Generation and mutational analysis of a transgenic mouse model of human SRY.

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage the pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive testis determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY's three domains (N-terminal, HMG box, and C-terminal) and performed a detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and present a versatile and powerful system in which to functionally analyze variations of SRY including known and novel pathogenic variants found in DSD.

SOX4 regulates gonad morphogenesis and promotes male germ cell differentiation in mice.

The group C SOX transcription factors SOX4, -11 and -12 play important and mutually overlapping roles in development of a number of organs. Here, we examined the role of SoxC genes during gonadal development in mice. All three genes were expressed in developing gonads of both sexes, predominantly in somatic cells, with Sox4 being most strongly expressed. Sox4 deficiency resulted in elongation of both ovaries and testes, and an increased number of testis cords. While female germ cells entered meiosis normally, male germ cells showed reduced levels of differentiation markers Nanos2 and Dnmt3l and increased levels of pluripotency genes Cripto and Nanog, suggesting that SOX4 may normally act to restrict the pluripotency period of male germ cells and ensure their proper differentiation. Finally, our data reveal that SOX4 (and, to a lesser extent, SOX11 and -12) repressed transcription of the sex-determining gene Sox9 via an upstream testis-specific enhancer core (TESCO) element in fetal gonads, raising the possibility that SOXC proteins may function as transcriptional repressors in a context-dependent manner.

Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

Functional Analysis of Mmd2 and Related PAQR Genes During Sex Determination in Mice.

INTRODUCTION: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. Mmd2 is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. METHODS: We used CRISPR to generate mouse strains deficient in Mmd2 and its 2 closely related PAQR family members, Mmd and Paqr8, which are also expressed during testis development. Following characterization of Mmd2 expression in the developing testis, we studied sex determination in embryos from single knockout as well as Mmd2;Mmd and Mmd2;Paqr8 double knockout lines using quantitative RT-PCR and immunofluorescence. RESULTS: Analysis of knockout mice deficient in Sox9 and Nr5a1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in Mmd2-null embryos. To determine if other genes might have compensated for the loss of Mmd2, we analyzed Paqr8 and Mmd-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. DISCUSSION: 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.

GADD45A and CDKN1A are involved in apoptosis and cell cycle modulatory effects of viscumTT with further inactivation of the STAT3 pathway.

ViscumTT, a whole mistletoe preparation, has shown synergistic induction of apoptosis in several pediatric tumor entities. High therapeutic potential has previously been observed in Ewing's sarcoma, rhabdomyosarcoma, ALL and AML. In this study, we analyzed modulatory effects on the cell cycle by viscumTT in three osteosarcoma cell lines with various TP53 statuses. ViscumTT treatment induced G1 arrest in TP53 wild-type and null-mutant cells, but S arrest in TP53 mutant cells. Blockage of G1/S transition was accompanied by down-regulation of the key regulators CDK4, CCND1, CDK2, CCNE, CCNA. However, investigations on the transcriptional level revealed secondary TP53 participation. Cell cycle arrest was predominantly mediated by transcriptionally increased expression of GADD45A and CDKN1A and decreased SKP2 levels. Enhanced CDKN1A and GADD45A expression further played a role in viscumTT-induced apoptosis with involvement of stress-induced MAPK8 and inactivation of MAPK1/3. Furthermore, viscumTT inhibited the pro-survival pathway STAT3 by dephosphorylation of the two sites, Tyr705 and Ser727, by down-regulation of total STAT3 and its direct downstream targets BIRC5 and C-MYC. Moreover, tests of the efficacy of viscumTT in vivo showing reduction of tumor volume confirmed the high therapeutic potential as an anti-tumoral agent for osteosarcoma.