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Abstract
Simple SummaryThe process of mammalian fertilization is dependent on many mechanisms mediated by regulatory genes and proteins expressed in the gametes and/or the female genital tract. This study aimed to determine the expression and location of the cytokine complex CXCL12:CXCR4 in the porcine gametes: oocytes and spermatozoa. This complex is known to play a pivotal role for sperm attraction towards the oocyte prior to internal fertilization in several mammalian species. Gene and protein expressions were analyzed in female and male porcine gametes. The results showed that the CXCL12 gene expression was higher in mature cumulus cells, and CXCR4 was higher in capacitated spermatozoa, both being requisites for gametes to accomplish fertilization. Moreover, for the first time, the CXCL12 protein was located in the cytoplasm of cumulus cells from mature COCs, and the CXCR4 protein was expressed in the midpiece and principal piece of uncapacitated spermatozoa and also in the sperm head of capacitated spermatozoa. These findings increase our current knowledge on porcine physiology of fertilization and reproduction, leading to possible improvements in the performance of reproductive technologies.Successful internal fertilization in mammals depends on several mechanisms, including those triggering the so-called “sperm attraction” towards the oocyte, which include some oocyte-derived sperm chemoattractants and interactive protein complexes, such as the cytokine C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12-CXCR4) receptor complex. The presence and precise localization of these crucial proteins was determined hereby, for the first time, in porcine cumulus–oocyte complexes (COCs) and spermatozoa. CXCL12 was overexpressed in the cumulus cells of in vitro matured, compared to immature COCs (p < 0.05), with its receptor (CXCR4) being up-regulated in capacitated spermatozoa (p < 0.03) compared to uncapacitated spermatozoa. The CXCR4 appeared specifically localized in the sperm tail of non-capacitated spermatozoa and also in the sperm head of capacitated spermatozoa, suggesting that the CXCL12-CXCR4 signaling complex would play a pivotal role in attracting capacitated spermatozoa towards the oocyte, facilitating fertilization in pigs.
Highlights
In vivo fertilization involves a series of different events that, in mammals, includes sperm capacitation, oocyte nuclear and cytoplasmic maturation, gamete transport to the site of fertilization in the oviduct, and sperm binding to the zona pellucida (ZP), among others [1,2]
The present study identified that the CXCL12 gene expression was significantly upregulated in the cumulus–oocyte complexes (COCs) with a mature oocyte compared to the immature COCs in the porcine species, which is in accordance with previous reports of this species [37,38], as well as in ovine species [23], which points out that CXCL12 signaling is contributing to oocyte maturation through the regulation of the expansion of the cumulus cell cloud
Our results show that CXCL12 is produced in the cumulus cells cytoplasm, from where it may act as a sperm chemoattractant prior to fertilization
Summary
In vivo fertilization involves a series of different events that, in mammals, includes sperm capacitation, oocyte nuclear and cytoplasmic maturation, gamete transport to the site of fertilization in the oviduct, and sperm binding to the zona pellucida (ZP), among others [1,2]. Chemotaxis is considered one of the most important factors guiding spermatozoa towards the oocyte–cumulus complex (COC) and involves hormones such as progesterone, glycosaminoglycans (GAGs), odorants, etc., it remains unclear how the final interaction steps occur [3,4,5]. Some of these “distant” chemoattractants are known to activate downstream pathways, increasing intracellular cAMP concentrations and the influx of Ca2+ in spermatozoa, issuing hyperactivation and somehow guiding the presumably capacitated cells towards the oocyte [6,7]. The cumulus cells might play dual roles by forming a massive cloud that spermatozoa can find or by mechanically entrapping them and releasing factors relevant to sperm–oocyte interaction [10,11]
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