Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat... Abstract Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes. Introduction If not fertilized or activated in time after ovulation, mammalian oocytes undergo a time-dependent process of aging. This postovulatory oocyte aging process can occur in vivo [1, 2] or in vitro [2, 3], and it is different from the ovarian aging, which refers to oocyte exposure to aged ovarian environment before ovulation [4]. Postovulatory oocyte aging have marked detrimental effects on embryo development [5, 6] and offspring [7, 8], and has been considered as one of the major causes for gradual decline in population size of several threatened mammalian species [9]. Thus, studies on the mechanisms and control of oocyte aging are very important for both normal and assisted reproduction. However, the mechanisms for oocyte aging are largely unknown. One of the earliest manifestations for aging oocytes is a spontaneous increase in the susceptibility to activation stimuli (STAS) [10, 11]. Many studies failed to obtain rat offspring following transfer of somatic cell nuclei [12, 13]. Unlike oocytes from other species, the rat oocytes undergo spontaneous activation (SA) soon after their release from the oviduct, due to the spontaneous increase in STAS [14, 15]. Premature chromosome condensation was not observed after transfer of somatic cell nuclei into enucleated rat oocytes [16], suggesting that these nuclei might not be properly reprogrammed due to oocyte SA [17]. Thus, understanding the mechanisms for SA and thence inhibiting SA of rat oocytes is of great importance for successful rat cloning. The activation of mammalian oocytes at fertilization [18] or at parthenogenetic activation [19] is always associated with intracellular Ca2+ oscillations. Furthermore, our recent studies observed cytoplasmic Ca2+ increases during SA of rat oocytes [13, 20]. It is known that free calcium ions from both extra- and intracellular sources can enter the cytoplasm through various calcium channels. Among the important calcium channels, the T- and L-type calcium channels are low- and high-voltage-activated calcium channels, respectively, which aid in mediating calcium influx into cells [21]. The ryanodine receptor (RyR) is a ligand-gated calcium channel, which causes release of calcium from intracellular calcium stores such as the sarcoplasmic reticulum when opened by ligand binding [22]. The Na+/Ca2+ exchanger (NCX) uses the electrochemical gradient of Na+ across the plasma membrane to exchange three Na+ ions into the cell for the extrusion of one Ca2+ ion [23]. The presence of L-type calcium channels was reported in rat oocytes [24, 25], and the T-type calcium channels was observed in mouse oocytes [26]. The RyR channels were reported in both pig [27] and Rhinella arenarum oocytes [28]. Furthermore, the NCX was found to play an important role in promoting calcium efflux in aging rat and mouse oocytes [13, 20]. The calcium-sensing receptor (CaSR) is a recently discovered G-protein-coupled receptor that senses extracellular Ca2+ levels. Extracellular Ca2+ elevations elicit a conformational change in CaSR, which activates phospholipase C (PLC) through a Gqα type of G protein [29]. The activated PLC hydrolyzes the phosphatidylinositol 4,5-bisphosphate (PIP2), leading to the production of inositol 1,4,5-trisphosphate (IP3). IP3 facilitates Ca2+ release from the internal Ca2+ store through interactions with the IP3 receptors on the endoplasmic reticulum (ER) [30]. Expression of CaSR has been observed in maturing oocytes of human [31], equine [32], and porcine species [33], and the beneficial effects of CaSR agonist and the detrimental effects of its antagonist on meiotic maturation have been reported in equine [32] and porcine oocytes [33]. However, neither the expression of CaSR in rat oocytes nor its role in aging oocytes of any species has been reported up to date. The objectives of the present study were to verify the expression of CaSR in rat oocytes and to determine its role in regulating STAS of postovulatory aging (POA) oocytes. Materials and methods Animal care and handling were conducted using the experimental procedures approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). Chemicals and reagents were purchased from Sigma Chemical Co. unless otherwise mentioned. Oocyte recovery Rats of the Sprague-Dawley strain were kept in a room with 14L:10D cycles, with the dark period starting from 20:00. Female rats, 23–26 days after birth, were induced to superovulate with 15 IU equine chorionic gonadotropin (eCG), followed 48 h later by 15 IU human chorionic gonadotropin (hCG). Both the eCG and hCG used in this study were from Ningbo Hormone Product Co., Ltd, China. The superovulated rats were euthanized at different times after hCG injection, and the oviductal ampullae were broken to release the oocytes. After being dispersed and washed three times in M2 medium, the oocytes were denuded of cumulus cells by pipetting with a thin pipette in a drop of M2 medium containing 0.1% hyaluronidase. Oocyte aging in vitro For in vitro aging, cumulus-denuded oocytes (DOs) were cultured for 6 h in the aging medium supplemented with different concentrations of agonist and inhibitors of CaSR. The aging medium used was modified rat one-cell embryo culture medium (mR1ECM). To prepare stock solutions, NPS-2143 (5 mM), Cinacalcet (1 mM), Nifedipine (200 mM), Ruthenium Red (10 mM) and ML218 (1.35 mM) were dissolved in dimethyl sulfoxide. All the stock solutions were stored in aliquots at −20°C and diluted to desired concentrations with the aging medium immediately before use. The aging culture was performed in wells of a 96-well culture plate (San-He Medical Instrument Factory, Haimen City, Zhejiang Province, China) at 37°C under 5% CO2 in humidified air; each well contained 200 μl of the aging medium and about 30 oocytes covered with mineral oil. Assessment of oocyte activation Spontaneous activation of rat oocytes was assessed immediately after the aging culture. To observe SA, oocytes were fixed with 3.7% paraformaldehyde in M2 for 30 min at room temperature before being stained with 10 μg/ml Hoechst 33342 and mounted on glass slides. The state of chromosomes was observed under an epifluorescence microscope (Leica DMLB) and was classified into two types. Oocytes with chromosomes compacted at the metaphase plate were considered to be at the metaphase II (MII) stage, whereas oocytes with chromosomes dispersed in the cytoplasm were classified as activated [13]. Measurement for cytoplasmic calcium Rat oocytes were loaded with Ca2+ probe by incubating at room temperature for 20 min in a loading medium. The loading medium used was Hepes-buffered mR1ECM (HR1) medium containing 1 μM Fura-2 AM and 0.02% pluronic F-127. The HR1 medium consisted of 76.7 mM NaCl, 3.2 mM KCl, 2 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 5 mM NaHCO3, 22 mM HEPES, 10 mM sodium lactate, 0.5 mM sodium pyruvate, 7.5 mM glucose, 1 g/L PVA, 0.1 mM glutamine, 2% (V/V) EAA, and 1% (V/V) NEAA. After loading, oocytes were transferred into a drop of HR1 medium in a Fluoro dish (FD35-100, World Precision Instruments) covered with mineral oil and observed