Expression, immunolocalization and processing of fertilins ADAM-1 and ADAM-2 in the boar (sus domesticus) spermatozoa during epididymal maturation

Fertilin alpha (ADAM-1) and beta (ADAM-2) are integral membrane proteins of the ADAM family that form a fertilin complex involved in key steps of the sperm-oocyte membrane interaction. In the present work, we analyzed the presence of ADAM-1 and ADAM-2 mRNAs, the spermatozoa proteins' processing and their sub-cellular localization in epididymal samples from adult boars. ADAM-1 and ADAM-2 mRNAs were highly produced in the testis, but also in the vas efferens and the epididymis. On immunoblots of sperm extracts, ADAM-1 subunit appeared as a main reactive band of ~50-55 kDa corresponding to occurrence of different isoforms throughout the epididymal duct, especially in the corpus region where isoforms ranged from acidic to basic pI. In contrast, ADAM-2 was detected as several bands of ~90 kDa, ~75 kDa, ~50-55 kDa and ~40 kDa. The intensity of high molecular mass bands decreased progressively in the distal corpus where lower bands were also transiently observed, and only the ~40 kDa was observed in the cauda. The presence of bands of different molecular weights likely results from a proteolytic processing occurring mainly in the testis for ADAM-1, and also throughout the caput epididymis for ADAM-2. Immunolocalization showed that fertilin migrates from the acrosomal region to the acrosomal ridge during the sperm transit from the distal corpus to the proximal cauda. This migration is accompanied by an important change in the extractability of a part of ADAM-1 from the sperm membrane. This suggests that the fertilin surface migration may be triggered by the biochemical changes induced by the epididymal post-translational processing of both ADAM1 and ADAM-2. Different patterns of fertilin immunolocalization then define several populations of spermatozoa in the cauda epididymis. Characterization of such fertilin complex maturation patterns is an important step to develop fertility markers based on epididymal maturation of surface membrane proteins in domestic mammals

The adult boar testicular and epididymal transcriptomes

<p>Abstract</p> <p>Background</p> <p>Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have acquired a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm gain their motility and fertility. Epididymis is a crescent shaped organ adjacent to the testis that can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda). It contains a long and unique convoluted tubule connected to the testis via the efferent ducts and finished by joining the <it>vas deferens </it>in its caudal part.</p> <p>Results</p> <p>In this study, the testis, the efferent ducts (<it>vas efferens</it>, VE), nine distinct successive epididymal segments and the deferent duct (<it>vas deferens</it>, 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 9 K 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 medoid (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue clustering defined seven transcriptomic units: testis, <it>vas efferens </it>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 over-expressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). 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.</p> <p>Conclusion</p> <p>This study described for the first time the complete transcriptomes of the testis, the epididymis, the <it>vas efferens </it>and the <it>vas deferens </it>on the same species. It described new genes or genes not yet reported over-expressed in these boar tissues, as well as new control mechanisms. It emphasizes and fulfilled the gap between studies 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.</p

Protéomique : apport dans la fertilité masculine

National audienc

New insights into epididymal function in relation to sperm maturation

Testicular spermatozoa acquire fertility only after 1 or 2 weeks of transit through the epididymis. At the end of this several meters long epididymal tubule, the male gamete is able to move, capacitate, migrate through the female tract, bind to the egg membrane and fuse to the oocyte to result in a viable embryo. All these sperm properties are acquired after sequential modifications occurring either at the level of the spermatozoon or in the epididymal surroundings. Over the last few decades, significant increases in the understanding of the composition of the male gamete and its surroundings have resulted from the use of new techniques such as genome sequencing, proteomics combined with high-sensitivity mass spectrometry, and gene-knockout approaches. This review reports and discusses the most relevant new results obtained in different species regarding the various cellular processes occurring at the sperm level, in particular, those related to the development of motility and egg binding during epididymal transit

Maturation des spermatozoïdes dans l'épididyme: un nouveau regard sur un problème ancien

La compréhension des mécanismes cellulaires et moléculaires que constitue la maturation épididymaire des spermatozoïdes est depuis de nombreuses années un enjeu dans le domaine de la fertilité mâle. C'est l'espoir de pouvoir intervenir efficacement pour traiter certaines fertilités chez l'homme sans recours à des techniques de procréations assistées. Dans le domaine de la reproduction animale, c'est la possibilité de trouver de nouveaux marqueurs de prédiction pour sélectionner les meilleurs animaux reproducteurs. Chez l'homme, c'est de trouver un moyen de contraception masculine non hormonal. Ce domaine de recherche a bénéficié des progrès technologiques spectaculaires de cette dernière décennie en biologie comme le séquençage des génomes, l'obtention d'animaux transgéniques et les analyses à haut débit comme la transcriptomique et la protéomique. D'importantes avancées dans la connaissance des modifications post-testiculaires des gamètes et de leurs environnements épididymaires ont été obtenues. Bien que ces différenciations gamétiques existent chez tous les mammifères (monotrèmes compris), il est évident maintenant que ces différenciations sont spécifiques de l'espèce. Grâce à ces nouvelles approches, il est possible que l'on soit capable prochainement, si l'on s'en donne les moyens, d'être capable de pouvoir contrôler le pouvoir fécondant des spermatozoïdes

Maturation des spermatozoïdes

Chapitre 6absen

Epididymal protein markers and fertility

The last stages of male gamete differentiation occur outside the gonad in a specific surrounding environment controlled by the epididymal epithelium. All the fundamental characteristics of a fertile spermatozoon are acquired sequentially during transit through the epididymal tubule. Full understanding of about the mechanisms involved in these gamete modifications is a key to understanding and controlling such important stages in male fertility. With the development of new large scale technologies, large amounts of information give hope to identifying the fundamental elements involved in such cellular events and to being able to obtain some predictive markers of male fertility that would be valuable both in human and/or animal reproduction

Identification, function and regulation of Train A (RNase 10): a new epididymal-specific RNase-A like protein

National audienc

The epididymal transcriptome and proteome provide some insights into new epididymal regulations

Present Adress: Jean-Luc Gatti, INRA-PACA, 06903 Sophia-Antipolisabsen