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.

Testicular development and spermatogenesis are critical for male reproduction, with histone (de)acetylation playing a key role in chromatin remodeling within germ cells. Sirt1, a key histone deacetylase, is implicated in chromatin remodeling, but its expression pattern and specific role in testicular development and spermatogenesis need further study. This study comprehensively analyzed Sirt1 expression in adult and juvenile mouse testicular tissues and across various male germ cells, utilizing RT-qPCR, Western blot, immunofluorescence, and cell transfection. GO and KEGG enrichment analyses were performed to elucidate the biological functions and pathways associated with Sirt1 and its related genes. Multiple miRNA databases were utilized to predict miRNAs targeting Sirt1, and their expression levels were validated using RT-qPCR. Lentiviral transfection was used to knockdown candidate miRNAs to assess their functional roles. The results revealed a significant downregulation of Sirt1 expression in adult mouse testicular tissues compared to juvenile tissues, with pronounced variation across diverse male germ cells. Sirt1 was highly expressed in spermatogonia and mature sperm, but comparatively lower in spermatocytes and spermatids. GO and KEGG enrichment analyses highlighted Sirt1's role in key biological processes, including chromatin organization, regulation of cell proliferation, and energy homeostasis, as well as its association with signaling pathways like cellular senescence, the FoxO signaling pathway, and the AMPK signaling pathway. Bioinformatic analysis and subsequent RT-qPCR validation identified miR-9-5p as a miRNA targeting Sirt1. The expression of miR-9-5p was significantly higher in adult mouse testicular tissues compared to juvenile tissues, inversely correlating with Sirt1 levels. Moreover, the knockdown of miR-9-5p led to a notable increase in Sirt1 mRNA and protein expression. In conclusion, Sirt1 is a key player in mouse testicular development and spermatogenesis. The discovery that miR-9-5p negatively regulates Sirt1 suggests a critical regulatory axis that may govern these processes, providing novel insights into male fertility and potential targets for therapeutic intervention.

Testicular development and spermatogenesis in mice involve complex and dynamic gene regulation and chromatin remodelling. In this study, Real-time fluorescence quantitative PCR (RT-qPCR), Western Blot (WB), Immunofluorescence (IF), transfection and other techniques were used to analyse the expression of Ino80d mRNA and its encoded proteins in mouse testicular tissue and mouse spermatogonial cells, and to further analyse the possible target-regulatory relationship and function of miR-92a-3p and Ino80d. We found that Ino80d mRNA and protein expression was up-regulated in adult mouse testis tissue relative to juvenile mouse testis tissue, whereas miR-92a-3p expression was down-regulated in adult mouse testis tissue. Immunofluorescence results showed that the Ino80d protein was mainly localized in the nucleus of male germ cells. Ino80d protein expression is higher in spermatogonia, spermatid and lower in primary spermatocytes, secondary spermatocytes and sperm. There is a decreasing trend in development from spermatogonia to secondary spermatocytes. The transfection results showed that the expression levels of Ino80d mRNA and protein were down-regulated after overexpression of miR-92a-3p in mouse spermatogonia. Increased miR-92a-3p may be a key factor in inhibiting the expression of Ino80d mRNA and proteins in the miR-92a-3p mimics group of mouse spermatogonial cells, whereas differential expression may be a result of the negative regulation of miR-92a-3p, which regulates testicular development and spermatogenesis in mice.

The INO80D protein, a component of the INO80 chromatin remodeling complex, plays a pivotal role in chromatin remodeling, gene expression, and DNA repair within mammalian sperm. In contrast to the condensed nuclear structure of mammalian sperm, Chinese mitten crab, Eriocheir sinensis, exhibits a distinctively decondensed sperm nucleus. The distribution and function of INO80D during the E. sinensis spermatogenesis were previously enigmatic. Our research endeavored to elucidate the distribution and function of INO80D, thereby enhancing our comprehension of sperm decondensation and the process of spermatogenesis in this species. Employing transcriptome sequencing, RT-qPCR, western blot analysis, and immunofluorescence techniques, we observed a pronounced upregulation of INO80D in the adult E. sinensis in comparison to the juvenile. The protein predominantly resides in the cellular nucleus, with high levels in spermatogonia and spermatocytes, less in stage I and III spermatids, and lowest in mature sperm. The results indicated that INO80D is initially instrumental in chromatin decondensation to facilitate gene accessibility and DNA repair during the early phases of spermatogenesis. Its role subsequently shifts to maintaining decondensed chromatin stability and genetic integrity during spermiogenesis. The sustained presence of INO80D during spermiogenesis is essential for the ultimate maturation of the decondensed sperm nucleus, imperative for preserving the unique decondensed state and the protection of genetic material in E. sinensis. Our study concludes that INO80D exerts a multifaceted influence on the spermatogenesis of E. sinensis, impacting chromatin decondensation, genetic integrity, and the regulation of early gene expression. This understanding could potentially improve crab breeding in aquaculture.

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.