with a Leica DMI6000 inverted microscope at 37.5°C. A Fura 2 fluorescence module was used for excitation, and a Leica LAS-AF calcium imaging module was used to calculate the F340/380 ratio, which represented the concentration of cytoplasmic calcium. The oocytes were monitored continuously for 60 min to record the F340/380 ratio. Immunofluorescence microscopy Unless otherwise specified, all the procedures were conducted at room temperature. Oocytes were washed three times in M2 medium between treatments. Denuded oocytes were (a) fixed with 3.7% paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 4 mM MgSO4, pH 7.0) for at least 30 min; (b) treated with 0.25% protease in M2 for 1–2 s to remove zona pellucida; (c) permeabilized with 0.1% Triton X-100 in PHEM for 5 min; (d) blocked for 1 h in PHEM containing 3% bovine serum albumin (BSA); (e) incubated at 4°C overnight with mouse monoclonal anti-CaSR (IgG, 1:200, Abcam) in 3% BSA in M2 medium; (f) incubated for 1 h with Cy3-conjugated goat-anti-mouse IgG (1:800, Jackson ImmunoResearch) in 3% BSA in M2; (g) incubated for 10 min with 10 μg/ml Hoechst 33342 in M2. Negative control samples in which the primary antibody was omitted were also processed. The oocytes were then mounted on glass slides and observed with a Leica laser scanning confocal microscope (TCS SP2). Blue diode (405 nm) and helium/neon (He/Ne; 543 nm) lasers were used to excite Hoechst and Cy3, respectively. Fluorescence was detected with 420–480 nm (Hoechst) and 560–605 nm (Cy3) bandpass emission filters, and the captured signals were recorded as blue and red, respectively. The relative content of CaSR was quantified by measuring the fluorescence intensities. For each experimental series, all high-resolution z-stack images were acquired with identical settings. The relative intensities were measured on the raw images using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD) under fixed thresholds across all slides. The values of freshly ovulated oocytes recovered 13 h post hCG injection were set as 1, and the other values were expressed relative to this quantity. Western blot analysis A total of 200 DOs were placed in a 1.5-ml microfuge tube containing 20-μl sample buffer (20 mM Hepes, 100 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.3 mM PMSF and 3 mg/ml leupetin, pH 7.5) and frozen at −80°C until use. To run the gel, 5 μl of 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to each tube and the tubes were heated at 100°C for 5 min. The samples were separated on a 6% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were washed twice in TBST (150 mM NaCl, 2 mM KCl, 25 mM Tris, 0.05% Tween-20, pH 7.4) and blocked for 1–1.5 h with TBST containing 3% BSA at room temperature. The membranes were then incubated at 4°C overnight with mouse monoclonal anti-CaSR (Abcam, ab19347) or mouse anti-β-tubulin (Merck Millipore, 05-661) at a dilution of 1:500 in 3% BSA-TBST. After being washed three times in TBST (5 min each), the membranes were incubated for 1 h at 37°C with goat anti-mouse IgG AP conjugated (CWBIO, cw0111 or cw0110) diluted to 1:1000 in 3% BSA-TBST. After three washings in TBST, the membranes were detected by a 5-Bromo-4-chloro-3-indolyl phosphate (BCIP)/NBT (Nitro blue tetrazolium) alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, China). The relative quantities of proteins were determined with Image J software by analyzing the sum density of each protein band image. The values of freshly ovulated oocytes were set as 1 and the other values were expressed relative to this quantity. β-tubulin was used as internal controls. Data analysis Each treatment contained at least three replicates. Percentage data were arcsine-transformed before being analyzed with Analysis of variance (ANOVA). A Duncan multiple comparison test was performed to find differences. The software used was SPSS (Statistics Package for Social Science). Data are expressed as mean ± SE and P < 0.05 was considered significant. Results Treatment with NPS-2143, an allosteric inhibitor of CaSR, suppressed both SA and elevation of cytoplasmic calcium in rat oocytes Rat oocytes recovered at 13 or 19 h post hCG injection were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. At the end of the culture, while some of the oocytes were examined for SA, others were placed in HR1 and monitored for cytoplasmic calcium levels. The results show that after culture without NPS-2143, SA rates were significantly higher in 19 h oocytes (69.2 ± 4.1%, n = 103) than in 13 h oocytes (34.6 ± 2.9%, n = 88) (Figure 1A, C13 vs. 0 μM NPS-2143), and that treatment with 2.5 or 5 μM NPS-2143 significantly suppressed SA (69.2 ± 4.1%, n = 103 vs. 30.9 ± 3.5%, n = 92 or 29.9 ± 2.7%, n = 90) and cytoplasmic calcium elevation (0.79 ± 0.04, n = 60 vs. 0.62 ± 0.01, n = 60) of rat oocytes (Figure 1B–D). Figure 1. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 on SA and level of cytoplasmic calcium of rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. C13 indicates that rat oocytes recovered at 13 h post hCG were cultured for 6 h in mR1ECM alone. Each treatment was repeated three times with each replicate containing about 30 oocytes. a, b: Values with a different letter above bars differ significantly (P < 0.02). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with (C) or without (D) 2.5 μM of NPS-2143 during the aging culture. Each treatment was repeated three times and each replicate included 20 oocytes. a, b: Values with a different letter above bars differ significantly (P = 0.04). Figure 1. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 on SA and level of cytoplasmic calcium of rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. C13 indicates that rat oocytes recovered at 13 h post hCG were cultured for 6 h in mR1ECM alone. Each treatment was repeated three times with each replicate containing about 30 oocytes. a, b: Values with a different letter above bars differ significantly (P < 0.02). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with (C) or without (D) 2.5 μM of NPS-2143 during the aging culture. Each treatment was repeated three times and each replicate included 20 oocytes. a, b: Values with a different letter above bars differ significantly (P = 0.04). Levels of CaSR protein in rat oocytes recovered at different times after hCG injection Rat oocytes recovered at 13, 19, 25, and 36 h post hCG injection were examined for CaSR levels using immunofluorescence microscopy and western blotting. When observed under a confocal microscope, CaSR was located both at the plasma membrane and within the cytoplasm in oocytes collected up to 25 h post hCG injection, but the CaSR density, particularly that in the cytoplasm, decreased significantly by 36 h after hCG injection (Figure 2A–D). Immunofluorescence quantification using the same microscopic parameters confirms that the level of total CaSR protein did not change significantly up to 25 h (1 ± 0.01 vs. 1.02 ± 0.11) but decreased significantly by 36 h post hCG injection (1 ± 0.01 vs. 0.53 ± 0.02) (Figure 2E). Western blotting showed one band at about 120 kDa representing the nonglycosylated form, two bands of about 130–140 kDa and 150–160 kDa, which correspond respectively to the