Leydig Cell Steroidogenesis Research Articles

Prohibitin-1 plays a regulatory role in Leydig cell steroidogenesis

SummaryMitochondria are essential for steroidogenesis. In steroidogenic cells, the initiation of steroidogenesis from cholesterol occurs on the matrix side of the inner mitochondrial membrane by the enzyme P450scc. This requires cholesterol import from the cytoplasm through the outer mitochondrial membrane, facilitated by the StAR protein. The subsequent steps leading to P450scc remain elusive. Here we report that the male transgenic mice that expressed a mutant form of a mitochondrial protein prohibitin-1 (PHB1Tyr114Phe) from the Fabp-4 gene promoter displayed smaller testes, higher testosterone, and lower gonadotropin levels compared with PHB1-expressing and wild-type mice. A subsequent analysis of the testis and Leydig cells from the mice revealed that PHB1 played a previously unknown regulatory role in Leydig cell steroidogenesis. This includes a role in coordinating cell signaling, cholesterol homeostasis, and mitochondrial biology pertaining to steroidogenesis. The implications of our finding are broad as the initial stages of steroidogenesis are indistinguishable across steroidogenic cells.

Macroautophagy involved in testosterone synthesis in Leydig cells of male dairy goat (Capra hircus)

Testosterone is an important steroid hormone that is indispensable for male sexual development and the reproductive system. Leydig cells (LCs), where autophagy is extremely active, reside in the testicular interstitium and are the major sites of testosterone production. However, the ultrastructural characteristics and the functional role of autophagy in LCs of livestock remain unknown. In this study, the LCs of the dairy goats were investigated to identify the steroidogenic activity and autophagy levels at different ages of development by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). Morphological results showed that the steroidogenic activity (3β-HSD staining) and ultrastructural characteristics of the LCs were changed with increasing age. TEM results demonstrated that the organelles involved in testosterone synthesis, e.g., smooth endoplasmic reticulum, mitochondria, and lipid droplets, were abundantly distributed within the cytoplasm of LCs in pubertal and adult testes. Moreover, autophagy activity was enhanced in the testes at pubertal and adult stages compared with that at the juvenile stage. Several different autophagic vacuoles, including pre-autophagosomes, autophagosomes, and autolysosomes, were observed within the cytoplasm of LCs from pubertal and adult testes. However, immunofluorescent staining and TEM results showed that no typical lipophagic or mitophagic vacuoles were observed in the cytoplasm of LCs. Furthermore, primary LCs from dairy goats were used to study the effect of autophagy on testosterone production. After treatment with 3-methyladenine (3-MA, an autophagy inhibitor), the primary LCs decreased testosterone production. In contrast, treatment with rapamycin (an autophagy activator), enhanced steroidogenesis in LCs. Collectively, these in vivo and in vitro results suggested that autophagy activity is related to steroidogenesis in LCs of dairy goats, which may ultimately influence the spermatogenesis and fertility of these animals.

Mechanisms of MEHP Inhibitory Action and Analysis of Potential Replacement Plasticizers on Leydig Cell Steroidogenesis.

Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.

The role of antioxidants in the treatment of infertile men with varicocele

Varicocele is a common disease that occurs in 35–40% of men with primary infertility and in 80% of men with secondary infertility Disturbance of microcirculation in the testicles, which occurs during varicocele, lead to ischemia and hypoxia of the testicles, which provoke degenerative changes in all testicular cell populations. Retrograde blood flow from the testicular vein and the external spermatic vein into pampiniform venous plexus leads to an increase of hydrostatic pressure in the testicular vascular system and rise of temperature, and it also contributes to the development of sterile inflammation. Oxidative stress, nitrosative stress, and hypoxia are generally recognized elements of the pathogenesis of infertility associated with varicocele. It is known that when the supraphysiological level of reactive oxygen species in the ejaculate is reached, their concentration disrupts the structure and function of spermatozoa. Nitric oxide (NO) is involved in the regulation of body temperature, NO-dependent mechanisms may contribute to the increased scrotal temperature during varicocele. The physiological concentration of NO pro duced by testicular macrophages stimulates steroidogenesis in Leydig cells. However, in the presence of certain reactive oxygen species, NO can transform into peroxynitrite and other strong cytotoxic metabolites, which lead to dysregulation of testosterone synthesis involved in spermatogenesis. Also, manifestations of varicocele among infertile men have decreased expression levels of the testis-specific heat-shock protein, HSPA2, and abnormal methylation of sperm DNA. Therefore, the use of combined anti oxidant supplements as part of adjuvant therapy after surgical treatment or as an independent application in some clinical cases appears to be an adequate strategy to optimize fertility in patients with varicocele. In this review, we evaluated the importance of various antioxidants, such as vitamin E, folic acid, carnitine, inositol, arginine, acetylcysteine, zinc, selenium in spermatogenesis and their effect on reproductive function during varicocele.

