ΔΨm, see below), human and mouse cumulus cell cultures and intact masses were treated with L-NAME for 8 h, followed by replacement with inhibitor-free medium containing JC-1. Four hours later, denuded oocytes were deposited on the surface of cumulus cell cultures or masses for 30 min, examined by fluorescence microscopy, and then returned to culture, but positioned at a distance from the masses or cumulus cell monolayers. Oocytes were examined by fluorescence microscopy 30–45 min later and the restoration of J-aggregate fluorescence in a low oxygen environment indicated that NOS inhibition was reversible. Denuded oocyte and COC culture under LOA or HOA conditions with normal medium, or medium containing SNP or L-NAME, was performed in modular incubators (Billups Rothenberg, Del Mar, CA) using defined (±0.1%), pre-mixed atmospheres. Results JC-1 staining of intact and denuded human oocytes Fluorescent microscopic analysis of denuded oocytes stained with JC-1 and examined as whole mounts (i.e. uncompressed) confirmed earlier findings (see below) by demonstrating that all GV (n = 26), GVB to MI (n = 87) and MII (n = 49) human oocytes (e.g. MII, Fig. 1A) exhibited a distinct subplasmalemmal domain of J-aggregate fluorescence (arrows, Fig. 1B). J-aggregates develop in high-polarized mitochondria (≥−140 mV), and fluoresce bright orange in the FITC channel (arrows, Fig. 1B, Van Blerkom et al. 2006) and red in the RITC channel (arrows, Fig. 1C). To investigate whether a similar pattern of J-aggregate fluorescence occurred in intact (i.e. cumulus-enclosed) oocytes, newly aspirated COCs containing GV (n = 7; Fig. 2A), GVB (n = 11, Fig. 1D and E), MI (Fig. 2C–F; n = 29) or MII oocytes (Fig. 1F–H; n = 9) were stained with JC-1 for up to 6 h (e.g. 1 h, Fig. 1E–H; 2 h Fig. 2E and F; 4 h Fig. 2C and D; 6 h, Fig. 2A) under LOA conditions. As discussed below, most JC-1 staining was performed in a low oxygen environment because it closely approximates intrafollicular conditions. Fluorescent microscopic analysis of COCs involved compression between two glass coverslips, which facilitated the detection of J-aggregate fluorescence (e.g. Figs 1D and E and 2C–F) and enabled the meiotic status of the oocyte to be confirmed by DAPI chromosomal fluorescence (e.g. Fig. 1H). Figure 1: View largeDownload slide (A–C) The typical subplasmalemmal location of high-polarized mitochondria (arrows, B and C) in MII human oocytes (first polar body, PB1, A) stained with JC-1 after denudation of cumulus and coronal cells and imaging in the living state by fluorescence microscopy in the FITC (B) and RITC (C) channels. (D and E (GVB) and F–H (MII)) are intact human COCs (D, light microscopic images) stained for mitochondria with JC-1 (FITC channel, F; RITC channel, G) and for DNA with DAPI (arrow indicates MII chromosomes). J-aggregate fluorescence that reports high-polarized mitochondria was detected in the cumulus oophorus (CO) and transzonal processes (arrows, E), but not in the ooplasm (O). Cytoplasmic JC-1 monomeric green fluorescence showed the typical distribution of mitochondria in normal MII human oocytes, including a subplasmalemmal domain of organelles (arrow, F). Figure 1: View largeDownload slide (A–C) The typical subplasmalemmal location of high-polarized mitochondria (arrows, B and C) in MII human oocytes (first polar body, PB1, A) stained with JC-1 after denudation of cumulus and coronal cells and imaging in the living state by fluorescence microscopy in the FITC (B) and RITC (C) channels. (D and E (GVB) and F–H (MII)) are intact human COCs (D, light microscopic images) stained for mitochondria with JC-1 (FITC channel, F; RITC channel, G) and for DNA with DAPI (arrow indicates MII chromosomes). J-aggregate fluorescence that reports high-polarized mitochondria was detected in the cumulus oophorus (CO) and transzonal processes (arrows, E), but not in the ooplasm (O). Cytoplasmic JC-1 monomeric green fluorescence showed the typical distribution of mitochondria in normal MII human oocytes, including a subplasmalemmal domain of organelles (arrow, F). Figure 2: View largeDownload slide In intact human COCs containing immature oocytes (A, germinal vesicle stage, GV) high-polarized mitochondria are observed in the somatic cells that surround the oocyte but not in the ooplasm. (B) The normal pattern of mitochondrial fluorescence in the cumulus and oocyte of a GV stage human oocyte stained with the mitochondria-specific fluorescent probe rhodamine 123 and imaged in the FITC channel. (C–F) are JC-1 stained human COCs with different degrees of cumulus expansion and imaged by light (GVB, C; MI, E) and fluorescent microscopy in the FITC channel (D and F) while slightly compressed between two glass coverslips to maximize detection of the oocyte. J-aggregate fluorescence indicative of high-polarized mitochondria was detected in the cells surrounding the oocyte, but not in the MI oocyte. However, partial mechanical removal of portions of corona and cumulus cells from the zona pellucida resulted in the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, H). This domain was detectable after complete denudation of the somatic cumulus and coronal cells (arrows, I) and shortly thereafter, extended throughout the subplasmalemmal cytoplasm (J, FITC channel; K, RITC channel). Figure 2: View largeDownload slide In intact human COCs containing immature oocytes (A, germinal vesicle stage, GV) high-polarized mitochondria are observed in the somatic cells that surround the oocyte but not in the ooplasm. (B) The normal pattern of mitochondrial fluorescence in the cumulus and oocyte of a GV stage human oocyte stained with the mitochondria-specific fluorescent probe rhodamine 123 and imaged in the FITC channel. (C–F) are JC-1 stained human COCs with different degrees of cumulus expansion and imaged by light (GVB, C; MI, E) and fluorescent microscopy in the FITC channel (D and F) while slightly compressed between two glass coverslips to maximize detection of the oocyte. J-aggregate fluorescence indicative of high-polarized mitochondria was detected in the cells surrounding the oocyte, but not in the MI oocyte. However, partial mechanical removal of portions of corona and cumulus cells from the zona pellucida resulted in the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, H). This domain was detectable after complete denudation of the somatic cumulus and coronal cells (arrows, I) and shortly thereafter, extended throughout the subplasmalemmal cytoplasm (J, FITC channel; K, RITC channel). While J-aggregate-positive cells were detected throughout the cumulus oophorus and corona radiata (FITC channel, Figs 1E and F and 2A, D and F), a similar signal was not observed in the corresponding oocyte(s) (e.g. Fig. 1G, RITC channel). However, the occurrence of green cytoplasmic fluorescence at intensities similar to levels observed in the denuded oocytes (Fig. 1B) indicates JC-1 uptake by mitochondria whose ΔΨm was below the threshold required to promote the potential-driven multimerization of JC-1 monomers into J-aggregates (≥−140 mV; see below), and that the presence of somatic cells did not impede uptake. The latter possibility was also suggested by (i) the finding that JC-1 detected a domain of subplasmalemmal mitochondria that are normally high polarized in denuded oocytes, but occur at a lower polarity in the intact oocytes (arrows in Fig. 1F) and (ii) patterns and intensities of mitochondrial fluorescence in intact oocytes stained with r123 (Fig. 2B) that were similar to their denuded counterparts (Van Blerkom, 2008). Proximity effect of cumulus masses on Δψm in the human oocyte Whether the presence of cumulus/coronal cells, rather than transzonal process (TZP)-mediated intercellular contact between these cells and the oolemma, could be regulatory with respect to the level of ΔΨm in the subplasmalemmal cytoplasm was examined in human COCs (n = 15) stained with JC-1 after varying portions of surrounding cells were removed mechanically. Figure 2G is an example of partial cellular denudation in which J-aggregate fluorescence was detected in the corresponding subplasmalemmal cytoplasm of a MI stage human oocyte after