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Short day lengths or reduced food availability are salient cues for small mammals that breed seasonally. Photoperiod-mediated gonadal regression in white-footed mice (Peromyscus leucopus) is a slow, orderly process that involves testicular apoptosis. Testicular regression in response to restricted caloric intake is relatively rapid, and it is generally reversed quickly by ad libitum (ad lib) feeding. To determine the contribution of apoptotic cell death during food restriction, and to examine possible interactions with photoperiod, mice housed in long (16L:8D) or short (8L:16D) photoperiods were fed either ad lib or 70% of their average ad lib intake. Testes were removed at 2, 4, 6, or 8 wk of experimental treatment. Apoptotic activity was determined by in situ TUNEL labeling and assessment of DNA laddering. Significant (P < 0.05) gonadal regression in response to short days was first detected at 8 weeks in mice fed ad lib. Food-restricted, long-day mice also showed significant testicular regression at 8 wk. Combined exposure to short day lengths and food restriction resulted in significant testicular regression at 6 wk (P < 0.05). TUNEL labeling was slightly, though significantly, elevated in germ cells at 2 and 4 wk in long-day food-restricted mice (P < 0.05). TUNEL labeling was also elevated in short-day food-restricted males at these early times but then increased nearly 5-fold at 6 and 8 wk in these mice (P < 0.001). DNA laddering confirmed elevated apoptosis. Overall apoptotic activity negatively correlated with paired testis mass, plasma testosterone, and spermatogenic index measurements in both ad lib and food-restricted males. Few histological markers of necrotic cell death were observed in any group. Taken together, these results suggest that testicular regression in response to limited caloric intake or short days is mediated by apoptosis.

Male aging is accompanied by reduced testosterone production by the Leydig cells, the testosterone-producing cells of the testis. The mechanism by which this occurs is unknown. Based on the observations that reactive oxygen is capable of damaging components of the steroidogenic pathway and that reactive oxygen is produced during steroidogenesis itself, we hypothesized that long-term suppression of steroidogenesis might inhibit or prevent age-related deficits in Leydig cell testosterone production. To test this, we administered contraceptive doses of testosterone to groups of young (3 months old) and middle-aged (13 months old) Brown Norway rats via Silastic implants to suppress endogenous Leydig cell testosterone production. After 8 months, the implants were removed, which rapidly (days) restores the ability of the previously suppressed Leydig cells to produce testosterone. Two months after removing the implants, when the rats of the two groups were 13 and 23 months of age, respectively, the Leydig cells in both cases were found to produce testosterone at the high levels of young Leydig cells, whereas significantly lower levels were produced by the 23-month-old controls. Thus, by placing the Leydig cells in a state of steroidogenic "hibernation," the reductions in Leydig cell testosterone production that invariably accompany aging did not occur. If hormonal contraception in the human functions the same way, the adverse consequences of reduced testosterone in later life (osteoporosis, reduced muscle mass, reduced libido, mood swings, etc. ) might be delayed or prevented.

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.

Many small, nontropical mammals stop breeding during winter. Chronic exposure of males to short days (<12.5 h light/day) causes the testes to atrophy and both steroidogenesis and gametogenesis to decrease. Male white-footed mice (Peromyscus leucopus) exposed to inhibitory short day lengths provide a natural animal model to study the cellular mechanisms regulating testicular regression. In the present study, the possible role of apoptosis was assessed during naturally occurring, short day-induced gonadal regression in white-footed mice by in situ terminal transferase-mediated end labeling (TUNEL), quantitative DNA 3'-end-labeling autoradiography (laddering) of DNA fragments, and quantification of Fas protein expression, an early initiator of apoptosis. Sexually mature male mice were exposed to short (8 h of light, 16 h of darkness) or long (16 h of light, 8 h of darkness) day lengths for 2, 4, 6, 8, or 10 weeks; gonads were then removed and processed for detection of apoptotic activity. In common with previous studies, the first significant reduction in relative testis mass was observed at week 10 of short day exposure. A 2- to 3-fold increase in apoptotic (TUNEL-positive) germ cells per seminiferous tubule was observed in the testes of mice exposed to short days for 4, 6, 8, or 10 weeks compared with the testes of long day animals. The extent of 3'-end labeling of low mol wt DNA increased with 4-8 weeks of short day exposure. Western blot analysis revealed an up-regulation of the Fas protein in the testes of short day males at 4, 8, and 10 weeks. Fas staining was primarily localized to spermatocytes and spermatids. Plasma testosterone concentrations decreased in short compared with long day animals after 6, 8, or 10 weeks. The increase in TUNEL positive-labeled germ cells, testicular DNA fragmentation, and up-regulation of the Fas protein before short day reductions of testis mass and function suggest that apoptosis is important for the mediation of photoperiod-induced testicular regression in white-footed mice.

We showed previously that exogenously administered testosterone caused age- and lobe-specific overgrowth of the prostate in Brown Norway rats. A common feature observed in testosterone-treated animals was cell hypertrophy in each of the ventral, dorsal, and lateral lobes of both young (6 mo old) and old (24 mo old) rats. By contrast, hyperplasia was seen only in the dorsal and lateral lobes of old rats treated with testosterone. These observations prompted us to examine whether age- and lobe-specific overgrowth might also occur in untreated rats as a consequence of the endogenous hormonal milieu. To this end, blood and prostates were collected from a large number (25-30 rats per group) of 4- to 6-mo-old (young) and 21- to 24-mo-old (old) Brown Norway rats. Both serum testosterone (-45%) and estradiol (-22%) concentrations decreased significantly with age, but the greater magnitude of the decrement in testosterone relative to estradiol led to a reduction in the serum testosterone:estradiol ratio. Paradoxically, although the prostate is androgen dependent, the wet weight, protein, and DNA contents increased significantly with age in the dorsal and lateral lobes of old rats despite the decrease in testosterone level. Histologic examination revealed that the increased weights and DNA contents of the dorsal and lateral lobes in old rats coincided with an increased number of epithelial cells in the distal and intermediate segments of these lobes, indicative of hyperplasia but independent of change in cell size. Taken together, these results show a spontaneous age-related overgrowth of cells in the dorsal and lateral prostatic lobes of old Brown Norway rats despite diminished serum testosterone concentrations. The aging Brown Norway rat, therefore, may be a useful model for studies of some aspects of the pathogenesis underlying spontaneous age-related prostatic hyperplasia.

