Molecular Biology of Germline Determination and Gametogenesis

Germ Cell Development and Male Infertility: Three percent of men are infertile because of severe defects in sperm production. In few cases has the cause of spermatogenic failure been identified. We found that a particular portion of the Y chromosome is deleted de novo in 13% of men with no sperm in semen. These deletions define a region in which should be found one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). These infertile men are otherwise healthy, suggesting that AZF is a "pure male sterile" locus. In the absence of AZF , spermatogenic output is diminished or extinguished, and in the more severe cases the testes contain no germ cells. We suspect that AZF may facilitate differentiation of primordial germ cells into spermatogonial stem cells or influence the destiny of these stem cells, which in normal males confront three alternative fates: proliferation, degeneration, or differentiation. The deletions encompass a gene, DAZ (Deleted in AZoospermia), which is expressed specifically in spermatogonia (and their immediate descendants, primary spermatocytes) and appears to encode an RNA binding protein. In Drosophila, a gene similar to DAZ is required for spermatogenesis. We are exploring the possibility that DAZ is AZF.

ZFX and ZFY: These genes, located on the X and Y chromosomes of all placental mammals, encode distinct but closely related proteins containing a highly acidic domain and 13 zinc fingers. The proteins likely function as sequence-specific activators of transcription. ZFY and ZFX were originally implicated in sex determination, but present evidence argues against such a role. Instead, our gene targeting experiments in mice suggest that ZFX contributes to early embryonic growth, animal size, and germ cell number in both males and females. Studies of these genes may also shed light on the evolution of X inactivation: while the mouse Zfx gene is X-inactivated, human ZFX is expressed on both "active" and "inactive" human X chromosomes.

Sex Determination: We are exploring mutations that cause gonadal sex reversal in humans or mice. By deletion studies of human XX males and XY females, we found that whether an embryo develops testes or ovaries is determined by the presence of less than 0.5% of the Y chromosome. SRY, a gene within this region, plays a pivotal role in gonadal sex determination. Other XY females, some human and some murine, appear to have intact Y chromosomes and are being studied for clues as to the identity of autosomal sex-determining genes operating upstream or downstream of SRY. In collaboration with Eva Eicher (Jackson Laboratory, Bar Harbor), we have carried out genetic linkage studies of XY sex reversal in mice. We have obtained evidence for and are presently localizing sex-determining genes on mouse chromosomes 2 and 4.

Turner Syndrome: Turner syndrome, classically associated with an XO karyotype, is a complex human phenotype of low viability in utero, short stature, ovarian failure, and somatic anatomic defects. Turner syndrome appears to be the result of monosomy for one or more genes common to the X and Y chromosomes. By deletion analysis, we identified a 100-kilobase-pair portion of the Y chromosome that probably contains one or more Turner genes. Within this region, we identified one gene, RPS4Y. A homolog on the X chromosome, RPS4X, escapes X inactivation. RPS4Y and RPS4X encode isoforms of ribosomal protein S4. Ribosomes from human male tissues contain both the X and Y-encoded protein isoforms, which are functionally interchangeable. We are testing the possibility that certain Turner features result from reduced protein synthetic capacity in embryos with only one RPS4 gene per cell.