Konstantin Svechnikov

Origin, Development and Regulation of Human Leydig Cells

Sex steroids are crucial regulators of sexual differentiation and the proper development of secondary sex characteristics and patterns of sexual behavior. Since Leydig cells are the primary major producers of these steroid hormones, maintenance of the normal functions of these cells determines the reproductive capacity and fertility of males. The present minireview discusses recent findings concerning endocrine and paracrine regulation of the proliferation, differentiation and involution of human Leydig cells. The physiology and function of the two distinct fetal and adult populations of human Leydig cells are described, with particular focus on the paracrine environment that triggers their differentiation and functional maturation. The roles of established and more recently discovered paracrine regulators of this maturation, including insulin-like factor 3, platelet-derived growth factor-α, desert hedgehog, ghrelin and leptin are considered. A brief description of the origin, ontogenesis and functional markers of human fetal and adult Leydig cells is presented.

The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats.

Phthalate esters are known to exert harmful effects on mammalian reproduction and fertility, but their potential adverse effects on the hormonal functions of the ovary have not yet been elucidated in detail. Here, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the hypothalamic-pituitary-gonadal axis of young developing female rats, as well as on ex vivo steroidogenesis by granulosa cells (GCs) and secretion of LH by gonadotropes. Exposure of 20-day-old female rats to 500 mg DEHP by oral gavage once daily for 10 days reduced their serum levels of progesterone and estradiol, while tending to enhance levels of LH. Furthermore, primary cultures of GCs isolated from these rats exhibited an attenuated capacity to produce progesterone in response to stimulation by LH and FSH, as well as a lower degree of transport of endogenous cholesterol into mitochondria. Moreover, the ability of primary cultures of pituitary cells isolated from DEHP-treated rats to produce and secrete LH in response to GnRH was significantly enhanced. In addition, 2-ethylhexanoic acid, a metabolite of DEHP, significantly potentiated GnRH-stimulated production of LH by cultures of pituitary cells isolated from untreated 20-day-old female rats. Together, these data indicate that DEHP exerts dual effects on the pituitary-gonadal axis, stimulating the hormonal function of the pituitary and, at the same time, by inhibiting steroidogenesis by GCs.

Endocrine Disruptors and Leydig Cell Function

During the past decades, a large body of information concerning the effects of endocrine disrupting compounds (EDCs) on animals and humans has been accumulated. EDCs are of synthetic or natural origin and certain groups are known to disrupt the action of androgens and to impair the development of the male reproductive tract and external genitalia. The present overview describes the effects of the different classes of EDCs, such as pesticides, phthalates, dioxins, and phytoestrogens, including newly synthesized resveratrol analogs on steroidogenesis in Leydig cells. The potential impact of these compounds on androgen production by Leydig cells during fetal development and in the adult age is discussed. In addition, the possible role of EDCs in connection with the increasing frequency of abnormalities in reproductive development in animals and humans is discussed.

Inhibitory effects of mono-ethylhexyl phthalate on steroidogenesis in immature and adult rat Leydig cells in vitro

Di-2-ethylhexyl (DEHP) phthalate, one of the phthalates most widely distributed in our general environment, causes reproductive toxicity that is attributable to the action of its primary metabolite, mono(2-ethylhexyl) phthalate (MEHP). Here, we have investigated the effects of MEHP on steroidogenesis by primary cultures of immature and adult rat Leydig cells. In both cases MEHP (250muM) was found to inhibit stimulation of androgen production evoked by human chorionic gonadotropin (hCG). This was associated with decreased expression of steroidogenic acute regulatory (StAR) protein and reduced transport of cholesterol into mitochondria but no detectable adverse effect on steroidogenic enzymes. Moreover, upon exposure to MEHP alone, 5alpha-reductase activity was decreased in immature, but not in adult Leydig cells. All together, our findings demonstrate that MEHP exerts suppressive effects on hCG-activated steroidogenesis in primary cultures of immature and adult rat Leydig cells and suppresses 5alpha-reductase activity in immature and not of adult rat cells. This may partly explain the anti-androgenic effects of DEHP in vivo and indicate a higher susceptibility in younger subjects.

Effects of resveratrol analogs on steroidogenesis and mitochondrial function in rat Leydig cells in vitro.

Resveratrol and its analogs are considered to be a promising drug candidate for treatment of cancer and different age‐associated diseases. In the present study we have investigated the effects of resveratrol and its synthetic analogs on steroidogenesis and mitochondrial function in primary cultures of rat Leydig cells. Our findings indicate that resveratrol and its analogs structure‐dependently attenuated hCG‐activated steroidogenesis in Leydig cells through suppression of the expression of steroidogenic acute regulatory protein and cytochrome P450c17. 3,5‐Diacetyl resveratrol was observed to modulate mitochondrial function in Leydig cells, suppressing polarization of inner mitochondrial membrane, and 3,4,4′‐trimethoxystilbene stimulated the overall activity of intracellular reductases involved in the reduction of WST‐1 to formazan. Thus, the inhibitory actions of resveratrol analogs on steroidogenesis in Leydig cells indicate novel mechanisms of action of these compounds, which may be of potential therapeutic interest, where suppression of androgen action is needed. Copyright

Ontogeny of gonadal sex steroids

Sex steroids are crucial hormones for the proper development and function of the body; they regulate sexual differentiation, the secondary sex characteristics, and sexual behaviour patterns. Gonads are the major sources of sex steroids, although adrenal cortex, placenta, and to a lesser extent other tissues contribute to their production in adult life and at various phases of development. Steroidogenesis of gonadal sex hormones is by definition sexually dimorphic, and involves differences not only in hormonal action but also in regulation and temporal patterns of production. This review focuses on the ontogeny and developmental regulation of steroid hormones in the gonads, with an attempt to detail these processes in humans.