Previous studies have shown that reductions in Leydig cell testosterone production occur with aging in the Brown Norway rat. The recent observation that changes in luteinizing hormone (LH) pulse interval and amplitude also occur with aging suggests the possibility that age-related reduced Leydig cell steroidogenesis might be related to changes in LH. We reasoned that, if this is the case, exogenously administered LH should restore testosterone production by aged rat Leydig cells to the higher levels produced by Leydig cells of young rats. To test this hypothesis, young (4-month-old) and aged (21-month-old) rats received testosterone- and estradiol-containing Silastic implants designed to suppress LH and, thus, endogenous Leydig cell testosterone production. At the same time, the rats received miniosmotic pumps programmed to deliver pulsatile ovine LH at a predetermined daily dose. In some experiments, treatment effects were determined by measuring testosterone production by testes perfused in vitro with maximally stimulating ovine LH. In others, Leydig cells were isolated by centrifugal elutriation and Percoll density gradient centrifugation, and their in vitro ability to produce testosterone in response to maximally stimulating LH was determined. Testes or isolated Leydig cells from untreated young rats produced about twice as much testosterone as that produced by Leydig cells from aged rats. The administration of testosterone- and estradiol-filled implants for 5 days reduced testosterone production significantly at both ages. In young rats administered 24 microg LH/day for 5 days, along with the implants, testosterone production was maintained at the high level of the young controls. Comparable treatment of aged rats resulted in testosterone production only at the low level of the aged controls. Indeed, even with higher LH doses (36 microg/day), testosterone production by the aged rat Leydig cells did not rise above the aged-control level. The inability of exogenously administered LH to increase testosterone production by testes and Leydig cells of aged rats suggests that Leydig cell steroidogenic deficits in the aged Brown Norway rat are unlikely to be the result of age-related changes in LH.

It has been clear for decades that testosterone is a necessary prerequisite for the maintenance of established spermatogenesis in the adult testes, and for the restoration of spermatogenesis in testes induced experimentally to become azoospermic. In contrast, the possible involvement of FSH in regulating spermatogenesis in the adult mammal, though also studied extensively, continues to be debated in the literature. The mechanisms by which testosterone and FSH regulate spermatogenesis remain uncertain as well. This review focuses on our current understanding of the involvement of testosterone and of FSH in the maintenance and restoration of spermatogenesis in the adult testes, and discusses the emerging concept that testosterone and FSH serve as cell survival factors., (Copyright 1998 Academic Press.)

We examined the temporal relationship between protein synthesis and testosterone production by rat Leydig cells in primary culture. Leydig cells were isolated from adult control Sprague-Dawley rats and from rats that had received LH-suppressive testosterone and estradiol (TE) implants in vivo for 10 days. The cells were incubated for 1-4 h with [35S]methionine in the presence or absence of maximally stimulating ovine LH, and newly synthesized proteins were examined by two-dimensional PAGE autoradiography. Approximately 800-900 newly synthesized polypeptides were readily visible on all autoradiograms, most of which did not differ in the cells from intact control and TE-treated rats. Incubation of cells from the control and treated rats with maximally stimulating LH for 4 h in both cases resulted in significant increases in testosterone production and in three newly synthesized polypeptides. These polypeptides, along with two others that changed little in response to LH, were similar in apparent molecular mass, 30 kDa, but differed in isoelectric point. Time-course studies revealed a temporal relationship between stimulation of the three 30-kDa proteins and of testosterone production. Western blot analysis identified the 30-kDa proteins as steroidogenic acute regulatory protein (StAR). The results of these studies, for the first time utilizing primary cultures of highly purified, testosterone-producing Leydig cells, provide further correlative evidence of a role for StAR protein in the acute regulation of Leydig cell testosterone biosynthesis by LH.

The prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-alpha (TGFalpha), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFalpha in the rat ventral prostate during postnatal development to adulthood. TGFalpha-immunopositive cells were located throughout the ductal epithelium from postnatal days 5-20. By day 45 and thereafter, regional variation in TGFalpha expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFalpha in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFalpha staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFalpha in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFalpha expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFalpha in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFalpha is not regulated by androgen in this region of the ventral prostate. In summary, TGFalpha expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFalpha may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.

Telomerase activity is essential for protection of cells against the telomere erosion that occurs with each round of cell replication, and thus appears to play a role in the indefinite replication potential of some, but not all, eukaryotic cells. In this regard, some tissues contain stem cells that have a long proliferative life-span and are capable of regenerating or renewing the somatic epithelial cell population within the tissue. Because the adult seminal vesicle exhibits the ability to regenerate during androgen-replacement after castration, we hypothesized that a pool of cells with regenerating potential is present in the adult seminal vesicle, which expresses telomerase activity. In this study, we used a highly sensitive PCR-based telomerase assay [the telomeric repeat amplification protocol (TRAP) assay] to detect telomerase activity in rat seminal vesicle. Our results show that telomerase activity is, indeed, present in the normal adult rat seminal vesicle, but that, in the presence of seminal vesicle fluid, telomerase activity cannot be detected. In fact, seminal vesicle fluid was found to contain some factor(s) that is inhibitory for the TRAP assay. In addition, we found that telomerase activity in the seminal vesicle changes with age and is regionally distributed within the distal, intermediate, and proximal segments of the duct. These results suggest that as is the case for the rat prostate, a population of telomerase-positive cells is present within the adult rat seminal vesicle, and thereby, this organ retains throughout life the potential to regenerate in response to androgen replacement following castration-induced apoptotic cell death.

It is generally established that telomerase activity is present in most cancer cells, germ line cells, and established cell lines but typically is not present in normal somatic cells. Among the few exceptions, telomerase activity has been detected in hematopoietic stem cells and in physiologically renewable epithelial cell populations (e.g. skin, small intestine). The adult rat prostate gland is a tissue that has the ability to regenerate in response to androgen. We hypothesized, therefore, that telomerase activity should be present in the normal adult rat prostate. In this study, using a highly sensitive PCR-based telomerase assay [the telomerase repeat amplification protocol (TRAP) assay], we tested this hypothesis by measuring telomerase activity in the ventral, dorsal, lateral, and anterior lobes of the adult rat prostate. We show herein that telomerase activity indeed is present in whole tissue extracts of the ventral, dorsal, and anterior lobes, though at differing levels, but not the lateral lobe. However, lateral lobe prostatic fluid was found to contain one or more factors inhibitory for the TRAP reaction, and therefore, telomerase activity was detected after removal of this fluid. Telomerase activities were not uniform within the prostatic ducts, but rather telomerase positive cells are regionally distributed within the distal, intermediate, and proximal ducts of the microdissected segments of each of the lobes. We speculate from these results that androgen-stimulated regeneration of rat prostate following androgen ablation may depend upon the telomerase-positive regenerating pool of cells present within specific regions of the ductal network.

The effect of testicular germ cell content on Leydig cell steroidogenic function in vivo in adult rats was examined. Three experimental paradigms were used to effect germ cell changes. First, a vitamin A-depletion/repletion regimen was used to achieve synchrony at different stages of the cycle of the seminiferous epithelium and thus produce testes with widely differing germ cell contents. Second, long term vitamin A depletion was used to effect germ cell, but not Leydig cell, loss. Third, Leydig cells and germ cells first were eliminated from the testes of adult rats by the administration of ethane 1,2-dimethane sulfonate (EDS) along with testosterone- and estradiol-filled Silastic capsules; Leydig cells were then restored to the germ cell-depleted testes by removal of the luteiniging hormone (LH)-suppressing capsules. Serum, interstitial fluid, and seminiferous tubule fluid testosterone concentrations did not differ between rats in which at least 70% of the seminiferous tubules contained germ cells at stages VII-VIII or at stages XII-III of the cycle. The capacity of the testes of these rats to produce testosterone, assessed by their in vitro perfusion with maximally stimulating LH, also showed no differences despite the differences in germ cell content. Elimination of germ cells throughout the testes by long term vitamin A depletion also did not affect the steroidogenic function of the testes. Finally, the steroidogenic function of Leydig cells restored to germ cell-depleted testes was indistinguishable from that of germ cell-containing controls. These results, taken together, provide evidence supporting the contention that the germ cell content of the testis has little or no effect on testicular steroidogenic function.

