Diverse effects of endocrine-disruptive chemicals on Leydig and adrenocortical cell steroidogenesis in rodents and humans

We are living in a man-made world and often exposed to different chemicals known as endocrine disrupting chemicals (EDCs) that negatively influence our hormonal system. These compounds have the potential to interrupt a normal steroidogenesis and result in harmful effects on the reproductive health of humans and among other organisms. Several studies have demonstrated a considerable decrease in fertility biomarkers such as sperm counts, and increase in malformations and undermasculinization of the male reproductive tract of mammals including humans. Increasing incidence of cryptorchidism, hypospadias and micropenis in male babies may be a consequence of the detrimental effect of environmental chemicals on male fetuses due to disruption of the steroidogenic machinery in the adrenal glands and gonads. The general aim of this thesis was to examine the impact of selected EDCs on hormonal function of steroidogenic cells from mice and human. In papers 1 and 2 we made use of primary cultures of immature mouse Leydig cells (LCs) to probe their response to bisphenol A and mono-phthalates. The cells were incubated with or without human chorionic gonadotropin (hCG) and a selection of chemicals ranging in concentrations over a certain period of time. It was uncovered that immature mouse LCs with varying abilities to produce testosterone were unaffected by estrogenic stimuli. Moreover, they exhibited a similar response to mono-phthalates. Our study showed that bisphenol A is able to stimulate testosterone production in the hCG pre-treated immature mouse LCs. However, among three mono-phthalates, only MEHP significantly increased basal androgen production via upregulation of StAR expression. In addition, mono-phthalates disturb a normal mitochondrial function, by attenuating ATP generation and increasing super oxide synthesis. In papers 3 and 4 we had possibility to obtain human abortion material from terminated pregnancies at gestational week 9-12. In study 3, we used a primary culture of human adrenocortical cells, stimulated with or without adrenocorticotropic hormone (ACTH) with resveratrol treatment, whereas in paper 4, the whole tissue of the human fetal adrenal gland (HFA) was taken for analysis. We observed that resveratrol had a suppressive effect on the synthesis of androstenedione, dehydroepiandrosterone (DHEA) and cortisol by the primary culture of human fetal adrenocortical cells through inhibition of lyase activity of cytochrome Cyp17 and Cyp21 expression in these cells. With the help of GC-MS/MS expertize was detected that the HFA is limited to produce androgens to testosterone and androsterone at the first trimester of pregnancy. This finding correlates with an observation of fully activated CYP17A1-POR-CYB5 complex and the concurrent upregulation of transcription factors (SF1, GATA-6) and downregulation of HSD3B2. Altogether, our results indicated that environmental endocrine disruptive chemicals have diverse effect on androgen production in steroidogenic cells in rodents and humans. Bisphenol A and MEHP should be taken into consideration as possible causes of premature maturation in boys. In contrast, pregnant women at an early gestational stage should try to avoid taking resveratrol due its ability to suppress steroidogenesis in the human fetal adrenals

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

Funding: The study was supported by the following grants: Chief Scientist Office (Scottish Executive, CZG/4/742) (PAF and PJOS) (http://www.cso.scot.nhs.uk/funding-2/); NHS Grampian Endowments 08/02 (PAF and PJOS) and 15/1/010 (PAF, PF, US, and PJOS) (https://www.nhsgcharities.com/); the Glasgow Children’s Hospital Research Charity Research Fund, YRSS/PHD/2016/05 (NW, MB, PJOS, and PAF) (http://www.glasgowchildrenshospitalcharity.org/research/glasgow-childrens-hospital-charity-research-fund); the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 212885 (PAF) (https://ec.europa.eu/research/fp7/index_en.cfm); Medical Research Council Grants MR/L010011/1 (PAF and PJOS) and MR/K501335/1 (MB, PAF, and PJOS) (https://mrc.ukri.org/); and the Kronprinsessan Lovisas Foundation, “Stiftelsen Gunvor och Josef Anérs,” the “Stiftelsen Jane och Dan Olssons,” and the “Stiftelsen Tornspiran” (KS and OS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

Basal and hCG-stimulated testosterone and estradiol production by Leydig cells from C57BL/6j and CBA/Lac mice as well as serum levels of LH.

<p>(A, B) Isolated Leydig cells were incubated with hCG or standard medium (control) for 17h, after which the concentrations of testosterone and estradiol were determined by RIA. Each experiment was performed four times independently obtaining similar results. (C). The data are expressed as means ± S.E.M (n = 23–27). *P<0.05, **P<0.01 compared to CBA/Lac; ♣P<0.05 compared to basal (untreated control).</p

Strain-related variations in sex hormone levels and their ratio in different mouse genotypes.

<p>The data are expressed as means ± S.E.M (n = 7–15). *P<0.05, **P<0.01, ***P<0.001compared to CBA/Lac; ♣♣P<0.01, ♣♣♣P<0.01 compared to C57BL/6j.</p

The effect of agonists of estrogen receptors and xenoestrogen BPA on basal and hCG-stimulated testosterone production by Leydig cells from C57BL/6j and CBA/Lac mice.

<p>Leydig cells were incubated with the agonists alone (A) or co-treated with hCG for 17 h (B). Each experiment was performed independently five times with similar results. **P<0.01 compared to hCG treatment.</p

qPCR primer sequences and running conditions, bp-base pair.

<p>qPCR primer sequences and running conditions, bp-base pair.</p

Comparative expression of the family of estrogen receptor genes in Leydig cells from CBA/Lac and C57BL/6j mice.

<p>Data are expressed as fold change ± S.E.M for four independent RNA preparations. *P<0.05 compared to CBA/Lac.</p

Comparative expression of steroidogenic genes in Leydig cells from CBA/Lac and C57BL/6j mice.

<p>Data are expressed as fold change ± S.E.M for four independent RNA preparations. *P<0.05 compared to CBA/Lac.</p