Mouse Sperm Acrosome Reaction Research Articles

Spermiogenesis in Caenorhabditis elegans: An Excellent Model to Explore the Molecular Basis for Sperm Activation.

C. elegans spermiogenesis converts non-motile spermatids into motile, fertilization-competent spermatozoa. Two major events include the building of a pseudopod required for motility and fusion of membranous organelles (MOs)-intracellular secretory vesicles-with the spermatid plasma membrane required for the proper distribution of sperm molecules in mature spermatozoa. The mouse sperm acrosome reaction-a sperm activation event occurring during capacitation-is similar to MO fusion in terms of cytological features and biological significance. Moreover, C. elegans fer-1 and mouse Fer1l5, both encoding members of the ferlin family, are indispensable for MO fusion and acrosome reaction, respectively. Genetics-based studies have identified many C. elegans genes involved in spermiogenesis pathways; however, it is unclear whether mouse orthologs of these genes are involved in the acrosome reaction. One significant advantage of using C. elegans for studying sperm activation is the availability of in vitro spermiogenesis, which enables combining pharmacology and genetics for the assay. If certain drugs can activate both C. elegans and mouse spermatozoa, these drugs would be useful probes to explore the mechanism underlying sperm activation in these two species. By analyzing C. elegans mutants whose spermatids are insensitive to the drugs, genes functionally relevant to the drugs' effects can be identified.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17β-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Starvation induces an increase in intracellular calcium and potentiates the progesterone-induced mouse sperm acrosome reaction.

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca2+ ionophore A23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca2+ ([Ca2+ ]i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca2+ ]i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca2+ ]i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features.

Knockout of family with sequence similarity 170 member A (Fam170a) causes male subfertility, while Fam170b is dispensable in mice†.

Families with sequence similarity 170 members A and B (FAM170A and FAM170B) are testis-specific, paralogous proteins that share 31% amino acid identity and are conserved throughout mammals. While previous in vitro experiments suggested that FAM170B, an acrosome-localized protein, plays a role in the mouse sperm acrosome reaction and fertilization, the role of FAM170A in the testis has not been explored. In this study, we used CRISPR/Cas9 to generate null alleles for each gene, and homozygous null (−/−) male mice were mated to wild-type females for 6 months to assess fertility. Fam170b−/− males were found to produce normal litter sizes and had normal sperm counts, motility, and sperm morphology. In contrast, mating experiments revealed significantly reduced litter sizes and a reduced pregnancy rate from Fam170a−/− males compared with controls. Fam170a−/−;Fam170b−/− double knockout males also produced markedly reduced litter sizes, although not significantly different from Fam170a−/− alone, suggesting that Fam170b does not compensate for the absence of Fam170a. Fam170a−/− males exhibited abnormal spermiation, abnormal head morphology, and reduced progressive sperm motility. Thus, FAM170A has an important role in male fertility, as the loss of the protein leads to subfertility, while FAM170B is expendable. The molecular functions of FAM170A in spermatogenesis are as yet unknown; however, the protein localizes to the nucleus of elongating spermatids and may mediate its effects on spermatid head shaping and spermiation by regulating the expression of other genes. This work provides the first described role of FAM170A in reproduction and has implications for improving human male infertility diagnoses.

Matrine compromises mouse sperm functions by a [Ca(2+)]i-related mechanism.

Matrine, a bioactive alkaloid widely used in Chinese medicine, inhibits mouse sperm functions in vitro. In this study, we investigated the reproductive toxicity of matrine to male mice in vivo. C57BL/6J mice were administered with daily doses of 0, 1, 10 and 50mg/kg matrine by intraperitoneal injection for 30 days. The results showed that matrine did not affect testis size, testis weight, sperm count and sperm viability, but it significantly inhibited total motility, progressive motility, linear velocity, capacitation and the progesterone-induced acrosome reaction of mouse sperm. Furthermore, the intracellular Ca(2+) concentration ([Ca(2+)]i), a key regulator of sperm function, was reduced in sperm of matrine-exposed mice. The current and gene expression of the sperm specific Ca(2+) channel, CatSper, which modulates Ca(2+) influx in sperm, were decreased in testes of matrine-exposed mice. These results indicate that matrine inhibits mouse sperm functions by a [Ca(2+)]i-related mechanism via CatSper channel.

