Project description:We performed bulk RNA-Seq on male fetal germ cells isolated from E13.5 and E15.5 testes from activin A-deficient mice (Inhba KO), and from E13.5 testes from mice with greater activin A bioactivity (Inha KO). The aim of this expereiment was to determine how chronic altered activin A bioactivity altered the germline transcriptome.
Project description:Background: The regular use of cannabis by young men has been associated with an increased incidence of testicular germ cell tumors (TGCT). TGCT, the most common cancer in young adults, is believed to arise from an alteration of testicular fetal germ cells differentiation during development, with a possible subsequent environmental trigger (eg drugs or other chemicals) during puberty or adulthood leading to cancer. Cannabis consumption by pregnant women is currently increasing worldwide, and legalization for its recreational and therapeutic purposes is debated in numerous countries. In this context, we aimed to determine whether cannabis exposure can affect development of the fetal testis. Methods: Since phytocannabinoids act on an endogenous system called the endocannabinoid system (ECS), we first investigated these signaling pathways in the human fetal testis, from 6 to 17 developmental weeks. We next investigated the effects of the two main components of cannabis, (−)-Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the human fetal testis ex vivo. Results: We show the presence of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), albeit with lower levels. The human fetal testis also expressed several enzymes and receptors of this signaling pathway. Human fetal testicular explants collected from first trimester were exposed to CBD, THC or CBD/THC [ratio 1:1] at concentrations ranging from 10-7 to 10-5M during 72h to 14 days. Phytocannabinoids treatments affected fetal testicular cell proliferation and viability, as well as testosterone secretion by Leydig cells and AMH secretion by Sertoli cells. Transcriptomic analysis performed by BRB-seq on exposed versus unexposed fetal testis explants showed 187 differentially expressed genes (DEGs), some of which involved in toxic substances response and steroid synthesis. Conclusions: Our study provides the first evidence of the presence of ECS in human fetal testis and support a potential adverse effect of cannabis consumption in pregnant women on the male reproductive function development.
Project description:In humans, the most common sex chromosomal disorder is Klinefelter syndrome (KS), caused by the presence of one or more extra X-chromosomes. The KS patients display a diverse adult phenotype with increased height, gynaecomastia, and hypergonadotropic hypogonadism as the most common symptoms. Men with KS are almost always infertile due to testicular degeneration, which accelerates during puberty. Very few studies investigated when the germ cell loss begins and whether it is caused by dysgenetic fetal development of the testes. We investigated a series of fetal KS testis tissue samples and found a marked reduction in MAGE-A4-positive pre-spermatogonia in the developing KS gonads compared to controls, indicating a failure of the gonocytes to differentiate into pre-spermatogonia. Transcriptome analysis by RNA sequencing of formalin-fixed and paraffin embedded gonads originating from 4 fetal KS samples and 5 age- and cellularity-matched controls revealed 211 differentially expressed transcripts in the fetal KS testis. We found a significant enrichment of upregulated X-chromosomal transcripts and validated the expression of the pseudoautosomal region 1 (PAR1) gene, AKAP17A. Moreover, we found enrichment of long non-coding RNAs in the KS testes (e.g. LINC01569 and RP11-485F13.1). In conclusion, our data indicates that the testicular phenotype observed among adult men with KS is initiated already in fetal life by failure of the gonocyte differentiation into pre-spermatogonia, which could be due to aberrant expression of long non-coding RNAs.
Project description:Identification of the embryonic germ cell Meioc-/- transcriptome, MEIOC targets in postnatal testis, and YTHDC2 targets in postnatal testis in mouse
Project description:Mammalian egg and sperm are produced by germ cells (GCs) that develop in the embryo long before reproductive maturity. Many GCs that begin this process do not contribute their genes to the next generation, although it remains unclear what determines GC fitness. Here, we examined how the composition of GCs in the fetal testis is affected by scheduled apoptosis as GCs transition between proliferation to sex differentiation. Using multicolored-lineage tracing, we find that apoptosis affects clonally-related GCs, suggesting that this fate decision occurs autonomously based on shared intrinsic properties. Single cell RNA-sequencing reveals extensive heterogeneity among GCs and identifies a Trp53-high subpopulation with elevated apoptotic susceptibility and diminished differentiation. By contrast, the most differentiated subpopulation is depleted for pro-apoptotic transcripts. These results indicate that cell-heritable differences in sex-differentiation segregate GCs into subpopulations with distinct fitness. The reciprocal relationship between sex-differentiation and apoptosis coordinates the removal of developmentally incompetent cells to improve male GC quality.
Project description:From fish to human, FOXL2 is considered one of the most conserved markers of ovarian granulosa cell identity. To determine if the sole expression of FOXL2 can determine ovarian differentiation, we created a mouse model that allows the conditional expression of FOXL2. Rosa26-CAG-LSL-Foxl2 mice were crossed to Sf1-Cre mice to induce the expression of FOXL2 in the SF1+ somatic cells of the fetal gonads.When FOXL2 was induced in the somatic cells of the undifferentiated testis, the Sertoli cells and consequently the other cell lineages composing the fetal gonads were feminized, resulting in a partial testis-to-ovary sex reversal We created a mouse genetic model that conditionaly express FOXL2 in the somatic cells of the fetal gonads. All embryos used in this study resulted from the crossing between Rosa26-CAG-LSL-Foxl2+/f and Sf1-cre+/Tg mice. XX and XY fetal gonads were collected at embryonic day E14.5. This microarray analysis led to the identification of the genes misregulated upon ectopic induction of FOXL2 in the fetal testis, and showed that FOXL2 expression resulted in feminization of both somatic and germ cells of the fetal gonad.
Project description:The goals of this study are to utilize high-throughput transcriptome sequencing of mutant and control fetal testis samples to identify changes in both transcript and repeat element abundance in tissues harboring a homozygous mutation for Glis3. 672 unique genes were differentially expressed in mutant versus wild-type samples. Of the downregulated genes, there was a strong enrichment for piRNA pathway members, while upregulated genes were associated with leydig cell differentiation, meiosis, and histone cluster genes. Differential expression of several repeat elements was also detected in mutant samples. Our findings provide valuable information on the potential mechanisms underlying the fetal germ cell loss observed in Glis3 mutant testes. Whole testis mRNA profiles of embryonic day 14.5 wild type (WT) and Glis3 mutant mice were generated by deep sequencing, using Illumina HiSeq2500