Previous studies have shown that the ability of Brown Norway rat Leydig cells to produce testosterone declines significantly with age. To address the possible mechanism(s) by which aging Leydig cells lose steroidogenic function, we determined the effect of age on the steady-state levels of the mRNAs for the steroidogenic enzymes P450 cholesterol side-chain cleavage (P450scc), delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), and 17 alpha-hydroxylase/C17-20 lyase (P450(17) alpha), and on the levels of immunoreactive steroidogenic enzyme proteins and enzyme activities. Northern blot analysis revealed that the levels of P450scc and P450(17) alpha mRNAs in Leydig cells isolated from the testes of aged (22-month-old) Brown Norway rats were reduced from their levels in young (4-month-old) rats, but that 3 beta-HSD mRNA was not reduced. Western blot analysis, however, revealed that cellular levels of each of the P450scc, P450(17) alpha, and 3 beta-HSD proteins were reduced with aging. The activities of the steroidogenic enzymes, assessed by incubating Leydig cells in culture with substrate and then summing all steroidogenic reaction products through testosterone, similarly revealed that P450scc, 3 beta-HSD, P450(17) alpha, and additionally 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), were all reduced with aging. We conclude that age-related loss of steroidogenic function results at least in part from reductions in the levels and activities of each of the steroidogenic enzymes responsible for converting cholesterol to testosterone, and not by differential regulation of these enzymes.

Using the in vitro perfused rat testis, the effects of testosterone (T) on its own biosynthesis, and in particular on the inhibition of specific steroidogenic step(s) in the biosynthetic pathway from cholesterol to T, were examined. Rat testes perfused in vitro for 1 hour with medium containing 1.5 microM T secreted significantly less T than control testes in response to physiologic or maximal luteinizing hormone (LH) stimulation. To locate the site(s) of this rapid inhibition, the effects of arterial T infusion on steroidogenesis by testes also infused with pregnenolone (PREG), progesterone (PROG), 17 alpha-hydroxyprogesterone (17-PROG), or androstenedione (ADIONE) were measured by summing all the possible reaction products from each substrate. This approach allowed us to examine the effect of T in situ on the reactions: LH-stimulated PREG secretion; PREG to PROG; PROG to 17-PROG; 17-PROG to ADIONE; and ADIONE to T. Only PROG to 17-PROG (17 alpha-hydroxylase activity) was inhibited by arterial T infusion. A kinetic examination of the PROG to 17-PROG reaction demonstrated that the specific inhibition by T was competitive. The apparent km for PROG in this system was 16.0 microM, whereas the apparent ki of T was 1.6 microM, indicating a relatively high degree of sensitivity of the reaction to T. Taken together, these data confirm that T is able to regulate its own synthesis and indicate that this autoregulation is the result of rapid, specific inhibition by T of 17 alpha-hydroxylase activity.

To facilitate investigations of the regulation of adult Sertoli cell function, we have established a Sertoli cell line from sexually mature Sprague-Dawley rats. The cells were immortalized with the temperature-sensitive mutant of the SV40 virus, tsA255. The tsA255 large T antigen is heat-labile and efficiently promotes propagation of cells at 33 degrees C (permissive temperature) but is inactive at 40 degrees C (nonpermissive temperature). The established clonal Sertoli cell line (ASC-17D) proliferates indefinitely at the permissive temperature. However, within 48 h at the nonpermissive temperature, cell proliferation ceases. ASC-17D cells show positive staining with antibodies to cytokeratin and vimentin, consistent with the Sertoli cell origin of these cells. Transferrin and sulfated glycoprotein (SGP)-2 mRNAs were nearly undetectable in ASC-17D cells cultured at the permissive temperature, but expression of both mRNAs was induced at the nonpermissive temperature. In contrast, SGP-1 was expressed equally at both the permissive and nonpermissive temperatures. There was no increase in either transferrin or SGP-2 with FSH or dibutyryl cAMP (db-cAMP) treatment at the permissive temperature or with FSH treatment at the nonpermissive temperature. However, the steady-state levels of both of these mRNAs were substantially increased in the presence of db-cAMP at the nonpermissive temperature. In contrast, SGP-1 mRNA was not affected by either FSH or db-cAMP. These results suggest that the ASC-17D cell line is derived from adult Sertoli cells and may be useful for the study of adult Sertoli cell function.

Previous studies have demonstrated that Leydig cell testosterone production diminishes with age in Brown Norway rats. The objective of the studies presented herein was to test the following possible explanations for age-related decline in steroidogenesis: (1) decline in Leydig cell number; (2) understimulation by luteinizing hormone (LH); (3) reduced ability of individual Leydig cells to produce testosterone; and (4) influence of loss of germ cells. Leydig cells isolated from the testes of young and aged rats by centrifugal elutriation and Percoll density gradient centrifugation were examined for their ability to produce testosterone when stimulated maximally with LH or with dibutyryl cyclic AMP (dbcAMP). Leydig cell number and volume were examined in situ using stereological methods. Serum LH levels were measured using a highly sensitive immunofluorometric assay. Average Leydig cell volume decreased with age, and consistent with this observation, individual Leydig cells isolated from aging rats produced significantly less testosterone than those from young rats whether the cells were cultured in vitro with maximally stimulating LH or with dbcAMP. The age-associated diminished testosterone production could not be explained by changes in Leydig cell number, serum LH levels, Leydig cell responsiveness to LH, or testicular germ cell content. These results, taken together, suggest that the reduced testosterone production seen in aged rats is related to defects in the steroidogenic pathway beyond the LH receptor-cAMP cascade. The nature of the initial age-related changes that cause reduced steroidogenesis is not known, and therefore it is not known whether such changes are intrinsic or extrinsic to the Leydig cells.

