TY - JOUR
AU - Han, Ching-Tack
AB - Abstract The objectives of this study were: (i) to investigate the possible role of T-type Ca2+ channels on the acrosome reaction (AR) of human spermatozoa; and (ii) to determine the sub-type of T-type calcium channels involved in the AR. The AR was induced in vitro by mannose–bovine serum albumin (BSA). The inhibitory effects of mibefradil (T-type Ca2+ channel blocker), NiCl2, or nifedipine (L-type Ca2+ channel blocker) on the mannose–BSA induced AR were evaluated in capacitated human spermatozoa. The AR was sensitively inhibited by low micromolar concentrations of mibefradil (IC50 = 1 μmol/l) in a dose-dependent manner. Low concentrations of Ni2+ (IC50 = 40 μmol/l) also inhibited the mannose–BSA induced AR. On the contrary, higher concentrations of nifedipine were required to block AR (IC50 = 60 μmol/l). Reverse transcription–polymerase chain reaction (RT–PCR) was performed to identify the sub-types of T-type channels present in human testes. Analysis of PCR products showed that only α1H subunits are expressed in testes. The expression of the α1H subunit may be tissue specific since its mRNA was not detected in the human ovary. The present study suggests that the AR of human spermatozoa is highly associated with T-type Ca2+ channels and is mainly mediated by calcium influx through α1H T-type Ca2+ channels. α1H, acrosome reaction, spermatozoa, testis, T-type Ca2+ channels Introduction The arosome reaction (AR), a Ca2+-dependent exocytotic event that is regulated by voltage-operated calcium ion channels (VOCCs) located in the plasma membrane of the sperm head, is an obligatory early step in the fertilization process, and must be completed prior to fusion with oocytes (Babcock and Pfeiffer, 1987; Florman et al., 1992; Florman, 1994; Arnoult et al., 1996). As in excitable somatic cells, sperm VOCCs are activated by membrane depolarization. In physiological conditions, activation of sperm VOCCs is usually mediated by progesterone (Foresta et al., 1993) and by contact with the zona pellucida (Florman et al., 1992; Arnoult et al., 1996). VOCCs can be basically classified into low voltage-activated (LVA) and high voltage-activated (HVA) calcium channels on the basis of thresholds for activation. HVA Ca2+ channels having high thresholds can be further classified by their pharmacological properties into L-, N-, P/Q-, and R-types (Birnbaumer et al., 1994; Dunlap et al., 1995). L-type (α1S, α1C, α1D, and α1F) and non-L-type (α1A, α1B, and α1E) Ca2+ channel α1 subunit genes have been isolated and analysed (for review, see Perez-Ryes and Schneider, 1994). The widely-used HVA Ca2+ channel antagonists are: nifedipine, diltiazem, and verapamil for L-type channels; ω-conotoxin GVIA for N-type channels; and ω-agaIVA for P/Q-type channels. There is no known block for R-type channels. Recently, three distinct Ca2+ channel α1 subunits (α1G, α1H, and α1I) of LVA channels (T-type) have been cloned and functionally analysed. The electrophysiological characteristics of currents from the artificially expressed subunits were identical to those of T-type currents described from isolated cells (Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999a). Recently, a new non-dihydropyridine Ca2+ channel antagonist, mibefradilTM (Hoffman-La Roche), was developed to selectively block T-type currents (Cribbs et al., 1998; McDonough and Bean, 1998; Gomora et al., 2000). Mibefradil has also been used (Blackmore and Eisoldt, 1999) to demonstrate the participation of T-type Ca2+ channels in the AR of human spermatozoa. Whether or not low doses of nickel can also selectively block T-type currents has been controversial. One group of workers (Lee et al., 1999b) reported that, of the three cloned T-type channel subunits, only α1H currents were selectively inhibited by low concentrations of Ni2+. ZP3, a sulphated glycoprotein from the zona pellucida, is the main mediator of sperm binding and the AR in mammals (Yanagimachi, 1994). ZP3 induces Ca2+ influx into cytoplasm, leading to increases of intracellular Ca2+ and pH, and resulting in acrosomal exocytosis (Babcock and Pfeiffer, 1987). Treatment of spermatozoa with either solubilized zona pellucida or recombinant ZP3 can evoke