Müllerian-inhibiting substance inhibits rat Leydig cell regeneration after ethylene dimethanesulphonate ablation

The postnatal development of Leydig cell precursors is postulated to be controlled by Sertoli cell secreted factors, which may have a determinative influence on Leydig cell number and function in sexually mature animals. One such hormone, Mullerian inhibiting substance (MIS), has been shown to inhibit DNA synthesis and steroidogenesis in primary Leydig cells and Leydig cell tumor lines. To further delineate the effects of MIS on Leydig cell proliferation and steroidogenesis, we employed the established ethylene dimethanesulphonate (EDS) model of Leydig cell regeneration. Following EDS ablation of differentiated Leydig cells in young adult rats, recombinant MIS or vehicle was delivered by intratesticular injection for 4 days (Days 11-14 after EDS). On Days 15 and 35 after EDS (1 and 21 days post-MIS injections), endocrine function was assessed and testes were collected for stereology, immunohistochemistry, and assessment of proliferation and steroidogenesis. Although serum testosterone and luteinizing hormone (LH) were no different, intratesticular testosterone was higher on Day 35 in MIS-treated animals. At both time points, intratesticular 5alpha-androstan-3alpha,17beta-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

Abnormal development of the testis after administration of the Leydig cell cytotoxic ethylene-1,2-dimethanesulphonate to the immature rat

Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDS-treated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3-5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDS-treated animals. These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood-testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.     

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.     

The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells

Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.     

Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS)

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.     

Structure and expression of the rat relaxin-like factor (RLF) gene.

The relaxin-like factor (RLF) is a novel member of the insulin-IGF-relaxin family of growth factors and hormones, and its mRNA is expressed very specifically in the Leydig cells of the testis and in the theca and luteal cells of the ovary. Here we report the cloning of the RLF gene and cDNA from the rat. The 0.8kb mRNA is produced from a small gene comprising two exons situated less than 1 kb downstream of the gene for the signalling factor JAK3. Northern hybridization confirms high RLF mRNA expression in the adult rat testis, and low expression in the ovary, but in no other tissues examined. Northern analysis of fetal and neonatal gonadal tissues showed that RLF mRNA is highly upregulated in the testes of day 19 embryos, but not in later neonatal stages, nor in any ovarian tissue from this period. This would indicate that RLF is a marker for the mature fetal as well as the adult-type Leydig cell, but is not expressed in premature, precursor, or dedifferentiated Leydig cells of either cell type. Finally, RNA was analysed from the testes of rats which had been treated with ethylene dimethane sulfonate (EDS), an alkylating agent that specifically destroys rat Leydig cells. RLF mRNA was absent from the acutely treated testes, but became detectable between 15 and 20 days post-treatment, concomitant with the repopulation of the testes by new Leydig cells. Continuous testosterone substitution of EDS-treated rats suppressed the production of gonadotropins, and LH-dependent Leydig cell differentiation, with the result that RLF mRNA remained undetectable throughout the study period. In conclusion, RLF is a very specific marker for the mature Leydig cell phenotype in both the adult-type and fetal Leydig cell populations of the rat testis.     

Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Physiological role of putative testicular gonadotrophin releasing hormone (GnRH)

Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non-existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate

Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.     

Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration.

Administration of ethane dimethane sulphonate (EDS) to adult rats results in the destruction of all Leydig cells, followed by a complete regeneration. We investigated this regeneration process in more detail, using different markers for precursor and developing Leydig cells: the LH receptor, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), transforming growth factor alpha (TGFalpha), and a new marker for Leydig cell maturation, relaxin-like factor (RLF). LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 +/- 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3beta-HSD, while some cells also stained positively for TGFalpha. After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased. The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.     

Environmental stress alters the developmental pattern of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of fetal rats: a quantitative cytochemical study

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Rat testicular endogenous steroids and number of Leydig cells between the fetal period and sexual maturity

Endogenous androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol), and some of their C21 precursors (pregnenolone, progesterone and 17-hydroxyprogesterone) were measured in rat testes between Day 18.5 of pregnancy and Day 64 postpartum, and correlated with numerical densities of Leydig cells. The latter parameter showed an early maximum on Day 19.5 of the fetal period, a nadir on Day 15 postpartum, and a gradual increase thereafter. The two dominating androgens, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol, had similar levels until 15 days of age, but the 5 alpha-diol predominated thereafter. The total steroid content per Leydig cell was highest on Day 18.5 of gestation (77 ng/10(6) cells). A decline started already in utero, and reached a nadir of 5 ng/10(6) cells on Day 29. Thereafter, a slight increase occurred with advancing age. It is concluded that: The fetal testis has highest Leydig cell and endogenous steroid concentrations. A nadir in these parameters is seen 2-4 wk postpartum. The Leydig cell concentration increases around puberty on Days 40-60, but only a slight concomitant increase occurs in steroids. A sharp decline in steroid content per Leydig cell occurs during the last fetal days, but the postnatal decline of testicular steroids is due to Leydig cell loss. The new Leydig cell generation after 15 days has a persistently low steroid concentration through puberty.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative protective actions of gonadotrophins and testosterone against the antispermatogenic action of ethane dimethanesulphonate

After a single dose of ethane dimethanesulphonate (EDS) (75 mg/kg) to rats the prolonged antispermatogenic action is due to a temporary elimination of the functional Leydig cell population. Replacement therapy with testosterone propionate (3 mg/day) maintains the spermatogenic epithelium but the EDS effect develops when hormone treatment is discontinued. In contrast, a short treatment with hCG (10-100 i.u./day) or LH (714 micrograms/day), starting before the EDS dose, permanently protects the spermatogenic epithelium. FSH treatment was completely ineffective. Although histological protection of spermatogenesis appeared complete with testosterone or hCG, effects on fertility remained but over different periods of time. Antispermatogenic and antifertility effects were produced in mice using much higher doses of EDS (5 X 250 mg/kg) but there was no protection from androgen or hCG. It is suggested that EDS binds to Leydig cells irreversibly, interfering with the action of gonadotrophin. At the dose level used the evidence suggests that the degree of reaction renders most of the Leydig cell population non-viable. A direct cytotoxic effect of the compound upon the spermatogenic epithelium might account for the inability of testosterone or hCG alone or in combination to maintain fertility at normal levels.