The prooxidant effect of X-irradiation on rat testis and liver tissue was studied with doses of 0.5 and 3.0 Gy; the latter dose kills the proliferating spermatogonia and causes a maturation-depletion process in the germ cells. The level of lipid peroxidation, measured by the formation of diene conjugates and thiobarbituric acid-reactive substances (TBARS) and the activities of the antioxidant enzymes were determined 0.5 hours, 1 day, 7 days, and 31 days after the exposure. In the liver, increased levels of diene conjugation (+36%, P < 0.05) in the group of 3.0 Gy at 0.5 hours indicated increased lipid peroxidation. At the same time, TBARS were increased (+25%, P < 0.05) in the group of 0.5 Gy, but not in the 3.0-Gy group. In the testis, diene conjugation was not determined at 0.5 hours postirradiation, and at day 1 it was at the control level. The level of TBARS in the testis was below control (-11%, P < 0.01) in the 3.0-Gy group at day 1. At day 31 after 3.0 Gy in the testis, an increase in the amount of conjugated dienes (+24%, P < 0.01) was observed in parallel with a decreased level of TBARS (-15%, P < 0.01). The activity of superoxide dismutase (SOD) was decreased in the testis at 0.5 hours postirradiation (-28%, P < 0.05, and -29%, P < 0.05, in the groups of 0.5 and 3.0 Gy), whereafter it returned to normal by day 7. In the liver, such inactivation of SOD was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)
The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.
Groups of eight adult male rats were given a single oral dose of 0 or 48 mg/kg of 1,3-dinitrobenzene and sacrificed at 1, 2, 4, 8, 16, 24, 32, 72, and 175 days posttreatment. The groups killed at 175 days were bred to untreated females during weeks 3, 4, 6, 9, 13, and 24. Decreased testis weight and testicular sperm numbers were observed by day 4; decreased cauda sperm reserves and epididymis weight occurred by day 8 and day 16, respectively. Reduced numbers of motile spermatozoa and abnormal sperm morphology were seen in spermatozoa from the cauda epididymidis by day 16. Fertilizing ability, as indicated by the presence of two pronuclei and a sperm tail in eggs flushed from the oviducts of inseminated females, was slightly reduced by week 4 and declined to zero by week 6. Group means for reproductive organ weights, sperm production, and sperm reserves failed to return to control levels although some individual animals approached full recovery. Normal fertilizing ability was restored in most animals by week 13, but two of seven remained infertile. Occlusion of some efferent ductules was observed in three of seven animals at 175 days. This study indicates that 1,3-dinitrobenzene is a potent testicular toxicant in the rat, capable of producing marked testicular damage, infertility, and possibly sterility from a single exposure.
This study determined the quantitative and qualitative histopathologic effects of a single oral dose of 1,3-dinitrobenzene (48 mg/kg) on the rat testis from 1 to 175 days postexposure. The testis was damaged severely by hour 24, as evidenced by increased numbers of regressive seminiferous tubules that exhibited degenerating pachytene spermatocytes, chromatin margination in spermatids, giant cells, deformed spermatid heads, retained spermatids, and reduced numbers of meiotic figures. The major effects during the first 48 hours posttreatment were degeneration or exfoliation of pachytene spermatocytes and round spermatids and the retention of step 19 spermatids. These regressive effects continued until 24 days, after which the tubules either recovered or became atrophic. At the end of the study (175 days), three males were normal, one had regressed testicles, and three males had atrophic tubules (15 to 45%). Several cellular abnormalities were common throughout the period. In addition, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermatogenesis. 1,3-Dinitrobenzene produced profound and specific lesions in the seminiferous tubules, and recovery was slow and incomplete. Atrophic tubules seemed to form if the normal cellular associations were not reestablished within 24 days, possibly due to the inability of Sertoli cells to reorganize the synchrony of germ cell development.
We have studied some characteristics of alpha-1,4-glucosidases in human male reproductive organs in order to obtain information on the origin of the enzyme in seminal plasma. Acid and neutral enzymes could be distinguished on the basis of their selective inhibition either by SDS (acid enzyme) or MTT (neutral enzyme). Only the epididymis contained a significant amount of SDS resistant neutral alpha-1,4-glucosidase which was comparable to what has been isolated in seminal plasma. The similarity of epididymal and seminal plasma neutral enzymes was further confirmed by ultracentrifugation on sucrose density gradients, which permitted a complete separation of neutral (11S) and acid (4S) iso-enzymes. The 11S form was present in epididymis and in seminal plasma, but was totally absent in seminal vesicles, prostates and testis. The epididymal enzyme also had some of the unique characteristics found in the seminal plasma enzyme: it precipitated upon dialysis against distilled water, and its mobility on SDS polyacrylamide gel electrophoresis was identical to that of form 1 in seminal plasma. These results, although they do not constitute absolute proof of the identity of epididymal and seminal plasma alpha-glucosidase, certainly provide strong support for this hypothesis.
Fractions obtained by gel filtration or ultrafiltration of dog serum were tested for their mitogenic activity on canine prostatic epithelial cells: two prostatic growth factor (PGF) entities were found, a major one of 150 kDa (PGF-I) and a minor one of 1.5-2.0 kDa (PGF-II). Treatment and/or extraction with acetic acid, hydrochloric acid, or acidified-ethanol or preparations enriched in PGF-I obtained either by ion-exchange chromatography, acetone precipitation, or retention by ultrafiltration membrane (cut-off 30 kDa) resulted, upon gel filtration, in the detection of a mitogenic activity eluting mainly at the position of PGF-II. Acid hydrolysis and proteolysis of PGF-II led to a loss of activity. It is proposed that, in dog serum, mitogenic peptides for prostatic epithelial cells of 1.5 kDa (PGF-II) are found in their free form and/or in association with proteins of 150 kDa (PGF-I).
The Cres (cystatin-related epididymal spermatogenic) gene encodes the defining member of a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Cres expression is highly tissue- and cell-specific, with messenger RNA (mRNA) present in the testicular round/elongating spermatids, proximal caput epididymal epithelium, gonadotroph cells in the anterior pituitary gland, and corpus luteum of the ovary. To begin to elucidate the molecular mechanisms controlling the tissue- and cell-specific expression of the Cres gene, transgenic mice were generated containing 1.6 kilobases (kb) of the mouse Cres promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. A CAT enzyme-linked immunosorbent assay detected CAT protein in the testis, epididymis, isolated cauda epididymal spermatozoa, and anterior pituitary gland from mice heterozygous and homozygous for the transgene. However, reverse transcription (RT)-PCR did not detect CAT mRNA in any regions of the epididymis, suggesting that the CAT protein detected in the epididymis was from spermatozoa. RT-PCR also did not detect CAT mRNA in the ovary. RT-PCR analysis of the testes from mice of different postnatal ages showed CAT mRNA first detected at day 22, which correlated with the first appearance of Cres mRNA and with the presence of round spermatids. These studies demonstrate that 1.6 kb of Cres promoter contains the DNA elements necessary for germ cell and pituitary gland-specific expression but lacks critical sequences necessary for expression in the epididymis and ovary.
