Molecular Human Reproduction, Vol. 5, No. 6, 507-512,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining
1 Departments of Obstetrics & Gynecology and 2 Department of Pathology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York, USA
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The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.
acrosome reaction/capacitation/flow cytometry/progesterone/spermatozoa
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The ability of sera obtained from different women to promote the penetration of zona-free hamster eggs by human spermatozoa has been investigated previously (Margalioth et al., 1988
). It was reported that incubation of spermatozoa in dilute luteal phase sera enhanced their egg penetrating ability while follicular phase serum did not. This effect was abrogated by charcoal treatment of serum, raising the issue that it was mediated by steroids. Furthermore, the addition of progesterone to a defined medium, Biggers–Whitten–Whittingham (BWW), promoted the egg penetrating ability of human spermatozoa. As evidence had been presented that progesterone binds to the sperm plasma membrane (Hyne et al., 1978), it was speculated that its effect might be through a receptor-mediated enhancement of capacitation (Margalioth et al., 1988). Subsequent work (Osman et al., 1989; Meizel et al., 1990) to investigate the ability of follicular fluid to promote the acrosome reaction (Suarez et al., 1986), demonstrated that the effect of follicular fluid was mediated in part by progesterone. Evidence exists that progesterone promotes the uptake of calcium by capacitated spermatozoa (Blackmore et al., 1990; Baldi et al., 1991; Shimizu et al., 1993), and that this increase in cytosolic calcium concentration is a prerequisite for the acrosome reaction. The promotion of calcium uptake appears to be mediated by a non-genomic progesterone receptor located within the plasma membrane of spermatozoa. A monoclonal antibody has been produced against human spermatozoa that appears to block the progesterone-mediated increase in spermatozoa of cytosolic calcium and their consequent acrosome reaction (Brucker et al., 1994).
Whether progesterone at physiological concentrations can, indeed, promote the acrosome reaction has remained controversial. Data have been presented suggesting that progesterone does not promote an acrosome reaction in human spermatozoa, as determined by flow cytometric analysis of the expression of membrane cofactor protein (CD46) by spermatozoa (Carver-Ward et al., 1996; Emiliozzi et al., 1996). Previously, this complement regulatory protein (Liscewsksi et al., 1991) had been shown to be present on the inner acrosomal membrane of human spermatozoa (Fenichel et al., 1989, 1990). While progesterone was shown to promote the uptake of calcium by spermatozoa, no increase in the percentage of acrosome-reacted spermatozoa was observed as judged by flow cytometric analysis. Prior results purporting to demonstrate an acrosome reaction-promoting effect of progesterone were criticized either on the basis of the use of non-specific techniques to detect acrosome-reacted spermatozoa, such as the triple stain (Talbot and Chacon, 1981), the failure of investigators to eliminate dead spermatozoa during enumeration of those acrosome-reacted, or an insufficient number of cells scored by visual methods.
We have performed similar flow cytometric studies, using a supravital dye to eliminate dead spermatozoa during flow cytometric analysis and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Confocal microscopy was used to confirm that CD46 staining was limited to the acrosomal region of the sperm head. When care was taken to ensure the adequacy of sperm capacitation conditions, as judged by two criteria, the ability of spermatozoa to acrosome react in response to mannosylated bovine serum albumin (BSA) (Benoff et al., 1997a) and to penetrate zona-free hamster eggs (Rogers et al., 1979), progesterone was shown to promote an acrosome reaction.
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Sperm preparation
Capacitation was performed as previously described (Bronson and Fusi, 1990
). Spermatozoa were obtained using a three-layer Isolate (Irvine Scientific, Santa Ana, CA, USA) centrifugation (90%, 1.5 ml; 70%, 1 ml; 40%, 1.5 ml). Semen (2 ml) was layered over a column of Isolate in human tubal fluid medium (HTF; Irvine Scientific) in 15 ml conical centrifuge tubes (Falcon; Becton Dickinson, Mountain View, CA, USA). Tubes were centrifuged at 200 g for 25 min, the pellet was collected and spermatozoa washed twice by centrifugation at 300 g for 5 min using BWW medium, containing 5 mg/ml human serum albumin (HSA, Fraction V, Lot. No.126H9322; Sigma, St Louis, MO, USA). Spermatozoa (20x106 cells/ml) were resuspended in BWW with 30 mg/ml HSA and capacitated by incubation overnight (18 h) at 37°C, 5% CO2.