a 20 min staining (arrows, Fig. 2H). The same oocyte is shown in Fig. 2I immediately following the removal of all cumulus and coronal cells. Within 15 min of complete denudation, J-aggregate fluorescence extended throughout the circumference of the oocyte (Fig. 2J; arrows, RITC channel, Fig. 2K). Similar results were obtained with COCs in which oocytes were at the GV (n = 6), GVB stages (n = 3), MI (n = 6) and MII (n = 3) stages. These findings indicate that the presence of cumulus cells, rather than their direct physical association with the oolemma, may influence the magnitude of ΔΨm in the subplasmalemmal cytoplasm. This notion was further investigated by co-culturing denuded immature oocytes (GV to MI, n = 48) and normal appearing, unfertilized MII oocytes (n = 43, e.g. Fig. 3B) with newly aspirated cumulus masses (Fig. 3A). As noted above, all denuded GV to MII human oocytes exhibited circumferential J-aggregate fluorescence after JC-1 staining. Prior to oocyte placement, cumulus masses were preloaded with JC-1 for 1 h, followed by location-specific deposition of oocytes as follows: (i) on the surface of the cumulus mass (arrows, Fig. 3A), (ii) into indentations in these structures or (iii) at intervals of ∼1–2 mm from the edge of the mass to cell-free locations (arrows, upper right, Fig. 3A). After 1 h, oocytes were extracted, pooled with respect to location and examined individually by fluorescence microscopy. Each co-culture study was repeated four times with cumulus cells and oocytes derived from different patients with a minimum of 10 oocytes/experiment. Figure 3: View largeDownload slide Co-culture of denuded human MI and MII human oocytes (B) with masses of cumulus oophorus retrieved from follicular aspirates showed a location-dependent influence on mitochondrial polarity (Δψm) in the subplasmalemmal cytoplasm. Oocytes placed on the surface or within indentations of cumulus masses (white arrows, A) were uniformly J-aggregate-negative (C and D), while those at a distance from the mass (black arrows, A), or within a few mm, were J-aggregate-positive throughout the subplasmalemmal cytoplasm (arrows, H). Oocytes in close proximity to, but not in direct contact with cumulus mass, showed asymmetric J-aggregate fluorescence (E–G). Where the initial orientation of the oocyte at the time of placement could be confirmed after staining, the J-aggregate-negative region in the subplasmalemmal cytoplasm had faced the cumulus during exposure to JC-1 (e.g. arrow, E). (I) The normal distribution of J-aggregate fluorescence imaged in the RITC channel in oocytes stained at a distance from cumulus masses. (J) The absence of detectable J-aggregate fluorescence in oocytes imaged in the RITC channel after JC-1 staining (see Fig. 1C) while on the surface or in close proximity to the edge to the mass. Normal subplasmalemmal J-aggregate fluorescence returned when J-aggregate-negative oocytes (e.g. C and D) were repositioned away from the cumulus mass (arrows, K–M). Culture of mouse (M) and human (H) oocytes in the presence of primary cultures of human cumulus cells (N, O1 and 2) maintained in vitro for as many as 21 days showed the same location-dependent response in ΔΨm in subplasmalemmal mitochondria as observed with intact masses. Human oocytes cultured on the surface of cumulus cell cultures were uniformly J-aggregate-negative (e.g. P) while those cultured at varying distances from the edge of the outgrowths exhibited asymmetric J-aggregate fluorescence (arrows, R–V), reduced intensity circumferential fluorescence (Q), or a normal subplasmalemmal signal (W). In all cases, placement of J-aggregate-negative oocytes in cell-free regions of the same culture was accompanied by the relatively rapid up-regulation of ΔΨm in the subplasmalemmal domain (X–Z1). Figure 3: View largeDownload slide Co-culture of denuded human MI and MII human oocytes (B) with masses of cumulus oophorus retrieved from follicular aspirates showed a location-dependent influence on mitochondrial polarity (Δψm) in the subplasmalemmal cytoplasm. Oocytes placed on the surface or within indentations of cumulus masses (white arrows, A) were uniformly J-aggregate-negative (C and D), while those at a distance from the mass (black arrows, A), or within a few mm, were J-aggregate-positive throughout the subplasmalemmal cytoplasm (arrows, H). Oocytes in close proximity to, but not in direct contact with cumulus mass, showed asymmetric J-aggregate fluorescence (E–G). Where the initial orientation of the oocyte at the time of placement could be confirmed after staining, the J-aggregate-negative region in the subplasmalemmal cytoplasm had faced the cumulus during exposure to JC-1 (e.g. arrow, E). (I) The normal distribution of J-aggregate fluorescence imaged in the RITC channel in oocytes stained at a distance from cumulus masses. (J) The absence of detectable J-aggregate fluorescence in oocytes imaged in the RITC channel after JC-1 staining (see Fig. 1C) while on the surface or in close proximity to the edge to the mass. Normal subplasmalemmal J-aggregate fluorescence returned when J-aggregate-negative oocytes (e.g. C and D) were repositioned away from the cumulus mass (arrows, K–M). Culture of mouse (M) and human (H) oocytes in the presence of primary cultures of human cumulus cells (N, O1 and 2) maintained in vitro for as many as 21 days showed the same location-dependent response in ΔΨm in subplasmalemmal mitochondria as observed with intact masses. Human oocytes cultured on the surface of cumulus cell cultures were uniformly J-aggregate-negative (e.g. P) while those cultured at varying distances from the edge of the outgrowths exhibited asymmetric J-aggregate fluorescence (arrows, R–V), reduced intensity circumferential fluorescence (Q), or a normal subplasmalemmal signal (W). In all cases, placement of J-aggregate-negative oocytes in cell-free regions of the same culture was accompanied by the relatively rapid up-regulation of ΔΨm in the subplasmalemmal domain (X–Z1). The principal finding was that proximity to cumulus masses had a pronounced effect on the relative magnitude of ΔΨm in the subplasmalemmal cytoplasm. The same results were obtained with JC-1 staining for 30 min (n = 23), 60 min (n = 38), 4 h (n = 25) or 6 h (n = 12). No J-aggregate fluorescence was observed in any oocyte placed within (Fig. 3C) or on the surface of the cumulus mass (Fig. 3D). For proximity studies, MII oocytes were initially positioned on the treated culture dish surface (see above) such that the first polar body stably faced the cumulus mass and the culture dishes were carefully handled to minimize potential disruption of this orientation. The findings demonstrate that the occurrence of J-aggregate fluorescence was distance dependant: oocytes directly adjacent to (≤∼1 mm), but not in direct contact with the mass, were all J-aggregate-negative (n = 35, similar to Fig. 3C and D), whereas those at a distance of ∼2 mm showed a partial domain of J-aggregate fluorescence (n = 27, e.g. arrow, Fig. 3E). This asymmetry diminished with distance such that at −3 mm, smaller regions of the subplasmalemmal cytoplasm were negatively affected (n = 16, e.g. Fig. 3F and G), and at ∼4 mm, the distribution and intensity of J-aggregate fluorescence was normal throughout the circumference of the subplasmalemmal cytoplasm and similar to oocytes in cell-free culture (n = 19; arrows, FITC channel, Fig. 3H: RITC channel, Fig. 3I). Figure 3J is a typical image of a J-aggregate-negative oocyte (RITC channel) that had been stained for 60 min with JC-1 while on the surface of a cumulus mass. The proximity effect of cumulus masses on Δψm is reversible Repositioning oocytes known to be J-aggregate-negative (e.g. Fig. 3J) away from the mass, or placing oocytes known to be J-aggregate-positive on the cumulus mass surface, demonstrated the second major finding of this study, that the apparent proximity effect was reversible. In all instances (n = 44), J-aggregate-positive oocytes became J-aggregate-negative within 30 min of placement on the surface of a cumulus mass. All J-aggregate-negative oocytes (n = 28, similar to Fig. 3C and D) showed a normal circumferential