Only a few studies have investigated the association between the severity of Peyronie disease (PD) and clinical parameters such as age and associated comorbidities. The aim of this study was to report the relationship between the degree of curvature of the penis and the clinical parameters among patients with PD. A total of 1001 patients with PD were evaluated retrospectively in terms of penile deformity, erectile status, and risk factors for systemic vascular diseases. The degree of curvature was assessed with a protractor during maximum erection in response to a combined injection and stimulation test and/or vacuum device. A modified Kelami classification was used to categorize penile deformities as follows: patients with deformities without curvature (notching, hourglass, and swan neck deformity, group 1), with mild curvature (≤ 30 degrees, group 2), with moderate curvature (31-60 degrees, group 3), or with severe curvature (> 60 degrees, group 4). Chi-square tests, 1-way analysis of variance, and univariate and multiple ordinal regression analyses were used for statistical analysis. Penile deformity without curvature was detected in 12.3% of the patients, whereas the curvature was less than 30 degrees in 39.5%, 30 to 60 degrees in 34.5%, and more than 60 degrees in 13.5% of the patients. Multiple ordinal regression analysis identified age (P = .013), side of deformity (P = .007), erectile dysfunction (P < .0001), and diabetes mellitus (P = .001) as significant independent predictors of the severity of penile curvature. In conclusion, patients' age, side of deformity, erectile function, and diabetes were significantly associated with the degree of curvature.
PDC-109 is the prevalent secretory protein from bovine seminal vesicles that binds to the midpiece of sperm once they pass the ampulla of the vas deferens during emission. Thereby, the protein changes biophysical membrane properties, eventually resulting in increased sperm motility. To elucidate the underlying biochemical mechanism, we have studied the ion-pumping activity (Ca(2+)-ATPase) in membrane preparations of bovine spermatozoa following in vitro incubation with the protein and analyzed whether PDC-109 influences sperm motility. PDC-109 was purified to homogeneity from bull seminal vesicle extracts using a newly described method. The effect of PDC-109 on sperm motility was analyzed using the CASA-method. These experiments clearly demonstrated that PDC-109 significantly increases sperm motility. Calcium-pumping mechanisms were analyzed by monitoring the effect of PDC-109 on various parameters of enzyme activity of Ca(2+)-ATPase in epididymal sperm plasma membranes and were compared with Ca(2+)-ATPase activities from other organs and from epididymal sperm of different species, respectively. Specificity studies were performed using different Ca(2+)-antagonists. Enzyme activities of both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPases increased in a dose-dependent manner following the addition of the PDC-109 (range 5-20 microg). Preincubation of PDC-109 at temperatures above 37 degrees C and pHs ranging from below 6.5 and above 8.5 led to the loss of the stimulatory effect. An analysis of enzyme kinetics pointed to irreversible, cooperative interaction of PDC-109 with the enzyme. The effect was organ-specific, that is, restricted to sperm ATPases, but it was not species-specific, as it could be elicited also in rat sperm.
Previous studies have suggested that glucocorticoid (GC) can directly affect testicular testosterone (T) biosynthesis by Leydig cells through a receptor-mediated mechanism. Interconversion of corticosterone (CORT), the active form in rodents, and 11-dehydroCORT, the biologically inert 11-keto form, is catalyzed by 11betaHSD1. This enzyme thus controls the intracellular concentration of active GC. We have postulated that elevated CORT levels resulting from stress exceed the Leydig cell's capacity for metabolic inactivation of CORT, resulting in suppressed T production. The present study tested whether inhibition of 11betaHSD1 in vivo, by the administration of glycyrrhetinic acid (GA), increases intracellular active GC concentration and thereby affects serum T concentration and Leydig cell T production. Adult Sprague-Dawley rats were treated with vehicle (corn oil), CORT, GA, or GA + CORT. Serum luteinizing hormone (LH), CORT, and T levels were measured, as were the steroidogenic capacities of purified Leydig cells. Twofold elevations of CORT were achieved by the administration of either CORT or GA alone, but in both cases there was no effect on serum T levels. However, when CORT and GA were administered in combination, serum CORT levels increased 3.5-fold (to 420 +/- 34 ng/mL) and serum T levels were reduced significantly (to 0.72 +/- 0.07 ng/mL; control, 2.12 +/- 0.23 ng/mL). Serum levels of LH were not affected by CORT, GA, or GA + CORT. Consistent with the reduced serum T levels following GA + CORT, steroidogenic enzyme expression and capacities were significantly reduced compared to control. These data support a role for 11betaHSD1 in modulating intracellular CORT concentrations and, in turn, for a direct effect of GC on Leydig cells in response to stress.
We examined the expression of claudin-11 (CLDN11) in the testes and male reproductive tracts of rabbits. The rabbit CLDN11 cDNA sequences were nearly identical with human, mouse, and bovine CLDN11. The levels of CLDN11 mRNA and protein (22 kDa) were markedly increased in the testis during adult development. On postnatal day (PND) 10, CLDN11 was colocalized with ZO-1 at the lateral contacts between adjacent Sertoli cells and was perpendicular to the basal lamina. In adult testis on PND 180, CLDN11 was codistributed with ZO1, and the pattern of immunoreactivity consisted of wavy linear tracts parallel to the basal lamina, which was different according to the spermatogenic stage. These results suggest that CLDN11 participates in inter-Sertoli cell tight junctions (TJs) at the blood-testis barrier in adult rabbits. CLDN11 was also found in the basal regions of Sertoli cells adjacent to the basal lamina in adult testis, suggesting that CLDN11 also participates in the adhesion between Sertoli cells and the basal lamina. CLDN11 mRNA and protein expressions were decreased in the adult epididymis compared with those in immature animals. In adults, CLDN11 mRNA levels were relatively high in the efferent duct, followed by those in the vas deferens, proximal corpus, and distal cauda, although low levels were observed in the initial segment and caput. On PND 10, CLDN11 immunoreactivity was identified at the apicolateral contacts between adjacent epithelial cells in the epididymis and vas deferens. In adults, CLDN11 was found in the nonciliated cells in the efferent duct and at the lateral contacts in the epithelial cells in the epididymal segments. In the caput, CLDN11 was found at the apicolateral contacts between adjacent epithelial cells, but expression was weak to negligible in the corpus of the vas deferens. CLDN11 may play an important role in TJs and cell adhesion in immature rabbit excurrent duct epithelia. In adult rabbits, CLDN11 in efferent duct epithelium and epididymal epithelium may contribute to the specific environment for sperm maturation.
