Elsevier

Gene

Volume 593, Issue 1, 15 November 2016, Pages 167-171
Gene

Research paper
Elevated expression of steroidogenesis pathway genes; CYP17, GATA6 and StAR in prenatally androgenized rats

https://doi.org/10.1016/j.gene.2016.07.067Get rights and content

Abstract

It is believed that excess androgen exposure of the fetus, via altered gene expression, causes hyperandrogenism a key feature of polycystic ovary syndrome (PCOS). The aim of this study was to evaluate expression of Cytochrome P450-17 (CYP17), GATA-binding protein (GAGT6) and Steroidogenic acute regulatory protein (StAR), genes of adult female rats prenatally exposed to androgen excess, closely reflect endocrine and ovarian disturbances of PCOS in women, by comparing them during different phases of estrus cycle with those of non-treated rats. Both the adult prenatally testosterone exposed and control rats (n = 23, each) were divided into four groups based on their observed vaginal smear (proestrus, estrus, metestrus and diestrus) and the relative expression of CYP17, GATA6 and StAR genes was measured in ovarian theca cells using Cyber-green Real-Time PCR. Serum sex steroid hormones and gonadotropins levels were measured using the ELISA method; a comparison of these two groups showed that there was an overall increase in the studied genes (CYP17; 2.39 fold change, 95% CI: 1.23–3.55; P < 0.05, GATA6; 2.08 fold change, 95% CI: 1.62–2.55; P < 0.0001, and StAR; 1.4 fold change, 95% CI: 1.02–1.78; P < 0.05), despite variations in different phases with maximum elevation for all genes in diestrus. The changes observed may impair the normal development of ovaries that mediate the programming of adult PCOS.

Introduction

Polycystic ovarian syndrome (PCOS), a complex hormonal disorder involving the reproductive and metabolic systems (Silva Dantas et al., 2013), affects 15%–20% of women in reproductive age (Sirmans and Pate, 2013). The syndrome is characterized by chronic anovulation and hyperandrogenism in the absence of specific diseases of the ovaries, adrenals, and the pituitary gland (Dunaif and Thomas, 2001). Based on familial clustering and studies on the prevalence of syndrome in twins, interaction of genetic and environmental factors in the pathogenesis of PCOS has been widely accepted (Lunde et al., 1989, Diamanti-Kandarakis et al., 2006, Vink et al., 2006).

Given the clinical heterogeneity and complex nature of the syndrome, development of animal models is the best approach to understand pathophysiologic mechanisms associated with the early etiology of PCOS that are difficult to resolve from human studies.

Emerging research findings confirm the role of excess fetal androgen as a main environmental factor, in development of the syndrome in adulthood (Barnes et al., 1994, Davies and Norman, 2002, Abbott et al., 2007). Therefore, several animal models have been developed in an attempt to understand the potential contribution of exposure to excess steroids on the development of this syndrome (Abbott et al., 2008, Walters et al., 2012). Useful animal model, should ideally replicate key features associated with human PCOS.

Ovarian hyperandrogenism is a key feature of PCOS (Carmina, 2002). PCOS theca cells showing remarkable increased androgen biosynthesis and elevated levels for expression of steroidogenic enzymes, including Cytochrome P450-17 (CYP17) and GATA-binding protein (GAGT6) (Nelson et al., 1999, Wood et al., 2003).

CYP17 enzyme mediates the conversion of pregnenolone or progesterone to 17α-OH pregnenolone or 17α-OH progesterone by 17α-hydroxylation, then cleavages the c17, 20 bonds of these to produce androgens DHEA or androstenedione in rodent gonads and androgens and corticosteroids in human gonads and adrenals (Miller, 2002, Rainey et al., 2002). The other function of CYP17 is 17, 20-lyase activity (Hall, 1986). GATA-6, as one of the six members of evolutionarily conserved GATA family of transcription factors, plays crucial roles in the development and differentiation of all eukaryotic organisms in vertebrates (Viger et al., 2008), accompanied by GATAs 4 and 5, mainly found in tissues of mesodermal and endodermal origin (heart, gut, and gonads) (Molkentin, 2000). Since, GATA6 specifically, is found in both post-mitotic germ cells and the ovarian epithelium of fetal rat ovary (LaVoie et al., 2004), it may have a role in primary ovarian morphogenesis, it also enhances transcription of steroid-metabolizing enzymes required for producing DHEA and the expression of enzymes needed to produce adrenal androgens in the adrenal (Jimenez et al., 2003). The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation, is facilitated by mediators such as the steroidogenic acute regulatory (StAR) protein (Rone et al., 2009). StAR, as a member of the high-affinity lipid and sterol carriers family (Stocco, 2001), is primarily present in all tissues producing steroids, including the adrenal cortex, gonads, brain and the nonhuman placenta (Bhangoo et al., 2006).

We have previously introduced a rat model for PCOS which convincingly resembling endocrine and ovarian disturbances similar to PCOS, with minimal morphological disorders in the reproductive system in response to a single dose of testosterone on gestational day 20 (Tehrani et al., 2014). Different aspects of the animal model have been described elsewhere (Daneshian et al., 2015, Noroozzadeh et al., 2015). In this study, we examined whether this prenatal androgen excess alters the expression of the aforementioned critical genes, involved in the steroidogenesis pathway that may lead to development of polycystic ovary syndrome.

Section snippets

Animals

Animals were handled in accordance with the ethical principles of laboratory animal care. This study approved by the local ethics committee of the Research Institute for Endocrine Sciences (RIES; 320 EC 90.09.07). Female adult Wistar rats (n = 16, age 75–95 days and body weight 170–180 g) were obtained from the RIES animal facility of the Shahid Beheshti University of Medical Sciences (Tehran, Iran) and Pasteur Institute, Karaj Production Center (Karaj, Iran). One male and one female rat were kept

Hormonal profile

Table 2, Table 3 show the results of comparison of hormone levels between the control and PNA adult female. Regardless of estrous cycle phases, in the PNA group testosterone and LH levels significantly increased while the FSH level decreased (P < 0.05). LH/FSH ratio proportionally had a significant increase in the PNA group. These trends were also observed in estrous cycle phases although in some phases the differences were not significant.

Relative gene expression

Compared to the control group, there was an overall

Discussion

Results of the present study show that prenatal androgen excess in rats causes increased expression of the CYP17, GATA6 and StAR, genes involved in steroidogenesis pathway in female offspring.

Despite the significant role of genetic factors in the development of PCOS, the etiologic details and key genes of this syndrome have not been clearly established. Animal models are one of the best, most useful methods to study of PCOS as many of them have demonstrated the presence of fetal programming and

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