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.

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).

Project description:Background. Primary cilia (PC) are solitary antennae present at the cell surface. These non-motile cilia play an important role in organ development and tissue homeostasis through the transduction of the Hedgehog (Hh) signaling pathway. We recently revealed the presence of PC in the epithelium of the developing epididymis, an organ of the male reproductive system whose dysfunction triggers male infertility. Acknowledging that systemic blockade of the Hh pathway trigger epididymal dysfunctions in vivo, our main goals were 1) to portray the epididymal Hh environment, 2) to determine the direct responsiveness of epididymal epithelial cells to Hh, and 3) to define the contribution of PC to the transduction of this pathway. Results. The Hh ligands Indian and Sonic hedgehog (Ihh and Shh) were respectively located in principal and clear cells of the mouse epididymis by immunofluorescent staining. The propensity of epididymal principal cells to respond to Hh signaling was assessed on immortalized epididymal DC2 cells by western-blot, confocal imaging and 3D-reconstruction. Our results indicate that epididymal principal cells secrete Ihh and expose PC that co-localize with the conventional acetylated tubulin/Arl13b ciliary markers, as well as with GLI3 Hh signaling factor. Gene expression microarray profiling indicated that the expression of 43 and 248 genes was respectively and significantly modified following pharmacological treatment of DC2 cells with the Hh agonist SAG (250 nM) or the Hh antagonist cyclopamine (20 µM) compared with the control. Among Hh target genes identified, 6.7 % presented perfect matches for GLI-transcription factor consensus sequences, and the majority belonged to interferon-dependent immune response and lipocalin 2 pathways. Finally, the contribution of epididymal PC to the transduction of canonical Hh pathway was validated by ciliobrevinD treatment, which induced a significant decrease of PC length and the expressional reduction of Hh signalling targets. Conclusions. All together our data indicate that PC from epithelial principal cells regulate gene expression profile through a possible autocrine Hh signaling. This provides new hypotheses regarding the potential contribution of PC and Hh signaling in intercellular cross-talk and immunological regulation of the epididymis.

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Effects of vasectomy on gene expression profiling along the human epididymis

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