Our objectives were to evaluate the effects of mono(2-ethylhexyl) phthalate (MEHP) on tight junctions (TJ) in cultured rat Sertoli cells (SC) and to investigate changes in the signal transduction pathways in SCs following MEHP treatment. SCs were isolated and purified from the testes of 18-day-old Sprague Dawley rats and incubated at 34°C for 3 days. After treatment of SCs with either the vehicle or MEHP for 0.5, 1, 3, 6 and 24 hours, whole cell lysates were isolated from each replicate to prepare RNA and protein. Expression levels of claudin-11, occludin, and zonula occludens-1 (ZO-1) mRNA were evaluated by quantitative real-time reverse transcription-polymerase chain reaction and changes in signal transduction pathways possibly induced by MEHP treatment were assessed by Western blot analyses. MEHP treatment led to significant decreases in the expression of claudin-11 and occludin mRNA, but not that of ZO-1, in rat SCs. Exposure of rat SCs to MEHP resulted in the marked induction of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), whereas other pathways examined in this study were not activated by MEHP. Furthermore, treatment of rat SCs with a specific inhibitor of p44/42 MAPK prevented the MEHP-induced down-regulation of claudin-11 and occludin. These findings demonstrate that MEHP exposure inhibited the expression of claudin-11 and occludin mRNA in rat SCs through the p44/42 MAPK pathway, suggesting the possible involvement of MEHP in spermatogenic function by regulating major components of TJs in SCs.
Glucocorticoid hormone controls Leydig cell steroidogenic function through a receptor-mediated mechanism. The enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) plays an important role in Leydig cells by metabolizing glucocorticoids, and catalyzing the interconversion of corticosterone (the active form in rodents) and 11-dehydrocorticosterone (the biologically inert form). The net direction of this interconversion determines the amount of biologically active ligand, corticosterone, available for glucocorticoid receptor binding. We hypothesize that 11betaHSD oxidative and reductive activities are controlled separately in Leydig cells, and that shifts in the favored direction of 11betaHSD catalysis provide a mechanism for the control of intracellular corticosterone levels. Therefore, in the present study, we tested the dependency of 11betaHSD oxidative and reductive activities on protein kinase C (PKC) and calcium-dependent signaling pathways. 11betaHSD oxidative and reductive activities were measured in freshly isolated intact rat Leydig cells using 25 nM radiolabeled substrates after treatment with protein kinase modulators. We found that PKC and calcium-dependent signaling had opposing effects on 11betaHSD oxidative and reductive activities. Stimulation of PKC using the PKC activator, 6-[N-decylamino]-4-hydroxymethylinole (DHI), increased 11betaHSD oxidative activity from a conversion rate of 5.08% to 48.23% with an EC50 of 1.70 +/- 0.44 microM (mean +/- SEM), and inhibited reductive activity from 26.90% to 3.66% conversion with an IC50 of 0.22 +/- 0.05 microM. This indicated that PKC activation in Leydig cells favors 11betaHSD oxidation and lower levels of corticosterone. The action of DHI was abolished by the PKC inhibitor bisindolylmaleimide I. In contrast, addition of calcium to Leydig cells increased 11betaHSD reductive activity while decreasing oxidative activity, thereby favoring reduction and conversion of inert 11-dehydrocorticosterone into active corticosterone. The opposite effect was seen after elimination of calcium-dependent signaling, including removal of calcium by EGTA or addition of the calmodulin (calcium binding protein) inhibitor SKF7171A, or the calcium/calmodulin-dependent protein kinase I (CaMK II) inhibitor, KN62. We conclude that 11betaHSD oxidative and reductive activities are separately regulated and that, in contrast to calcium-dependent signaling, PKC stimulates 11betaHSD oxidation while inhibiting 11betaHSD reduction. Maintenance of a predominantly oxidative 11betaHSD could serve to eliminate adverse glucocorticoid-induced action in Leydig cells.
The human sperm antigen SP-10 is a testis-specific, intra-acrosomal protein associated with the membranes of the acrosomal vesicle. The molecule has been designated a "primary vaccine candidate" by a World Health Organization (WHO) Taskforce on Contraceptive Vaccines. cDNA cloning and sequencing have indicated that SP-10 is encoded by a 795-base-pair (bp) reading frame that predicts a 265-amino acid protein of 28.3 kd. In this study, we used a 634-bp fragment (bp 68 through 700, amino acids 3 through 222) of the SP-10 sequence to probe, by Southern blotting, EcoRI-digested DNA from 33 mouse/human somatic cell hybrids involving 16 unrelated human cell lines and 4 mouse cell lines. The hybrids were characterized by karyotypic analysis and by mapped enzyme markers. The presence or absence of positive human bands was scored on the blots and the percent of concordance and discordance with a specific human chromosome was determined. The DNA probe for SP-10 showed a concordance of 31 and a discordancy of 0 for human chromosome 11, mapping SP-10 unequivocally to this chromosome. The hybrid XER-7 with the 11/X translocation: 11p12 or 11p11----11qter:: Xq11----Xqter and the hybrid EXR-5CSAZ with the X/11 translocation: Xpter----Xq22::11q13----11qter localized the SP-10 gene to the p12----q13 region. The SP-10 locus has been assigned the gene symbol ACRV1 (acrosomal vesicle protein-1).
The potent androgens dimethandrolone 17β-undecanoate (DMAU) and 11β-methyl-19-nortestosterone 17β-dodecylcarbonate (11β-MNTDC) are in development for androgen replacement therapy and hormonal contraception in men. They can be delivered either orally or as long-acting injectables. In the current study, their long-term effects on body composition (percentage lean and fat mass); bone mineral density (BMD); serum gonadotropin levels; and weights of the prostate, seminal vesicles, and levator ani muscle were assessed. Four-week-old male rats were sham-operated (intact) or castrated (Cx) and treated subcutaneously for 16 weeks postsurgery with vehicle (Cx, intact), DMAU, or 11β-MNTDC every 4 weeks; testosterone enanthate (TE) every 2 weeks; or a testosterone (T) implant. There were significant differences in body weights over time with a general trend of intact = Cx + T = Cx + TE > Cx + 11β-MNTDC > Cx > Cx + DMAU. At week 18, rats were evaluated by dual-energy x-ray absorptiometry using the whole-body function of the Hologic software. The percentage lean body mass and BMD were lower (P < .05) in Cx rats than intact rats but equivalent in all groups of androgen-treated Cx rats and intact rats (P > .05). The highest percentage body fat was observed in Cx rats. Only DMAU- and 11β-MNTDC-treated rats had lower percentage body fat compared with Cx rats (P < .05). Prostate, seminal vesicles, and levator ani muscle weights, corrected for final body weight, were decreased (P < .05) in Cx compared with intact rats and increased to varying extents in androgen-treated Cx rats compared with Cx rats (P < .05). The most marked increases were observed in the DMAU-treated rats in which prostate and seminal vesicle weights/kg body weight were 2.4 to 2.7 times those of intact rats, and levator ani muscle weights were increased approximately 1.5-fold. Blood was collected from the tail vein at weeks 4, 8, 12, and 16 for measurement of serum levels of androgens and at necropsy at week 18 for measurement of serum gonadotropins. Serum levels of luteinizing hormone and follicle-stimulating hormone were greatly elevated in Cx rats at week 18 and suppressed to levels comparable to those in intact rats by DMAU, 11β-MNTDC, and T implants (P > .05). Collectively, our data indicate that androgen replacement with DMAU or 11β-MNTDC in Cx rats resulted in favorable changes in body composition and maintenance of BMD comparable to those of T.