We examined the effects of age and of increasing concentrations of testosterone on the wet weight, protein content, cell number, and cell size of the ventral, dorsal, and lateral lobes of the Brown Norway rat prostate. Young (3 mo of age) and aged (15, 17, and 21 mo of age) rats received implants of increasing sizes of testosterone-filled Silastic capsules for 3 mo. Wet weights of the prostate were the same in untreated young (6-mo-old) and aged (18-24-mo-old) rats. Testosterone administration resulted in serum testosterone concentrations ranging from physiologic to superphysiologic. Dose-dependent increases in wet weights and protein contents were seen in the ventral, dorsal, and lateral prostatic lobes of both young and aged rats. For each given dose of testosterone, including doses that resulted in serum testosterone concentrations within the physiologic range, the weights and protein contents of the dorsal and lateral lobes were greater in old (24-mo-old) than in young (6-mo-old) rats, indicating an effect of age in these lobes. In contrast, ventral prostate weights and protein contents increased equivalently in young and aged rats with increasing testosterone concentration. DNA content, a measure of cell number, increased significantly in the dorsal and lateral lobes as a function of testosterone dose and age, but in the ventral lobe did not differ with testosterone dose or age. Quantitative morphologic analyses showed significant hypertrophy of epithelial cells throughout each of the three lobes in both young and aged rats treated with testosterone. Taken together, these results indicate that the prostate of Brown Norway rats shows age and lobe-specific responses to androgen with respect to wet weight, protein content, cell number, and cell morphology.

The efficacy and toxicity of a new method for chronic direct intratesticular drug infusion were assessed in a rat model. To this end, luteinizing hormone (LH) or buffer was infused via miniosmotic pumps for 14 days directly into the parenchyma of Copenhagen rat testes. The surgical manipulation and direct infusion of buffer did not have any apparent adverse effect upon either spermatogenesis or steroidogenesis as measured by testis weight, homogenization-resistant spermatid count, and in vitro response of the testes to a maximally stimulating concentration of LH. Histologic studies revealed only a localized inflammatory response in the testis around the Silastic tubing leading from the mini-osmotic pump to the testis. The biologic efficacy of direct infusion into the testis was assessed by determining the ability of mini-osmotic pump-infused LH to maintain steroidogenesis for 14 days in animals whose pituitary function was suppressed by simultaneous subcutaneous placement of testosterone/estradiol capsules. Steroidogenesis was found to be maintained quantitatively in testes infused with an appropriate dose of LH. At a given LH dose, the directly infused testes were found to produce fivefold more testosterone than contralateral testes and 10-fold more testosterone than testes from rats receiving systemic administration of the same dose of LH. We conclude that the miniosmotic pump system is a useful means to chronically administer a high concentration of LH, and presumably other agents, to the testis without any significant adverse effects.

It is well established that testosterone (T) is required to maintain spermatogenesis in the mammalian testis, presumably acting through a receptor mechanism, as elsewhere. It is puzzling, however, that the T concentration that is required to maintain spermatogenesis quantitatively in intact adult rats (15-20 ng/ml) is well in excess of the concentration required to saturate testicular androgen receptors. If, indeed, T regulation of spermatogenesis is mediated through the androgen receptor, 5 alpha-dihydrotestosterone (DHT) would be expected to more effectively maintain spermatogenesis than T because of its greater affinity for, and slower rate of dissociation from, the androgen receptor. To test this hypothesis experimentally, the quantitative relationship between the intratesticular concentration of exogenously administered DHT and the maintenance of germ cell production was examined in intact rats in which endogenous T production was suppressed. To this end, rats received increasing doses of DHT via polydimethylsiloxane implants of 6-36 cm in length for 8 weeks. In each DHT-treated rat, serum luteinizing hormone (LH) concentration was reduced to below the limit of detectability by radioimmunoassay (RIA), and intratesticular T concentration was reduced by > 90%. Serum and intratesticular DHT concentrations increased with increasing implant size. The serum and intratesticular concentrations of 5 alpha-androstane-3 alpha, 17 beta-diol(3 alpha-Diol) were consistently higher than DHT concentrations because of conversion of DHT to 3 alpha-Diol. Spermatogenesis was maintained qualitatively in some rats that received implants of 6 cm, in most rats that received implants of 12 cm, and in all rats that received implants of 18 cm.(ABSTRACT TRUNCATED AT 250 WORDS)

We have shown previously that cell proliferation occurs throughout the ducts of the rat ventral prostate during its postnatal development. Our objectives herein were to identify and quantify proliferating cells in the dorsal and lateral lobes, to quantify DNA synthetic activity in the distal and proximal segments of these lobes, and to compare the results obtained to the results of our previous studies of the ventral lobe. [3H]-Thymidine, administered in vivo, was incorporated into both the distal and proximal segments of day 10-60 rat dorsal and lateral prostates. Quantitative analyses of autoradiographs revealed that the percentages of labeled cells in the distal and proximal segments of the dorsal and lateral lobes were not significantly different at days 45 and 60, and they were only slightly (though significantly) different at days 10 and 20. Biochemical analyses of [3H]-thymidine incorporation similarly revealed no significant differences in the distal and proximal segments at days 45 and 60, and only small differences at days 10 and 20. The results of analyses of mitotic figures were consistent with the results of autoradiographic analyses, showing clearly that cell proliferation occurs throughout the rat dorsal and lateral prostates. These results show for the first time that cell proliferation occurs throughout the dorsal and lateral lobes, strikingly reminiscent of the proliferation pattern that we reported previously for the ventral lobe.

We have previously demonstrated that ethane-1,2-dimethane-sulfonate (EDS) kills adult, but not immature, rat Leydig cells in vivo and in vitro. The mechanism responsible for this selective toxicity is not known. Here we report that the cytotoxic effects of EDS on adult rat Leydig cells were not dependent upon new protein synthesis or cytochrome P450 enzyme activity. To determine whether inhibition of glutathione synthesis protects Leydig cells from the cytotoxic effects of EDS, adult rat Leydig cells were cultured in the presence or absence of 4 mM buthionine sulfoximine (BSO; 2 hr), a specific inhibitor of glutathione synthesis, and subsequently with increasing doses of EDS (3 hr). Following EDS addition, the ability of the cells to produce testosterone in response to LH stimulation, and to synthesize protein ([35S]methionine incorporation) were evaluated. In both cases, Leydig cells cultured in the presence of BSO were far less sensitive than Leydig cells cultured in medium alone (control) to EDS effects on testosterone production (control: EC50 = 60 micrograms EDS/ml; BSO: EC50 > 1500 micrograms EDS/ml) and [35S]methionine incorporation (control: EC50 = 95 micrograms EDS/ml; BSO: EC50 = 1560 micrograms EDS/ml). This protective effect of BSO was abolished by restoring intracellular glutathione levels with glutathione ethyl ester (8 mM; GSHEE). Interestingly, none of these treatments altered the viability (i.e., [35S]methionine incorporation) of immature rat Leydig cells (EC50 = 420 micrograms EDS/ml for control, GSHEE, and BSO groups). Taken together, these data suggest that the mechanism by which EDS kills adult rat Leydig cells may involve Leydig cell glutathione.

