Haolin Chen

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.

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.

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.

Vitamin E, aging and Leydig cell steroidogenesis

Previous studies have suggested that oxidant-induced damage may play a role in the reduced ability of aged Brown Norway rat Leydig cells to produce testosterone. We reasoned that if this was the case, antioxidants such as vitamin E (VE) would be expected to have protective effects on steroidogenesis. To test this hypothesis, the effects of VE on Leydig cell steroidogenesis were examined both in vitro and in vivo. In vitro studies were conducted using Leydig cells isolated from the testes of young adult Brown Norway rats. In one experiment, isolated cells were incubated with luteinizing hormone (LH) alone or with LH plus VE (1.3-40 microg/ml). At each of 3, 5 and 7 days thereafter, the ability of the cells to produce testosterone was greater in the presence of VE than in its absence, and depended upon VE dose. Culturing the Leydig cells with the antioxidants melatonin or N-tert-butyl-alpha-phenylnitrone also protected Leydig cell steroidogenic function. Additionally, VE was found to suppress Fe2+/sodium ascorbate-induced lipid peroxidation in Leydig cells. These studies strongly supported the contention that VE has a protective effect on Leydig cell steroidogenesis. These in vitro results prompted us to ask whether, in vivo, VE also would affect steroidogenesis as Leydig cells age. To this end, rats were provided one of three diets, begun when the rats were 6 months of age and carried out through age 25 months: VE-deficient, VE-control, or VE-supplemented. The VE-deficient diet had no effect on the age-related reductions in Leydig cell testosterone production observed in VE-control rats. The VE-supplemented diet did not prevent age-related reductions in steroidogenesis, but the reductions at ages 23 and 25 months were significantly less than those seen in Leydig cells from VE-control or VE-deficient rats. Taken together, the results of the in vitro and in vivo studies reported herein are consistent with the conclusion that vitamin E exerts a protective effect on Leydig cell steroidogenesis.

Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate on hormone-stimulated steroidogenesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate, a widely used plasticizer, and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to exert adverse effects on the reproductive tract in developing and adult animals. As yet, however, the molecular mechanisms by which they act are uncertain. In the present study, we address the molecular and cellular mechanisms underlying the effects of MEHP on basal and human chorionic gonadotropin (hCG)-stimulated steroid production by MA-10 Leydig cells, using a systems biology approach. MEHP induced dose-dependent decreases in hCG-stimulated steroid formation. Changes in mRNA and protein expression in cells treated with increasing concentrations of MEHP in the presence or absence of hCG were measured by gene microarray and protein high-throughput immunoblotting analyses, respectively. Expression profiling indicated that low concentrations of MEHP induced the expression of a number of genes that also were expressed after hCG stimulation. Cross-comparisons between the hCG and MEHP treatments revealed two genes, Anxa1 and AR1. We suggest that these genes may be involved in a new self-regulatory mechanism of steroidogenesis. The MEHP-induced decreases in hCG-stimulated steroid formation were paralleled by increases in reactive oxygen species generation, with the latter mediated by the Cyp1a1 gene and its network. A model for the mechanism of MEHP action on MA-10 Leydig cell steroidogenesis is proposed.

Stem Leydig cells: From fetal to aged animals

Leydig cells are the testosterone-producing cells of the testis. The adult Leydig cell (ALC) population ultimately develops from undifferentiated mesenchymal-like stem cells present in the interstitial compartment of the neonatal testis. Distinct stages of ALC development have been identified and characterized. These include stem Leydig cells (SLCs), progenitor Leydig cells, immature Leydig cells, and ALCs. This review describes our current understanding of the SLCs in the fetal, prenatal, peripubertal, adult, and aged rat testis, as well as recent studies of the differentiation of steroidogenic cells from the stem cells of other organs.

Oxidative stress and phthalate-induced down-regulation of steroidogenesis in MA-10 Leydig cells

Previous studies have shown that phthalate exposure can suppress steroidogenesis. However, the affected components of the steroidogenic pathway, and the mechanisms involved, remain uncertain. We show that incubating MA-10 Leydig cells with mono-(2-ethylhexyl) phthalate (MEHP) resulted in reductions in luteinizing hormone (LH)-stimulated cAMP and progesterone productions. cAMP did not decrease in response to MEHP when the cells were incubated with cholera toxin or forskolin. Incubation of MEHP-treated cells with dibutyryl-cAMP, 22-hydroxycholesterol or pregnenolone inhibited the reductions in progesterone. Increased levels of reactive oxygen species (ROS) occurred in response to MEHP. In cells in which intracellular glutathione was depleted by buthionine sulfoximine pretreatment, the increases in ROS and decreases in progesterone in response to MEHP treatment were exacerbated. These results indicate that MEHP inhibits MA-10 Leydig cell steroidogenesis by targeting LH-stimulated cAMP production and cholesterol transport, and that a likely mechanism by which MEHP acts is through increased oxidative stress.

Stem Leydig Cell Differentiation: Gene Expression During Development of the Adult Rat Population of Leydig Cells

ABSTRACT Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.