Leydig cells (LCs) are thought to differentiate from spindle-shaped precursor cells that exhibit some aspects of differentiated function, including 3␤-hydroxysteroid dehydrogenase (3␤HSD) activity. The precursor cells ultimately derive from undifferentiated stem LCs (SLCs), which are postulated to be present in testes before the onset of precursor cell differentiation. We searched for cells in the neonatal rat testis with the abilities to: (i) proliferate and expand indefinitely in vitro (self renew); (ii) differentiate (i.e., 3␤HSD and ultimately synthesize testosterone); and (iii) when transplanted into host rat testes, colonize the interstitium and subsequently differentiate in vivo. At 1 week postpartum, spindle-shaped cells were seen in the testicular interstitium that differed from the precursor cells in that they were 3␤HSD-negative, luteinizing hormone (LH) receptor (LHR)-negative, and platelet-derived growth factor receptor ␣ (PDGFR␣)-positive. These cells were purified from the testes of 1-week-old rats. The cells contained proteins known to be involved in LC development, including GATA4, c-kit receptor, and leukemia inhibitory factor receptor. The putative SLCs expanded over the course of 6 months while remaining undifferentiated. When treated in media that contained thyroid hormone, insulin-like growth factor I, and LH, 40% of the putative SLCs came to express 3␤HSD and to synthesize testosterone. When transplanted into host rat testes from which LCs had been eliminated, the putative SLCs colonized the interstitium and subsequently expressed 3␤HSD, demonstrating their ability to differentiate in vivo. We conclude that these cells are likely to be the sought-after SLCs.c-kit ͉ leukemia inhibitory factor ͉ platelet-derived growth factor receptor ␣ ͉ puberty ͉ steroidogenesis L eydig cells (LCs) are the primary source of testosterone in the male, and their differentiation in the testes during puberty is a signature event in the development of the male body plan. It is hypothesized, but far from proven, that LCs first arise from undifferentiated stem cells [stem LCs (SLCs)] (1-3). It has been suggested that, in rats, the putative SLCs are present in the testis at birth, and that by 11 days postpartum, at least some of their progeny express LC-specific genes and thus become committed to the LC lineage (4, 5).The committed cells subsequently undergo phased transitions through progenitor and immature stages and ultimately to terminally differentiated adult LC stage (6). In particular, progenitor LCs (PLCs) form during days 12-28 postpartum (presumably from SLCs). The PLCs proliferate and also exhibit some aspects of differentiated function, including 3␤-hydroxysteroid dehydrogenase (3␤HSD) activity (7). Luteinizing hormone (LH) receptors (LHRs) first appear as the PLCs differentiate, suggesting that SLCs are likely to be independent of LH control (8). The development of the steroidogenic capacity of PLCs requires stimulation by LH (9). The mitotic activity of PLCs gradually is reduced, and the cells enlarge...

Herein we summarize important discoveries made over many years about Leydig cell function and regulation. Fetal Leydig cells produce the high levels of androgen (testosterone or androstenedione, depending upon the species) required for differentiation of male genitalia and brain masculinization. Androgen production declines with loss of these cells, reaching a nadir at postpartum. Testosterone then gradually increases to high levels with adult Leydig cell development from stem cells. In the adult, luteinizing hormone (LH) binding to Leydig cell LH receptors stimulates cAMP production, increasing the rate of cholesterol translocation into the mitochondria. Cholesterol is metabolized to pregnenolone by the CYP11A1 enzyme at the inner mitochondrial membrane, and pregnenolone to testosterone by mitochondria and smooth endoplasmic reticulum enzymes. Cholesterol translocation to the inner mitochondrial membrane is mediated by a protein complex formed at mitochondrial contact sites that consists of the cholesterol binding translocator protein, voltage dependent anion channel, and other mitochondrial and cytosolic proteins. Steroidogenic acute regulatory protein acts at this complex to enhance cholesterol movement across the membranes and thus increase testosterone formation. The 14-3-3γ and ε adaptor proteins serve as negative regulators of steroidogenesis, controlling the maximal amount of steroid formed. Decline in testosterone production occurs in many aging and young men, resulting in metabolic and quality-of-life changes. Testosterone replacement therapy is widely used to elevate serum testosterone levels in hypogonadal men. With knowledge gained of the mechanisms involved in testosterone formation, it is also conceivable to use pharmacological means to increase serum testosterone by Leydig cell stimulation.