Identification of novel genes and pathways regulated by the orphan nuclear receptor COUP-TFII in mouse MA-10 Leydig cells†.

In males, Leydig cells are the main producers of testosterone and insulin-like 3 (INSL3), two hormones essential for sex differentiation and reproductive functions. Chicken ovalbumin upstream promoter-transcription factors I (COUP-TFI/NR2F1) and COUP-TFII (NR2F2) belong to the steroid/thyroid hormone nuclear receptor superfamily of transcription factors. In the testis, COUP-TFII is expressed and plays a role in the differentiation of cells committed to give rise to fully functional steroidogenic adult Leydig cells. Steroid production has also been shown to be diminished in COUP-TFII-depleted Leydig cells, indicating an important functional role in steroidogenesis. Until now, only a handful of target genes have been identified for COUP-TFII in Leydig cells. To provide new information into the mechanism of action of COUP-TFII in Leydig cells, we performed microarray analyses of COUP-TFII-depleted MA-10 Leydig cells. We identified 262 differentially expressed genes in COUP-TFII-depleted MA-10 cells. Many of the differentially expressed genes are known to be involved in lipid biosynthesis, lipid metabolism, male gonad development, and steroidogenesis. We validated the microarray data for a subset of the modulated genes by RT-qPCR. Downregulated genes included hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), prolactin receptor (Prlr), nuclear receptor subfamily 0, group B, member 2 (Shp/Nr0b2), ferredoxin 1 (Fdx1), scavenger receptor class B, member 1 (Scarb1), inhibin alpha (Inha), and glutathione S-transferase, alpha 3 (Gsta3). Finally, analysis of the Gsta3 and Inha gene promoters showed that at least two of the downregulated genes are potentially new direct targets for COUP-TFII. These data provide new evidence that further strengthens the important nature of COUP-TFII in steroidogenesis, androgen homeostasis, cellular defense, and differentiation in mouse Leydig cells.

Sertoli cell-derived exosome-mediated transfer of miR-145-5p inhibits Leydig cell steroidogenesis by targeting steroidogenic factor 1.

In the mammalian testis, two distinct populations of Sertoli cells (SCs), the immature SCs (ISCs) and adult SCs (ASCs), play significant roles in regulating the development and function of Leydig cells. However, the effect of different SC types on the function of Leydig cells is poorly understood. Here, our study showed that miR-145-5p expression was significantly different in SCs at different stages, with the highest expression observed in ISCs. Exosomes mediate the transfer of miR-145-5p from ISCs to Leydig cells. Overexpression of miR-145-5p in Leydig cells significantly downregulated steroidogenic gene expression and inhibited testosterone synthesis. Additionally, miR-145-5p functioned by directly targeted steroidogenic factor-1 (Sf-1) and downregulated the expression of SF-1, which further downregulated the expression of steroidogenic genes, induced accumulation of lipid droplets, and eventually suppressed testosterone production. These findings demonstrate that SC-derived miR-145-5p plays a significant role in regulating the functions of Leydig cells and may therefore serve as a diagnostic biomarker for male hypogonadism developmental abnormalities during puberty.

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.

FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis.

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3β). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.

Slit/Robo signaling regulates Leydig cell steroidogenesis

BackgroundFirst identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored.MethodsImmunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice.ResultsSlit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism.ConclusionTaken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis.4VdBjKyUK_zz54UEEeomAMVideo

Testicular STAC3 regulates Leydig cell steroidogenesis through potentiating mitochondrial membrane potential and StAR processing

