Nanos RNA-binding proteins are critical factors of germline development throughout the animal kingdom and their dysfunction causes infertility. During evolution, mammalian Nanos paralogues adopted divergent roles in germ cell biology. However, the molecular basis behind this divergence, such as their target mRNAs, remains poorly understood. Our RNA-sequencing analysis in a human primordial germ cell model-TCam-2 cell line revealed distinct pools of genes involved in the cell cycle process downregulated upon NANOS1 and NANOS3 overexpression. We show that NANOS1 and NANOS3 proteins influence different stages of the cell cycle. Namely, NANOS1 is involved in the G1/S and NANOS3 in the G2/M phase transition. Many of their cell cycle targets are known infertility and cancer-germ cell genes. Moreover, NANOS3 in complex with RNA-binding protein PUM1 causes 3′UTR-mediated repression of FOXM1 mRNA encoding a transcription factor crucial for G2/M phase transition. Interestingly, while NANOS3 and PUM1 act as post-transcriptional repressors of FOXM1, FOXM1 potentially acts as a transcriptional activator of NANOS3, PUM1, and itself. Finally, by utilizing publicly available RNA-sequencing datasets, we show that the balance between FOXM1-NANOS3 and FOXM1-PUM1 expression levels is disrupted in testis cancer, suggesting a potential role in this disease.

46,XY gonadal dysgenesis, characterised by absent or defective testicular development in individuals with a 46,XY karyotype, results from disruptions in the genetic programme governing testis determination and differentiation during embryogenesis. While monogenic causes explain approximately 50% of cases, emerging evidence suggests an oligogenic basis in some patients. However, many cases remain without a definitive molecular diagnosis. In this study, we investigated a patient with 46,XY gonadal dysgenesis to explore the underlying genetic aetiology. Whole-exome sequencing in the patient did not reveal any pathogenic variants in genes previously associated with this condition. Instead, it detected rare variants in STARD9 and CDK5RAP2, which encode centrosomal proteins known to interact with each other. Gene expression analysis of embryonic gonads revealed that STARD9 is sexually dimorphic, with the highest expression in testis-specific Sertoli cells, while CDK5RAP2 is ubiquitously expressed, including in Sertoli cells. These findings suggest a role for both genes in Sertoli cell development, implicating them in the pathogenesis of 46,XY gonadal dysgenesis. To evaluate the functional relevance of the identified variants, we performed molecular dynamics simulations, which suggest that these variants may impair the individual and/or combined functions of STARD9 and CDK5RAP2 proteins. This study is the first to propose a role for STARD9 and CDK5RAP2 genes in human Sertoli cell development and highlights their potential contribution to 46,XY gonadal dysgenesis.

46,XY gonadal dysgenesis (46,XY GD) results from disruptions in the genetic program that governs testicular differentiation during gonadal sex determination, presenting as either complete (46,XY CGD) or partial (46,XY PGD) forms. While monogenic defects account for approximately 50% of cases, recent evidence suggests an oligogenic basis for some 46,XY GD cases. In this study, we investigated a case of 46,XY CGD and performed whole-exome sequencing (WES) on the patient and her parents to explore the genetic basis of the patient’s condition. Although no pathogenic variants were identified in known 46,XY GD-associated genes, we detected rare variants in the STARD9 and CDK5RAP2 genes. Previous study in mice indicate that the orthologues of these genes are highly expressed in Sertoli cells during gonadal sex determination, with Cdk5rap2 playing a critical role in Sertoli cell polarization. Notably, the human STARD9 and CDK5RAP2 proteins interact with each other. Structural analysis suggests that the variants in STARD9 and CDK5RAP2 may alter their protein-protein interactions. Based on these findings, we propose that STARD9 and CDK5RAP2 variants may act together to impair Sertoli cell function, leading to 46,XY CGD, consistent with an oligogenic mode of inheritance. These results suggest that STARD9 and CDK5RAP2 should be considered as candidate genes for 46,XY GD and included in genetic panels for this condition.