RESUMO
CONTEXT: Phthalates are ubiquitous environmental chemicals. Fetal exposure to certain phthalates [e.g. di-n-butyl phthalate (DBP)] causes masculinization disorders in rats, raising concern for similar effects in humans. We investigated whether DBP exposure impairs steroidogenesis by the human fetal testis. OBJECTIVE: The aim of the study was to determine effects of DBP exposure on testosterone production by normally growing human fetal testis xenografts. DESIGN: Human fetal testes (14-20 wk gestation; n=12) were xenografted into castrate male nude mice that were treated for 4-21 d with vehicle, or 500 mg/kg·d DBP, or monobutyl phthalate (active metabolite of DBP); all mice were treated with human chorionic gonadotropin to mimic normal human pregnancy. Rat fetal testis xenografts were exposed for 4 d to DBP as a positive control. MAIN OUTCOME MEASURES: Testosterone production was assessed by measuring host serum testosterone and seminal vesicle (SV) weights at termination, plus testis gene expression (rats). RESULTS: Human fetal testis xenografts showed similar survival (â¼80%) and total graft weight (8.6 vs. 10.1 mg) in vehicle and DBP-exposed hosts, respectively. Serum testosterone (0.56 vs. 0.64 ng/ml; P>0.05) and SV weight (67.2 vs. 81.9 mg; P>0.05) also did not differ. Exposure to monobutyl phthalate gave similar results. In contrast, exposure of rat fetal xenografts to DBP significantly reduced SV weight and testis Cyp11a1/StAR mRNA expression and lowered testosterone levels, confirming that DBP exposure can inhibit steroidogenesis in xenografts, further validating the negative findings on testosterone production in the human. CONCLUSIONS: Exposure of human fetal testes to DBP is unlikely to impair testosterone production as it does in rats. This has important safety and regulatory implications.
Assuntos
Dibutilftalato/farmacologia , Testículo/efeitos dos fármacos , Testosterona/biossíntese , Animais , Feto , Humanos , Masculino , Camundongos , Camundongos Nus , Testículo/embriologia , Testículo/metabolismo , Transplante HeterólogoRESUMO
Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.
Assuntos
Esgotos/efeitos adversos , Espermatogênese/efeitos dos fármacos , Animais , Feminino , Humanos , Masculino , Saúde Reprodutiva , Síndrome de Células de Sertoli/epidemiologia , Carneiro Doméstico , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
BACKGROUND: The neonatal period of pituitary-testicular activity (NPTA) in human males has been hypothesized to play a role in germ cell proliferation and differentiation and to be defective in cryptorchid testes. The present study was carried out to establish in the marmoset if suppression of the NPTA, by treatment with a GnRH antagonist, results in impaired germ cell proliferation and/or differentiation. METHODS: Comparison of germ cell (GC) numbers and differentiation from gonocytes to pre-spermatogonia and spermatogonia, at birth (in controls) and at the end of the NPTA in marmoset co-twin males treated from birth to age 14 weeks with vehicle or GnRH antagonist. RESULTS: From birth to age 18-24 weeks, testis weight increased approximately 5-fold and GC number approximately 10-fold, including increased numbers of gonocytes and pre-spermatogonia and the first appearance of spermatogonia. Treatment with GnRH antagonist attenuated the increase in testis weight and GC numbers, but the effect was only partial (24-30% reduction), and the relative proportions of gonocytes, pre-spermatogonia and spermatogonia in the GnRH antagonist-treated group were unchanged from control values. CONCLUSIONS: The NPTA plays only a minor, if any, role in GC proliferation and differentiation in the marmoset. The changes in GnRH antagonist-treated co-twins may reflect impaired GC survival due to withdrawal of gonadotrophin support for Sertoli cells. These findings do not support a pivotal role for the NPTA in neonatal GC development in primates.
