Functional Analysis of <b><i>Mmd2</i></b> and Related PAQR Genes During Sex Determination in Mice

Abstract

<b><i>Introduction:</i></b> Sex determination in eutherian mammals is controlled by the Y-linked gene <i>Sry</i>, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking <i>Sry</i> action and male sex determination remain largely unknown. Here, we focused on <i>Mmd2</i>, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. <i>Mmd2</i> is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. <b><i>Methods:</i></b> We used CRISPR to generate mouse strains deficient in <i>Mmd2</i> and its 2 closely related PAQR family members, <i>Mmd</i> and <i>Paqr8</i>, which are also expressed during testis development. Following characterization of <i>Mmd2</i> expression in the developing testis, we studied sex determination in embryos from single knockout as well as <i>Mmd2;Mmd</i> and <i>Mmd2;Paqr8</i> double knockout lines using quantitative RT-PCR and immunofluorescence. <b><i>Results:</i></b> Analysis of knockout mice deficient in <i>Sox9</i> and <i>Nr5a1</i> revealed that <i>Mmd2</i> operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in <i>Mmd2</i>-null embryos. To determine if other genes might have compensated for the loss of <i>Mmd2</i>, we analyzed <i>Paqr8</i> and <i>Mmd</i>-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated <i>Mmd2</i>;<i>Mmd</i> and <i>Mmd2</i>;<i>Paqr8</i> double-null embryos and again observed normal testis development. <b><i>Discussion:</i></b> These results may reflect functional redundancy among PAQR factors, or their dispensability in gonadal development. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.