We reported previously that stem cells associated with adult rat testis seminiferous tubules are able to give rise to differentiated Leydig cells in vitro. The regulatory mechanisms by which they do so, however, are uncertain. Herein, we hypothesized that the proliferation and differentiation of Leydig cell stem cells (stem Leydig cells, SLCs) depend upon locally produced factors from the seminiferous tubules. Microarray analysis revealed that platelet-derived growth factor receptor alpha (PDGFRalpha) is up-regulated and PDGFRbeta is down-regulated with postnatal differentiation of SLCs. This suggested that their ligands, PDGF-AA and PDGF-BB, respectively, might have important roles in SLC proliferation and differentiation. To test this, we developed a unique in vitro culture system in which SLCs proliferate on the surfaces of cultured seminiferous tubules largely during Week 1 of culture and their progeny subsequently differentiate to testosterone-forming Leydig cells during Weeks 2 through 4. Using this system, seminiferous tubules from adult rat testes were cultured with PDGF-AA or PDGF-BB for up to 4 wk. Both ligands stimulated SLC proliferation during the first week of culture, with PDGF-BB significantly more potent than PDGF-AA. Furthermore, PDGF-AA had a stimulatory effect on SLC differentiation from Weeks 2 through 4 of culture. In contrast, PDGF-BB, which stimulated cell proliferation during Week 1, had a significant inhibitory effect on differentiation during Weeks 2 through 4. These findings, made possible by the development of the seminiferous tubule culture system, reveal distinct roles by locally produced PDGFs in SLC regulation.
A rat varicocele model using partial occlusion of the left renal vein was described previously. Reproducibility in creating this model has met with varied success. Alternate routes of testicular venous drainage may negate the effect of partial renal vein occlusion on varicocele creation. We hypothesized that varicocele induction would be more effective if microsurgical ligation of the gonadal venous drainage to the common iliac vein was combined with partial occlusion of the left renal vein.
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.
We observed previously that after long-term suppression of luteinizing hormone (LH) and thus of Leydig cell steroidogenesis, restimulation of the Leydig cells by LH resulted in significantly higher testosterone production than by age-matched cells from control rats. These studies suggest that stimulation over time may elicit harmful effects on the steroidogenic machinery, perhaps through alteration of the intracellular oxidant-to-antioxidant balance. Herein we compared the effects of LH stimulation on stress response genes, formation of intracellular reactive oxygen species (ROS), and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and in aged cells. Microarray analysis indicated that LH stimulation resulted in significant increases in expression of genes associated with stress response and antiapoptotic pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in transiently increased ROS levels compared to controls. Aged Leydig cells also showed increased ROS soon after LH stimulation. However, in contrast to the young cells, ROS production peaked later and the time to recovery was increased. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage but significantly more so in the aged cells. DNA damage levels in response to LH and the levels of intracellular ROS were highly correlated. Taken together, these results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells and that while DNA damage occurs in cells of both ages, there is greater damage in the aged cells.
An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17?-estradiol (E(2)), and 5?-androstan-3?,17?-diol (3?-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-?L samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E(2). Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E(2) and 3?-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.
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.
Previous studies in MA-10 tumor Leydig cells demonstrated that disruption of the mitochondrial electron-transport chain (ETC), membrane potential (??(m)), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ??(m), and ATP synthesis cooperatively affected steroidogenesis. These results suggest significant differences between the two systems and call into question the extent to which results from tumor Leydig cells relate to primary cells. Thus, to further understand the similarities and differences between the two systems as well as the impact of ATP disruption on steroidogenesis, we performed comparative studies of MA-10 and primary Leydig cells under similar conditions of mitochondrial disruption. We show that mitochondrial ATP synthesis is critical for steroidogenesis in both primary and tumor Leydig cells. However, in striking contrast to primary cells, perturbation of ??(m) in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ??(m) powers the mitochondrial ATP synthase. Further studies revealed that a significant proportion of cellular ATP in MA-10 cells derives from glycolysis. In contrast, primary cells appear to be almost completely dependent on mitochondrial respiration for their energy provision. Inhibitor studies also suggested that the MA-10 ETC is impaired. This work underscores the importance of mitochondrial ATP for hormone-stimulated steroid production in both MA-10 and primary Leydig cells while indicating that caution must be exercised in extrapolating data from tumor cells to primary tissue.
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.
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.
The free radical, or oxidative stress, theory posits that imbalance in cells between prooxidants and antioxidants results in an altered redox state and, over time, an accumulation of oxidative damage. We hypothesized herein that cells with an increasingly prooxidant intracellular environment also might be particularly susceptible to acute oxidative stress. To test this hypothesis, MA-10 cells were used as a model because of their well-defined, measurable function, namely progesterone production. We first experimentally altered the redox environment of the cells by their incubation with buthionine sulfoximine (BSO) or diethyl maleate (DEM) so as to deplete glutathione (GSH), and then exposed the GSH-depleted cells acutely to the prooxidant tert-butyl hydroperoxide (t-BuOOH). Neither BSO nor DEM by themselves affected progesterone production. However, when the GSH-depleted cells subsequently were exposed acutely to t-BuOOH, intracellular reactive oxygen species concentration was significantly increased, and this was accompanied by significant reductions in progesterone production. In striking contrast, treatment of control cells with t-BuOOH had no effect. Depletion of GSH and subsequent treatment of the cells with t-BuOOH-induced the phosphorylation of each of ERK1/2, JNK and p38, members of the MAPK family. Inhibition of p38 phosphorylation largely prevented the t-BuOOH-induced down-regulation of progesterone production in GSH-depleted cells. These results suggest that, as hypothesized, alteration of the intracellular GSH redox environment results in the increased sensitivity of MA-10 cells to oxidative stress, and that this is mediated by activation of one or more redox-sensitive MAPK members.
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.
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 cells, the testosterone-producing cells of the adult testis, rarely turn over. However, their elimination with ethane dimethanesulfonate (EDS) is followed by the appearance of new, fully functional adult Leydig cells. The cells that give rise to the new Leydig cells have not been well characterized, and little is known about the mechanism by which they are regulated. We isolated cells expressing platelet-derived growth factor receptor-?, but not 3?-hydroxysteroid dehydrogenase (3?-HSD(neg)) from the testes of EDS-treated adult rats. Depending on conditions, these cells proliferated indefinitely or differentiated and produced testosterone. To localize these cells and to determine the effect of the testicular environment on their function, the seminiferous tubules and testicular interstitium were physically separated and cultured. During the first 72 h in culture, 3?-HSD(neg) cells on the tubule surfaces underwent divisions. Some of these cells later expressed 3?-HSD and produced testosterone. Removal of the newly formed 3?-HSD(pos) cells from the tubule surfaces with EDS, followed by further culture of the stripped tubules, resulted in the reappearance of testosterone-producing cells. These results, taken together, suggest that the precursors for newly formed Leydig cells are stem cells, with many if not all situated on the surfaces of the seminiferous tubules. Although normally quiescent, the stem cells are capable of self-renewal and differentiation. The development of the tubule culture system should provide a valuable in vitro approach to assess the role(s) of niche components on the function of adult Leydig stem cells despite their residing in a complex mammalian tissue.
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