Iuliia Savchuk

Mouse leydig cells with different androgen production potential are resistant to estrogenic stimuli but responsive to bisphenol a which attenuates testosterone metabolism.

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E2) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E2 ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.

Phthalates Exert Multiple Effects on Leydig Cell Steroidogenesis

Humans are significantly exposed to phthalates via food packaging, cosmetics and medical devices such as tubings and catheters. Testicular Leydig cells (LCs) are suggested to be among the main targets of phthalate toxicity in the body. However, their sensitivity to phthalates is species-dependent. This paper describes the response of the LCs from different species (mouse, rat and human) to phthalate exposure in different experimental paradigms (in vivo, ex vivo and in vitro), with particular focus on mechanisms of phthalate action on LC steroidogenesis. A comprehensive analysis of the impact of phthalate diesters and phthalate monoesters on LCs in different stages of their development is presented and possible mechanisms of phthalates action are discussed. Finally novel, not yet fully elucidated sites of action of phthalate monoesters on the backdoor pathway of 5α-dihydrotestosterone biosynthesis in immature mouse LCs and their effects on steroidogenesis and redox state in adult mouse LCs are reported.

Mono-2-ethylhexyl phthalate stimulates androgen production but suppresses mitochondrial function in mouse Leydig cells with different steroidogenic potential

Numerous studies have reported on testicular toxicity of phthalates in different experimental paradigms and showed that Leydig cells (LCs) were one of the main targets of phthalate actions. Adverse effects of phthalates on LCs steroidogenesis have been attributed to their metabolites, monophthalates. This study focuses on investigation whether LCs responsiveness to monophthalates action is associated with their potential to produce androgens. We found that of 3 monophthalates investigated [ie, mono-2-ethylhexyl phthalate (MEHP), mono-n-butyl phthalate, and mono-n-benzyl phthalate] only MEHP caused biological effects on the mouse LCs function. This monophthalate stimulated basal steroidogenesis associated with upregulation of StAR protein expression with no effect on hCG-stimulated androgen production by LCs from CBA/Lac and C57BL/6j mouse genotypes were observed. Further, MEHP attenuated ATP production and increased superoxide generation by both phenotypes of mouse LCs that indicated on mitochondrial dysfunction induced by the monophthalate. All together, our data indicate that MEHP-mediated stimulation of steroidogenesis and perturbation in mitochondrial function are not associated with the capacity of the LCs to synthesize androgens. We suggest that this effect of MEHP observed in LCs of rodent origin needs to be taken into consideration in analysis of earlier start of puberty in boys and may highlight a possible influence of phthalates on reproductive health in males.

Prepubertal onset of obesity negatively impacts on testicular steroidogenesis in rats.

Obesity is a global health problem and impacts negatively on levels of testosterone and quality of sperm production. At present little is known about mechanisms that attenuate testicular function in obese males. Our study characterized testicular steroidogenesis and explored levels of relevant paracrine and hormonal factors in rats with short- and long-term obesity. We have found that obesity state increased serum levels of estradiol and leptin in both groups of obese rats and inhibited the expression of StAR and Cyp11a1 associated with low levels of intratesticular testosterone in rats with long-term obesity. Further, long-term obesity reduced the number of Leydig cells, increased the testicular levels of the proinflammatory adipocytokine TNFα and the number of testicular macrophages. All together, our data indicate that long-term obesity may cause chronic inflammation in the testis and negatively impacts on Leydig cell steroidogenesis.

Resveratrol inhibits steroidogenesis in human fetal adrenocortical cells at the end of first trimester

SCOPE Resveratrol has a diverse array of healthful effects on metabolic parameters in different experimental paradigms but has also potential to inhibit steroidogenesis in rodent adrenals. The aim of the present study was to characterize the effects of resveratrol on human fetal adrenal steroidogenesis at gestational weeks (GW) 9-12. METHODS AND RESULTS Adrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/mL) with or without resveratrol (10 μM) for 24 h. The production of steroids by HFAC was analyzed by gas and liquid chromatography coupled to tandem/mass spectrometry. The expression of steroidogenic enzymes at GW 9-12 was quantified by automated Western blotting. We observed that resveratrol significantly suppressed synthesis of dehydroepiandrosterone (DHEA), androstenedione and 11-deoxicortisol by ACTH-activated and unstimulated HFAC, which was associated with inhibition of the activities and expression of cytochromes 17α-hydroxylase/17,20 lyase (CYP17) and 21-hydroxylase (CYP21) in these fetal adrenocortical cells. CONCLUSION Our in vitro findings on the sensitivity of human fetal adrenal steroidogenesis to resveratrol at GW9-12 suggest that intake of this polyphenol at high doses by women who are at early stages of pregnancy is undesirable.