the AR in spermatozoa. However, neither of these methods can be used in human studies, because of the difficulty in acquiring enough human material. Instead, several bovine serum albumin (BSA) neoglycoproteins were shown to be able to stimulate the AR in recent studies. For example, N-acetyl-α-d-glucosamine (gluNAc) and α-d-mannose could elicit the AR by interacting with the putative receptor for ZP3 in human spermatozoa (Brandelli et al., 1996; Blackmore and Eisoldt, 1999). The types of VOCCs participating in the AR remain to be fully characterized. Some pharmacological and molecular studies have suggested a possible involvement of L-type channels in the sperm AR (Florman et al., 1992; Florman, 1994; Goodwin et al., 1997). However, several features of the Ca2+ entry pathway were inconsistent with the characteristics of L-type Ca2+ channels. Firstly, inhibition of the AR by L-type channel antagonists required 10–100-fold higher concentrations than those needed to inhibit L-type currents found in excitable cells (Florman et al., 1992; Florman, 1994). Secondly, electrophysiological recordings of rodent spermatogenic cells revealed that spermatogenic cells expressed only T-type Ca2+ channels (Arnoult et al., 1996; Liévano et al., 1996; Santi et al., 1996). These studies suggested that the major VOCCs in spermatozoa are T-type Ca2+ channels through which calcium influx might play a pivotal role in the AR. However, the molecular identification of T-type Ca2+ channels in mammalian testis remains to be solved. To study the functional roles of VOCCs in the AR of human spermatozoa, we examined the sensitivity of mannose–BSA mediated ARs to inhibition by mibefradil, Ni2+, or nifedipine. Using reverse transcription–polymerase chain reaction (RT–PCR), we also demonstrated the expression of mRNA transcripts corresponding to T-type Ca2+ channels in human testis. Materials and methods Human sperm preparation and capacitation Human semen samples were obtained from healthy donors (n = 12) with normal sperm density, motility, and morphology according to World Health Organization criteria (WHO, 1992) after 3 days sexual abstinence. Samples were allowed to liquefy for 30–60 min at room temperature. The spermatozoa were washed twice (by centrifugation at 300 g for 5 min) with Ham's F-10 medium. The supernatants were discarded, and motile spermatozoa (>90%) were then isolated using a simple swim-up method with 5% CO2 at 37°C for 1 h. The sperm concentration was adjusted to 2×107 cells/ml. Spermatozoa were capacitated by incubating in Ham's F-10 supplemented with 3 mg/ml BSA under an atmosphere of 5% CO2 at 37°C for 3 h. Induction and evaluation of the acrosome reaction To determine an optimal concentration of mannose–BSA for AR induction, a stock solution of mannose–BSA (10 mmol/l in Ham's F10) was added to the capacitated sperm suspensions to give final concentrations of 10, 50, 75, and 100 μmol/l. The optimal concentration was 100 μmol/l at which the AR was saturated. To evaluate the AR, mannose–BSA treated sperm suspensions were prepared as follows. Sperm suspensions were incubated at 37°C with 5% CO2 for 20 min. The spermatozoa were washed with PBS, collected by centrifugation at 300 g for 10 min, and resuspended in 250 μl of hypo-osmotic swelling medium (7.35 g sodium citrate, 13.51 g fructose/l dry weight). After incubation for 1 h at 37°C, the spermatozoa were washed with 0.9% NaCl and smeared on the slides. The sperm samples were fixed in a 95% ethanol solution at room temperature and were air-dried. The slides were covered with a droplet of Pisum sativum agglutinin–fluorescein isothicyanate (PSA–FITC; Sigma Chemical Co, St Louis, MO, USA) at a concentration of 5 μg/ml in PBS and incubated in a moist chamber in the dark for 1 h. After rinsing the slides with water, the AR of spermatozoa (>100) was scored for FITC fluorescence using fluorescence microscope (Nikon, Tokyo, Japan) (Tesarik et al., 1993). Effects of mibefradil, nifedipine, and Ni2+ on the acrosome reaction The maximum AR was evoked by adding mannose–BSA to give a final concentration of 100 μmol/l to sperm suspensions. To determine inhibitory effects of calcium channel blockers, capacitated human spermatozoa were pre-treated (for 