Abstract: Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze-thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk-Tris-glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus-oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+0.3% polyvinyl alcohol (PVA); control), then maintained at 23 degrees C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4 degrees C (EYTG-4), and the third treatment was held at 23 degrees C (EYTG-23) for 4 hours. The fourth treatment was cooled to 4 degrees C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen-thawed). After the 4-hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; +0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9-11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh-extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 micrograms/ml), three times more frozen-thawed than fresh-extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG-4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2x more than spermatozoa preexposed to EYTG but held at 23 degrees C (EYTG-23; P < 0.0001). No differences were observed among fertilization rates for fresh-extended (EYTG-4) and frozen-thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.

Abstract: An investigation was undertaken to determine whether the germ-cell loss associated with exposure of the testis to abdominal temperature occurs by apoptosis. Using an adult-mouse model of experimental unilateral cryptorchidism, it was observed that DNA fragmentation, consistent with apoptosis, was observed on day 6 in the cryptorchid testis, with subsequent loss of testicular weight, histologic evidence of germ-cell loss, and histochemical staining of apoptotic germ cells observed on day 7. Vacuolization of the germinal epithelium and the appearance of multinucleated giant cells was noted synchronously with the onset of germ-cell loss. Histochemical staining for apoptosis was noted most frequently among the primary spermatocytes and round spermatids. These results indicate that the testicular germ-cell loss observed with exposure to abdominal heat stress occurs by apoptosis. Further investigation of the biochemical mechanisms involved in testicular apoptosis may provide strategies to address a variety of male reproductive issues such as contraception and infertility.

Abstract: The number and magnitude of studies involving testosterone-supplementation therapy in older men are limited. In addition, many studies to date have not been blinded or controlled, were reported in abstract form only, and had involved a variety of androgen-replacement regimens and outcomes measurements. Nonetheless, an overview of the data suggests there is real potential for supplementation therapy to improve bone mass and muscle mass and strength in this age group. Affects on mood, sexual function, and cognition are less clear but may be meaningful in certain men. Questions still remain, however, on the magnitude and longevity of the beneficial effects of testosterone supplementation in the older man and whether only certain subgroups of men would truly benefit from therapy. More importantly, the long-term risks of androgen therapy in this age group really are not known, especially in the areas of cardiovascular disease and prostate diseases. Presently, men who use androgen-supplementation therapy for age-related "testosterone deficiency" should consider this as a gamble.

Abstract: We investigated the relationship between psychological stress and sperm concentration, motility, and morphometry in a prospective study of 157 volunteers who were enrolled in a prepaid health plan We measured psychological job stress and life-event stress by telephone interview Sperm-kinematic and nuclear-morphometric variables were measured using computer-assisted image analyses Sperm concentration, percent motility, and semen volume were determined by objective visual methods We performed multiple linear regression for each semen variable to examine its relationship to stress, controlling for potential confounders Stress at work and total number of life events were not related to differences in semen quality However, the recent death of a close family member was associated with a reduction in straight-line velocity (P = 0002) and percent of progressively motile sperm (P = 002); it was also marginally associated with an increase in the fraction of sperm with larger and more tapered nuclei These findings suggest that the fecundity of men experiencing the stress of a family member's death might be temporarily diminished

Abstract: Scrotal regions of mice were exposed to a 38.0, 40.0, or 42.0 degrees C (+/-0.1) H2O bath for 60 minutes to determine the effects of elevated temperatures on testicular cells and sperm chromatin structure. Mice were killed on various days after exposure, and ratios of acridine orange-stained testicular cell populations were determined by flow cytometry. Testicular weights of mice exposed to 42.0 degrees C decreased significantly day 1 (P < 0.01) through 35 (P < 0.001). Also, a significant relative decrease in testicular haploid cells was seen on days 3-35 (P < 0.001) with a corresponding increase in the diploid population (P < 0.001). Testicular analyses of mice exposed to 38.0 degrees C were not significantly different from control values. Testis weights of mice exposed to 40.0 degrees C were not affected, but a relative decrease in percent haploid cells occurred on days 11 and 14 (P < 0.001). The sperm chromatin structure assay (SCSA) was used to measure the susceptibility of cauda epididymal sperm DNA to in situ denaturation at low pH. Caudal epididymides of mice exposed to 42.0 degrees C had no sperm. Caudal epididymal sperm from mice exposed to 40.0 degrees C were most susceptible to acid-induced DNA denaturation on days 3 (P < 0.05), 7, 11, and 14 (all P < 0.001). The 38.0 degrees C exposed mice showed some minor sperm chromatin abnormalities at later time points (days 11-35). When compared to sperm head morphology measurements, SCSA parameters were more sensitive indicators of heat-induced sperm abnormalities. These results show that mouse spermatogenesis is disrupted by scrotal exposure to environmental temperatures several degrees over normal physiological temperature and, of more biological interest, that some thermal ranges above normal allowed production of sperm with compromised nuclear chromatin structure.