Aging of the mammalian testis is often accompanied by loss of germ cells and, as a result, decreased daily sperm production. It is currently unknown whether cell loss is the result of aging-related changes in germ cells or whether there are also aging-related changes in the Sertoli cells that normally support germ development and differentiation. To begin to compare the effects of age on germ cells and on Sertoli cells, we examined brown Norway rats of 6, 12, 18, 21, and 24 months of age for the frequency of seminiferous tubule regression and total testis contents of transcripts for three Sertoli cell products: SGP-2, transferrin, and cyclic protein-2 (CP-2)/cathepsin L. Histological analysis revealed no changes in the seminiferous epithelium from 6 to 12 months of age. However, from 12 to 24 months of age, the percentage of normal tubules gradually decreased from 95% to 15% of the total while the percentage of fully regressed tubules (containing no germ cells per tubule cross section) increased from 0% to 78%. In our analysis of specific Sertoli cell transcripts, we noted no change in total testis content of SGP-2 mRNA from 6 to 24 months. However, total testis content of transferrin mRNA was unchanged from 6 to 18 months, but increased by 24 months to 368% of the content of a 6-month-old testis. In contrast, total testis content of CP-2/cathepsin L mRNA was unchanged from 6 to 12 months, but decreased by 24 months to 58% of the content of a 6-month-old testis.(ABSTRACT TRUNCATED AT 250 WORDS)

In seeking an animal model of age-associated changes in the male reproductive tract, we examined the effects of age on the health and testicular steroidogenic activity of the Brown Norway rat, with comparisons made to the Sprague-Dawley rat. When perfused in vitro under conditions of maximally stimulating luteinizing hormone significant age-associated reductions were seen in testosterone production by testes of Sprague-Dawley rats of 21-24 months of age and by testes of Brown Norway rats of 18-30 months of age. Decreases in the capacity of the testes to produce testosterone were reflected in age-associated decreases in both serum testosterone and in testosterone concentration within the seminiferous tubule fluid. In contrast to the Sprague-Dawley rat, changes in steroidogenic activity in the Brown Norway rat were not accompanied by the occurrence of pituitary adenomas, obesity, or testicular tumors. This along with its longevity, make the Brown Norway strain a highly promising model for testicular aging.

We previously used autoradiographic methods to show that cells proliferate throughout the rat ventral prostate during its postnatal development (Biol Reprod 1991; 45:773-782). The objective of the present study was to quantify the extent to which DNA synthesis, and thus growth, occur in the distal and proximal segments of the rat ventral prostate. To this end, rats were administered 3H-thymidine in vivo. Subsequently, 3H-thymidine incorporation per prostatic segment and per micrograms DNA was determined, as was the number of labeled cells in the proximal and distal segments. We report that 3H-thymidine was incorporated into both the distal and proximal segments of the ventral prostate of rats 10, 20, 45, and 60 days of age, with significantly greater amounts (6-21 times) incorporated into the distal segment at each age. Consistent with this, the distal segment contained 5-20 times more labeled cells than the proximal segment. However, when 3H-thymidine incorporation was expressed per micrograms DNA, only relatively small, though significant, differences were seen between the distal and proximal segments of Days 10 and 20; and no differences were seen at Days 45 and 60. Taken together, these results strongly support the contention that DNA synthesis occurs throughout the rat ventral prostate, and suggests therefore that prostatic growth is not limited to the distal tips of the gland.

Testes from flight rats on COSMOS 2044 and simulated-launch, vivarium, or caudal-elevation control rats (5/group) were analyzed by subjective and quantitative methods. On the basis of observations of fixed tissue, it was evident that some rats had testicular abnormalities unassociated with treatment and probably existing when they were assigned randomly to the four treatment groups. Considering rats without preexisting abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (P less than 0.05) in flight than in simulated-launch or vivarium rats. However, ratios of germ cells to each other or to Sertoli cells and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. Expression of testis-specific gene products was not greatly altered by flight. Furthermore, there was no evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rat luteinizing hormone in testicular tissue and surface density of smooth endoplasmic reticulum in Leydig cells were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20% of values for simulated-launch or vivarium controls. Thus spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Exposure to microgravity for greater than 2 wk might result in additional changes. Sequelae of reduced androgen production associated with microgravity on turnover of muscle and bone should be considered.

We have examined the effects of decreasing intratesticular testosterone concentration and of decreasing germ cell number on levels of transferrin mRNA and sulfated glycoprotein (SGP)-2 mRNA in the adult rat testis. Intact rats received implants of testosterone- and estradiol-filled capsules to suppress LH secretion from the pituitary, thereby suppressing Leydig cell testosterone production. The levels of intratesticular testosterone declined 70% to 20 ng/ml within 3 days, were reduced further to approximately 15 ng/ml by 14 days, and subsequently reached a minimum of about 10 ng/ml. In contrast, the number of elongated spermatids per testis remained unchanged through 14 days, then declined to fewer than 20% of normal between 14 and 28 days, and reached zero by 56 days postimplantation. Likewise, both pachytene spermatocytes and round spermatids declined only after 14 days postimplantation. Northern blots of testicular RNA showed that Sertoli cell transferrin mRNA per testis decreased markedly between 14 and 28 days postimplantation. However, SGP-2 mRNA per testis was unchanged over the time course of the experiment. The decrease in transferrin mRNA, concomitant with germ cell loss, suggests that this mRNA is regulated by the number of germ cells in the testis and not directly by testosterone. In contrast, the constant level of SGP-2 mRNA in the face of reduced intratesticular testosterone and the subsequent loss of germ cells suggests that this mRNA is constitutively maintained in the adult rat testis.

The purpose of this study was to determine whether the levels of sulfated glycoprotein-2 (SGP-2) and/or transferrin mRNA in Sertoli cells responds to testosterone in the adult rat testis and, if so, to distinguish between the effects of testosterone and those of changing populations of germ cells. To this end we have examined changes in the steady state levels of these two mRNAs in relationship to changes in intratesticular testosterone concentration and germ cell numbers after treatment of adult hypophysectomized rats with testosterone. Hypophysectomy for 4 weeks caused intratesticular testosterone concentrations to become reduced from 50 to 3 ng/ml and caused significant reductions in the numbers of testicular spermatozoa (undetectable), round spermatids (nearly undetectable), and pachytene spermatocytes (12% of normal). Intratesticular testosterone concentrations rose to 30 ng/ml within 3 days after implantation of testosterone-containing polydimethylsiloxane capsules into the hypophysectomized rats. Three days after the initiation of testosterone treatment, increases were seen in the numbers of round spermatids (10% of normal) and pachytene spermatocytes (29% of normal). The major increases in the numbers of these cells and of spermatozoa occurred between days 14-56 of testosterone treatment, with pachytene spermatocytes reaching a maximum of 75% of the control level by day 28, and round spermatids and spermatozoa reaching 75% and 21% of the control levels, respectively, by 56 days. The steady state levels of SGP-2 mRNA were not affected by hypophysectomy, testosterone administration, or subsequent increases in germ cell numbers. In contrast, transferrin mRNA levels were reduced by hypophysectomy. As found for SGP-2, transferrin mRNA levels were unresponsive to increased testosterone concentration, but, unlike SGP-2 mRNA, transferrin mRNA increased as germ cells were restored after testosterone administration. These results suggest that SGP-2 mRNA is not regulated by testosterone or germ cells, but that the level of transferrin mRNA is influenced by the interaction of Sertoli and germ cells in the adult rat testis.