Localization of Tektin 1 at Both Acrosome and Flagella of Mouse and Bull Spermatozoa

Tektins (TEKTs) are constitutive filamentous proteins of microtubules in cilia, flagella, basal bodies, and centrioles. In mammals, five TEKTs (TEKT1, 2, 3, 4, and 5) have been identified in testis and spermatozoa. With the exception of TEKT1, these TEKTs have been reported to be present in spermatozoa with predominant localization at the peri-axoneme structures of flagella, i.e., mitochondria and outer dense fibers. In the present study, we produced an antibody against TEKT1 to examine the localization of TEKT1 in mouse, bull, and rat spermatozoa. By immunoblot analyses and immunofluorescence microscopy, we found TEKT1 to be present in sperm flagella and at the apical region of acrosome cap in spermatozoa of all these species. Acrosome-associated TEKT1 disappeared after in vitro acrosome reaction in mouse spermatozoa. These observations suggest another potential role for TEKT1 as a cytoskeletal element in the sperm head, or as a molecule involved in acrosome-related phenomena, such as acrosome reaction.

Capacitation and acrosome reaction differences of bovine, mouse and porcine spermatozoa in responsiveness to estrogenic compounds.

BackgroundEndocrine disruptors are exogenous substance, interfere with the endocrine system, and disrupt hormonal functions. However, the effect of endocrine disruptors in different species has not yet been elucidated. Therefore, we investigated the possible effects of 17ß-estradiol (E2), progesterone (P4), genistein (GEN) and 4-tert-octylphenol (OP), on capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. In this in vitro trial, spermatozoa were incubated with 0.001-100 μM of each chemical either 15 or 30 min and then assessed capacitation status using chlortetracycline staining.ResultsE2 significantly increased capacitation and the acrosome reaction after 30 min, while the acrosome reaction after 15 min incubation in mouse spermatozoa. Simultaneously, capacitation and the acrosome reaction were induced after 15 and 30 min incubation in porcine spermatozoa, respectively. Capacitation was increased in porcine spermatozoa after 15 min incubation at the lowest concentration, while the acrosome reaction was increased in mouse spermatozoa after 30 min (P <0.05). E2 significantly increased the acrosome reaction in porcine spermatozoa, but only at the highest concentration examined (P <0.05). P4 significantly increased the acrosome reaction in bovine and mouse spermatozoa treated for 15 min (P <0.05). The same treatment significantly increased capacitation in porcine spermatozoa (P <0.05). P4 significantly increased capacitation in mouse spermatozoa treated for 30 min (P <0.05). GEN significantly increased the acrosome reaction in porcine spermatozoa treated for 15 and 30 min and in mouse spermatozoa treated for 30 min (P <0.05). OP significantly increased the acrosome reaction in mouse spermatozoa after 15 min (P <0.05). Besides, when spermatozoa were incubated for 30 min, capacitation and the acrosome reaction were higher than 15 min incubation in E2 or GEN. Furthermore, the responsiveness of bovine, mouse and porcine spermatozoa to each chemical differed.ConclusionsIn conclusion, all chemicals studied effectively increased capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. Also we found that both E2 and P4 were more potent than environmental estrogens in altering sperm function. Porcine and mouse spermatozoa were more responsive than bovine spermatozoa.