10 min) with various concentrations of mibefradil (Hoffman-La Roche, Nutley, NJ, USA) (0.1, 1, 3, 10 μmol/l), Ni2+ (1, 10, 30, 100 μmol/l), or nifedipine (1, 10, 30, 100 μmol/l) and then treated with 100 μmol/l mannose–BSA. Sperm suspensions were incubated at 37°C in a humid atmosphere of 5% CO2, 95% air for 20 min to allow the AR to proceed. RNA isolation and RT–PCR Human testis tissues were obtained from azoospermic patients who had given their consent for this study. After biopsy, testis tissues were fixed immediately in Bouin's solution and then paraffin-embedded, sectioned, and stained with haematoxylin and eosin (H&E) for pathological evaluation. After pathological evaluation, the tissues of testes with normal spermatogenesis and Sertoli cell-only syndrome were used in this study. Fresh testis tissues were stored in liquid nitrogen immediately for future studies. Total RNAs from testes were isolated using a RNeasy RNA isolation kit (Qiagen, Chatsworth, CA, USA). Prior to each reverse transcription (RT) reaction, 1 μg of total RNA was dissolved in 10 μl water and the trace of DNA in RNA sample was digested with 1 IU RNase-free DNase I (Life Technologies, Gaithersburg, MD, USA) in the transcription buffer for 15 min at room temperature. After the incubation, the DNase was heat-inactivated at 65°C for 15 min. RT was performed with the Superscript preamplification system (Life Technologies), according to the manufacturer's protocol. Degenerative PCR primers were designed to amplify any of the α1G, α1H or α1I subunits: forward primer, 5′-GGCGT(G/C)GT(G/C)GT(G/C)GAGAACTT-3′; reverse primer, 5′-GATGATGGTGGG(A/G)TTGAT-3′ (Lambert et al., 1998). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was chosen as an endogenous internal control for normalization of PCR products. The sequences for the GAPDH primers were as described previously (Wong et al., 1994). PCR reactions were carried out in a final volume of 50 μl having 200 μmol/l dNTP, 20 pmol forward and reverse primers, 1.5 mmol/l MgCl2, and 2.5 IU Taq polymerase. The PCR reaction was first denatured at 94°C for 5 min, and then 30 cycles consisting of denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s were carried out. The annealing temperature was started from 68°C for the first cycle and then decreased 1°C every cycle until 59°C (cycle 10). The annealing temperature of the following 20 cycles was kept at 58°C. The RT–PCR products from testes were cloned in pMOSBLUE vector (Amersham, Piscataway, NJ, USA), and transformed. In all, 20 PCR products in the vector were sequenced and compared with the reported T-type channel sequences. With a semi-quantitative RT–PCR for which GAPDH mRNA was used as a control, relative amounts of T-type Ca2+ channel mRNA were compared between testes with normal spermatogenesis and Sertoli cell-only syndrome. Results Pharmacological characterization of the mannose–BSA initiated AR Mannose–BSA is known to act as a surrogate for ZP3, a native inducer of the AR. We examined whether the mannose–BSA-induced AR could be a proper assay method to evoke AR in human spermatozoa. Mannose–BSA evoked AR in a dose-dependent manner, reaching 93% AR at 100 μmol/l (data not shown). This result suggested that mannose–BSA treatment is a proper method to induce AR, and 100 μmol/l mannose–BSA was used in the following experiments. The effects of various concentrations of mibefradil, NiCl2, or nifedipine were tested on mannose–BSA induced AR. These calcium channel blockers inhibited the AR. The inhibitory dose–response curves of mibefradil, NiCl2, or nifedipine to the AR are shown in Figure 1. AR was sensitively inhibited by very low micromolar concentrations mibefradil (IC50 = 1 μmol/l) in a dose-dependent manner (Figure 1A). Low concentrations of Ni2+ (IC50 = 40 μmol/l) also reduced the mannose–BSA induced AR (Figure 1B). On the contrary, much higher concentrations of nifedipine were required to block the AR (IC50 = 60 μmol/l) (Figure 1C), in comparison with the concentrations reported to block L-type channels (~1 μmol/l; Mikami et al., 1989). Identification of T-type calcium channels in human testes by RT–PCR To identify the types of T-type Ca2+channels expressed in human spermatozoa, we performed RT–PCR