A male infertility mutation reverts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression ofGADD45A,GADD45B,GADD45GandRHOBgenes

AbstractBackgroundWhile Nanos-mediated maintenance of germ cells inDrosophilaand mice has been related to regulation of apoptosis, the relevance of these findings to human physiology is uncertain. Previously we have described the p.[(Pro34Thr);(Ser83del)] doubleNANOS1mutation as associated with an absence of germ cells in the testes of infertile patients. The aim of this study was to identify the mechanism underlying infertility phenotype of patients bearing theNANOS1mutation.MethodsConstructs encoding a wild-type or mutated NANOS1 protein were used for transfection of TCam-2 cell line, representing male germ cells in culture. Influence of this mutation on cell proliferation was performed using MTS assay while apoptosis and cell cycle were measured by flow cytometry. RNA-Seq analysis including quantitative RT-PCR was conducted for selecting pro-apoptotic genes, repressed by the wild-type NANOS1. Influence of the p.[(Pro34Thr);(Ser83del)] NANOS1 mutation on that repression was investigated by quantitative RT-PCR.ResultsWe show here that the p.[(Pro34Thr);(Ser83del)] doubleNANOS1mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line TCam-2. This mutation disrupts repression of mRNAs encoding pro-apoptotic GADG45A, GADD45B, GADD45G and RHOB factors, which could contribute to an increase in apoptosis.ConclusionsThis report underscores the conservation of Nanos from flies to humans as a repressor of pro-apoptotic mRNAs in germ cells, and provides a basis for understanding NANOS1 functions in human reproductive health.