The effect of group X secreted phospholipase A2 on fertilization outcome is specific and not mimicked by other secreted phospholipases A2 or progesterone

Mouse group X sPLA2 (mGX) is an acrosomal protein playing an important role in fertilization and controlling acrosome reaction (AR) occurring during capacitation. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential invitro. We also showed that treatment of wild-type sperm with recombinant mGX during capacitation improved fertilization outcome. This interesting property suggests that sPLA2s could be used to improve fertilization in assisted reproductive technologies (ART). However the molecular mechanism explaining the mGX-dependent enhancing effect on fertilization outcome remains unclear so far. Interestingly, like progesterone (P4), mGX is a very potent activator of AR and the role of mGX-induced AR in fertilization outcome was not evaluated so far. To assess the role of sPLA2-induced AR in IVF, we first tested the potency of 9 mouse and 2 human sPLA2s and P4 to trigger AR of mouse sperm. We then tested the ability of 6 of these molecules (mouse Group IIA, mouse Group IID, mouse Group X, human Group V, human Group X and P4) to improve the yield of 2-cell embryos obtained by IVF in mouse. We showed that in the mouse neither P4 nor any of the other sPLA2s tested were able to mimic the IVF improvement produced by mGX-treatment. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that their utilisation in other species, is subjected to the identification of probably unique species-specific active sPLA2.

Fucose, Mannose, and β-N-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm Acrosome Reaction through Convergent Signaling Pathways

The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca2+-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

Xenoestrogenic chemicals effectively alter sperm functional behavior in mice

Xenoestrogenic compounds (XCs) can disrupt endogenous hormone function and affect sperm function by binding to receptors on sperm membrane. Albeit spermatozoa are potentially a useful model for screening estrogenic activities of endocrine disruptors, high-quality in vitro test system that examination of the XCs effects on sperm function is required. The objective of this study was to compare the effects of XCs (genistein and 4-tert-octylphenol) to those of steroids (estrogen and progesterone) and heparin on in vitro capacitation and acrosome reaction (AR) in mouse spermatozoa. Mouse spermatozoa were incubated with various concentrations (0.001–100 μM) of each chemical for 15 or 30 min, and then capacitation and AR were assessed using chlortetracycline. All chemicals studied effectively alter capacitation and/or AR in mouse spermatozoa with different manner. Therefore, we believed that our system will provide a good in vitro model system to characterize the physiological effect of XCs especially when compared with steroids.

The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida

The acrosome reaction (AR), an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca²(+) into the spermatozoa through voltage-dependent Ca²(+) channels and store-operated channels. Maitotoxin (MTx), a Ca²(+)-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida (ZP), possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 (rhZP3), mouse ZP and two MTx channel blockers (U73122 and U73343), we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca²(+) ([Ca²(+)](i)) in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 (inactive analogue), can block phospholipase C (PLC). Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

182. ROLE OF CRISP4 IN ION CHANNEL REGULATION AND MALE REPRODUCTION

CRISPs are a group of 3 proteins found in mammals (4 in the mouse) which show a strong expression bias in the male reproductive organs. Whilst the function of most CRISPs are yet to be elucidated, mouse CRISP2 is a known regulator of the ion channel, ryanodine receptor. CRISP4 is most abundantly produced by the principal cells of the epididymis and are secreted into the lumen, where they adhere to sperm during epididymal transit. In this study we examined the role of CRISP4 ion channel regulation in mouse spermatozoa through cell assays and mouse models. The identification of the Transient Receptor Potential (TRP) ion channel, TRPM8 to interact with CRISP4 was confirmed using stably-transfected CHO cell lines. Production of CRISP4 KO mouse model, whilst males are fertile, they exhibit a subtle infertility phenotype characterized by a reduced ability to capacitate and undergo the acrosome reaction. This data is further emphasized by the ability of TRPM8 agonists, icillin and menthol, to inhibit the acrosome reaction in mouse spermatozoa that could be prevented by the addition of recombinant CRISP4 crisp domain. Corresponding to these data, CRISP4 is localized to the tail and head of mouse spermatozoa. In conclusion, we have demonstrated that CRISP4 is a regulator of TRPM8 in mouse spermatozoa, and due to its expression and localization pattern is an important protein in sperm epididymal maturation.

Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.

This study evaluates the use of two fluorescein-labelled (FITC) plant lectins, Pisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA), in order to determine the most accurate and reliable method to experimentally detect and assess the acrosome reaction in mouse spermatozoa. PNA-FITC labelling was restricted to the acrosome and was not influenced by the fixation procedure; either absolute methanol or paraformaldehyde. In contrast, PSA-FITC not only labelled the acrosome, but also the whole head and the flagellum. This aspect was especially marked after methanol fixation. The cytoplasmic droplet, when present, was also stained by PSA-FITC. Incubation with the calcium ionophore ionomycin induced a concentration and time-dependent increase in the number of acrosome reactions. Compared to spotted preparations, smear samples exhibited a high proportion of spermatozoa with damaged acrosome. In conclusion, PNA-FITC labelling was more accurate than PSA-FITC labelling to detect the acrosome of mouse spermatozoa. The fixation method (methanol vs. paraformaldehyde) had no influence on the staining pattern of PNA-FITC labelling, but spotted preparations are recommended to avoid mechanical damage to the acrosome. Ionophore challenge confirmed the existence of a calcium-dependent acrosome reaction in mouse spermatozoa and validated the use of PNA-FITC to quantify this physiological process. The present study illustrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome reaction.

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.

Synaptotagmin VI and VIII and Syntaxin 2 Are Essential for the Mouse Sperm Acrosome Reaction

The sperm acrosome is a large secretory granule that undergoes calcium-stimulated exocytosis by a mechanism analogous to neuronal secretion. In neurons the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, composed of syntaxin (Stx), SNAP-25, and VAMP2, mediates vesicle fusion, whereas calcium regulation is thought to be accomplished by the synaptotagmin (Syt) family, some of which exhibit calcium-dependent binding to syntaxin and SNAP-25. Sperm express Syt VI and VIII and Stx2, which are co-localized to the acrosomal compartment where they might mediate exocytosis in response to calcium influx. Therefore, we examined the calcium dependence and isoform-specific interaction of Syt and Stx. We found that Stx2 binds to Syt I, VI, and VIII in a calcium-dependent manner with EC(50) values of 175, 233, and 96 mum calcium, respectively. We also determined that the EC(50) for calcium of the acrosome reaction in streptolysin O-permeabilized sperm is 87 mum, which closely coincides with the calcium sensitivity of Stx2 and Syt VIII interaction. Consistent with this is the greater potency of recombinant Syt VIII, VI, and Stx2 compared with other isoforms in inhibiting the acrosome reaction in streptolysin O-permeabilized sperm. Similarly, introduction of Syt VIII-specific antibodies was equally effective in inhibiting the acrosome fusion. Taken together, our data suggest a critical role for Syt VIII and Stx2 in membrane fusion and acrosome reaction in the sperm.

Ketone bodies could support the motility but not the acrosome reaction of mouse sperm.

Ketone bodies, D-beta-hydroxybutyrate and acetoacetate, produced by the metabolism of fatty acids, are an important energy source for many organs, especially the heart, kidney and brain. They are utilized by the body with the help of succinyl CoA transferase (SCOT), which is ubiquitously expressed in various organs. Previously, we identified a novel SCOT-t specifically expressed in testicular germ cells and sperm, substituting somatic cell-type SCOT, however the physiological role of SCOT-t had not then been clarified. In the present study, we investigated the effects of ketone bodies, the substrate of SCOT-t, on the motility and acrosome reaction of mouse sperm. D-beta-hydroxybutyrate and acetoacetate both stimulated the motility of sperm as glucose or pyruvate. The glycolysis inhibitor stopped the motility of sperm mediated by glucose but not by D-beta-hydroxybutyrate. In contrast, ketone bodies did not stimulate the activation of the acrosome reaction of sperm, different from the effect of glucose. These results indicate that ketone bodies could be involved in sperm movement but not the acrosome reaction and the SCOT-t enzyme we have identified in sperm mitochondria may have important roles in the activity of sperm, resulting in male infertility when its function is disabled.

