Effect of genotype on hormonal function formation of Leydig cells during mouse postnatal development

Changes in in vitro testosterone production by Leydig cells induced by chorionic gonadotropin, dibutyryl-cAMP, and pregnenolone have been studied during postnatal development of four inbred mouse strains BALB/c, PT, CBA/Lac, and A/He, with contrast hormonal activity of testes in sexually mature males. The interlinear differences significantly change with age of the males by all studied indices indicating genotype-dependent formation of hormonal activity of Leydig cells during postnatal development. Coordinated interlinear variability between all indices of Leydig cells reactivity has been established for each studied period of postnatal development. Hence, we have established coordinated interlinear genetic variability of hormonal function of Leydig cells, which was confirmed by considerable changes in it during postnatal development at puberty. Definitive genotypic differences in hormonal activity of Leydig cells appeared by late pubertal and early postpubertal development (day 60) and coincided with termination of morphological differentiation of Leydig cells and appearance of the differentiated cell population.     

Effect of Genotype on Formation of Hormonal Function of Leydig Cells during Mouse Postnatal Development

Changes in in vitro testosterone production by Leydig cells induced by chorionic gonadotropin, dibutyryl-cAMP, and pregnenolone have been studied during postnatal development of four inbred mouse strains BALB/c, PT, CBA/Lac, and A/He, with contrast hormonal activity of testes in sexually mature males. The interlinear differences significantly change with age of the males by all studied indices indicating genotype-dependent formation of hormonal activity of Leydig cells during postnatal development. Coordinated interlinear variability between all indices of Leydig cells reactivity has been established for each studied period of postnatal development. Hence, we have established coordinated interlinear genetic variability of hormonal function of Leydig cells, which was confirmed by considerable changes in it during postnatal development at puberty. Definitive genotypic differences in hormonal activity of Leydig cells appeared by late pubertal and early postpubertal development (day 60) and coincided with termination of morphological differentiation of Leydig cells and appearance of the differentiated cell population.     

A role for kit receptor signaling in Leydig cell steroidogenesis

Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo.     

Trends of reproductive hormones in male rats during psychosocial stress: role of glucocorticoid metabolism in behavioral dominance

Stress in socially subordinate male rats, associated with aggressive attacks by dominant males, was studied in a group-housing context called the visible burrow system (VBS). It has been established that subordinate males have reduced serum testosterone (T) and higher corticosterone (CORT) relative to dominant and singly housed control males. The relationship of the decreased circulating T levels in subordinate males to changes in serum LH concentrations has not been evaluated previously. Since decreases in LH during stress may cause reductions in Leydig cell steroidogenic activity, the present study defined the temporal profiles of serum LH, T, and CORT in dominant and subordinate males on Days 4, 7, and 14 of a 14-day housing period in the VBS. The same parameters were followed in serum samples from single-housed control males. Leydig cells express glucocorticoid receptors and may also be targeted for direct inhibition of steroidogenesis by glucocorticoid. We hypothesize that Leydig cells are protected from inhibition by CORT at basal concentrations through oxidative inactivation of glucocorticoid by 11beta-hydroxysteroid dehydrogenase (11betaHSD). However, Leydig cell steroidogenesis is inhibited when 11betaHSD metabolizing capacity is exceeded. Therefore, 11betaHSD enzyme activity levels were measured in Leydig cells of VBS-housed males at the same time points. Significant increases in LH and T relative to control were observed in the dominant animals on Day 4, which were associated with the overt establishment of behavioral dominance as evidenced by victorious agonistic encounters. Serum LH and T were lower in subordinate males on Day 7, but T alone was lower on Day 14, suggesting that lowered LH secretion in subordinates may gradually be reversed by declines in androgen-negative feedback. Serum CORT levels were higher in subordinate males compared to control at all three time points. In contrast, oxidative 11betaHSD activity in Leydig cells of dominant males was higher relative to control and unchanged in subordinates. These results suggest the following: 1) failure of Leydig cells of subordinate males to compensate for increased glucocorticoid action during stress, by increasing 11betaHSD oxidative activity, potentiates stress-mediated reductions in T secretion; and 2) an inhibition of the reproductive axis in subordinate males at the level of the pituitary.     