J-aggregate signal (arrows, Fig. 3K–M) when restained at a distance of ∼5 mm from the mass (e.g. upper arrows, Fig. 3A). The extent to which ΔΨm could be repeatedly up- or down-regulated in the subplasmalemmal domain was determined by sequentially repositioning oocyte(s) with respect to cumulus masses in the same JC-1 containing culture, with fluorescent examinations of short duration (∼2–3 s) made at each transition. Shifts in the magnitude of ΔΨm reported by J-aggregate fluorescence could be documented for the same oocytes (n = 61) during three cycles of repositioning. For example, Figures 4A–E show a typical cycle of proximity-related transitions in ΔΨm, from high (Fig. 4A) to low (Fig. 4B) to high (FITC channel, Fig. 4C; RITC channel, Fig. 4D), and back to low (Fig. 4E), that was specifically related to position with respect to a cumulus mass, as described above. These results indicate that the level of ΔΨm in the subplasmalemmal domain is not regulated by direct intercellular contact between the oocyte and somatic cell compartment but can be directly influenced by proximity to cumulus cells. Figure 4: View largeDownload slide The influence of cumulus masses or cumulus cell cultures on ΔΨm in the subplasmalemmal cytoplasm was demonstrated by several cycles of culture of the same oocyte(s) at areas distant from these cells (A and C, FITC channel; D, RITC channel), on their surface (B), or in close proximity (E). Human oocytes co-cultured with mouse cumulus masses or on the surface of primary cultures of cumulus cells (arrows, F and G) were uniformly J-aggregate-negative (H) but a normal subplasmalemmal signal returned when these oocytes were repositioned to cell-free areas in the same culture (I). The arrow in (I) denotes normal J-aggregate fluorescence in a human 2-cell embryo that was J-aggregate-negative when co-cultured with mouse cumulus cells. Newly ovulated MII mouse oocytes (O, J) stained with JC-1 showed J-aggregate fluorescence in the cumulus oophorus and corona radiata (CR, J), but not in the oocyte (O) when viewed in a slightly compressed state (K) in the FITC (L) or RITC channel (O, M). That uptake of JC-1 was not impeded by the enveloping cumulus and coronal cells was indicated by normal cytoplasmic monomeric green fluorescence, which occurred at higher intensity around the MII spindle (arrow, L), and by normal mitochondrial and chromosomal fluorescence after staining with r123 (N) and DAPI (O). (P) The normal subplasmalemmal distribution of J-aggregate fluorescence in denuded mouse oocytes detected between the GV and MII stages. Partial removal of the cumulus oophorus (CO) and corona radiata from the surface of the zona pellucida in GV stage oocytes was accompanied by the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, Q), which increased in intensity as more of this cellular layer was extracted (R). The focal domain of positive J-aggregate fluorescence remained detectable after most of these cells were removed (S, FITC channel; T, RITC channel), and involved the entire subplasmalemmal cytoplasm shortly after complete denudation (U). The differential effects on ΔΨm of culturing denuded MII mouse oocytes at different locations with respect to human or mouse cumulus masses or primary cell cultures (V) is shown in figures W–X. Oocytes at a distance from mouse cells were J-aggregate-positive (FITC, channel. W); those in close proximity showed asymmetric J-aggregate fluorescence (RITC channel, Y); those on the surface of cell cultures were uniformly J-aggregate-negative (X). Figure 4: View largeDownload slide The influence of cumulus masses or cumulus cell cultures on ΔΨm in the subplasmalemmal cytoplasm was demonstrated by several cycles of culture of the same oocyte(s) at areas distant from these cells (A and C, FITC channel; D, RITC channel), on their surface (B), or in close proximity (E). Human oocytes co-cultured with mouse cumulus masses or on the surface of primary cultures of cumulus cells (arrows, F and G) were uniformly J-aggregate-negative (H) but a normal subplasmalemmal signal returned when these oocytes were repositioned to cell-free areas in the same culture (I). The arrow in (I) denotes normal J-aggregate fluorescence in a human 2-cell embryo that was J-aggregate-negative when co-cultured with mouse cumulus cells. Newly ovulated MII mouse oocytes (O, J) stained with JC-1 showed J-aggregate fluorescence in the cumulus oophorus and corona radiata (CR, J), but not in the oocyte (O) when viewed in a slightly compressed state (K) in the FITC (L) or RITC channel (O, M). That uptake of JC-1 was not impeded by the enveloping cumulus and coronal cells was indicated by normal cytoplasmic monomeric green fluorescence, which occurred at higher intensity around the MII spindle (arrow, L), and by normal mitochondrial and chromosomal fluorescence after staining with r123 (N) and DAPI (O). (P) The normal subplasmalemmal distribution of J-aggregate fluorescence in denuded mouse oocytes detected between the GV and MII stages. Partial removal of the cumulus oophorus (CO) and corona radiata from the surface of the zona pellucida in GV stage oocytes was accompanied by the rapid appearance of J-aggregate fluorescence in the corresponding subplasmalemmal cytoplasm (arrows, Q), which increased in intensity as more of this cellular layer was extracted (R). The focal domain of positive J-aggregate fluorescence remained detectable after most of these cells were removed (S, FITC channel; T, RITC channel), and involved the entire subplasmalemmal cytoplasm shortly after complete denudation (U). The differential effects on ΔΨm of culturing denuded MII mouse oocytes at different locations with respect to human or mouse cumulus masses or primary cell cultures (V) is shown in figures W–X. Oocytes at a distance from mouse cells were J-aggregate-positive (FITC, channel. W); those in close proximity showed asymmetric J-aggregate fluorescence (RITC channel, Y); those on the surface of cell cultures were uniformly J-aggregate-negative (X). The proximity effect of cumulus cells is not species-specific Human oocytes (n = 17) placed on the surface of mouse cumulus masses were all J-aggregate-negative (similar to Fig. 3C), but high polarity was re-established when repositioned away from the mass (similar to Fig. 3H). MII stage mouse oocytes (n = 60) placed on/in close proximity to human cumulus oophorus were all J-aggregate-negative (Fig. 4H), but all returned to a positive state when repositioned ≥4 mm distant (Fig. 4I). Whether cumulus cells directly influence ΔΨm was examined by simultaneously culturing mouse (M) or human oocytes (H) in primary cultures of human (Fig. 3O1 and 2) or mouse cumulus cells (Fig. 3N). Human oocytes stained with JC-1 while on the surface of the human cumulus cell layer (n = 10, Fig. 3O1) or at the immediate margin (n = 14; Fig. 3O2) were all J-aggregate-negative (Fig. 3P). In contrast, those located at a distance from the cumulus cells were all J-aggregate-positive (similar to Fig. 3W). Human oocytes cultured on the surface of mouse cumulus cells were J-aggregate-negative (n = 11, similar to Fig. 3P), but all returned to a positive state (Fig. 3Q) when repositioned to acellular regions of the same culture (H, Fig. 3N). Human oocytes placed at varying distances from the margins of mouse or human cumulus cells showed asymmetries in J-aggregate fluorescence that were proximity related (n = 27; arrows, Fig. 3R–V). Figure 3W is a typical image of subplasmalemmal J-aggregate fluorescence in MII oocytes that were the furthest from the cumulus cells. In all instances, repositioning J-aggregate-negative oocytes to regions distant from the cumulus cell layer resulted in the appearance of normal J-aggregate fluorescence (arrows, Fig. 3X–Z1). The suppressive effect of cumulus cells did not diminish for mouse or human cultures maintained for 21 days. For example, the arrows in Fig. 4F and G show different regions of a 5-day-old mouse cumulus cell culture in which human and mouse oocytes were placed in regions of comparatively high or low cell density, respectively. Similar to cultures up to Day 21, no detectable J-aggregate signal occurred