Developmental profiles of progesterone receptor transcripts and molecular responses to gestagen exposure during Silurana tropicalis early development

Abstract

Environmental gestagens are an emerging class of contaminants that have been recently measured in surface water and can interfere with reproduction in aquatic vertebrates. Gestagens include endogenous progestogens, such as progesterone (P4), which bind P4-receptors and have critically important roles in vertebrate physiology and reproduction. Gestagens also include synthetic progestins, which are components of human and veterinary drugs, such as melengestrol acetate (MGA). Endogenous progestogens are essential in the regulation of reproduction in mammalian species, but the role of P4 in amphibian larval development remains unclear. This project aims to understand the roles and the regulatory mechanisms of P4 in amphibians and to assess the consequences of exposures to environmental gestagens on the P4-receptor signaling pathways in frogs. Here, we established the developmental profiles of the P4 receptors: the intracellular progesterone receptor (ipgr), the membrane progesterone receptor β (mpgrβ), and the progesterone receptor membrane component 1 (pgrmc1) in Western clawed frog (Silurana tropicalis) embryos using real-time qPCR. P4-receptor mRNAs were detected throughout embryogenesis. Transcripts for ipgr and pgrmc1 were detected in embryos at Nieuwkoop and Faber (NF) stage 2 and 7, indicative of maternal transfer of mRNA. We also assessed the effects of P4 and MGA exposure in embryonic and early larval development. Endocrine responses were evaluated through transcript analysis of a suite of gene targets of interest, including: ipgr, mpgrβ, pgrmc1, androgen receptor (ar), estrogen receptor α (erα), follicle stimulating hormone β (fshβ), prolactin (prl), and the steroid 5-alpha reductase family (srd5α1, 2, and 3). Acute exposure (NF 12-46) to P4 caused a 2- to 5-fold change increase of ipgr, mpgrβ, pgrmc1, and ar mRNA levels at the environmentally relevant concentration of 195 ng/L P4. Acute exposure to MGA induced a 56% decrease of srd5α3 at 1140 ng/L MGA. We conclude that environmental exposure to P4 induced multiple endocrine-related transcript responses in amphibians; however, the differential responses of MGA suggest that the effects of MGA are not mediated through the classical P4 signaling pathway in S. tropicalis.

Agrochemicals disrupt multiple endocrine axes in amphibians

“…This study demonstrates a decrease of serum testosterone levels in mice after trenbolone exposure together with the downregulation of the expression of three genes (3b-HSD, P450scc and Cyp17a1) involved in steroidogenesis (Zhang S et al, 2019). Progesterone (P4) and its synthetic analogue, melengestrol acetate, a veterinary pharmaceutical used to suppress estrus and promote growth in cattle (Orlando and Ellerstad, 2014), were shown to alter expression of sex-steroid-related genes in S. tropicalis embryos (Thomson and Langlois, 2018). Relatively high concentrations of P4 have been implicated in malebiased sex ratios in amphibians (reviewed in Hayes, 1998).…”

NTP Research Report on the Scoping Review of Prenatal Exposure to Progestogens and Adverse Health Outcomes

“…No congenital malformations were observed following prenatal exposure to 17OHPC (five studies) (Carbone and Brent 1993;Hendrickx et al 1987;Johnstone and Franklin 1964;Schardein et al 2012;Seegmiller et al 1983), similar to its absence of effect in human studies. In contrast to the bird and fish models, no congenital malformations were reported in the studies evaluating progesterone in nonhuman mammalian models (Foote et al 1968;Harini et al 2009;Pointis et al 1987;Wharton and Scott 1964) or in an amphibian model (Thomson and Langlois 2018).…”

Steroid levels in frog eggs: Manipulations, developmental changes, and implications for maternal steroid effects

“…One consequence of this process appears to be that frog eggs contain relatively high amounts of various progestogens (Haccard et al, 2012; this study). From the perspective of embryonic development, progestogens might influence development via a number of direct (Bayaa et al, 2000; Thomson & Langlois, 2018) or indirect mechanisms. For progesterone specifically, direct effects may be mediated by the classical progesterone receptor (PGR) (Bayaa et al, 2000) or other receptors such as the progesterone receptor membrane component 1 (PGRMC1), both of which are present in at least some frog embryos (Thomson & Langlois, 2018).…”

“…From the perspective of embryonic development, progestogens might influence development via a number of direct (Bayaa et al, 2000; Thomson & Langlois, 2018) or indirect mechanisms. For progesterone specifically, direct effects may be mediated by the classical progesterone receptor (PGR) (Bayaa et al, 2000) or other receptors such as the progesterone receptor membrane component 1 (PGRMC1), both of which are present in at least some frog embryos (Thomson & Langlois, 2018). Indirect effects of progestogens may result from their conversion to other steroids.…”

“…After characterizing the abundance of steroids in eggs (see Section 3), we attempted a manipulation of initial pregnenolone and progesterone levels by immersing R. variabilis eggs in a steroid solution (Auperin & Geslin, 2008; Stratholt et al, 1997; Thomson & Langlois, 2018; Tokmakov et al, 2011). We selected R. variabilis for this manipulation because these frogs produce clutches more frequently than R. imitator and O. pumilio and we had more pairs available than for P. vittatus .…”