Abstract
Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.
Highlights
Despite the recent insights into the regulation of mammalian germ cell development and the advances in assisted reproductive technologies, infertility remains a common problem in modern society affecting ∼15% of couples in industrialized countries (Agarwal et al, 2015)
While our results have revealed a moderate reduction in the number of Trim71-deficient PGC-like cells (PGCLCs) upon induction, we believe that this cannot fully explain the dramatic phenotype observed in Trim71 conditional knockout mouse (cKO) mice
Our data do not provide evidence for a reduction in Primordial germ cells (PGCs) proliferation in vivo, these results suggest that Tripartite Motif Containing 71 (TRIM71) may be important for the expansion of the germ cell pool during embryogenesis, which could be an explanation for the reduction of male gonocytes observed in neonatal Trim71 cKO mice
Summary
Despite the recent insights into the regulation of mammalian germ cell development and the advances in assisted reproductive technologies, infertility remains a common problem in modern society affecting ∼15% of couples in industrialized countries (Agarwal et al, 2015). BLIMP1 activates the expression of Tfap2c (Kurimoto et al, 2008), and together BLIMP1, TFAP2C and PRDM14 establish the transcriptional program required for PGC specification (Wang and Cao, 2016) which is completed at E7.5. At this time point, about 40 cells in the proximal epiblast express early PGC-specific markers such as Nanos (Ginsburg et al, 1990; Tsuda et al, 2003). Unraveling the processes controlling germline development will contribute to our understanding of infertility, but may offer unique opportunities for the treatment of GCT
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