Stress hormone and male reproductive function

The Leydig cell is the primary source of testosterone in males. Levels of testosterone in circulation are determined by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Stress-induced increases in serum glucocorticoid concentrations inhibit testosterone-biosynthetic enzyme activity, leading to decreased rates of testosterone secretion. It is unclear, however, whether the excessive glucocorticoid stimulation also affects total Leydig cell numbers through induction of apoptosis and thereby contributes to the stress-induced suppression of androgen levels. Exposure of Leydig cells to high concentrations of corticosterone (CORT, the endogenously secreted glucocorticoid in rodents) increases their frequency of apoptosis. Studies of immobilization stress indicate that stress-induced increases in CORT are directly responsible for Leydig cell apoptosis. Access to glucocorticoid receptors in Leydig cells is modulated by oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase (11βHSD). Under basal levels of glucocorticoid, sufficient levels of glucocorticoid metabolism occur and there is likely to be minimal binding of the glucocorticoid receptor. We have established that Leydig cells express type 1 11βHSD, an oxidoreductase, and type 2, a unidirectional oxidase. Generation of redox potential through synthesis of the enzyme cofactor NADPH, a byproduct of glucocorticoid metabolism by 11βHSD-1, may potentiate testosterone biosynthesis, as NADPH is the cofactor used by steroidogenic enzymes such as type 3 17β-hydroxysteroid dehydrogenase. In this scenario, inhibition of steroidogenesis will only occur under stressful conditions when high input amounts of CORT exceed the capacity of oxidative inaction by 11βHSD. Changes in autonomic catecholaminergic activity may contribute to suppressed Leydig cell function during stress, and may explain the rapid onset of inhibition. However, recent analysis of glucocorticoid action in Leydig cells indicates the presence of a fast, non-genomic pathway that will merit further investigation.     

Leydig cell number and function in the adult cynomolgus monkey (Macaca fascicularis) is increased by daily hCG treatment but not by daily FSH treatment

Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.     

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100ng/ml) for 18h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.     

Effects of the transmethylation inhibitor S-adenosyl-homocysteine and of the methyl donor S-adenosyl-methionine on rat Leydig cell function in vitro

In purified rat Leydig cells, the methyl donor S-adenosyl-methionine (SAM), increases significantly in a dose dependent manner the [125I]hCG binding as well as the productions of cAMP and of testosterone; the competitive inhibitor of methylations S-adenosyl-homocysteine (SAH), has an opposite effect. Associated to oLH, SAM further enhances the cAMP synthesis while SAH inhibits significantly the adenylate cyclase activity. With regard to testosterone synthesis, SAM potentiates the stimulating roles of oLH and dbcAMP (27 and 38% increases, respectively) although SAH diminishes testosterone productions (48 and 35%, respectively under oLH and dbcAMP stimulations). Scatchard analysis has shown that SAM (1.4 mM) increases the number of LH/hCG binding sites on Leydig cells while SAH (1.4 mM) decreases it; LH/hCG Ka values are not modified neither by SAM nor by SAH. These data suggest that the in vitro regulation of steroidogenesis in purified rat Leydig cells may involve methylation processes (presumably phospholipids are the potential substrates of these reactions) which modulates the transmission of the hormonal signal through the membrane and affects the testosterone synthesis at a step beyond the adenylate cyclase.     

Alterations in mitogenic and steroidogenic activities in rat testicular interstitial fluid after administration of ethane dimethane sulphonate

A single dose of EDS was given to mature male rats and interstitial fluid (IF) was collected to determine the potency of mitogenic and steroidogenic activities therein. The potency of the factor stimulating testosterone secretion in vitro by Percoll-purified Leydig cells was significantly elevated 2 weeks after EDS, whilst the potency of mitogenic activities (stimulation of DNA synthesis by BALB/c 3T3 cells) was not elevated until 4 weeks after EDS treatment. This study suggests that two separate factors, one with mitogenic and the other steroidogenic activity, may be involved in the response of Leydig cells after EDS administration. The mitogenic factor may play a role in Leydig cell regeneration whereas the testosterone-stimulating factor may be involved in testicular testosterone homeostasis.     

In vitro interactions between Sertoli cells and steroidogenic cells

Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells.     

