ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling

Abstract

SignificanceSex determination involves antagonistic interactions between the testis-determining (SRY-SOX9-FGF9) and ovary-promoting (RSPO1-WNT/β-catenin-FOXL2) pathways, but the underlying molecular mechanisms remain unclear. We show that ZNRF3, an E3 ubiquitin ligase that inhibits WNT signaling and is a direct target of RSPO1-mediated membrane clearance, is testis-determining in mice. Testis determination defects in the absence of ZNRF3 arise due to ectopic canonical WNT signaling in XY gonads at the sex-determining stage. We identify human ZNRF3 sequence variants in cases of 46,XY disorders of sex development with XY female presentation. In vitro functional assays show that these variants disrupt ZNRF3 function. Our data reveal a sex-determining role for ZNRF3 and indicate that interactions between ZNRF3 and RSPO1 regulate mammalian sex determination.

Disorders of Sex Development—Novel Regulators, Impacts on Fertility, and Options for Fertility Preservation

“…In accordance, three human ZNRF3 variants were identified in rare cases of 46,XY female DSD, thereby identifying a testis-determining function for ZNRF3 in humans and suggesting an antagonistic relationship between ZNRF3 and RSPO1 also in human gonadal sex differentiation [101]. In the five patients with ZNRF3 variants and 46,XY DSD varying degrees of gonadal dysgenesis was reported, but no information about fertility was included [101].…”

“…Interestingly, the WNT signaling antagonist ZNRF3, which is also a direct target of RSPO1-mediated inhibition, was recently shown to be required for testicular differentiation in mice, with XY mice lacking Znrf3 exhibiting complete or partial gonadal sex reversal [101]. In accordance, three human ZNRF3 variants were identified in rare cases of 46,XY female DSD, thereby identifying a testis-determining function for ZNRF3 in humans and suggesting an antagonistic relationship between ZNRF3 and RSPO1 also in human gonadal sex differentiation [101]. In the five patients with ZNRF3 variants and 46,XY DSD varying degrees of gonadal dysgenesis was reported, but no information about fertility was included [101].…”

Male Hypogonadism and Disorders of Sex Development

“…In newborns with androgen levels above but AMH within the female range, virilization is not related to hypogonadal states but rather to adrenal or placental dysfunction. In chimeric forms of DSD, e.g., 46,XX/46,XY or variants, ovotesticular differentiation usually occurs, characterized by androgen and AMH levels that are above the female but below the male range, indicating that there is a whole testicular tissue dysfunction. All these are primary forms of fetal hypogonadism.…”

“…Loss-of-function variants of MAP3K1 lead to an increased expression of pro-ovarian factors, like β-catenin, and reduced expression of pro-testicular factors, like SOX9/FGF9 (45). A similar pathogenesis has been proposed for mutations in ZNRF3 (46). Dysgenetic DSD can also be attributed to mutations in DHX37 (47, 48) particularly those individuals exhibiting the embryonic testicular regression syndrome and rearrangements involving SOX8 (49).…”

A genetic system for biasing the sex ratio in mice

“…Manipulated animals that produce only one sex are impossible to sustain by self-29 3 crossing, because either the male or female is absent, or due to sub-fertility or infertility of 30 the manipulated animal. Thus, despite the identification of genetic factors such as Sry, 31Sox9, Foxl2, and Wnt4, that determine the sex of animals, and the success of reversing 32 animal's sex, fertility and other genetic restrictions precluded a system that reliably 33 produces a single sex progeny [13][14][15][16][17] . 34 We chose to provide a proof of concept for an approach that produces single-sex 35 mouse progeny while retaining a reproductive reservoir of males and females.…”