Abstract: Prior studies from this laboratory, using untreated castrated (CASTRATE) rats and testosterone-treated castrated (TES-TO) rats, have shown that the magnitude of the intracavernosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavernosal NO and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of the nitric oxide synthase gene (nNOS). The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause of the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of nitric oxide synthase enzyme protein (NOS) resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavernosal pressure in CASTRATE rats but not in TESTO rats, suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.

Abstract: The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.

Abstract: Cancer of the prostate is the leading cancer among American men, yet few risk factors have been established. Hair growth and development are influenced by androgens, and it has long been suspected that prostate cancer also is responsive to these hormones. A blinded, case-control study was undertaken to determine if hair patterning is associated with risk of prostate cancer, as well as specific hormonal profiles. The study accrued 315 male subjects who were stratified with regard to age, race, and case-control status (159 prostate cancer cases/156 controls). Hair-patterning classification and serum levels of total and free testosterone (T), sex hormone binding globulin, and dihydrotestosterone (DHT) were performed. Data indicate that hair patterning did not differ between prostate cancer cases and controls; however, significant hormonal differences were detected between the two groups. Free T was greater among cases than in controls (16.4 +/- 6.1 vs. 14.9 +/- 4.8 pg/ml, P = 0.02). Conversely, DHT-related ratios were greater among controls (P = 0.03 for DHT/T and P = 0.01 for DHT/free T). Several strong associations also were found between hormone levels and hair patterning. Men with vertex and frontal baldness had higher levels of free T (16.5 +/- 5.5 and 16.2 +/- 8.0 pg/ml, respectively) when compared to men with either little or no hair loss (14.8 +/- 4.7 pg/ml) (P = 0.01). Data suggest that increased levels of free T may be a risk factor for prostatic carcinoma. In addition, although no differences in hair patterning were detected between cases and controls within this older population, further research (i.e., prospective trials or case-control studies among younger men) may be necessary to determine if hair patterning serves as a viable biomarker for this disease, especially given the strong association between free T levels and baldness.

Abstract: Rat penile erection is an androgen-dependent process with castration leading to a loss of potency. The present study was designed to determine if one of the mechanisms by which androgens maintain the erectile response is the regulation of the alpha-adrenergic responsiveness of cavernosal smooth muscle. Electrical stimulation of the major pelvic ganglion (MPG) was used to elicit erection in untreated, castrated rats (CASTRATE) or castrated rats given testosterone replacement (TESTO). The effects of phenylephrine (an alpha 1-adrenergic agonist) and prazosin (an alpha 1-adrenergic antagonist) on the erectile response were investigated. Phenylephrine, when administered to both TESTO and CASTRATE animals during erection, resulted in a dose-dependent decrease in the intracavernosal pressure (CCP) with an ED50 value of 1.8 +/- 0.48 micrograms/kg BW for TESTO rats; in the CASTRATE animals, the ED50 was significantly reduced to 0.29 +/- 0.08 microgram/kg BW. The increases in mean arterial pressure (MAP) resulting from phenylephrine injection in TESTO and CASTRATE animals were of similar magnitude and were not significantly different. Prazosin administration resulted in an enhancement of the erectile response in CASTRATE but not in TESTO animals. Taken together these results demonstrate that the cavernosal vasculature in CASTRATE animals possesses increased reactivity to alpha-adrenergic stimulation as compared to the sensitivity in TESTO rats. Based on these findings, we conclude that one of the mechanisms by which androgens maintain erectile function is by regulating the alpha 1-adrenergic responsiveness of the cavernosal smooth muscle.

