Abstract
BackgroundOviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.Methods & resultsIn the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.ConclusionOur study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.
Highlights
Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species
Identification of proteins in the equine oviduct fluid using surface plasmon resonance In order to analyze whether osteopontin and Atrial Natriuretic Peptide A (ANP A) are expressed in the equine oviduct, we used a surface plasmon resonance biosensor
No significant difference in in vitro fertilization (IVF) rates was observed between stallion A and stallion B
Summary
Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Some studies show that oviduct epithelial cells (OEC) co-culture promotes in vitro production of embryos in human [5,6], bovine[7,8,9], porcine [10,11], deer [12,13] and dromedary [14]. The oviduct proteins have been shown to interact with gametes and to improve efficiency of in vitro fertilization (IVF) in porcine [10,15], bovine [9] and human [16]. Some of these proteins have been identified: osteopontin [17,18,19] in bovine and porcine, Atrial Natriuretic Peptide A (ANP A), [20,21,22] and oviductin [[9,23] for review;[10]] in bovine, porcine and human
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