Involvement of Histone Acetyltransferase 1 (HAT1) in the Spermatogenesis of Non-Condensed Nuclear Sperm in Chinese Mitten Crab, Eriocheir sinensis.

Chinese mitten crab, Eriocheir sinensis, is a decapod crustacean with a special, non-condensated nucleus in the sperm. Studies have shown that the nuclear compact state of male germ cells during the spermatogenesis is closely related to histone modification. To explore the possible role of histone acetyltransferase 1 (HAT1) in the chromatin organization during the E. sinensis spermatogenesis, we took the testis tissues of both adult and juvenile crabs as the materials of study and analyzed the biological functions of HAT1 by whole transcriptome sequencing and bioinformatics, then further analyzed the expression and distribution of HAT1 using the methods of RT-qRCR, western blotting, and immunofluorescence location. The results showed that HAT1 is an alkaline-unstable hydrophilic protein. It was predicted to interact with a variety of histones and chromosome assembly proteins, including Asf1b, Chaf1b, and Hist1h3f, and is involved in many biological functions pertaining to chromatin dynamics such as chromatin organization, DNA dependent nucleosome assembly, DNA conformational changes, and so on. HAT1 was up-regulated in the adult testes compared to the juvenile (n = 3, P < 0.05). HAT1 was mainly located in the nuclei of male germ cells of E. sinensis. As spermatogenesis proceeded, the expression of HAT1 decreased and even disappeared in the nuclei (n = 3, P < 0.05). HAT1 is an important player in histone acetylation, which facilitates chromatin alteration in a three-dimensional conformation. The expression of HAT1 in different male germ cells might indicate the chromatin dynamics at the diversity stages of spermatogenesis. The high expression of HAT1 at the early stages of E. sinensis spermatogenesis hints the active involvement in chromatin organization, while its progressively reduced expression accompanied by the progression of spermatogenesis suggests a relatively gradual stabilization and stereotyping of chromatin. As for the disappearance of HAT1 in mature sperm with non-condensed nuclei, the reduction in histones targeted by HAT1 or histone acetylation may be an important initiator.

miR-124-3p regulates the involvement of Ptpn1 in testicular development and spermatogenesis in mouse.

Testicular development and spermatogenesis in mouse are a complex process in which phosphorylation modifications and regulation of genes by non-coding RNAs play an important role. However, protein tyrosine phosphatase, non-receptor type 1 (Ptpn1) is widely expressed in mammalian tissues. In this study, we analyzed the expression of Ptpn1 mRNA and its encoded proteins in testicular tissues of juvenile and adult mice by using experimental techniques such as biological information, real-time fluorescence quantitative PCR (RT-qPCR), western blot (WB), immunofluorescence (IF) and transfection, and further analyzed the possible target-regulatory relationship and regulatory mechanisms of miR-124-3p and Ptpn1. We found that Ptpn1 mRNA and its encoded protein were up-regulated in adult mouse testis compared to juvenile mouse testis. The expression trend of miR-124-3p was opposite to that of Ptpn1. In other cell types, Ptpn1 protein is localized in cell membrane, cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. Immunofluorescence showed that Ptpn1 protein was mainly localized in the cytoplasm of male germ cells and was expressed at a high level in early-stage cells (spermatogonia) and at a low level in late-stage cells (sperm). Transfection results showed that the expression levels of Ptpn1 mRNA and its protein were significantly down-regulated after miR-124-3p overexpression in mouse spermatogonia. Bioinformatics analysis showed that Ptpn1 can involved in biological processes such as protein kinase inactivation through peptidyl tyrosine dephosphorylation. The reduction of miR-124-3p may be a key factor in promoting the high expression of Ptpn1 in testicular tissues of adult mice. Increased miR-124-3p may be a key factor in suppressing Ptpn1 expression in the mouse spermatogonia mimics group. The differential expression results from the negative regulation of miR-124-3p.

Role of cytoskeleton-related proteins in the acrosome reaction of Eriocheir sinensis spermatozoa.

Cytoskeleton-related proteins are essential for cell shape maintenance and cytoskeleton remodeling. The spermatozoa of Eriocheir sinensis (Chinese mitten crab) have a unique cellular structure, and the mechanism of spermatozoal metamorphosis during the acrosome reaction is not well understood. In this study, the E. sinensis spermatozoa were induced using calcium ionophore A23187 to undergo the acrosome reaction in vitro, and the acrosome-reacting and fresh (non-reacting) spermatozoa were collected separately. The differential expression of cytoskeleton-related protein genes in acrosome-reacting and fresh spermatozoa of E. sinensis was analyzed by whole transcriptome sequencing and bioinformatics analysis, and PPI network and miRNA-mRNA regulation network were constructed to analyze their possible function and regulation mechanism. The results showed that numerous differentially expressed cytoskeleton-related protein genes, miRNAs and lncRNAs were found in acrosome-reacting and fresh spermatozoa of E. sinensis; 27 cytoskeleton-related protein genes were down regulated and 687 miRNAs were up regulated in acrosome-reacting spermatozoa; 147 miRNAs target these 27 cytoskeleton-related protein genes. In the PPI networks, RAC1, BCAR1, RDX, NCKAP1, EPS8, CDC42BPA, LIMK1, ELMO2, GNAI1 and OCRL were identified as hub proteins. These proteins are mainly involved in the regulation of cytoskeleton organization, actin cytoskeleton organization, microtubule skeleton organization and small GTPase-mediated signal transduction and other biological processes, and play roles in pathways such as actin cytoskeletal regulation and axon guidance. miR-9, miR-31 and two novel miRNAs in the miRNA-mRNA regulatory network are the core miRNAs targeting cytoskeleton-related protein genes. miR-9 targets and regulates OBSCN, CDC42BPA, ELMO2, BCAS3, TPR and OCRL; while miR-31 targets and regulates CDC42BPA and TPR. This study provides a theoretical basis for revealing the mechanism of acrosome reaction under the special spermatozoa morphology of E. sinensis.