experiments, using degenerative primers derived from α1G, α1H, and α1I sequences (Lambert et al., 1998). Figure 2A shows the electophoretic pattern of the PCR products amplified from normal human testis mRNA. Comparison of the sequences of 20 separate PCR products with the reported T-type channel sequences, showed that only α1H sequences (fragment size 489 bp) were detected from testes. Using semi-quantitative RT–PCR, the amounts of T-type Ca2+ channel mRNAs were compared between testes with normal spermatogenesis (Figure 2B, lane 2) and with Sertoli cell-only syndrome (Figure 2B, lane 3). T-type channel expression was predominantly detected in normal testes, and expressed at a lower level with testes of Sertoli cell-only syndrome. The similar results were obtained repeatedly three times from tissues of other infertile patients. However, T-type Ca2 channel mRNA was not detected in human ovary (Figure 2B, lane 4). The conspicuous pattern of GAPDH mRNA quantities, as an internal control, was uniform among the tissues (Figure 2B). Discussion We identified T-type Ca2 channels in human testis and demonstrated their involvement in the AR. Molecular studies suggested that the VOCCs expressed in human testis are α1H T-type Ca2+ channels and pharmacological studies showed their high sensitivity to mibefradil. Fertilization is a multiple-step process among which the AR is a mandatory step for a spermatozoon to penetrate into an oocyte. The AR is a calcium-mediated event for which calcium entry through VOCCs is essential to trigger exocytosis of the acrosome. One of the basic questions is what kinds of calcium entry pathways evoke the AR? Several studies have suggested that L-type Ca2+ channels participate in triggering the AR, since nifedipine and verapamil blocked the AR (Brandelli et al., 1996; Goodwin et al., 1997). In our study, the mannose–BSA-induced AR was also inhibited by nifedipine, an L-type blocker. However, the concentration of nifedipine (IC50 = 60 μmol/l) required to inhibit the AR was much higher than that needed to inhibit Ca2+ influx through L-type Ca2+ channels in somatic cells (~1 μmol/l; Mikami et al., 1989). It has been reported that high concentrations of dihydropyridines are able to block low threshold T-type currents (Bean, 1989; Hess, 1990; Liévano et al., 1994). Thus, we speculate that the blocking effect by high concentrations of nifedipine might be due to non-selective blocking effects of nifedipine on T-type Ca2+ channels rather than selective blocking of L-type Ca2+ channels. Consistently, the mannose–BSA initiated AR was specifically inhibited by low concentrations of a T-type blocker, mibefradil (IC50 = 1 μmol/l). The IC50 value for mibefradil inhibition of the AR was comparable with previous results (Blackmore and Eisoldt, 1999), which showed that an increase in intracellular Ca2+ in human spermatozoa was inhibited by mibefradil. These results lead us to conclude that T-type Ca2+ channel might directly mediate the mannose–BSA induced AR. The α1E and α1A mRNA signals were detected in mouse spermatogenic cells by RT–PCR (Liévano et al., 1996). Other authors (Goodwin et al., 1997, 1998, 2000; Benoff, 1998) have reported the detection of messages for α1C in rat testis, human testis, and human spermatozoa, claiming involvement of L-type calcium channels in the AR (Goodwin et al., 1998). However, these molecular studies on the VOCCs of male germ cells were not consistent with the electrophysiological and pharmacological investigations. Although transcription of these HVA Ca2+ channel genes occurs during spermatogenesis, those HVA currents were not significantly detected in electrophysiological studies (Arnoult et al., 1996; Liévano et al., 1996; Santi et al., 1996). Patch clamp recordings and pharmacological studies showed that immature spermatogenic cells express only low voltage-activated (T-type) calcium currents (Arnoult et al., 1996, 1998; Liévano et al., 1996; Santi et al., 1996; Linares-Hernanadez et al., 1998). One possible explanation of why only HVA calcium channel mRNA messages were detected by Liévano and Benoff's groups might be the extremely high sensitivity of PCR, rather than the abundance of their messages in spermatozoa or spermatogenic