Abstract Chinese mitten crab, Eriocheir sinensis, is a decapod crustacean with a special, non-condensated nucleus in the sperm. Studies has shown that this is closely related to histone modification. To explore the possible role of histone acetyltransferase 1 (HAT1) in the non-condensation mechanism of sperm nucleus and the spermatogenesis of E. sinensis, we took the testis tissues of adult and juvenile crabs as the objects of study and analyzed their biological functions by whole transcriptome sequencing and bioinformatics, then further analyzed the expression and distribution of HAT1 through RT-qRCR, western blotting, and immunofluorescence. The results showed that HAT1 was up-regulated in adult testes compared with in the juvenile ones (n = 3, P < 0.05). Besides, HAT1 was mainly located in the nucleus of the male germ cell of E. sinensis. With the development of spermatogonia, the expression of HAT1 decreased (n = 3, P < 0.05), and the sperm nucleus of E. sinensis also gradually become non-condensed. Bioinformatics analysis shows that HAT1 and proteins such as Asf1b, Chaf1b and Hist1h3f are involved in biological processes such as DNA conformational changes and DNA replication-dependent nucleosome assembly. It follows that HAT1 is an important regulator of maintaining histone acetylation at specific levels. During the spermatogenesis of E. sinensis, HAT1 first loosens the chromatin structure of sperm nucleus by acetylating histone, while the reduction of HAT1 expression in the later stage effectively avoids excessive acetylation, and maintains histone acetylation at a specific level, which protects the nuclear chromatin state of non-condensed sperm and is important in the spermatogenesis of E. sinensis.

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.

The aim of this study was to investigate the regulatory role of miR-133b on the expression of Sin3a associated protein 30 (SAP30) during sperm nuclear decondensation. The expressions of both SAP30 at mRNA and protein levels and miRNA targeted into SAP30 were analyzed in the testis tissues of adult and juvenile Eriocheir sinensis, to explore the distribution of SAP30 and the possible regulatory relationship of miRNAs with SAP30, using the high-throughput sequencing, real-time fluorescence quantitative (RT-qPCR), western blot(WB), Immunofluorescence (IF), and other techniques. The results showed that SAP30 was differentially expressed in decondensed nuclei of E. sinensis testis tissue (P < 0.05), and was up-regulated in adult crab testis tissue. The expression at the mRNA level was consistent with the expression at the protein level. miR-133b targeting SAP30 is differentially expressed in adult and juvenile crab testis tissues, but compared with juvenile crab testis tissue, they are all down-regulated in adult crab testis tissue, indicating that SAP30 and miR-133b are highly likely to have Targeting regulatory relationships. SAP30 was mainly located in the nucleus in the testis tissue of E. sinensis, and its expression level was the highest in spermatogonia. With the development of cells, the expression of SAP30 decreased. The SAP30 gene encodes a protein that is a component of the histone deacetylase complex. SAP30 may be involved in the regulation of chromatin dynamics by regulating the level of histone deacetylation, and thus plays an important role in the transition from the relatively condensed state in the early stage to the decondensed state in the late stage of the sperm nucleus of E. sinensis. SAP30 may be a key protein involved in sperm nuclear decondensation, and the reduction of miR-133b may be a key factor promoting the high expression of SAP30 in adult crab testis tissue.

目的 探讨免疫球蛋白μ结合蛋白2(Ighmbp2)在小鼠肝细胞癌(HCC)中上调表达的可能机制.方法 20 只小鼠注射生理盐水,收集其肝组织作为SL组.80 只小鼠注射H22 肝癌细胞系,分别收集注射3d的小鼠的肝组织、30d未成瘤小鼠的肝组织及成瘤小鼠的癌旁组织和瘤组织,分别作为H3L组、H30NL组、H30L组和H30T组.采用全转录组测序和生物信息学分析Ighmbp2 基因在各组中的表达,并进一步采用华大基因多组学系统筛选差异表达的Ighmbp2 相关基因,构建差异表达相关基因的PPI、Target以及ceRNA网络,分析其功能.结果 与SL组相比,H3L组、H30NL组、H30L组和H30T组Ighmbp2基因表达均上调,其中H30T组中表达最高;组间比较显示,仅H30T组与其他四组间Ighmbp2 基因的表达差异具有统计学意义(P＜0.05).PPI、Target和ceRNA调控网络显示,与Ighmbp2 存在密切关系的转录本有5 个、基因编码蛋白有43 个,互为竞争性ceRNA的分子有 143 个,其中miR-107-5p、miR-3064-5p和miR-1968-5p在H30T组中的表达显著低于其他各组,转录本XM_006531702.1 大量表达于各组,并且在H30T组的表达远高于其他组.GO和KEGG富集分析发现,Ighmbp2 及其相关基因共同参与的功能包括核苷酸结合、ATP结合等,其中部分基因与Ighmbp2 基因一样属于核、核仁等细胞组分.结论 Ighmbp2基因的上调表达主要是转录本XM_006531702.1 的上调表达;miR-107-5p、miR-3064-5p和miR-1968-5p这3 种miRNA可能在瘤体组织中低表达从而减弱了抑制效应进而间接促进Ighmbp2 在瘤体组织中的上调表达,其中miR-1968-5p可能是影响Ighmbp2表达的关键因素,多种相关分子可能与以上3 种miRNA结合形成ceRNA调控机制进而进一步精细调控Ighmbp2 的表达.