Comparison of gene expression of the oncogenic Wnt/ß-catenin signaling pathway components in the mouse and human epididymis.

ß-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high ß-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the ß-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/ß-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of ß-catenin and the high concordance of this pathway's components' gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of ß-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of ß-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.

Why are epididymal tumours so rare?

Epididymal tumour incidence is at most 0.03% of all male cancers. It is an enigma why the human epididymis does not often succumb to cancer, when it expresses markers of stem and cancer cells, and constitutively expresses oncogenes, pro-proliferative and pro-angiogenic factors that allow tumour cells to escape immunosurveillance in cancer-prone tissues. The privileged position of the human epididymis in evading tumourigenicity is reflected in transgenic mouse models in which induction of tumours in other organs is not accompanied by epididymal neoplasia. The epididymis appears to: (i) prevent tumour initiation (it probably lacks stem cells and has strong anti-oxidative mechanisms, active tumour suppressors and inactive oncogene products); (ii) foster tumour monitoring and destruction (by strong immuno-surveillance and -eradication, and cellular senescence); (iii) avert proliferation and angiogenesis (with persistent tight junctions, the presence of anti-angiogenic factors and misplaced pro-angiogenic factors), which together (iv) promote dormancy and restrict dividing cells to hyperplasia. Epididymal cells may be rendered non-responsive to oncogenic stimuli by the constitutive expression of factors generally inducible in tumours, and resistant to the normal epididymal environment, which mimics that of a tumour niche promoting tumour growth. The threshold for tumour initiation may thus be higher in the epididymis than in other organs. Several anti-tumour mechanisms are those that maintain spermatozoa quiescent and immunologically silent, so the low incidence of cancer in the epididymis may be a consequence of its role in sperm maturation and storage. Understanding these mechanisms may throw light on cancer prevention and therapy in general.

Vitamin K requirement and reproduction in bromadiolone-resistant Norway rats.

BACKGROUND: Nucleotide polymorphisms in the VKORC1 gene can be linked to anticoagulant rodenticide resistance in Norway rats (Rattus norvegicus Berkenhout). This provides a fitness advantage to rats exposed to anticoagulant actives, but may also cause fitness costs. The vitamin K requirement and reproductive parameters of bromadiolone-resistant rats (Westphalian resistant strain; VKOR variant Tyr139Cys) and bromadiolone-susceptible Norway rats were compared. RESULTS: At vitamin K deficiency, blood clotting times increased in all homozygous resistant males within 8 days and in 80% of homozygous resistant females within 15 days. There was little effect on blood clotting in heterozygous males and no effect in heterozygous females and VKOR wild-type individuals. Litter size was about 20% higher in sensitive pairs compared with resistant pairs. Testes growth, male gonad weight, sperm motility and testis cell concentration were unaffected by the mutation. CONCLUSIONS: The VKOR variant Tyr139Cys causes considerable physiological cost in Norway rats in terms of vitamin K requirement and reproduction. This may affect the distribution and spread of resistant individuals in the wild. Decreased litter size of resistant parents seems to be due to lowered female reproductive performance, as there was no significant effect of the mutation on any aspects of male reproduction considered, but this requires further study.

Aquaporins in spermatozoa and testicular germ cells: identification and potential role.

Mammalian spermatozoa have relatively high water permeability and swell readily, as in the hypo-osmotic swelling test used in the andrology clinic. Physiologically, spermatozoa experience changes in the osmolality of the surrounding fluids in both the male and the female tracts on their journey from the testis to the ovum. Sperm volume regulation in response to such osmotic challenges is important to maintain a stable cell size for the normal shape and function of the sperm tail. Alongside ion channels for the fluxes of osmolytes, water channels would be crucial for sperm volume regulation. In contrast to the deep knowledge and numerous studies on somatic cell aquaporins (AQPs), the understanding of sperm AQPs is limited. Among the 13 AQPs, convincing evidence for their presence in spermatozoa has been confined to AQP7, AQP8 and AQP11. Overall, current findings indicate a major role of AQP8 in water influx and efflux for sperm volume regulation, which is required for natural fertilization. The preliminary data suggestive of a role for AQP7 in sperm glycerol metabolism needs further substantiation. The association of AQP11 with the residual cytoplasm of elongated spermatids and the distal tail of spermatozoa supports the hypothesis of more than just a role in conferring water permeability and also in the turnover and recycling of surplus cellular components made redundant during spermiogenesis and spermiation. This would be crucial for the maintenance of a germinal epithelium functioning efficiently in the production of spermatozoa.