Induction of the acrosome reaction with progesterone
Acrosome reactions were induced in capacitated spermatozoa with progesterone, as previously described (Osman et al., 1989; Fusi et al., 1996). A stock solution of water-soluble progesterone (5 mg/ml) (Sigma p-7556) was stored in aliquots at –70°C and was never thawed more than once. The stock solution was diluted in BWW with 30 mg/ml HSA to a concentration of 500 µg/ml and added to the capacitated spermatozoa to a final concentration of 5 µg/ml or 15 µg/ml. After 1 h incubation at 37°C in 5% CO2, spermatozoa were washed by two centrifugations at 500 g for 5 min, in phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA) or BWW with 5 mg/ml HSA and used subsequently for incubation with monoclonal antibodies and flow cytometry or Pisum sativum agglutinin (PSA)–fluorescent isothiocyanate (FITC) staining (Cross et al., 1986).
Scoring of the acrosome reaction by direct immunofluorescence and flow cytometry
Spermatozoa were washed with 2 ml of PBS. Cells were pelleted at 500 g for 5 min and resuspended in PBA (PBS + 0.5% BSA) at a concentration of 107/ml. Appropriate amount of antibodies was added: 10 µl of anti-CD46–FITC mouse monoclonal antibody (Serotec, Raleigh, NC, USA) per 106 cells or 5 µl (1 µg) of anti-CD59–PE mouse monoclonal antibody per 106 cells (Caltag, Burlingame, CA, USA). Cells were incubated for 30 min at room temperature in the dark. Cells were pelleted at 500 g for 5 min. Supernatant was discarded and cells were washed twice with 2 ml PBA. Cells were resuspended in 500 µl of 1% paraformaldehyde in PBS for fixation. Tubes were stored in the dark at 4°C until analysed by flow cytometry (FACScan; Becton Dickinson). When CD46 was determined, only live cells were scored. Fluorescence detection of cell viability were performed in all specimens using the dye 7-aminoactinomycin D (7-AAD) (Molecular Probes, Eugene, OR, USA), which stains non-viable cells. 25 µl of 7-AAD (1 µg/ml) were added to 975 µl of PBA to form a working solution. 20 µl of working solution were added to 106 cells together with the FITC-labelled anti-CD46 antibody. The matching isotype murine myeloma proteins for the CD46 and CD59 monoclonal antibodies, MOPC21 [immunoglobulin (Ig)G1, Sigma] and UPC10 (IgG2a; Becton Dickinson), were used as controls for non-specific immunoglobulin binding. Following labelling, the specimens were gated by light scatter properties for spermatozoa and analysed for dual colour fluorescence. 7AAD-stained dead cells were excluded and CD46 was detected only among live cells.
Visual measurement of the acrosome reaction in human spermatozoa
The percentage of fresh and capacitated spermatozoa that had spontaneously acrosome-reacted, as well as the percentage of capacitated spermatozoa which had acrosome-reacted after exposure to progesterone, was determined by means of PSA–FITC staining (Cross et al., 1986). Dead spermatozoa that could have undergone degenerative loss of acrosomal contents were identified with a supravital stain H258 (Hoechst 33258; bisbenzimide; Sigma). Spermatozoa (20x106/ml in BWW with 5 mg/ml HSA) were incubated with H258, working solution 10 µg/ml in PBS for 5 min at 37°C and 5% CO2, and freed from the excess stain by stratification over 2% polyvinylpyrrolidone (PVP, 40000; Sigma) in BWW for 20 min at 900 g. The pellet was resuspended in 100 µl of ethanol 95% in PBS and incubated at 4°C for at least 30 min. Thereafter, a drop of sperm suspension was placed on a slide at room temperature and, when ethanol had evaporated in air, covered with 20 µl of 100 µg/ml PSA–FITC and incubated in the dark in a humid chamber 5 min. The slides were then carefully rinsed in distilled water and mounted. Only live spermatozoa, which had not appeared fluorescent through a 480 nm filter for excitation of H258, were scored under fluorescent illumination using a 520–540 nm filter and was divided into acrosome-intact and acrosome-reacted respectively, when the acrosome exhibited uniform green fluorescence and when no fluorescence or only an equatorial green band was evident. When confocal microscopy was used, Slow Fade reagent (Molecular Probes) was used for mounting the slides to avoid bleaching.
When confocal microscopy was used, ~25x106 cells after incubation with anti-CD46 antibody, fixed in 100 µl of 1% paraformaldehyde, were placed on a slide at room temperature. After drying, slides were rinsed with PBS or with equilibration buffer from the Slow Fade kit (Molecular Probes, Cat.No.S-2828) and mounted with Slow Fade to avoid bleaching.