immature and mature glycosylated form, and one band at 170–180 kDa representative of the dimeric and active form of the CaSR (Figure 2G). Quantification showed that the functional dimer CaSR protein increased significantly at 19 h (1.54 ± 0.09) and 25 h (1.37 ± 0.17) post hCG injection compared to that in oocytes recovered 13 h post hCG (1.0 ± 0.0) (Figure 2F). By 36 h post hCG injection, the dimer CaSR protein (0.41 ± 0.02) decreased significantly. Figure 2. View largeDownload slide Levels of CaSR in rat oocytes recovered at different times after hCG injection. Micrographs A to D are merged confocal images with DNA and CaSR protein colored blue and red, respectively; A, B, C, and D show oocytes recovered at 13, 19, 25, and 36 h post hCG injection, respectively. Bar is 13 μm. Graph E shows quantification of total proteins of CaSR by immunofluorescence. Each treatment was repeated three times with each replicate containing 30 oocytes. The values of oocytes recovered 13 h post hCG injection were set as 1 and the other values were expressed relative to this quantity. a, b: Values with different letters above bars differ significantly (P < 0.01). Graph F shows quantification of CaSR dimmer by western blotting. Each treatment was repeated three times and each replicate included 200 oocytes. a–c: Values with different letters above bars differ significantly (P < 0.05). Panel G shows full blots of western blotting displaying bands of dimmer, mature glycosylated (Mat-G), immature glycosylated (Imm-G), and nonglycosylated (Non-G) CaSR proteins. Figure 2. View largeDownload slide Levels of CaSR in rat oocytes recovered at different times after hCG injection. Micrographs A to D are merged confocal images with DNA and CaSR protein colored blue and red, respectively; A, B, C, and D show oocytes recovered at 13, 19, 25, and 36 h post hCG injection, respectively. Bar is 13 μm. Graph E shows quantification of total proteins of CaSR by immunofluorescence. Each treatment was repeated three times with each replicate containing 30 oocytes. The values of oocytes recovered 13 h post hCG injection were set as 1 and the other values were expressed relative to this quantity. a, b: Values with different letters above bars differ significantly (P < 0.01). Graph F shows quantification of CaSR dimmer by western blotting. Each treatment was repeated three times and each replicate included 200 oocytes. a–c: Values with different letters above bars differ significantly (P < 0.05). Panel G shows full blots of western blotting displaying bands of dimmer, mature glycosylated (Mat-G), immature glycosylated (Imm-G), and nonglycosylated (Non-G) CaSR proteins. Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA and cytoplasmic calcium levels of rat oocytes Because our previous studies demonstrated that activating NCX with NaCl could inhibit SA of rat oocytes by facilitating calcium efflux from the oocyte [13, 20], this experiment tested whether SA of rat oocytes could be completely suppressed by inhibiting CaSR while activating NCX. Rat oocytes recovered 19 h post hCG injection were cultured for 6 h in mR1ECM alone or with 2.5 μM NPS-2143 or 50 mM NaCl [13, 20] or both. At the end of the treatment, some oocytes were examined for SA, and others were monitored for cytoplasmic calcium levels. Although NaCl inhibited SA to a greater extent (86.2 ± 2.1%, n = 81 vs. 16.6 ± 2.4%, n = 92) than NPS-2143 did (86.2 ± 2.1%, n = 81 vs. 33.6 ± 4.9%, n = 82) (Figure 3), treatment with both NaCl and NPS-2143 did not decrease SA (11.4 ± 2.7%, n = 86 vs. 16.6 ± 2.4%, n = 92) or calcium level (0.44 ± 0.01, n = 60 vs. 0.45 ± 0.01, n = 60) further compared to that achieved with NaCl alone (P > 0.05), suggesting that calcium sources other than the CaSR-controlled channels are contributing to the elevation of cytoplasmic calcium in aging rat oocytes. Figure 3. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA rates and levels of cytoplasmic calcium in rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM medium alone (Ctrl), with 2.5 μM NPS-2143 (NPS) or 50 mM NaCl, or with both NPS-2143 and NaCl (NP + Na). Each treatment was repeated three times with each replicate containing about 30 oocytes. a–c: Values with a different letter above bars differ significantly (P < 0.01). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with NaCl alone (C) or with both NaCl and NPS-2143 (D) during the aging culture. In panel B, each treatment was repeated three times and each replicate included 20 oocytes. a: Values with the same letter above bars do not differ significantly (P = 0.66). Figure 3. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA rates and levels of cytoplasmic calcium in rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM medium alone (Ctrl), with 2.5 μM NPS-2143 (NPS) or 50 mM NaCl, or with both NPS-2143 and NaCl (NP + Na). Each treatment was repeated three times with each replicate containing about 30 oocytes. a–c: Values with a different letter above bars differ significantly (P < 0.01). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with NaCl alone (C) or with both NaCl and NPS-2143 (D) during the aging culture. In panel B, each treatment was repeated three times and each replicate included 20 oocytes. a: Values with the same letter above bars do not differ significantly (P = 0.66). Roles of calcium channels other than CaSR and NCX in controlling STAS of aging rat oocytes Rat oocytes recovered at 19 h post hCG injection were aged for 6 h in mR1ECM containing different concentrations of various calcium channel blockers before examination for SA. All the blockers tested including Nifedipine (a L-type channel blocker), ML218 (a T-type channel blocker), and Ruthenium Red (a RyR channel inhibitor) significantly decreased the SA rates of rat oocytes when used at optimal concentrations (Figure 4A–C). When Nifedipine, ML218, and NPS-2143 were used in combination, SA rates decreased to minimum (70.19 ± 2.8%, n = 97 vs. 11.6 ± 1.0%, n = 94) (Figure 4D). When Ruthenium Red was added to the combination, however, the SA rate did not decreased further (11.6 ± 1.0%, n = 94 vs. 9.0 ± 0.4%, n = 101) suggesting that Ruthenium Red had a mild effect on SA of rat oocytes in the presence of other calcium channel blockers. Figure 4. View largeDownload slide Rates of SA after rat oocytes recovered at 19 h post hCG injection were cultured for 6 h with different concentrations of various calcium channel blockers. Graphs A, B, and C show percentages of SA oocytes after treatment with various concentrations of Ruthenium Red, Nifedipine, and ML218, respectively. Graph D shows SA rates after oocytes were treated with Nifedipine, ML218, and NPS-2143 in different combinations. In graph D, optimal concentrations of Nifedipine (Nife, 200 μM), ML218 (M218, 0.85 μM), NPS-2143 (NPS, 2.5 μM), and Ruthenium Red (RuR, 15 μM) were used. Each treatment was repeated three times with each replicate containing about 30 oocytes. a–e: Values without a common letter above bars differ significantly (P < 0.05). Figure 4. View largeDownload slide Rates of SA after rat oocytes recovered at 19 h post hCG injection were cultured for 6 h with different concentrations of various calcium channel blockers. Graphs