Purpose Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. Methods Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, N G -nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. Results Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters ( P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments ( P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. Conclusions These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine. Electronic supplementary material The online version of this article (10.1007/s10815-019-01526-6) contains supplementary material, which is available to authorized users.

Several in vitro studies have pointed to the importance of nitric oxide (NO) in the female and male reproductive system in mammals. Its functions vary from preventing oocyte aging, improving the integrity of the microtubular spindle apparatus in aged oocytes, modulating the contraction of the oviduct, to regulating sperm physiology by affecting the motility, inducing chemotaxis in spermatozoa, regulating tyrosine phosphorylation, enhancing the sperm-zona pellucida binding ability, and modulating the acrosomal reaction. In spermatozoa, NO exerts its functions in different ways, which involve key elements such as the soluble isoform of guanylate cyclase, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), adenylate cyclase, and the extracellular signal-regulated kinase (ERK) pathway. Furthermore, NO leads to the S-nitrosylation of several sperm proteins, among them a substantial group associated with energy generation and cell movement, but also with signal transduction, suggesting a role for S-nitrosylation in sperm motility and in modulating the sperm function, respectively. In this chapter, an overview of how NO modulates the sperm physiology is presented, based on the knowledge acquired to this day.

Recent studies have shown that extracellular vesicles may play an important role in modulating the fertilization capacity of sperm during their journey through the female reproductive tract. Extracellular vesicles (EVs), exosomes and micro vesicles, are a type of heterogeneous structures present in most body fluids, including bovine oviductal fluid. EVs contain various compounds derived from the original cell, such as proteins, lipids, mRNA, miRNA and DNA. EVs in the oviduct are produced by epithelial cells and their functions include interaction with spermatozoa, maintenance of their viability, participation in oocyte maturation and in the fertilization process. During the in vitro fertilization process and in order to improve it by mimicking in vivo conditions, numerous researchers have used bovine oviductal epithelial cell (BOEC) cultures with remarkable improvements. These cells produce, among others components, VEs, for this reason, in this work we have proposed a comparative study of the EVs present in the bovine oviductal fluid (OF) collected at times close to ovulation (in vivo) and those produced in BOEC cultures after 7 days of culture (in vitro) comparing the size, population distribution and protein concentration in both types. The EVs were identified by electron microscopy, their size by laser light scattering and their protein concentration by Bradford's method. The results showed that the EVs size evaluated per intensity were similar between both experimental groups. On the other hand, differences were observed in terms of protein concentration. EVs obtained in vivo contained a greater amount of protein in their cargo than the EVs obtained in vitro.Regarding the identification of VEs by transmission electron microscopy, only those obtained in vivo could be observed. This fact could be due to the place where they have been collected, to the method of culture of bovine oviductal epithelial cells or the shortage in their production. Las vesículas extracelulares (VEs), exosomas y micro vesículas son un tipo de estructuras heterogéneas presentes en la mayoría de los fluidos orgánicos incluyendo el fluido oviductal. Las VEs contienen varios compuestos derivados de la célula original, como proteínas, lípidos, ARNm, miARN y ADN. Las VEs en el oviducto son producidas por las células epiteliales y entre sus funciones se encuentran: interacción con los espermatozoides, mantener la viabilidad de estos, participar en la maduración de los ovocitos y en el proceso de fecundación. Durante la fecundación in vitro y, con el fin de mejorarla imitando las condiciones in vivo, numerosos investigadores han utilizado cultivos de células del epitelio oviductal bovino (CEOB) con notables mejoras. Estas células producen, entre otros componentes VEs, por ello, en este trabajo hemos planteado un estudio comparativo de VEs presentes en el fluido oviductal (FO) bovino recogido en momentos próximos a la ovulación (in vivo) y de aquellas VEs producidas en cultivos de CEOB a los 7 días de cultivo (in vitro) comparando el tamaño, la distribución de la población y la concentración de proteína en ambos tipos. Las VEs se identificaron mediante microscopía electrónica, su tamaño mediante dispersión de luz láser y la concentración de proteínas mediante el método Bradford. Los resultados mostraron que el tamaño de las VEs fue similar entre ambos grupos experimentales. Por otro lado, sí que se observaron diferencias en cuanto a la concentración de proteínas. Las VEs obtenidas in vivo contenían mayor cantidad de proteína en su cargo que en las VEs obtenidas in vitro. En cuanto a identificación de las VEs mediante microscopía electrónica de transmisión, solo pudieron ser observadas aquellas obtenidas in vivo. Este hecho podría deberse al lugar de dónde han sido recogidas, al método de cultivo de células epiteliales oviductales bovinas o la escasez en su producción.