Induction of the acrosome reaction with mannose
The acrosome reaction was induced in capacitated spermatozoa with mannose (75 mM mannose + mannosylated BSA; Sigma), using a modification of a previously published method (Benoff et al., 1997a). Capacitated spermatozoa were washed three times at 500 g for 5 min with a 75 mM (D+) mannose (Sigma) in a calcium-containing buffer (30 mM HEPES, pH 7.0, 0.5 mM MgCl2, 0.5 mM NaCl, 10 mg/ml BSA, 20 mM CaCl2 and 75 mM mannose). Washed spermatozoa were incubated in the same buffer with 100 µg/ml of mannosylated BSA (Sigma, A4664) for 45 min at 37°C, 5% CO2, and then washed twice (500 g, 5 min) with calcium- and mannose-free buffer (30 mM HEPES, pH 7.0, 0.5 mM MgCl2, 0.5 NaCl, 10 mg/ml BSA). Washed spermatozoa were scored for CD46 staining by direct immunofluorescence and flow cytometry and/or by PSA–FITC staining, as described.
Zona-free hamster egg penetration test
The method of Rogers et al. (Rogers et al., 1979), as modified by Bronson et al. (Bronson et al., 1981) was used to assess the penetrating ability of capacitated human spermatozoa. In brief, zona-free hamster eggs obtained from a pool of three or four animals were denuded enzymatically of their vestments with hyaluronidase and trypsin and 20–30 eggs inseminated with spermatozoa (5x106/ml ) from each semen donor that had been capacitated by overnight incubation in BWW with 30 mg/ml HSA. Following a 3 h gamete co-incubation, eggs were washed free of spermatozoa and stained as whole mounts with Acridine Orange. The numbers of penetrating spermatozoa and the percentage of eggs penetrated were determined by epifluorescence microscopy.
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Results |
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The ability of progesterone to promote the acrosome reaction in capacitated human spermatozoa were studied utilizing spermatozoa from three men whose spermatozoa penetrated zona-free hamster eggs within the fertile range and from 19 men from infertile couples, whose ejaculates exhibited normal semen parameters by World Health Organization criteria (WHO, 1992). The egg penetration frequencies (mean ± SD) for donors 134, 139, and 141 were 93.9 ± 9.7% (n = 13 tests), 64.5 ± 26.7% (n = 11), and 84.2 ± 17.8% (n = 5) respectively. Between 12 and 30 eggs were inseminated with donor spermatozoa for each test. Capacitation was associated with an increase in both the percentage of acrosome-reacted (CD46-positive) spermatozoa and the geometric mean of fluorescence intensities (Figure 1
). Progesterone stimulated a further increase. The percentage of capacitated spermatozoa exhibiting CD46 increased following their exposure to progesterone, as did the geometric mean, while there was no change in the percentage of spermatozoa displaying CD59 staining incubated under similar conditions (Table I). The progesterone-promoted increase in percentage of acrosome-reacted spermatozoa in five ejaculates of donor 134 varied from 10.1 to 36.3% (18.1 ± 10.5, mean ± SD) and for donor 141 from 4.6 to 12% (7.5 ± 2.7, mean ± SD). Spermatozoa from one ejaculate of donor 139 failed to respond to progesterone, while the percentage of acrosome-reacted spermatozoa following exposure of spermatozoa from a second ejaculate of the same donor to progesterone increased from 2.4 to 13.4%. Capacitated spermatozoa in 18 out of 19 specimens from the infertile men exhibited a response to progesterone (10.4 ± 12.3), although a wide range in response was noted (Table I).
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When we compared the response of capacitated spermatozoa to progesterone versus mannose, which has also been shown to promote an acrosome reaction (Benoff et al., 1997a
), the mean percentages of acrosome-reacted spermatozoa in eight separate experiments using three different known fertile men were not significantly different for progesterone (7.7 ± 4.7) and mannose (13.8 ± 11.5), although a wider variability of response to mannose was noted, as reflected in the SD. In a preliminary study using donors 134 and 141, we compared the percentage of acrosome-reacted spermatozoa, as scored visually by epifluorescence microscopy, using fluorescein-conjugated PSA staining of acrosomal contents with the percentage CD46-positive cells, as determined by flow cytometry. In each case, dead cells were excluded by staining with supervital dyes (Hoescht 33258) for visual scoring and 7ADD for FAScan. There was no significant difference in scores. The increment in the percentage of acrosome-reacted spermatozoa was 15% for CD46 staining versus 15.8% for PSA lectin staining for donor 134 and 9.4% versus 11.5% for donor 141.
