VOLTAGE DEPENDENT CALCIUM CHANNELS IN MAMMALIAN SPERMATOZOA

Susan Benoff

Division of Human Reproduction, Department of Obstetrics and Gynecology, North Shore University Hospital-New York University School of Medicine, Manhasset, New York

Received 7/1/98 Accepted 11/10/98

4. ROLE OF L-TYPE VDCC IN THE ACROSOME REACTION AND HUMAN MALE INFERTILITY

My impetus to characterize the calcium channels expressed in mammalian sperm was derived from an unexpected IVF fertilization failure with normospermic semen (88). The patient's sperm exhibited a reduced ability to undergo agonist-stimulated acrosome exocytosis. The patient was medicated with nifedipine, a calcium ion channel blocker (156,157), for hypertension control. As (1) agonist-stimulated calcium influx initiates the physiological acrosome reaction (e.g., 4,69,79,158) and (2) fertility was restored following discontinuation of nifedipine (136), these observations suggested that the observed inhibition of sperm fertilizing potential was causally related to administration of nifedipine. Subsequently, 19 additional cases of human male infertility directly attributable to calcium entry antagonists were identified (88,135,136; A. Hershlag and S. Benoff, unpublished observations).

The alpha-1 subunit of the L-type VDCC contains binding sites for nifedipine and other calcium channel blockers (43,47-49,159,160). Rat and human sperm express antigenic epitopes on their equatorial/post-acrosomal head plasma membranes which are shared with those of the alpha-1 subunit and dihydropyridine receptor of the rabbit skeletal muscle VDCC (126,161-163; see figures 3A-D). Taken together, these observations suggested that: (1) the pharmacological blockade of L-type VDCC contributed to the production of male infertility in patients taking calcium entry antagonists, and (2) PCR primers derived from somatic L-type VDCC alpha-1 sequences could be used to clone the L-type VDCC expressed in sperm.

Figure 3. Rat and human sperm bind antibodies directed against L-type VDCC and phosphotyrosine residues. Identification of antigenic epitopes expressed by mammalian sperm. Indirect immunocytochemical labeling was performed following previously published protocols (4,126,178,250). Labeled sperm were viewed at X600 magnification with UV-epifluorescence illumination and photographed at X1500 magnification on 400 ASA film with an exposure time of 50 seconds. Paired phase (A,C,E) and epifluoresence (B,D,F) are shown. (A,B) Unfixed non-permeabilized rat sperm display unique staining with polyclonal sheep antibody 006 limited (anti-skeletal muscle dihydropyridine receptor; 251) to the post-acrosomal region of the head (126). No staining is observed with control aliquots not exposed to primary antibody (not shown). (C,D) Unfixed Triton-permeabilized preparations of motile human sperm from fertile donors bind monoclonal antibody IIF7 (anti-skeletal muscle dihydropyridine receptor; 161) over the equatorial/post-acrosomal regions of their heads (126,178). This binding pattern differs signficantly from that observed with anti-actin or anti-myosin antibodies (176,178,219,250) and is not observed in the absence of primary antibody (not shown). (E,F) Anti-phosphotyrosine monoclonal antibody clone 1G2 (Boehringer Mannheim Corp., Indianapolis, IN) binds to the tails of capacitated motile human sperm, confirming results from another laboratory (252). In addition, this antibody binds to the human sperm head in two distributions: (i) over the acrosome cap, or (ii) limited to the equatorial/post-acrosome regions (4,178). The percentages of sperm displaying clone 1G2 binding in the equatorial/post-acrosome regions is significantly increased after induction of the acrosome reaction with model zona ligand. containing mannose while the percentages of sperm displaying clone 1G2 binding over the acrosome cap was unaffected by this treatment (4,178). Typical results are shown with the arrows pointing to sperm binding clone 1G2 in the equatorial/post-acrosome regions. This distribution coincides with that observed for antibodies to L-type VDCC alpha-1 subunit (see Part D).