Abstract: In order to identify a possible threshold for a serum testosterone level below which sleep-related erections are impaired and to compare this threshold with the normal laboratory range of testosterone serum levels, we studied 201 men, including hypogonadal and eugonadal subjects. The protocol included nocturnal penile tumescence and rigidity monitoring and the assay of basal testosterone, prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) serum levels. The subjects were assigned to eight groups according to their testosterone serum levels. Group 1 had testosterone between 0 ng/dl and 99 ng/dl; the following seven groups had testosterone levels increased by 100 ng/dl per group. The groups of subjects with higher testosterone serum levels showed almost constantly higher values for the erectile parameters we studied than the subjects with serum testosterone < or = 99 ng/dl. On the contrary, subjects with higher testosterone serum levels showed higher values for only some erectile parameters compared to the subjects with serum testosterone between 100 and 199 ng/dl, without any significant difference among the groups with testosterone serum levels in the normal range. Our data suggest that the serum testosterone threshold for sleep-related erections is lower than the low end of the normal laboratory male range and is about 200 ng/dl. Further efforts are needed to find the precise serum testosterone ranges related to normal sleep-related erections and to normal sexual behavior, the testosterone ranges of which will probably not coincide.

Abstract: In a previous study, we found that ethane dimethanesulphonate (EDS) compromised the fertilizing ability of proximal cauda epididymal sperm from the rat within 4 days of exposure, an effect that persisted in castrated, testosterone (T)-implanted animals, establishing direct action on the epididymis. This EDS-induced reduction in fertilizing ability was highly correlated with a quantitative decrease in specific sperm protein. Here we sought to determine whether the fertility of proximal cauda epididymal sperm recovered from animals exposed to a variety of male reproductive toxicants could be predicted by assessing quantitative changes in specific sperm protein(s), or whether more common endpoints (e.g., sperm motility, sperm morphology, serum and epididymal tissue T, cauda epididymal sperm reserves) also are required to predict fertility. Intact adult male rats were dosed with EDS (25 or 50 mg/kg), chloroethylmethanesulphonate (CEMS; 12.5 or 18.75 mg/kg), or epichlorohydrin (EPI; 3 or 6 mg/kg) daily for 4 days. Castrated, T-implanted rats were dosed with hydroxyflutamide (HFLUT; 12.5 or 25 mg/kg) daily for 5 days. On day 5, proximal cauda epididymal sperm were inseminated in utero into receptive, cervically stimulated adult females, and on day 9, fertility (implants/corpora lutea) was assessed. Fertility-was decreased by the higher dose of each toxicant (P 50% fertility) and subfertile (< 50 fertility) animals, respectively, when discriminant analysis was performed. Thus, the amount of SP22 in a cauda epididymal sperm sample may be a useful predictor of fertility in toxicant-treated animals.

Abstract: PH-20 is a sperm plasma-membrane protein that has been shown to have hyaluronidase activity in several mammalian species including nonhuman primates. In this investigation, the PH-20 protein was characterized in noncapacitated human sperm and in capacitated human sperm. Two forms of PH-20 were observed in immunoblots of sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS PAGE) using a polyclonal antibody to recombinant PH-20: a major band of 64 kDa appeared in noncapacitated and capacitated sperm extracts and a 53-kDa band that appeared only in the acrosome-reaction supernatant of acrosome-reacted sperm. Using hyaluronic acid substrate gel analysis, we demonstrated that noncapacitated sperm extracts, capacitated sperm extracts, and the acrosome-reaction supernatant had hyaluronidase activity at neutral pH (pH 7) and acid pH (pH 4). The 64-kDa form in all samples had hyaluronidase activity at both neutral and acid pH, but the 53-kDa form was only active at acid pH. Total hyaluronidase activity, as measured by a microplate assay, was higher at pH 7 than at pH 4. Very low hyaluronidase activity was detected in the acrosome-reaction supernatant. Transmission electron microscopy and immunogold labeling showed that PH-20 of acrosome-intact human sperm was located on the plasma membrane over the entire head but not on the sperm midpiece and tail. After the acrosome reaction, PH-20 was also located on the inner acrosomal membrane. The biochemical characteristics and the ultrastructural localization of PH-20 in human sperm suggest that this protein is the human sperm hyaluronidase and, therefore, has an important function during fertilization.

