Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles

Abstract

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.

Zebrafish Androgen Receptor: Isolation, Molecular, and Biochemical Characterization1

“…To minimize variation due to differences in RNA loading, each sample was normalized to the expression level of the control gene, beta actin (bactin). As the expression of bactin varied over developmental time, to account for differences in its expression during early stages and adult liver and muscle, the level of expression within each group of samples was corrected by previously described formula [47][48][49]. The formula is as follows: individual value within a group/(mean value within a group/mean value of randomly selected control group).…”

Use of Early Juvenile Zebrafish Danio Rerio for In-Vivo Assessment of Endocrine Modulation by Xenoestrogens

“…Filby et al [11] also showed down-regulation of star mRNA in adult male fathead minnow exposed to 10 ng EE2 L -1 but no significant changes in exposed females. In vitro cultures of zebrafish ovarian follicles at different maturation stages exposed to human chorionic gonadotropin revealed an increase in expression of star, cyp11a1 and hsd3b1 with mature stages, in contrast with a decreasing pattern without exposure [12]. In this study, the mRNA levels of cyp11a1 and hsd3b1 were relatively lower for all exposure concentrations with lowest relative expression for 25 ng EE2 L -1 at 59% and 45% respectively (Figure 2b).…”

“…Key enzymes in steroidogenesis include steroidogenic acute regulatory (Star) protein, cytochrome P450 side-chain cleavage (P450scc) or Cyp11a1, 3-beta-hydroxysteroid dehydrogenase (Hsd3b1), and cytochrome P450 aromatase (P450arom) or Cyp19a1 ( Figure 1). Other studies have also discussed regulation in the steroidogenic pathway with gene expression analyses with adult fathead minnow [11] and zebrafish (in vitro, [12]; in vivo, [13,14]. Immunohistochemical studies showed the significance of (endogenous) sex steroids on gonadal sex differentiation in teleosts [9,15].…”

Cloning, characterization, and expression analysis of a putative 17 beta-hydroxysteroid dehydrogenase 11 in the abalone, Haliotis diversicolor supertexta

“…They summarized some of the steroid-related genes in mollusks, such as 17␤-HSD types 3, 5, and 9. However, the other members of the 17␤-HSD family (for example, type 11) have not been functionally characterized and their genes have not been cloned. In our previous work, we demonstrated that 17␤-HSD type 12 participates in steroid-mediated physiological processes in abalones (a marine mollusk) [15].…”

“…In recent years, 17␤-HSDs have also been found to participate in biological functions that are important for steroid metabolism in aquatic fish, such as the zebrafish and Japanese eel [11,12]. Increasing numbers (or isoforms) of the 17␤-HSD family are being isolated and identified because of their diversity [13], but, to the best of our knowledge, relatively little information is known about 17␤-HSDs in mollusks.…”