Assuntos
Animais Recém-Nascidos/fisiologia , Callithrix/fisiologia , Hipófise/fisiologia , Espermatozoides/citologia , Testículo/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Senescência Celular/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Masculino , Hipófise/efeitos dos fármacos , Espermatogônias/citologia , Espermatogônias/fisiologia , Espermatozoides/fisiologia , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , GêmeosRESUMO
BACKGROUND: Inexplicably, boys treated with some therapies for cancer at age 2-10 years, a time of supposed 'testicular quiescence', are at risk of low sperm counts/infertility in adulthood. Our aims were to use the marmoset as a surrogate for man to establish testicular cell function/activity during 'quiescence' between the neonatal period and puberty, and to test if any cell activity could be suppressed by prior treatment with a GnRH antagonist. METHODS AND RESULTS: Based on immunoexpression studies, functional development of Sertoli cells (SGP-2, androgen receptor) and Leydig cells (3 beta-hydroxysteroid dehydrogenase) was detectable at an age (35 weeks) when the testis is considered to be quiescent, and in advance of the pubertal rise in blood testosterone levels (50-60 weeks). Other changes at 35 weeks were the appearance of focal seminiferous tubule lumens and proliferating germ cells [indicated by immunoexpression of proliferating cell nuclear antigen (PCNA)]. Treatment from 25 to 35 weeks with GnRH antagonist largely (>85%) prevented these changes. However, the PCNA-labelling index of spermatogonia in GnRH antagonist-treated animals did not differ from controls (41.3 versus 43.6%) though total spermatogonia volume per testis was reduced by 41%. Some protein markers (inhibin-alpha, estrogen receptor-beta) showed little change with age or treatment. Beyond 35 weeks, GnRH antagonist-treated animals showed a delay in the pubertal rise in plasma testosterone levels. CONCLUSIONS: These findings reinforce the view that the 'childhood' testis is not quiescent. This may explain the damaging effects of some cancer therapies on subsequent fertility of boys and raises the issue of protective intervention. The present studies suggest that GnRH antagonist-based intervention might be only partially successful. Identification of the factors regulating spermatogonial development in the infant marmoset may aid in the design of such strategies.
Assuntos
Envelhecimento/fisiologia , Animais Recém-Nascidos/fisiologia , Gonadotropinas/fisiologia , Testículo/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Antineoplásicos/farmacologia , Callithrix , Senescência Celular/fisiologia , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Células Intersticiais do Testículo/fisiologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Células de Sertoli/fisiologia , Maturidade Sexual/fisiologia , Espermatozoides/fisiologia , Testículo/citologia , Testículo/efeitos dos fármacos , Testosterona/antagonistas & inibidores , Testosterona/sangueRESUMO
In this study we evaluated the effect of manipulating the estrogen and androgen environment of the neonatal male rat on subsequent immunoexpression of sex steroid receptors in the seminal vesicles (SVs) at age 18 days. The aim was to establish to what extent such changes were associated with and predictive of changes in SV structure/composition. Treatments were either diethylstilbestrol (DES; 10, 1, or 0.1 microg/injection), ethinyl estradiol (EE; 10 microg/injection), tamoxifen (2 mg/kg/day), flutamide (50 mg/kg), a gonadotropin-releasing hormone antagonist (GnRHa; 10 mg/kg), genistein (4 mg/kg/day), octylphenol (2 mg/injection), or bisphenol A (0.5 mg/injection). Compared with controls, treatment with DES (10 microg) induced loss of epithelial and stromal androgen receptor (AR) immunoexpression coincident with induction of stromal progesterone receptor (PR) immunoexpression and upregulation of stromal immunoexpression of estrogen receptor-alpha (ERalpha). These changes were associated with gross distortion (increase) of the normal stromal:epithelial tissue proportions in the SVs. DES (1 microg) and EE induced similar but less pronounced changes, and DES (0.1 microg) had no noticeable effect. Tamoxifen and flutamide induced PR and slightly upregulated ERalpha immunoexpression but had only a minor or no effect on AR expression and the stromal:epithelial ratio, though flutamide retarded normal development of the SVs. The latter was also evident in GnRHa-treated males, but otherwise this treatment had no effect on AR and PR immunoexpression. None of the foregoing treatments had any detectable effect on the immunoexpression of ERss in stromal or epithelial cells. The major treatment-induced changes in immunoexpression of AR, PR, and ERalpha and lack of change in ERss were confirmed by Western blots of SV protein extracts. None of the three weak (environmental) estrogens tested caused any detectable change in sex steroid receptor immunoexpression or SV tissue composition. We conclude that treatment-induced loss of AR is a prerequisite for altered stromal:epithelial proportions in the SVs and that such loss is always associated with induction of PR and upregulation of ERalpha; the latter two changes are insufficient on their own to bring about such a change. Nevertheless, induction of PR expression was always associated with altered SV development and is a potentially useful marker because it is not normally expressed in male reproductive tissues.
