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Background: The mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques., Methods: Interstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks., Results: Seven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week., Conclusion: Seven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Huang, Wang, Wang, Hu, Li, Xu, Qin, Wen, Cao, Guan, Duan, Chen and Chen.)

Background: The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability., Methods: Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic-activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non-steroidogenic (25-OH-vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24 h., Results: Reanalysis of scRNA-seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 10 6 cells/testis), high purity (about 95%) and viability (> 93%). Purified LCs retained high steroidogenic and non-steroidogenic functions in responding to maximal LH stimulations, with more than 10-fold increases in T production in 3 h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24 h., Discussion and Conclusion: We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro., (© 2022 American Society of Andrology and European Academy of Andrology.)

Spermatogenesis is an efficient, complex, and highly organized proliferation and differentiation process that relies on multiple factors including testosterone produced by the Leydig cells. Although the critical role played by testosterone in spermatogenesis is well recognized, the mechanism by which it works is still not completely understood, partially due to the inability to specifically and precisely monitor testosterone-dependent changes within developing germ cells. Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells after the rats were treated with ethanedimethanesulfonate, which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration cycle. The dataset, which includes all developing germ cells from spermatogonia to spermatozoa, should prove useful for characterizing developing germ cells, their regulatory networks, and novel cell-specific markers. The dataset should be particularly useful for exploring the effects of the androgen environment on the regulation of spermatogenesis. As this is the first single-cell RNA-Seq dataset for rat testes, it can also serve as a reference for future studies., (© 2022. The Author(s).)

Previous studies reported that, with aging, Leydig cell intracellular antioxidants are reduced in concentration and intracellular ROS levels increase, suggesting that oxidant/antioxidant imbalance may contribute to the reduced testosterone production that characterizes the aging cells. As yet, little is known about how the Leydig cell oxidant/antioxidant environment is regulated. Sirt1, an enzyme that deacetylates transcription factors, and the transcription factor Nrf2, have been shown to be associated with cellular response to oxidative stress. We hypothesized that Sirt1 and/or Nrf2 might be involved in regulating the oxidant/antioxidant environment of Leydig cells, and therefore, the testosterone production. We found that Sirt1 and Nrf2 are present in the Leydig cells of Brown Norway rats, though reduced in aged cells. In MA-10 cells in which Sirt1 or Nrf2 were suppressed by nicotinamide (NAM) or ML385, respectively, or in which siRNAs were used for knockdown of Sirt1 or Nrf2, increased ROS levels and decreased progesterone production occurred. In rat Leydig cells, inhibition of Sirt1 by culturing the cells with NAM resulted in increased ROS and reduced testosterone production, and subsequent removal of NAM from the culture medium resulted in increased testosterone production. Activation of rat Leydig cells Sirt1 with honokiol or of Nrf2 with sulforaphane resulted in the maintenance of testosterone production despite the exposure of the cells to oxidizing agent. These results, taken together, suggest that Sirt1 and Nrf2 are involved in maintaining the Leydig cell oxidant/antioxidant environment, and thus in maintaining steroid production., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis., Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1-30 μM) was added to the culture medium, and the effects on LC development were assayed., Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1-5 μM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15-30 μM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway., Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Ji, Wen, Chen, Huang, Guan, Tian, Xie, Shao, Wang, Huang, Lin, Ye and Chen.)

Peritubular stem Leydig cells (SLCs) have been identified from rat testicular seminiferous tubules. However, no stem cells for peritubular myoid cells have been reported in the adult testis so far. In the present study, we tested the hypothesis that the peritubular SLCs are multipotent and able to form either Leydig or myoid cells. Using cultured tubules, we show that in the presence of PDGFAA and luteinizing hormone, SLCs became testosterone-producing Leydig cells, while in the presence of PDGFBB and TGFB, the cells formed α-smooth muscle actin-expressing myoid cells. This multipotency was also confirmed by culture of isolated CD90 + SLCs. These results suggest that these stem cells outside the myoid layer are multipotent and give rise to either Leydig or myoid cells, depending on the inducing factors. These cells may serve as a common precursor population for maintaining homeostasis of both Leydig and myoid cell populations in the adult testis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Perfluoroundecanoic acid (PFUnA) is one of long-chain perfluoroalkyl carboxylic acids. However, the effect of PFUnA on pubertal development of Leydig cells remains unclear. The goal of this study was to investigate the effect of PFUnA on Leydig cell development in pubertal male rats. We orally dosed male Sprague-Dawley rats (age 35 days) with PFUnA at doses of 0, 1, 5, and 10 mg/kg/day from postnatal day (PND) 35 to PND 56. Serum testosterone and luteinizing hormone levels were remarkably reduced by PFUnA at ≥1 mg/kg while serum follicle-stimulating hormone levels were lowered at 5 and 10 mg/kg. PFUnA down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Nr5a1, Fshr, Dhh, Sod1, and Sod2 and their proteins in the testis and the expression of Lhb and Fshb in the pituitary. PFUnA reduced Leydig cell number at 5 and 10 mg/kg. PFUnA induced oxidative stress and increased autophagy. These may result from the inhibition of phosphorylation of mTOR, AKT1, AKT2, and ERK1/2 in the testis. In conclusion, PFUnA exhibits inhibitory effects on pubertal Leydig cell development possibly via inducing oxidative stress and increasing autophagy., (Copyright © 2021. Published by Elsevier Inc.)