A, B, and C show percentages of SA oocytes after treatment with various concentrations of Ruthenium Red, Nifedipine, and ML218, respectively. Graph D shows SA rates after oocytes were treated with Nifedipine, ML218, and NPS-2143 in different combinations. In graph D, optimal concentrations of Nifedipine (Nife, 200 μM), ML218 (M218, 0.85 μM), NPS-2143 (NPS, 2.5 μM), and Ruthenium Red (RuR, 15 μM) were used. Each treatment was repeated three times with each replicate containing about 30 oocytes. a–e: Values without a common letter above bars differ significantly (P < 0.05). Discussion In this study, our immunofluorescence microscopy showed that CaSR were localized both at the plasma membrane and in the cytoplasm of rat oocytes. By immunofluorescence microscopy, Dell’Aquila et al. [31] observed CaSR expression in human GV, MI, and MII oocytes, and they found an ubiquitous distribution of CaSR along the oolemma and within the cytoplasm. De Santis et al. [32] demonstrated CaSR at the plasma membrane and, more pronounced, within the cytoplasm of equine oocytes at GV, MI, and MII stages of meiosis. Thus, CaSR are localized both at the plasma membrane and in the cytoplasm of mammalian oocytes. Our western blotting revealed that rat oocytes contained four bands of 120, 130–140, 150–160, and 170–180 kDa, representing the nonglycosylated, immature, and mature glycosylated and dimeric forms of CaSR, respectively. The results were exactly similar to those reported in human vascular smooth muscle cells [34]. By western blot analysis, Dell’Aquila et al. [31] and De Santis et al. [32] revealed a single CaSR protein of 130 kDa in human and equine MII oocytes, respectively, and Liu et al. [33] observed a 160 kDa CaSR protein in pig oocytes. It is known that CaSR present at the plasma membrane functions as a homodimer in performing its central role of sensing minute alterations in extracellular calcium [35, 36]. For example, studies have suggested that the constitutive CaSR dimerization occurs in the ER and is necessary for exit of the receptor from the ER and trafficking to the cell surface [36]. Studies using natural CaSR mutations have also emphasized the functional importance of CaSR dimerization [37]. Furthermore, it has been reported that activation of CaSR promotes its expression, trafficking, and membrane insertion. For example, the agonist-driven maturation and plasma membrane insertion of CaSR were found to dynamically control signal amplitude [38]. In the present study, although our immunofluorescence indicated that the density of total CaSR protein remained constant up to 25 h post hCG injection, our western blotting showed that the functional dimer protein of CaSR increased significantly from 13 to 25 h after hCG injection in aging rat oocytes. This suggested that STAS in aging oocytes was positively correlated with the level of the functional dimer protein rather than its total protein of CaSR, and that the activation of CaSR by certain stimuli had facilitated the dimerization of CaSR monomers. Both previous studies [10, 39] and the present results have demonstrated that aged oocytes are more susceptible to activating stimuli than newly ovulated oocytes. Possible pathways leading to cytoplasmic calcium elevation and SA have been proposed for rat oocytes recovered at 19 h post hCG injection (Figure 5). Undefined stimuli concomitant with oocyte release from the oviduct activate CaSR as well as the L- and T-channels. While the activated T- and L-channels open to allow a direct influx of extracellular calcium into the oocyte [21], the activated CaSR facilitates Ca2+ release from the internal Ca2+ store into the cytoplasm [30]. The increased calcium is then pumped out of the oocyte by NCX [13, 20], which further activates CaSR and promotes calcium release from calcium store, leading to a second elevation in cytoplasmic calcium. The resultant repeated increases in cytoplasmic calcium lead to SA in aged rat oocytes. Thus, the present results that the level of the functional dimer protein of CaSR increased with postovulatory oocyte aging have provided new evidence that activation of CaSR is a major factor causing the difference in STAS between newly ovulated and aged oocytes. Figure 5. View largeDownload slide A diagram showing the possible pathways leading to cytoplasmic calcium elevation and SA of rat oocytes recovered at 19 h post hCG injection. Numbers in brackets indicate the precedence of the calcium events. For detailed explanations, please refer to text in the Discussion section. Figure 5. View largeDownload slide A diagram showing the possible pathways leading to cytoplasmic calcium elevation and SA of rat oocytes recovered at 19 h post hCG injection. Numbers in brackets indicate the precedence of the calcium events. For detailed explanations, please refer to text in the Discussion section. The current results demonstrated that treatment with T- or L-type calcium channel blockers significantly reduced SA of rat oocytes, and that suppression of CaSR together with these major calcium channels reduced SA to minimum. This suggested that the control of cytoplasmic calcium involves multiple channels in aging rat oocytes. It is known that free calcium ions from both extra- and intracellular calcium stores can enter the cytoplasm of oocytes. For example, during A23187-induced activation of pig oocytes, it was the influx of extracellular calcium that gave rise to the intraoocyte calcium elevations [40]. Yoo and Smith [24] observed that SA of rat oocytes was significantly decreased in calcium-free medium or in calcium-containing medium supplemented with L-type calcium channel blocker or IP3R inhibitor, suggesting that both the L-type calcium channel and CaSR are involved in the control of SA in rat oocytes. Extracellular Ca2+ elevations activate CaSR, which promotes the production of IP3 by hydrolyzing PIP2 [29]. IP3 is a Ca2+-releasing second messenger, which causes the release of intracellular calcium by interacting with IP3 receptors [41]. It has been demonstrated that the IP3-induced intracellular calcium release plays an important role in the regulation of meiosis in rabbit [42] and cattle [43] oocytes. In summary, we have studied the expression of CaSR and its role in regulating STAS of POA rat oocytes. The results indicate that CaSR is expressed in rat oocytes, and the STAS of aging rat oocytes was positively correlated with the level of the functional dimer protein of CaSR. Suppression of all the calcium channels tested reduced SA to minimum, suggesting that blocking multiple calcium channels might be used to control SA of aging rat oocytes. This is the first report on expression of CaSR in rat oocytes and on the role of CaSR in regulating oocyte aging. The data are important not only for understanding the mechanisms for oocyte POA, but also for controlling SA of rat oocytes. Footnotes † Grant Support: This study was supported by grants from the National Key R&D Program of China (Nos. 2017YFC1001601 and 2017YFC1001602), the National Basic Research Program of China (Nos. 2014CB138503), the China National Natural Science Foundation (Nos. 31772599 and 31702114), the Research Foundation of Northeast Agricultural University (No. 106518001), and the Funds of Shandong Double Tops Program (No. SYL2017YSTD12). Notes Edited by Dr. Melissa E. Pepling, PhD, Syracuse University. References 1. Yanagimachi R, Chang MC. 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Google Scholar PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of Reproduction Oxford University Press