Assuntos
Poluentes Ambientais/efeitos adversos , Estrogênios/farmacologia , Regulação da Expressão Gênica , Receptores de Esteroides/biossíntese , Glândulas Seminais/ultraestrutura , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Masculino , Ratos , Ratos Wistar , Glândulas Seminais/efeitos dos fármacos , Regulação para CimaRESUMO
The aims of this study were: (i) to investigate the cellular immunoexpression of androgen receptor and oestrogen receptor beta in the testes of the common marmoset (Callithrix jacchus) during neonatal life compared with their expression at later ages; (ii) to establish whether neonatal marmoset Sertoli cells are targets for androgens or oestrogens or both; and (iii) to investigate the relationship between neonatal plasma testosterone concentrations and androgen receptor immunoexpression by abolishing the neonatal testosterone surge with a potent GnRH antagonist. Androgen receptor and oestrogen receptor beta immunoexpression were evaluated in neonatal animals aged 1-4 days, 4 weeks and 6 weeks, and compared with immunoexpression in animals aged 18-22 weeks (early infancy), 35 weeks (late infancy), 58-62 weeks (late pubertal) and > 100 weeks (adult). Immunoexpression of androgen receptor in the reproductive tract was also evaluated at each age. Sertoli cell immunoexpression of androgen receptor was weak or absent in neonatal animals, but increased substantially in infant animals, reaching adult levels by the end of infancy. In contrast, immunoexpression of androgen receptor during the neonatal period was strong in testicular interstitial cells and very strong in epithelial cell nuclei throughout the reproductive tract, and did not change greatly with age in these cells or tissues. Similarly, immunoexpression of oestrogen receptor beta was prominent in many Sertoli cells and in the germ cells of neonatal animals, and was relatively constant throughout life. Weak immunoexpression of androgen receptor in neonatal Sertoli cells was associated with high plasma testosterone concentrations (2.7-5.5 ng ml(-1)), whereas strong Sertoli cell immunoexpression was associated with baseline (approximately 0.12 ng ml(-1)) testosterone concentrations in infant animals and with > 10 ng ml(-1) in late pubertal and adult animals. Immunoexpression of androgen receptor and oestrogen receptor beta was also evaluated in co-twin males aged 4 and 35 weeks, after treatment from birth to 4 weeks or from week 25 to week 35, respectively, with either vehicle or with GnRH antagonist at a dose known to suppress the neonatal testosterone surge completely. Only GnRH antagonist treatment during weeks 25-35 reduced androgen receptor immunoexpression, whereas immunoexpression of oestrogen receptor beta was unaffected by treatment during either period. On the basis of these findings it is suggested that: (i) neonatal marmoset Sertoli cells may be targets primarily for oestrogens rather than androgens; (ii) androgen receptor expression in the testes of neonatal and infant marmosets is not regulated in a straightforward way by testosterone; and (iii) high neonatal concentrations of plasma testosterone are not absolutely necessary for expression of androgen receptor in marmoset testes at this time.
Assuntos
Animais Recém-Nascidos/metabolismo , Callithrix/metabolismo , Receptores Androgênicos/análise , Receptores de Estrogênio/análise , Testículo/química , Testosterona/sangue , Envelhecimento , Animais , Receptor beta de Estrogênio , Imuno-Histoquímica , Masculino , Células de Sertoli/químicaRESUMO
The effects on reproductive tract development in male rats, of neonatal exposure to potent (reference) oestrogens, diethylstilboestrol (DES) and ethinyl oestradiol (EE), with those of two environmental oestrogens, octylphenol and hisphenol A were systematically compared. Other treatments, such as administration of a gonadotrophin-releasing hormone antagonist (GnRHa) or the anti-oestrogen tamoxifen or the anti-androgen flutamide, were used to aid interpretation of the pathways involved. All treatments were administered in the neonatal period before onset of puberty. The cellular sites of expression of androgen receptors (AR) and of oestrogen receptor-alpha (ERalpha) and ERbeta were also established throughout development of the reproductive system. The main findings were as follows: (i) all cell types that express AR also express one or both ERs at all stages of development; (ii) Sertoli cell expression of ERbeta occurs considerably earlier in development than does expression of AR; (iii) most germ cells, including fetal gonocytes, express ERbeta but not AR; (iv) treatment with high, but not low, doses of potent oestrogens such as DES and EE, induces widespread structural and cellular abnormalities of the testis and reproductive tract before puberty; (v) the latter changes are associated with loss of immunoexpression of AR in all affected tissues and a reduction in Leydig cell volume per testis; (vi) none of the effects in (iv) and (v) can be duplicated by treating with high-dose octylphenol or bisphenol A; (vi) none of the reproductive tract changes in (iv) and (v) can be induced by simply suppressing androgen production (GnRHa treatment) or action (flutamide treatment); and (vii) the adverse changes induced by high-dose DES (iv and v) can be largely prevented by co-administration of testosterone. Thus, it is suggested that many of the adverse changes to the testis and reproductive tract induced by exposure to oestrogens result from a combination of high oestrogen and low androgen action. High oestrogen action or low androgen action on their own are unable to induce the same changes.