Studies have shown that phthalates are capable of affecting the development and functions of male reproductive system. The effect of phthalates on Leydig cell functions is well documented. However, little is known about their potential effects on the functions of stem Leydig cells (SLC). In the present study, we have examined the effects of mono-(2-ethylhexyl) phthalate (MEHP) on SLC functions in vitro by culturing seminiferous tubules and isolated SLCs. The results indicate that MEHP can significantly inhibit the proliferation and differentiation of SLCs in both the organ and cell culture systems. Interestingly, the minimal effective concentration that is able to affect SLC function was lower in the tubule culture system (1 μM) than in the isolated cells (10 μM), suggesting a possible involvement of the niche cells. Also, MEHP appeared to affect both the efficiency of SLCs to form Leydig cells and a selected group of Leydig cell-specific genes, including Lhcgr, Scarb1, Hsd3b1, Cyp17a1, Star, Srd5a1, Akr1c14, Insl3, Hao2 and Pah. Since SLCs are multipotent, we also tested the effect of MEHP on the differentiation of SLCs to adipocytes. Though MEHP by itself can not specify SLCs into adipocyte lineage, it indeed significantly increased the adipogenic activity of SLCs if used with an adipocyte inducing medium by up-regulation of multiple adipogenic-related genes, including Pparg and Cebpa. Overall, the results indicate that MEHP inhibits SLCs differentiating into Leydig lineage while stimulates the differentiating potential of SLCs to adipocytes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Cholesterol import into the mitochondria of steroid-producing cells is the rate-determining step in steroidogenesis. Numerous studies have provided evidence that the cholesterol-binding translocator protein (18 kDa TSPO) plays an important role in cholesterol translocation into mitochondria and that it also might act on cholesterol homeostasis. Several TSPO-specific ligands have been shown to increase steroid production in vitro and in vivo., Objectives: The present study assessed the effects of the TSPO drug ligand FGIN-1-27 on cholesterol accumulation and lipid droplet formation in relationship to steroid formation., Materials and Methods: Using MA-10 and primary Leydig cells, immunocytochemical and molecular methods were used to examine cholesterol accumulation, the formation of lipid droplets, and steroid formation in response to LH and FGIN-1-27. Additionally, we determined the effects of Tspo knockout by CRISPR/Cas9, and of siRNA knockdowns of Tspo and Plin2 (Perilipin 2; also known as adipose differentiation-related protein, ADFP) on LH- and FGIN-1-27-induced steroidogenesis., Results: In response to LH and FGIN-1-27, cultured MA-10 cells and primary Leydig cells increased steroid formation, cholesterol accumulation, and lipid droplet formation. Cholesterol accumulation in the lipid droplets also was increased in Tspo knockout cells. Knockout of Tspo or its knockdown in MA-10 cells resulted in reduced progesterone formation in response to both LH and FGIN-1-27, as did knockdown of Plin2. Steroid production also was inhibited by the cholesteryl ester hydrolase inhibitor diethylumbelliferyl phosphate., Discussion and Conclusion: These results support the conclusion that FGIN-1-27 stimulates steroid formation by increasing TSPO-mediated cholesterol translocation into the inner mitochondria for steroidogenesis, as well as into the cytosol for lipid droplet formation. FGIN-1-27 also increased steroid formation at least in part by inducing the conversion of cholesteryl ester located in lipid droplets to cholesterol, thus making available more substrate for steroid formation., (© 2019 American Society of Andrology and European Academy of Andrology.)