Morphological and functional variations of Leydig cells in testis of the domestic pig during the different biological stages of development

The relationship of morphometrical and androgen receptor evaluations of the main testicular interstitium cellular element (Leydig cells) in the domestic pig provided interesting numerical and morphological features during the different aging stages. As early as 25 days (a period in which the pig is sexually immature) there was a low number of Leydig cells (1.46 x 10(8)) with respect to a 78% and 35% increase in the adult (2.48 x 108) and aged (1.78 x 10(8)) animal, respectively. Interestingly, when the volume density of Leydig cells was considered, the average volume of these cells seemed to be high (75%) in the aged pig with respect to the young immature animal whereas a lower increase (27%) was observed for the adult animal. Moreover, the evaluation of testosterone receptor binding sites in the testis at the various stages of development also displayed a differentiated pattern since elevated testosterone receptor binding levels of the high dissociation affinity type were obtained for the adult pig. Thus, from the combined morphological variations of Leydig cells and testosterone receptor binding activity, it appears that this androgenic receptor component exerts distinct autocrine effects on the different functional features of some testicular tissue constituents at the different aging stages of the domestic pig.     

Luteinizing hormone induces expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells

Background  

Leydig cells are the primary source of testosterone in male vertebrates. The biosynthesis of testosterone in Leydig cells is strictly dependent on luteinizing hormone (LH). On the other hand, it can be directly inhibited by excessive glucocorticoid (Corticosterone, CORT, in rats) which is beyond the protective capability of 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and type 2 (11beta-HSD2; encoded by gene Hsd11b2 in rats) in Leydig cells. Our previous study found that LH increases 11beta-HSD1 expression in rat Leydig cells, but the effect of LH on the expression and activity of 11beta-HSD2 is not investigated yet.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

Regulation of Leydig cell steroidogenic function during aging

This article summarizes a talk on Leydig cell aging presented at the 1999 Annual Meeting of the Society for the Study of Reproduction. In the Brown Norway rat, serum testosterone levels decrease with aging, accompanied by increases in serum FSH. The capacity of Leydig cells to produce testosterone is higher in young than in old rats. Binding studies with hCG revealed reduced receptor number in old vs. young Leydig cells. In response to incubation with LH, cAMP production was found to be reduced in old vs. young Leydig cells, indicating that signal transduction mechanisms in the old cells are affected by aging. Steroidogenic acute regulatory protein and mRNA levels are reduced in old Leydig cells, suggesting that there may be deficits in the transport of cholesterol to the inner mitochondrial membrane of aged cells. The activity of P450 side-chain cleavage enzyme is reduced in old vs. young cells, as are the activities of each of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/C17-20 lyase, and 17-ketosteroid reductase. Serum LH levels do not differ between young and old rats, and the administration of LH failed to induce old Leydig cells to produce high (young) testosterone levels, suggesting that the cause of age-related reductions in steroidogenesis is not LH deficits. We hypothesized that reactive oxygen, produced as a by-product of steroidogenesis itself, might be responsible for age-related reductions in testosterone production by the Leydig cells. Consistent with this, long-term suppression of steroidogenesis was found to prevent or delay the reduced steroidogenesis that accompanies Leydig cell aging. A possible explanation of this finding is that long-term suppression of steroidogenesis prevents free radical damage to the cells by suppressing the production of the reactive oxygen species that are a by-product of steroidogenesis itself.     

Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Testicular function and Leydig cell ultrastructure in long-term bilaterally cryptorchid rams

This study was conducted to investigate the effects of bilateral cryptorchidism induced in adult rams on testicular function and Leydig cell ultrastructure. The results indicated that long-term bilateral cryptorchidism resulted in decreased testicular size, degeneration of seminiferous tubules, elevated serum LH levels, maintenance of normal testosterone concentrations in peripheral and spermatic vein serum, impairment of the magnitude and duration of androgen response to exogenous luteinizing hormone (LH), a 13-fold reduction in total number of Leydig cells/paired testes, and a 3-fold hypertrophy in the average size of remaining Leydig cells. Based on quantitative morphometry, the hypertrophied Leydig cells exhibited significant increases in the volume of intracellular organelles, including the cell nucleus, mitochondria, smooth and rough endoplasmic reticulum, lysosome-like bodies and lipid vesicles. Quantitatively, the hypertrophy alone was not enough to offset the loss in number of Leydig cells and was insufficient to explain the maintenance of normal levels of testosterone in jugular and spermatic venous blood. The additional mechanisms responsible for production of normal serum testosterone levels in the cryptorchid ram remain to be elucidated.     

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.     

Effect of seasonal changes in Leydig cell number on the volume of smooth endoplasmic reticulum in Leydig cells and intratesticular testosterone content in stallions

Testes from 47 adult (4-20 years) stallions obtained in November-January (non-breeding season) and 41 adult stallions obtained in May-July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0.5 micron sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P less than 0.05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.     

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.