ZD7288 inhibits low-threshold Ca 2+ channel activity and regulates sperm function

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca 2+ channels have. Interestingly, ZD7288 blocked native T-type Ca 2+ currents in mouse spermatogenic cells with an IC 50 of about 100 μM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca 2+ channels in the mouse sperm AR.

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Zona pellucida (ZP)–induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCδ4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCδ4−/− sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCδ4−/− sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCδ4−/− sperm. These results indicate that PLCδ4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Evidence suggesting that the mouse sperm acrosome reaction initiated by the zona pellucida involves an alpha7 nicotinic acetylcholine receptor.

The mammalian sperm acrosome reaction (AR) is essential to fertilization and is believed to be initiated in vivo by ZP3, a glycoprotein component of the egg zona pellucida (ZP). Recently, we reported the results of antagonist studies suggesting that a nicotinic acetylcholine receptor (nAChR) containing an alpha7 subunit (alpha7nAChR) plays a role in the human sperm AR initiated by recombinant human ZP3 or by acetylcholine (ACh). Here, we show that ACh can initiate the mouse sperm AR and that antagonists of the nAChR inhibit the AR initiated by ACh or by ZP obtained from ovarian oocytes (isolated heat-solubilized mouse ZP). Preincubation with three antagonists of the nAChR, alpha-bungarotoxin (100 nM), alpha-conotoxin IMI (100 nM), and methyllycaconitine (100 nM), significantly blocked AR initiation by ACh or by isolated heat-solubilized mouse ZP (P </= 0.002). Because the only nAChR subunit known to bind all three antagonists is the alpha7, an alpha7nAChR appears to be involved in the mouse sperm AR initiated by mouse ZP or by ACh. The nAChR antagonists did not inhibit the AR initiated by calcium ionophore A23187, suggesting that the role of alpha7nAChR is upstream from Ca2+ influx. Pertussis toxin (PTX, 100 ng/ml) did not inhibit the AR initiated by ACh, suggesting that the alpha7nAChR might be a candidate for the PTX-insensitive, poorly selective cation channel shown previously to play a role in ZP-initiated mouse sperm AR. These studies with mouse sperm and ovary-derived ZP strongly support our previous conclusion that activation of an alpha7nAChR is important to the mammalian AR initiated by the egg ZP.

Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells: EXPRESSION OF MULTIPLE ISOFORMS

Low voltage activated, voltage-operated Ca(2+) channels are expressed in rodent male germ cells and are believed to be pivotal in induction of the acrosome reaction in mouse spermatozoa. However, in humans, very little is known about expression of voltage-operated Ca(2+) channels in male germ cells or their function. We have used reverse transcription-polymerase chain reaction, in situ hybridization, and patch clamp recording to investigate the expression of low voltage activated voltage-operated Ca(2+) channels in human male germ cells. We report that full-length transcripts for both alpha(1G) and alpha(1H) low voltage activated channel subunits are expressed in human testis. Multiple isoforms of alpha(1G) are present in the testis and at least two isoforms of alpha(1H), including a splice variant not previously described in the human. Transcripts for all the isoforms of both alpha(1G) and alpha(1H) were detected by reverse transcription-polymerase chain reaction on mRNA isolated from human spermatogenic cells. In situ hybridization for alpha(1G) and alpha(1H) localized transcripts both in germ cells and in other cell types in the testis. Within the seminiferous tubules, alpha(1H) was detected primarily in germ cells. Using the whole cell patch clamp technique, we detected T-type voltage-operated Ca(2+) channel currents in isolated human male germ cells, although the current amplitude and frequency of occurrence were low in comparison to the occurrence of T-currents in murine male germ cells. We conclude that low voltage activated voltage-operated Ca(2+) channels are expressed in cells of the human male germ line.