Novel regulators of human gonadal development

Abstract

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.