The Leydig cells of the mammalian testis produce testosterone (T) in response to luteinizing hormone (LH). In rats and men with reduced serum T levels, T replacement therapy (TRT) will raise T levels, but typically with suppressive effects on sperm formation. The rate-determining step in T formation is the translocation of cholesterol to the inner mitochondrial membrane, mediated by protein-protein interactions of cytosolic and outer mitochondrial membrane proteins. Among the involved proteins is cholesterol-binding translocator protein (TSPO) (18 kDa TSPO). We hypothesized that in contrast to TRT, the administration of the TSPO agonist N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27), by stimulating the ability of the Leydig cells to produce T, would result in the elevation of serum T levels while maintaining intratesticular T concentration and therefore without suppression of spermatogenesis. Age-related reductions in both serum and intratesticular T levels were seen in old Brown Norway rats. Both exogenous T and FGIN-1-27 increased serum T levels. With exogenous T, serum LH and Leydig cell T formation were suppressed, and intratesticular T was reduced to below the concentration required to maintain spermatogenesis quantitatively. In contrast, FGIN-1-27 stimulated Leydig cell T formation, resulting in increased serum T without reductions in intratesticular T concentrations or in testicular sperm numbers. FGIN-1-27 also significantly increased serum and intratesticular T levels in rats made LH-deficient by treatment with the gonadotropin-releasing hormone antagonist cetrorelix. These results point to a possible approach to increasing serum T without negative effects on spermatogenesis, based upon stimulating T production by the Leydig cells themselves rather than administering T exogenously., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Androgen deficiency (hypogonadism) affects males of all ages. Testosterone replacement therapy (TRT) is effective in restoring serum testosterone and relieving symptoms. TRT, however, is reported to have possible adverse effects in part because administered testosterone is not produced in response to the hypothalamic-pituitary-gonadal (HPG) axis. Progress in stem cell biology offers potential alternatives for treating hypogonadism. Adult Leydig cells (ALCs) are generated by stem Leydig cells (SLCs) during puberty. SLCs persist in the adult testis. Considerable progress has been made in the identification, isolation, expansion and differentiation of SLCs in vitro. In addition to forming ALCs, SLCs are multipotent, with the ability to give rise to all 3 major cell lineages of typical mesenchymal stem cells, including osteoblasts, adipocytes, and chondrocytes. Several regulatory factors, including Desert hedgehog and platelet-derived growth factor, have been reported to play key roles in the proliferation and differentiation of SLCs into the Leydig lineage. In addition, stem cells from several nonsteroidogenic sources, including embryonic stem cells, induced pluripotent stem cells, mature fibroblasts, and mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord have been transdifferentiated into Leydig-like cells under a variety of induction protocols. ALCs generated from SLCs in vitro, as well as Leydig-like cells, have been successfully transplanted into ALC-depleted animals, restoring serum testosterone levels under HPG control. However, important questions remain, including: How long will the transplanted cells continue to function? Which induction protocol is safest and most effective? For translational purposes, more work is needed with primate cells, especially human., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Adult testicular Leydig cells arise from stem cells in the neonatal and adult testis. The nature of these stem Leydig cells (SLCs) have not been well characterized. We have found previously that a group cells expressing CD90, a cell surface glycoprotein that may play roles in cell-cell and cell-matrix interactions and associated with the seminiferous tubule surface, have the ability to form Leydig cells. As yet, the relationship between this CD90+ cell population and SLCs reported previously by other groups is still unknown. In the present study, we systematically characterized these CD90+ cells by their ability to express multiple potential SLC markers and to proliferate and differentiate into Leydig cells in vitro. First, we have found by qPCR and immunohistochemical staining that the CD90+ cells do not express any of the markers of the common seminiferous tubular cells, including myoid, Sertoli, germ and Leydig cells, as well as macrophages. Moreover, when the CD90+ cells were isolated by fluorescent-sorting, the cells expressed high levels of all the potential SLC marker genes, including Nestin, Cd51, Coup-tf2, Arx, Pdgfra and Tcf21. Also, CD90-positive, but not -negative, cells were able to form Leydig cells in vitro with the proper inducing medium. Overall, the results indicated that the tubule-associated CD90+ cells represent a population of SLC in adult testis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