Abstract: Prior studies have demonstrated that the erectile response in the rat penis is androgen dependent and is mediated by nitric oxide (NO), the neurotransmitter synthesized by the enzyme nitric oxide synthase (NOS). The present studies used L-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, to determine if androgens also regulate alternative pathways leading to the erectile response but not mediated by NO. Castrated rats that were treated with L-NAME (L-NAME CASTRATE) exhibited little or no increase in intracavernosal pressure in response to stimulation of the major pelvic ganglion. This ganglion controls blood flow into the penis and, when stimulated, normally leads to erection. However, when castrated animals were treated with testosterone along with L-NAME (L-NAME TESTO), the animals responded to the ganglionic stimulation with increased intracavernosal pressure. This finding suggests that there are other androgen-dependent pathways that lead to penile erection but are not mediated by NO. Erection occurred in both L-NAME CASTRATE and L-NAME TESTO rats in response to intracavernosal injection of sodium nitroprusside (an NO donor drug), proving that the NO responsive mechanisms were unaffected by the inhibition of NOS activity. To investigate further the nature of this NO independent pathway, L-NAME CASTRATE and L-NAME TESTO rats were treated with either zaprinast (a specific phosphodiesterase 5 inhibitor), which would block the breakdown of cGMP to 5'GMP, or methylene blue (an inhibitor of guanylate cyclase) to prevent the synthesis of cGMP. Zaprinast treatment led to increased erectile response in L-NAME TESTO rats but not in L-NAME CASTRATE rats, demonstrating that androgen-sensitive alternative pathways increased guanylate cyclase activity. Methylene blue inhibited the erectile response in all treatment groups, showing that cyclic GMP is critical to the NO-independent pathway as well as the NO-dependent pathway. Taken together, these results support the hypothesis that androgens maintain the erectile response by alternate pathways, including one that is independent of NO but involves the synthesis of cyclic GMP.

Abstract: Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 x 10(6) spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 x 10(6) spermatozoa/0.5 ml French straw. One-half of the straws for each dilution series was stored 24 hours at 5 degrees C, while the other half was cryopreserved. Spermatozoa were stained with SYBR-14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red-stained spermatozoa (PI), viable green-stained spermatozoa (SYBR-14), and moribund spermatozoa that stained both red and green (doubly-stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR-14-stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR-14-stained spermatozoa in that semen when it was cryopreserved and diluted to < 10 x 10(6) spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR-14-stained spermatozoa in the 24-hour-stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa < 10 x 10(6) spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.

Abstract: Our recent discovery that testicular genn cells and epi­ didymal spenn ClOI11U't active P450 aroma1ase suggests that the repro­ ductive tract may be a target for estrogen Therefore, the objective of this study was to detennine if estrogen receptors (ER) are present in the avian epididymis using immunocytochemistry, northern blot analysis, and in situ hybridization lmrnunoperoxidase staining for ER was found principally In nuclei of nonclliated epithelial cells of proximal and distal efferent ductules and the epididymis c1Jct The ciliated cells also 8Jr peared to be slightly positive In the efferent ductules Weak immuno­ stalnlng was also obaerYed in the connecllve tissue of the epididymis duel lmmunostaining was more intense In epithelial cells of the efferent T he avian epididymis, consisting of the efferent ducts (ductuli efferentes), connecting ducts (ductuli conjugen­ tes), and the epididymis duct (ductus epididymidis), plays an important role in the resorption of lwninal fluids from the testis (Qulow and Jones, 1988) Although spenn tra­ verse this region of the male reproductive tract rapidly, these ducts resorb nearly 90% of the testicular plasma output be­ fore the spenn are stored for an extended period in the duc­ tus deferens Androgens are considered the major hormones involved in the regulation of epididymal functions in mammals (Ro­ baire and Henno, 1988) However, there is considerable ev­ idence that structure and function of the efferent ductules and the initial segment of the epididymis are also dependent on other lwninal factors derived from the testis (Fawcett and Hoff, 1979; Douglass et al, 1991 ) The avian species con­ tains homologous structures to the mammalian epididymis, ie, a fluid-resorbing epithelium with ciliated cells (Tmgari, 1972; Qulow and Jones, 1988); therefore, it is possible that some regions of the avian epididymis are less responsive to androgens and are regulated by other testicular factors One of these nonandrogen factors may be estrogen In

