Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

Abstract

26The nuclear receptor NR5A1 (also known as Ad4BP, or SF1) is essential for the initial steps 27 of mammalian gonadal development. The Nr5a1 gene is equally expressed in XX and XY 28 gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in 29 XX gonads. We recently reported a case of 46, XX disorder of sex development (DSD) in 30 which ectopically expressed NR5A1 in XX gonads led to an ovo-testicular phenotype, 31 suggesting that excess NR5A1 can direct the development of immature XX gonads towards 32 testicular formation. However, a direct causal relationship has not been demonstrated in an 33 animal model. Here, using a Wt1-BAC (bacterial artificial chromosome) transgene system, 34 we generated two lines of mice overexpressing Nr5a1 in the fetal gonads at different levels.35 One of these lines (Tg-S), highly expressing Nr5a1, revealed that enforced Nr5a1 expression 36 alone is insufficient to switch the fate of the 46,XX gonads toward testicular formation in 37 mice. In the other line (Tg-A) expressing Nr5a1 at lower level, ovarian development was 38 compromised, with multi-oocyte follicles, reduced number of matured follicles, and impaired 39 expression of Wnt4, resulting in late onset infertility at 20 weeks after birth. The phenotype 40 was similar to that of genetically modified mice with impaired Notch signaling. Indeed, the 41 expression level of Notch2 and 3 was significantly reduced in Tg-A mice, and the ovarian 42 phenotype in Tg-A mice was almost completely rescued by in utero treatment with a Notch2 43 agonist HMN2-29. We conclude that suppression of Nr5a1 during the fetal period optimizes 44 ovarian development by fine tuning of Notch signaling levels. 45( 257 words) 46 47 AUTHOR SUMMARY 48 Sexual development is a process of differentiation from undifferentiated bipotential gonads, 3 50 understanding of organogenesis. The nuclear receptor NR5A1 which is essential for 51 mammalian gonadal development, is equally expressed in both gonadal primordia, but after 52 sex determination, is up-regulated in XY and down-regulated in XX gonads. We have 53 recently demonstrated that this down-regulation is mediated by ovarian transcription factor, 54 Forkhead box L2 (FOXL2). This finding raised two key questions, whether Nr5a1 can 55 function as a male sex-determining factor, and whether the repression is essential for 56 appropriate ovarian development. By generating two lines Tg mice in XX gonads with 57 different enforced expression levels of Nr5a1, our present study revealed that alterations in 58 Nr5a1 dosage, either reduced or excessive, result in pathological effects in ovarian 59 development and female fertility, indicating that the precise control of Nr5a1 at the 60 transcriptional level is essential for optimal ovarian development. We envisage that the 61 improved understanding of how this pathway regulates ovarian development and female 62 fertility would aid the development of artificial somatic ovarian cells, which in turn may 63 provide a valuable treatment option...