Polyclonal antibodies produced against a peptide derived from the Fer tyrosine kinase sequence also specifically recognized a 110-kd protein (p110) up-regulated in dividing versus resting dog prostate epithelial cells in vitro. In vivo in the dog prostate, p110 expression was detected when basal cell metaplasia was induced by estrogens after castration but not when renewing the differentiated epithelium with androgens. It was also detected in extracts from the human prostatic carcinoma cell lines LNCaP, DU145, and PC-3, and from 6 out of 11 human prostate cancer tissues analyzed, but not from normal or hyperplastic glands. The tyrosine kinase Src was shown by coimmunoprecipitation to associate with p110, and this interaction was positively modulated by bombesin stimulation of PC-3 cells. However, p110 was not tyrosine phosphorylated. Moreover, it was mainly distributed in the nuclear fraction. This nuclear p110 protein, expressed in dividing prostate epithelial cells and in human prostate cancer cells and tissues, could thus be a downstream mediator of bombesin-signaling pathways, acting via its association with Src.
Objectives: Testicular cancer is the most common cancer in young men. Several studies have reported an alteration in semen quality in non-seminoma tumors but this result has not been confirmed in all of the published data. We performed a retrospective study in a population of 1158 men with testicular cancer who banked sperm between 1999 and 2003 in 11 French Centre d'Etude et de Conservation des Oeufs et du Sperme humain (CECOS) laboratories. Our study evaluated pre-freeze and post-thaw sperm parameters according to patient medical history, tumor histological type and disease stage. Findings: Pure seminomas were found in 48% of our population. Testicular cancer was generally diagnosed at stage I. In cases of a history of unilateral cryptorchidism, testicular cancer occurred preferentially in the mal-descended testis. Semen samples were preferentially collected after orchiectomy. The sperm concentration and total sperm number were significantly lower before orchiectomy in seminomas compared with non-seminoma tumors (p<0.001). After orchiectomy, these parameters decreased for non-seminoma tumors and did not vary for seminomas. Semen parameters were more severely impaired for stage III tumors, and when patients had a history of cryptorchidism or when they were less than 20 years of age. Azoospermia was more frequently observed before than after orchiectomy. Conclusion: In this study, we determined that sperm cryobanking should preferentially be performed before orchiectomy and that testicular sperm extraction concurrent with orchiectomy should be used in severe spermatogenesis impairment. Our study highlights that seminomas alter sperm production more significantly than non-seminoma tumors and seem to preferentially impair spermatogenesis in tumor-bearing testes.
Glucocorticoids suppress testosterone production in Leydig cells. The level of glucocorticoid action is set within the Leydig cell by the number of glucocorticoid receptors and by the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD). This enzyme acts either as an oxidase inactivating glucocorticoid or as a reductase amplifying its action. It is currently unknown whether extracellular conditions might cause 11betaHSD oxidative and reductive activities in Leydig cells to change inversely or independently. The aim of the present study was to determine whether extracellular conditions set in vitro by various culture time and media components, such as glucose and pyruvate, affect the relative rates of 11betaHSD oxidation and reduction. Primary rat Leydig cells and cell lines (COS1 and CHOP cells) transfected with 11betaHSD-I complementary DNA (cDNA), were incubated with 25 nmol/L (physiologic range) or 500 nmol/L (stress range) concentrations of radiolabeled substrates, corticosterone or 11-hydrocorticosterone, for 0 to 24 hours. Oxidative activity predominated over reductive activity under initial conditions when product formation increased linearly with time. For example, in Dulbecco's modified Eagle medium/F12 medium (containing 5.5 mmol/L glucose), the peak ratio of oxidation to reduction (with 1 denoting equivalence of oxidative and reductive activities) was 5.5 in rat Leydig cells, 19.7 in COS1 cells, and 20.8 in CHOP cells. Glucose stimulated reductive activity but did not change the predominant direction of 11betaHSD catalysis at earlier times. In COS1 cells transfected with 11betaHSD-I cDNA, oxidative activity rapidly increased during the first 2 hours of the incubation, then gradually decreased while reductive activity increased steadily. The relative ratio of oxidation to reduction rapidly declined to less than 0.5 at 6 hours, and thus the favored direction of catalysis changed from oxidation to reduction. However, in transfected CHOP cells, 11betaHSD oxidative activity rapidly increased during the first 2 hours and continued to increase for 24 hours. The ratio of oxidative to reductive activity rapidly declined but kept above 1 in CHOP cells for 24 hours, and the favored direction of catalysis remained predominantly oxidative. These results revealed that 11betaHSD-I is a predominant oxidase initially in Leydig cells and 2 cell lines, and that the oxidative activity is gradually lost over time. The data suggest that type I 11betaHSD is a predominant oxidase in Leydig cells in vivo.
The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ([125I]hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of [125I]hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the [125I]hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of [125I]hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval. Likewise, an attempt to correlate silver grains with small coated or uncoated pits, the stacks of saccules of the Golgi apparatus and other Golgi-related elements including GERL, proved unsuccessful, since these structures were mostly unlabeled. These in vivo experiments thus demonstrate the specific binding of [125I]hCG to the plasma membrane of Leydig cells predominantly on their microvillar processes, and the subsequent internalization of the labeled hCG to secondary lysosomes. In addition, binding and internalization of hCG persisted for long periods of time.
The present study was undertaken to evaluate the effectiveness of an avian chemosterilant, 20, 25-diazacholesterol dihydrochloride (SC-12937), on the rat testis. Adult male rats were injected intraperitoneally with 10 mg (Group 1) or 30 mg (Group 2) of SC-12937/kg/d or with vehicle alone (Group 3) for 10 days, and were killed 24 hours after the last injection. A wide range of variation in the appearance of affected seminiferous tubules was observed in the testis of SC-12937-treated rats at both dose levels. This ranged from apparently normal-looking seminiferous tubules to almost completely atrophied tubules with no cells. Affected tubules exhibited intraepithelial vacuoles of varying size, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. The presence of severely damaged and entirely normal seminiferous tubules adjacent to one another in the same section was noteworthy. The changes appeared to be dose-related. A greater number (34.6%) of affected tubules were observed in rats receiving 30 mg of SC-12937 compared with the ones receiving 10 mg of this compound (19.6%). The Sertoli cells also were affected by this drug and exhibited cytoplasmic vacuolation, a marked increase in the accumulation of lipid droplets and myeloid bodies. Necrotic Sertoli cells also were observed in the severely affected tubules. The possible mechanism of antispermatogenic action of SC-12937 in rats has been discussed briefly.
A series of complex processes takes place during the first meiotic division, including pairing, synapsis, recombination, and segregation of homologous chromosomes. When any of these processes is altered, cellular checkpoints arrest the progression of meiosis and induce cell loss, causing a severe reduction in fertility, or even sterility. In this study, we report on a 29-year-old, healthy, but severe oligozoospermic male with a supernumerary, ring-neocentric 13q12.3 → 13q22 chromosome and a reciprocal deletion, which interfere with the meiotic pairing of chromosomes 13, causing spermatogenesis failure.