It was reported previously that adult mouse stem Leydig cells (SLCs) express CD51 (integrin α-chain V). However, it is still unclear whether all CD51 +  cells are SLCs. In the present study, we found that CD51 +  cells can be classified into two sub-groups, a weakly-staining group (CD51 + ) and a strongly-staining group (CD51 ++ ). The CD51 +  cells expressed common SLC marker genes, including Nestin, Pdgfra and Coup-tf2, while CD51 ++ cells did not express these genes. Instead, they expressed macrophage markers, such as F4/80, Cd115 and Tnfa. When these cells were induced to differentiate in vitro, the CD51 +  cells, but not CD51 ++ cells, formed Leydig cells. Overall, our results showed that although SLCs expressed CD51, not all CD51-expressing cells are SLCs. The cells that expressed high levels of CD51 are actually macrophages., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We reported that FGIN-1-27 (N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide, FGIN), a synthetic ligand for translocator protein (TSPO, 18 kDa), increased serum testosterone levels in young and aged Brown Norway rats after its administration daily for 10 days. It is not known, however, how soon after treatment with FGIN serum testosterone rises, how long levels remain elevated after cessation of treatment, or whether the drug acts solely through TSPO. Adult Sprague-Dawley male rats received a single ip dose of FGIN (1 mg/kg BW). Serial blood samples were collected, and serum testosterone and luteinizing hormone (LH) were assessed hourly throughout 24 h. Testosterone concentration was maximal by 3 h, remained significantly higher than the controls at 10 h, and returned to the control level by 24 h. Consistent with the in vivo study, culturing isolated Leydig cells with either FGIN (40 μM) or LH (0.1 ng/ml) resulted in significantly increased testosterone production by 30 min, and the stimulatory effects persisted through 48 h. At a very early (15 min) treatment time, however, FGIN significantly increased testosterone production but LH had not yet done so. Surprisingly, in vivo treatment with FGIN not only increased serum testosterone but also serum LH concentration, raising the possibility that FGIN may increase serum testosterone concentration by dual mechanisms., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

We reported previously that stem Leydig cells (SLC) on the surfaces of rat testicular seminiferous tubules are able to differentiate into Leydig cells. The proliferation and differentiation of SLCs seem likely to be regulated by niche cells, including nearby germ and Sertoli cells. Due to the cyclical nature of spermatogenesis, we hypothesized that the changes in the germ cell composition of the seminiferous tubules as spermatogenesis proceeds may affect tubule-associated SLC functions. To test this hypothesis, we compared the ability of SLCs associated with tubules at different stages of the cycle to differentiate into Leydig cells in vitro. SLCs associated with stages IX-XI were more active in proliferation and differentiation than SLCs associated with stages VII-VIII. However, when the SLCs were isolated from each of the two groups of tubules and cultured in vitro, no differences were seen in their ability to proliferate or differentiate. These results suggested that the stage-dependent local factors, not the SLCs themselves, explain the stage-dependent differences in SLC function. TGFB, produced in stage-specific fashion by Sertoli cells, is among the factors shown in previous studies to affect SLC function in vitro. When TGFB inhibitors were included in the cultures of stages IX-XI and VII-VIII tubules, stage-dependent differences in SLC development were reduced, suggesting that TGFB may be among the paracrine factors involved in the stage-dependent differences in SLC function. Taken together, the findings suggest that there is dynamic interaction between SLCs and germ/Sertoli cells within the seminiferous tubules that may affect SLC proliferation and differentiation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The inability of cultured primary Leydig cells to maintain luteinizing hormone (LH)-responsive testosterone formation in vitro for more than 3-5 days has presented a major challenge in testing trophic effects of regulatory factors or environmental toxicants. Our primary objective was to establish culture conditions sufficient to maintain LH-responsive testosterone formation by Leydig cells for at least a month. When isolated rat adult Leydig cells were cultured in DMEM/F12 and M199 culture medium containing insulin (10μg/ml), PDGFAA (10 ng/ml), lipoprotein (0.25 mg/ml), horse serum (1%) and a submaximal concentration of LH (0.2 ng/ml), the cells retained the ability to produce testosterone in vitro for at least 4 weeks. By using the longer-term culture conditions of this system, we were able to detect suppressive effects on testosterone production by low levels of the toxicant MEHP (mono-(2-ethylhexyl) phthalate), an active metabolite of the plasticizer DEHP, that were not detected by short-term culture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Serum testosterone (TS) levels decrease with aging in both humans and rodents. Using the rat as a model system, it was found that age-related reductions in serum TS were not due to loss of Leydig cells, but rather to the reduced ability of the Leydig cells to produce TS in response to luteinizing hormone (LH). Detailed analyses of the steroidogenic pathway have suggested that two defects along the pathway, LH-stimulated cAMP production and cholesterol transport to and into the mitochondria, are of particular importance in age-related reductions in TS production. Although the mechanisms involved in these defects are far from certain, increasing oxidative stress appears to play a particularly important role. Interestingly, increased oxidative stress also appears to be involved in the suppressive effects of endocrine disruptors on Leydig cell TS production., (© 2017 Society for Reproduction and Fertility.)