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes

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Abstract

Abstract Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes. Introduction If not fertilized or activated in time after ovulation, mammalian oocytes undergo a time-dependent process of aging. This postovulatory oocyte aging process can occur in vivo [1, 2] or in vitro [2, 3], and it is different from the ovarian aging, which refers to oocyte exposure to aged ovarian environment before ovulation [4]. Postovulatory oocyte aging have marked detrimental effects on embryo development [5, 6] and offspring [7, 8], and has been considered as one of the major causes for gradual decline in population size of several threatened mammalian species [9]. Thus, studies on the mechanisms and control of oocyte aging are very important for both normal and assisted reproduction. However, the mechanisms for oocyte aging are largely unknown. One of the earliest manifestations for aging oocytes is a spontaneous increase in the susceptibility to activation stimuli (STAS) [10, 11]. Many studies failed to obtain rat offspring following transfer of somatic cell nuclei [12, 13]. Unlike oocytes from other species, the rat oocytes undergo spontaneous activation (SA) soon after their release from the oviduct, due to the spontaneous increase in STAS [14, 15]. Premature chromosome condensation was not observed after transfer of somatic cell nuclei into enucleated rat oocytes [16], suggesting that these nuclei might not be properly reprogrammed due to oocyte SA [17]. Thus, understanding the mechanisms for SA and thence inhibiting SA of rat oocytes is of great importance for successful rat cloning. The activation of mammalian oocytes at fertilization [18] or at parthenogenetic activation [19] is always associated with intracellular Ca2+ oscillations. Furthermore, our recent studies observed cytoplasmic Ca2+ increases during SA of rat oocytes [13, 20]. It is known that free calcium ions from both extra- and intracellular sources can enter the cytoplasm through various calcium channels. Among the important calcium channels, the T- and L-type calcium channels are low- and high-voltage-activated calcium channels, respectively, which aid in mediating calcium influx into cells [21]. The ryanodine receptor (RyR) is a ligand-gated calcium channel, which causes release of calcium from intracellular calcium stores such as the sarcoplasmic reticulum when opened by ligand binding [22]. The Na+/Ca2+ exchanger (NCX) uses the electrochemical gradient of Na+ across the plasma membrane to exchange three Na+ ions into the cell for the extrusion of one Ca2+ ion [23]. The presence of L-type calcium channels was reported in rat oocytes [24, 25], and the T-type calcium channels was observed in mouse oocytes [26]. The RyR channels were reported in both pig [27] and Rhinella arenarum oocytes [28]. Furthermore, the NCX was found to play an important role in promoting calcium efflux in aging rat and mouse oocytes [13, 20]. The calcium-sensing receptor (CaSR) is a recently discovered G-protein-coupled receptor that senses extracellular Ca2+ levels. Extracellular Ca2+ elevations elicit a conformational change in CaSR, which activates phospholipase C (PLC) through a Gqα type of G protein [29]. The activated PLC hydrolyzes the phosphatidylinositol 4,5-bisphosphate (PIP2), leading to the production of inositol 1,4,5-trisphosphate (IP3). IP3 facilitates Ca2+ release from the internal Ca2+ store through interactions with the IP3 receptors on the endoplasmic reticulum (ER) [30]. Expression of CaSR has been observed in maturing oocytes of human [31], equine [32], and porcine species [33], and the beneficial effects of CaSR agonist and the detrimental effects of its antagonist on meiotic maturation have been reported in equine [32] and porcine oocytes [33]. However, neither the expression of CaSR in rat oocytes nor its role in aging oocytes of any species has been reported up to date. The objectives of the present study were to verify the expression of CaSR in rat oocytes and to determine its role in regulating STAS of postovulatory aging (POA) oocytes. Materials and methods Animal care and handling were conducted using the experimental procedures approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). Chemicals and reagents were purchased from Sigma Chemical Co. unless otherwise mentioned. Oocyte recovery Rats of the Sprague-Dawley strain were kept in a room with 14L:10D cycles, with the dark period starting from 20:00. Female rats, 23–26 days after birth, were induced to superovulate with 15 IU equine chorionic gonadotropin (eCG), followed 48 h later by 15 IU human chorionic gonadotropin (hCG). Both the eCG and hCG used in this study were from Ningbo Hormone Product Co., Ltd, China. The superovulated rats were euthanized at different times after hCG injection, and the oviductal ampullae were broken to release the oocytes. After being dispersed and washed three times in M2 medium, the oocytes were denuded of cumulus cells by pipetting with a thin pipette in a drop of M2 medium containing 0.1% hyaluronidase. Oocyte aging in vitro For in vitro aging, cumulus-denuded oocytes (DOs) were cultured for 6 h in the aging medium supplemented with different concentrations of agonist and inhibitors of CaSR. The aging medium used was modified rat one-cell embryo culture medium (mR1ECM). To prepare stock solutions, NPS-2143 (5 mM), Cinacalcet (1 mM), Nifedipine (200 mM), Ruthenium Red (10 mM) and ML218 (1.35 mM) were dissolved in dimethyl sulfoxide. All the stock solutions were stored in aliquots at −20°C and diluted to desired concentrations with the aging medium immediately before use. The aging culture was performed in wells of a 96-well culture plate (San-He Medical Instrument Factory, Haimen City, Zhejiang Province, China) at 37°C under 5% CO2 in humidified air; each well contained 200 μl of the aging medium and about 30 oocytes covered with mineral oil. Assessment of oocyte activation Spontaneous activation of rat oocytes was assessed immediately after the aging culture. To observe SA, oocytes were fixed with 3.7% paraformaldehyde in M2 for 30 min at room temperature before being stained with 10 μg/ml Hoechst 33342 and mounted on glass slides. The state of chromosomes was observed under an epifluorescence microscope (Leica DMLB) and was classified into two types. Oocytes with chromosomes compacted at the metaphase plate were considered to be at the metaphase II (MII) stage, whereas oocytes with chromosomes dispersed in the cytoplasm were classified as activated [13]. Measurement for cytoplasmic calcium Rat oocytes were loaded with Ca2+ probe by incubating at room temperature for 20 min in a loading medium. The loading medium used was Hepes-buffered mR1ECM (HR1) medium containing 1 μM Fura-2 AM and 0.02% pluronic F-127. The HR1 medium consisted