Previous studies showed that interleukin-6 (IL-6) was expressed in human Leydig and Sertoli cells and that it inhibited sperm motility. The aim of this study was to compare the expression of IL-6, IL-6R, and GP130 in ejaculated spermatozoa between normozoospermic and asthenozoospermic men. Human spermatozoa in the semen were purified by Percoll gradient technique to separate the seminal plasma and other round cells. RT-PCR, immunocytochemistry, and Western blot were used to detect the expression of IL-6, IL-6R, and GP130 in spermatozoa. With RT-PCR, only GP130 mRNA but not IL-6 and IL-6R mRNA was expressed in human ejaculated spermatozoa. The expression of GP130 mRNA was significantly lower in asthenozoospermic men than in normozoospermic men. The protein expression of GP130 was further confirmed by both immunocytochemistry and Western blot. Again, GP130 protein levels were significantly lower in asthenozoospermic men than in normozoospermic men. The results suggested that the decreased expression of GP130 in ejaculated spermatozoa could be associated with low sperm motility in asthenozoospermic men.
The isoforms of the highly conserved and ubiquitously expressed 14-3-3 family of proteins function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways. Low levels of 14-3-3 isoforms appear to be expressed in most tissues, but specific isoforms or combinations have been shown to be overexpressed in a cell-specific manner. In the present study we show that the theta isoform of 14-3-3 is expressed in Sertoli cells. Although previous reports have shown the presence of a 14-3-3 theta isoform in mouse testicular germ cells, this report demonstrates the presence of the 14-3-3 theta isoform in rat Sertoli cells. The 14-3-3 theta isoform isolated from rat Sertoli cells appears to have a truncated 3' UTR, which makes the transcript shorter by 244 bp, compared with its brain counterpart. Northern blot analysis suggests that the 14-3-3 theta isoform may also be present in other testicular cell types and tissues. The truncation of the 3' UTR suggests a potential role in regulating cell-specific expression of 14-3-3 theta. The expression of 14-3-3 theta in Sertoli cells was confirmed by Northern blot, polymerase chain reaction, Western blot, and immunocytochemical analysis. The levels of 14-3-3 theta mRNA and protein in Sertoli cells remained unchanged in response to the gonadotropin, FSH. Consistent with the absence of the effect of FSH on the expression of 14-3-3 theta, an antisense oligonucleotide to 14-3-3 theta had no effect on FSH-induced activation of the transferrin promoter in Sertoli cells. The widespread expression of 14-3-3 theta in testis and the lack of effect of FSH on levels of its expression suggest that 14-3-3 theta influences Sertoli cell function in an FSH-independent manner.
A new membrane-permeant DNA stain, SYBR-14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours at 36 degrees C to assess changes in cell viability. As cell death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.
The basis for cell-cell adhesion in the seminiferous epithelium of the developing testis is doubtless critical in supporting events that are essential for the onset and maintenance of normal spermatogenesis. In this study, we applied immunoblotting and immunolocalization approaches for the following reasons: 1) to ask whether neural cell adhesion molecule (NCAM) underlies cell-cell interactions in vivo, as we previously showed for cells in vitro, 2) to characterize the isoform or isoforms of NCAM expressed during testicular development, and 3) to study NCAM expression in long-term Sertoli cell-gonocyte cocultures and to compare and contrast this pattern of expression with that in vivo. Our findings indicate that NCAM is found ubiquitously at cell-cell interfaces within the seminiferous cord from birth through day 10 and thereafter is restricted to interstitial cells. Moreover, only polysialic acid-negative 140-kDa NCAM is expressed in the testis or in coculture, an isoform whose properties are compatible with the concept of NCAM as both a direct modifier of cell function and an indirect influence on cell responses mediated by other external factors. In addition, we found that germ cells, potentially gonocytes or Type A spermatogonia, persist in long-term cocultures maintained for 15 days after isolation from 5-day-old rat pups and that NCAM continues to be expressed at high levels in these cultures. This observation is in marked contrast to our observation that NCAM gradually decreases and eventually disappears in vivo by postnatal day 15. Thus, our findings indicate that 140-kDa NCAM is prominent in neonatal testes but is down-regulated by as yet unidentified mechanisms thereafter. Our findings also indicate that down-regulation of NCAM fails to occur in hormone- and serum-free Sertoli cell-germ cell cocultures.
Paired testicular volumes and weights, as well as age, height, and weight, were recorded from a series of 1056 consecutive necropsies on adult males ranging in age from 18 to 96 years. These data were analyzed to examine the effects of age, nutritional state (standardized body weight), and illness on testicular size. Testicular volume and weight were related by a constant density of 1.038 g/ml, regardless of testicular size, age or illness. Mean testicular volume was correlated with height (r = 0.470), weight (r = 0.504), body surface area (r = 0.549) and standardized body weight (r = 0.152). Advancing age, malnutrition, alcoholism, malignancy, and a chronic, terminal illness were each individual risk factors for reduced testicular size, whereas diabetes, narcotic or other drug usage, and pelvic injury were not associated with reduced testicular volume. Since advancing age, reduced standardized body weight, and some disease states were all associated with diminution of testicular size, the interaction of age, malnutrition, and illness on testicular size were examined by statistical modeling, using multivariate logistic regression and covariance analysis. The associations of alcoholism, malignancy, and chronic, terminal illness with decreased testicular volume were independent of aging or nutritional state. The effects of chronic, terminal illness were mostly explained by the concurrent effects of reductions in standardized body weight (malnutrition). After exclusion of men with diseases shown to be associated with decreased testicular size, he specific effects of age alone demonstrated a reduction in testicular volume only in the 8th decade of life.(ABSTRACT TRUNCATED AT 250 WORDS)
The radical surgical option we propose for Peyronie's disease consists in removing the sclerohyalinotic focus of disease and replacing it by an autologous dermal graft taken from the upper outer thigh area. Between 1981 and 1991, we operated on 335 patients with Peyronie's disease, 152 of whom underwent plaque excision and dermal graft. All could be assessed with a 2-year follow-up. Two main complications were observed: mild penile flexure due to scar retraction of the graft (35% of cases), and partial erectile deficit with decreased corporal rigidity (17% of cases). The degree of graft retraction is linked to the individual's histologic response. A mild deviation of the penis can occur some months after surgery and is not a relapse flexure due to disease progression, but is mere scar retraction and will spontaneously regress. Because the patient will date the onset of a postoperative erectile deficit from the time of the operation, it is advisable to assess preoperatively the erectile ability of all patients. Furthermore, an impaired erectile response could result from hypoaesthesia of the glans, postsurgical stress, and fibrosis of the erectile tissue. A retrospective assessment of radical surgery cases involving plaque excision and dermal graft led us to propose this option where precise indications apply, providing that other alterations of the erectile function are preoperatively assessed.