Adult Leydig cells develop from undifferentiated mesenchymal-like stem cells (stem Leydig cells, SLCs) present in the interstitial compartment of the early postnatal testis. Putative SLCs also have been identified in peritubular and perivascular locations of the adult testis. The latter cells, which normally are quiescent, are capable of regenerating new Leydig cells upon the loss of the adult cells. Recent studies have identified several protein markers to identify these cells, including nestin, PDGFRα, COUP-TFII, CD51 and CD90. We have shown that the proliferation of the SLCs is stimulated by DHH, FGF2, PDGFBB, activin and PDGFAA. Suppression of proliferation occurred with TGFβ, androgen and PKA signaling. The differentiation of the SLCs into testosterone-producing Leydig cells was found to be regulated positively by DHH (Desert hedgehog), lithium-induced signaling and activin; and negatively by TGFβ, PDGFBB, FGF2, Notch and Wnt signaling. DHH, by itself, was found to induce SLC differentiation into LH-responsive steroidogenic cells, suggesting that DHH plays a critical role in the commitment of SLC into the Leydig lineage. These studies, taken together, address the function and regulation of low turnover stem cells in a complex, adult organ, and also have potential application to the treatment of androgen deficiency., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In the testis, KIT ligand (KITL, also called stem cell factor) is expressed by Sertoli cells and its receptor (c-kit, KIT) is expressed by spermatogonia and Leydig cells. Although KITL-KIT signaling is critical for the spermatogenesis, its roles in Leydig cell development during puberty are not clear. In the present study, we investigated effects of KITL on stem Leydig cell proliferation and differentiation. Using an in vitro culture system of seminiferous tubules from Leydig cell-depleted testis, we found that KITL increased the proliferation activity of putative stem Leydig cells at higher concentration (10 and 100 ng/ml). Low concentration (1 ng/ml) of KITL significantly induced the differentiation of stem Leydig cells via increasing the expression level of steroidogenic acute regulatory protein (Star). In contrast, higher concentration (100 ng/ml) of KITL inhibited the differentiation of stem Leydig cells via inhibiting the steroidogenic enzyme (Cyp11a1, Cyp17a1, and Hsd17b3) expression levels. We cultured rat progenitor Leydig cells with KITL for 48 h and did not find any influence of KITL on the proliferation and androgen production of these cells. In conclusion, KITL is a growth factor that regulates the development of the stem Leydig cell., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Leydig cells (LCs) play crucial roles in producing testosterone, and their dysfunction leads to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Here, we report our success in reprogramming mice fibroblasts into LCs by expressing three transcriptional factors, Dmrt1, Gata4, and Nr5a1. The induced Leydig-like cells (iLCs) expressed steroidogenic genes, had a global gene expression profile similar to that of adult LCs, and acquired androgen synthesis capabilities. When iLCs were transplanted into rats or mice testes that were selectively depleted of endogenous LCs, the transplanted cells could survive and function in the interstitium of testis, resulting in the restoration of normal levels of serum testosterone. These findings demonstrate that the fibroblasts were able to be directly converted into iLCs by few defined factors, which may facilitate future applications in regenerative medicine., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vivo and in vitro studies were conducted to determine whether testosterone-producing Leydig cells are able to develop from cells associated with rat seminiferous tubules, interstitium, or both. Adult rat seminiferous tubules and interstitium were isolated, encapsulated separately in alginate, and implanted subcutaneously into castrated rats. With implanted tubules, serum testosterone increased through two months. Tubules removed from the implanted rats and incubated with LH produced testosterone, and cells on the tubule surfaces expressed steroidogenic enzymes. With implanted interstitial tissue, serum levels of testosterone remained undetectable. However, co-culture of interstitium plus tubules in vitro resulted in the formation of Leydig cells by both compartments. These results indicate that seminiferous tubules contain both cellular and paracrine factors necessary for the differentiation of Leydig cells, and that the interstitial compartment contains precursor cells capable of forming testosterone-producing Leydig cells but requires stimulation by paracrine factors from the seminiferous tubules to do so., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.

