Functions and mechanism of noncoding RNA in regulation and differentiation of male mammalian reproduction.

Noncoding RNAs (ncRNAs) are active regulators of a wide range of biological and physiological processes, including the majority of mammalian reproductive events. Knowledge of the biological activities of ncRNAs in the context of mammalian reproduction will allow for a more comprehensive and comparative understanding of male sterility and fertility. In this review, we describe recent advances in ncRNA-mediated control of mammalian reproduction and emphasize the importance of ncRNAs in several aspects of mammalian reproduction, such as germ cell biogenesis and reproductive organ activity. Furthermore, we focus on gene expression regulatory feedback loops including hormones and ncRNA expression to better understand germ cell commitment and reproductive organ function. Finally, this study shows the role of ncRNAs in male reproductive failure and provides suggestions for further research.

Altered G-Protein Transduction Protein Gene Expression in the Testis of Infertile Patients with Nonobstructive Azoospermia.

Recent studies have shown that several members of the G-protein-coupled receptors (GPCR) superfamily play crucial roles in the maintenance of ion-water homeostasis of the sperm and Sertoli cells, development of the germ cells, formation of the blood barrier, and maturation of sperm. The GPCR, guanyl-nucleotide exchange factor, membrane traffic protein, and small GTPase genes were analyzed by microarray and bioinformatics (3513 sperm and Sertoli cell genes). In the microarray analyses of three human cases with different nonobstructive azoospermia sperm, the expression of GOLGA8IP, OR2AT4, PHKA1, A2M, OR56A1, SEMA3G, LRRC17, APP, ARHGAP33, RABGEF1, NPY2R, GHRHR, LTB4R2, GRIK5, OR6K6, NAPG, OR6C65, VPS35, FPR3, and ARL4A was upregulated, while expression of MARS, SIRPG, OGFR, GPR150, LRRK1, and NGEF was downregulated. There was an increase in GBP3, GBP3, TNF, TGFB3, and CLTC expression in the Sertoli cells of three human cases with NOA, whereas expression of PAQR4, RRAGD, RAC2, SERPINB8, IRPB1, MRGPRF, RASA2, SIRPG, RGS2, RAP2A, RAB2B, ARL17, SERINC4, XIAP, DENND4C, ANKRA2, CSTA, STX18, and SNAP23 were downregulated. A combined analysis of Enrich Shiny Gene Ontology (GO), STRING, and Cytoscape was used to predict proteins' molecular interactions and then to recognize master pathways. Functional enrichment analysis showed that the biological process (BP), regulation of protein metabolic process, regulation of small GTPase-mediated signal transduction were significantly expressed in up-/downregulated differentially expressed genes (DEGs) in sperm. In molecular function (MF) experiments of DEGs that were up-/downregulated, it was found that GPCR activity, guanyl ribonucleotide binding, GTPase activity and nucleoside-triphosphatase activity were overexpressed. An analysis of GO enrichment findings of Sertoli cells showed BP and MF to be common DEGs. When these gene mutations have been validated, they can be used to create new GPCR antagonists or agonists that are receptor-selective.

OCT4 protein and gene expression analysis in the differentiation of spermatogonia stem cells into neurons by immunohistochemistry, immunocytochemistry, and bioinformatics analysis.

BACKGROUND: Spermatogonia Stem Cells (SSCs) are potential candidates for reprogramming and regeneration. Recent studies have revealed that differentiated cells can be reverted to pluripotent by overexpressing a set of pluripotent transcription factors. OCT4 (encoded by pou5f1), a POU transcription factor family member, is essential to the potential that controls pluripotency, and it is widely expressed in pluripotent stem cells, although it decreased or suppressed after differentiation. METHODS: In this investigated research, we examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein-protein interaction and enrichment analysis. RESULTS: We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming. CONCLUSION: Our findings can help us better understand the process of differentiation of spermatogonia stem cells into neurons, and it can be effective in finding new and more efficient treatments for neurogenesis and repair of neurons. We examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein-protein interaction and enrichment analysis. We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming.

VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis.

VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein-protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Whole Exome Sequencing and In Silico Analysis of Human Sertoli in Patients with Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is a serious cause of male infertility. The Sertoli cell responds to androgens and takes on roles supporting spermatogenesis, which may cause infertility. This work aims to enhance the genetic diagnosis of NOA via the discovery of new and hub genes implicated in human NOA and to better assess the odds of successful sperm extraction according to the individual's genotype. Whole exome sequencing (WES) was done on three NOA patients to find key genes involved in NOA. We evaluated genome-wide transcripts (about 50,000 transcripts) by microarray between the Sertoli of non-obstructive azoospermia and normal cells. The microarray analysis of three human cases with different non-obstructive azoospermia revealed that 32 genes were upregulated, and the expressions of 113 genes were downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluations were applied to predict the functional and molecular interactions of proteins and then recognize the master pathways. The functional enrichment analysis demonstrated that the biological process (BP) terms "inositol lipid-mediated signaling", "positive regulation of transcription by RNA polymerase II", and "positive regulation of DNA-templated transcription" significantly changed in upregulated differentially expressed genes (DEGs). The BP investigation of downregulated DEGs highlighted "mitotic cytokinesis", "regulation of protein-containing complex assembly", "cytoskeleton-dependent cytokinesis", and the "peptide metabolic process". Overrepresented molecular function (MF) terms in upregulated DEGs included "ubiquitin-specific protease binding", "protease binding", "phosphatidylinositol trisphosphate phosphatase activity", and "clathrin light chain binding". Interestingly, the MF analysis of the downregulated DEGs revealed overexpression in "ATPase inhibitor activity", "glutathione transferase activity", and "ATPase regulator activity". Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiologies of germ cell abnormalities and infertility.

