Ren-Shan Ge

Modulation of Rat Leydig Cell Steroidogenic Function by Di(2-Ethylhexyl)Phthalate

Abstract Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg−1 kg−1 day−1 DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg−1 kg−1 day−1 DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg−1 kg−1 day−1 DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21–48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg−1 kg−1 day−1 and 10 mg−1 kg−1 day−1, respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg−1 kg−1 day−1 DEHP for 28 days (Postnatal Days 62–89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

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.

Trends of Reproductive Hormones in Male Rats During Psychosocial Stress: Role of Glucocorticoid Metabolism in Behavioral Dominance

Abstract 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 11β-hydroxysteroid dehydrogenase (11βHSD). However, Leydig cell steroidogenesis is inhibited when 11βHSD metabolizing capacity is exceeded. Therefore, 11βHSD 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 11βHSD 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 11βHSD 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.

Identification of a Kinetically Distinct Activity of 11β-Hydroxysteroid Dehydrogenase in Rat Leydig Cells1

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11β-hydroxysteroid dehydrogenase (11β-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11β-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11β-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11β-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether th...

Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero.

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long–Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2–20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6–30 cells increased in all treatment groups, with 29 ± 2% in control vs. 37 ± 3, 35 ± 3, and 56 ± 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.

Mechanisms of glucocorticoid-induced Leydig cell apoptosis

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.

Phthalate-induced testicular dysgenesis syndrome: Leydig cell influence

Phthalates, the most abundantly produced plasticizers, leach out from polyvinyl chloride plastics and disrupt androgen action. Male rats that are exposed to phthalates in utero develop symptoms characteristic of the human condition referred to as testicular dysgenesis syndrome (TDS). Environmental influences have been suspected to contribute to the increasing incidence of TDS in humans (i.e. cryptorchidism and hypospadias in newborn boys and testicular cancer and reduced sperm quality in adult males). In this review, we discuss the recent findings that prenatal exposure to phthalates affects Leydig cell function in the postnatal testis. This review also focuses on the recent progress in our understanding of how Leydig cell factors contribute to phthalate-mediated TDS.

A Metabolite of Methoxychlor, 2,2-Bis(p-Hydroxyphenyl)-1,1,1-Trichloroethane, Reduces Testosterone Biosynthesis in Rat Leydig Cells Through Suppression of Steady-State Messenger Ribonucleic Acid Levels of the Cholesterol Side-Chain Cleavage Enzyme

Abstract Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and cholesterol utilization. The reduced steady-state mRNA level for P450scc in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.

Hormonal Regulation of Oxidative and Reductive Activities of 11β-Hydroxysteroid Dehydrogenase in Rat Leydig Cells1

We have proposed that the 11β-hydroxysteroid dehydrogenase (11β-HSD) of Leydig cells protects against glucocorticoid-induced inhibition of testosterone (T) production. However, Leydig cells express type I 11β-HSD, which has been shown to be reductive in liver parenchymal cells. Because reduction would have the opposite effect of activating glucocorticoid, the present study was designed to determine: 1) whether Leydig cell 11β-HSD is primarily oxidative or reductive; and 2) whether oxidative and reductive activities are separately modified by known regulators of Leydig cell steroidogenic function. Leydig cells and liver parenchymal cells were purified from mature male Sprague-Dawley rats (250 g BW), and 11β-HSD oxidative and reductive activities were measured using radiolabeled substrates and TLC of triplicate media samples from 1-h incubations immediately after cell isolation. Enzyme activities also were examined in purified Leydig cells at the end of 3 days of culture in vitro in the presence of LH (10 n...

Developmental Changes in Glucocorticoid Receptor and 11β-Hydroxysteroid Dehydrogenase Oxidative and Reductive Activities in Rat Leydig Cells1

Glucocorticoids directly regulate testosterone production in Leydig cells through a glucocorticoid receptor (GR)-mediated repression of the genes that encode testosterone biosynthetic enzymes. The extent of this action is determined by the numbers of GR within the Leydig cell, the intracellular concentration of glucocorticoid, and 11β-hydroxysteroid dehydrogenase (11βHSD) activities that interconvert corticosterone (in the rat) and its biologically inert derivative, 11-dehydrocorticosterone. As glucocorticoid levels remain stable during pubertal development, GR numbers and 11βHSD activities are the primary determinants of glucocorticoid action. Therefore, in the present study, levels of GR and 11βHSD messenger RNA (mRNA) and protein were measured in rat Leydig cells at three stages of pubertal differentiation: mesenchymal-like progenitors (PLC) on day 21, immature Leydig cells (ILC) that secrete 5α-reduced androgens on day 35, and adult Leydig cells (ALC) that are fully capable of testosterone biosynthesi...