The presence of CC chemokines and their aberrant role in the porcine corpus luteum.

The process of luteal regression is tightly regulated by the immune system and chemokines-small cytokines responsible mostly for the activation and migration of immune cells. The role of chemokines in porcine corpus luteum (CL) function is still not well understood. The aim of this study was to investigate the expression profile and distribution of CC chemokines in the porcine CL during the natural oestrous cycle and early pregnancy. Additionally, the effect of PGF2α on the expression of selected chemokines and their luteotropic and apoptotic influence on CL cells were studied in vitro. The expression levels of the chemokines CCL2, CCL4, and CCL5 and the chemokine receptor CCR5 were time-dependent (low on Days 8-10 and high on Days 12-14 of the oestrous cycle). Moreover, CCL8 and CCR2 transcript levels were also elevated during the period of luteolysis. The immunolocalization of CCL2, CCL4, CCL5, CCR1, CCR2 and CCR5 was determined using CL sections obtained from cycling and pregnant pigs. The immunofluorescence signals were localized mainly in luteal cells. PGF2α treatment of CL cells caused increased mRNA expression of CCL2 and CCR1. CCL2 treatment alone upregulated the expression of genes BAX, BCL2 and StAR in CL cells in vitro, but additional experiments showed that the chemokines CCL2, CCL4 and CCL5 alone do not cause apoptosis in a mixed population of CL cells. The chemokine CCL4 increased the transcript levels of StAR and HSD3-ß1. Additionally, CCL5 led to the inhibition of BAX gene expression. The differential spatiotemporal expression of CCL2, CCL4, CCL5 and CCR5 throughout the oestrous cycle and the direct but aberrant effect of these three chemokines on genes associated with apoptosis and progesterone synthesis indicate the complicated involvement of these factors in the regulation of luteolysis in pigs.

Efficient isolation, biophysical characterisation and molecular composition of extracellular vesicles secreted by primary and immortalised cells of reproductive origin.

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50-150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy.