Identification of Ovarian Genes Regulated by Follicle-Stimulating Hormone During Early Secondary Oocyte Growth in Salmon.

Abstract

It is well established in mammals that follicle-stimulating hormone (FSH) plays a major role in regulating oocyte growth and development by modulating the expression of follicle cell and oocyte factors. In fishes, however, little is known about the function(s) of FSH and regulation of early oocyte growth, a period of development that may affect later egg quality and fecundity. In species that exhibit synchronous gonad development (e.g., salmonids, catfish, striped bass), plasma FSH and/or pituitary gonadotropin subunit expression increases significantly during early secondary oocyte growth and continues to rise during vitellogenesis, suggesting FSH may play a role in regulating follicle development during this period. The goal of this study was to gain a fundamental understanding of how FSH acts at the level of the fish ovary by identifying FSH regulated ovarian genes. To examine time course effects of FSH, coho salmon (Oncorhynchus kisutch) previtellogenic ovarian follicles were cultured with or without 500 ng salmon FSH for 12-72 h. Culture media was collected for estradiol-17beta (E2) measurement, ovarian RNA was isolated, and abundance of candidate oocyte and follicle cell mRNAs was measured by quantitative PCR. Medium E2 levels were significantly elevated in response to FSH by 12 h and plateaued after 48 h. Messenger RNAs encoding steroidogenic enzymes, such as steroid acute regulatory protein (star) and 3beta-hydroxysteroid (hsd3b) were upregulated by 12 h, while P450 aromatase (cyp19a1a) showed no change in response to FSH. Other putative follicle cell transcripts such as FSH receptor (fshr) and lipoprotein lipase (lpl) were depressed initially by FSH treatment and later recovered to control levels. Putative oocyte transcripts such as zona pellucida glycoproteins, alveolin, and a C-type egg lectin showed no change over the 72-h experiment. Additional FSH regulated ovarian genes were subsequently identified through construction of four suppression subtractive hybridization (SSH) libraries with RNA isolated from FSH-treated and untreated whole ovarian follicles, as well as follicle/interstitial cell enriched ovarian tissues. Many of the genes identified by SSH are involved in signal transduction, cell cycle regulation, cell growth and proliferation, apoptosis, cholesterol metabolism and steroidogenesis. Experiments utilizing steroidogenesis inhibitors are underway to distinguish ovarian genes directly regulated by FSH versus those regulated by sex steroids produced in response to FSH. As evident by the rapid and significant increase in E2 release and changes in steroidogenic enzyme gene expression within the ovary, we can conclude that FSH strongly influences steroidogenesis of the salmon ovary during early secondary oocyte growth and may be a critical signal for puberty onset. This is the first comprehensive assessment of the biological actions of FSH in the fish ovary. Discoveries will aid in developing methods to reduce common reproductive dysfunctions in aquaculture, such as delayed puberty and poor egg quality. This project was supported in part by National Research Initiative Competitive Grant no. 2007-35203-18082 from the USDA Cooperative State Research, Education, and Extension Service to JAL. (platform)