Abstract: Germ cell and Sertoli cell numbers were estimated in six normal adult monkeys (Macaca fascicularis) using a contemporary unbiased and efficient stereological method--the optical disector. The data was used to assess the efficiency of spermatogenesis from type B spermatogonia to elongated spermatids. Animals underwent orchidectomy, and the right testis (volume 17.5 +/- 1.7 cm3 [mean +/- SEM], range 13.2-25.1 cm3) was fixed in Bouin's fluid. Blocks were embedded in methacrylate resin and germ cells were counted in thick (25 microm) sections using the optical disector in conjunction with a systematic uniform random-sampling protocol. The total numbers of Sertoli cells and all germ cells per testis were 566 +/- 43 (419-683) million and 12.8 +/- 1.6 (9.0-20.2) billion, respectively. On average, one Sertoli cell supported 12.4 +/- 1.9 (range 8.2-18.4) step 1-12 spermatids, 3.1 +/- 0.4 (2.3-4.5) pachytene spermatocytes, and 23.7 +/- 4.1 (15.0-39.0) total germ cells. Sertoli cell number correlated poorly with both testicular size (correlation coefficient r = -0.12) and germ cell numbers (r = -0.35 with total germ cell number). However, testicular size had a consistent and significant correlation with germ cell numbers (r = 0.97 with total germ cell number). The conversion ratio of pachytene spermatocytes to step 1-12 spermatids was 3.94 +/- 0.19, which is close to the theoretical maximum of 4. Similarly, the conversion between other cell types was consistently close to the maximum theoretical value. We conclude that the efficiency of spermatogenesis in the adult monkey is high, with stepwise conversion being consistently close to the maximal values. The capacity of Sertoli cells to support a cohort of germ cells varies widely between monkeys. Although absolute number of cells per testis is always the preferred parameter, it cannot always be obtained in an experimental situation where cost and ethical constraints mean that biopsies, rather than whole testes, are collected. Thus, if absolute data on germ cell numbers are not available, experimental outcomes impacting on cells beyond preleptonene spermatocytes may be best expressed in terms of changes in germ cell conversion rather than the traditional germ cell: Sertoli cell ratio.

Abstract: A cDNA probe that exhibits specificity for the rat Y chromosome was generated by using a set of primers specific to the murine Sry gene, the sex-determining region of the Y chromosome. A 459-base pair (bp) DNA fragment was obtained by polymerase chain reaction (PCR) amplification from male, but not female, rat genomic DNA (EMBL Nucleotide Sequence Database accession number X89730). This DNA fragment was purified, cloned using a vector, and digested with EcoR1 to yield a 270-bp DNA fragment. This 270-bp cDNA fragment, when used as a probe in Southern blot analysis of rat DNA, was observed to bind to three separate bands of approximately 2.3, 5.0, and 7.0 kb in size. The binding was demonstrated with male, but not female, genomic DNA. Another set of primers was generated to sequences within the 270-bp fragment that produced a PCR product of 104 bp. This DNA fragment, when used as a probe in Southern blot analysis, enabled PCR detection of at least 0.1% male cells in a mixed population of female cells. These cDNA probes should prove useful in studies designed to track cell populations (e.g., tumor metastasis and hemopoietic cells after bone marrow transplantation) in syngeneic male/female pairs. In addition, a cDNA probe that is specific for the rat Sry gene might be valuable in studies of fetal male sexual development or the study of spermiogenesis.