Structural chromosomal abnormalities in gonadal tissue represent an important category of parentally transmittable unbalanced chromosomal abnormalities to the offspring. A child with multiple anomalies was sent for cytogenetic analysis, and his karyotype was 46,XY,der(17)t(15;17)(q21; q25). This abnormality was transferred from his grandfather to his father and to the proband. In this family, 5 persons (1 female and 4 male) are the carriers of this abnormality. In this study, fluorescence in situ hybridization (FISH) on sperm nuclei of 4 male carriers was studied to determine the distribution of segregation patterns of the balanced translocation 15q;17q. The segregation results showed that the segregation products in the third carrier (the grandfather) were different, but they were not statistically significant. The segregation patterns in the other carriers were similar. Overall, 50.3% of the sperm nuclei (mean value for 4 carriers) analyzed were the result of alternate segregation; 36.9%, of adjacent I segregation; 9.0%, of adjacent II segregation; and 2.4%, of 3:1 segregation; the remaining 1.3% could be diploid sperm nuclei or of 4:0 segregation. Multicolor FISH analysis appears to be a rapid and reliable method for the direct analysis of segregation patterns in sperm nuclei of carriers of balanced reciprocal translocation, and it also provides interesting information for determining the possible risks for the offspring.
In vitro studies suggest that the protease activity of PSA might play a functional role to facilitate the growth and spreading of cancerous prostatic cells. hK2 may have similar properties. Further investigation to prove their in vivo effects is required. Regulation of PSA and hKLK2 gene expression is mediated not only by androgens, but also by a number of autocrine and paracrine factors, suggesting that the control mechanisms for expression of these genes are complex and multifaceted. Such factors may also be integral for the growth and differentiation of prostate cells. Thus PSA and hKLK2 may serve as useful markers to study the regulation of gene expression during cell growth and differentiation of the prostate.
In addition to other known markers of the human prostate, it was shown that the prostatic fraction of the split ejaculate was rich in a 16-kDa protein with properties not described previously. This protein was purified from human seminal plasma using ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and gel filtration on Sephadex G-100. The purified protein showed a single prominent spot on two-dimensional gel electrophoresis. The sequence of the first 40 amino acids that could be positively identified was identical to that of a prostatic secretory protein of 94 amino acids (PSP94) previously designated as beta-inhibin. Antibodies produced in rabbits against the purified protein were used to develop a radioimmunoassay. These antibodies appeared to recognize only the NH2-terminal portion of the native molecule since they did not react with a synthetic peptide composed of the 28 C-terminal residues. The radioimmunoassay showed that the concentration of the protein was 1320 +/- 183 micrograms/ml in the seminal plasma of adult fertile men and 1134 +/- 136 micrograms/ml in vasectomized patients. In hypertrophic and adenocarcinomatous prostates, the concentrations were 326 +/- 156 and 104 +/- 23 micrograms/ml, respectively, while values were lower than 0.060 micrograms/ml in the testis, epididymis, vas deferens and liver. The blood plasma concentration was 0.019 +/- microgram/ml in 23 asymptomatic men 45 to 65 years old and 0.115 +/- 0.036 microgram/ml in eight patients with prostate cancer.(ABSTRACT TRUNCATED AT 250 WORDS)
LHRH agonist analogs have been investigated as potential male contraceptives. It has been shown that the LHRH agonistic analog [D-Trp6,Pro9-NEt] LHRH (LHRHA) given to men in single doses up to 500 micrograms daily for up to 20 weeks with the coadministration of testosterone enanthate produces reversible oligozoospermia. Individual responses to the treatment, however, were variable. In this study, we gave the same analog to eight normal male volunteers as a continuous infusion of 500 micrograms daily for 16 weeks. Testosterone enanthate, 100 mg, was given by injection every second week. Six of the subjects became oligozoospermic but the other two retained sperm counts that were greater than 20 million/ml, although their treatment continued for 20 weeks. The reasons for this variability of response are not clear. Serum immunoreactive LH values increased during the infusion period whereas testosterone declined. FSH values fell during treatment in all subjects except the two non-responders. The acute pituitary response to LHRHA during the treatment or shortly thereafter (48 h) was completely abolished, and bioactive LH values were suppressed totally. FSH, LH, testosterone and sperm counts returned to normal in all subjects following discontinuation of LHRHA infusion. Continuous infusion of 500 micrograms of LHRHA daily for 16 weeks with 100 mg of testosterone enanthate every 2 weeks induced desensitization of the pituitary, loss of LH bioactivity, and decreases of FSH and testosterone. This mode of administration, however, did not improve sperm density results obtained earlier by single daily injections of the analog. Heterogeneity of sperm density profiles still persists for reasons that are not yet clear.
Cytochrome P450 17alpha-hydroxylase/17, 20-lyase (CYP17) is crucial for cortisol and sex steroid biosynthesis. In a previous study we examined CYP17 function by generating mice with a targeted CYP17 deletion. We found that in addition to its role in steroid biosynthesis, CYP17 is present in germ cells. In the present study we examined the effect of CYP17 on sperm morphology. Disorganization of the sperm midpiece, small sperm mitochondria with reduced inner membranes and matrix, and irregular sperm shape were found to be associated with the CYP17 gene deletion. Treating the mice carrying the CYP17 deletion with testosterone did not alleviate the observed sperm phenotypes, suggesting that CYP17 acts in a testosterone-independent manner. These results suggest that CYP17, in addition to its role in androgen formation, is critical for proper mitochondrial architecture and sperm morphology and thus for sperm function and normal fertility.
This article reports an isotope dilution mass spectrometric method for the simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in human plasma and prostatic tissue. After addition of tri-deuterated steroids as internal standards to prostatic tissue homogenates or plasma samples, extraction was performed with diethylether. The extracts were purified by two chromatographic steps (Sep-Pak C 18 cartridge and Sephadex LH-20) and injected into a gas chromatograph coupled with a mass spectrometer after derivatization with heptafluorobutyric acid. This method was highly specific and showed good precision, accuracy, reproducibility and sensitivity. T, DHT, and 3 alpha-diol were measured in human plasma and in prostatic tissue of seven patients with benign prostatic hyperplasia (BPH) treated for 3 months with a long acting GnRH analog before surgery. Plasma levels of T, DHT, and 3 alpha-diol were reduced by GnRH analog treatment to castrate levels. The tissue concentrations of the same steroids, compared with those obtained from 19 untreated patients, showed a mean reduction of about 90% for DHT and 3 alpha-diol, and about 75% for T. These results suggest that about 90% of prostatic DHT and 3 alpha-diol depend on testicular activity because they are dramatically reduced after pharmacologic castration.
