Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells

Abstract

The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis.

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

“…In the mouse testis E2F2 and E2F3 have been shown to be partially expressed in the same germ‐cell types as E2F1 (El‐Darwish et al ., 2006; Rotgers et al ., 2014). To our surprise there were no other E2Fs among the differentially expressed genes in E2F1 −/− ‐testis.…”

“…Immunofluorescence double labeling of apoptotic cells with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or proliferating cells with 5‐bromo‐2′‐deoxyuridine (BrdU) coupled to detection of specific cell types with marker antibodies were done as described previously (Rotgers et al ., 2014). Spermatogonia were detected using Magbee antibody (a kind gift from V. Thane) and Sertoli cells using Wilm's tumor 1 (WT1) antibody from P10 and P40 testes, n  = 4 per group.…”

“…The stage‐specific analysis was done to decrease the variation caused by the stage‐specific pattern of apoptosis and to increase the sensitivity of the assay as there is no almost apoptosis in the normal testis in these stages. Double immunofluorescence labeling of γH2AX‐S139 and Plzf antibodies was performed on P40 testes using a standard immunofluorescence protocol (Rotgers et al ., 2014). …”

Experimental evidence of proliferation and reproduction of highly differentiated Sertoli cells

“…With aging, the distur bances in the SC organization and in spermatogenesis as a whole in RBcKO mice became more severe, lead ing to male sterility. The results obtained by Nalam et al (2009) were largely confirmed in another essen tially similar study (Rotgers et al, 2014), which was also performed using a model of mice knockout for the RB gene in SCs (SCs RbKO). The analysis of prolifer ative activity showed that the number of BrdU posi tive SCs in 30 day old SCs RbKO mice significantly increased.…”

“…These authors also found that the testes of 10 and 12 week old mutant mice expressed cytokeratin 18, a marker of immature fetal SCs, and that the levels of RNA that are normally used as a tem plate for retinoblastoma protein synthesis were greatly reduced. In addition, it was shown (Nalam et al, 2009;Rotgers et al, 2014) that, in adult mice knock out for the RB gene, apoptotic signaling cascades were triggered in the SCs that resumed active proliferation. These conclusions were made by these authors on the basis of enhanced expression of 47 genes associated with apoptosis (upregulation), which was detected by the microarray technique, and the appearance of TUNEL positive apoptotic nuclei in the SC popula tion.…”