SH3 and cysteine-rich protein 3 (STAC3), a small adapter protein originally identified as a core component of excitation–contraction coupling machinery, regulates the voltage-induced Ca2+ release in skeletal muscle. However, the possibility of additional, as yet unknown, non-muscle effects of STAC3 cannot be ruled out. Herein, we provide the evidence for the expression and functional involvement of STAC3 in spermatogenesis. STAC3 expression was localized in the testicular interstitium of rodent and human testes. By using the cytotoxic drug ethylene dimethane sulfonate (EDS), STAC3 expression was observed to be decreased sharply in rat testis after selective withdrawal of Leydig cells (LCs), and reappeared immediately after LCs repopulation, indicating that testicular expression of STAC3 mainly stems from LCs. From a functional standpoint, in vivo lentiviral vector–mediated suppression of STAC3 resulted in a significant decrease in testosterone production, and thereafter caused impairment of male fertility by inducing oligozoospermia and asthenospermia. The indispensible involvement of STAC3 in testicular steroidogenesis was validated using the in vivo knockdown model with isolated primary LCs as well as in vitro experiments with primary LCs. By generating the TM3Stac3−/− cells, we further revealed that STAC3 depletion attenuated mitochondrial membrane potential and StAR processing in db-cAMP-stimulated LCs. Thus, the inhibitory effect of STAC3 deficiency on testicular steroidogenesis may be ascribed to a disturbed mitochondrial homeostasis. Collectively, the present results strongly suggest that STAC3 may function as a novel regulator linking mitochondrial homeostasis and testicular steroidogenesis in LCs. Our data underscore an unexpected reproductive facet of this muscle-derived factor.

Testis Toxicants: Lesson from Traditional Chinese Medicine (TCM).

The testis is one of the organs in the mammalian body that is sensitive to toxicants. Accumulating evidence has shown that human exposure to toxic ingredients in Traditional Chinese Medicine (TCM), such as triptolide, gossypol, cannabidol, piperine, α-solanine, matrine, aristolochic acid, and emodin, lead to testis injury and reproductive dysfunction. The most obvious phenotype is reduced sperm counts due to defects in spermatogenesis. Studies have also shown that Sertoli cells in the seminiferous tubule, the functional unit of the testis that supports spermatogenesis, are the cell type that is most sensitive to the disruptive effects of toxicants. Since Sertoli cells are the "mother cells" that nurture germ cell development, Sertoli cell injury thus leads to failure in germ cell development in the seminiferous epithelium. Mounting evidence has shown that the Sertoli cell cytoskeletons, mitochondria function, Leydig cells steroidogenesis pathways and sperm ion channels are some of the prime targets of toxicants from TCM. We carefully evaluate recent findings in this area of research herein, and to provide a summary of these findings, including someinsightful information regarding the underlying molecular basis of toxicant-induced testis injury that impede spermatogenesis.

Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells.

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3β-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.

Environmental doses of arsenic exposure are associated with increased reproductive-age male urinary hormone excretion and in vitro Leydig cell steroidogenesis

Humans are ubiquitously exposed to arsenic from multiple sources, and chronic arsenic exposure may be associated with male reproductive health. Although association regarding arsenic exposure and sex hormone secretion in blood has been reported, sex hormone excretion in urine studies is lacking. Urinary sex hormone excretion has emerged as a complementary strategy to evaluate gonadal function. Herein, we determined the associations between environmental exposure to arsenic and urinary sex hormone elimination and in vitro Leydig cell steroidogenesis. Concentrations of arsenic and testosterone (T), estradiol (E2) and progesterone (P) in repeated urine samples were determined among 451 reproductive-age males. Moreover, an in vitro Leydig cell MLTC-1 steroidogenesis experiment was designed to simulate real-world scenarios of low human exposure. Multivariable linear regression models were used to assess the associations of urinary arsenic levels with urinary hormones. Urinary arsenic concentrations were positively associated with urinary sex hormone (T, E2, and P) levels. An in vitro test further demonstrated that a population-based environmental exposure range (0.01–5 μM) of arsenic induced Leydig cell steroidogenesis potency. Our results indicate that low-dose arsenic exposure exhibits an endocrine disrupting effect by stimulating Leydig cell steroidogenesis and accelerating urinary steroid excretion, which extends previous knowledge of the inverse association of high-dose arsenic exposure with sexual steroid production that is assumed to be anti-androgen.

Endocrine-disrupting chemicals rapidly affect intercellular signaling in Leydig cells

