Microvesicles Isolated from Follicular Fluid Impact In Vitro Maturation of Pig Oocytes.

Abstract

Oocyte maturation is a dynamic process characterized by a lack of oocyte transcriptional activity following the breakdown of the germinal vesicle during the progression from prophase I until metaphase II arrest. During follicular development, intercellular communication between the oocyte and the surrounding cumulus cells can influence oocyte maturation and oocyte quality. One potential mechanism for molecule transfer into and between cells of the cumulus oocyte complex (COC) is through microvesicles found in follicular fluid. The objective of this project was to identify the expression of a specific miRNA (MIR21) in microvesicles isolated from porcine follicular fluid and to determine their impact on in vitro oocyte maturation in media lacking follicle stimulating hormone (FSH) and luteinizing hormone (LH). Microvesicles were isolated from follicular fluid of large (8-12 mm) follicles from ovaries collected from a local abattoir. Following ultracentrifugation of follicular fluid, microvesicles were utilized for total RNA extraction or were resuspended in in vitro maturation media with or without gonadotropins. Total RNA was utilized to conduct quantitative RT-PCR for MIR21. In vitro maturation conditions included: control maturation media (MM) with LH and FSH, MM media without either LH and FSH or microvesicles, MM without LH and FSH but with microvesicles, and MM with LH and FSH and microvesicles, (n= 4 replications per treatment). Utilization of stem-loop quantitative RT-PCR demonstrated the presence of MIR21 in total RNA extracted from microvesicles. Following in vitro maturation, the percentage of oocytes achieving MII arrest in the control treatment was 64.5 ± 2.8%. Removal of both LH and FSH reduced maturation rate (37.2 ± 4.0%, P < 0.001) compared to control conditions. Interestingly, addition of microvesicles increased maturation rate (52.2 ± 2.9%; P < 0.01) when added to MM lacking LH and FSH despite being significantly less than control conditions (P < 0.03). Addition of microvesicles to MM containing FSH and LH did not improve maturation rate (65.5 ± 5.9%) compared to control conditions. Collectively, these data demonstrate the ability of follicular fluid microvesicles containing miRNA to improve in vitro maturation of porcine oocytes in the absence of gonadotropins. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-05309 and 2008-35205-18712 from the USDA National Institute of Food and Agriculture.