Project description:A study utilizing next-generation sequencing provided a comprehensive expression profile of murine spermatozoa, revealing the transcriptional landscape of mature sperm isolated from the corpus and cauda epididymides of C57BL/6 mice. By analyzing RNA content, we identified a distinct set of retained transcripts, including those encoding sperm-specific ion channels, as well as transcripts associated with sperm motility, capacitation, and the acrosome reaction. In addition to spermatozoa, the study also reported the transcriptional profiling of murine leukocytes, providing comparative insights into cell-type-specific gene expression. This study advances our understanding of sperm transcriptomics and its implications for male fertility
Project description:Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate with the loss of sperm function during cryopreservation remains unsolved. The present study attempted to clarify this issue evaluating differential changes in the proteome of pig spermatozoa retrieved from the cauda epididymis and the ejaculate, with clear differences in cryotolerance, comparing fresh and frozen-thawed cells. Sperm samples were collected from 10 healthy, sexually mature and fertile boars, and cryopreserved using a standard 0.5 mL straw protocol. Total and progressive motility, viability and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) cauda epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT sperm samples were analyzed using a LC-ESI-MS/MS-based SWATH approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins directly involved in mitochondrial functionality among those quantitatively altered in ejaculated spermatozoa, which would explain the worse post-thaw quality of ejaculated pig spermatozoa.
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 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:In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development leading to an increased appreciation of the importance of RNA interference pathways, and in particular microRNAs (miRNAs), as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon we employed next generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the dynamic post-testicular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Since sperm are not capable of de novo transcription these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to condition the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring. Examination of the microRNA expression profile in sperm thoughout the mouse epididymis and mouse using next generation sequencing in duplicate.
Project description:In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development leading to an increased appreciation of the importance of RNA interference pathways, and in particular microRNAs (miRNAs), as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon we employed next generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the dynamic post-testicular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Since sperm are not capable of de novo transcription these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to condition the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.
Project description:Background - Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm acquire their motility and fertility. Epididymis can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda), containing a long and unique convoluted tubule connecting the testis to the vas deferens. Results - In this study, the testis, the efferent ducts (vas efferens, VE), nine distinct successive epididymal segments and the deferent duct (vas deferens, VD) of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9K nylon microarray (AGENAE program; GEO accession number: GPL3729) spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoïds (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue samples analysis defined seven transcriptomic units: testis, vas efferens and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically overexpressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). Among these markers some well-known epididymal genes were retrieved while some were new genes or genes not yet reported in these boar tissues. The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information. Conclusions - This study fulfilled the gap between those done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa. Keywords: tissue type comparaison 96 samples - 12 tissue samples from 4 boars
Project description:Spermatozoa acquire fertilization potential during passage through a highly specialized region of the extra-testicular ductal system known as the epididymis. In the absence of de novo gene transcription or protein translation, this functional transformation is extrinsically driven via the exchange of varied macromolecular cargo between spermatozoa and the surrounding luminal plasma. Key among these changes is a substantive remodeling of the sperm proteomic architecture, the scale of which has yet to be fully resolved. Here, we have exploited quantitative mass spectrometry-based proteomics to define the extent of changes associated with the maturation of mouse spermatozoa; reporting the identity of an unprecedented >6,000 proteins, encompassing the selective loss and gain of several hundred proteins. Further, we demonstrate epididymal driven activation of RHOA mediated signaling pathways is an important component of sperm maturation. These data contribute molecular insights into the complexity of proteomic changes associated with epididymal sperm maturation.