A decline in male fertility possibly caused by environmental contaminants, namely endocrine-disrupting chemicals (EDCs), is a topic of public concern and scientific interest. This study addresses a specific role of testicular gap junctional intercellular communication (GJIC) between adjacent prepubertal Leydig cells in endocrine disruption and male reproductive toxicity. Organochlorine pesticides (lindane, methoxychlor, DDT), industrial chemicals (PCB153, bisphenol A, nonylphenol and octylphenol) as well as personal care product components (triclosan, triclocarban) rapidly dysregulated GJIC in murine Leydig TM3 cells. The selected GJIC-inhibiting EDCs (methoxychlor, triclosan, triclocarban, lindane, DDT) caused the immediate GJIC disruption by the relocation of gap junctional protein connexin 43 (Cx43) from the plasma membrane and the alternation of Cx43 phosphorylation pattern (Ser368, Ser279, Ser282) of its full-length and two N-truncated isoforms. After more prolonged exposure (24 h), EDCs decreased steady-state levels of full-length Cx43 protein and its two N-truncated isoforms, and eventually (triclosan, triclocarban) also tight junction protein Tjp-1. The disturbance of GJIC was accompanied by altered activity of mitogen-activated protein kinases MAPK-Erk1/2 and MAPK-p38, and a decrease in stimulated progesterone production. Our results indicate that EDCs might disrupt testicular homeostasis and development via disruption of testicular GJIC, a dysregulation of junctional and non-junctional functions of Cx43, activation of MAPKs, and disruption of an early stage of steroidogenesis in prepubertal Leydig cells. These critical disturbances of Leydig cell development and functions during a prepubertal period might be contributing to impaired male reproduction health later on.

BMI1 promotes steroidogenesis through maintaining redox homeostasis in mouse MLTC-1 and primary Leydig cells

ABSTRACT In males, aging is accompanied by decline in serum testosterone levels due to impairment of testicular Leydig cells. The polycomb protein BMI1 has recently been identified as an anti-aging factor. In our previous study, BMI1 null mice showed decreased serum testosterone and Leydig cell population, excessive oxidative stress and p16/p19 signaling activation. However, a cause-and-effect relationship between phenotypes and pathways was not investigated. Here, we used the rescue approach to study the role of oxidative stress or p16/p19 in BMI1-mediated steroidogenesis. Our results revealed that treatment with antioxidant NAC, but not down-regulation of p16/p19, largely rescued cell senescence, DNA damage and steroidogenesis in BMI1-deficient mouse MLTC-1 and primary Leydig cells. Collectively, our study demonstrates that BMI1 orchestrates steroidogenesis mainly through maintaining redox homeostasis, and thus, BMI1 may be a novel and potential therapeutic target for treatment of hypogonadism.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

SUN-719 The Impact of FOXA3 on Testicular Steroidogenesis

The Forkhead box(Fox) transcription factors are evolutionarily conserved in organisms and regulate diverse biological processes during development as well as adult life. Among the Fox family, FoxA subfamily members Foxa1-3 have been termed `pioneer’ transcription factors as they bind both nucleosome-bound and nucleosome-free DNA targets with the same recognition site. Foxa3 is the only member of FoxA subfamily that is expressed in both male and female gonads. In the testis, Foxa3 is expressed in spermatids and interstitial Leydig cells. We focused our study to elucidate the role of FOXA3 in Leydig cells and its impact on testicular steroidogenesis. Expression of FOXA3 dramatically decreased in mouse Leydig cells during testicular development. In addition, the time-dependent expression of FOXA3 showed an opposite pattern to that of steroidogenic genes in cAMP-induced primary Leydig cells. Meanwhile, Nur77 is among the prime regulators of steroidogenesis in the testicular Leydig cells. Overexpression of FOXA3 in MA-10 cells (mouse Leydig tumor cell line) repressed the cAMP-induced Nur77 promoter activity, which further resulted in the reduced activity of Nur77-target steroidogenic gene promoters (StAR, CYP17A1and 3β-HSD). Similar to above results, the expression of Nur77 and its target genes,StAR, 3β-HSD and CYP11A1, were repressed by adenovirus-mediated overexpression of FOXA3 in mouse primary Leydig cells, although the expression of CYP17A1, another steroidogenic gene, was differentially affected. These results suggest that FOXA3 locally regulates the expression of steroidogenic genes through Nur77 during testicular development.

Prohibitin‐1 plays a multifaceted role in mediating luteinizing hormone‐induced steroidogenesis in Leydig cells.

