Abstract
Simple SummaryBoth in natural breeding and some assisted reproduction technologies, spermatozoa are deposited into the uterus. The journey the spermatozoa must take from the place of semen deposition to the fertilization site is long, hostile, and selective of the best spermatozoa. For the fertilization to succeed, spermatozoa are guided by chemical stimuli (chemoattractants) to the fertilization site, mainly secreted by the oocyte, cumulus cells, and other substances poured into the oviduct in the periovulatory period. This work studied some sources of chemotactic factors and their action on spermatozoa functionality in vitro, including the fertility. A special chemotactic chamber for spermatozoa selection was designed which consists of two wells communicated by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. This study focuses on the use of follicular fluid (FF), periovulatory oviductal fluid (pOF), conditioned medium from the in vitro maturation of oocytes (CM), and progesterone (P4) as chemoattractants to sperm. The chemotactic potential of these substances is also investigated as related to their action on CatSper which is a calcium channel in the spermatozoa known to be sensitive to chemoattractants and essential for motility.Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (p < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (p < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.
Highlights
Assisted reproductive technologies (ART) have been widely used in humans and farm animals in the last decades
The present study investigated the chemotactic ability of the female reproductive fluids (FF, periovulatory oviductal fluid (pOF), Conditioned Medium (CM)) and P4 on the selection of porcine spermatozoa and the fertility of those selected spermatozoa (Figure 1)
Despite chemotaxis being mainly attributed to P4, other components have been suggested as potential chemoattractants in the porcine species
Summary
Assisted reproductive technologies (ART) have been widely used in humans and farm animals in the last decades. The efficiency of many ART is still low, in part due to spermatozoa quality which directly influences fertilization and embryo development. In standard methodologies of in vitro fertilization (IVF) in mammals, both male and female gametes are co-cultured without barriers regulating their interaction which causes a high number of spermatozoa to be simultaneously at the place of fertilization. Increased rates of polyspermy (more than one spermatozoon penetrating the oocyte) are achieved, compromising the embryo development. This is of particular interest to the porcine species, representing one of the major detrimental factors for an efficient and successful porcine IVF (reviewed by Romar et al [2])
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