cells. Another possibility is that, up to now, PCRs to detect LVA calcium channel messages have not been extensively attempted. Expression of α1G, a subunit proved to generate T-currents in neurons, was not detected in rat testis by Northern blot analysis (Perez-Reyes et al., 1998). The other two T channels, α1H and α1I, have also been cloned and expressed (Cribbs et al., 1998; Lee et al., 1999a), but molecular identification of their presence or absence in male germ cells has not been studied. In this study, therefore, we attempted to identify mRNAs corresponding to T-type currents which have been electrophysiologically recorded from the mammalian spermatogenic cells. The primers derived from conserved amino acid sequences of domain IIIS6 and domain IVS6 amplified PCR products of which all turned out to be α1H sequences. This molecular identification supported the pharmacological evidence that AR was sensitively inhibited a T-type calcium channel blocker, mibefradil. According to recent reports (Lee et al., 1999b; Williams et al., 1999), α1H currents were sensitively blocked by Ni2+ (IC50 = 10 μmol/l), while α1G currents were insensitive to Ni2+ (IC50 = 250 μmol/l). Under an assumption that the AR is proportional to calcium influx through only T-type calcium channels, we attempted to calculate a fractional percentage of α1H contributing to AR using Ni2+ sensitivities of α1H and α1G currents as follows:   \[IC_{50}\ for\ AR\ =\ A{\times}IC_{50}\ ({\alpha}1G)\ +\ B{\times}IC_{50}\ ({\alpha}1H),\ where\ A\ +\ B\ =\ 100\%,\ IC_{50}\ ({\alpha}1H)\ =\ 10\ {\mu}mol/l),\ IC_{50}\ ({\alpha}1G)\ =\ 250\ {\mu}mol/l\ IC_{50}\ for\ AR\ (in\ this\ experiment)\ =\ 40\ {\mu}mol/l\] From the above equation, contribution of α1H currents was calculated to be 88%. This result is consistent with the results of RT–PCR in which all of the 20 PCR products in transformed clones were α1H sequences. Therefore, we propose that α1H T-type calcium channels are mainly involved in the AR. However, we cannot rule out the possibility that other calcium channels, e.g. α1G, HVA calcium channels α1C or α1E, non-selective cation channels, or a combination of these may contribute in part to the AR. Additionally, we examined male germ cell specificity on mRNA expression of T-type calcium channels. In semi- quantitative RT–PCR, the amount of α1H mRNA was significantly higher in testis with normal spermatogenesis than in testis with Sertoli cell-only. In the ovary, α1H mRNA was not detected. These results imply that α1H may be specifically expressed in the male germ cell. However, further analyses are necessary to examine the cellular pattern and the developmental change of T-type Ca2+ channel mRNA expression in testicular epithelium, which is under investigation. In conclusion, this study suggests that the AR of human spermatozoa is associated with T-type Ca2+ channels and mainly triggered by calcium influx through α1H T-type Ca2+ channels. The significance of these observations is that the molecular identification of α1H T-type Ca2+ channels in testis was consistent with pharmacological properties associated with T-type Ca2+ channels, in terms of a sensitive blockade of the AR by mibefradil. In addition, molecular detection of α1H may provide an explanation for why T-type currents have been recorded from spermatogenic cells (Arnoult et al., 1996; Liévano et al., 1996; Santi et al., 1996). Figure 1. View largeDownload slide Dose–response curves of mibefradil, Ni2+, and nifedipine for inhibition of mannose–bovine serum albumin (BSA)-induced acrosome reaction (AR) in human spermatozoa. (A) Inhibitory effects of varying doses of mibefradil on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting those data with a sigmoidal dose–response equation. The IC50 = 1 μmol/l/l. Each value is the mean ± SD from six separate sperm preparations. (B) Inhibitory effects of varying doses of Ni2+ on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting the data with a sigmoidal dose–response equation. The IC50 = 40 μmol/l. Each value is the mean ± SD from six sperm preparations. (C) Inhibitory effects of varying doses of nifedipine on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting with a sigmoidal dose–response. The