in human or mouse oocytes stained with JC-1 while on the cellular surface (e.g. mouse oocytes, Fig. 4H). When repositioned to cell-free areas, all oocytes showed normal subplasmalemmal J-aggregate fluorescence (Fig. 4I). A multinucleated 2-cell human embryo (arrow, Fig. 4I) was included in this culture and showed normal J-aggregate fluorescence when removed from the immediate vicinity of mouse cumulus cells (see below). JC-1 staining of denuded mouse oocytes and intact COCs Intact mouse COCs retrieved from the ampullary region of the oviduct were stained with JC-1 for up to 4 h (n = 65, e.g. Fig. 4J, shown uncompressed). While high ΔΨm in the cumulus and coronal cells (Fig. 4K, compressed) was indicated by yellow-orange fluorescence in the FITC channel (Fig. 4L), the corresponding oocyte cytoplasm fluoresced green, indicating a ΔΨm below the threshold required to form J-aggregates. A pronounced accumulation of green fluorescence surrounding the MII spindle (arrow, Fig. 4L) demonstrated uptake of the JC-1 monomer and confirmed previous findings that mitochondria in this location were low polarized (Van Blerkom et al., 2002). When compressed COCs were viewed in the RITC channel, no detectable J-aggregate fluorescence was observed in the ooplasm (O, Fig. 4M). Similar to results from human studies, mouse COCs (n = 25) stained with r123 showed green mitochondrial fluorescence for both ooplasm and granulosa cells (FITC channel, Fig. 4N). After staining with DAPI, fluorescent nuclei in the cumulus oophorus and corona radiata, and MII chromosomes in the oocyte (arrow, Fig. 4O) were clearly evident. Staining with JC-1, r123 and DAPI in the examples shown was for 30 min, indicating that similar to human COCs, the presence of cumulus and coronal cells in the mouse did not impede the uptake of these stains by the oocyte. The typical pattern of subplasmalemmal J-aggregate fluorescence detected in all denuded mouse oocytes between the GV and MII stages (n = 155) is shown in Fig. 4P (see also, Van Blerkom et al., 2002). Similar to findings from manipulated human COCs, partial removal of the surrounding cumulus and coronal cells was spatially associated with J-aggregate formation. The arrows in Fig. 4Q denote J-aggregate fluorescence that developed in the exposed region of the subplasmalemmal cytoplasm in all GV stage mouse oocytes (n = 40) within 15 min after partial denudation of the cumulus oophorus (CO) and corona radiata. As more of the cellular envelope was removed, the corresponding exposed cell margins showed J-aggregate fluorescence (arrow, Fig. 4R, 10 min after denudation, n = 30). After complete denudation, the entire circumference of the subplasmalemmal cytoplasmic showed a normal J-aggregate signal (Fig. 4U). Similar findings were obtained for all oocytes at the MI (n = 30, Fig. 4S and T) and MII stages (n = 50; similar to Fig. 4W). These results indicate that the capacity of subplasmalemmal mitochondria in the mouse oocyte to assume a higher state of polarization is influenced by cumulus and coronal cells in a manner similar to their human counterparts. This conclusion was tested by co-culture of denuded MII mouse oocytes with intact mouse or human cumulus masses and primary cumulus cell cultures. Similar to findings with human oocytes, all MII oocytes (n = 70) stained with JC-1 while on the surface of, or deposited within fresh mouse (n = 25) or human cumulus masses (n = 45) were J-aggregate-negative (Fig. 4X). In both mouse (Fig. 3N) and human cumulus cell cultures (Fig. 4V), all mouse oocytes (n = 85) placed at a distance from the monolayer were J-aggregate-positive throughout the subplasmalemmal cytoplasm (similar to Fig. 4U), and J-aggregate-negative when deposited on the surface of the cumulus cells (similar to Fig. 4X). Mouse oocytes that remained fixed in position ∼1–2-mm from cumulus masses (n = 110) or cumulus monolayers (n = 70) showed asymmetries in the spatial distribution of J-aggregate fluorescence (Fig. 4Y, RITC channel) that were similar to the proximity-dependent patterns described above for human oocytes cultured under similar conditions. These findings indicate for two species that cumulus cells can influence the magnitude of ΔΨm in the subplasmalemmal cytoplasm of each other’s oocyte, regardless of stage of meiotic maturity. The suppression of high Δψm in the oocyte involves NO of cumulus-cell origin Media from 1–5-day-old cultures of mouse and human cumulus masses, or from primary cumulus cell cultures maintained for up to 1 week, and which were shown to suppress ΔΨm in fresh oocytes, were used for oocyte culture and JC-1 staining of human (n = 9) and mouse oocytes (n = 65). No effect on the normal intensity and distribution of J-aggregate fluorescence (similar to Figs 3H and 4I) was found under LOA or HOA conditions. This result suggested that putative regulators of ΔΨm were either short-lived, labile or did not accumulate in culture medium at levels that could detectably suppress ΔΨm under the conditions used. The observed rapidity with which polarity in the subplasmalemmal domain was up- or down-regulated, and the proximity effects described above, suggested that putative regulatory factors were short-lived, low molecular weight molecule(s) that could rapidly and freely diffuse through the zona pellucida and oolemma. For reasons described below, the gaseous lipophilic free radical NO was considered a likely candidate. To determine whether NO could influence levels of ΔΨm in a manner similar to the one observed in the cumulus co-culture studies described above, denuded MI and MII human oocytes were exposed to the NO donor SNP for 30 min under LOA (n = 38) or HOA (n = 19) conditions. The typical pattern of JC-1, J-aggregate fluorescence in human oocytes cultured in the absence of this NO donor is shown in Fig. 5A (LOA) and D (HOA), respectively, and as presented, closely reflect the actual intensity of fluorescence observed. Exposure to SNP at concentrations up to 1 mM/l in HOA conditions had no detectable effect on the relative intensity or distribution of subplasmalemmal J-aggregate fluorescence (1 mM/l, Fig. 5B; 500 µM/l, Fig. 5C). In contrast, culture in LOA conditions completely abolished detectable J-aggregate fluorescence in all oocytes at 150 µM/ml (Fig. 5E). Comparatively high SNP levels that had no evident effect on the intensity of JC-1 monomer or J-aggregate fluorescence in a HOA (e.g. 300 µM/l) showed a clear reduction in the relatively intensity of JC-1 monomer (green) fluorescence in LOA culture (Fig. 5G), indicating that uptake of JC-1 by mitochondria distant from the oolemma and their relative ΔΨm were likely being affected. Figure 5: View largeDownload slide (A and D) J-aggregate fluorescence in denuded MI and MII human oocytes cultured in an atmosphere containing ~20 or 4.5% O2, respectively. (B and C) Images of J-aggregate fluorescence in oocytes cultured in the presence of a nitric oxide (NO) donor at ~20% O2. J-aggregate fluorescence was absent in oocytes cultured in the presence of a NO donor (SNP) at 4.5% O2 (E) but returned to normal (F) during culture in SNP-free medium. At higher SNP concentrations, levels of cytoplasm JC-1 fluorescence were reduced (G), but returned to normal in the absence of the donor (H). Exposure of mouse COCs to L-NAME, an inhibitor of nitric oxide synthase for 1–4 h (I,K and N) showed a progressive increase in subplasmalemmal J-aggregate fluorescence (L and M, 2 h; FITC and RITC channels, respectively; O, 3 h). At 4 h, the intensity of the fluorescent signal in oocytes observed under compression (P, FITC channel; Q, RITC channel), was similar to levels observed in denuded oocytes (see Fig. 4U) examined as whole mounts after a 30 min staining with JC-1. (R) The intensity and distribution of J-aggregate fluorescence at 4 h in untreated mouse COCs in LOA conditions. (S–Z) The differential effects on ΔΨm of culturing cleavage stage embryos (human, S–V) and blastocyst (mouse, W–Z) in the presence or absence of cumulus cells (masses or cell cultures). Normal subplasmalemmal J-aggregate fluorescence (T) is undetectable in the presence of cumulus cells (U) and restored when embryos were relocated