Assuntos
Anormalidades Induzidas por Medicamentos , Animais Recém-Nascidos/crescimento & desenvolvimento , Exposição Ambiental , Estrogênios/farmacologia , Genitália Masculina/anormalidades , Genitália Masculina/efeitos dos fármacos , Androgênios/fisiologia , Animais , Estrogênios/metabolismo , Masculino , RatosRESUMO
This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities. Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas. In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis. In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group). To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis). It is concluded that 1) reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume; 2) these changes are largely absent with a lower dose of DES (1 microg); 3) treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES; 4) a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.
Assuntos
Antagonistas de Androgênios/farmacologia , Androgênios/fisiologia , Animais Recém-Nascidos , Dietilestilbestrol/efeitos adversos , Genitália Masculina/efeitos dos fármacos , Animais , Western Blotting , Relação Dose-Resposta a Droga , Genitália Masculina/anormalidades , Imuno-Histoquímica , Masculino , Ratos , Ratos WistarRESUMO
This study in rats sought to 1) characterize immunoexpression of estrogen receptor alpha (ERalpha) and ERss in the efferent ducts, epididymis, and vas deferens during postnatal development; 2) establish whether ER expression changed after neonatal treatment with diethylstilbestrol (DES); and 3) determine whether ER changes coincided with abnormal epididymal/vas development. Rats were administered 10 microg DES or vehicle on days 2, 4, 6, 8, 10, and 12 and were sampled on days 10, 18, 25, 35, and 90+. At all ages, ERalpha was immunoexpressed intensely in the efferent ducts. On day 10, immunoexpression of ERalpha was absent from the epididymis and vas, but was detectable on day 18 in epithelial cells in the caput, corpus, and proximal cauda. Epithelial expression of ERalpha was absent from the distal cauda and in the proximal and distal vas was confined to a band of periductal stromal cells. Thus, on day 18, the site of ERalpha expression delineated the epididymis-vas boundary. On days 25-35, epithelial expression of ERalpha was absent, but stromal expression persisted in the vas and distal cauda. In adults, immunoexpression of ERalpha in the epididymis and vas was absent. In contrast, ERbeta was immunoexpressed in epithelial cells and some stromal cells in the efferent ducts, epididymis, and vas at all ages. In the vas, stromal expression of ERalpha and ERbeta was in different layers. DES treatment caused 1) underdevelopment of the epididymal duct and reduced epithelial height in epididymis and vas; 2) coiling of the extraepididymal vas; 3) thickening of the periductal actin-free stromal layer in the distal cauda and vas; and 4) reduced cell proliferation on day 18 in the epididymis and vas, based on incorporation of bromodeoxyuridine, especially in the epithelium. These changes coincided with abnormalities in cell- and region-specific immunoexpression of ERalpha, but not ERbeta. Thus, in DES-treated rats on day 18, epithelial expression of ERalpha occurred in all regions of the epididymis and vas instead of being confined to the caput, corpus, and proximal cauda as in controls. Similarly, stromal ERalpha expression in the vas of DES-treated rats was not confined to a periductal layer as in controls, but occurred diffusely in the muscle layer. It is suggested that 1) estrogens play a role in peripubertal development of the epididymis and vas; 2) the cellular site of expression of ERalpha either plays a role in or reflects demarcation of the epididymal/vas boundary; and 3) blurring of this boundary in DES-treated rats coincides with altered ERalpha immunoexpression.