IC50 = 60 μmol/l (n = 6). Each value is the mean ± SD from six sperm preparations. Figure 1. View largeDownload slide Dose–response curves of mibefradil, Ni2+, and nifedipine for inhibition of mannose–bovine serum albumin (BSA)-induced acrosome reaction (AR) in human spermatozoa. (A) Inhibitory effects of varying doses of mibefradil on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting those data with a sigmoidal dose–response equation. The IC50 = 1 μmol/l/l. Each value is the mean ± SD from six separate sperm preparations. (B) Inhibitory effects of varying doses of Ni2+ on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting the data with a sigmoidal dose–response equation. The IC50 = 40 μmol/l. Each value is the mean ± SD from six sperm preparations. (C) Inhibitory effects of varying doses of nifedipine on the AR induced by 100 μmol/l mannose–BSA. A smooth dose–response curve was obtained from fitting with a sigmoidal dose–response. The IC50 = 60 μmol/l (n = 6). Each value is the mean ± SD from six sperm preparations. Figure 2. View largeDownload slide Reverse transcription–polymerase chain reaction (RT–PCR) to detect T-type Ca2+ mRNA signals in human testes and ovary. (A) Separation of PCR products on an agarose gel with size markers. M = DNA size marker; lane 1 = negative control; lane 2 = testis with normal spermatogenesis. (B) M = DNA size marker; lane 1 = negative control; lane 2 = testis with normal spermatogenesis; lane 3 = testis with Sertoli cell-only syndrome; lane 4 = ovary. Figure 2. View largeDownload slide Reverse transcription–polymerase chain reaction (RT–PCR) to detect T-type Ca2+ mRNA signals in human testes and ovary. (A) Separation of PCR products on an agarose gel with size markers. M = DNA size marker; lane 1 = negative control; lane 2 = testis with normal spermatogenesis. (B) M = DNA size marker; lane 1 = negative control; lane 2 = testis with normal spermatogenesis; lane 3 = testis with Sertoli cell-only syndrome; lane 4 = ovary. 3 To whom correspondence should be addressed at: Department of Life Science, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121–742, Korea. E-mail: cthan@ccs.sogang.ac.kr References Arnoult, C., Cardullo, R.A., Lemos, J.R. et al. ( 1996) Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida. Proc. Natl Acad. Sci. USA , 93, 13004–13009. Google Scholar Arnoult, C., Villaz, M. and Florman, H.M. ( 1998) Pharmacological properties of the T-type Ca2+ channel current of mouse spermatogenic cells. Mol. Pharmacol. , 53, 1104–1111. Google Scholar Babcock, D.F., Pfeiffer, D.R. ( 1987) Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms. J. Biol. Chem. , 262, 15041–15047. Google Scholar Bean, B.P. ( 1989) Classes of calcium channels in vertebrate cells. Ann. Rev. Physiol. , 51, 367–384. Google Scholar Benoff, S. ( 1998) Modeling human sperm–egg interactions in vitro: signal transduction pathways regulating the acrosome reaction. Mol. Hum. Reprod. , 4, 453–471. Google Scholar Birnbaumer, L., Campbell, K.P., Catterall, W.A. et al. ( 1994) The naming of voltage-gated calcium channels. Neuron , 13, 505–506. Google Scholar Blackmore, P.F. and Eisoldt, S. ( 1999) The neoglycoprotein mannose–bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa. Mol. Hum. Reprod. , 5, 498–506. Google Scholar Brandelli, A., Miranda, P.V. and Tezon, J.G. ( 1996) Voltage-dependent calcium channels and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosaminyl/mannosyl neoglycoproteins. J. Androl. , 17, 522–529. Google Scholar Cribbs, L.L., Lee, J.H., Yang, J. et al. ( 1998) Cloning and characterization of α1H from human heart, a member of the T-type Ca2+ channel gene family. Circ. Res. , 83, 103–109. Google Scholar Dunlap, K., Luebke, J.I. and Turner, T.J. ( 1995) Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci ,, 18, 89–98. Google Scholar Florman, H.M. ( 1994) Sequential focal and global elevations of sperm intracellular calcium are initiated by the zona pellucida during acrosomal exocytosis. Dev. Biol. , 165, 152–164. Google Scholar Florman, H.M., Corron, M.E., Kim, T.D.H. et al. ( 1992) Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis. Dev. Biol. , 152, 304–314. Google Scholar Foresta, C., Rossatao, M., and Di Virgilio, F. ( 