A NANOS3 mutation linked to protein degradation causes premature ovarian insufficiency

Abstract

Primary ovarian insufficiency (POI), or premature ovarian failure, is defined as the cessation of ovarian function before the age of 40. An insufficient ovarian follicle pool derived from primordial germ cells (PGCs) is an important cause of POI. Although the Nanos gene family is known to be required for PGC development and maintenance in diverse model organisms, the relevance of this information to human biology is not yet clear. In this study, we screened the coding regions of the NANOS1, NANOS2 and NANOS3 genes in 100 Chinese POI patients and identified four variants in the coding regions of these three genes, including one synonymous variant in NANOS3, one missense variant in each of NANOS1 and NANOS2 and one potentially relevant mutation (c.457C>T; p.Arg153Trp, heterozygous) in NANOS3. We demonstrated that the p.Arg153Trp substitution decreases the stability of NANOS3, potentially resulting in a hypomorph. Furthermore, an investigation of the relationship between the number of PGCs and the dosage of NANOS3 in mouse models showed that the population of PGCs is controlled by the level of NANOS3 protein. Taken together, our results provide new insight into the properties of the NANOS3 protein and establish that NANOS3 mutation is one possible cause of POI.

Figure 1

Characterisation of the properties of wild-type and mutant hNANOS3 proteins. (a) The alignment of amino-acid sequences of NANOS3 homologues in different species shows that the arginine (grey shadow) is conserved. (b) Turnover of hNANOS3 and hNANOS3 R153W proteins. (c) The levels of hNANOS3 and hNANOS3 R153W proteins were quantified and plotted relative to the corresponding hNANOS3 levels at 0 h. Data represent the means±S.E. of the results from at least three independent experiments. Two stars represent statistical significance, P<0.001. (d) Turnover of hNANOS3 protein with R153 substituted with Ala, Phe, Leu, Met, Pro and Val. (e) Turnover of mNANOS3 and NANOS3 R133W with or without T132 substituted with Val. (f) Turnover of hNANOS3 protein with MG132 (20 mM), chloroquine (100 mM) or NH₄Cl (50 mM) treatment. Treatment with MG132 obviously slowed the turnover of both wild-type and mutant NANOS3 proteins. (g) The increased accumulation of hNANOS3 R153W in the insoluble fraction

Figure 2

Neo cassette insertion into the promoter region decreased Nanos3 expression level. (a) A PGK-Neo cassette flanked by two flippase recognition target (FRT) sites and followed by a LoxP site was inserted in the Nanos3 promoter region 1476 bp upstream of the Nanos3 initiator. Another LoxP site was inserted 906 bp downstream of the Nanos3 3′UTR. (b) Quantitative real-time PCR analysis of Nanos3 mRNA from E12.5 female gonad in indicated genotype mice. (c) Immunofluorescence analysis of NANOS3 expression in the E12.5 female gonad. OCT4 is used as a marker of PGCs. NANOS3 is in the cytoplasm of germ cells in Nanos3^Neo/+ mice, whereas NANOS3 is hardly detected in Nanos3^Neo/Neo mice. Representative PGCs are magnified (insets). (d) Quantification of NANOS3 expression in OCT4-positive cells. Data represent the means±S.E. of the results from three independent sections. Two stars represent statistical significance, P<0.001

Figure 3

The causal link between the dosage of NANOS3 and the number of PGCs. (a) Immunofluorescence analysis of MVH expression in the gonadal ridges at E12.5 from mice of the indicated genotype. (b) Quantification of germ cell number in gonadal ridges at E12.5 from mice of the indicated genotype. (c) Immunofluorescence analysis of MVH expression in the ovaries at postnatal day 4 from mice of the indicated genotype. (d) Quantification of ovaries at postnatal day 4 from mice of the indicated genotype. Two stars represent statistical significance, P<0.001