to cell-free areas in the same culture (V). The typical high-intensity J-aggregate fluorescence detected in the mural trophectoderm (mTR, W and X), and to a lesser extent in polar trophectoderm (pTR, W and X) of the expanded mouse blastocyst was abolished during co-culture with mouse or human cumulus masses or primary cumulus cultures (Y), but reappeared within minutes when embryos were cultured in cell-free areas (Z). BN, binucleated blastomeres. Figure 5: View largeDownload slide (A and D) J-aggregate fluorescence in denuded MI and MII human oocytes cultured in an atmosphere containing ~20 or 4.5% O2, respectively. (B and C) Images of J-aggregate fluorescence in oocytes cultured in the presence of a nitric oxide (NO) donor at ~20% O2. J-aggregate fluorescence was absent in oocytes cultured in the presence of a NO donor (SNP) at 4.5% O2 (E) but returned to normal (F) during culture in SNP-free medium. At higher SNP concentrations, levels of cytoplasm JC-1 fluorescence were reduced (G), but returned to normal in the absence of the donor (H). Exposure of mouse COCs to L-NAME, an inhibitor of nitric oxide synthase for 1–4 h (I,K and N) showed a progressive increase in subplasmalemmal J-aggregate fluorescence (L and M, 2 h; FITC and RITC channels, respectively; O, 3 h). At 4 h, the intensity of the fluorescent signal in oocytes observed under compression (P, FITC channel; Q, RITC channel), was similar to levels observed in denuded oocytes (see Fig. 4U) examined as whole mounts after a 30 min staining with JC-1. (R) The intensity and distribution of J-aggregate fluorescence at 4 h in untreated mouse COCs in LOA conditions. (S–Z) The differential effects on ΔΨm of culturing cleavage stage embryos (human, S–V) and blastocyst (mouse, W–Z) in the presence or absence of cumulus cells (masses or cell cultures). Normal subplasmalemmal J-aggregate fluorescence (T) is undetectable in the presence of cumulus cells (U) and restored when embryos were relocated to cell-free areas in the same culture (V). The typical high-intensity J-aggregate fluorescence detected in the mural trophectoderm (mTR, W and X), and to a lesser extent in polar trophectoderm (pTR, W and X) of the expanded mouse blastocyst was abolished during co-culture with mouse or human cumulus masses or primary cumulus cultures (Y), but reappeared within minutes when embryos were cultured in cell-free areas (Z). BN, binucleated blastomeres. Whereas J-aggregate fluorescence was undetectable after exposure to SNP in an LOA, the typical pattern and intensity of J-aggregate fluorescence was completely restored after transfer to normal JC-1-containing medium (in the same atmosphere), including all oocytes that had been exposed to SNP at high concentration (15 min, Fig. 5F). For example, the oocyte in Fig. 5H is the same as in Fig. 5G, and shows the restoration of subplasmalemmal J-aggregate fluorescence after a 30 min culture in normal medium. Similar to the oocyte transfer/repositioning studies with cumulus masses and primary cumulus cell cultures described above, the magnitude of ΔΨm in subplasmalemmal cytoplasm of human oocytes could be reversibly down- (similar to Fig. 5E) and up-regulated (similar to Fig. 5H) over several cycles with sequential culture in the presence or absence of SNP under conditions of LOA. These findings indicate that NO may have a significant influence on ΔΨm and that the magnitude of the effect may be related to O2 concentration, as indicated by the differential response to SNP under LOA and HOA conditions. Effects of NOS inhibition on Δψm in subplasmalemmal mitochondria Figure 5I, K and N are light microscopic images of newly ovulated mouse COCs containing MII oocytes and observed under compression after 1 (n = 15), 2 (n = 16) and 4 h (n = 12) of culture in an LOA, respectively, in the presence of the nitric oxide synthase (NOS) inhibitor, L-NAME; Figure 5J, L and O are the corresponding JC-1 fluorescent patterns seen in the FITC channel. All images closely approximate the actual intensity of JC-1 (green) and J-aggregate (orange) fluorescence observed under compression, which as described above, detects punctate J-aggregate fluorescence throughout the subplasmalemmal cytoplasm in the same approximate plane of focus. J-aggregate fluorescence was undetectable in all oocytes (n = 40) during the first hour of treatment with L-NAME. However, within 2 h, J-aggregate fluorescence was evident in the subplasmalemmal cytoplasm (n = 40; arrows, Fig. 5L, FITC channel; Fig. 5M, RITC channel). At 4 h (n = 25), levels of J-aggregate fluorescence in the subplasmalemmal cytoplasm of intact oocytes were similar to those observed in denuded oocytes examined under compression (FITC channel, Fig. 5P; RITC channel, Fig. 5Q). Under HOA conditions, J-aggregate fluorescence was detectable in intact mouse oocytes (n = 20) within the first hour of culture in the presence of L-NAME (similar to Fig. 5M) and by 2 h, was similar to patterns observed at 4 h under LOA conditions (i.e. similar to Fig. 5P and Q). This finding indicates that (i) suppression of NOS activity in COCs is associated with the development of high polarity in the subplasmalemmal mitochondria of the corresponding oocyte, and (ii) the apparent rate at which high ΔΨm occurs is influenced by the concentration of ambient oxygen. The specific effect of L-NAME-treated cumulus cells on oocyte ΔΨm was confirmed by culturing denuded MII human (n = 12) and mouse (n = 60) oocytes in the presence of L-NAME for 4–6 h, under LOA and HOA conditions. As expected, the intensity and distribution of J-aggregate fluorescence in the subplasmalemmal cytoplasm was the same as observed in their untreated (denuded) siblings (similar to Figs 1B and 4U). After exposure to L-NAME (LOA culture), mouse and human cumulus masses were rinsed through several changes of inhibitor-free medium and cultured in the same medium for 5 h, at which time the medium was exchanged with freshly equilibrated medium containing JC-1. After 1 h of culture, mouse MII (n = 30) and human MI (n = 18) and MII human oocytes (n = 12) were deposited on the surface of the masses, as described above, and stained for 60 min. None of the oocytes exhibited J-aggregate fluorescence (similar to Figs 3C and 4X). When repositioned away from the mass, a normal subplasmalemmal signal was evident in all oocytes within 30 min (similar to Figs 1B for human and 4P for mouse). This finding indicates that the suppression of NOS activity by L-NAME in human and mouse cumulus oophorus masses is reversible. The timing and extent of spontaneous J-aggregate formation in untreated mouse COCs was examined at 1–2 h intervals during a 16-h culture under LOA (n = 8 COCs/time point) and HOA conditions (n = 5 COCs/time point). Under HOA conditions, the pattern of subplasmalemmal J-aggregate fluorescence at 3 h was similar to (i) sibling COCs exposed to L-NAME for 1 h (similar to Fig. 5P and Q) and (ii) denuded oocytes stained with JC-1 for 30 min. Under LOA conditions, subplasmalemmal J-aggregate fluorescence remained relatively scant for as long as the cumulus oophorus remained intact (e.g. asterisk, Fig. 5R, 6 h). The spontaneous detachment of portions of the cumulus oophorus began at ∼8 h of culture and was accompanied by an increased density of J-aggregate fluorescence that reached levels similar to those shown in Fig. 5M and Q. This finding suggests that that the suppression of high polarity is associated with the degree to which an intact corona radiata and cumulus oophorus persists. The possibility that NO produced by cultured cumulus cells directly influences ΔΨm in the oocyte was examined by treating primary mouse and human cumulus cell cultures with L-NAME (for 8 h under LOA conditions), followed by the placement of denuded MII mouse (n = 30) and human (n = 12) oocytes on the surface a confluent monolayer. As described above, oocyte co-culture under conditions of LOA (but not HOA conditions) is associated with the absence of J-aggregate fluorescence. The occurrence of circumferential J-aggregate fluorescence in all oocytes (similar to Figs 3H and 4U) in L-NAME-treated cultures supports the suppression of NOS activity and