Unknown,Transcriptomics,Genomics,Proteomics

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The microRNA signature of mouse spermatozoa is dynamically modified during epididymal maturation

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Brett Nixon

PROVIDER: E-GEOD-70198 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract. Examination of the microRNA expression profile in the whole mouse epididymis and mouse epididymal epithelial cells using next generation sequencing in duplicate.

Project description:BACKGROUND: The molecular mechanisms resulting in the establishment of regionalized gene expression in the human epididymis involve numerous and complex regulatory elements that have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis that could be involved in the regulation of mRNA stability and post-transcriptionnal expression in this organ. METHODS: Using a global miRNA microarray approach, we investigated the correlation between the miRNA signature and the gene expression profile found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed in silico using TargetScan and Miranda software’s. To experimentally verify some of these data, FHCE1 immortalized epididymal cell lines were cotransfected with mimics microRNAs and plasmid constructs containing the 3’UTR of target genes downstream of the luciferase gene. RESULTS: We identified 35 miRNAs differentially expressed between the different segments of the epididymis (fold change > 2, P < 0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same cluster located on the X chromosome and specifically expressed in epididymal tissues, are significantly more expressed in the corpus and cauda regions compared to the caput. Interestingly, a strong negative correlation (r= -0,89, P <0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma), with miR-145 and Cldn10 mRNA (r= -0,92, P<0.001) and with miR-145 and Cftr mRNA (r= -0,69, P<0.05). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3’ UTRs respectively, whereas miR-145 does not affect the expression of luciferase after its co-transfection with the plasmid containing the Cftr 3’UTR. CONCLUSION: Overall, our study shows that the expression of miRNAs is segmented along the different regions of the human epididymal duct and correlates with the pattern of target gene expression in these regions. Therefore, epididymal miRNAs may be in charge of the maintenance of gene expression profile in the epididymis that dictate segment-specific secretion of proteins and establish physiological compartments that directly or indirectly affect sperm maturation and fertility. Normal human epididymides from three donors of 26–50 years of age, were collected and dissected into 3 anatomical regions (caput, corpus and cauda).A cross-comparison between the miRNA signature found in different segments was performed using GeneChip miRNA Array 1.0 (Affimetrix, Santa Clara, CA).

Project description:BACKGROUND: Vasectomized men who wish for restoration of fertility commonly face problems to conceive despite high surgical success rates of vasovasostomy, a surgery routinely used to retrieve duct patency. In human, vasectomy has consequences on the epididymal transcriptome and is responsible for non-reversible changes that affect the biochemical parameters and functions of ejaculated sperm in vasovasotomized men. Since microRNAs (miRNAs) are key regulators of post-transcriptional gene expression, we explored the consequences of vasectomy on miRNA pattern in the human epididymis and determined the repercussion and reversibility of these changes in seminal microvesicles (SMVs) from semen of vasovasostomized men. METHOD: miRNA signatures from a unique set of human epididymal segments (caput, corpus, cauda) provided by controls and vasectomized donors as well as miRNA content of SMVs isolated from semen of normal, vasectomized and vasovasostomized donors were determined by using high-throughput microarray technology. Epididymal miRNA signature was correlated with gene expression profiles by employing an integrated genomic approach. RESULTS: While the segmented pattern of miRNA expression was maintained along the human epididymis following vasectomy, the array of miRNAs from each anatomical region significantly differed between normal and vasectomized donors. Greatest changes were observed in cauda epididymidis and were negatively correlated with transcription factors. Vasectomy also impacted expression of miRNAs found in SMVs from normal donors, including miRNAs of epididymal origin contained in epididymosomes. Among seminal miRNAs sequelae, 52 were reversible according to miRNA expression level following duct patency retrieval. In contrast, 66 of miRNAs found in SMVs were not retrieved in SMVs after vasovasostomy. CONCLUSION: We showed that vasectomy has a substantial impact on the miRNA signature of the epididymis and SMVs, and that vasovasostomy does not restore all molecular players found in normal fertile individuals. According to the critical role played by miRNAs in the control of male fertility, we believe that miRNA sequelae occurring upstream and downstream of the vasectomy site may be in part responsible for the reduced fertility outcome reported after surgically successful vasectomy reversal. Human epididymides from 3 control (26, 47 and 50 years old. GEO accession number GSE35522) and 3 vasectomized donors (45, 47 and 48 years old), were collected and dissected into 3 anatomical regions (caput, corpus and cauda). Seminal microvesicles (SMVs) were isolated from the semen of normal (pool of 3 donnors), vasectomized (pool of 3 donnors) and vasovasostomized donors (pool of 5 donnors). A cross-comparison between the miRNA signature found in different segments and seminal vesicles was performed using GeneChip miRNA Array 2.0 (Affimetrix, Santa Clara, CA).

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The microRNA signature of mouse spermatozoa is dynamically modified during epididymal maturation

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