This study was designed to determine the effects of 17 beta-estradiol (E2) on Leydig cell development in the rat. Mature (60 to 65 days old) male rats received a single intraperitoneal injection of ethane dimethylsulfonate (EDS, 100 mg/kg body weight); untreated rats served as controls. In one series of experiments, groups of EDS-treated rats also received daily injections of either E2 (25 micrograms/100 g body weight), human chorionic gonadotropin (hCG, 20 IU/day), a combination of the two, or vehicle only (EDS controls). Animals were killed on days 2, 4, 10, 16, 24, 30, and 36 after EDS treatment. In another series of experiments, groups of EDS-treated rats received daily injections of hCG and E2 during days 0 through 5, 5 through 30, or 16 through 30 after EDS treatment, and were killed on day 30. In both series of experiments, the steroidogenic capacity and hCG binding capacity of the Leydig cells were examined in short-term in vitro incubations using collagenase-dispersed interstitial cells. Testes were also prepared and examined histologically by light and electron microscopy. E2 treatment of animals during the initial 5 days after EDS administration had no effect on the regeneration of interstitial cells and Leydig cells. Treatment with E2 during days 5 to 30 post-EDS blocked the regeneration of Leydig cells and thereby significantly reduced the increase in interstitial cell numbers. Finally, when E2 treatment was delayed until 16 days post-EDS, there was no significant reduction in the regeneration of interstitial or Leydig cells. These data suggest that an important developmental process that is necessary for Leydig cell regeneration occurs between days 5 and 16 post-EDS.(ABSTRACT TRUNCATED AT 250 WORDS)
Dimethandrolone undecanoate (DMAU: 7α,11β-dimethyl-19-nortestosterone 17β-undecanoate) is a potent orally active androgen with progestational activity that is in development for therapeutic uses in men. We hypothesized that because of its dual activity, DMAU might have potential as a single-agent oral hormonal contraceptive. To test this possibility, adult male rabbits (5/group) of proven fertility were treated orally with vehicle or DMAU at 1.0, 2.5, 5.0, or 10.0 mg/kg/d for 12 or 13 weeks. Semen and blood samples were collected every other week through week 30. Sperm were decreased (P < .05) in semen samples from DMAU-treated rabbits at 2.5 and 5.0 mg/kg/d at weeks 12, 14, 16, 18, and 20 compared to week 0 (prior to treatment). The percentage of forward progressive motile sperm in those rabbits that still had measurable sperm was also reduced by DMAU treatment at 2.5 mg/kg/d at weeks 14, 16, 18, and 20 and at 5.0 mg/kg/d at week 18 (P < .05). At 1.0 mg/kg/d only 1 rabbit had reduced sperm numbers and motility. A mating trial was performed at week 15. The number of bred males that were fertile was 4 of 4 in the vehicle-treated group and 4 of 5, 0 of 4, and 2 of 5 in the 1.0, 2.5, and 5.0 mg/kg/d DMAU treatment groups. By week 22, sperm numbers and forward progressive motility increased, and they returned to pretreatment levels in all DMAU-treated rabbits by week 30. All bred males were fertile at week 31. Serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were significantly suppressed in DMAU (1.0, 2.5, or 5.0 mg/kg/d)-treated rabbits during the 12-week dosing interval, but were comparable to pretreatment levels after cessation of dosing. These data indicate that DMAU suppressed the hypothalamic-pituitary-gonadal axis, resulting in severe oligospermia in the majority of rabbits in the 2.5 and 5.0 mg/kg/d dosing groups. Infertility was observed when sperm numbers decreased to about 10% of pretreatment levels. In rabbits dosed with DMAU at 10.0 mg/kg/d, no effect on sperm numbers or motility was observed by week 12. Dosing continued for another week, and the rabbits underwent a gross necropsy on week 13 with removal of testes and epididymides for histology and preparation of testicular cytosol. Serum testosterone, FSH, and LH levels were considerably suppressed in these rabbits as in the lower-dose groups. The lack of oligospermia in the 10.0 mg/kg group as well as in the 2 fertile males in the 5.0 mg/kg group may have been due to high intratesticular levels of 7α,11β-dimethyl-19-nortestosterone, the active metabolite of DMAU. Hence, as observed previously for testosterone, DMAU has a biphasic effect on spermatogenesis. Collectively, these data indicate that DMAU has the potential to be an orally active single-agent male hormonal contraceptive at an appropriate dose level and should be tested for contraceptive efficacy in nonhuman primates.
Cocaine hydrochloride (30, 15, or 0 mg/kg) was administered daily subcutaneously to male rats for a minimum of 72 days. Animals receiving the 15-mg/kg and 0-mg/kg doses were pair-fed with animals receiving the higher dose. A fourth group served as a nontreated ad libitum-fed control to assess the role of cocaine-associated decreases in food and water intake. Administration of cocaine resulted in dose-related decreases in body weights, increases in locomotor activity, and decreases in estradiol levels. The high dose of cocaine was also associated with an increase in the percentage of spermatozoa with heads separated from tails. Cocaine did not affect sexual behavior, relative weights of the testis or accessory organs, histology of the testis, number of implantations, resorptions, fetal or newborn weight of offspring, or offspring weight at 21 days of age but did result in hyperactivity and increased perseverance in a T maze.
We have investigated the effect of acute immobilization (3 hours) stress on testicular steroidogenesis in the adult rat. Immobilization did not alter plasma luteizing hormone (LH) levels, but plasma testosterone (T) levels were reduced by 82%. Plasma levels of corticosterone in stressed rats were elevated more than ninefold over control levels. After 3 hours of stress, testicular levels of progesterone were elevated 33%, and levels of 17 alpha-hydroxyprogesterone and T were reduced 47% and 37%, respectively, compared to controls. Immobilization for 3 hours had no effect on the association or dissociation rate constants of LH/human chorionic gonadotropin (hCG) receptors of testicular interstitial cells and did not alter specific hCG binding. The effect of 3 hours of immobilization on testicular 17 alpha-hydroxylase and 17,20-lyase was assessed by incubating testicular microsomes from stressed and control animals in the presence of 21[14C]progesterone and [3H]17 alpha-hydroxyprogesterone. Immobilization of rats reduced the Vmax values of 17 alpha-hydroxylase and 17,20-lyase by 47% and 48%, respectively, but had no effect on the Km values. These results support the hypothesis that stress for 3 hours disrupts rat testicular steroidogenesis via a mechanism that is independent of changes in circulating levels of LH and the binding characteristics of LH/hCG receptors. The effects of immobilization on the content of testicular steroids and on the activities of 17 alpha-hydroxylase and 17,20-lyase suggest that stress inhibits the activities of both 17 alpha-hydroxylase and 17,20-lyase.
Human Sertoli cell parameters, namely lactate, estradiol-17 beta, and transferrin production, were determined after a 24-hour incubation with either human follicle stimulating hormone (FSH) or dbcAMP in the presence or absence of testosterone plus a phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine; MIX). Testicular tissues were obtained from 10 young patients (mean age, 29 years); using a 3-step enzymatic treatment, Sertoli cell enriched preparations (> 92%) were studied after 4 days as primary cultures. No significant changes in lactate, estradiol-17 beta, and transferrin outputs have been observed according to age in patients ranging in age from 16 years to 47 years. Sertoli cell production of the compounds is controlled by testosterone plus MIX; FSH (or dbcAMP) treatment only slightly improves their synthesis. It is suggested that human Sertoli cell function, as far as the parameters measured in this study are concerned, is likely regulated by cAMP-dependent and independent pathways.