Assuntos
Envelhecimento/metabolismo , Animais Recém-Nascidos/metabolismo , Dietilestilbestrol/farmacologia , Epididimo/metabolismo , Estrogênios não Esteroides/farmacologia , Receptores de Estrogênio/metabolismo , Ducto Deferente/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Divisão Celular/efeitos dos fármacos , Epididimo/efeitos dos fármacos , Epididimo/crescimento & desenvolvimento , Epididimo/patologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Distribuição Tecidual , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/crescimento & desenvolvimento , Ducto Deferente/patologiaRESUMO
This study investigated whether neonatal exposure of male rats to estrogenic compounds altered pubertal spermatogenesis (days 18 and 25) and whether the changes observed resulted in long-term changes in testis size, mating, or fertility (days 90-100). Rats were treated neonatally with a range of doses (0.01-10 microg) of diethylstilbestrol (DES; administered on alternate days from days 2-12), a high dose of octylphenol (OP; 2 mg administered daily from days 2-12) or bisphenol A (Bis-A; 0.5 mg administered daily from days 2-12), or vehicle, while maintained on a standard soy-containing diet. The effect on the same parameters of rearing control animals on a soy-free diet was also assessed as was the effect of administering such animals genistein (4 mg/kg/day daily from days 2-18). Testis weight, seminiferous tubule lumen formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to characterize pubertal spermatogenesis. Compared with (soy-fed) controls, DES administration caused dose-dependent retardation of pubertal spermatogenesis on day 18, as evidenced by decreases in testis weight, lumen formation, and spermatocyte nuclear volume per unit Sertoli cell and elevation of the germ cell apoptotic index. However, the two lowest doses of DES (0.1 and 0.01 microg) significantly increased spermatocyte nuclear volume per unit Sertoli cell. Similarly, treatment with either OP or Bis-A significantly advanced this and some of the other aspects of pubertal spermatogenesis. Maintenance of control animals on a soy-free diet also significantly advanced lumen formation and spermatocyte nuclear volume per unit Sertoli cell compared with controls fed a soy-containing diet. Administration of genistein reversed the stimulatory effects of a soy-free diet and significantly retarded most measures of pubertal spermatogenesis. In general, plasma FSH levels in the treatment groups changed in parallel to the spermatogenic changes (reduced when pubertal spermatogenesis retarded, increased when pubertal spermatogenesis advanced). By day 25, although the changes in FSH levels largely persisted, all of the stimulatory effects on spermatogenesis seen on day 18 in the various treatment groups were no longer evident. In adulthood, testis weight was decreased dose dependently in rats treated neonatally with DES, but only the lowest dose group (0.01 microg) showed evidence of mating (3 of 6) and normal fertility (3 litters). Animals treated neonatally with OP or Bis-A had normal or increased (Bis-A) testis weights and exhibited reasonably normal mating/fertility. Animals fed a soy-free diet had significantly larger testes than controls fed a soy-containing diet, and this difference was confirmed in a much larger study of more than 24 litters, which also showed a significant decrease in plasma FSH levels and a significant increase in body weight in the males kept on a soy-free diet. Neonatal treatment with genistein did not alter adult testis weight, and although most males exhibited normal mating and fertility, a minority did not mate or were infertile. It is concluded that 1) neonatal exposure of rats to low levels of estrogens can advance the first wave of spermatogenesis at puberty, although it is unclear whether this is due to direct effects of the estrogen or to associated elevation of FSH levels; 2) the effect of high doses of OP and Bis-A on these processes is essentially benign; and 3) the presence or absence of soy or genistein in the diet has significant short-term (pubertal spermatogenesis) and long-term (body weight, testis size, FSH levels, and possibly mating) effects on males.
Assuntos
Animais Recém-Nascidos/fisiologia , Estrogênios/farmacologia , Fertilidade/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/anatomia & histologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Apoptose/fisiologia , Dieta , Exposição Ambiental , Hormônio Foliculoestimulante/sangue , Inibinas/sangue , Masculino , Tamanho do Órgão , Ratos , Ratos Wistar , Túbulos Seminíferos/efeitos dos fármacos , Células de Sertoli/citologia , Glycine max , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/fisiologiaRESUMO
Oestrogen exposure of the male during fetal/neonatal life can fundamentally alter the structure and function of the reproductive system, though how is unknown. This study examined whether such treatment was able to induce a 'female' characteristic, namely immunoexpression of progesterone receptor (PR), in the reproductive system of the male. Rats were treated on postnatal days 2, 4, 6, 8, 10 and 12 with either 10, 1 or 0.1 microg diethystilbestrol (DES) or with the vehicle (20 microl corn oil). Groups of control and treated rats were killed on days 18, 25, 35 and 90 (= adults) and tissues fixed in Bouins for immunolocalisation studies using antisera to PR (recognises A and B forms) and oestrogen receptor-beta (ER beta). PR immunoexpression was absent from all tissues studied in control rats at all ages with the exception of the parasympathetic ganglia of the prostate. In rats treated with 10 microg DES, intense immunoexpression of PR was detected in the nuclei of stromal, but not epithelial, cells of the caput and cauda epididymis, the vas deferens, seminal vesicles and at the base of the dorsolateral prostatic complex (DLPC) at day 18, but was absent from the ventral prostate and from the testis. DES induction of PR immunoexpression was evident after a single injection (on day 3) and at 18-35 days the intensity of immunoexpression was DES dose-dependent; rats treated neonatally with 0.1 microg DES showed no detectable PR immunoexpression at any age. These findings were confirmed by Western analysis which indicated that most of the PR induced was probably the B form. Co-localisation studies, using confocal microscopy, demonstrated that PR and ER beta frequently co-localised to the same stromal cells in the DLPC, epididymis and seminal vesicles of DES-treated rats at day 18, whereas epithelial cells, which also expressed ER beta, did not express PR. In the tissues studied, only occasional stromal cells expressed ER alpha in comparison to the more widespread expression of ER beta, although epithelial cell expression of ER alpha was also detected in the epididymis on day 18 (but not on day 10). In DES-treated rats, immunoexpression of PR in the reproductive tract decreased progressively in intensity from days 18-35 and was non-detectable in adulthood. In conclusion, these findings are interpreted as evidence that neonatal oestrogen treatment exerts pervasive 'reprogramming' effects throughout the reproductive system of the developing male as indicated by the induction of PR immunoexpression. This induction was restricted to stromal tissue even though both stromal and epithelial cells at most sites expressed ER beta and/or ER alpha.
