Project description:Spermatogenesis has been well studied in rodents and invertebrates, but remains poorly understood in humans. As a step towards illuminating human spermatogenesis, we used single-cell RNA-sequencing (scRNAseq) analysis to analyze neonatal and adult human testes. Clustering analysis of neonatal testes revealed 3 germ subsets, including cells with characteristics of primordial germ cells (PGCs), and more differentiated cells with gene expression profiles similar with adult spermatogonial stem cells (SSCs). We identified markers for these neonatal subsets, including protein markers for the PGC-like (PGCL) subset. Clustering analysis of the adult testis revealed 9 germ and 3 somatic cell subsets. Among the germ cell clusters are 4 undifferentiated spermatogonia (SPG) states, each marked by specific genes. One of the SPG states has characteristics suggesting it is enriched for SSCs. We identified protein markers specific for this state, including cell-surface proteins that we used to enrich for these cells. We mapped the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also defined somatic cell subsets in the human testis and traced their developmental trajectories. Together, our data provides a blueprint for understanding the development of the male germline and supporting somatic cells in humans. The germ cell subset markers we identified are candidates to be used for clinical applications, including SSC therapy for treating infertility.
Project description:WIN 18,446/RA treatment of neonatal male mice was used to synchronize spermatogenesis to 2-3 different stages of the cycle of the seminiferous epithelium in the adult testis
Project description:Todate, the investigation into the molecular mechanisms of testis development and spermatogenesis has largely focused on the role of protein-coding genes and small noncoding RNA including microRNAs and piRNAs. In recent years, long noncoding RNAs (lncRNAs) have been shown to play critical regulatory roles in mammalian development. To understand the role of lncRNAs in development of the mammalian testis and spermatogenesis, we firstly utilized commercial microarray to systematically investigate lncRNAs expression profiles of neonatal (6-day-old) and adult (8-week-old) mouse testis. By comparison the lncRNAs expression profiles of two developmental stage of testes, we obtained the differentially expressed lncRNAs and examined their genomic context, promoter characteristics, neighbored protein-coding genes, provide an important foundation for future research on molecular mechanisms of lncRNAs in mammalian testis development and spermatogenesis.
Project description:Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility.
Project description:WIN 18,446/RA treatment of neonatal male mice was used to synchronize spermatogenesis to 2-3 different stages of the cycle of the seminiferous epithelium in the adult testis 6 different groups of synchronized adult mouse testis samples (N = 2 per group), each representing only 2-3 stages of the cycle, were collected and analyzed using GeneChip Mouse Gene ST 1.0 Arrays
Project description:The sequence of gene regulatory events that drive neonatal germ cell development in the mammalian testis is not yet clear. We assessed changes in mRNA utilization in the neonatal testis at 1 and 4 dpp, times when the testis contains quiescent gonocytes (1 dpp) and proliferating spermatogonia (4 dpp). There are not thought to be major changes in the nature or number of somatic cells over that interval. We used microarrays to detail the global expression levels of mRNA distribution between non-translating mRNAs and efficiently translating mRNAs during testis development at 1 dpp and 4 dpp Extracts of mouse testes from 1 dpp and 4 dpp CD-1 mice were fractionated over a sucrose gradient in triplicate. Gradients were fractionated using an ISCO gradient fractionation system equipped with a UA-6 detector. The position of the 80S peak defined the boundary between translating and non-translating mRNAs. Pooled non-polysomal (non-translating; fractions 1-3) and heavy polysomal (efficiently translating, fractions 7-13) RNAs were isolated from sucrose gradient fractions for hybridization on Affymetrix 420 2.0 microarrays.
Project description:The development of the testis involves a large number of tissue-specific proteins, possibly because that sperms in it are the most divergent of all cell types. Although the proteome of the larval testis of Bombyx mori has been reported, no comprehensive data on adult testis proteome is available for silkworm. In this study, Liquid chromatography-tandem mass spectrometry was employ to investigate the protein compositions of the adult testis of silkworm. A total of 14431 peptides were identified in the adult testis of B. mori, which were match to 2292 proteins. Thirty-two heat shock proteins constitute a group of most abundant proteins in the adult testis, suggested that they are critical for the development, differentiation and survival of germ cells. Other proteins in this analysis were also involved in testis-specific processes mainly including sperm motility, meiosis, germ cell development, and spermatogenesis.
Project description:An excess in thyroid hormone during rodent neonatal life causes abnormal prolifration and maturation of testicular cells, leading to reduced testis size and impairments in steroidogenesis, Sertoli cell function, spermatogenesis and fertility. The high expression of type 3 deiodinase in the neonatal testis protects this tissue from premature exposure to thyroid hormones, since this gene (Dio3) function is to degrade thyroid hormones. DIO3-deficient mice (Dio3KO) exhibit a marked reduction in Sertoli cell proliferation and testis size, abnormalities in the reproductive axis and impaired fertility (Martinez et al. 2016, Endocrinology157:1276). To identify genes that are untimely regulated by thyroid hormone in the developing testes, we have performed RNAseq in testis total RNA from wild type and DIO3KO mouse neonates.
Project description:The sequence of gene regulatory events that drive neonatal germ cell development in the mammalian testis is not yet clear. We assessed changes in mRNA utilization in the neonatal testis at 1 and 4 dpp, times when the testis contains quiescent gonocytes (1 dpp) and proliferating spermatogonia (4 dpp). There are not thought to be major changes in the nature or number of somatic cells over that interval. We used microarrays to detail the global expression levels of mRNA distribution between non-translating mRNAs and efficiently translating mRNAs during testis development at 1 dpp and 4 dpp