NO production. After JC-1 staining, cumulus cultures were washed through several changes of medium, and then cultured for 12 h in the same atmosphere in the absence of L-NAME. The medium was exchanged for fresh medium containing JC-1 and denuded mouse (n = 40) and human (n = 7) oocytes were deposited on the surface of the cumulus cell monolayers. Similar to findings obtained with washed cumulus masses described above, all mouse (similar to Fig. 4H) and human oocytes (similar to Fig. 3C) were J-aggregate-negative, indicating that the effect of L-NAME on NOS activity in primary cumulus cell cultures was reversible. Influence of cumulus cells on Δψm during the preimplantation stages Whether the apparent depressive influence of NO on ΔΨm in the subplasmalemmal cytoplasm of the oocyte extended to mitochondria in preimplantation stage embryos, especially at the blastocyst stage, where mitochondrial development is well advanced, was examined by culturing human and mouse cleavage (2—4-cell; human, n = 11; mouse, n = 90), morula (human, 8—16-cell, n = l5; mouse, n = 70) and expanded blastocyst stage embryos (mouse, n = 35) in the presence of mouse or human cumulus masses, or cumulus cell cultures, as described above. The normal subplasmalemmal J-aggregate fluorescence observed in cleavage and morula stage blastomeres (Van Blerkom et al., 2002; human, Fig. 5S and T) was abolished when embryos were cultured on the surface of, or in close proximity to, intact cumulus masses or primary cell cultures (Fig. 5U), but returned when the same embryos were relocated to cell-free areas in the same culture dish (Fig. 5V). Binucleated (BN) human blastomeres are indicated by arrows in Fig. 5T and V. A similar depressive effect was observed for mouse blastocysts (Fig. 5W), where high intensity J-aggregate fluorescence typical of the trophectoderm (mural trophectoderm, mTR; polar trophectoderm, pTR, FITC channel, Fig. 5X) was reduced or abolished (Fig. 5Y). Restoration of subplasmalemmal J-aggregate fluorescence in trophectodermal cells occurred within 15 min of repositioning embryos away from the cumulus masses or high-density regions of cumulus cell cultures (Fig. 5Z). The positional manipulations had no effect on the inner cell mass (ICM, Fig. 5Y), whose mitochondria are normally low polarized (Fig. 5X, see below). These findings indicate that the influence of cumulus cells on ΔΨm is not stage-specific, species-specific or related to state of mitochondrial development. Discussion The association between mitochondria and developmental competence in the oocyte and early embryo has focused primarily on the relationship between bioenergetic capacity and the normality of stage-specific developmental processes (Muller-Hocker et al., 1996; Dumollard et al., 2007; Harvey et al., 2007; Van Blerkom, 2004, 2008, for reviews). The adverse developmental consequences of a bioenergetic deficit that may be associated with a subnormal mitochondrial complement in the oocyte (Santos et al., 2006; Zeng et al., 2007) or between blastomeres during early cleavage (resulting from disproportionate mitochondrial inheritance: Van Blerkom et al., 2000; Katayama et al., 2006; Shourbagy et al., 2006), indicate apparent numerical thresholds for these organelles that may be central to the normality of meiotic maturation, fertilization and preimplantation embryogenesis (May-Panloup et al., 2007; Shoubridge and Wai, 2007). While mitochondria in the oocyte and early embryo are viewed as largely homogenous with respect to morphology, function and activity, a spatially stable subplasmalemmal domain of organelles characterized by a high ΔΨm has been suggested to be competence-related (Van Blerkom, 2004, 2008; Van Blerkom and Davis, 2007). The bioenergetic contribution of mitochondria in this domain to the total cytoplasmic ATP appears to be marginal (Van Blerkom and Davis, 2007). However, their unique spatial position and the known influences of ΔΨm on mitochondrial functions suggest specialized or local regulatory functions during early development (Van Blerkom, 2008). For example, if they are spontaneously eliminated by minor fragmentation or subject to disproportionate segregation between blastomeres, the domain is not reconstituted (Van Blerkom and Davis, 2006), and in the human, affected blastomeres (with negligible loss of cytoplasm and mitochondria to fragments) cease to divide. A role for high-polarized mitochondria in fertilization was suggested by the failure of sperm penetration and cortical granule exocytosis (but not attachment to the oolemma) to occur in zona-free mouse oocytes when the magnitude of ΔΨm was experimentally reduced; these early events in the fertilization process took place when ΔΨm returned to normal levels (Van Blerkom and Davis, 2007). High-polarized mitochondria are detected with potentiometric probes that report differences in inner mitochondrial transmembrane potential (ΔΨm, polarity), and in this regard, the fluorescent stain JC-1 has been especially useful (Salvioli et al., 1997; Van Blerkom et al., 2002, 2003). After incorporation into the mitochondrial matrix and excitation at appropriate frequencies, mitochondria whose ΔΨm −100 mV or less fluorescence green, whereas those ≥140 mV fluoresce red (Smiley et al., 1991; Cossarizza et al., 1996). A shift to longer wavelengths (green to red) results from the potential-dependent multimerization of the JC-1 monomer into so-called J-aggregates (Reers et al., 1995). Is the level of Δψm in the oocyte, suppressed by communication with the corona radiata and cumulus oophorus? Our previous studies suggested that gap-junction-mediated communication between the oocyte and TZPs extending from the corona radiata and cumulus oophorus (Albertini, 2004) may be associated with a constitutive down-regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte (Van Blerkom et al., 2002; Van Blerkom, 2004). We proposed that the depressive influence was relieved after the detachment of TZPs at the outset of the pre-ovulatory maturation (i.e. resumption of at the GV stage) and may focally increase mitochondrial activities, such as ATP generation, regulation of free calcium levels or their ability to participate in certain signal transduction pathways (Van Blerkom et al., 2002, 2003). The notion of a specialized or local regulatory role for high-polarized mitochondria was also suggested by stage-specific mitochondrial translocations during mouse oocyte maturation (Van Blerkom and Runner, 1985) that did not seem to include high-polarized organelles localized to the subplasmalemmal domain (Van Blerkom et al., 2002). Here, the finding that J-aggregate fluorescence occurred in the subplasmalemmal cytoplasm GV stage oocytes where the overlying corona radiata and cumulus oophorus had been mechanically removed supports an earlier contention that direct intercellular communication between the oocyte and somatic cell compartments in COCs suppresses a shift to higher ΔΨm (Van Blerkom et al., 2002). Indeed, the present findings showed that the partial domain of J-aggregate fluorescence remained evident after complete cellular denudation, but within ∼15 min, high polarity was detectable throughout the subplasmalemmal cytoplasm. The possibility that a focal up-regulation of ΔΨm could be associated with the detachment of TZPs is an attractive one because prior to the resumption of meiosis, these cellular extensions provide a continuous conduit for the bidirectional flow of regulatory and signaling factors between the somatic and germ cell compartments, including secondary messages such as cyclic AMP, that maintain meiosis in an arrested state. If increased ΔΨm in the subplasmalemmal cytoplasm is an early aspect of pre-ovulatory maturation, exogenous regulation of ΔΨm may involve a similar mechanism of intercellular communication. The present studies confirm previous findings that described a distinct domain of subplasmalemmal high-polarized mitochondria, reported by coincident J-aggregate fluorescence, in denuded mouse and human oocytes between the GV and MII stages (Van Blerkom et al., 2002). In contrast, while J-aggregate fluorescent mitochondria were clearly detectable