of 76.7 mM NaCl, 3.2 mM KCl, 2 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 5 mM NaHCO3, 22 mM HEPES, 10 mM sodium lactate, 0.5 mM sodium pyruvate, 7.5 mM glucose, 1 g/L PVA, 0.1 mM glutamine, 2% (V/V) EAA, and 1% (V/V) NEAA. After loading, oocytes were transferred into a drop of HR1 medium in a Fluoro dish (FD35-100, World Precision Instruments) covered with mineral oil and observed with a Leica DMI6000 inverted microscope at 37.5°C. A Fura 2 fluorescence module was used for excitation, and a Leica LAS-AF calcium imaging module was used to calculate the F340/380 ratio, which represented the concentration of cytoplasmic calcium. The oocytes were monitored continuously for 60 min to record the F340/380 ratio. Immunofluorescence microscopy Unless otherwise specified, all the procedures were conducted at room temperature. Oocytes were washed three times in M2 medium between treatments. Denuded oocytes were (a) fixed with 3.7% paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 4 mM MgSO4, pH 7.0) for at least 30 min; (b) treated with 0.25% protease in M2 for 1–2 s to remove zona pellucida; (c) permeabilized with 0.1% Triton X-100 in PHEM for 5 min; (d) blocked for 1 h in PHEM containing 3% bovine serum albumin (BSA); (e) incubated at 4°C overnight with mouse monoclonal anti-CaSR (IgG, 1:200, Abcam) in 3% BSA in M2 medium; (f) incubated for 1 h with Cy3-conjugated goat-anti-mouse IgG (1:800, Jackson ImmunoResearch) in 3% BSA in M2; (g) incubated for 10 min with 10 μg/ml Hoechst 33342 in M2. Negative control samples in which the primary antibody was omitted were also processed. The oocytes were then mounted on glass slides and observed with a Leica laser scanning confocal microscope (TCS SP2). Blue diode (405 nm) and helium/neon (He/Ne; 543 nm) lasers were used to excite Hoechst and Cy3, respectively. Fluorescence was detected with 420–480 nm (Hoechst) and 560–605 nm (Cy3) bandpass emission filters, and the captured signals were recorded as blue and red, respectively. The relative content of CaSR was quantified by measuring the fluorescence intensities. For each experimental series, all high-resolution z-stack images were acquired with identical settings. The relative intensities were measured on the raw images using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD) under fixed thresholds across all slides. The values of freshly ovulated oocytes recovered 13 h post hCG injection were set as 1, and the other values were expressed relative to this quantity. Western blot analysis A total of 200 DOs were placed in a 1.5-ml microfuge tube containing 20-μl sample buffer (20 mM Hepes, 100 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.3 mM PMSF and 3 mg/ml leupetin, pH 7.5) and frozen at −80°C until use. To run the gel, 5 μl of 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to each tube and the tubes were heated at 100°C for 5 min. The samples were separated on a 6% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were washed twice in TBST (150 mM NaCl, 2 mM KCl, 25 mM Tris, 0.05% Tween-20, pH 7.4) and blocked for 1–1.5 h with TBST containing 3% BSA at room temperature. The membranes were then incubated at 4°C overnight with mouse monoclonal anti-CaSR (Abcam, ab19347) or mouse anti-β-tubulin (Merck Millipore, 05-661) at a dilution of 1:500 in 3% BSA-TBST. After being washed three times in TBST (5 min each), the membranes were incubated for 1 h at 37°C with goat anti-mouse IgG AP conjugated (CWBIO, cw0111 or cw0110) diluted to 1:1000 in 3% BSA-TBST. After three washings in TBST, the membranes were detected by a 5-Bromo-4-chloro-3-indolyl phosphate (BCIP)/NBT (Nitro blue tetrazolium) alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, China). The relative quantities of proteins were determined with Image J software by analyzing the sum density of each protein band image. The values of freshly ovulated oocytes were set as 1 and the other values were expressed relative to this quantity. β-tubulin was used as internal controls. Data analysis Each treatment contained at least three replicates. Percentage data were arcsine-transformed before being analyzed with Analysis of variance (ANOVA). A Duncan multiple comparison test was performed to find differences. The software used was SPSS (Statistics Package for Social Science). Data are expressed as mean ± SE and P < 0.05 was considered significant. Results Treatment with NPS-2143, an allosteric inhibitor of CaSR, suppressed both SA and elevation of cytoplasmic calcium in rat oocytes Rat oocytes recovered at 13 or 19 h post hCG injection were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. At the end of the culture, while some of the oocytes were examined for SA, others were placed in HR1 and monitored for cytoplasmic calcium levels. The results show that after culture without NPS-2143, SA rates were significantly higher in 19 h oocytes (69.2 ± 4.1%, n = 103) than in 13 h oocytes (34.6 ± 2.9%, n = 88) (Figure 1A, C13 vs. 0 μM NPS-2143), and that treatment with 2.5 or 5 μM NPS-2143 significantly suppressed SA (69.2 ± 4.1%, n = 103 vs. 30.9 ± 3.5%, n = 92 or 29.9 ± 2.7%, n = 90) and cytoplasmic calcium elevation (0.79 ± 0.04, n = 60 vs. 0.62 ± 0.01, n = 60) of rat oocytes (Figure 1B–D). Figure 1. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 on SA and level of cytoplasmic calcium of rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. C13 indicates that rat oocytes recovered at 13 h post hCG were cultured for 6 h in mR1ECM alone. Each treatment was repeated three times with each replicate containing about 30 oocytes. a, b: Values with a different letter above bars differ significantly (P < 0.02). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with (C) or without (D) 2.5 μM of NPS-2143 during the aging culture. Each treatment was repeated three times and each replicate included 20 oocytes. a, b: Values with a different letter above bars differ significantly (P = 0.04). Figure 1. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 on SA and level of cytoplasmic calcium of rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM with different concentrations of NPS-2143. C13 indicates that rat oocytes recovered at 13 h post hCG were cultured for 6 h in mR1ECM alone. Each treatment was repeated three times with each replicate containing about 30 oocytes. a, b: Values with a different letter above bars differ significantly (P < 0.02). