Barry R. Zirkin

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.

The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo.

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.

Regulation of Leydig Cell Steroidogenic Function During Aging

Abstract 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 tranduction 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 3β-hydroxysteroid dehydrogenase, 17α-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.

Quantitative pathologic changes in the human testis after vasectomy. A controlled study

To determine whether there are any deleterious changes in the human testis after vasectomy, we obtained testicular biopsy specimens from 31 healthy men undergoing vasectomy reversal and from 21 healthy, fertile volunteers. Morphometric analyses of these specimens revealed a 100 per cent increase in the thickness of the seminiferous tubular walls (P less than 0.001), a 50 per cent increase in the mean cross-sectional tubular area (P less than 0.001), and a significant reduction in the mean number of Sertoli cells (P less than 0.01) and spermatids (P less than 0.01) per tubular cross section in the post-vasectomy group, as compared with the control group. Focal interstitial fibrosis was observed in 23 per cent of the specimens from the post-vasectomy group and in none from the control group. There was a significant correlation (P less than 0.01) between interstitial fibrosis and infertility in patients who underwent a surgically successful vasectomy reversal (sperm in the ejaculate). None of the other measured characteristics correlated with infertility after vasectomy reversal. We conclude that significant morphologic changes occur in the human testis after vasectomy. The presence of focal interstitial fibrosis was associated with a high incidence of infertility in this series.

Leydig cells: From stem cells to aging.

Leydig cells are the testosterone-producing cells of the testis. The adult Leydig cell population ultimately develops from undifferentiated mesenchymal-like stem cells present in the interstitial compartment of the neonatal testis. Four distinct stages of adult Leydig cell development have been identified and characterized: stem Leydig cells, progenitor Leydig cells, immature Leydig cells and adult Leydig cells. The stem Leydig cells are undifferentiated cells that are capable of indefinite self-renewal, differentiation, and replenishment of the Leydig cell niche. Progenitor Leydig cells are derived from the stem Leydig cells. These spindle-shaped cells are luteinizing hormone (LH) receptor positive, have high mitotic activity, and produce little testosterone but rather testosterone metabolites. The progenitor Leydig cells give rise to immature Leydig cells which are round, contain large amounts of smooth endoplasmic reticulum, and produce some testosterone but also very high levels of testosterone metabolites. A single division of these cells produces adult Leydig cells, which are terminally differentiated cells that produce high levels of testosterone. As men age, serum testosterone levels decline, and this is associated with alterations in body composition, energy level, muscle strength, physical, sexual and cognitive functions, and mood. In the Brown Norway rat, used extensively as a model for male reproductive aging, age-related reductions in serum testosterone result from significant decline in the ability of aged Leydig cells to produce testosterone in response to LH stimulation. This review describes Leydig cell development and aging. Additionally, the molecular mechanisms by which testosterone synthesis declines with aging are discussed.

In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat

We examined the effects of fetal exposure to a wide range of di-(2-ethylhexyl) phthalate (DEHP) doses on fetal, neonatal, and adult testosterone production. Pregnant rats were administered DEHP from Gestational Day (GD) 14 to the day of parturition (Postnatal Day 0). Exposure to between 234 and 1250 mg/kg/day of DEHP resulted in increases in the absolute volumes of Leydig cells per adult testis. Despite this, adult serum testosterone levels were reduced significantly compared to those of controls at all DEHP doses. Organ cultures of testes from GD20 rats exposed in utero to DEHP showed dose-dependent reductions in basal testosterone production. Surprisingly, however, no significant effect of DEHP was found on hCG-induced testosterone production by GD20 testes, suggesting that the inhibition of basal steroidogenesis resulted from the alteration of molecular events upstream of the steroidogenic enzymes. Reduced fetal and adult testosterone production in response to in utero DEHP exposure appeared to be unrelated to changes in testosterone metabolism. In view of the DEHP-induced reductions in adult testosterone levels, a decrease in the expression of steroidogenesis-related genes was anticipated. Surprisingly, however, significant increases were seen in the expression of Cyp11a1, Cy17a1, Star, and Tspo transcripts, suggesting that decreased testosterone production after birth could not be explained by decreases in steroidogenic enzymes as seen at GD20. These changes may reflect an increased number of Leydig cells in adult testes exposed in utero to DEHP rather than increased gene expression in individual Leydig cells, but this remains uncertain. Taken together, these results demonstrate that in utero DEHP exposure exerts both short-term and long-lasting effects on testicular steroidogenesis that might involve distinct molecular targets in fetal and adult Leydig cells.

