# Functional Study of Four Histone Genes Involved in the Spermatogenesis of Cynoglossus semilaevis

## TL;DR

This study explores how four histone genes influence spermatogenesis in Chinese tongue sole, a fish species where females can become pseudomales that produce only Z sperm.

## Contribution

The study identifies four histone genes and their regulatory roles in gonadal development and spermatogenesis in Chinese tongue sole.

## Key findings

- The four histone genes show peak expression during late gonadal development in Chinese tongue sole.
- siRNA knockdown of these genes affects spermatogenesis-related genes like dmrt1, tesk1, and neurl3.
- Promoter analysis suggests YY1A, YY1B, C-JUN, and JUNB may negatively regulate these histone genes.

## Abstract

The genetic females of Chinese tongue sole (ZW sex chromosomes) can sex reverse to phenotypic males, designated pseudomales, while the pseudomale produces only Z sperm with W sperm missing. Previous studies have shown that histone plays an important role in spermatogenesis; thus, we selected four histone genes, h1.1-like, h1.2-like, h3, and h3.3-like, for further analysis. Their expression reached their highest levels at 1.5–2 years post-hatching, indicating that its role began during the late stage of gonadal development. The promoter of the four genes was located approximately 2000 bp upstream, and transcription factor sites were predicted. Among them, YY1A, YY1B, C-JUN, and JUNB may have negative regulatory effects on h1.1-like, h1.2-like, h3, and h3.3-like; AR and ETS-2 may have positive regulatory effects on h3 and h3.3-like. In situ hybridization showed mRNAs of these four genes were located mainly in the germ cells in gonads. siRNA knockdown of the four histone genes could affect the genes related to spermatogenesis. These results provide a genetic basis for the molecular mechanism of gonadal development and spermatogenesis in Chinese tongue sole.

Chinese tongue sole (Cynoglossus semilaevis) is an important mariculture fish in China, and female individuals present a growth advantage. However, genetic females (ZW) can sex reverse to phenotypic males, designated pseudomales. The pseudomale shows abnormal spermatogenesis and produces only Z sperm. Histone is pivotal in spermatogenesis, and post-translational modification could regulate its function. A comparison of testis phosphorylated and ubiquitinated proteins revealed 8 and 12 differentially phosphorylated and ubiquitinated histones in the testes of male and pseudomale Chinese tongue soles, respectively, but there was no difference in the translation level of these proteins. We selected four histone genes, h1.1-like, h1.2-like, h3, and h3.3-like, for further analysis. The expression levels of the h1.1-like, h3, and h3.3-like genes reached their highest levels at 2 years post-hatching (yph), and the expression level of h1.2-like reached its highest level at 1.5 years post-hatching (1.5 yph), indicating that its role began during the late stage of gonadal development. Promoter activity verification revealed that the promoters of the h1.1-like, h1.2-like, h3, and h3.3-like genes were located approximately upstream 2000 bp and six histone-related transcription factor sites were predicted. YY1A, YY1B, C-JUN, and JUNB may have negative regulatory effects on h1.1-like, h1.2-like, h3, and h3.3-like; AR and ETS-2 may have positive regulatory effects on h3 and h3.3-like. The ISH results revealed that h1.1-like, h1.2-like, h3, and h3.3-like mRNAs were located mainly in the sperm cells in the testes and the oocytes at various stages in the ovaries. After siRNA knockdown, the expression of dmrt1 in testis cell lines and the expression of tesk1 and neurl3 in males was downregulated, suggesting that the h1.1-like, h1.2-like, h3, and h3.3-like genes may have a negative regulatory role in spermatogenesis. The regulatory role in female fish remains to be explored. Mass spectrometry analysis revealed that histones have an important role in chromosome remodeling. These results provide a genetic basis for the molecular mechanism of gonadal development and spermatogenesis in Chinese tongue sole.