The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction industry, and di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment. Daily human exposure to DEHP in the U.S. is significant, and occupational and clinical exposures from DEHP-plasticized medical devices, e.g., blood bags, hemodialysis tubing, and nasogastric feeding tubes, increase body burden levels. We investigated the effects of chronic exposures to low environmentally relevant DEHP levels on testicular function. Our data show that prolonged exposures to this agent induced high levels of the gonadotropin luteinizing hormone and increased the serum concentrations of sex hormones [testosterone and 17␤-estradiol (E2)] by >50%. Increased proliferative activity in Leydig cells was evidenced by enhanced expression of cell cycle proteins, as determined by RT-PCR. The numbers of Leydig cells in the testis of DEHP-treated rats were 40 -60% higher than in control rats, indicating induction of Leydig cell hyperplasia. DEHP-induced elevations in serum testosterone and E2 levels suggest the possibility of multiple crosstalks between androgen, estrogen, and steroid hormone receptors, whereas the presence of estrogen receptors in nonreproductive tissues, e.g., cardiovascular system and bones, implies that the increases in serum E2 levels have implications beyond reproduction, including systemic physiology. Analysis of the effects of phthalate exposures on gonadotropin and steroid hormone levels should form part of overall risk assessment in human populations. R eports of a higher incidence of urogenital anomalies of the newborn, such as cryptorchidism, hypospadias, and reproductive abnormalities in wild life exposed to high levels of chemicals in the environment, have generated public concern that these agents may impair human reproductive health (1, 2). Phthalates are used as plasticizers in certain infant toys and consumer products (e.g., containers for soaps, shampoos, and perfumes) and medical devices such as tubings and catheters. The U.S. Department of Health and Human Services in 1985 (3) estimated the total daily human consumption of di-(2-ethylhexyl) phthalate (DEHP) from all sources of exposure at 5.8 mg in the U.S. In a report just published by the Center for Disease Control and Prevention, the urinary levels of mono-(ethylhexyl) phthalate (MEHP) (micrograms per liter), which is the chief metabolite of DEHP, ranged from 3.26 to 4.15 in males and 2.93 to 3.51 in females; these levels are thought to represent only one-tenth of the ingested DEHP dose within the previous 24 h (4). In a recent review of laboratory studies, the U.S. National Toxicology Program's Center for the Evaluation of Risks to Human Reproduction Expert Panel concluded that DEHP has the potential to produce adverse reproductive effects in humans (5). Indeed, several propo...

Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.

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 17beta-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 objective of this study was to determine whether postnatal increases in rat Leydig cell number result from differentiation of precursor cells, division of existing Leydig cells, or both. Our approach was 1) to examine changes in the absolute number of Leydig cells and potential precursor cells (macrophages, pericytes, and mesenchymal, endothelial, and myoid cells) per testis on day 19 of gestation (day -2) and days 7, 14, 21, 28, and 56 postpartum; 2) to examine the frequency with which mesenchymal and Leydig cells divide during prenatal and postnatal development; and 3) to identify and examine the fate of the progeny of Leydig and mesenchymal cell divisions during prenatal and postnatal development. Stereological methods were used to show that mesenchymal cells comprised 44% of the total interstitial cell population and Leydig cells 16% on day -2, whereas by day 56 postpartum the relationship had reversed; mesenchymal cells comprised 3% and Leydig cells 49%. These results suggested a precursor-product relationship between mesenchymal and Leydig cells because no such reciprocal relationship was observed between Leydig cells and macrophages, pericytes, endothelial, or myoid cells. Autoradiographic analysis of [3H]thymidine incorporation into mesenchymal and Leydig cells was consistent with this interpretation. In a series of pulse-chase experiments, the percentage of labeled mesenchymal and Leydig cells was measured after a single injection of [3H]thymidine on days 2, 14, 28, and 56 postpartum, each followed by sampling at timed intervals (between 1 h and 14 days) thereafter. Starting on day 14, the percentage of labeled Leydig cells was approximately 1% immediately after injection of [3H]thymidine and increased significantly to approximately 6% by 6 days after injection. No such increase was observed when rats were similarly injected starting on days 2, 28, and 56 postpartum. The rise in Leydig cell labeling between days 14 and 28 postpartum did not result in a decline in the number of silver grains over labeled Leydig cell nuclei, indicating that the increase in the percentage of labeled cells was not caused by Leydig cell division. These observations led us to conclude that the increase in Leydig cell labeling from days 14 to 28 was the result of recruitment from a compartment of labeled mesenchymal cells. In contrast, our analysis indicated that from day 28 postpartum and thereafter until the mature number of Leydig cells is attained, Leydig cells are generated by division of morphologically recognizable Leydig cells.

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.

A B S T R A C T Spontaneous prostatic hyperplasia in the beagle appears to progress with age from a glandular to a cystic histological appearance. Prostatic hyperplasia can be induced in young beagles with intact testes by treatment for 4 mo with either dihydrotestosterone or 5a-androstane-3a,17,8-diol, alone, or with either of these steroids in combination with 17,3-estradiol.In contrast, the induction of prostatic hyperplasia in young castrated beagles, in which the gland had been allowed to involute for 1 mo, requires the administration of both 17,3-estradiol and either 5a-androstane-3a, 17,8-diol or dihydrotestosterone. Testosterone and 17,8-estradiol, either singly or in combination, did not produce the hyperplastic condition in intact or castrated beagles. The experimentally induced prostatic hyperplasia is identical in pathology to the glandular hyperplasia that occurs naturally in the aging dog with intact testes. However, cystic hyperplasia was not produced by any of the treatments tested in young animals.