Effects of cryoprotectant treatments on bovine sperm function and osmolyte content.

The hypothesis that addition and removal of cryoprotectants to and from spermatozoa would initiate regulatory volume decrease, and lead to osmolyte loss and reduced sperm function, was tested. Common cryoprotectants, in the absence of freezing and thawing, affected bovine ejaculated spermatozoa by lowering their total and progressive motility in medium, reducing their migration through surrogate cervical mucus, damaging sperm head membranes and inducing sperm tail coiling. Sperm function was slightly better maintained after cryoprotectants were added and removed in multiple small steps rather than in a single step. The intracellular content of the polyol osmolytes, D-sorbitol and myo-inositol, exceeded that of the zwitterion osmolytes, L-carnitine and L-glutamate. Certain cryoprotectants reduced intracellular L-carnitine and L-glutamate concentration but not that of myo-inositol or D-sorbitol. Multistep treatments with some cryoprotectants had advantages over one-step treatments in mucus penetration depending on the original amount of intracellular carnitine and glutamate in the spermatozoa. Overall, sperm quality was best maintained by multistep treatment with glycerol and propanediols that were associated with decreased intracellular glutamate concentration. Bovine spermatozoa seem to use glutamate to regulate cryoprotectant-induced cell swelling.

New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells.

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in vitro spermatogenesis has resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional (3D) in vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotrophin support. For the first time, we demonstrate that pre-meiotic male germ cells [revealed by the absence of meiotic marker expression (e.g. Boule)] obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotrophins but appeared independent from the matrix in a 3D culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

Multistep and single-step treatment of human spermatozoa with cryoprotectants.

OBJECTIVE: To compare the impact of multi- and single-step addition of cryoprotectants on human spermatozoa. DESIGN: Human semen was subjected to cryoprotective agents (CPAs) in both one-step and multistep protocols before removal of the CPAs by multi-step and single-step methods. SETTING: Infertility clinic. PATIENT(S): Healthy normozoospermic volunteers. INTERVENTION(S): Single- and multistep addition and removal of a range of CPAs. MAIN OUTCOME MEASURE(S): Analysis of tail morphology and total and progressive motility, as well as kinematic parameters, in medium and sperm penetration into artificial mucus. RESULT(S): While no CPA drastically affected sperm vitality, some were more deleterious than others. Propane-1,3-diol was the least damaging to sperm function, whereas glycerol and ethyl(hydroxymethyl)propane were particularly disruptive. For all CPAs, multistep addition and removal of CPAs proved less damaging to sperm function than the single-step protocol. CONCLUSION(S): Optimal CPAs may be those that minimize osmotic stresses to spermatozoa during prefreeze and post-thaw procedures.

Aquaporin isoforms involved in physiological volume regulation of murine spermatozoa.

Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K(+)-channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl(2), a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K(+)-ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl(2). Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg(2+)-insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7(-/-) mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg(2+) were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7(-/-) sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg(2+)-sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.

Hepatic selenoprotein P (SePP) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice.