The capacity of Brown Norway rat Leydig cells to produce testosterone (T) decreases with aging. In a previous study, we reported that a new generation of Leydig cells can be restored in both young and old rat testes after a single injection of ethane dimethanesulfonate (EDS), and that the abilities of the new Leydig cells in young and old rats to produce T were equivalent. Our objective herein was to compare the steroidogenic fate of the new Leydig cells over time. Young (3 month-old) and old (18 month-old) rats were injected with EDS to eliminate the existing Leydig cells. Ten weeks after EDS, Leydig cells had been restored and T production by the new Leydig cells isolated from young and old rat testes was equivalent. Thirty weeks after EDS treatment of young rats, the ability of the new Leydig cells to produce T had not diminished from 10 weeks post-EDS. In contrast, at 30 weeks post-EDS, T production by new cells in old rat testes was reduced significantly from the 10-week level. Serum T levels at 10 and 30 weeks were consistent with Leydig cell T production. Serum LH levels did not differ in any group. Thus, although the Leydig cells restored to both young and old rats after EDS initially produced T at high, equivalent levels, the cells in the old testes did not maintain this ability. These results suggest that: 1) the cells from which new populations of Leydig cells are derived may differ depending upon the age of the rat; and/or 2) factors extrinsic to the new Leydig cells in young and old testes differ, and it is these differences that are responsible for reductions in T by the newly formed Leydig cells in the testes of old rats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aging in rodents and men is associated with reduced serum levels of testosterone and Leydig cell testosterone productions. To further investigate the mechanism by which Leydig cell testosterone production declines, the effect of knocking out Nrf2, a master regulator of phase 2 antioxidant genes, was examined. In wild-type mice, testosterone production and serum testosterone levels remained unchanged through middle age (8 months), but then were reduced significantly by old age (21-24 months). In contrast, serum testosterone levels and Leydig cell testosterone production were reduced significantly in the Nrf2-/- mice as early as middle age, and were reduced further in the aged mice. Reduced steroidogenesis in the knockout mice was associated with reduced antioxidant capacity, and increased expression of protein nitrotyrosine residues, a marker of ROS. These results support the hypothesis that, over time, increases in oxidative stress contribute to or cause the reduced testosterone production that characterizes Leydig cell aging., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Testosterone deficiency is associated with sickle cell disease (SCD), but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle) exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH) levels compared with wild type (WT) mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol)- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR), but not cholesterol side-chain cleavage enzyme (P450scc), in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi) exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function.

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., (© 2014 by the Society for the Study of Reproduction, Inc.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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-α, but not 3β-hydroxysteroid dehydrogenase (3β-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, 3β-HSD(neg) cells on the tubule surfaces underwent divisions. Some of these cells later expressed 3β-HSD and produced testosterone. Removal of the newly formed 3β-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.

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 (ΔΨ(m)), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ΔΨ(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 ΔΨ(m) in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ΔΨ(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., (2010 Wiley-Liss, Inc.)

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., (2010 Elsevier Ireland Ltd. All rights reserved.)

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.

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 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.

Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70% and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl-alpha-phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, whereas 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month old) and old (24 month old) rats were injected with BSO twice a day for 7 d, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70% and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and 17alpha-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone.

Studies of MA-10 Leydig cells have shown that intact mitochondria with active respiration are essential for LH-induced Leydig cell steroidogenesis. To further elucidate the role played by mitochondria in steroidogenesis, we examined the effects of the perturbation of the mitochondrial electron transport chain with myxothiazol (MYX) on testosterone production by primary cultures of Brown Norway rat Leydig cells. Analysis of the steroidogenic pathway revealed that cAMP production and the activities of each of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/C17-20 lyase, and 17beta-hydroxysteroid dehydrogenase were inhibited by MYX and that LH-stimulated testosterone production was suppressed. In contrast to the inhibition of LH-stimulated testosterone production by MYX, the incubation of Leydig cells with MYX in the absence of LH stimulated testosterone production. Although testosterone production was increased, steroidogenic acute regulatory protein was decreased in response to MYX, not increased as could be expected. Additional electron transport chain inhibitors had stimulatory effects on testosterone production that were similar to those of MYX, strongly suggesting that the effect of MYX on basal testosterone production is related to its effect on the mitochondrial electron transport chain. Finally, incubation of the cells with a combination of MYX and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrakis acetoxymethyl ester suppressed MYX-mediated increased basal steroidogenesis but had no effect on hydroxycholesterol-mediated steroidogenesis. Taken together, these results indicate that inhibition of the mitochondrial electron transport chain can block LH-stimulated testosterone production through suppression of a number of steps of the steroidogenic pathway but also stimulates basal testosterone production through a calcium-mediated mechanism.

Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF-alpha or IL-1beta also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

Previous studies have shown that testosterone production by the Leydig cells of aged Brown Norway rats is reduced from the relatively high levels produced by Leydig cells of young rats and that this reduction is not secondary to decreased serum luteinizing hormone concentration. The free radical theory of aging proposes that imbalance between pro-oxidants and the antioxidant defense system ultimately results in oxidative damage to cellular processes. With this in mind, we hypothesized herein that age-related reductions in steroidogenesis by Brown Norway rat Leydig cells may be associated with the impairment of the antioxidant defense system of these cells. To begin to test this hypothesis, we compared the activities and steady-state mRNA and protein levels of the antioxidant enzymes copper zinc (CuZn) superoxide dismutase (CuZnSOD, SOD1), manganese (Mn) superoxide dismutase (MnSOD, SOD2), and glutathione peroxidase (GPx) and the levels of reduced and oxidized glutathione in Leydig cells isolated from the testes of young (4-month-old) and aged (20-month-old) Brown Norway rats. For some studies, Leydig cells were isolated separately from aged testes that either had regressed because of age-related losses of germ cells or that were nonregressed. SOD (total) and GPx activities were found to decrease significantly with age whether or not the testes were regressed. CuZnSOD and MnSOD mRNA levels decreased with aging, though the magnitude of the decreases were considerably lower than the respective decreases in enzyme activities. GPx mRNA levels also decreased, which is consistent with the decreases seen in enzyme activity. MnSOD protein expression declined with age, and to a lesser extent, CuZnSOD did as well. Reduced and oxidized glutathione also exhibited age-related reductions in cells from both normal and regressed aged testes. The age-related decreases in Leydig cell antioxidant enzyme activities, gene expression, and protein levels and in glutathione were consistent with the hypothesis that the loss of steroidogenic function that accompanies Leydig cell aging may result in part from a decrease in the fidelity of the cellular antioxidant defense system.

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.

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 alpha 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., 11beta-hydroxysteroid dehydrogenase 2 (Hsd11b2); neurotransmitter receptors, e.g., acetylcholine receptor nicotinic alpha 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.

We have shown previously that testosterone concentration in the blood serum of Brown Norway rat becomes reduced with aging, and that this results from reduced testosterone production by individual Leydig cells. Herein we examine the effects of caloric restriction (CR), an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on Leydig cell steroidogenic function. CR (40%) was initiated in 4 month-old Brown Norway rats, and continued through age 34 months. Serum testosterone concentration in the ad libitum (AL)-fed controls was reduced by 30% from 5 to 13 months, by another 67% through 25 months, and then was sustained through 34 months. For the CR rats, the serum testosterone level was reduced to 45% of AL controls by 5 months, only 6 weeks after the initiation of the CR regimen. There was no further reduction through 25 months, at which time serum testosterone concentration in CR animals was significantly higher than in AL controls. By age 28-34 months, there was no significant difference between the two diets. The weights of prostate and seminal vesicle, two biomarkers of serum androgen levels, were consistent with the changes in serum testosterone concentration in both AL and CR animals. The ability of isolated Leydig cells to produce testosterone in vitro also paralleled the age- and CR-related changes in serum testosterone concentration. CR resulted in a rapid, 36% reduction in testosterone production from control by age 5 months. In contrast to cells from the AL rats, there were no further decreases in testosterone production through age 25 months. Indeed, Leydig cells from the 25 month-old CR rats produced significantly greater amounts of testosterone than cells from the 25 month-old AL rats. These results indicate that short-term CR results in the suppression of Leydig cell function and in reduction in serum testosterone levels. The significantly higher concentrations of serum testosterone concentration, and increased Leydig cell testosterone production, elicited by CR in 25 month-old rats compared to AL controls suggest that long-term CR can transiently suppress the reductions in steroidogenesis that are characteristic of aging.

Previous studies have shown that the capacity of Leydig cells from aged (21-24-month-old) Brown Norway rats to produce testosterone is reduced from young (4-month-old) levels, and that this is correlated with reductions in steroidogenic acute regulatory protein (StAR), peripheral benzodiazapine receptor (PBR), and the levels and activities of the steroidogenic enzymes. The age(s) at which particular changes in the steroidogenic pathway occur, and the relationship of particular changes to reduced testosterone production, are not known. We examined 3 critical components of the steroidogenic pathway, cyclic adenosine monophosphate (cAMP) production, StAR, and P450 side-chain cleavage enzyme (P450scc) in relationship to age-related decreases in testosterone production. Leydig cells isolated from Brown Norway rats of increasing ages (4, 9, 15, and 20 months) were evaluated. The ability of Leydig cells to produce testosterone was reduced at 9 months, although not significantly. Significant reductions in testosterone production were first seen in cells isolated from rats of 15 months of age, and further reductions occurred thereafter. Reduced testosterone was correlated with reductions in StAR, P450scc mRNA, and protein. Significant decline in luteinizing hormone-stimulated intracellular cAMP levels was seen by 9 months, before significant reductions in testosterone, StAR, and P450scc. Further declines in cAMP levels were seen at 15 and 20 months. These studies suggest that age-related reductions in intracellular cAMP may lead to the reduced testosterone production that characterizes aged Leydig cells. This suggestion is supported by recent studies from our lab demonstrating that long-term (3 days) culture of old Leydig cells with dbcAMP restored testosterone production to levels approximating those of young cells.

