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Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders

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Sources of Funding: none

Introduction

Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood.

Methods

Testicular biopsy specimens from 30 men (mean age: 35 years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. The transcriptome of the testicular homogenate using the Illumina platform were expressed as fragments per kilobase. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin’s solution-fixed sections. Apoptosis of germ cells was assessed by TdT-mediated digoxygenin-dUTP nick end labelling (TUNEL). Expression and localization of H3.5 were compared with patient background, germinal stage and PCNA expression.

Results

In normal spermatogenesis testes, the H3.5 protein was mainly localized in leptotene spermatocytes, independent of germinal stage. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p<0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p<0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy.

Conclusions

Different from other testis-specific histones, which are often expressed during the histone to protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders.

Funding

none

Authors
Koji Shiraishi
Aya Shindo
Akihito Harada
Yasuyuki Ohkawa
Hitoshi Kurumizaka
Hiroshi Kimura
Hideyasu Matsuyama
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