Using such methodology, we have cloned and characterized the entire mRNA encoding the L-type VDCC alpha-1_C subunit that is expressed in rat and human testis (31,126,164,165). The complete cDNA sequence and the deduced amino acid sequence of the rat testes alpha-1_C subunit appears in Goodwin et al. (31). We have observed four main regions in which the rat testis-specific VDCC alpha-1_C subunit differs from that of the L-type VDCC alpha-1_C of cardiac muscle. The observed diversity is due, in part, to alternative exon usage from a single primary transcript, which we have directly demonstrated by genomic PCR analysis encompassing multiple exons and the intervening intronic sequences (164): (1) domain I segment 6 (IS6) (preferential usage of exon 8A vs. exon 8 in cardiac muscle), (2) domain III segment 2 (IIIS2) (preferential use of exon 21 vs. exon 22 in cardiac muscle), and (3) domain IV segment 3 (IVS3) (preferential use of exon 31 vs. exon 32 in cardiac muscle) (e.g., see figures 1 and 4) (31,126,164). Exon 8A has not previously been described in the gene encoding the cardiac alpha-1 subunit (166). Additional diversity is derived from the use of alternative promotor sequences, resulting in use of alternate exon 1A and a truncated 5' end in the testis-specific sequence (31). All the differences we have identified occur in regions of the VDCC alpha-1 subunit that regulate the gating kinetics and dihydropyridine sensitivity of the VDCC (19,22,47-49).

The regions of alternative splicing observed in the testis-specific alpha-1_C subunit conform to those observed in somatic tissues (7,8,13). The amino terminus is the only region which is spliced in a tissue-restricted manner (8). The amino terminus of the testis-specific alpha-1_C subunit is also expressed in brain, smooth muscle from lung and fibroblasts (167-169). The IS6 sequence observed in the testis-specific alpha-1_C subunit is found in the alpha-1_C subunit expressed in smooth muscle from lung (167). The IIIS2 segment of the testis-specific alpha-1_C subunit is detected in brain (48,168,170). The IVS3 segment of the testis-specific alpha-1_C subunit is expressed in skeletal muscle where, however, the alpha-1 subunit is the product of a separate gene (alpha-1_S; 13).

It is likely that two of the alternate splicing events detected, i.e., transmembrane segment IS6 and the amino terminus, could directly affect the electrophysiological characteristics of the calcium current in sperm. With regard to the former event, electrophysiological characterization of the L-type VDCC alpha-1_C expressed in smooth muscle indicates that this subunit deactivates slowly, in a manner ascribed to T-type channels (171,172). This is probably the result of expression of an alternately spliced alpha-1_C transcript in the region encoding exon 8 (IS5-IS6 linker/IS6) in smooth muscle and fibroblasts as compared to cardiac muscle (167), which modulates voltage-dependent inactivation (22). As the IS5-IS6 linker contributes to ion selectivity (7,173), this alternate splicing event could provide an explanation as to why the testis-specific VDCC is more sensitive to nickel and less sensitive to cadmium than classical L-type VDCC (69,79; also see Section 6). Additional changes in channel inactivation kinetics could result from the alternatively expressed amino terminus (47). The amino terminus is cytoplasmic (see figure 1). Molecular modeling using related potassium channels (174,175) indicates that the amino terminus can directly occlude the ion pore of the alpha-1 subunit.

These findings indicate that the electrophysiological properties of alpha-1_C subunit expressed in rat (and human testis and sperm; see below) would not exactly fit those expected of the prototype L-type VDCC alpha-1_C. This conclusion is supported by studies in excitable somatic cells in culture, where divergent genotypic and phenotypic expression of alpha-1 subunits has been reported (30). Our data is also consistent with observations on calcium currents in smooth muscle in vivo. These cells exclusively express alpha-1_C subunits (167) but display both typical L-type currents and a nifedipine-sensitive current with slow tail current decay, e.g., T-like (171,172). Nevertheless, we needed to rule out the possibility that "T-type" alpha-1_G subunits were also expressed in these tissues.