Androgenic potential of human fetal adrenals at the end of the first trimester

The onset of steroidogenesis in human fetal adrenal glands (HFA) during the first trimester is poorly investigated. An unresolved question is the capacity of the HFA to produce potent androgen DHT via conventional and/or the backdoor pathway(s) at the end of first trimester, when androgen-responsive organs are developed. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes and transcription factors in HFA at gestational weeks (GW) 9–12 with focus on their androgenic potential. Steroids in the HFA were analyzed by gas chromatography/mass spectrometry. The expression of steroidogenic enzymes and transcription factors in the HFA at GW9–12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that during GW9–12 HFA produced steroids of the ∆5, ∆4 and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17α-OH-dihydroprogesterone, androsterone and androstanedione without further conversion to DHT. The only androgens identified in the HFA were testosterone and androsterone, a precursor in the biosynthesis of DHT. We also observed higher levels of CYP17A1 but low expression of 3βHSD2 at GW11–12 in the HFA. Elevated levels of CYP17A1 were associated with an increased expression of SF-1 and GATA-6. Altogether, our data demonstrate that of those steroids analyzed, the only potent androgen directly produced by the HFA at GW9–12 was testosterone. The onset of steroidogenesis in the HFA is a complex process that is regulated by the coordinated action of related transcription factors.

Alternative (backdoor) androgen production and masculinization in the human fetus

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production but little is known about the synthesis of backdoor androgens despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human male fetuses using multi-dimensional and high-resolution mass-spectrometry. Results show that androsterone is the principal backdoor androgen in the fetal circulation and that DHT is undetectable (<1ng/ml). Backdoor pathway intermediates are found primarily in the placenta and fetal liver with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are mostly undetectable in the fetal testes. This is consistent with transcript levels of enzymes involved in the backdoor pathway (SRD5A1, AKR1C2/4, CYP17A1), as measured by qPCR. These data identify androsterone as the predominant backdoor androgen in the human fetus and show that it is formed primarily in non-gonadal tissue with placental progesterone the likely substrate. Masculinization of the human fetus depends, therefore, on androgen synthesis by both the fetal testes and non-gonadal tissues leading to DHT formation at the genital tubercle. Our findings provide, for the first time, a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans

Adipose Tissue is a Potential Source of Hyperandrogenism in Obese Female Rats: Testosterone Production in Adipose Tissue

Obesity in females is often associated with metabolic complications and hyperandrogenism, but the sources of androgens are not completely understood. Therefore, this study investigated whether adipose tissue could be a source of androgens promoting hyperandrogenism development in obese female rats.

The human genital tubercle is steroidogenic organ at early pregnancy

It is generally accepted that androgens produced by fetal Leydig cells (FLC) control proper masculinization of the male external genitalia. Here, we hypothesized that the human genital tubercle (GT) has potential to synthesize androgens independently of FLC at early pregnancy. We observed that human GT of both genders have capacity to synthesize steroids of the Δ4, Δ5 and alternative pathway of DHT synthesis including the androgen itself. The presence of steroids in the GT was associated with the expression of corresponding steroidogenic enzymes. Levels of steroids and the expression of steroidogenic enzymes were similar in the GT from male and female fetuses. In contrast to the GT, the human fetal testis synthesized DHT from testosterone but not via the alternative pathway. Our findings strongly suggest that the human GT at early pregnancy can synthesize DHT via the alternative pathway, which may play an important role in organogenesis of the urethra.

Impact of uteroplacental insufficiency on postnatal rat male gonad.

Prenatal events such as intrauterine growth restriction can affect gonadal development of the offspring and have an impact on reproductive health. To investigate the effects of intrauterine growth restriction induced by uterine artery ligation on the postnatal rat testis. Pregnant rats underwent uterine artery ligation at day 19 of gestation. Offspring were killed at 5, 20 and 40 days post-partum (dpp). At killing, one gonad was snap-frozen in liquid nitrogen and processed for RNA and steroid extraction. The other gonad was formalin-fixed for histology. Gene expression was analyzed by TaqMan Low-Density Array. Intratesticular testosterone, estradiol and serum gonadotrophins were measured. Thirty genes were dysregulated in intrauterine growth-restricted rats compared to controls, among which markers of Sertoli cell and Leydig cell function, cell metabolism and growth factors. Testis weights were significantly reduced at 5 and 20 dpp in intrauterine growth-restricted rats and caught-up by 40 dpp. Accordingly, Sertoli cell number was significantly lower in 5 dpp intrauterine growth-restricted rats. At 20 dpp, intratesticular testosterone was significantly increased in intrauterine growth-restricted rats, whereas serum gonadotrophins were unchanged. IUGR altered the gene expression in the rat testes up to peripubertal age and reduced testis size and Sertoli cell number in neonatal age. Multiple mechanisms encompassing genetic changes and steroid production by the testis may be involved in the catch-up growth phase that restored testis size by 40 dpp. Permanent consequences on organ function and gamete integrity cannot be excluded and deserve further investigations.