Similar causes of various reproductive disorders in early life

During the past few decades, scientific evidence has been accumulated concerning the possible adverse effects of the exposure to environmental chemicals on the well-being of wildlife and human populations. One large and growing group of such compounds of anthropogenic or natural origin is referred to as endocrine-disrupting chemicals (EDCs), due to their deleterious action on the endocrine system. This concern was first focused on the control of reproductive function particularly in males, but has later been expanded to include all possible endocrine functions. The present review describes the underlying physiology behind the cascade of developmental events that occur during sexual differentiation of males and the specific role of androgen in the masculinization process and proper organogenesis of the external male genitalia. The impact of the genetic background, environmental exposures and lifestyle factors in the etiology of hypospadias, cryptorchidism and testicular cancer are reviewed and the possible role of EDCs in the development of these reproductive disorders is discussed critically. Finally, the possible direct and programming effects of exposures in utero to widely use therapeutic compounds, environmental estrogens and other chemicals on the incidence of reproductive abnormalities and poor semen quality in humans are also highlighted.

Human testicular peritubular cells host putative stem Leydig cells with steroidogenic capacity.

AIM We aim to examine the steroidogenic phenotype and the differentiation potential of human testicular peritubular cells (HTPCs) and to explore their possible relationship to the adult Leydig cell lineage. BACKGROUND The cells of the adult Leydig cell lineage may reside in the peritubular compartment of the testis. This suggestion is supported by the facts that the rodent peritubular cells can be differentiated toward this lineage and that cAMP enhances their steroidogenic potential. METHODS Human testicular biopsies, and derived HTPCs, were analyzed by immunohistochemistry, RT-PCR, and Western blotting. After stimulation by forskolin or platelet-derived growth factor-BB, quantitative RT-PCR was used to compare the levels of mRNAs encoding proteins involved in steroidogenesis and steroid production was analyzed by liquid chromatography and tandem mass spectrometry. RESULTS Immunohistochemical analysis revealed that the peritubular cells that form the outer part of the tubular wall express platelet derived growth factor receptor-α. Furthermore, the pluripotency markers (POU domain class 5 transcription factor 1, GATA-binding protein 4), stem Leydig cell markers (platelet derived growth factor receptor-A, leukemia inhibitory factor receptor), and mRNAs encoding proteins involved in steroidogenesis (nuclear receptor subfamily 5, group A, member 1; steroidogenic acute regulatory protein; CYP11A1; CYP17A1; 3β-hydroxysteroid dehydrogenase) were expressed by the HTPCs. Stimulation with forskolin increased the expression of the steroidogenic markers, which was accompanied by the production of pregnenolone and progesterone by HTPCs in vitro. Treatment with platelet-derived growth factor-BB induced expression of steroidogenic acute regulatory protein. CONCLUSIONS Our results indicate that the tubular wall of the human testis is a reservoir for cells of the adult Leydig cell lineage and that the steroidogenic potential of these cells can be activated in culture.

Phenotype and steroidogenic potential of PDGFRα-positive rat neonatal peritubular cells.

Platelet-derived growth factor receptor α (PDGFRα)-positive peritubular cells (PTCs) are suggested to be putative stem Leydig cells. At present little is known about their phenotype and steroidogenic potential. We isolated highly purified PDGFRα-positive neonatal PTCs by magnetic cell sorting (MACS) from 8dpp rat testes and characterized them in vitro. We have demonstrated that PDGFRα-positive PTCs have a mixed phenotype. They expressed PTC-specific genes (αSma, Myh11), pluripotency markers (Pou5f1, nestin, Lifr) and genes encoding steroidogenic enzymes. Treatment with the cAMP-analog (Bu)2cAMP for 7 days upregulated steroidogenic enzyme gene expression and significantly increased their steroidogenic potential. The main end-point steroid was progesterone due to rapid inactivation of CYP17 and 17βHSD. Long-term culturing of PDGFRα-positive PTCs increased the expression of Myh11, and treatment with (Bu)2cAMP attenuated this process. All together, our findings support the hypothesis that neonatal PDGFRα-positive PTCs are steroidogenic competent progeny of stem Leydig cells (SLCs) which give rise to the adult Leydig cell lineage.

In vitro spermatogenesis - optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats

Although three-dimensional testicular cell cultures have been demonstrated to mimic the organization of the testis in vivo and support spermatogenesis, the optimal culture conditions and requirements remain unknown. Therefore, utilizing an established three-dimensional cell culture system that promotes differentiation of pre-meiotic murine male germ cells as far as elongated spermatids, the present study was designed to test the influence of different culture media on germ cell differentiation, Leydig cell functionality, and overall cell survival. Single-cell suspensions prepared from 7-day-old rat testes and containing all the different types of testicular cells were cultured for as long as 31 days, with or without stimulation by gonadotropins. Leydig cell functionality was assessed on the basis of testosterone production and the expression of steroidogenic genes. Gonadotropins promoted overall cell survival regardless of the culture medium employed. Of the various media examined, the most pronounced expression of Star and Tspo, genes related to steroidogenesis, as well as the greatest production of testosterone was attained with Dulbecco’s modified eagle medium + glutamine. Although direct promotion of germ cell maturation by the cell culture medium could not be observed, morphological evaluation in combination with immunohistochemical staining revealed unfavorable organization of tubules formed de novo in the three-dimensional culture, allowing differentiation to the stage of pachytene spermatocytes. Further differentiation could not be observed, probably due to migration of germ cells out of the cell colonies and the consequent lack of support from Sertoli cells. In conclusion, the observations reported here show that in three-dimensional cultures, containing all types of rat testicular cells, the nature of the medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation, due to the lack of proper organization of the Sertoli cells.