in the cumulus oophorus, corona radiata and within TZPs (at the GV stage), indicating that JC-1 was reporting high ΔΨm in the somatic cell compartment, high polarity in the subplasmalemmal domain of the corresponding oocyte was not evident until MII, well after TZPs had detached from the oolemma. This finding is inconsistent with the model of ΔΨm regulation by cell contact. Studies with r123 and DAPI, and prolonged staining with JC-1 at higher concentrations, support an earlier conclusion that the absence of J-aggregate fluorescent in the oocyte was not uptake related (Van Blerkom et al., 2002, 2003). Δψm in the subplasmalemmal cytoplasm of the oocyte is regulated by a freely diffusible factor originating from cumulus cells We report that the presence of cumulus cells alone had a pronounced suppressive effect on ΔΨm in the subplasmalemmal cytoplasm of denuded oocytes, and that the magnitude of the effect was proximity-dependent. Transitions in ΔΨm from high and low, and low and high, could be repeated several times for the same oocyte(s) under identical culture conditions, with the only variable being proximity to intact cumulus masses or confluent cumulus cell cultures. Exposure of denuded oocytes to medium extracted from micro-cultures of cumulus cell masses or primary cultures showed no detectable effect on ΔΨm, suggesting that a putative factor was likely of relatively low molecular weight, freely diffusible across the zona pellucida and oolemma, and either short-lived or unstable. The apparent influence of cumulus cells on ΔΨm is not species-specific, as human and mouse oocytes responded in a similar manner when exposed to the others cumulus masses or cultured cells. The possibility that a cumulus-derived factor capable of depressing ΔΨm in the subplasmalemmal cytoplasm is gaseous in nature was suggested by the rapidity with which levels of ΔΨm could be repeatedly up- or down-regulated, and the ‘proximity effect’ described above. NO, a ubiquitous gaseous lipophilic free radical that results from the conversion of L-arginine to NO and L-citrulline by NOS, was considered a likely candidate involved in the regulation of ΔΨm for the reasons set out in Table I. Table I. Properties of NO compatible with a role in the regulation of mitochondrial polarity. (i) NO effects mitochondrial respiration by competing with O2 binding to the ‘oxygen sensor,’ the heme moiety of the terminal acceptor in the electron transport chain, cytochrome C oxidase. (ii) Cytochrome C oxidase is located on the inner mitochondrial membrane and catalyzes the oxidation of cytochrome C and the reduction of O2 to water, a process coupled with the outward pumping of protons from the mitochondrial matrix (Erusalimsky and Moncada, 2007). Outward proton pumping across the inner mitochondrial membrane creates a proton gradient that has two components, a ΔΨm and a pH gradient, and the energy stored in either component drives the conversion of ADP to ATP by respiratory chain enzymes. (iii) NO has stimulatory or depressive effects on ΔΨm related to metabolic state and the local redox environment of the cytoplasm, and competition with oxygen at the level of the inner mitochondrial membrane is regulative with respect to respiration (Erusalimsky and Moncada, 2007). (iv) NO has a half-life measured in seconds and is rapidly and freely diffusible across cell membranes (Fostermann et al., 1984; Huet-Hudson, 2007). (i) NO effects mitochondrial respiration by competing with O2 binding to the ‘oxygen sensor,’ the heme moiety of the terminal acceptor in the electron transport chain, cytochrome C oxidase. (ii) Cytochrome C oxidase is located on the inner mitochondrial membrane and catalyzes the oxidation of cytochrome C and the reduction of O2 to water, a process coupled with the outward pumping of protons from the mitochondrial matrix (Erusalimsky and Moncada, 2007). Outward proton pumping across the inner mitochondrial membrane creates a proton gradient that has two components, a ΔΨm and a pH gradient, and the energy stored in either component drives the conversion of ADP to ATP by respiratory chain enzymes. (iii) NO has stimulatory or depressive effects on ΔΨm related to metabolic state and the local redox environment of the cytoplasm, and competition with oxygen at the level of the inner mitochondrial membrane is regulative with respect to respiration (Erusalimsky and Moncada, 2007). (iv) NO has a half-life measured in seconds and is rapidly and freely diffusible across cell membranes (Fostermann et al., 1984; Huet-Hudson, 2007). View Large NO is an important intracellular and intercellular signaling molecule that regulates gene expression (Bogdan, 2001) and redox signal transduction pathways (Huang et al., 1999; Kozhukhar et al., 2006). NO is produced in the ovarian follicle by cell-type-specific NOS isoforms (Zackisson et al., 1996; Jablonka-Shariff and Olson, 1997, 1998; Yamagata et al., 2002; Bu et al., 2003; Mitchell et al., 2004), and has pleiotropic influences on follicular biology that include the regulation of steroidogenesis, survival of pre-ovulatory granulosa cells and ovulation (Olson et al., 1996; Jablonka-Shariff and Olson, 1998; Nakamura et al., 2002; Maul, 2003; Mitchell et al., 2004). NO may have a direct effect on the normality of oocyte maturation via cGMP, cAMP and mitogen-activated protein kinase signaling pathways (Lander et al., 1996; Jablonka-Shariff and Olson, 1998; Sengoku et al., 2001; Nakamura et al., 2002; Bu et al., 2003; 2004; Tao et al., 2004; Viana et al., 2007). NO, O2 and Δψm Here, the main finding is that cumulus cells can modulate the level of ΔΨm in the oocyte, and results with SNP and L-NAME suggest that the putative regulatory agent is NO (summarized in Fig. 6). Because NO modulation of respiration occurs in an oxygen-dependent manner associated with competition between these gases at the mitochondrial level, interpretations of the effects of NO on mitochondrial activity can be problematic when studies are performed under normal atmospheric conditions. In vivo, cells normally experience O2 tensions ≤6% (Palacios-Callender et al., 2004; Erusalimsky and Moncada, 2007), and this level is especially relevant here because the pre-ovulatory intrafollicular milieu in women is hypoxic, with O2 concentrations between ∼1 and ≤4% typical of gonadotrophin-stimulated follicles (Van Blerkom et al., 1997; Huey et al., 1999). The importance of O2 concentration was indicated in the present study. When oocytes were treated with SNP in 95% air, there was little, if any, detectable effects on J-aggregate fluorescence, even at relatively high concentration. In contrast, culture in a 4.5% O2 atmosphere abolished detectable J-aggregate fluorescence, which was restored when the NO donor was removed. For intact COCs exposed to L-NAME, the rapidity with which high ΔΨm was detected in the subplasmalemmal ooplasm was related to atmospheric O2 concentration. These findings suggest that the level of ambient O2 at the surface of the oolemma may mediate the suppressive influence of NO on ΔΨm and possibly, corresponding levels of organelle activity. Figure 6: View largeDownload slide Diagrammatic summary of experimental evidence for the role of nitric oxide (NO) in the regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte. Cumulus-enclosed oocytes (top centre) showed no J-aggregate staining for polarized mitochondria while a band of polarized mitochondria (red) appeared in areas which had been partially denuded (top right). Cumulus-enclosed oocytes cultured in the presence of the nitric oxide synthase (NOS) inhibitor L-NAME developed J-aggregate staining (red) similar to fully denuded oocytes (centre) implicating a role for nitric oxide. Denuded oocytes cultured in close proximity to cumulus cells lost J-aggregrate staining (middle left) and this property was abolished by treating the cumulus cells with L-NAME (bottom left). Further evidence comes from treatment of denuded oocytes with the nitric oxide donor SNP in low oxygen resulting in no J- aggregate staining (middle right) although this effect was not seen in high oxygen (bottom right). Figure 6: View largeDownload slide Diagrammatic