The Role of TRIM71 in Male Germ Cell Development

Abstract

Germ cells are the key to continuation of life as they are the unique cell lineage capable to transfer the complete genetic information to the next generation. Both, infertility and testicular cancers are usually originating from errors during embryonic germ cell development. Germ cell development is a very precisely regulated process – spatially and temporally – with cell proliferation, differentiation and apoptosis being involved. <br /> An evolutionary conserved role in the regulation of embryonic development has been described for TRIM71 and its orthologs. In the somatic lineage, TRIM71 is restrictively expressed in stem and progenitor cells and regulates the balance between stem cell renewal and differentiation. Besides the expression of TRIM71 being validated in gonocytes and spermatogonial stem cells (SSCs) in mouse testes, the function of TRIM71 in the germ cell lineage is only poorly understood. Therefore, this study aimed at elucidating the role of TRIM71 in male germ cell development.<br /> In mice, TRIM71 deficiency causes infertility which is associated with reduced testis size and weight. By histological analysis of testes from germline-specific TRIM71-deficient adult mice, an almost complete lack of germ cells was observed with the testes resembling symptoms of a Sertoli-cell-only (SCO) syndrome. This loss of germ cells was found to be predominantly of embryonic origin and becoming more pronounced after birth, when mitosis is reinitiated in germ cells. To better understand the molecular functions of TRIM71 in germ cell maintenance, we used the germ cell tumour cell line NCCIT as an <em>in vitro</em> surrogate model to analyse the growth behaviour of NCCIT cell populations with CRISPR/Cas9-mediated deletion of the N-terminal TRIM71 RING domain (ΔRING) or functional deletion of the C-terminal amino acids within the last NHL repeat (Δ6NHL). Highly innovative growth competition analysis revealed that the RING domain and particularly the last NHL repeat are necessary for proper growth behaviour. For both TRIM71 truncations a decline in the respective NCCIT cell population was present, though being more pronounced in the NCCIT TRIM71 Δ6NHL population. Moreover, while stemness markers were unaltered, proliferation was decreased and sensitivity towards apoptosis was enhanced in NCCIT TRIM71 ΔRING and again more strongly in NCCIT TRIM71 Δ6NHL cells. In this context, two factors and known TRIM71 targets, namely the cell cycle inhibitor CDKN1A as well as the tumour suppressor and ‘guardian of the genome’ p53, were found to be involved in regulating proliferation and apoptosis in NCCIT cells, respectively.<br /> Overall, our findings strongly indicate that TRIM71 is indispensable for sustaining and building the germ cell niche by promoting proliferation combined with the inhibition of apoptosis. Thus, we advance the understanding on genetic factors that cause testicular cancer and male infertility. Consequently, TRIM71 might be a putative pharmacological target paving the way for improved testicular cancer treatment and reproductive medicine.