Project description:Analysis of gene expression and alternate splicing effects of retinoic acid treatment on gestational day 15 rat fetal testes in whole testis culture Retinoic acid exposure in cultured fetal testis has previously been demonstrated to have significant effects on the histology of the fetal testis in multiple species, as well as to alter the meiotic states of germ cells. However, previous experiments have not analyzed the mechanisms by which retinoic acid exposure leads to altered tubulogenesis and loss of seminiferous cord structure. This experiment demonstrated that retinoic acid exposure activated signaling pathways that promote the ovary development program and oppose normal testis development in mid-gestational rat fetal testes.
Project description:We report the results of an RNA-seq analysis conducted as part of an experiment investigating the effects of the phthalate, mono-(2-ethylhexyl) phthalate (MEHP), and all-trans retinoic acid (ATRA) on cultured fetal mouse testes. The goal of the study was to determine whether fetal testis toxicity of MEHP is partially driven by disruption of retinoic acid signaling.
Project description:Analysis of LBNF1 rat testes from controls, containing both somatic and all germ cell types and from irradiated rats in which all cells germ cells except type A spermatgogonia are eliminated. Results provide insight into distinguishing germ and somatic cell genes and identification of somatic cell genes that are upregulated after irradiation.
Project description:Spermatogenesis is an intricate developmental process occurring in testes by which spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm. The molecular mechanisms for SSC self-renewal and differentiation, while have been well studied in mice, may differ between mice and domestic animals including pigs. To gain knowledge about the molecular mechanisms for porcine SSC self-renewal and differentiation that have to date been poorly understood, here we isolated and enriched primitive spermatogonia from neonatal porcine testes, and exposed the cells to retinoic acid, a direct inducer for spermatogonial differentiation. We then identified that retinoic acid could induce porcine primitive spermatogonial differentiation into leptotene spermatocyte-like cells, which was accompanied by a clear transcriptomic alteration, as revealed by the RNA-sequencing analysis. We also compared retinoic acid-induced in vitro porcine spermatogonial differentiation with the in vivo process, and compared retinoic acid-induced in vitro spermatogonial differentiation between pigs and mice. Furthermore, we analyzed retinoic acid-induced differentially expressed long non-coding RNAs (lncRNAs), and demonstrated that a pig-specific lncRNA, lncRNA-106504875, positively regulated porcine spermatogonial proliferation by targeting the core transcription factor ZBTB16. Taken together, these results would help to elucidate the roles of retinoic acid in porcine spermatogonial differentiation, thereby contributing to further knowledge about the molecular mechanisms underlying porcine SSC development and, in the long run, to optimization of both long-term culture and induced differentiation systems for porcine SSCs.
Project description:Aims: To obtain small non-coding RNA expression profiles of short-term activated and long-term activated hepatic stellate cells (HSCs). To obtain small non-coding RNA expression profiles of small extracellular vesicles (sEVs) from short-term activated and long-term activated HSCs. Methods: Primary rat HSCs were isolated and cultured in vitro. To isolate HSC-derived sEVs, culture media (CMs) from Day 0 to Day 3 and Day 7 to Day 14 were collected. The sEVs from Day 0 to Day 3 HSC CMs were referred to as short-term activated HSC-sEVs (3dHSC-sEVs), and those from Day 7 to Day 14 HSC CMs were referred to as long-term activated HSC-sEVs (14dHSC-sEVs). Day 3 HSCs, Day 14 HSCs, and HSC-sEVs were collected and lysed in TRIzol (Life Technologies) for RNA sample preparation at the indicated time points.
Project description:Analysis of hormone effects on irradiated LBNF1 rat testes, which contain only somatic cells except for a few type A spermatgogonia. Rats were treated for 2 weeks with either sham treatment (group X), hormonal ablation (GnRH antagonist and the androgen receptor antagonist flutamide, group XAF), testosterone supplementation (GnRH antagonist and testosterone, group XAT), and FSH supplementation ((GnRH antagonist, androgen receptor antagonist, and FSH, group XAFF). Results provide insight into identifying genes in the somatic testis cells regulated by testosterone, LH, or FSH.
Project description:This study aimed to investigate the direct mechanism(s) of action ofMono-(Ethyl-Hexyl) phthalate (MEHP) on the rat fetal testis with particular emphasis to Leydig cell steroidogenesis, by means of an in vitro system based on culture for 3 days of fetal testes from 14.5 days post-coïtum old rats. We first confirmed that an exposure to MEHP led to a dose-dependent decrease of testosterone and demonstrated that dihydrotestosterone (DHT) production was also inhibited. We then demonstrated that in 10µM MEHP-exposed testis basal 17alpha-hydroxy-progesterone (17OHP) production was increased (+40%) while androstenedione levels decreased (-55%). Furthermore, the addition of the latter steroid but none of the other precursors rescued testosterone thus establishing further that MEHP specifically blocked steroidogenesis at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), which converts 17OHP to androstenedione. Furthermore and accordingly, both levels of testicular CYP17 protein (western blot) and cyp17a1 gene (qPCR, microarrays) were found to decrease under MEHP exposure. The microarray analysis also showed that among the few other testicular genes cyp17a1 whose expression was inhibited by MEHP were the genes encoding for the Leydig cell insulin-like peptid 3 (INSL3), involved in the control of testicular descent, and for inhibin A (INHA), that regulates follicular-stimulating hormone secretion.Under in vitro conditions where MEHP is not metabolized and remains at low intratesticular concentration, our findings show in particular that this phthalate directly inhibits several important Leydig cell factors involved in testis development and function. Transcription profiling of rat fetal testes explants, vehicle or monoethylhexylphthalate (MEHP)-treated (1uM, 10uM) 3 condition experiment, 1-3 biological replicates per treatment. 2 technical replicates per sample.