summary of experimental evidence for the role of nitric oxide (NO) in the regulation of ΔΨm in the subplasmalemmal cytoplasm of the oocyte. Cumulus-enclosed oocytes (top centre) showed no J-aggregate staining for polarized mitochondria while a band of polarized mitochondria (red) appeared in areas which had been partially denuded (top right). Cumulus-enclosed oocytes cultured in the presence of the nitric oxide synthase (NOS) inhibitor L-NAME developed J-aggregate staining (red) similar to fully denuded oocytes (centre) implicating a role for nitric oxide. Denuded oocytes cultured in close proximity to cumulus cells lost J-aggregrate staining (middle left) and this property was abolished by treating the cumulus cells with L-NAME (bottom left). Further evidence comes from treatment of denuded oocytes with the nitric oxide donor SNP in low oxygen resulting in no J- aggregate staining (middle right) although this effect was not seen in high oxygen (bottom right). Intrafollicular hypoxia may enhance the suppressive effect of cumulus-derived NO owing to the competition between these gases, as described above. This may explain the relatively rapid appearance of high polarity in denuded oocytes cultured in an LOA and the absence of a SNP effect on high ΔΨm in near normal atmospheric conditions, which may reflect reduced competition with NO at a non-physiological O2 concentration. The findings also indicate that the concentration of NO at the surface of intact cumulus masses or confluent cumulus cell cultures is sufficient to suppress ΔΨm to levels below the threshold (≤140 mV) required for J-aggregate formation (Smiley et al., 1991; Reers et al., 1995) in a low oxygen environment. The relatively short half-life of NO may confine the reduction of ΔΨm to the subplasmalemmal domain if this region of the ooplasm is the site of maximal intracellular competition. In this regard, recent mathematical modeling of O2 distribution within COCs suggests that levels at surface of the zona pellucida are likely the same as in the follicular fluid (Clark et al., 2006). The dynamic balance between NO and O2 at the surface of the oolemma may change during the pre-ovulatory period if higher O2 concentrations occur in follicular fluid as a result of increased rates of diffusion from the perifollicular microvasculature (Van Blerkom and Trout, 2007, for review). Whether the diffusion of exogenous NO extends deeper into the cytoplasm and occurs internally at levels sufficient to influence the polarity of mitochondria distant from the oolemma could not be determined under the conditions used. However, as noted above, the ΔΨm of these mitochondria is normally below the level required for J-aggregate formation. We are currently investigating other methods to assess whether the ratio between NO and O2 influences polarity in mitochondria located in the interior of the ooplasm (Lemasters and Ramshesh, 2007). Possible developmental relevance of exogenous regulation of Δψm by NO It has been suggested that high-polarized mitochondria may be involved in the focal generation of elevated levels of ATP or may be more active in calcium release and sequestration (homeostasis) than their lower polarized counterparts and as such, could have spatially related regulatory roles in the oocyte and early embryo (Van Blerkom and Davis, 2007; Van Blerkom, 2008). Based on the evidence described above, the detectable influence of cumulus cells on ΔΨm in the subplasmalemmal domain appears to be directly mediated by NO. In the intact state, NO may enter the perivitelline space or the immature oocyte through TZPs. After detachment, these cellular extensions may still provide a conduit for NO as they persist at high density on the inner surface of the zona pellucida and remain associated with functional coronal and cumulus cells (Makabe and Van Blerkom, 2006). If mitochondrial activity in the subplasmalemmal cytoplasm is ΔΨm related, it could be under exogenous control prior to ovulation and progressively relaxed during the peri-ovulatory and early post-ovulatory periods. Because NO can de-energize mitochondria at the low O2 concentrations normally experienced in cells and tissues (Schweizer and Richter, 1994), the suppression of high polarity observed in vitro (under low O2 conditions) may be experienced in vivo and associated with a ‘metabolic homeostasis’ that depresses ATP generation and other energy requiring processes localized to the subplasmalemmal domain of the follicular oocyte. In this respect, the ‘quiet rules’ for metabolism that Leese et al. (2007) relate to viability for the preimplantation embryo may extend to the follicular oocyte. The up-regulation of ΔΨm in the ovulated MII oocyte may temporally coincide with, or facilitate, early events in fertilization (Van Blerkom and Davis, 2007). Two isoforms of NOS, endothelial (eNOS) and inducible (iNOS), have been identified by immunofluorescence in MII oocytes and preimplantation stage mouse embryos (Nishikimi et al., 2001). However, if the very weak staining detected by these investigators in the oocyte is indicative of low NOS of activity, NO regulation of ΔΨm may be largely, if not entirely, exogenous in origin. In contrast, up-regulation of NOS activity after fertilization suggests an endogenous source of NO in the early embryo that could influence ΔΨm in a manner similar to the depression observed here with cumulus masses or cumulus cell cultures. If modulation of mitochondrial activity in vivo involves endogenously produced NO, the stage-specific timing of increased NOS activity (Manser et al., 2004) may coincide with the natural loss of an exogenous source. Such a transition could influence patterns of gene expression in the early embryo, and may become particularly relevant at the blastocyst stage, where NOS activity is localized to the ICM (Nishikimi et al., 2001), whose structurally developed mitochondria normally occur in a low-polarized state (Van Blerkom et al., 2006). The present findings demonstrate that similar to their counterparts in the oocyte, high polarity in the developing mitochondria of the preimplantation stage embryo is also sensitive to suppression by cumulus cells that we propose involves exogenous NO. Whether spatially related changes in ΔΨm have specific coincident (local) effects on developmentally significant processes in the oocyte requires further study. Confirmation of such an association may also provide new insights into the origins of differential oocyte/embryo competence and its relationship to specific intrafollicular conditions (Van Blerkom and Trout, 2007, for review). In the same respect, the extent to which early events in embryonic development are associated with an elevation of ΔΨm in the subplasmalemmal cytoplasm needs to be investigated in the context of the present findings, because penetration, fertilization and early cleavage occur while the oocyte is still enclosed by or associated with residual cumulus and coronal cells, which as shown here, retain the capacity to depress ΔΨm. However, studies of fertilization in zona-free mouse oocytes showed that penetration and cortical granule exocytosis occurred in regions of the oolemma where the subjacent mitochondria were high polarized (Van Blerkom and Davis, 2007) and as reported here, this state develops in the MII oocyte after ovulation. References Albertini D. Van Blerkom J, Gregory L. Oocyte-granulosa cell interactions, Essential IVF: Basic Research and Clinical Applications. , 2004 Boston Kluwer Academic Publishers(pg. 43- 58) Bogdan C. 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Molecular Human Reproduction – Oxford University Press

Published: Jun 30, 2008

Keywords: Keywords mitochondria mitochondrial polarity nitric oxide cumulus cells regulation of mitochondrial activity

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Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

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Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

Regulation of mitochondrial polarity in mouse and human oocytes: the influence of cumulus derived nitric oxide

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