Ubiquitin Ligase Huwe1 Modulates Spermatogenesis by Regulating Spermatogonial Differentiation and Entry into Meiosis

Rohini Bose,Teruko Taketo,Gurpreet Manku,Kai Sheng,Cristian O’Flaherty,Simon S Wing,Martine Culty,Adel R Moawad,Kin Lam Fok

doi:10.1038/s41598-017-17902-0

Abstract

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1’s critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.

Highlights

Haploid spermatids undergo a process of cellular remodeling and condensation, referred to as spermiogenesis
The Stra8-Cre KO testis had fewer Stra8+ pre-leptotene spermatocytes, which indicated that there was a defect in the process of spermatogonial differentiation. This was confirmed by measuring the expression of markers of spermatogonial differentiation (Stra[8], Dazl, Sohlh2) by Q-PCR, which were markedly decreased in the Stra8-Cre KO testis (Fig. 2b). Since these results suggested a defect in spermatogonial differentiation, we focused on this process by sacrificing the animals at 8 days post RA injection (dpRA) when the WT testis tubules will contain either pre-leptotene or leptotene spermatocytes from the first round of differentiation or A1 or A2 spermatogonia from the second round of differentiation
Huwe[1] is dispensable for the completion of meiosis and for spermatid maturation since activating Cre recombinase in leptotene spermatocytes or elongating spermatids using the Spo[11] or Prm promoters respectively had no effects on spermatogenesis or fertility (Fig. 5 and data not shown)

Summary

Introduction

Haploid spermatids undergo a process of cellular remodeling and condensation, referred to as spermiogenesis. To explore for additional roles of Huwe[1], we inactivated the enzyme using Cre-recombinase driven by the Stra[8] promoter which turns on in type A1 spermatogonia and is active up to the pre-leptotene spermatocytes[19] and by the Spo[11] promoter which is activated in spermatocytes that have initiated meiosis[20]. With this approach, we show that Huwe[1] has critical roles in spermatogonial differentiation leading up to meiosis

Methods

Results

Conclusion