Assuntos
Dietilestilbestrol/administração & dosagem , Estrogênios não Esteroides/administração & dosagem , Genitália Masculina/fisiologia , Receptores de Progesterona/biossíntese , Envelhecimento/fisiologia , Animais , Imuno-Histoquímica , Masculino , Ratos , Células Estromais/fisiologiaRESUMO
The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1-14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18-22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18-22 but was comparable to control values in adulthood. However, seminiferous epithelium volume was reduced significantly in adult marmosets treated neonatally with GnRHa, and there was a tendency for reduced germ cell volume per Sertoli cell. In the same animals, there was significant expansion of the interstitium and an increase in Leydig cell volume per testis when compared with co-twin controls; a similar increase in Leydig cell volume was evident in adult rats treated neonatally with GnRHa. Comparison of Sertoli cell numbers in 6 infantile (18-24 wk) and 10 adult marmosets showed that adult numbers of Sertoli cells were present by the start of infancy but, unlike rats, marmosets were still able to replicate Sertoli cells beyond this period. However, marmoset Sertoli cells supported only approximately 20% of the germ cell volume supported by rat Sertoli cells, indicative of poor efficiency of spermatogenesis, as shown previously in the human. This finding, together with the demonstration of a temporal pattern of Sertoli cell replication similar to that in the human, supports the use of marmosets as a model for human male testicular development and function.
Assuntos
Animais Recém-Nascidos , Callithrix/crescimento & desenvolvimento , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Células de Sertoli/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Envelhecimento , Animais , Contagem de Células , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Ratos , Testículo/efeitos dos fármacos , Testosterona/sangue , GêmeosRESUMO
This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.
Assuntos
Animais Recém-Nascidos , Estrogênios/farmacologia , Hormônio Foliculoestimulante/sangue , Células de Sertoli , Espermatogênese/efeitos dos fármacos , Testosterona/sangue , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Dietilestilbestrol/farmacologia , Estrogênios/administração & dosagem , Etinilestradiol/farmacologia , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Inibinas/sangue , Masculino , Oligopeptídeos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Testículo/crescimento & desenvolvimentoRESUMO
Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.
Assuntos
Envelhecimento/sangue , Inibinas/sangue , Células de Sertoli/citologia , Animais , Animais Recém-Nascidos , Hormônio Foliculoestimulante/sangue , Hipotireoidismo/induzido quimicamente , Masculino , Propiltiouracila/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.
