RESUMO
Follicular fluid (FF), a product of vascular transudate and granulosa and thecal cell secretions, is the milieu that has evolved to support oocyte growth and maturation which plays a central role in oocyte quality determination. Therefore, a suboptimal FF composition may be reflected in compromised oocyte progression through maturation, fertilization or embryo development. To date, the composition of bovine FF remains understudied. To address this, we comprehensively characterized the metabolomic constituency of bovine FF in the period during which the oocyte undergoes meiotic maturation. More specifically, FF from pre (-24 h) and peri (-2 h) -ovulatory follicles was profiled by high-throughput untargeted ultra-high-performance liquid chromatography tandem mass spectroscopy. A total of 634 metabolites were identified, comprising: lipids (37.1%), amino acids (30.0%), xenobiotics (11.5%), nucleotides (6.8%), carbohydrates (4.4%), cofactors and vitamins (4.4%), peptides (3.6%) and energy substrates (2.1%). The concentrations of 67 metabolites were significantly affected by stage of follicle development, 33.3% (n=21) were reduced (P≤0.05) by a mean of 9.0-fold, whereas 46 were elevated (P≤0.05) by a mean of 1.7-fold in peri vs. pre -ovulatory FF. The most pronounced individual metabolite concentration decreases were hypoxanthine (98.9-fold), xanthine (65.7-fold), 17ß-oestradiol (12.4-fold), and inosine (4.6-fold). In contrast, the greatest increases were in retinal (4.9-fold), 1-methyl-5-imidazoleacetate (2.7-fold), and isovalerylcarnitine (2.7-fold). This global metabolomic analysis of bovine FF temporal dynamics provides new information for understanding the environment supporting oocyte maturation and facilitating ovulation, that has the potential for improving oocyte quality both in vivo and in vitro.
RESUMO
Ovulation has been described as an inflammatory event, characterized by an influx of leukocytes into the ovulatory follicle and changes in the expression profile of immune factors in both the theca and granulosa tissue layers. Since information on this process is limited in cattle, our objective was to elucidate the contribution of the immune system to dominant follicle luteinization, ovulation and corpus luteum (CL) formation in cattle. Beef heifers (n = 50) were oestrous synchronized, slaughtered and ovarian follicular or luteal tissue collected during a 96 h window around ovulation. Follicular fluid cytokine concentration, temporal immune cell infiltration and inflammatory status were determined by Luminex multiplex analysis, immunohistochemistry and quantitative real-time PCR-analysis, respectively, in pre- and peri-ovulatory follicular tissues. The concentrations of IL10 and VEGF-A were highest in pre-ovulatory and the concentration of CXCL10 was highest in peri-ovulatory follicular fluid samples. The pre and peri-ovulatory follicles play host to a broad repertoire of immune cells, including T-cells, granulocytes and monocytes. Dendritic cells were the most abundant cells in ovulatory follicular and luteal-tissue at all times. The mRNA expression of candidate genes associated with inflammation was highest in pre- and peri-ovulatory tissue, whereas tissue growth and modelling factors were highest in the post-ovulatory follicular and early luteal tissue. In conclusion, ovulation in cattle is characterized by the presence of neutrophils, macrophages and dendritic cells in the ovulatory follicle, reflected in compartmentalized cytokine and growth factor expression. These findings indicate a tightly regulated sterile inflammatory response to the LH surge in the ovulatory follicle which is rapidly resolved in advance of CL formation.
