Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line

Abstract

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.

Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

“…Downregulation of this phase accelerates both cell proliferation and S phase [ 39 ]. These results are in line with our previous findings that NANOS1 downregulates GADD45 family members which are involved in G1/S phase DNA damage checkpoint [ 20 ]. In turn, in the case of NANOS3 overexpression, we observed a higher number of cells in the G2/M phase compared to control cells ( Figure 3 B bottom panel).…”

“…Namely, a mutation of NANOS1 is associated with male infertility caused by absence of germ cells in the seminiferous tubules of patients [ 19 ]. Moreover, when this NANOS1 gene mutation was tested in a hPGCs model, TCam-2 cell line, it resulted in a functional switch of NANOS1 from anti-apoptotic to pro-apoptotic, providing an explanation of the infertility phenotype of the patients [ 20 ]. In turn, NANOS3 gene mutations were found to be associated with premature ovarian insufficiency, a female infertility phenotype [ 21 , 22 ].…”

Emerging Roles of NANOS RNA-Binding Proteins in Cancer

“…Additionally, it demonstrated that NANOS1 caused a significant decrease in apoptosis. It was further demonstrated by transcriptomic data that the NANOS1-mediated decrease in apoptosis was accompanied by the downregulation of pro-apoptotic genes such as RHOB , MYC , and GADD45 family genes [ 26 ]. These genes are downstream targets of p53 and are involved in p53-mediated DNA-damage checkpoint pathway.…”

“…With the emergence of multiple Nanos paralogues during evolution, mammalian Nanos paralogues (Nanos1, Nanos2, and Nanos3) diverged in their functions and adopted distinct roles in germline development. For example, NANOS1 represses apoptosis in human germ cell line TCam-2 [ 26 ], while mouse Nanos2 is crucial for inducing mitotic arrest during male germ cell development [ 27 ]. Lastly, Nanos3 is important for the maintenance of the male germline stem cells [ 28 ] and suppresses premature spermatogonial differentiation [ 29 ].…”

Nanos2+cells give rise to germline and somatic lineages in the sea anemoneNematostella vectensis

“…The nanos gene family codes for zinc-finger containing RNA-binding proteins, with apoptosis and differentiation promoting mRNA targets, thereby inhibiting those processes (21,22). The activity of both gene families is especially conserved in the germline of vertebrates and insects, where they ensure genomic integrity and keep gametes in a stem cell-like state (23)(24)(25)(26). In Nematostella, we found three piwi genes (one of which appears to be a pseudogene) (27) and two nanos paralogs, nanos1 and nanos2.…”