Assuntos
Dietilestilbestrol/farmacologia , Estrogênios/fisiologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/fisiologia , Envelhecimento , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Contagem de Células , Hormônio Foliculoestimulante/sangue , Masculino , Oligopeptídeos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Espermatogênese/efeitos dos fármacos , Testículo/crescimento & desenvolvimentoRESUMO
The aim of this study was to investigate the effect of the absence of elongate spermatids (ES) from the rat seminiferous epithelium on the quantitative secretion and synthesis of the three major Sertoli cell secretory proteins--SGP-1, SGP-2 and CP-2. Seminiferous tubules (ST) were isolated (a) from normal 28-day-old rats, in which the most mature germ cell type is the round spermatid, (b) from normal adult rats at stages IX-XIV of the spermatogenic cycle, i.e. after spermiation, or at stages I-V and VI-VIII, when ES are still attached to the Sertoli cell, and (c) at stages VI-VIII from normal adult rats and from rats treated with methoxyacetic acid (MAA) in order to specifically deplete ES at these stages. Two-dimensional SDS PAGE combined with computerized image analysis was used to analyse 35S-methionine-labelled intracellular and secreted proteins. In the case of SGP-1 and SGP-2, almost all of the protein synthesized by ST was secreted. The total amount of both SGP-1 and CP-2 secreted by unstaged ST from immature rats was significantly lower than that secreted by unstaged ST from adult rats. The total amount of SGP-1 and CP-2 secreted by adult ST at stages IX-XIV of the spermatogenic cycle also declined dramatically compared to ST at earlier stages. The proportion of the total CP-2 synthesized by ST which was secreted also declined in all situations in which ES were absent from the seminiferous epithelium. The synthesis of only SGP-2 was changed by ES depletion from ST at stages VI-VIII, which was almost doubled compared to synthesis of this protein by ST from control rats. Our results suggest strongly that the secretion of SGP-1 and SGP-2 is via the constitutive pathway, and that regulation of these two proteins by ES is at the level of protein synthesis. In contrast, the regulation of CP-2 by ES is predominantly at the level of secretion, suggesting that this protein is secreted via a regulated pathway. Our findings add to the evidence showing that ES play a major role in the regulation of Sertoli cell function.
Assuntos
Catepsinas/biossíntese , Endopeptidases , Glicoproteínas/biossíntese , Chaperonas Moleculares , Células de Sertoli/fisiologia , Espermátides/fisiologia , Acetatos/farmacologia , Animais , Catepsina L , Catepsinas/metabolismo , Clusterina , Cisteína Endopeptidases , Precursores Enzimáticos/biossíntese , Epitélio , Glicoproteínas/metabolismo , Masculino , Precursores de Proteínas/biossíntese , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Saposinas , Túbulos Seminíferos/fisiologia , Células de Sertoli/metabolismo , Espermátides/citologia , Espermátides/efeitos dos fármacos , EspermatogêneseRESUMO
The aim of the present study was to assess whether proteins secreted by the seminiferous tubules (ST) can be detected in testicular interstitial fluid (IF) and testicular (TV), spermatic (SV), and peripheral venous (PV) plasma from adult rats. An antiserum was raised against seminiferous tubule-conditioned medium (STCM) prepared from adult rats and used in conjunction with Western blot analysis to screen IF and blood samples resolved by one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of IF and PV were analyzed from control adult rats and rats exposed to scrotal heating (43 degrees C for 30 minutes) 24 hours earlier to ascertain whether damage to spermatogenesis would affect 'leakage' of proteins from the seminiferous tubules. In all control rats, the STCM antiserum specifically detected three proteins in testicular IF with molecular weights of 24, 16, and 14 kDa, respectively. Heat treatment increased the abundance of these proteins and induced the appearance of several other less-abundant proteins, all with molecular masses below 25 kDa. Two of the proteins present in IF were identified, the 24-kDa protein as phosphatidylethanolamine-binding protein (PEBP), and the 14-kDa protein as an androgen-regulated protein (ARP-2). Both of these proteins have been shown in previous studies to be secreted by round spermatids. Our results suggest that germ cell secretory products can gain access to the interstitium under both normal physiological conditions and more easily after induction of damage to spermatogenesis. The antiserum was unable to detect any ST-derived proteins in blood, although it is likely that this result may be due to insensitivity of the presently used techniques. The development of specific immunoassays for germ cell-secreted proteins (e.g., PEBP and ARP-2) should enable more definitive assessment of whether proteins secreted by the seminiferous epithellum can be measured in blood and thus provide a potential means of monitoring spermatogenesis.