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with (C) or without (D) 2.5 μM of NPS-2143 during the aging culture. Each treatment was repeated three times and each replicate included 20 oocytes. a, b: Values with a different letter above bars differ significantly (P = 0.04). Levels of CaSR protein in rat oocytes recovered at different times after hCG injection Rat oocytes recovered at 13, 19, 25, and 36 h post hCG injection were examined for CaSR levels using immunofluorescence microscopy and western blotting. When observed under a confocal microscope, CaSR was located both at the plasma membrane and within the cytoplasm in oocytes collected up to 25 h post hCG injection, but the CaSR density, particularly that in the cytoplasm, decreased significantly by 36 h after hCG injection (Figure 2A–D). Immunofluorescence quantification using the same microscopic parameters confirms that the level of total CaSR protein did not change significantly up to 25 h (1 ± 0.01 vs. 1.02 ± 0.11) but decreased significantly by 36 h post hCG injection (1 ± 0.01 vs. 0.53 ± 0.02) (Figure 2E). Western blotting showed one band at about 120 kDa representing the nonglycosylated form, two bands of about 130–140 kDa and 150–160 kDa, which correspond respectively to the immature and mature glycosylated form, and one band at 170–180 kDa representative of the dimeric and active form of the CaSR (Figure 2G). Quantification showed that the functional dimer CaSR protein increased significantly at 19 h (1.54 ± 0.09) and 25 h (1.37 ± 0.17) post hCG injection compared to that in oocytes recovered 13 h post hCG (1.0 ± 0.0) (Figure 2F). By 36 h post hCG injection, the dimer CaSR protein (0.41 ± 0.02) decreased significantly. Figure 2. View largeDownload slide Levels of CaSR in rat oocytes recovered at different times after hCG injection. Micrographs A to D are merged confocal images with DNA and CaSR protein colored blue and red, respectively; A, B, C, and D show oocytes recovered at 13, 19, 25, and 36 h post hCG injection, respectively. Bar is 13 μm. Graph E shows quantification of total proteins of CaSR by immunofluorescence. Each treatment was repeated three times with each replicate containing 30 oocytes. The values of oocytes recovered 13 h post hCG injection were set as 1 and the other values were expressed relative to this quantity. a, b: Values with different letters above bars differ significantly (P < 0.01). Graph F shows quantification of CaSR dimmer by western blotting. Each treatment was repeated three times and each replicate included 200 oocytes. a–c: Values with different letters above bars differ significantly (P < 0.05). Panel G shows full blots of western blotting displaying bands of dimmer, mature glycosylated (Mat-G), immature glycosylated (Imm-G), and nonglycosylated (Non-G) CaSR proteins. Figure 2. View largeDownload slide Levels of CaSR in rat oocytes recovered at different times after hCG injection. Micrographs A to D are merged confocal images with DNA and CaSR protein colored blue and red, respectively; A, B, C, and D show oocytes recovered at 13, 19, 25, and 36 h post hCG injection, respectively. Bar is 13 μm. Graph E shows quantification of total proteins of CaSR by immunofluorescence. Each treatment was repeated three times with each replicate containing 30 oocytes. The values of oocytes recovered 13 h post hCG injection were set as 1 and the other values were expressed relative to this quantity. a, b: Values with different letters above bars differ significantly (P < 0.01). Graph F shows quantification of CaSR dimmer by western blotting. Each treatment was repeated three times and each replicate included 200 oocytes. a–c: Values with different letters above bars differ significantly (P < 0.05). Panel G shows full blots of western blotting displaying bands of dimmer, mature glycosylated (Mat-G), immature glycosylated (Imm-G), and nonglycosylated (Non-G) CaSR proteins. Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA and cytoplasmic calcium levels of rat oocytes Because our previous studies demonstrated that activating NCX with NaCl could inhibit SA of rat oocytes by facilitating calcium efflux from the oocyte [13, 20], this experiment tested whether SA of rat oocytes could be completely suppressed by inhibiting CaSR while activating NCX. Rat oocytes recovered 19 h post hCG injection were cultured for 6 h in mR1ECM alone or with 2.5 μM NPS-2143 or 50 mM NaCl [13, 20] or both. At the end of the treatment, some oocytes were examined for SA, and others were monitored for cytoplasmic calcium levels. Although NaCl inhibited SA to a greater extent (86.2 ± 2.1%, n = 81 vs. 16.6 ± 2.4%, n = 92) than NPS-2143 did (86.2 ± 2.1%, n = 81 vs. 33.6 ± 4.9%, n = 82) (Figure 3), treatment with both NaCl and NPS-2143 did not decrease SA (11.4 ± 2.7%, n = 86 vs. 16.6 ± 2.4%, n = 92) or calcium level (0.44 ± 0.01, n = 60 vs. 0.45 ± 0.01, n = 60) further compared to that achieved with NaCl alone (P > 0.05), suggesting that calcium sources other than the CaSR-controlled channels are contributing to the elevation of cytoplasmic calcium in aging rat oocytes. Figure 3. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA rates and levels of cytoplasmic calcium in rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM medium alone (Ctrl), with 2.5 μM NPS-2143 (NPS) or 50 mM NaCl, or with both NPS-2143 and NaCl (NP + Na). Each treatment was repeated three times with each replicate containing about 30 oocytes. a–c: Values with a different letter above bars differ significantly (P < 0.01). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with NaCl alone (C) or with both NaCl and NPS-2143 (D) during the aging culture. In panel B, each treatment was repeated three times and each replicate included 20 oocytes. a: Values with the same letter above bars do not differ significantly (P = 0.66). Figure 3. View largeDownload slide Effects of inhibiting CaSR with NPS-2143 while activating NCX with NaCl on SA rates and levels of cytoplasmic calcium in rat oocytes. Panel A shows SA percentages after rat oocytes recovered 19 h post hCG were cultured for 6 h in mR1ECM medium alone (Ctrl), with 2.5 μM NPS-2143 (NPS) or 50 mM NaCl, or with both NPS-2143 and NaCl (NP + Na). Each treatment was repeated three times with each replicate containing about 30 oocytes. a–c: Values with a different letter above bars differ significantly (P < 0.01). Panels B, C, and D show levels of cytoplasmic calcium (F340/F380) in rat oocytes treated with NaCl alone (C) or with both NaCl and NPS-2143 (D) during the aging culture. In panel B, each treatment was repeated three times and each replicate included 20 oocytes. a: Values with the same letter above bars do not differ significantly (P = 0.66). Roles of calcium channels other than CaSR and NCX in controlling STAS of aging rat oocytes Rat oocytes recovered at 19 h post hCG injection were aged for 6 h in mR1ECM containing different concentrations of various calcium channel blockers before examination for SA. All the blockers tested including Nifedipine (a L-type channel blocker), ML218 (a T-type channel blocker), and Ruthenium Red (a RyR channel inhibitor) significantly decreased the SA rates of rat oocytes when used at optimal concentrations (Figure 4A–C). When Nifedipine, ML218, and NPS-2143 were used in combination, SA rates decreased to minimum (70.19 ± 2.8%, n = 97 vs. 11.6 ± 1.0%, n = 94) (Figure 4D). When Ruthenium Red was added to the combination, however, the SA rate did not decreased further (11.6 ± 1.0%, n = 94 vs. 9.0 ± 0.4%, n = 101) suggesting that Ruthenium Red had a mild effect on SA of rat oocytes in the presence of other calcium channel blockers. Figure 4. View largeDownload slide Rates of SA after rat oocytes recovered at 19 h post hCG injection were cultured for 6 h with different concentrations of various calcium channel blockers. Graphs A, B, and C show percentages of SA oocytes after treatment with various concentrations of Ruthenium Red, Nifedipine, and ML218, respectively. Graph D shows SA rates after oocytes were