Abstract: Testosterone enanthate (TE) administration is associated with a failure rate (3.4/100 person-years) considerably lower than that of other currently available reversible male contraceptive methods. However acceptability of this regimen is limited by the need for weekly injections the lack of complete azoospermia and the decrease in high-density lipoprotein (HDL)-cholesterol. Under investigation is the concurrent administration of a progestin that can act synergistically with TE in the suppression of gonadotropins. Because of the additive effects the dose of each steroid can be decreased minimizing androgen-related side effects. Third-generation progestins and hybrid progestins seem to offer the most potential for such an approach. In particular the combination of cyproterone acetate (CPA) and TE has been found to result in a rapid profound suppression of spermatogenesis without side effects through its ability to block both the follicle-stimulating hormone and androgen effects at the testis level. The anti-androgenic action of CPA is based on its ability to competitively inhibit the binding of testosterone and dehydrotestosterone to androgen receptors. The block of intratesticular testosterone effect by CPA is presumed to cause a decrease in cell-adhesion molecule concentrations and contribute to the premature sloughing of permatids from the seminiferous epithelium thereby resulting in the early disappearance of spermatozoa from the ejaculate. This regimen did not cause any changes in plasma HDL or other lipoproteins or affect liver function test results.

Abstract: The effect of seminal plasma, as well as some seminal plasma fractions, on the heterogeneity and viability of frozen-thawed ram spermatozoa was studied Fresh and frozen sperm samples were simultaneously assessed by a routine semen-quality assay and centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system analysis All samples used for this study were obtained from Salz rams by an artificial vagina and frozen according to the Fiser method Sperm samples, consisting of either raw or washed semen, were frozen in the presence of either whole ram seminal plasma (RSP) or > 10 kDa seminal plasma fraction Two control samples were considered: one diluted in Fiser's extender and an undiluted sample Cell-membrane integrity (viability), hypoosmotic swelling (HOS) test, and sperm motility were assessed in all cases after collection, before freezing (at 5 degrees C), after thawing, and after removing the diluent before CCCD analysis Our results show that sperm cell partition behavior was dependent on the sperm treatment and that the freezing-thawing process accounted for a clear loss of sperm heterogeneity and membrane integrity These losses were less apparent when seminal plasma had been removed from semen samples before freezing In addition, washed semen frozen in the presence of either > 10 kDa seminal plasma fraction or whole re-added seminal plasma maintained a higher rate of sperm heterogeneity and viability

Abstract: The induction of neonatal hypothyroidism from day 1 to day 25 postpartum using 6-propyl-2-thiouracil (PTU) resulted in a 45% and 77% reduction of testis weight on days 20 and 30, respectively, and an increase in Sertoli cell number. The present study evaluated the effect of neonatal hypothyroidism on the developing germ cell population during the first 30 days postnatally. Qualitative and quantitative studies using the optical disector method were undertaken on the testes of control and hypothyroid rats, on days 10, 20, and 30 after birth. Germ cell development was obviously impaired in the hypothyroid rats, as shown by decreased primary spermatocyte and round spermatid numbers in day 20 and day 30 testes, and the persistence of gonocytes on days 10 and 20. These reductions obviously accounted for the reduced testis weight, absolute volume, and diameter of the seminiferous cords/tubules, especially on days 20 and 30. The failure to establish a seminiferous tubule lumen in hypothyroid rats probably reflects decreased fluid production and a functional immaturity of the Sertoli cells. The delay in germ cell maturation and increased degeneration may be because of the immature state of the Sertoli cell or result from the low follicle-stimulating hormone and thyroxine levels known to occur in the hypothyroid rats.

Abstract: Hematospermia is a disconcerting symptom that produces extreme anxiety in sexually active male patients. To understand the pathophysiology of hematospermia, the anatomy of the ejaculatory system and neurophysiology of emission and ejaculation is essential. Emission and ejaculation must be present for hematospermia to occur. Hematospermia may be the result of inflammation, infection, ductal obstruction or cysts, neoplasms, vascular abnormalities, and systemic or iatrogenic factors. Most patients promptly consult a urologist after an episode of hematospermia. History and physical examination are often unrevealing and the judicious use of imaging modalities, such as transrectal ultrasound, MRI, and rigid or flexible endoscopy may be diagnostic. Unless the specific etiology is defined, most cases are managed expectantly. We review the etiology of hematospermia and an algorithm is provided for the diagnosis and management.