Using three sets of primers derived from the rat brain alpha-1_G sequence (9) and rat brain RNA as template, robust products of the expected size and sequence were obtained (176; see figure 2B). In contrast, we were unable to amplify the sequences encoding the pore of the alpha-1_G subunit in rat testis RNA. Positive control reactions using actin primers and either template gave similar results. Thus, to date, there is no direct evidence for the expression of alpha-1_G subunits in mammalian testis.

Importantly, analysis of the human testis-specific VDCC sequence (165) reveals additional diversity that we did not catalog in the rat (31). In contrast to the rat where essentially only two VDCC isoforms were detected in testis, 12 different alternative splicing events in alpha-1_C transcripts have found in human testis (table 1), which in combinations produce at least 16 different VDCC isoforms. For example, in the IVS3 alternatively expressed region, we have observed a 72% use of exon 31 in the population (testis poly A+ RNA source pooled from 10 male subjects 10-60 yrs.). However, we have also found that 10% of the time there is a deletion following exon 31, which corresponds with exon skipping of exon 33 (figure 4). This exon acts as a linker from IVS3 to IVS4. Upon depolarization, the charged residues in S4 are thought to rotate toward the extracellular face of the bilayer. If there is a restriction on the size and/or increased rigidity of the S3 to S4 linker segments, this may have an influence on the kinetics of channel opening.

Figure 4. Comparison of the splicing patterns of the alpha-1_C subunit mRNA observed in human testis and cardiac muscle (165). Transmembrane region IVS3 participates both in dihydropyridine binding and in voltage gating by forming salt bridges with the S4 voltage sensor. IVS Exons 31 and 32 encode alternate IVS3 segments in the alpha-1_C gene. Only exon 32 is detected in cardiac muscle. In contrast, exon 31 is expressed in testis. In 10% of the clones examined from testis RNA as template, a deletion of exon 33 was observed (see table 1). Exon 33 encodes a short linker segment between IVS3 and its voltage sensor.

RT-PCR, cloning of the PCR products and DNA sequencing were performed using previously published protocols (31,126,164). P>Results from RT-PCR in situ and from immunocytochemistry indicate that VDCC alpha-1_C subunit mRNA and protein are expressed in rat testis sections throughout the seminiferous epithelium, both in all stages of the germ cell lineage and in Sertoli cells, but not in the interstitial space (164). Examination of mRNA from cultured Sertoli cells confirms that Sertoli cells express the testis-specific VDCC alpha-1_C isoform (31). VDCC alpha-1_C transcripts have recently been detected in RNA from ejaculated human sperm, suggesting post-meiotic VDCC gene expression (177).

In addition, we now have documented inter-individual variability in testis-specific L-type VDCC alpha-1_C isoform expression. In a preliminary study of ten men, we found that each man reproducibily expressed, in ejaculated sperm, RNA populations encoding only one of the 16 isoforms (L.O. Goodwin, D.S. Karabinus, R.G. Pergolizzi and S. Benoff, submitted). In two of these cases, we detected a deletion of exons 31 through 33 (see Table 1), suggesting that the calcium ion pore would be non-functional in these men. These data suggest the existence of human sperm VDCC isoform diversity relevant to sperm fertilizing potential. The examination of this potential VDCC isoform diversity in fertile and infertile men is in progress.

Finally, we have identified two mechanisms regulating VDCC function which are unique to sperm (4).