SePP (selenoprotein P) is central for selenium transport and distribution. Targeted inactivation of the Sepp gene in mice leads to reduced selenium content in plasma, kidney, testis and brain. Accordingly, activities of selenoenzymes are reduced in Sepp(-/-) organs. Male Sepp(-/-) mice are infertile. Unlike selenium deficiency, Sepp deficiency leads to neurological impairment with ataxia and seizures. Hepatocyte-specific inactivation of selenoprotein biosynthesis reduces plasma and kidney selenium levels similarly to Sepp(-/-) mice, but does not result in neurological impairment, suggesting a physiological role of locally expressed SePP in the brain. In an attempt to define the role of liver-derived circulating SePP in contrast with locally expressed SePP, we generated Sepp(-/-) mice with transgenic expression of human SePP under control of a hepatocyte-specific transthyretin promoter. Secreted human SePP was immunologically detectable in serum from SEPP1-transgenic mice. Selenium content and selenoenzyme activities in serum, kidney, testis and brain of Sepp(-/-;SEPP1) (SEPP1-transgenic Sepp(-/-)) mice were increased compared with Sepp(-/-) controls. When a selenium-adequate diet (0.16-0.2 mg/kg of body weight) was fed to the mice, liver-specific expression of SEPP1 rescued the neurological defects of Sepp(-/-) mice and rendered Sepp(-/-) males fertile. When fed on a low-selenium diet (0.06 mg/kg of body weight), Sepp(-/-;SEPP1) mice survived 4 weeks longer than Sepp(-/-) mice, but ultimately developed the neurodegenerative phenotype. These results indicate that plasma SePP derived from hepatocytes is the main transport form of selenium supporting the kidney, testis and brain. Nevertheless, local Sepp expression is required to maintain selenium content in selenium-privileged tissues such as brain and testis during dietary selenium restriction.

Male congenital hypothyroid Pax8-/- mice are infertile despite adequate treatment with thyroid hormone.

Severe forms of congenital hypothyroidism lead to serious clinical symptoms if thyroid hormone replacement therapy is not instituted immediately after birth. In this study, Pax8(-/-) mice that are born without a thyroid gland were used as an animal model to study the consequences of congenital hypothyroidism. As expected, adequate treatment of these animals with thyroxine restored the general deficits of congenital hypothyroidism; however, Pax8-deficient male mice were infertile. We report here that in these mice, the efferent ducts and epididymides are either absent or the efferent ducts exhibit a reduced lumen and extensive connective tissue, which appears to impair testicular drainage and subsequently leads to complete absence of spermatozoa from the epididymis. The results suggest that, starting with the onset of pubertal testicular fluid secretion, a backpressure is created in the testis by the absence of efferent ducts or constriction of their tubule lumen when present. This subsequently leads to secondary disorganization of the seminiferous epithelium that increases with age, resulting in mixed atrophy of the testis in the adult. Serum testosterone levels as well as mRNA expression of anterior pituitary hormones are in the normal range, indicating that the observed infertility is not due to hormonal imbalance, but rather to a developmental defect of the efferent ducts. The demonstration of Pax8 expression in the epithelia of the epididymis and the efferent ducts suggests a direct morphogenic role of Pax8 in the development of these organs. It remains to be elucidated whether congenital hypothyroid male patients with mutations in the Pax8 gene are similarly affected.

Concentrations of carnitine, glutamate and myo-inositol in epididymal fluid and spermatozoa from boars.

This study examines the concentrations of carnitine, glutamate and myo-inositol in fluid and spermatozoa from six epididymal regions. Samples were taken from six post-pubertal boars, and the sperm concentration, the protein concentration in epididymal fluid and the concentrations of carnitine, myo-inositol and glutamate in the epididymal fluid and spermatozoa were analysed. In epididymal fluid the concentration of myo-inositol decreased in a proximo-distal direction, whereas intraluminal concentrations of L-carnitine and L-glutamate increased distally. As changes in the concentration of these solutes did not parallel changes in sperm concentration, this may reflect secretion or absorption of theses solutes. The sperm content of inositol fell as they moved from the distal caput whereas glutamate content increased from the distal caput to more distal regions and carnitine content remained unchanged during epididymal transit. This is the first attempt to elucidate the changes in the content of glutamate and inositol in epididymal spermatozoa of mammals and in the fluid from different epididymal regions of boars.

Changes in endocrine profile and reproductive organs during puberty in the male marmoset monkey (Callithrix jacchus).

