mouse testis RNA

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq)...

The branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential for animal growth and metabolic health. However, the effect of valine on male reproduction and its underlying molecular mechanism remain largely unknown. Here, we showed that l-valine supplementation (0.30% or 0.45%, water drinking for 3 weeks) did not change body and testis weights, but significantly altered morphology of sertoli cells and germ cells within seminiferous tubule, and enlarged the space between seminiferous tubules within mouse testis...

Long noncoding RNAs (lncRNAs) are transcribed in complex, overlapping, sense- and antisense orientations from intronic and intergenic regions of mammalian genomes. Transcription of genome in mammalian testis is more widespread compared to other organs. LncRNAs are involved in gene expression, chromatin regulation, mRNA stability and translation of proteins during diverse cellular functions. We report molecular cloning of two novel lncRNAs (mLINC-RBE and mLINC-RSAS) and their expression by RT-PCR as well as cellular localization by RNA in-situ hybridization in the mouse testes...

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development...

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 (Orc6), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence...

Eif2s3y (eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked, Eif2s3y) is an essential gene for spermatogenesis. Early studies have shown that Eif2s3y can promote the proliferation of spermatogonial stem cells (SSCs) and can replace the Y chromosome together with sex-determining region Y (Sry) to transform SSCs into round spermatozoa. We injected lentiviral particles into the seminiferous tubules of mouse testes by sterile surgery surgically to establish overexpressing Eif2s3y testes...

Controversy continues over the functional prevalence of long non-coding RNAs (lncRNAs) despite their being widely investigated in all kinds of cells and organisms. In animals, lncRNAs have aroused general interest from exponentially increasing transcriptomic repertoires reporting their highly tissue-specific and developmentally dynamic expression, and more importantly, from growing experimental evidence supporting their functionality in facilitating organogenesis and individual fitness. In mammalian testes, while a great multitude of lncRNA species are identified, only a minority of them have been shown to be useful, and even fewer have been demonstrated as true requirements for male fertility using knockout models to date...

Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes 1, 2 . Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation 3, 4 . In C. elegans , inherited small RNA precursors have poly (UG) tails 5 , but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs...

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros...

Proliferation and meiotic division of spermatogonial stem cells are highly regulated biological processes that occur during spermatogenesis. In addition to protein coding genes, mammalian genome encodes thousands of lncRNAs which are spatio-temporally expressed and play key role(s) in cellular differentiation and development. Mrhl lncRNA is one such mono-exonic polyadenylated non-coding RNA encoded within the 15th intron of the mouse Phkb gene. Mrhl lncRNA is expressed in mouse testis amongst other tissues...

Maintenance and self-renewal of the spermatogonial stem cell (SSC) population in the testis are dictated by the expression of a unique suite of genes. In manipulating gene expression through loss-of-function approaches, we can identify important regulatory mechanisms that dictate spermatogonial fate decisions. One such approach is RNA interference (RNAi), which uses natural cellular responses to small interfering RNAs to decrease levels of a targeted transcript. RNAi is performed in primary cultures of undifferentiated spermatogonia, and can be paired with techniques such as spermatogonial transplantation to assess the functional consequences of downregulated expression of the target gene on stem cell maintenance...

Single-molecule fluorescence in situ hybridization (smFISH) enables the detection and localization of individual mRNAs in tissue sections with single-molecule resolution while preserving spatial context, and thus, is a useful tool for examining gene expression in biological systems. In particular, the growing reliance on single-cell RNA sequencing (scRNA-seq) to explore cellular heterogeneity has reinvigorated this approach as a validation tool to spatially re-map mRNA expression patterns described in isolated cells to their parent tissue...

BACKGROUND: The Asian lineage Zika virus (ZIKV) emerged as a public health emergency in 2016 causing severe neurological pathologies with no apparent historical correlate to the mild, disease-causing innocuous member of the mosquito-borne flavivirus genus that was discovered in Africa in 1947. Replication error rate of RNA viruses combined with viral protein/RNA structural plasticity can lead to evolution of virus-induced pathogenicity that is critical to identify and validate. METHODS: Infection studies in cells and A129 interferon alpha/beta receptor deficient mice with ZIKV French Polynesian H/PF/2013 clinical isolate, plaque-purified isogenic clone derivatives as well as infectious cDNA clone derived wild-type and site-specific mutant viruses, were employed together with Next-Generation Sequencing (NGS) to pin-point the contributions of specific viral variants in neurovirulence recapitulated in our ZIKV mouse model...

CTCF is an architectonic protein that organizes the genome inside the nucleus in almost all eukaryotic cells. There is evidence that CTCF plays a critical role during spermatogenesis as its depletion produces abnormal sperm and infertility. However, defects produced by its depletion throughout spermatogenesis have not been fully characterized. In this work, we performed single cell RNA sequencing in spermatogenic cells with and without CTCF. We uncovered defects in transcriptional programs that explain the severity of the damage in the produced sperm...

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation...

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here we characterize the function of a testis-specific IQ motif containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Through immunohistochemistry of testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS)...

Antineoplastic treatments for cancer and other non-malignant disorders can result in long-term or permanent male infertility by ablating spermatogonial stem cells (SSCs). SSC transplantation using testicular tissue harvested before a sterilizing treatment is a promising approach for restoring male fertility in these cases, but a lack of exclusive biomarkers to unequivocally identify prepubertal SSCs limits their therapeutic potential. To address this, we performed single-cell RNA-seq on testis cells from immature baboons and macaques and compared these cells with published data from prepubertal human testis cells and functionally-defined mouse SSCs...

GRTH/DDX25 is a testis-specific DEAD-box family of RNA helicase, which plays an essential role in spermatogenesis and male fertility. There are two forms of GRTH, a 56 kDa non-phosphorylated form and a 61 kDa phosphorylated form (pGRTH). GRTH-KO and GRTH Knock-In (KI) mice with R242H mutation (lack pGRTH) are sterile with a spermatogenic arrest at step 8 of spermiogenesis due to failure of round spermatids (RS) to elongate. We performed mRNA-seq and miRNA-seq analysis on RS of WT, KI, and KO to identify crucial microRNAs (miRNAs) and mRNAs during RS development by establishing a miRNA-mRNA network...

Testis-specific transcript 10 (Tex10) is a critical factor for pluripotent stem cell maintenance and preimplantation development. Here, we dissect its late developmental roles in primordial germ cell (PGC) specification and spermatogenesis using cellular and animal models. We discover that Tex10 binds the Wnt negative regulator genes, marked by H3K4me3, at the PGC-like cell (PGCLC) stage in restraining Wnt signaling. Depletion and overexpression of Tex10 hyperactivate and attenuate the Wnt signaling, resulting in compromised and enhanced PGCLC specification efficiency, respectively...

Tributyltin chloride (TBTCL), a commonly used antiseptic substance, is commonly found in the environment. Human exposure to TBTCL through the consumption of contaminated seafood, fish, or drinking water has aroused concern. It is well-characterized that TBTCL has multiple detrimental effects on the male reproductive system. However, the potential cellular mechanisms are not fully elucidated. Here, we characterized molecular mechanisms of TBTCL-induced cell injury in Leydig cells, a critical supporter for spermatogenesis...