Assuntos
Proteínas/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Proteínas Sanguíneas/análise , Western Blotting , Líquidos Corporais/química , Meios de Cultivo Condicionados/química , Temperatura Alta/efeitos adversos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Wistar , Túbulos Seminíferos/química , Testículo/lesõesRESUMO
The aim of this study was to investigate the effect of testosterone manipulation on the quantitative synthesis and secretion of a number of specific proteins produced by seminiferous tubules (ST) isolated at stages VI-VIII of the spermatogenic cycle from adult rats. The proteins selected were derived from different cellular sources. ST were isolated from control rats, from rats treated 4 days earlier with ethane dimethane sulfonate (EDS) to induce complete testosterone withdrawal by the destruction of the Leydig cells, and from EDS-treated rats injected with testosterone esters (TE) in order to maintain quantitatively normal spermatogenesis. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, combined with computerized image analysis, was used to analyze 35S-methionine-labeled intracellular and secreted proteins. Testosterone withdrawal did not affect to any significant degree the total synthesis of any of the proteins studied. Similarly, the secretion of the major known Sertoli cell proteins SGP-1 and SGP-2, together with a third putative Sertoli cell protein, all of which appeared to be secreted constitutively, was also not affected to any major degree by EDS treatment. In contrast, the secretion of another probable Sertoli cell protein, together with six proteins found to be secreted by germ cells and one protein that appeared to derive from more than one cellular source, was reduced dramatically by testosterone withdrawal, but was maintained by treatment with EDS+TE. All of the affected proteins appeared to be secreted in a regulated manner. Our results confirm that testosterone manipulation has little or no effect on either total protein synthesis by ST, or on the secretion of the major Sertoli cell secretory proteins, at stages VI-VIII of the spermatogenic cycle, but suggest strongly that testosterone regulation of ST protein secretion at these stages is mediated by an effect on the regulated secretory pathways. Our findings also demonstrate that the secretion, not only of Sertoli cell proteins, but also of those secreted by germ cells, is androgen-regulated.
Assuntos
Androgênios/fisiologia , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testosterona/fisiologia , Actinas/química , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Líquidos Corporais/metabolismo , Eletroforese em Gel de Poliacrilamida , Células Germinativas/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Rede do Testículo/metabolismo , Túbulos Seminíferos/citologia , Células de Sertoli/metabolismoRESUMO
The aims of this study were to provide an overall comparison of the in-vitro protein secretory profile of seminiferous tubules (ST) isolated from man and the rat, and to identify specific proteins secreted by both species. Two-dimensional SDS PAGE was used to compare the profile of proteins secreted by cultured ST from 15 men undergoing orchidectomy with those secreted by ST isolated at stages VI-VIII of the spermatogenic cycle from normal adult rats, and by ST at the same stages isolated from rats pretreated with methoxyacetic acid (MAA) in order to deplete both pachytene spermatocytes and round spermatids. Two abundant groups of proteins not present in the medium of rat ST were secreted consistently by human ST, though the profile of proteins secreted by human ST was otherwise more variable than in the rat. Twelve proteins secreted by ST isolated from humans were identified as possible homologues of rat ST-secreted proteins, including the major rat Sertoli cell products SGP-1 and SGP-2, and an androgen-regulated protein which derives in the rat from round spermatids. Otherwise, the majority of the 12 potential homologues corresponded to proteins which in the rat are secreted by ST from both normal and germ cell-depleted testes or by ST from germ cell-depleted testes only, suggesting that they are probably secreted by Sertoli cells and/or peritubular cells. Based on the potential homology of the proteins identified, our results suggest, first, that these proteins may play an important role in spermatogenesis and second, that the profile of proteins secreted by human ST is more akin to that secreted by ST isolated from germ cell-depleted rats.
Assuntos
Proteínas/metabolismo , Túbulos Seminíferos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
The aim of this study was to explore whether pituitary adenylate cyclase activating polypeptide (PACAP) could regulate protein synthesis by enriched preparations of spermatocytes and spermatids from the adult rat testis. Spermatocytes and spermatids were incubated for 8 h or 24 h in the absence (control) or presence of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP) or dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP). Total synthesis of intracellular and secreted proteins, during the incubation periods, was assessed and selected samples were analysed by 2-D SDS-PAGE. PACAP-38 (200 nM), VIP (200 nM) and db-cAMP (1 mM) significantly increased the synthesis of spermatocyte-secreted and intracellular proteins by 8 h and 24 h. Synthesis of both intracellular and secreted proteins by spermatids was significantly inhibited at 8 h and 24 h with PACAP, VIP and db-cAMP. The abundance of four germ cell-secreted proteins (GSP1, GSP2, GSP3 and phosphatidyethanolamine-binding protein (PEBP), which can be identified in both spermatocyte and spermatid culture medium, and beta-actin, which can only be identified in spermatid culture medium, was analysed. PACAP-38 and db-cAMP significantly increased the incorporation of label into GSP1, GSP2, GSP3 and PEBP, derived from spermatocyte culture medium, at 8 h and 24 h. In contrast PACAP-38 inhibited the incorporation of label into GSP1 and beta-actin, derived from spermatid culture medium, at 24 h. The results show that PACAP can regulate synthesis of both secreted and intracellular proteins by spermatids and spermatocytes in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)