The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for -KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no -KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/-KTS ratio.

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

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.

Androgen insensitivity syndrome (AIS), manifesting incomplete virilization in 46,XY individuals, is caused mostly by androgen receptor (AR) gene mutations. Therefore, a search for AR mutations is a routine approach in AIS diagnosis. However, some AIS patients lack AR mutations, which complicates the diagnosis. Here, we describe a patient suffering from partial androgen insensitivity syndrome (PAIS) and lacking AR mutations. The whole exome sequencing of the patient and his family members identified a heterozygous FKBP4 gene mutation, c.956T>C (p.Leu319Pro), inherited from the mother. The gene encodes FKBP prolyl isomerase 4, a positive regulator of the AR signaling pathway. This is the first report describing a FKBP4 gene mutation in association with a human disorder of sexual development (DSD). Importantly, the dysfunction of a homologous gene was previously reported in mice, resulting in a phenotype corresponding to PAIS. Moreover, the Leu319Pro amino acid substitution occurred in a highly conserved position of the FKBP4 region, responsible for interaction with other proteins that are crucial for the AR functional heterocomplex formation and therefore the substitution is predicted to cause the disease. We proposed the FKBP4 gene as a candidate AIS gene and suggest screening that gene for the molecular diagnosis of AIS patients lacking AR gene mutations.

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins (RBPs) with wide-ranging roles. They are involved in germ cell development, which has functional implications in development and fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity. To address that problem, first we used RIP-Seq and RNA-Seq approaches, and identified mRNA pools regulated by PUM1 and PUM2 proteins in the TCam-2 cell line, a human male germ cell model. Second, applying global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-interacting putative protein cofactors, most of them involved in RNA processing. Third, combinatorial analysis of RIP and RNA-Seq, mass spectrometry, and RNA motif enrichment analysis revealed that PUM1 and PUM2 form partially varied RNP-regulatory networks (RNA regulons), which indicate different roles in human reproduction and testicular tumorigenesis. Altogether, this work proposes that protein paralogues with very similar and evolutionary highly conserved functional domains may play divergent roles in the cell by combining with different sets of protein cofactors. Our findings highlight the versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.