Abstract
Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.
Highlights
One of the problems that affect in vitro fertilization (IVF) in mammals is polyspermy [1]
When the Cumulus-oocyte complexes (COCs) diameters from the AG and L-NAME groups were compared to those measured in immature COCs before starting the maturation process (167.72¡2.63 mm), no significant difference was found, meaning that AG and L-NAME completely inhibited cumulus expansion
The inhibition produced by L-NMMA was only partial because the level of COC expansion in this group was significantly higher than that assessed in the immature oocytes (p,0.05, Fig. 2)
Summary
One of the problems that affect in vitro fertilization (IVF) in mammals is polyspermy [1] In porcine this problem is especially important [2] and, as a consequence, the in vitro production of embryos is extremely low, with respect to other species. For this reason most work to date has focused on studying the conditions affecting IVF [3]. Oocyte in vitro maturation (IVM) is another important step that could be related to polyspermic fertilization and low embryo production. NO has been suggested to act as an intracellular signal that triggers the activation of the oocyte [8]
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