The idea that varicocele plays a detrimental role in fertility is supported by the presence of a higher frequency of affected men among the infertile population than among men with normal semen parameters. In this research we examined ejaculates from a large group of selected men affected by varicocele by light and electron microscopy. The effect of varicocele on chromosome meiotic segregation was investigated by fluorescence in situ hybridization (FISH). The potential benefits of varicocelectomy on sperm quality were evaluated by analyzing sperm characteristics before and after surgical correction of varicocele. Transmission electron microscopy (TEM) analysis, elaborated previously, showed that the incidence of immaturity, apoptosis, and necrosis was higher in the varicocele group than in controls. FISH analysis performed on sperm nuclei from selected patients with varicocele showed that the mean frequencies of disomies and diploidies were generally out of the normal range, indicating a severe disturbance in meiotic segregation. Sperm characteristics evaluated before and after varicocele repair showed a general improvement. As a consequence, the varicocele seem to affect sperm morphology and function concomitantly with meiotic segregation derangement. In consideration of these data, we suggest that TEM and FISH analyses should be performed for all varicocele patients.
A questionnaire was designed to assess the effects of renal transplantation in men of reproductive age on marital status and fertility. The study sought to correlate recipients' marital status and fertility with the health of the recipients after the transplantation, the health of children they fathered after the procedure, and the functioning of the transplanted kidney. Male recipients (n = 243) who were single and of reproductive age before renal transplantation were selected from 2007 recipients of a renal transplant recorded in the authors' hospitals in China. Of the 243 surveyed, 185 completed the questionnaire and participated in follow-up in the clinic or by telephone. Their marital status and fertility were investigated. Of the 185 recipients, 69 got married 12-88 months (mean, 32.19 +/- 14.30 months) after renal transplantation, and 62 of 69 couples were actively attempting to become pregnant. Fifty-three patients fathered 54 children, including 1 pair of twins, 9-72 months (mean, 25.81 +/- 15.33 months) after marriage. The birth weights of the newborns ranged from 2500 to 4600 g (mean, 3395 +/- 456.80 g). These children developed well. Nine patients did not father any children, and 3 of these 9 cases were attributable to infertility in the wife. Seven patients were using contraceptives. Three recipients suffered from chronic graft rejection and resumed hemodialysis 2-11 years after they fathered children. In addition, 2 patients died after fathering 1 child: 1 from dysfunction of the transplanted kidney 9 years after birth of his child, and another in an accident 1 year after his child's birth. Our findings suggest that, like men without renal transplants, male recipients of renal transplants can get married and father children, and the transplantation procedure appears to have no significant effect on the children fathered afterwards, on the recipients' health, or on the functioning of the transplanted kidney. It is very important to indicate that, in addition to needing contraception if they do not conceive, male renal transplant recipients should expect fertility rates that are similar to those of the general population.
Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.
ABP, a Sertoli cell secretory product, was identified in the seasonal rodent Octodon degus (Molina, 1872). It was shown to be present in cytosols from the testis and epididymis. It migrated with an Rf of 0.37 on nondenaturing polyacrylamide gels. Ligation of the vas efferens caused the disappearance of ABP from the epididymis and its accumulation in the testis, indicating its testicular origin. Binding to [3H]5 alpha-DHT was specific and completely reversible, with an apparent Kd of 3.5 +/- 0.4 X 10(-9) M. Half-times of association and dissociation were at 15 and 120 minutes, respectively. Binding equilibrium was achieved at 120 minutes. Steroid affinity relative to the best competitor, 5 alpha-DHT, was 0.27 for testosterone, 0.06 for 17 beta-estradiol, and 0.01 for cyproterone acetate. The presence and similar characteristics of ABP in a wide variety of mammals, including those with special reproductive strategies such as seasonal breeding, suggests that this protein may play a general role in the mechanisms regulating spermatogenesis, probably affecting the transport and concentration of androgens in the testis and epididymis.
Testosterone with a progestogen can suppress spermatogenesis for contraception. The synthetic androgen 7alpha-methyl-19-nortestosterone (MENT) may offer advantages because it is resistant to 5alpha-reduction and is therefore less active at the prostate. This study aimed to investigate MENT implants in combination with etonogestrel on spermatogenesis, gonadotropins, and androgen-dependent tissues in comparison with a testosterone/etonogestrel regimen. Healthy men (n = 29) were recruited and randomized to receive 2 etonogestrel implants with either 600-mg testosterone pellets repeated every 12 weeks or 2 MENT implants for up to 48 weeks. Testosterone concentrations in the testosterone group remained in the normal range. Subjects with 2 MENT implants showed peak MENT levels at 4 weeks with testosterone concentrations of 2 nmol/L. Sperm concentrations fell rapidly to less than 1 x 10(6)/mL at 12 weeks in 8 of 10 subjects in the MENT group and 13 of 16 subjects in the testosterone group with equally suppressed gonadotropins. Thereafter, suppression was not maintained in the MENT group, and 6 men noted loss of libido. Fourteen men completed 48 weeks of testosterone treatment, and all became azoospermic. Hemoglobin concentrations rose, and high density lipoprotein-cholesterol (HDL-C) fell in both groups. The MENT group showed a fall in prostate-specific antigen with no change in bone mass. MENT with a progestogen can achieve rapid suppression of spermatogenesis similar to testosterone, but this promising result was not sustained due to a decline in MENT release from the implants. This dose of testosterone, compared with previous studies using a lower dose with a higher dose of etonogestrel, had nonreproductive side effects without any increase in spermatogenic suppression. These data indicate the importance of the doses of progestogen and testosterone for optimum spermatogenic suppression while minimizing side effects.
Testosterone and its esters are widely used for androgen replacement therapy. In the prostate, testosterone ins 5 alpha-reduced to dihydrotestosterone (DHT), which leads to an amplification of its stimulatory activity in this and other tissues that have significant 5 alpha-reductase activity. While this amplification is essential during fetal development, it has potentially undesirable consequences during adult life. 7 alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that does not undergo 5 alpha reduction and is therefore being investigated for long-term clinical use because it is expected to be less stimulatory to the prostate. Since we anticipate using MENT acetate (MENT Ac) rather than MENT as the form of this androgen in humans, the bioavailability of MENT following the administration of MENT and MENT Ac was investigated in cynomolgus monkeys. Equimolar concentrations of MENT or MENT Ac were administered as a continuous subcutaneous infusion via Alzet osmotic pumps. Serum MENT levels were measured by radioimmunoassay (RIA) in blood samples collected daily for 4 days during steady state. The serum MENT levels were not significantly different in the two groups (11.3 +/- 1.6 vs. 13.1 +/- 1.2 nmol/L). This suggested that MENT Ac was rapidly converted to MENT in circulation. The hydrolysis of MENT Ac to MENT was confirmed by the in vitro incubation of MENT Ac with blood or plasma and the demonstration of MENT in products following separation by high-performance liquid chromatography (HPLC). Following the demonstration of the safety of MENT Ac in subchronic toxicity studies in rats and rabbits, a pharmacokinetic study was performed in men. In normal men, a single intravenous bolus of 500 micrograms of MENT led to peak serum MENT levels at 3 minutes after dosing (when the first samples were collected), followed by an exponential decline, reaching undetectable levels by 180 minutes. The average terminal half-life and the metabolic clearance rate (MCR) were calculated to be 40 minutes and 2,360 L/day, respectively. The results of the pharmacokinetic studies show that in both men and monkeys, the MCR of MENT is much faster than the values reported for testosterone. The faster MCR can be attributed, in part, to the finding that, in contrast to testosterone, MENT showed no binding to sex hormone binding globulin (SHBG).