1993) Ion fluxes through the progesterone-activated channel of the sperm plasma membrane. Biochem. J. , 294, 279–283. Google Scholar Gomora, J.C., Xu, L., Enyeart, J.A. et al. ( 2000) Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca(2+) channels in cortisol-secreting cells. J. Pharmacol. Exp. Ther. , 292, 96–103. Google Scholar Goodwin, L.O., Leeds, N.B., Hurley, I. et al. ( 1997) Isolation and characterization of the primary structure of testis-specific L-type calcium channel: implications for contraception. Mol. Hum. Reprod. , 3, 255–268. Google Scholar Goodwin, L.O., Leeds, N.B., Hurley, I. et al. ( 1998) Alternative splicing of exons in the alpha1 subunit of the rat testis L-type voltage-dependent calcium channel generates line-specific dihydropyridine binding sites. Mol. Hum. Reprod. , 4, 215–226. Google Scholar Goodwin, L.O., Karabinus, D.S., Pergolizzi, R.G. et al. ( 2000) L-type voltage-dependent calcium channel α1C subunit mRNA is present in ejaculated human spermatozoa. Mol. Hum. Reprod. , 6, 127–136. Google Scholar Hess, P. ( 1990) Calcium channels in vertebrate cells. Annu. Rev. Neurosci. , 13, 337–356. Google Scholar Lambert, R.C., Mckenna, F., Maulet, Y. et al. ( 1998) Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (α1G/H) in rat primary sensory neurons. J. Neurosci. , 18, 8605–8613. Google Scholar Lee, J.H., Daud, A.N., Cribbs, L.L. et al. ( 1999) Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J. Neurosci. , 19, 1912–1921. Google Scholar Lee, J.H., Gomora, J.C., Cribbs, L.L. et al. ( 1999) Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys. J. , 77, 3034–3042. Google Scholar Liévano, A., Bolden, A., and Horn, R. ( 1994) Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits. Am. J. Physiol. , 267, C411–C424. Google Scholar Liévano, A., Santi, C.M., Serrano, C.J. et al. ( 1996) T-type channels and 1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction. FEBS Letts. , 388, 150–154. Google Scholar Linares-Hernandez, L., Guzman-Grenfell, A.M., Hicks-Gomez et al. ( 1998) Voltage-dependent calcium influx in human sperm assessed by simultaneous detection of intracellular calcium and membrane potential. Biochim. Biophys. Acta , 1372, 1–12. Google Scholar McDonough, S.I., and Bean, B.P. ( 1998) Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons. Mol. Pharmacol. , 54, 1080–1087. Google Scholar Mikami, A., Imoto, K., Tanabe, T. et al. ( 1989) Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature , 340, 230–233. Google Scholar Perez-Reyes, E., Cribbs, L.L., Daud, A. et al. ( 1998) Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature , 391, 896–900. Google Scholar Perez-Reyes, E., and Schneider, T. ( 1994) Calcium channels: structure, function, and classification. Drug Dev. Res. , 33, 295–318. Google Scholar Santi, C.M., Darszon, A., and Hernandez-Cruz, A. ( 1996) A dihydropyridine-sensitive T-type Ca2+ current is the main Ca2+ current carrier in mouse primary spermatocytes. Am. J. Physiol. , 271, C1583–C1593. Google Scholar Tesarik, J., Mendoza, C., and Carreras, A. ( 1993) Fast acrosome reaction measure: a highly sensitive method for evaluating stimulus-induced acrosome reaction. Fertil. Steril. , 59, 424–430. Google Scholar Williams M.E., Washburn, M.S., Hans, M. et al. ( 1999) Structure and functional characterization of a novel human low-voltage activated calcium channel. J. Neurochem. , 72, 791–799. Google Scholar Wong, H., Anderson, W.D., Cheng, T. et al. ( 1994) Monitoring mRNA expression by polymerase chain reaction: the `primer-dropping' method. Anal. Biochem. , 223, 251–258. Google Scholar World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK. Google Scholar Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill, J.D. (eds), The Physiology of Reproduction. Raven Press, New York, USA, pp. 189–317. Google Scholar © European Society of Human Reproduction and Embryology
TI - Acrosome reaction of human spermatozoa is mainly mediated by α1H T-type calcium channels
JO - Molecular Human Reproduction
DO - 10.1093/molehr/6.10.893
DA - 2000-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/acrosome-reaction-of-human-spermatozoa-is-mainly-mediated-by-1h-t-type-w0ce4y0YkN
SP - 893
EP - 897
VL - 6
IS - 10
DP - DeepDyve
ER -