Resumen de Physiological role of sperm and seminal plasma proteins along the female reproductive tract in livestock
Semen is composed by sperm and seminal plasma, the fluid that mixes with those cells during ejaculation. Physiological processes that take place after ejaculation are mediated by proteins, including sperm capacitation and modulation of maternal tract environment by paternal factors. Understanding the regulation of these processes is crucial to improve the reproductive performance of livestock, such as pigs and cattle. In this context, the present Dissertation sought to investigate the regulatory role of sperm and seminal plasma proteins in sperm capacitation and interaction with female tract. For this purpose, the first three studies were focused on in vitro capacitation of pig sperm. In these, sperm were incubated in capacitation medium, and subsequently, sperm motility and a variety of cell parameters indicative of capacitation (plasma and acrosome membrane integrity, lipid membrane disorder, intracellular calcium levels, and mitochondrial membrane potential) were evaluated by flow cytometry. Analyses were performed after 0, 120, and 240 min of incubation, as well as 5, 30, and 60 min after induction of acrosome exocytosis by progesterone exposure. On the one hand, the first study evaluated the impact of blocking potassium channels on these parameters. The second one aimed to study whether relative levels of PARK7, measured by Western Blot, as well as its localisation assessed by immunofluorescence, were related to the indicated parameters, in addition to intracellular levels of peroxides and superoxides. The third study of this Dissertation investigated how prolonged exposure (48 or 72 h) to seminal plasma (15% or 30%) during liquid storage affects the ability of pig sperm to elicit capacitation by evaluating the aforementioned parameters and the tyrosine phosphorylation levels of GSK3α/β by Western Blot. Finally, the fourth study sought to elucidate the effects of seminal plasma and/or sperm on the modulation of the female tract environment in species with intravaginal ejaculation, using the cattle as a model. To this end, heifers were mated to intact or vasectomised bulls and, 24 h after mating, changes in the endometrial transcriptome were analysed by RNA sequencing. Additionally, the expression of a selection of genes (IL6, IL1A, IL8, TNFA, PLA2G10, CX3CL1, C4BPA, PRSS2, BLADQB, and CEBPD) was also assessed by real-time PCR in vagina and oviduct tissues. Results obtained in this Dissertation indicated that potassium channels play a key role in the regulation of mitochondrial activity, sperm motility, and intracellular calcium levels during capacitation. In addition, lower mortality, higher tolerance to superoxide generation, and a more progressive capacitation process were observed in sperm with higher relative levels of PARK7. Moreover, seminal plasma exposure reduced the capacity of sperm to undergo acrosomal exocytosis, lower mitochondrial activity, and a decrease in phosphorylation levels of GSK3α/β. With regard to the effects of seminal plasma on the female tract, whereas no changes after mating to vasectomised bulls were observed, mating to intact bulls resulted in 24 and 22 differentially expressed genes compared to unmated animals and heifers mated to vasectomised bulls, respectively. Despite the lack of differences between treatments, mating to intact and vasectomised bulls induced an increase in the expression of IL1A and TNFA in the vagina compared with the oviduct. Taken collectively, it can be concluded that sperm proteins, as well as those present in the seminal plasma, play a crucial role in the modulation of sperm capacitation. Conversely, in species with intravaginal ejaculation, sperm seem to play a more relevant function in the modulation of the female tract environment. It is, however, needed to determine whether this function is performed by intrinsic proteins or those acquired from seminal plasma. Although further studies are still needed to fully understand the modulatory role of sperm and seminal plasma proteins in order to give them a practical application, the results obtained in this Dissertation positively contribute with new insights that could be useful in assisted reproduction field.