Previous studies have provided evidence that the rat ventral prostate grows primarily, if not exclusively, at its distal tips. However, as yet there have been no analyses in which individual cells in defined regions of the prostatic ductal system have been resolved and quantified. Moreover, the possibility that the prostate might grow differently at different times of postnatal development has received little attention. Our objectives were to identify and quantify the proliferating epithelial and stromal cells in defined regions of the rat ventral prostate during its postnatal development. To this end, 3H-thymidine was administered in vivo to rats of ages 10-60 days. A dissection technique was then used by which the distal, intermediate, and proximal segments of the prostatic ductal system were physically isolated from each other without removing the stromal tissue. Longitudinal sections of these segments were examined for cellular composition and DNA synthetic activity. Regional heterogeneity with respect to cell composition and cell proliferation was seen. In rats of all ages, DNA synthetic activity was seen in epithelial and stromal cells throughout the prostate, rather than only in the distal segment. At Days 10 and 20, significantly higher percentages of epithelial and stromal cells were labeled in the distal than in the proximal segments; but at Days 45 and 60, the percentages of labeled epithelial and stromal cells in the distal, intermediate, and proximal segments were similar. Thus, in all segments, and at all ages, substantial labeling was seen throughout the prostate. These data suggest that the prostate grows in both length and width throughout postnatal development, reminiscent of the growth of a tree.

Leydig cells from immature rat testes appear to be insensitive to doses of ethane-1,2-dimethanesulfonate (EDS) which eliminate Leydig cells from adult rat testes. We sought to determine whether this differential response to EDS is intrinsic to the Leydig cell or mediated by other intra- or extratesticular differences between adult and immature rats. To differentiate among these possibilities, Leydig cells were exposed to EDS (1) in vivo, (2) through in vitro testicular perfusion, or (3) in highly purified Leydig cell primary cultures. Four days after ip injections of 85 mg EDS/kg body wt Leydig cells were eliminated from testes of adult, but not immature rats. Total androgen production by testes perfused in vitro with 94 micrograms EDS/ml was dramatically reduced in adult, but not immature rats. Highly purified adult, but not immature, rat Leydig cells were far more sensitive to the effects of EDS on luteinizing hormone-stimulated androgen production (functional effects; apparent EC50 = 94 for adult and 407 micrograms/ml for immature rat Leydig cells) and on [35S]methionine incorporation (cytotoxic effects; apparent EC50 = 140 for adult and 1000 micrograms/ml for immature rat Leydig cells). Finally, the in vitro effects of EDS were both cell type and chemical specific. Since the differential response of adult and immature rat Leydig cells to EDS was manifest in vivo, during in vitro testicular perfusion, and in highly purified Leydig cell primary cultures, we conclude that immature rat Leydig cells are intrinsically less sensitive to the specific cytotoxic effects of EDS than adult rat Leydig cells.

The objective of this study was to determine whether estradiol has a direct effect on progesterone secretion by the rabbit corpus luteum. Empty or estradiol-filled Silastic capsules were implanted sc into pseudopregnant rabbits (day 0). Ten days later (day 10), peripheral blood was obtained via the marginal ear vein, and Silastic capsules were removed. Twenty-four hours after capsule removal (day 11), blood samples were obtained and ovaries removed for in vitro perfusion. The artery and vein of each ovary were individually cannulated, and ovaries were perfused in vitro for 6 h. Mean progesterone secretion rates were determined from perfusate samples taken every 30 min. On day 10, serum progesterone concentrations were similar in control and estradiol-treated animals. On day 11, 24 h after withdrawal of Silastic capsules, serum progesterone concentration in the estradiol-treated rabbits decreased significantly compared to controls. The withdrawal of estradiol also significantly reduced the secretion of progesterone by in vitro perfused ovaries in estradiol-withdrawn rabbits compared to empty capsule controls. Addition of estradiol or 25-hydroxycholesterol (25-OH) to the perfusion medium significantly increased progesterone secretion by ovaries from estradiol-withdrawn rabbits but not to control values. In contrast, a combination of estradiol plus 25-OH restored progesterone secretion to control levels. Although estradiol together with 25-OH stimulated progesterone secretion 24 h after estradiol withdrawal, progesterone secretion in vitro was unaffected 48 h after capsule removal, whereas pregnenolone stimulated secretion 5-fold. These results demonstrate that estradiol has a direct and acute stimulatory effect on progesterone secretion by the rabbit corpus luteum.

The aim of the present study was to investigate the effects of a single injection of LH on rat Leydig cell peroxisome volume and peroxisomal sterol carrier protein-2 (SCP2) content. Sexually mature Sprague-Dawley rats (n = 5) were injected sc with 500 micrograms LH and euthanized, and trunk blood was collected at 0, 0.5, 1, 2, and 3 h. Additionally, LH-treated rats were whole body perfused-fixed, and their testes were processed for qualitative and quantitative histochemical and immunocytochemical studies at 0, 0.5, 1, and 2 h. Peroxisomes were identified by cytochemical staining for catalase activity with the alkaline 3,3'-diaminobenzidine tetrahydrochloride method. Catalase and SCP2 were immunolocalized in Leydig cell organelles via 10-nm AuroProbe EM protein-A gold particles. Peak plasma testosterone concentrations were observed 1 and 2 h after the single sc LH injection. The average volume of a Leydig cell was unchanged by the LH treatment at all time points tested. Similarly, the absolute volumes of smooth endoplasmic reticulum and mitochondria per Leydig cell were unchanged at all time points tested. By contrast, the absolute volume of peroxisomes per Leydig cell increased 3-fold 0.5 h after LH injection (P less than 0.01) and then returned to control values by 2 h. The absolute volume of negative bodies (single membrane-bound cytoplasmic organelles lacking catalase) per Leydig cell was elevated above the control value 0.5 and 1 h after LH injection. Western blot analysis demonstrated a single protein at 14 and 60 kDa with anti-SCP2 and anticatalase, respectively, for both homogenates obtained from liver and purified Leydig cells. Quantitative immunocytochemical studies demonstrated that the gold particle density representing SCP2 over peroxisomes increased 5-fold 0.5 h after the LH injection (P less than 0.01) and then returned to control values by 2 h. In contrast, the gold particle density representing catalase over peroxisomes was not different in control and LH-injected groups. We conclude that a single sc injection of LH causes a rapid, specific, and transient increase in both the volume of peroxisomes and the peroxisomal content of SCP2 in Leydig cells.

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.