treated with Nifedipine, ML218, and NPS-2143 in different combinations. In graph D, optimal concentrations of Nifedipine (Nife, 200 μM), ML218 (M218, 0.85 μM), NPS-2143 (NPS, 2.5 μM), and Ruthenium Red (RuR, 15 μM) were used. Each treatment was repeated three times with each replicate containing about 30 oocytes. a–e: Values without a common letter above bars differ significantly (P < 0.05). Figure 4. View largeDownload slide Rates of SA after rat oocytes recovered at 19 h post hCG injection were cultured for 6 h with different concentrations of various calcium channel blockers. Graphs A, B, and C show percentages of SA oocytes after treatment with various concentrations of Ruthenium Red, Nifedipine, and ML218, respectively. Graph D shows SA rates after oocytes were treated with Nifedipine, ML218, and NPS-2143 in different combinations. In graph D, optimal concentrations of Nifedipine (Nife, 200 μM), ML218 (M218, 0.85 μM), NPS-2143 (NPS, 2.5 μM), and Ruthenium Red (RuR, 15 μM) were used. Each treatment was repeated three times with each replicate containing about 30 oocytes. a–e: Values without a common letter above bars differ significantly (P < 0.05). Discussion In this study, our immunofluorescence microscopy showed that CaSR were localized both at the plasma membrane and in the cytoplasm of rat oocytes. By immunofluorescence microscopy, Dell’Aquila et al. [31] observed CaSR expression in human GV, MI, and MII oocytes, and they found an ubiquitous distribution of CaSR along the oolemma and within the cytoplasm. De Santis et al. [32] demonstrated CaSR at the plasma membrane and, more pronounced, within the cytoplasm of equine oocytes at GV, MI, and MII stages of meiosis. Thus, CaSR are localized both at the plasma membrane and in the cytoplasm of mammalian oocytes. Our western blotting revealed that rat oocytes contained four bands of 120, 130–140, 150–160, and 170–180 kDa, representing the nonglycosylated, immature, and mature glycosylated and dimeric forms of CaSR, respectively. The results were exactly similar to those reported in human vascular smooth muscle cells [34]. By western blot analysis, Dell’Aquila et al. [31] and De Santis et al. [32] revealed a single CaSR protein of 130 kDa in human and equine MII oocytes, respectively, and Liu et al. [33] observed a 160 kDa CaSR protein in pig oocytes. It is known that CaSR present at the plasma membrane functions as a homodimer in performing its central role of sensing minute alterations in extracellular calcium [35, 36]. For example, studies have suggested that the constitutive CaSR dimerization occurs in the ER and is necessary for exit of the receptor from the ER and trafficking to the cell surface [36]. Studies using natural CaSR mutations have also emphasized the functional importance of CaSR dimerization [37]. Furthermore, it has been reported that activation of CaSR promotes its expression, trafficking, and membrane insertion. For example, the agonist-driven maturation and plasma membrane insertion of CaSR were found to dynamically control signal amplitude [38]. In the present study, although our immunofluorescence indicated that the density of total CaSR protein remained constant up to 25 h post hCG injection, our western blotting showed that the functional dimer protein of CaSR increased significantly from 13 to 25 h after hCG injection in aging rat oocytes. This suggested that STAS in aging oocytes was positively correlated with the level of the functional dimer protein rather than its total protein of CaSR, and that the activation of CaSR by certain stimuli had facilitated the dimerization of CaSR monomers. Both previous studies [10, 39] and the present results have demonstrated that aged oocytes are more susceptible to activating stimuli than newly ovulated oocytes. Possible pathways leading to cytoplasmic calcium elevation and SA have been proposed for rat oocytes recovered at 19 h post hCG injection (Figure 5). Undefined stimuli concomitant with oocyte release from the oviduct activate CaSR as well as the L- and T-channels. While the activated T- and L-channels open to allow a direct influx of extracellular calcium into the oocyte [21], the activated CaSR facilitates Ca2+ release from the internal Ca2+ store into the cytoplasm [30]. The increased calcium is then pumped out of the oocyte by NCX [13, 20], which further activates CaSR and promotes calcium release from calcium store, leading to a second elevation in cytoplasmic calcium. The resultant repeated increases in cytoplasmic calcium lead to SA in aged rat oocytes. Thus, the present results that the level of the functional dimer protein of CaSR increased with postovulatory oocyte aging have provided new evidence that activation of CaSR is a major factor causing the difference in STAS between newly ovulated and aged oocytes. Figure 5. View largeDownload slide A diagram showing the possible pathways leading to cytoplasmic calcium elevation and SA of rat oocytes recovered at 19 h post hCG injection. Numbers in brackets indicate the precedence of the calcium events. For detailed explanations, please refer to text in the Discussion section. Figure 5. View largeDownload slide A diagram showing the possible pathways leading to cytoplasmic calcium elevation and SA of rat oocytes recovered at 19 h post hCG injection. Numbers in brackets indicate the precedence of the calcium events. For detailed explanations, please refer to text in the Discussion section. The current results demonstrated that treatment with T- or L-type calcium channel blockers significantly reduced SA of rat oocytes, and that suppression of CaSR together with these major calcium channels reduced SA to minimum. This suggested that the control of cytoplasmic calcium involves multiple channels in aging rat oocytes. It is known that free calcium ions from both extra- and intracellular calcium stores can enter the cytoplasm of oocytes. For example, during A23187-induced activation of pig oocytes, it was the influx of extracellular calcium that gave rise to the intraoocyte calcium elevations [40]. Yoo and Smith [24] observed that SA of rat oocytes was significantly decreased in calcium-free medium or in calcium-containing medium supplemented with L-type calcium channel blocker or IP3R inhibitor, suggesting that both the L-type calcium channel and CaSR are involved in the control of SA in rat oocytes. Extracellular Ca2+ elevations activate CaSR, which promotes the production of IP3 by hydrolyzing PIP2 [29]. IP3 is a Ca2+-releasing second messenger, which causes the release of intracellular calcium by interacting with IP3 receptors [41]. It has been demonstrated that the IP3-induced intracellular calcium release plays an important role in the regulation of meiosis in rabbit [42] and cattle [43] oocytes. In summary, we have studied the expression of CaSR and its role in regulating STAS of POA rat oocytes. The results indicate that CaSR is expressed in rat oocytes, and the STAS of aging rat oocytes was positively correlated with the level of the functional dimer protein of CaSR. 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Biology of ReproductionOxford University Press

Published: Feb 1, 2018

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