Soy of dietary source plays a preventive role against the pathogenesis of prostatitis in rats

This study examined the effects of diet on the development of prostatitis in male rats. Adult male rats were placed on either of two specially formulated diets which differed from one another by the presence or absence of soy as the protein source. A third group of rats (control) was fed standard laboratory rat chow which also includes soy as a source of protein. After 11 weeks, it was found that rats maintained on soy-free diet developed prostatitis mainly in the lateral lobe of the prostate. Increased severity and incidence of prostatitis in rats maintained on the soy-free diet coincided with a significant decrease in urinary excretion of various phytoestrogens. There was no evidence of prostatitis in rats maintained on soy-containing diets. Urinary excretion of phytoestrogens in rats maintained on soy-containing diet was also not different from controls. These results suggest that soy as a dietary source plays a protective role against the development of prostatitis in rats, and indicate that the ventral, lateral and dorsal lobes of the rat prostate have different sensitivities to alterations in dietary factors.

Leydig cell aging and the mechanisms of reduced testosterone synthesis

In males, serum testosterone levels decline with advancing age. Though part of a complex process, this age-related decline in testosterone appears to occur, in part, due to a significant decline in the ability of aged Leydig cells to produce testosterone maximally in response to luteinizing hormone (LH). The structure of the molecular machinery responsible for the synthesis of testosterone is described, and placed in the context of Leydig cell biology. Multiple parameters related to the synthesis of testosterone by the Leydig cell have been observed to change with age. Relationships among these changes are reviewed. A discussion of potential causes of the age-related decline in Leydig cell steroidogenic capacity presents a model in which the inability of aged cells to adequately respond to hormonal stimulation results in cellular regression with concomitant decline in maximal testosterone output.

Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function?

Aging in Brown Norway rats is accompanied by the reduced production of testosterone by the Leydig cells, the testicular cells responsible for synthesizing and secreting this essential steroid. As yet, the mechanism by which Leydig cell steroidogenesis is reduced is unknown. Herein we assess the production of mitochondrial reactive oxygen species by intact Leydig cells isolated from the testes of young and old rats. To this end, Leydig cells were incubated with lucigenin (bis-N-methylacridinium nitrate), a probe that enters cells, localizes to mitochondria, and yields a significant chemiluminescent response following its reaction with intramitochondrial superoxide. Leydig cells from old rats elicited significantly greater lucigenin-derived chemiluminescence (LDCL) than those from young rats. Electron microscopic stereological analysis revealed that the absolute volume of mitochondria in the old cells was reduced from that in the young. These results, taken together, suggest that there are age-related changes in the production of reactive oxygen species by the mitochondria of Leydig cells, with those of old Leydig cells producing significantly greater levels than those of young Leydig cells. The results are consistent with the proposal that mitochondrial-derived reactive oxygen may play a role in the irreversible decline in the ability of old Leydig cells to produce testosterone.

Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis

Abstract The postnatal development of Leydig cells can be divided into three distinct stages: initially they exist as fibroblast-like progenitor Leydig cells (PLCs) appearing in the testis by Days 14–21; subsequently, by Day 35, they become immature Leydig cells (ILCs) acquiring steroidogenic organelle structure and enzyme activities but metabolizing most of the testosterone they produce; finally, as adult Leydig cells (ALCs) by Day 90, they actively produce testosterone. The factors controlling proliferation and differentiation of Leydig cells remain largely unknown, and the aim of the present study was to identify changes in gene expression during development through cDNA array analysis of PLCs, ILCs, and ALCs. By cluster analysis, it was determined that the transitions from PLC to ILC to ALC were associated with downregulation of mRNAs corresponding to 107 genes. The downregulated genes included cell-cycle regulators, e.g., cyclin D1 (Ccnd1); growth factors, e.g., basic fibroblast growth factor (Fgf2); growth-factor-related receptors, e.g., platelet-derived growth factor α receptor (Pdgfra); oncogenes, e.g., kit oncogene (Kit); and transcription factors, e.g., early growth response 1 (Egr1). Conversely, expression levels of 264 genes were increased by at least twofold. Most of these were related to differentiated function and included steroidogenic enzymes, e.g., 11β-hydroxysteroid dehydrogenase 2 (Hsd11b2); neurotransmitter receptors, e.g., acetylcholine receptor nicotinic α 4 (Chrna4); stress response factors, e.g., glutathione transferase 8 (Gsta4); and protein turnover enzymes, e.g., tissue inhibitor of metalloproteinase 2 (Timp2). The detection of Hsd11b2 mRNA in the array was the first indication that this gene is expressed in Leydig cells, and parallel increases in Hsd11b2 mRNA and enzyme activity were recorded. Thus, gene profiling demonstrates that postnatal development is associated with changes in the expression levels of several different clusters of genes consistent with the processes of Leydig cell growth and differentiation.