The secretions of oviduct epithelial cells increase the equine in vitrofertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

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.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

“…This result is interesting, considering that conventional IVF has only limited success in equine species (Palmer et al 1991, Dell'aquila et al 1996, Hinrichs et al 2002, McPartlin et al 2009, Mugnier et al 2009). We chose the technique developed by McPartlin et al (2009) in which spermatozoa were hyperactivated using procaine.…”

“…In equine species, IVF rates are low and no repeatable IVF technique is available yet (Palmer et al 1991, Dell'aquila et al 1996, Alm et al 2001, Hinrichs et al 2002, McPartlin et al 2009). We have shown a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates (Mugnier et al 2009). Thus, we studied the role of DMBT1 in gamete interaction during equine and porcine fertilization.…”

“…Oviduct epithelial cells co-culture promotes in vitro production of embryos in human (Bongso et al 1991, Kervancioglu et al 1997, bovine (Chian & Sirard 1995, Way et al 1997, Martus et al 1998, porcine (Romar et al 2001, McCauley et al 2003, deer (Locatelli et al 2005), and dromedary (Khatir et al 2004) species. Moreover, some oviductal proteins have been shown to interact with gametes and to improve efficiency of IVF in porcine (McCauley et al 2003, bovine (Martus et al 1998), human (O' Day-Bowman et al 1996), and equine (Mugnier et al 2009) species. Some of these proteins, such as osteopontin, have been identified (Hao et al 2006, Goncalves et al 2008, Monaco et al 2009) in cattle and pigs and atrial natriuretic peptide A (Anderson et al 1994, Zamir et al 1995, Zhang et al 2006 and oviductin (oviduct-specific glycoprotein; Martus et al 1998, Buhi 2002, McCauley et al 2003 in cattle, pigs, and humans.…”

Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus)

“…After IVM, jennies oocytes were preincubated with oviductal fluid. Preincubation of equine oocytes with oviductal fluid or oviductal cells significantly increases the fertilization rate after treatment of sperm with calcium ionophore [28] or with procaine [36]. Based on our previously published review on the beneficial effect of oviductal cells and secretions on equine IVF [47], we anticipated a beneficial effect of preincubation of jennies oocytes with oviductal fluid.…”

“…The third step is IVF of oocytes. Several attempts to establish an efficient conventional IVF technique in the equine have been performed, but IVF rates remained quite low and IVF techniques were not repeatable [24][25][26][27][28][29][30]. Intracytoplasmic Sperm Injection (ICSI) has been widely adopted to generate horse embryos in vitro, both for scientific purposes and in the horse breeding industry [11,31].…”