Sohlh1 and Sohlh2 Double Knockouts Reveal Cooperative Function in the Male and Female Germ Cell Differentiation Pathways.

Abstract

Proper maintenance of germ cells and their differentiation are essential determinants of reproductive life. We discovered two novel germ cell specific transcriptional regulators, Sohlh1 and Sohlh2. Sohlh1 and Sohlh2 encode basic helix-loop-helix transcriptional regulators and are essential in early folliculogenesis and spermatogonial differentiation, as shown by individual knockouts. Sohlh1 and Sohlh2 individual knockouts show remarkably similar phenotypes. Here we examined the phenotype and gene expression in mice that lack both Sohlh1 and Sohlh2 transcriptional regulators. Antibodies against Sohlh1 and Sohlh2 show similar pattern of expression in both male and female germ cells. In males, Sohlh1 and Sohlh2 are expressed in spermatogonia, and co-expressed with Plzf and c-Kit. In females, antibodies against Sohlh1 and Sohlh2 label the same population of oocytes. In order to determine whether Sohlh1 and Sohlh2 physically interact, we utilized testes extracts to show that Sohlh1 and Sohlh2 co-immunoprecipitate, sugessting that Sohlh1 and Sohlh2 heterodimerize. In addition, Sohlh2 expression is not completely abrogated in Sohlh1-/- mice and vice versa. These data imply that residual Sohlh1 and Sohlh2 proteins were produced in each others mutant, possibly alleviating a more severe phenotype. We therefore analyzed Sohlh1-/-Sohlh2-/- double knockout mice. Sohlh1-/-Sohlh2-/- newborn ovary retains as many oocytes as wild-type. By 3-week of age, the double mutant ovary contains few if any oocytes as single mutants. We also analyzed testes development in double mutants. In postnatal day 7 double mutant testes, there is no obvious difference as compared to the wild type but spermatogonial differentiation is disrupted at 2-week of age and beyond, a finding similar to single gene mutants. The spermatogonias of single or double mutant retain Plzf expression, and continue to proliferate as shown by BrdU incorporation. Thus histological analysis revealed that male and female double mutants are a phenocopy of Sohlh1-/- and Sohlh2-/-. However, microarray analysis of double mutant and single mutants show a synergistic effect of the combined Sohlh1 and Sohlh2 deficiency on gene expression. In testis, although there is statistically no significant difference between Sohlh1-/- and Sohlh2-/-, Sohlh1-/-Sohlh2-/- versus wild-type shows nearly 4-fold probe change as compared to the case of single mutants versus wild-type. These studies indicate that Sohlh1 and Sohlh2 are purely essential for oocyte and spermatogonia differentiation, and function either cooperatively or independently in those pathways. (platform)