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. 2021 Nov 15;105(5):1154-1159.
doi: 10.1093/biolre/ioab132.

RNF8 is not required for histone-to-protamine exchange in spermiogenesis†

Hironori Abe  1   2 , Rajyalakshmi Meduri  3 , Ziwei Li  3 , Paul R Andreassen  4 , Satoshi H Namekawa  1   2
Affiliations

RNF8 is not required for histone-to-protamine exchange in spermiogenesis†

Hironori Abe et al. Biol Reprod. .

Abstract

While an E3 ubiquitin ligase, RNF8, was initially reported to be required for histone-to-protamine exchange in spermiogenesis, we subsequently demonstrated that RNF8 is not involved in this process. Nevertheless, reflecting a lingering misunderstanding in the field, a growing number of studies have continued to postulate a requirement for RNF8 in the histone-to-protamine exchange. For example, a recent study claimed that a mouse PIWI protein, MIWI, controls RNF8-mediated histone-to-protamine exchange. Here, confirming our earlier conclusions, we show that RNF8 is required neither for the establishment of histone H4K16 acetylation, which is an initial step in histone removal during spermiogenesis, nor for the incorporation of two protamine proteins, PRM1 and PRM2. Thus, whereas RNF8 mediates ubiquitination of H2A on the sex chromosomes in meiosis, during the prior stage of spermatogenesis, our genetic evidence underscores that RNF8 is not involved in histone-to-protamine exchange.

Keywords: MIWI; RNF8; histone-to-protamine exchange; sperm; spermiogenesis.

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Figures

Figure 1
Figure 1
RNF8 is not required for H4K16 acetylation or for histone-to-protamine exchange in spermiogenesis. (A–D) Testis sections from WT and Rnf8-KO mice immunostained with antibodies shown in the panels (H4K16ac: H4K16 acetylation; H4ac: pan-acetyl H4; FK2: ubiquitination). Dashed squares are magnified in panels to the right. Scale bars: 100 μm unless specified.
Figure 2
Figure 2
RNF8-independent incorporation of PRM1 and PRM2 in epididymal sperm. (A and B) WT and Rnf8-KO epididymal sperm immunostained with antibodies against PRM1 or PRM2. Upper panels: photos of representative sperm; Scale bars: 5 μm. Bottom panels: photos of multiple sperm; Scale bars: 25 μm.
Figure 3
Figure 3
Failure of an anti-RNF8 antibody to specifically recognize RNF8. Western blot of WT and Rnf8-KO lysates from whole mouse testes probed with an anti-RNF8 antibody. 40 μg protein of sample was loaded in each lane. Two independent samples for WT and Rnf8-KO testes are shown. Loading control: Lamin B1. The expected molecular weight of RNF8 is 56–60 kDa. Of critical importance, the band most proximal to the expected size of RNF8 was equally present in extracts of WT and Rnf8-KO testes, and, additionally, no other prominent band displayed a clear difference between these genotypes.
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