Data on pubertal maturation in male marmoset, a model for human reproduction, are scant and conflicting. We collected data on novel parameters to characterize puberty. Twenty-five marmoset monkeys were assigned to five age groups by weeks (wk): 21 (pre-pubertal), 43 (onset of puberty), 52 (fully pubertal), 70 (mature), and 116 (fully adult). Serum and intratesticular testosterone and pituitary bioactive chorionic gonadotropin (bioCG) were measured. Testicular development was assessed by ultrasonography, histology, and flow cytometry. Three consecutive blood samples revealed extreme fluctuations in testosterone concentrations, suggesting an erratic secretion. Age-related changes in serum testosterone and pituitary bioCG concentrations were observed. Intratesticular androgens (ITAs) showed high fluctuations within groups at all ages and were high in some animals by 21 wk. Unexpectedly, no correlation between pituitary bioCG and serum testosterone or ITAs was found, but these parameters significantly correlated with testicular weight and volume. These observations were consistent a dependence on the testis growth on bioCG. Unfortunately, the low serum levels of bioCG were not measurable in this study. At 43 wk, the animals reached puberty. At 52 wk of age, animals attained maximum body and epididymal weights and qualitatively normal spermatogenesis, but testes continued growing, reaching a maximum of all parameters at 70 wk of age, without further major changes at the age of 116 wk. It is concluded that (1) gonadal activation is evident at wk 21, (2) the male marmoset reaches the pubertal threshold around 43 wk of age, attains qualitative parameters at 52 wk, matures further to sexual maturity at 70 wk, and (3) serum testosterone and ITAs are highly variable without any identifiable correlation with pituitary bioCG.

Localization and characterization of an orphan receptor, guanylyl cyclase-G, in mouse testis and sperm.

We recently identified a novel testis-enriched receptor guanylyl cyclase (GC) in the mouse, designated mGC-G. To further investigate its protein expression and function, we generated a neutralizing antibody specifically against the extracellular domain of this receptor. RT-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both the mRNA and protein levels. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell surface protein restricted to the plasma membrane overlying the acrosome and midpiece of the flagellum in mature sperm. Interestingly, Western blot analysis demonstrates that testicular mGC-G is approximately 180 kDa but is subject to limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on the mature sperm surface. On Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that serum albumin-induced elevation of sperm intracellular Ca(2+) concentration, protein tyrosine phosphorylation, and progressive motility associated with capacitation are markedly reduced by preincubation of the anti-mGC-G neutralizing antibody. Together, these results indicate that mGC-G is proteolytically modified in mature sperm membrane and suggest that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology.

Antimicrobial drug ornidazole inhibits hamster sperm capacitation, in vitro.

To be fertilization competent, spermatozoa undergo a series of changes in the female reproductive tract collectively referred to as capacitation. In an attempt to understand, if ornidazole, a known anti-fertility drug, adversely affects sperm functions by targeting capacitation, we designed experiments to study the influence of this drug on hyperactivation (HA), capacitation-associated protein tyrosine phosphorylation (pY) and the acrosome reaction (AR). Addition of ornidazole at 0 h, inhibited the onset of HA and total pY in a dose dependent manner. However, when ornidazole was added at 3.5h, severe effects were still seen on HA and pY of high molecular weight proteins but, pY of lower M(r) proteins (50-56 kDa) was affected only marginally. Further, lower doses of ornidazole (5 and 10 mM) had greater inhibitory effect when added at 0 h, while addition of ornidazole at 3.5 h required higher doses of ornidazole (25 mM) to cause significant inhibition of acrosome reaction. Collectively, through in vitro studies, we demonstrate that ornidazole affects the onset and progression of hamster sperm hyperactivation, capacitation associated protein tyrosine phosphorylation and acrosome reaction, and the severity depends on the dose (5, 10 or 25 mM) and the time of addition (0 or 3.5 h) of the drug to the spermatozoa.

Study of the polyol pathway in the porcine epididymis.

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.

Computer-aided evaluation of assessment of "grade a" spermatozoa by experienced technicians.