Confirmation of the specificity of CD46 binding to the sperm surface was shown by confocal microscopy. Fluorescence was limited to the acrosomal region of the spermatozoon head (Figure 2), consistent with prior ultrastructural observations that CD46 is localized on the inner acrosomal membrane (Fenichel et al., 1990). There was a range of CD46 reactivity, from completely unstained spermatozoa (acrosome intact) to completely stained (acrosome-reacted), with other cells showing intermediate patchy staining over their acrosomal region. These observations were correlated with our reciprocal findings during staining of acrosomal contents with fluoresceinated PSA, which was lost in a patch-like manner, as judged by epifluorescence microscopy. They are also consistent with the known mechanism of acrosome reaction, during which time the plasma membrane fuses with the outer acrosomal membrane, creating fenestrations exposing the inner acrosomal membrane present over the rostral portion of the spermatozoon head. As the amount of CD46 antibody binding to spermatozoa, as reflected in the geometric mean of fluorescence intensities during flow cytometric analysis, increased following addition of progesterone, this suggests that greater numbers of spermatozoa had undergone completion of the acrosome reaction.
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Discussion |
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Mammalian spermatozoa obtained fresh from ejaculates lack the ability to fertilize eggs, but acquire this capability during their residence within the female reproductive tract (Yanagmachi, 1994). The alterations leading to this ability to fertilize have been termed capacitation. Only spermatozoa that have undergone capacitation are capable of undergoing the acrosome reaction, during which time the plasma membrane of the rostral portion of the sperm head fuses with the outer acrosomal membrane, leading to the release of acrosomal contents and the shedding of these membranes (Yanagimachi, 1994
). Only acrosome-reacted spermatozoa are capable of penetrating the zona pellucida, fusing with the oolemma, and entering the cortical ooplasm.
Our results provide further evidence that progesterone promotes the acrosome reaction. Capacitated spermatozoa, from both known fertile men and men from infertile couples, exhibited increases in the proportion of acrosome-reacted cells following progesterone exposure. The variation in the increase in the acrosome reacted spermatozoa was wider within the infertile group and the increment tended to be lower than in the known fertile men, as expected. We also noted differences in the response of spermatozoa to progesterone between ejaculates of the same fertile men, despite using standard incubation conditions. These results suggest that the ability of spermatozoa to be capacitated and undergo an acrosome reaction may vary through time. The ability of spermatozoa to penetrate zona-free hamster eggs has also been shown to vary through time (Bronson et al., 1998). Whether this variation was related to the period of sexual abstinence, diet, stress or other external factors remains unknown.
These results support previous work (Fenichel et al., 1989, 1990), which indicated that the detection of membrane cofactor protein (CD46) is a reliable marker of spermatozoa that have undergone the acrosome reaction. Our observations using confocal microscopy demonstrating that staining by FITC anti-CD46 monoclonal antibody was localized to the acrosomal region of spermatozoa are consistent with its immunogold localization by transmission electron microscopy on the inner acrosomal membrane but not the plasma membrane of human spermatozoa (Fenichel et al., 1990). The specificity of immunostaining was further validated in our experiments comparing anti-CD46 staining with that of against CD59, a complement regulatory protein present on both the plasma membrane and inner acrosomal membranes (Rooney et al., 1993; Simpson and Holmes, 1994). CD46 exhibited an increased expression following addition of progesterone to the medium while CD59 did not. When supra-vital dyes were used to exclude dead spermatozoa that may have undergone a senescent acrosome reaction, the proportion of CD46-positive cells as determined by flow cytometry was correlated with that observed by loss of FITC–PSA staining of acrosomal contents as judged visually using epifluorescence microscopy.
There is increasing evidence (Cross, 1998), that the loss of sperm plasma membrane cholesterol plays an essential role in capacitation. The capacitation of human spermatozoa is dependent upon the addition of a protein source to the culture medium, usually bovine or human serum albumin. Different lots of albumin have been found to vary in their ability to promote capacitation, judged by the penetration of zona-free hamster eggs (Bronson and Rogers, 1988). Observation of the egg penetrating ability of spermatozoa from three known fertile donors after their concurrent incubation of spermatozoa in a chemically-defined medium (BWW) containing human serum albumin obtained from each of two different sources (Sigma versus Miles Laboratories) demonstrated widely different penetration frequencies. The range of percentage of eggs penetrated was 98.4–100% following incubation in Sigma HSA compared with 52.1–73.4% in Miles HSA, for the same spermatozoa co-incubated with zona-free sister eggs. The mean number of penetrating spermatozoa per egg varied from 4.05–13.6 for spermatozoa incubated in Sigma HSA versus 1.51–2.1 for Miles HSA. The varying ability of different lots of HSA to support sperm capacitation, as judged by penetration frequencies of zona-free hamster eggs, was correlated with their lipid transfer activity (Ravnick and Mueller, 1993), which was shown to be dependent upon their content of lipid transfer protein I.