Leydig cells (LCs) present in the interstitium between seminiferous tubules in the testis are responsible for the biosynthesis of testosterone from cholesterol in response to luteinizing hormone (LH) from the pituitary. The principal pathways of testosterone biosynthesis, the identities of the main steroidogenic enzymes, and genetic disorders of most of these enzymes have been elucidated. However, many important questions related to LH signaling and cholesterol transport remain unknown. For example, the mechanisms of StAR‐mediated cholesterol transport from the outer mitochondrial membrane to cytochrome P450 side chain cleavage (P450scc or Cyp11a1) enzyme in the inner mitochondrial membrane, which is a critical step in steroidogenesis, remain elusive. It is conceivable that we are missing some important players involved in this process.Prohibitin‐1 (PHB1) is a pleiotropic protein with cell compartment‐specific functions, including the phosphorylation‐dependent membrane signaling and mitochondrial chaperone, which appears to require heterodimerization with its homologous protein prohibitin‐2 (PHB2) in the inner mitochondrial membrane. Previously, we have reported transgenic mouse models expressing PHB1 (PHB‐Tg) or Y114F‐PHB1 (mPHB‐Tg) from the fatty acid binding protein‐4 (Fabp4) gene promoter. During their phenotypic characterization, unexpectedly, we found that male mPHB‐Tg mice, but not PHB‐Tg mice, have high testosterone levels. Subsequent analysis revealed that it was due to leaky overexpression of m/PHB1 in LCs. This prompted us to investigate whether LH regulates PHB1 in LCs. A dose and time‐dependent effect of human chorionic gonadotropin, hCG (pituitary analog of LH) was found on PHB1 expression levels in LCs. To define the cell compartment‐specific role of PHB1 in LCs, we investigated the functional status of LH signaling in testes/LCs and ultrastructure of LCs from transgenic mice. A difference in phospho‐ERK levels was found between PHB‐Tg and mPHB‐Tg, which was higher in mPHB1 expressing cells. A similar effect was found in PHB1 manipulated MA‐10 cells (a model LC line). A parallel change in ultrastructural features of LCs (mitochondrial structure and lipid droplets) was found in PHB and mPHB overexpressing cells. Moreover, co‐immunoprecipitation experiments showed that PHB1 and PHB2 interact with P450scc and/or StAR. Analysis of PHBs protein sequences revealed the presence of multiple CRAC and/or CARC motifs (cholesterol recognition motifs) in both proteins. In aggregate, this finding suggests that PHB1 is a LH regulated protein in LCs and plays a multifaceted role in LH‐induced steroidogenesis, including ERK activation and potentially in the functional coupling of StAR and P450scc across mitochondrial membrane for steroidogenesis. The scope of our findings is broad because the basics of cholesterol transport across mitochondrial membranes are similar in different steroidogenic tissues.

Improvement of Testicular Steroidogenesis Using Flavonoids and Isoflavonoids for Prevention of Late-Onset Male Hypogonadism.

Androgen production, being important for male fertility, is mainly accomplished by the Leydig cells from the interstitial compartment of the testis. Testosterone plays a critical role in testis development, normal masculinization, and the maintenance of spermatogenesis. Within seminiferous tubules, appropriate Sertoli cell function is highly dependent on testicular androgen levels and is essential to initiate and maintain spermatogenesis. During aging, testosterone production by the testicular Leydig cells declines from the 30s in humans at a rate of 1% per year. This review outlines the recent findings regarding the use of flavonoids and isoflavonoids to improve testosterone production, contributing to normal spermatogenesis and preventing age-related degenerative diseases associated with testosterone deficiency. With the cumulation of information on the actions of different flavonoids and isoflavonoids on steroidogenesis in Leydig cells, we can now draw conclusions regarding the structure-activity relationship on androgen production. Indeed, flavonoids having a 5,7-dihydroxychromen-4-one backbone tend to increase the expression of the steroidogenic acute regulatory protein (StAR), being critical for the entry of cholesterol into the mitochondria, leading to increased testosterone production from testis Leydig cells. Therefore, flavonoids and isoflavonoids such as chrysin, apigenin, luteolin, quercetin, and daidzein may be effective in delaying the initiation of late-onset hypogonadism associated with aging in males.

Dioxin male rat reproductive toxicity mode of action and relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran characterized by fetal pituitary and testis transcriptome profiling

Fetal rat exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces epididymal sperm number involving altered pituitary-testicular hormonal signaling as the proposed mode-of-action (MOA). To evaluate this MOA and compare TCDD to 2,3,7,8-tetrachlorodibenzofuran (TCDF), an in utero rat exposure and study was conducted. Endpoints included congener tissue levels and transcriptomes of maternal liver and fetal liver, testis, and pituitary. Decreased gonadotropin subunit mRNAs levels (Lhb and Fshb) and enriched signaling pathways including GNRH Signaling and Calcium Signaling were observed in fetal pituitary after TCDD (but not TCDF) exposure. TCDD (but not TCDF) decreased fetal testis cholesterologenic and steroidogenic pathway genes. TCDD tissue concentrations in dam liver, dam adipose, and whole fetus were approximately 3- to 6-fold higher than TCDF. These results support a MOA for dioxin-induced rat male reproductive toxicity involving key events in both the fetal pituitary (e.g., reduced gonadotropin production) and fetal testis (e.g., reduced Leydig cell cholesterologenesis and steroidogenesis).