Gonad development and male gametogenesis

Abstract

Testicular and ovarian organogenesis involve a pathway of developmental genes which are differentially regulated in males and females. In mammals, sex determination is controlled by a dominant gene, SRY, located on the Y chromosome. Once testes are formed, they produce two major hormones, testosterone and anti-Müllerian hormone (AMH), allowing male sexual differentiation to take place. The female pathway is thought to be the default pathway i.e in the absence of SRY, the formation of ovaries takes place, followed by female sex differentiation.

The first evidence of sexual development is the appearance of primordial germ cells (PGCs) in the early postimplantation stages of mouse development around 7 dpc. These are derived from a small population of epiblastic cells, which migrate from the base of the allantois into the gonads. While migrating, and within the gonad, germ cells increase in number mitotically, so that by embryonic day 13.5 dpc there may be about 25,000 PGCs present. The male germ cells then arrest in the G0/G1 phase of the cell cycle until after birth when they resume mitosis and differentiate into spermatogonia. At puberty, these cells proliferate during spermatogenesis, go through meiosis and finally differentiate into mature spermatozoa. In the female gonad, meiosis is induced during the fetal stage and the oocytes are arrested in the diplotene stage. Little is known about the molecules involved in the interplay between somatic cells and germ cells, but the sex differentiation of the germ cells depends of the sex of the gonadal somatic cells.

In this thesis, the isolation and characterization of two novel mouse genes, Testatin and Mage-b4, is presented. Testatin is expressed mainly in pre-Sertoli cells of the early testis and has the characteristics of a secreted protein. According to its spatial and temporal expression pattern, testatin may be involved in different aspects of early testis development downstream of Sry. Mage-b4 expression is confined to fetal and adult germ cells. The normal function of the MAGE proteins is unknown but several members of this family of proteins are reported to have growthsuppressive functions. Based on the expression pattern, we hypothesized that Mage-b4 and its human counterparts, the human MAGE B genes were involved in germ cell differentiation. Therefore, infertile men were screened for mutations/deletions in MAGE B 1, B2 and B4. None of the patients (n=21) showed deviations from the reported sequence in any of the three genes.

For testis development, the only necessary gene on theY chromosome is the SRY gene. However, for normal spermatogenesis to take place, it has been postulated that at least three regions on the human Y chromosome are necessary, named azoospermia factor (AZF) a, b and c, each containing at least one functional spermatogenesis gene. To establish the prevalence of clinically relevant Y chromosome microdeletions among infertile men, we screened 192 men with disturbed spermatogenesis. Of these 192, 139 were azoospermic and 53 oligozoospermic (<5 million spermatozoal ejaculate). None of the oligozoospermic men and only four, less than three percent, of the azoospermic men, had deletions of the Y chromosome. Further, the expression pattern of the human AZFb candidate gene, RBMY l was investigated in order to elucidate its function during spermatogenesis. The protein was localized to the nucleus of Sertoli cells and in all stages of germ cells except elongating spermatids.