OBJECTIVE: To determine how well technicians assess progressively motile spermatozoa. DESIGN: Comparison of computerized and technician assessments of "grade a" spermatozoa. SETTING: University fertility clinic. PATIENT(S): Infertile men of barren marriages. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Threshold velocities of spermatozoa categorized as "grade a." RESULT(S): The ability of andrology technicians to distinguish "grade a" from "grade b" spermatozoa was examined monthly. Computer-aided sperm analysis (CASA) provided the threshold velocities used by technicians to categorize "grade a" spermatozoa. The mean cutoff value for grades a/b (25.5 microm/s) was close to the designated velocity, but the cutoff for grades b/c sperm (9.9 microm/s) was higher. Cutoff values for "grade a" spermatozoa were significantly, positively, and linearly related with the mean velocity of spermatozoa obtained by CASA, and deviated from it in a systematic way. The percentage of "grade a" spermatozoa judged by technicians was poorly correlated with the percentage "grade a" indicated by CASA and the cutoff velocity of "grade a" spermatozoa depending on the overall sperm quality of the semen sample. CONCLUSION(S): Consideration should be given to replacing the subdivision of progressing spermatozoa into two categories with one of progressive spermatozoa, or the use of CASA to distinguish "grade a" sperm reliably should be insisted upon.

Relationship of cytoplasmic droplets to motility, migration in mucus, and volume regulation of human spermatozoa.

A significantly greater percentage of motile than immotile spermatozoa bore droplets at the osmolality of semen and cervical mucus. The percentage of spermatozoa with droplets was not significantly correlated with the osmolality of semen or the extent of cell swelling in response to quinine in hypotonic medium. Droplets appeared slightly more frequently (ie, were more obvious) in the presence of quinine, which blocks regulatory volume decrease, indicating that they are the major site of volume expansion. There was no selection for or against droplet-bearing spermatozoa migrating through viscous surrogate mucus at high or low osmolality. Sperm swelling in response to quinine at mucus osmolality was significantly greater in fathers than in patients whose partners had no fertility problem. Therefore, cytoplasmic droplets are not deleterious to sperm motility and may be related to physiological volume regulation, which may be predictive of some forms of human male infertility.

Norethisterone enanthate has neither a direct effect on the testis nor on the epididymis: a study in adult male cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE. METHODS: Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated. RESULTS: There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected. CONCLUSIONS: Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.

The role of anion channels and Ca2+ in addition to K+ channels in the physiological volume regulation of murine spermatozoa.

Studies in the human, transgenic mice, and cattle indicate that sperm cell volume regulation plays an important role in male fertility as spermatozoa encounter a hypo-osmotic challenge upon ejaculation into the female tract. Physiological regulatory volume decrease (RVD) was examined using flow cytometry in murine sperm released into incubation medium mimicking uterine osmolality and including putative channel inhibitors. The involvement of K+ channels was indicated by the recovery of volume regulation by the K+ ionophore valinomycin in defective sperm from infertile transgenic mice, and from blockage of RVD by quinine in normal sperm. However, in neither case was the recovery complete. The involvement of volume-sensitive osmolyte and anion channels (VSOAC) were investigated using blockers effective in other cell types. NPPB (5-nitro-2(3-phenylpropylamino) benzoic acid) and tamoxifen inhibited RVD but SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) at 0.4 and 1 mM had no effect whereas DIDS (di-isothiocyanato-stilbene-2,2'-disulphonic acid) at 1 mM enhanced RVD. Verapamil, but not another P-glycoprotein antagonist cyclosporin, caused sperm swelling which persisted in the presence of valinomycin, in Ca2+-free medium and in the presence of thapsigargin, but swelling was abolished by the Ca2+ ionophore A23187. Nifedipine was slightly effective in blocking RVD. Analysis by Western blotting failed to reveal ClC-2 and ClC-3 members of the chloride channel family in murine or rat sperm proteins despite signal bands in positive tissue controls. These findings implicate the involvement of some unidentified VSOAC in sperm volume regulation, which is probably Ca+-dependent.