Abstract: The biological effects of ethane dimethanesulphonate (EDS) are unique since cytotoxicity in the adult rat is almost exclusively confined to the Leydig cells For this reason, EDS has been used extensively to investigate the physiological role of the Leydig cell and its products Experiments were conducted to determine whether the Leydig cell will undergo apoptosis in response to EDS or methylprednisolone (MP), a glucocorticoid known to cause apoptosis in a number of other cell types Percoll-purified Leydig cells were incubated for 24 hours with EDS (750 micrograms/ml), at which time the cells attached to the culture plate became rounded up while control cells were flattened and polyhedral Following incubation with EDS or MP (10 microM), cells that became detached from the plate were characteristically apoptotic when stained with the fluorescent DNA dye, acridine orange These cells had shrunk and the nuclear chromatin had become condensed, which is an early characteristic of apoptosis in other cells; eventually, apoptotic bodies formed, reflecting a later apoptotic stage Electrophoresis of DNA extracted from the treated Leydig cells exhibited the characteristic ladder of the apoptotic process Increasing the concentration of EDS or MP resulted in a dose-dependent increase in the incidence of apoptosis that reached a maximum of 25% (EDS) or 12% (MP) of detached cells Administration of EDS in vivo caused a 20-fold increase in the number of apoptotic cells observed in interstitial cell preparations In conclusion, the data indicates that programmed cell death, apoptosis, can occur in the Leydig cell and that this is the likely mechanism by which EDS kills the cells in vivo and in vitro

Abstract: Clusterin is the major protein produced by rat Sertoli cells and is deposited onto sperm membranes; however, its function is unknown. In order to gain insight into the regulation of clusterin in Sertoli cells, the objective of the present study was to develop a model where the expression of clusterin could be affected in Sertoli cells in vitro. Rat Sertoli cells and mouse Sertoli cells (MSC1) were cultured under heat stress conditions (41 degrees C) for up to 48 hours. The mRNA for clusterin in Sertoli cells was compared to that in human epitheliod cancer cells (A431) to determine if clusterin expression was regulated in a lestis-specific manner. The mRNA for heat shock protein 70 (HSP70) was also examined as it is a known stress-regulated gene. Expression of HSP70 mRNA was increased in all three cell types by 4 hours after the start of heat stress. Clusterin mRNA was increased over that of controls by 4 hours in heat-stressed A431 cells but did not significantly increase in MSC1 or Sertoli cells until 12 hours (P < 0.05). The induction of clusterin mRNA in MSC1 cells continued for at least 48 hours and required the sustained exposure of cells to the 41 degrees C temperature. The increase in the amount of clusterin mRNA was not due to an increase in transcript half-life, as determined by the addition of actinomycin D to the media of control vs. heat-stressed MSC1 cells. From the development of this in vitro model, we have seen that the timing of induction of clusterin by heat stress is Sertoli cell specific and is different than that of HSP70. This response in surviving cells during heat stress may be protective in that clusterin would bind to toxic compounds or solubilize cellular debris released by degenerating cells.

Abstract: Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.

Abstract: This study provides quantitative information on the early (up to 3 months) effects of vasectomy on apoptosis in the hamster testis. Groups of five adult male golden hamsters were either bilaterally vasectomized or sham-operated and sacrificed at intervals of 3, 6, and 12 weeks after surgery. In all three postvasectomy groups, testis weight and testicular and plasma testosterone (T) levels were not different from controls. Spermatogenic alterations, ranging from tubules with mild intraepithelial vacuoles to almost completely atrophied tubules, were detected in samples of 1 of 5 testes both at 3 and 12 weeks after vasectomy. Histometric analysis of testicular tissues at 3, 6, and 12 weeks in the postvasectomy groups showed no discernible effect of vasectomy on the absolute volumes of seminiferous tubules, tubular lumen, and total Leydig cells when compared to respective controls. In situ analysis of germ-cell apoptosis, characterized by 3'-end-labeling immunocytochemistry, revealed a significant increase (2.5-fold) in germ-cell apoptosis at stages XIII-I, involving primarily the dividing spermatocytes after 3 weeks of vasectomy. Apoptotic index was not changed from sham-operated animals at 6 and 12 weeks postvasectomy. Interestingly, a very high incidence of macrophage apoptosis was detected in the samples of three out of five testes in the 12 weeks postvasectomy group (39.3%) compared to that of controls (0.8%). These results demonstrate that vasectomy has little or no detrimental effect on the morphologic characteristics of the spermatogenesis or intratesticular concentrations of testosterone in the majority of the animals studied up to 12 weeks postsurgery, although vasectomy transiently (3 weeks postsurgery) activated germ-cell apoptosis, involving dividing spermatocytes at stages XIII-I.

