Project description:Total RNAs were extracted from the purified caput epididymal spermatozoa of mouse (C57BL/6, 2 months old) and rats (Sprague Dawley, 450g young adult), and the 18 - 45 nt fraction small RNAs were subjected to library construction and deep sequencing, using Illumina HiSeq 2000.
Project description:Total RNAs were extracted from the purified cauda epididymal spermatozoa of mouse (C57BL/6, 2 months old) and rats (Sprague Dawley, 450g young adult), and the 18 - 45 nt fraction small RNAs were subjected to library construction and deep sequencing, using Illumina GAIIx.
Project description:Total RNAs were extracted from the Testis and Epididymal Caput, Corpus and Cauda tissues of 2-month and 13-month-old WT and Gpx5 KO mice (C57BL/6). The 18 - 40 nt fraction small RNAs and transcriptomes were subjected to library construction and deep sequencing, using Illumina GAIIx or Hiseq 2000.
Project description:Following their production in the testis, spermatozoa enter the epididymis to gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence the progeny outcome. While recent studies underscored the dynamics of small non-coding RNAs in the maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. To map out the sperm methylome dynamics, we purified spermatozoa by FACS from the testis and the different epididymal segments (i.e. caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice. Reduced-Representation Bisulfite Sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5546 entries meeting our threshold values (q value < 0.01, methylation difference above 25 %). Most of these changes were transitory during epididymal sperm maturation according to the low number of entries identified between spermatozoa from cauda vs. testis.
Project description:The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.
Project description:Between the testes and the vas deferens lies the epididymis, a highly convoluted tubule whose unique environmental characteristics are crucial for the functional maturation of spermatozoa. Within the epididymal epithelium, the secretory system releases small non-coding RNA molecules (sncRNAs) and proteins housed within extracellular vesicles (epididymosomes) that are destined for delivery to recipient sperm cells which play key roles in fertility success and act as major conduits of epigenetic information delivered to the oocyte. The epithelial cells of the epididymis have proven to be highly sensitive to environmental stressors which can influence the sperm epigenome. Utilizing a label-free mass spectrometry proteomic approach we sought to characterize an immortalized mouse caput epididymal epithelial cell line (mECap18) and sequenced >5,100 proteins. When compared to a previous in-vivo mouse caput epithelial proteomic profile, a significant overlap (>75%) and proportionally similar patterns of protein classification were observed. Furthermore, key pathways associated with protein synthesis (e.g. EIF2 signaling) and cellular protection in the male reproductive tract (e.g. sirtuin signaling) were enriched in both proteomes. Leveraging this comparison supports mECap18 cells as a promising in-vitro model for recapitulating the in-vivo environment, providing a platform for testing therapeutic invention.
Project description:Previously the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, and adult pachytene spermatocytes, round spermatids, caput epididymal sperm, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed.
Project description:In previous studies, the pesticide DDT was shown to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs), also called epimutations, which can mediate this epigenetic inheritance. In the current study, the developmental origins of the transgenerational DMRs during gametogenesis have been investigated. Male control and DDT lineage F3 generation rats were used to isolate embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus DDT lineage samples were determined at each developmental stage. The top 100 statistically significant DMRs at each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed.
Project description:Background Chronic preconception paternal alcohol use modifies the sperm epigenome, inducing fetoplacental growth defects in the offspring of exposed males. A crucial outstanding question in the field of paternal epigenetic inheritance concerns the resilience of the male reproductive tract and the germline's capacity to recover and correct sperm-inherited epigenetic errors after stressor withdrawal. Objectives We set out to determine if measures of the sperm-inherited epigenetic program revert to match the control treatment one month after withdrawing daily alcohol treatments. Materials and Methods Using a voluntary access model, we exposed C57Bl6/J males to 10% alcohol for ten weeks, withdrew alcohol treatment for four weeks, and used RNA sequencing to examine gene expression patterns in the caput section of the epididymis. We then compared the abundance of sperm small RNA species between treatments. Results In the caput section of the epididymis, chronic alcohol exposures induced changes in the transcriptional control of genetic pathways related to mitochondrial function, oxidative phosphorylation, the generalized stress response (EIF2 signaling), and Sirtuin signaling. Subsequent analysis identified region-specific, alcohol-induced changes in mitochondrial DNA copy number across the epididymis, which correlated with increases in the mitochondrial DNA content of alcohol-exposed sperm. Notably, in the corpus section of the epididymis, increases in mitochondrial DNA copy number persisted one month after alcohol cessation. Analysis of sperm noncoding RNAs between Control and Alcohol-Exposed males one month after alcohol withdrawal revealed a ~100-fold increase in mir-196a, a microRNA induced as part of the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-driven cellular antioxidant response. Discussion and Conclusion Our data reveal that alcohol-induced epididymal mitochondrial dysfunction and differences in sperm noncoding RNA content persist after alcohol withdrawal. Further, differences in mir-196a and sperm mitochondrial DNA copy number may serve as viable biomarkers of adverse alterations in the sperm-inherited epigenetic program.