Microarray and in silico analysis of DNA repair genes between human testis of patients with nonobstructive azoospermia and normal cells.

DNA repair processes are critical to maintaining genomic integrity. As a result, dysregulation of repair genes is likely to be linked with health implications, such as an increased prevalence of infertility and an accelerated rate of aging. We evaluated all the DNA repair genes (322 genes) by microarray. This study has provided insight into the connection between DNA repair genes, including RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, INO80, PMS2L1, CHEK2, TRIP13, and POLD4. The microarray analysis of six human cases with different nonobstructive azoospermia revealed that RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, and INO80 were upregulated, and expression of PMS2L1, CHEK2, TRIP13, and POLD4 was downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluation was applied to predict proteins' functional and molecular interactions and then performed to recognize the master pathways. Functional enrichment analysis revealed that the biological process (BP) terms "base-excision repair, AP site formation," "nucleotide-excision repair, DNA gap filling," and "nucleotide-excision repair, preincision complex assembly" was significantly overexpressed in upregulated differentially expressed genes (DEGs). BP analysis of downregulated DEGs highlighted "histone phosphorylation," "DNA damage response, detection DNA response," "mitotic cell cycle checkpoint signaling," and "double-strand break repair." Overrepresented molecular function (MF) terms in upregulated DEGs included "Oxidized base lesion DNA N-glycosylase activity," "uracil DNA N-glycosylase activity," "bubble DNA binding" and "DNA clamp loader activity." Interestingly, MF investigation of downregulated DEGs showed overexpression in "heterotrimeric G-protein complex," "5'-deoxyribose-5-phosphate lyase activity," "minor groove of adenine-thymine-rich DNA binding," and "histone kinase activity." Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

A review of protein-protein interaction and signaling pathway of Vimentin in cell regulation, morphology and cell differentiation in normal cells.

The Vimentin intermediate filament (VIF) is an essential cytoskeleton component. It shows dynamically changing expression patterns throughout various phases of the differentiation process, suggesting that the protein is physiologically important. Vimentin's essential functions have recently been clear, so Vimentin-deficient of animals was described as a change of morphology and signaling pathway. Recent research has discovered many vital roles for Vimentin that were previously unknown. VIF emerges as an organizer of many essential proteins involved in movement and cell signaling. The highly dynamic and complicated phosphorylation of VIF seems to be a regulator mechanism for various activities. Changes in IF expression patterns are often linked with cancer progression, especially those leading to enhanced invasion and cellular migration. This review will discuss the function of Vimentin intermediate filaments in normal cell physiology, cell adhesion structures, cell shape, and signaling pathways. The genes interaction and gene network linked with Vimentin will be discussed in more studies. However, research aimed at understanding the function of Vimentin in different signaling cascades and gene interactions might offer novel methods for creating therapeutic medicines. Enrichr GEO datasets used gene ontology (GO) and pathway enrichment analyses. STRING online was used to predict the functional connections of proteins-proteins, followed by Cytoscape analysis to find the master genes. Cytoscape and STRING research revealed that eight genes, Fas, Casp8, Casp6, Fadd, Ripk1, Des, Tnnc2, and Tnnt3, were required for protein-protein interactions with Vimentin genes involved in cell differentiation.

Testicular Localization and Potential Function of Vimentin Positive Cells during Spermatogonial Differentiation Stages.

Vimentin is a type of intermediate filament (IF) and one of the first filaments expressed in spermatogenesis. Vimentin plays numerous roles, consisting of the determination of cell shape, differentiation, cell motility, the maintenance of cell junctions, intracellular trafficking, and assisting in keeping normal differentiating germ cell morphology. This study investigated the vimentin expression in two populations of undifferentiated and differentiated spermatogonia. We examined vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), and Fluidigm real-time polymerase chain reaction. IMH data showed that the high vimentin expression was localized in the middle of seminiferous tubules, and low expression was in the basal membrane. ICC analysis of the colonies by isolated differentiated spermatogonia indicated the positive expression for the vimentin antibody, but vimentin's expression level in the undifferentiated population was negative under in vitro conditions. Fluidigm real-time PCR analysis showed significant vimentin expression in differentiated spermatogonia compared to undifferentiated spermatogonia (p < 0.05). Our results showed that vimentin is upregulated in the differentiation stages of spermatogenesis, proving that vimentin is an intermediate filament with crucial roles in the differentiation stages of testicular germ cells. These results support the advanced investigations of the spermatogenic process, both in vitro and in vivo.