Abstract: There are numerous tools available to modify the genetic makeup of animals. They are being used to good advantage for studying basic biological phenomena. Within the decade, biomedical products derived from transgenic animals will be available, but the use of this technology for enhancing the quality and efficiency of livestock production will await further refinements in the technology.

Abstract: The rat cauda epididymidis receives sympathetic innervation from the inferior mesenteric ganglion (IMG). We have previously demonstrated that surgical removal of the IMG and proximal hypogastric nerves (IMG denervation) results in significant and cauda-specific changes in epididymal sperm transport, sperm motility, luminal fluid protein composition, and tissue histology. In the present study we used natural mating trials and intrauterine insemination (IUI) techniques to determine whether or not IMG denervation affects male fertility and reproductive capacity. For the initial studies, adult male Sprague Dawley rats were mated with estrous females 1 and 4 weeks following IMG denervation. Nine days after mating, uterine implantation sites and corpora lutea (CL) were counted. In females mated with sham-operated control males, 85.8% of ovulated oocytes were fertilized and subsequently implanted. In contrast, females mated with IMG-denervated males 1 or 4 weeks following surgery had 0% and 3.5%, respectively, of ovulated oocytes fertilized and implanted. For rats maintained 21 days after mating, an average of 13 ± 1 pups were delivered by each of nine females mated with sham-operated control male rats; whereas, only seven morphologically normal pups were delivered by one of 14 females mated with IMG-denervated male rats. Additional experiments demonstrated that the decrement in offspring was, in part, due to a significant decrease in the number of spermatozoa in the female uterus following mating with IMG-denervated males. To determine whether IMG denervation exerted an additional effect directly on the fertilizing ability of spermatozoa, IUI experiments were performed. Six million cauda epididymal spermatozoa from 1- or 4-week IMG-denervated males were inseminated into the uterine horns of luteinizing hormone-releasing hormone (LHRH)-synchronized females and 9 days later implantation sites and CL were counted. Implantations were observed for 78%, 28%, and 25% of ovulated oocytes following IUI with spermatozoa from sham-operated controls and from 1 - and 4-week IMG-denervated rats, respectively. To determine whether the reduction in implantation sites following IUI with spermatozoa from IMG-denervated rats resulted from impaired oocyte fertilization, studies were performed in which oocytes were retrieved and stained 24 hours after IUI. Comparable fertilization rates of 76.5% and 89.0% were observed using cauda epididymal spermatozoa from IMG-denervated and sham-operated control males, respectively, indicating that oocyte fertilization was not affected by the loss of innervation. These studies establish the importance of innervation from the IMG for ejaculatory competence and sperm reproductive capacity in the male rat. These data further suggest that sympathetic innervation in the epididymis critically influences paternal factors associated with embryonic development.

Abstract: The mammalian sperm acrosome reaction (AR) is essential to fertilization. It can be initiated in vitro by progesterone, a putative physiological initiator that helps to activate sperm GABA(A) receptor/chloride channels and by glycine, a substitute for the egg zona pellucida, which activates sperm glycine receptor/chloride channels. Even at 1 nM (0.41 ng/ml or 0.41 ppb), chlordane and endosulfan, chlorinated cyclodiene blockers of insect neuronal GABA(A) receptor/chloride channels, strongly inhibited the AR initiated by progesterone or glycine. Inhibition of the latter was also seen at 0.1 nM chlordane and endosulfan, but neither cyclodiene inhibited either AR initiator at 0.01 nM. Inhibitory concentrations of these cyclodienes are well within the range detected in human and wildlife tissue and fluids as a result of environmental contamination.