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: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:While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by FASP digestion were analysed by LC-MS/MS with SWATH acquisition. A total of 4,304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus and cauda spermatozoa had strong enrichment for tubulins and included 4 histone proteins. The most significant proteomic remodelling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1,131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias towards protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.
Project description:Purpose: The goal of this study is to detect differentially expressed genes, between wild-type (WT) and Adgrg2-/Y (Adgrg2-KO) caput epididymis by RNA sequencing Methods: Caput epididymal mRNA profiles of 8-week-old WT and Adgrg2-KO mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000. Results: RNA-seq data identified differentially expressed transcripts. Conclusions: Our results show that the expression of many genes was slightly downregulated in Adgrg2-KO caput epididymis compared with WT one.
Project description:Sperm small RNAs have emerged as important non-genetic contributors to embryogenesis and offspring health. A subset of sperm small RNAs are thought to be acquired during epididymal transit. However, the transfer of RNAs from the somatic epididymis to the sperm has been questioned, and the identity of the specific small RNAs transferred remains unclear. Here, we employ Cre/Lox genetics to generate germline- and epididymal-specific Dgcr8 conditional knockout mice to investigate the dynamics of sperm microRNAs and their function in the early embryo. Interestingly, sperm from germline specific Dgcr8 knockout males restored the levels of 58 of the 98 (59%) miRNAs that were lost in testicular sperm during epididymal transit. Conversely, sperm from epididymal Dgcr8 knockouts displayed a 5-fold reduction in 25 miRNAs. This substantial loss of epididymal miRNAs in sperm was accompanied by transcriptomic changes in the embryo which was rescued by microinjection of epididymal miRNAs. These findings ultimately demonstrate the acquisition of miRNAs by sperm during epididymal transit and their regulation of post-fertilization embryonic gene expression.
Project description:Mammalian spermatogenesis involves a series of events during which germ cells differentiate into immature sperm cells. Following spermatogenesis in the seminiferous tubules, sperm undergo post-testicular maturation where they suffer extensive remodelling of their proteome culminating in the acquisition of forward motility and fertilizing ability. Despite the advances, the proteomic landscape of mammalian testicular and epididymal sperm cells remains largely uncharacterized. Shotgun proteomics was used to compare proteomes of bull testicular, caput and cauda epididymal spermatozoa. This is crucial for understanding the physiological mechanisms that lead to sperm competence.
Project description:Purpose: The goal of this study is to detect differentially expressed genes, between wild-type (WT) efferent duct ligation (EDL)-treated, and W/Wv caput epididymis by RNA sequencing Methods: The EDL treatment of WT mice was performed from 10 weeks old and continued for 4 weeks until tissue sampling at 14 weeks old. Caput epididymal mRNA profiles of 14-week-old WT, EDL, and W/Wv mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000. Results: RNA-seq data identified differentially expressed transcripts. Conclusions: Our results show that the expression of many genes was downregulated in EDL or W/Wv caput epididymis compared with WT one.
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.