The major objective of the studies presented herein was to compare the extent to which exogenously administered testosterone is able to restore spermatogenesis in adult rats made azoospermic by withdrawal of all pituitary hormones (hypophysectomy for 4 weeks) vs. withdrawal of LH alone [testosterone- and estradiol-filled (TE) polydimethylsiloxane implants of 2.5 and 0.1 cm, respectively, for 8 weeks]. In hypophysectornized (Hypox) rats, serum LH and FSH were both undetectable; in the rats that received TE implants, serum LH was undetectable, but FSH was unaffected compared to control values. Seminiferous tubule fluid testosterone concentrations were reduced significantly from their control values of 60-65 to 1.4-1.7 ng/ml in the azoospermic Hypox and TE rats. These rats then received testosterone-filled implants of 4, 12, 18, or 24 cm and were killed 2 months later. In both the Hypox and TE rats, seminiferous tubule fluid testosterone concentrations rose linearly with increasing capsule sizes, and with each of the implant sizes, these concentrations did not differ significantly between the Hypox and TE rats. This made it possible for the first time to examine the effects of comparable intratesticular testosterone concentrations on the numbers of advanced spermatids per testis that could be restored in the azoospermic testes of rats lacking all pituitary factors vs. those lacking only LH. The results that we present demonstrate that the numbers of restored advanced spermatids were consistently and significantly lower in Hypox than in TE rats despite equivalent seminiferous tubule fluid testosterone concentrations. These results provide quantitative conclusive evidence to support the contention that pituitary factors in addition to LH are required for the quantitative restoration of spermatogenesis in the adult rat testis.

The aims of this study were to differentially identify peroxisomes and lysosomes in Leydig cells of the sexually mature rat using cytochemical techniques, to describe the size and shape of peroxisomal profiles, and to localize catalase and sterol carrier protein-2 (SCP2) in Leydig cell peroxisomes. Peroxisome profiles, identified by cytochemical staining for catalase activity using 3,3'-diaminobenzidine tetrahydrochloride (DAB) were categorized according to their longest diameter as small (less than 0.18 microns), intermediate (0.18-0.45 microns), and large (more than 0.45 microns); and according to their shape, which were designated as circular, oval, and dumbbell. Together these peroxisomal profiles occupied 11.2 microns 3/Leydig cell. Lysosomes, identified in the same tissue sections as acid phosphatase positive organelles, occupied 12.9 microns 3/Leydig cell. Negative bodies with morphology identical to cytochemically unstained peroxisomes also were detected. These organelles occupied 14.5 microns 3/Leydig cell. Catalase was immunolocalized exclusively in Leydig cell peroxisomes using AuroProbe EM protein A G10 (ie, 10 nm gold particles). Sterol carrier protein-2 was immunolocalized in Leydig cell peroxisomes by AuroProbe EM protein A G15 (ie, 15 nm gold particles). Immunolocalization of catalase and SCP2 using 10 nm and 15 nm gold particles in the same peroxisomes confirmed that Leydig cell peroxisomes contain SCP2. Taken together, these results show conclusively that adult rat Leydig cell peroxisomal profiles occur in different shapes and sizes, which suggests the existence of a network of peroxisomes, rather than peroxisomes occurring as separate isolated organelles. More importantly, the present study demonstrates for the first time that Leydig cell peroxisomes contain SCP2.(ABSTRACT TRUNCATED AT 250 WORDS)

In a previous study it was demonstrated that spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult intact rats that lack LH. The studies described herein were designed to examine the extent to which spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult rats that lack all pituitary hormones. Adult male rats were hypophysectomized and testosterone was administered at the time of hypophysectomy via sustained release polydimethylsiloxane (PDS) capsules of increasing lengths. We used the PDS capsules to clamp testosterone at defined concentrations within the seminiferous tubule fluid over a 2- to 3-month treatment period. Mean testis weights and advanced spermatid numbers per testis stabilized by 8 weeks of testosterone treatment regardless of testosterone dose. Both testis weight and advanced spermatid number responded to testosterone dose, reaching plateaus of 1.2 g and 170 x 10(6) per testis, respectively. These values were 60% of, and significantly less than, the respective control values. This result was in striking contrast to the results of our previous study of LH-suppressed intact rats, in which exogenously administered testosterone resulted in testis weights and advanced spermatid numbers that plateaued at values not significantly different from those in controls. These different effects of testosterone in intact and hypophysectomized rats occurred despite the fact that the seminiferous tubule fluid testosterone concentrations achieved in the hypophysectomized rats (up to 25 ng/ml) were greater than the minimal testosterone concentration found previously to be required to maintain spermatogenesis quantitatively in LH-suppressed intact rats (13 ng/ml). Taken together, these results demonstrate clearly that intratesticular testosterone doses that are as high as or higher than those that maintain spermatogenesis quantitatively in intact rats lacking LH fail to maintain spermatogenesis quantitatively in rats lacking all pituitary hormones.

Synthetic trypsin inhibitors known to inhibit acrosin were incubated with capacitated guinea pig spermatozoa to determine their possible effects on the membrane vesiculation and acrosomal matrix dispersion steps of the acrosome reaction. As seen by phase-contrast microscopy, the inhibitors nitrophenyl p-guanidinobenzoate, benzamidine and p-aminobenzamidine delayed acrosome reactions induced synchronously in capacitated spermatozoa. Electron microscopy revealed that the membrane vesiculation step of the acrosome reaction was unaffected by treatment of the spermatozoa with trypsin inhibitors, but that dispersion of the acrosomal matrix was blocked in the inhibitor-treated spermatozoa. These results suggest that in the guinea pig, proteinase activity (most likely that of acrosin) is involved in the dispersion of the acrosomal matrix following the membrane vesiculation step of the acrosome reaction, but not in membrane vesiculation.

The studies described herein were designed to examine whether there is a threshold concentration of testosterone (T) within the seminiferous tubules that is required to maintain spermatogenesis in the rat, or alternatively, whether there is a dose-response relationship between the intratesticular T concentration and the maintenance of spermatogenesis. T was administered to intact adult male rats via sustained release polydimethylsiloxane capsules in order to experimentally clamp T at well defined concentrations within the seminiferous tubules. Implantation of T-filled capsules of increasing sizes resulted in linear increases in T concentrations in serum, interstitial fluid, and seminiferous tubule fluid (STF). We examined the effect of step decreases in intratesticular T concentration on the numbers of advanced spermatogenic cells maintained by the testis over a 2-month period. Quantitatively complete spermatogenesis was maintained despite an 80% reduction in the STF T concentration (to approximately 13 ng/ml) from control values. The ability of the testis to maintain complete spermatogenesis was extremely sensitive to further decreases in STF T concentration. Thus, reduction of the STF T concentration from approximately 13 to 9 ng/ml resulted in a reduction in the number of advanced spermatids that were maintained in the testis by approximately 100 x 10(6). Reduction of the STF T concentration to approximately 4 ng/ml resulted in a further reduction in the number of advanced spermatids per testis by 100 x 10(6). Taken together, these data support the contention that there is far more T present within the seminiferous tubules of intact rat testes than is required to maintain quantitatively normal spermatogenesis and reveal for the first time that there is a dose-response relationship between the STF T concentration and the quantitative maintenance of advanced spermatogenic cells in the rat testis.