We documented the adequacy of capacitating conditions in the present experiments by two independent criteria. A mannose binding ligand has been shown to be expressed by human spermatozoa during their capacitation; its expression is dependent upon cholesterol efflux, and mannosylated BSA has promoted the acrosome reaction in these human spermatozoa (Benoff et al., 1997a). This interaction has been postulated to mimic the promotion of the acrosome reaction known to occur following binding of spermatozoa to the zona pellucida (Benoff et al., 1997b). In our experiments, the proportion of spermatozoa that underwent an acrosome reaction following exposure to mannosylated BSA versus progesterone was roughly equivalent for the two moieties. Indeed, mannose-binding ligands and progesterone receptors appear to be co-expressed during capacitation of human spermatozoa (Benoff et al., 1995). Spermatozoa were also documented to be capacitated by their ability to penetrate zona-free hamster eggs (Yanagimachi et al., 1976).
As we have addressed the other authors' concerns (Carver-Ward et al., 1996; Emiliozzi et al., 1996), one must conclude that their negative results were related to inefficient capacitation of spermatozoa. While we documented that spermatozoa were capacitated by their ability to both acrosome react in response to an independent stimulus (mannosylated BSA) and to penetrate zona-free hamster eggs, this was not done in either of the prior studies. In the former study (Carver-Ward et al., 1996), ejaculates were obtained from known fertile men, spermatozoa recovered by Percoll density gradient centrifugation and capacitated in vitro by incubation for 4 or 24 h in HTF–BSA. A similar concern can be raised in analysing the data from the latter study (Emiliozzi et al., 1996), where they measured cytosolic calcium of human spermatozoa using the calcium indicator dye Indo-1. As noted by the authors, progesterone induced a rapid, transient, dose-dependent change in cytosolic calcium in human spermatozoa incubated for 5 h in B2 medium containing 10 mg/ml BSA. However, a similar response was observed in spermatozoa before their capacitating incubation in B2, following their Percoll gradient separation and washing. These findings are in contrast with those of other authors (Baldi et al., 1991), as well as our own studies (Shimizu et al., 1993), in which the steady state cytosolic calcium (Ca2+)i and progesterone-evoked calcium transients were shown to increase in magnitude following a capacitating incubation in BWW medium containing 30 mg/ml of HSA. It has been proposed that a threshold of calcium uptake is required to induce the acrosome reaction, and we suggest that this was not achieved in the experiments of Emiliozzi et al. (Emiliozzi et al., 1996). As the calcium transients observed in their study were depicted on a relative scale, it was not possible for us to compare the magnitude of results between laboratories. However, these authors have noted in their discussion, that other studies (Osman et al., 1989; Yang et al., 1994) found increases in the proportion of acrosome-reacted spermatozoa induced by progesterone in human spermatozoa that had been capacitated in BWW and HSA. In conclusion, we believe that the present study has provided convincing evidence that progesterone can promote the acrosome reaction in capacitated human spermatozoa.
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3 To whom correspondence should be addressed at: Dept. of Ob/Gyn, Health Sciences Center T9-080, S.U.N.Y., Stony Brook, New York 11794–8091, USA
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Submitted on December 15, 1998; accepted on March 11, 1999.
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 C. V. Harper, C. L.R. Barratt, S. J. Publicover, and J. C. Kirkman-Brown Kinetics of the Progesterone-Induced Acrosome Reaction and Its Relation to Intracellular Calcium Responses in Individual Human Spermatozoa Biol Reprod, December 1, 2006; 75(6): 933 - 939. [Abstract] [Full Text] [PDF]
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A. A. El-Ghobashy and C. R. West The Human Sperm Head: A Key for Successful Fertilization J Androl, March 1, 2003; 24(2): 232 - 238. [Abstract] [Full Text] [PDF]
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S. Gadkar, C.A. Shah, G. Sachdeva, U. Samant, and C.P. Puri Progesterone Receptor as an Indicator of Sperm Function Biol Reprod, October 1, 2002; 67(4): 1327 - 1336. [Abstract] [Full Text] [PDF]
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 S. Jagannathan, E. L. Punt, Y. Gu, C. Arnoult, D. Sakkas, C. L. R. Barratt, and S. J. Publicover Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells. EXPRESSION OF MULTIPLE ISOFORMS J. Biol. Chem., March 1, 2002; 277(10): 8449 - 8456. [Abstract] [Full Text] [PDF]
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