First, channel opening is regulated by ligand-stimulated phosphorylation of unique tyrosine residues in IS6 and/or IIIS2, which is inhibited in vivo (178) and in vitro by dihydropyridines (4; e.g., see figures 3F and 5), suggesting a mechanism by which calcium transport is abrogated by these drugs. The human sperm mannose receptor plays an important role in this process: binding of mannose moieties is associated with VDCC tyrosine phosphorylation (figure 5; 4,82). The deduced amino acid sequence of the testis-specific alpha-1_C subunit provides no evidence for an intrinsic tyrosine kinase activity and, thus, autophosphorylation. Rather, we suggest (4) that this phosphorylation is effected by the ZRK/hu9 tyrosine kinase activity of the sperm head which is postulated to play a regulatory role in induction of the acrosome reaction (179,180).

Figure 5. Nifedipine, an L-type dihydropyridine calcium channel anatagonist, inhibits protein tyrosine phosphorylation induced by exposure of human sperm to model zona ligands containing mannose. Typical results are shown for motile sperm populations from three fertile donors incubated overnight in capacitation media supplemented with 5 uM nifedipine (4,88,178). Control aliquots were not exposed to the drug. Acrosome loss was induced by mannose treatment (61,63,131,250) and sperm were then labeled anti-phosphotyrosine monoclonal antibody clone 1G2 (Boehringer Mannheim Corp., Indianapolis, IN) (4,178). At least 300 sperm in each aliquot were examined for antibody binding ("ANTI-PTYR ABS") in the equatorial region of the sperm head (see Figure 3F). Exposure to nifedipine resulted in a significant inhibition of the both acrosome loss (not shown) and anti-phosphotyrosine antibody binding (t-test, P <0.05). Although only one specimen from each fertile donor was analyzed, these observations confirm prior in vitro (4,178) and in vivo (178) findings. Second, proteolysis appears to be involved in the formation of the active channel (4). Western blot of membrane proteins extracted from fertile human donor sperm and probed with antibodies specific for the L-type VDCC alpha-1 subunit indicate the presence of two antigenic protein species (figure 6, lane 2). The first species migrates between 165 to 175 kDa, consistent with the size reported for the alpha-1 subunit expressed in somatic tissues (161,163). The second species, which migrates at approximately 60 kDa, has never been reported for VDCC from somatic tissues, and was not observed in the rat germ line (164). Preliminary studies suggest that this lower molecular weight protein is not detectable in extracts from sperm from infertile men (4; figure 6, lane 1). The loss of this protein species is likely to be of clinical importance as about 50% of cases of reduced or failed fertilization in IVF exhibit normal levels of expression of receptors for human zona ligands containing mannose but a reduced ability to undergo a mannose-stimulated acrosome reaction (131,181).

Figure 6. Expression of the alpha-1_C subunit protein differs between fertile and some infertile men. Sperm plasma membrane extracts, SDS-polyacrylamide gel electrophoresis of sperm membrane proteins in the presence of pre-stained molecular weight size standards (Bio-Rad Laboratories, Hercules, CA), transfer of size-separated proteins to nitrocellulose membranes, reaction with monoclonal antibody IIF7 (anti-skeletal muscle dihydropyridine receptor; 161) and detection of antibody binding using Renaissance Chemiluminescence Reagent (NEN Dupont, Boston, MA) were performed as previously described (4,164). Typical results are shown. The migration of the size standards (203 kDa, 126 kDa and 42 kDa) is indicated to the left. The arrows identify the positions of protein species which bind antibody. (Lane 1) In a specimen from a patient who exhibited complete failure of fertilization in vitro as a result of an acrosome reaction insufficiency (131) only one protein species, M_r 165-175 kDa, was reactive with monoclonal antibody IIF7. (Lane 2) In contrast, two proteins species, M_r 165-175 kDa and 60 kDa, which contain antigenic epitopes which are recognized by monoclonal antibody IIF7 are observed in plasma membrane protein extracts prepared from fertile donor sperm. These data confirm prior findings (4,176).

These data unequivocally demonstrate that an L-type VDCC alpha-1_C subunit is expressed in mammalian testis and sperm and provide evidence for a role for this subunit in the mammalian sperm acrosome reaction.