We have shown previously that administration of an orally active competitive aromatase inhibitor 4-(5,6,7,8-tetrahydroimidazo [1,5a] pyridin-5-yl) benzonitrile monohydrochloride to adult male beagles increases peripheral blood LH and testosterone concentrations, and that testes from treated dogs produce more testosterone when perfused in vitro than age-matched controls. In the present study we posed the question of whether the increased testosterone secretion by testes from these same aromatase-treated dogs was due to Leydig cell hypertrophy or hyperplasia, and if the latter, whether cytoplasmic organelles are increased. Beagles were treated with the inhibitor at a dosage of 2.5 mg/kg.day for 25 weeks and were euthanized by an overdose of iv sodium pentobarbital; testes were perfusion-fixed, embedded, and sectioned for stereological analysis. There were no significant differences in testis volume and absolute volumes of seminiferous tubules, blood vessels, lymphatic space, macrophage cells, and mesenchymal cells between the control and treated dogs. In contrast absolute interstitium volume and the absolute volume of Leydig cells per testis were significantly increased (P less than 0.05) in treated dogs. This increased Leydig cell volume per testis was due to increased volume of individual Leydig cells rather than to increases in Leydig cell number per testis. Additional studies showed that the surface area per Leydig cell of smooth endoplasmic reticulum, outer and inner mitochondrial membranes, and membranes of lipid droplets per testis were significantly higher (P less than 0.05) in the treated dogs as compared to the controls. In summary, the results of this study lead us to conclude that aromatase inhibition in the mature dog causes Leydig cell hypertrophy rather than hyperplasia and increased surface area per Leydig cell of subcellular organelles that contain enzymes involved in steroid biosynthesis.

We examined the effect of exogenously administered testosterone (T) on the quantitative restoration of advanced spermatogenic cells in adult rat testes rendered azoospermic by treating rats with polydimethylsiloxane (PDS) implants of T and estradiol (E). Experimental rats received PDS-TE implants for an initial 8-week period; control rats received empty implants. By 8 weeks of PDS-TE treatment, rats became severely oligospermic, and the T concentration within the seminiferous tubule fluid (STF) was reduced approximately 80% (from 57.8 ng/ml in controls to 9.6 ng/ml). After the initial 8-week PDS-TE treatment, PDS-TE implants were removed from one group of rats; a second group of PDS-TE-implanted rats received an additional PDS-T implant of 24 cm. Eight weeks after the removal of PDS-TE implants or the implantation of additional T, testis weight and numbers of advanced spermatogenic cells were restored to those of control rats. The STF T concentration 8 weeks after the removal of PDS-TE implants also was restored to that in control rats. In contrast, the STF T concentration increased to only 40% of control values in the rats that received an additional T implant. Despite this 60% reduction in T concentration compared to the control value, advanced spermatogenic cell number was restored to a value indistinguishable from that of intact controls. These observations indicate that spermatogenesis can be quantitatively restored in PDS-TE-implanted rats with exogenously administered T, and moreover, that this restoration does not require the high T concentration found in the STF of intact control rats.

The male ACI rat has a congenital Wolffian duct defect manifested by unilateral agenesis of the kidney, ureter, seminal vesicle, ductus deferens, and most of the epididymis. In the adult the testis on the affected side is markedly smaller than the contralateral testis and spermatogenesis is absent. In addition, in vitro perfusion of the affected testis revealed significantly less testosterone secretion than did the contralateral testis. Moreover, electron microscopy revealed that the smooth endoplasmic reticulum of the Leydig cell was significantly decreased. Thus, the affected testes clearly showed deranged spermatogenesis and Leydig cell structure and function. The ACI rat model may provide insight into the potential effects of congenital epididymal abnormalities on the testis of the human.

Stereological methods at the light and electron microscopic levels were used to examine the morphological changes in the beagle prostate associated with increasing age and with the occurrence of spontaneous benign hyperplasia. Analyses of semithin sections of tissue embedded in plastic revealed that proliferation of prostatic glandular and stromal components, increase in epithelial cell numbers, and increase in epithelial cell size all contributed to the dramatic increase in prostate weight seen in aging dogs. Analyses at the electron microscopic level revealed that the volume densities of rough endoplasmic reticulum and secretion granules were significantly lower in epithelial cells of the immature prostate than in the prostate of mature dogs (greater than 1.5 years of age). In contrast, no significant differences were seen in the volume densities of rough endoplasmic reticulum, free ribosomes, secretion granules, smooth endoplasmic reticulum, or mitochondria in randomly selected prostatic epithelial cells of dogs 1.5 to 9 years of age, whether or not the prostates were hyperplastic.

These studies were designed to test the hypothesis that sperm nuclear decondensation and male pronuclear formation during hamster fertilization depend upon the ability of the fertilized oocyte to reduce sperm nuclear disulfide bonds. In a first series of experiments, treatment of mature oocytes with the sulfhydryl blocking agent iodoacetamide or the glutathione oxidant diamide caused a dose-dependent inhibition of decondensation in microinjected sperm nuclei. Inhibition of decondensation was not observed, however, when sperm nuclei were treated in vitro with dithiothreitol (DTT) to reduce disulfide bonds prior to their microinjection. In a second series of experiments, germinal vesicle (GV)-intact oocytes and pronuclear eggs, in which mature, disulfide-rich sperm nuclei do not decondense, were found to support the decondensation of disulfide-poor DTT-treated sperm nuclei or testicular spermatid nuclei. The decondensed sperm nuclei were not, however, transformed into male pronuclei. The results of these studies suggest: (1) that sperm nuclear decondensation in the hamster requires disulfide bond reduction, (2) that GV-intact oocytes and pronuclear eggs lack sufficient reducing power to effect sperm nuclear decondensation, and (3) that disulfide bond reduction is required but not sufficient for pronuclear formation.

In this study, we examined changes in luteal cell structure that accompany estradiol-altered progesterone production by the rabbit corpus luteum. To stimulate progesterone production, polydimethylsiloxane (Silastic) capsules containing 17 beta-estradiol were inserted sc into superovulated New Zealand White rabbits. Luteal progesterone production, assessed by measurement of progesterone in peripheral serum, was high after the estradiol-filled capsules were inserted, declined within 24 h after the capsules were removed, and increased within 32 h after reinsertion of the capsules. Stereological analyses at the light microscopic level revealed that the number of luteal cells and the volume of an average luteal cell did not differ significantly between estradiol-stimulated and estradiol-deprived rabbits over the time period employed. In contrast, stereological analyses at the electron microscopic level demonstrated that the surface areas of smooth endoplasmic reticulum and inner mitochondrial membrane declined with estradiol deprivation, but were restored by reimposition of estradiol stimulation. These changes in the surface area per cell of smooth endoplasmic reticulum and inner mitochondrial membrane were strongly correlated (r = 0.94 and r = 0.88, respectively) with changes in progesterone concentrations in peripheral serum. Changes in the surface area of lipid droplets per luteal cell also occurred, but were inversely correlated (r = -0.87) with progesterone levels. No significant changes were seen in the surface areas per cell of outer mitochondrial membrane or rough endoplasmic reticulum. These results demonstrate that estradiol stimulation and deprivation cause reversible quantitative changes in the rabbit luteal cell organelles known to be directly involved in progesterone biosynthesis. This leads to the conclusion that the steroidogenic activity of the luteal cell is tightly coupled to its subcellular structure.

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.