La simulación del ambiente oviductal modula la capacitación espermática y la fertilidad in vitro en la especie porcina

Abstract

Fertilization is a complex biological process in which innumerable factors are directly and/or indirectly involved. Although the efficiency of in vitro fertilization (IVF) is, for healthy animals, lower than in vivo, in some mammalian species such as bovine or murine a high yield has been achieved. However, in porcine species the IVF is still suboptimal. Currently, the polyspermic penetration of oocytes remains the main obstacle in porcine IVF, and the in vitro simulation of in vivo conditions of sperm capacitation and fertilization is postulated as the best strategy to improve the IVF output. The concentration of bicarbonate (HCO3-), one of the spermatozoa capacitating effectors which acts through activating the soluble adenylyl cyclase (sAC)/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, varies greatly in the different environments that mature spermatozoa go through from the cauda epididymis (~3-4 mM) to the oviduct (from 10 mM in the isthmus to between 25 and 90 mM in the fertilization site, depending on the stage of the estrous cycle). In the oviduct, the low HCO3- concentration in the caudal portion of the isthmus allows the spermatozoa to remain in a quiescent state, attached to the oviductal epithelial cells and forming the sperm reservoir (SR). At ovulation, there is an increase in HCO3- concentration, which triggers the hyperactivation of spermatozoa that allows their release from the SR. The HCO3- secretion provokes a great increase in pH in the periovulatory phase from around 6.5 in the isthmus and to around 8.0 in the oviductal ampulla. The increasing flow of HCO3- and pH enable the spermatozoa transport to the fertilization site at the ampullary-isthmic junction (AIJ), the development of the acrosome reaction (AR) to penetrate the egg vestments and oocyte fertilization. The periovulatory oviductal environment in which these phenomena take place is quite different from the in vitro characteristics of the medium usually used for sperm capacitation and IVF which contains a standard concentration of HCO3- of 25 mM and pH 7.4. These biological processes in vivo take place in specific fluids of the female genital tract, the uterine fluid (UF) and oviductal fluid (OF). However, in vitro sperm capacitation and IVF are presently produced in static systems in chemically defined media, with diverse grades of complexity but of limited composition and physical characteristics (probably distant from those encountered in the reproductive secretions), and plastic tubes or dishes in which gametes make contact randomly, rather than by selection and/or sperm competition. And none has been widely used in reproduction laboratories or eliminated the great problem of polyspermy in pigs. This work focuses on the effect on IVF of factors such as HCO3- concentration and pH of the medium (extracellular pH, pHe), the effect of periovulatory oviductal secretions and a device in which gametes are located physically separated and spermatozoa must swim to contact the oocytes. In chapter 1, the effects of different HCO3- concentrations (0, 5, 15 and 25 mM) were studied on the capacitation of mature spermatozoa and different functionality parameters were determined, including in vitro fertility. The pHi increased in spermatozoa after HCO3- uptake but only extracellular concentrations of 15 mM and above activated the cAMP/PKA capacitation cascade in the spermatozoa. The time required for the high phosphorylation of PKA substrates (PKAs-P) was HCO3- concentration-dependent, since at 15 mM it took PKAs-P significantly longer than with the usual concentration of 25 mM. 15 mM of HCO3- also stimulated sperm linear motility and tyrosine phosphorylation (Tyr-P). To evaluate spermatozoa fertility according to the HCO3- concentration of the medium, two different IVF systems were contemplated: i) a one-phase IVF system (monophasic IVF), in which spermatozoa were directly inseminated without any previous treatment and co-cultured with the oocytes in an IVF medium with the different HCO3- concentrations, and ii) a two-phases IVF system (biphasic IVF) in which spermatozoa were first pre-incubated for 60 min in a capacitating medium with different HCO3- concentrations and then inseminated in an IVF medium with the oocytes that contained the same or higher HCO3- concentration than in pre-incubation. The results of monophasic IVF showed that 15 mM led to higher efficiency (26.2%) than the rest of concentrations. In the biphasic IVF, both pre-incubation and IVF at 15 mM achieved 33.9% of viable zygotes (oocytes penetrated by only one spermatozoa), which represented in a significant increase of 25.3%. Chapter 2 combined a medium with the pH found in the oviduct during the periovulatory stage (pHe 8.0), a mixture of oviductal periovulatory components formed by COC secretions, follicular fluid and oviductal periovulatory fluid (OFCM) and a device that interposes a physical barrier between gametes (an inverted screw cap of a Falcon¿ tube, S) in which spermatozoa must to swim towards the encounter with the oocytes. The results showed that the combined use of the three factors studied (pHe 8.0, OFCM addition and S device) reduced polyspermy and increased the final efficiency of viable zygotes obtained to 48.7% compared with the classical system at pHe 7.4, lacking OFCM and a W device (18.9%). The spermatozoa functionality study under these IVF conditions showed that both pHe and OFCM modulate sperm capacitation. The motility parameters and capacitation status through Tyr-P was lower at pHe 8.0 and with OFCM. PKAs-P and AR were lower in the presence of OFCM, independently of the pHe. We concluded that the higher efficiency of IVF with pHe 8.0, in the presence of OFCM and using an S device could be a direct consequence of the action of OFCM components on gametes. The results obtained in this work suggest that the current suboptimal IVF conditions in porcine species can be improved by simulating the in vivo periovulatory milieu in the oviduct. Adjusting the HCO3- concentration and acid-base equilibrium of the porcine IVF systems, adding periovulatory oviductal fluids and using a device in which gametes are located physically separated but with a means of contact, increased the efficiency of IVF by reducing polyspermy and obtaining higher proportion of potentially viable zygotes. Despite the innumerable known and unknown factors that influence the capacitation and fertilization processes we identify new paths towards improving IVF in porcine and probably a new model for ARTs in all species.