The wealth of knowledge about the function and regulation of adult Leydig cells, the cells within the mammalian testis that produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian aging is decreased serum testosterone concentration. In the Brown Norway rat, this has been shown to be associated with the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing the aged cells with dibutyryl cAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic acute regulatory protein and P450scc, the proteins involved in the rate-limiting steps of steroidogenesis. These results strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and that bypassing signal transduction reverses the steroidogenic decline by the aged cells.

In mammals, the concentration of testosterone in blood serum typically becomes reduced with aging. We have shown that, in rats, this results from reduced testosterone production by individual Leydig cells. To gain an understanding of the mechanisms by which Leydig cell steroidogenic function changes with aging, we compared the gene expression profiles in Leydig cells isolated from young and old Brown Norway rats, focusing on possible changes in genes involved in, or associated with, steroidogenesis. We also explored the effects of caloric restriction, an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on the expression of these genes. Of the total of 1176 genes arrayed on each of the five membranes per group that we used, about 500 (45%) were detectable in Leydig cells isolated from young and old rat testes, with the same genes detectable in cells from both ages. An ANOVA model was applied to statistically analyze the 500 detectable genes in the four treatment groups. The primary criterion by which interesting genes were selected was a P value of 1.5. However, when P1.5. Among the 500 genes, 17 were found to decrease in expression with aging, the most prominent among which were genes involved in steroidogenesis and stress response/free radical scavenging. Long-term caloric restriction had little effect on the 45 genes altered by age. In contrast to the effects of caloric restriction as seen in many other cell types and systems, the vast majority (44/45) of the Leydig cell genes that changed with age were not 'rescued' by this intervention.

The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.

Aging in Brown Norway rats is associated with reduced Leydig cell T production. To address the mechanism by which aging Leydig cells become steroidogenically hypofunctional, Leydig cells from young and old rat testes were isolated and cultured long-term with LH. Leydig cells isolated from young rats that had received LH-suppressive T implants served as positive controls. The ability of young control Leydig cells to produce T at high levels was sustained over a 3-d culture period. T production by cells from young LH-suppressed rats increased over this period, almost to control levels. In contrast, culture of the steroidogenically hypofunctional old Leydig cells with LH failed to increase their T production, suggesting that LH stimulation, by itself, is unable to reverse the steroidogenic deficits of old Leydig cells. Reduced numbers of LH binding sites characterized Leydig cells from old rats and LH-suppressed young rats. However, whereas Leydig cells from young LH-suppressed rats produced cAMP at the high levels of young control cells, the old cells produced far less cAMP, suggesting that old Leydig cells have defects in the LH-cAMP signaling cascade. When stimulated with forskolin, old cells produced the same amount of cAMP as young control and young LH-suppressed cells, suggesting that adenylate cyclase is maintained in the old cells. Taken together, these results suggest that inefficient signal transduction may explain the reduced steroidogenesis that characterizes old Leydig cells.

Simple Summary: Platelet-derived growth factor B (PDGFB) serves as an important cytokine for cell growth and development. Tibetan sheep (Ovis aries), a unique local breed in the Qinghai–Tibet Plateau, have poor reproductive physiological characteristics, including long developmental cycles, late sexual maturity, and low fecundity, yet the mechanisms behind remain elusive. Spermatogenesis and sperm maturation are highly dynamic and multifactorial developmental processes occur in the male testis and epididymis, respectively, and thus are crucial for maintaining male reproductive function and species continuity. Despite the importance of PDGFB, its role in maintaining the development and function of the testes and epididymides in sheep remains obscure. Herein, we report, for the first time, the cloning and molecular characterization of Tibetan sheep PDGFB. Meanwhile, we investigated the gene expression pattern and subcellular localization of PDGFB in developmental testes and epididymides in Tibetan sheep. The results revealed that the Tibetan sheep PDGFB gene has a high degree of evolutionary conservation and suggested that it participates in the development and function of testicular and epididymal red blood cells, Leydig cells as well as epididymal principal cells and ciliated epithelial cells, thus contributing to the spermatogenesis and sperm maturation of Tibetan sheep. Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep. [ABSTRACT FROM AUTHOR]