Protein profile of follicular fluid during folliculogenesis of the mare

Follicular fluid (FF) is the only environment in which the avascular compartment of the follicle (granulosa cells and oocyte) are exposed and provides the microenvironment within which the cumulus-oocyte complex (COC) matures and granulosa cells differentiate. This experiment aims to compare the FF protein profile of the largest follicle (F1) and compare the largest and second largest follicle (F2) during different stages of follicular development (emergency, deviation, dominance and pre-ovulatory). Ovaries from 20 non-pregnant and cyclic mares were collected in an abattoir. Before slaughter, the mares were examined by transrectal palpation and ultrasound examination of the genital tract in order to evaluate ovaries and uterus. Blood samples were collected by jugular venipuncture. The diameter of the two largest follicles (F1 and F2) and the corpus luteum (CL) were obtained from each mare. Echotexture of the endometrium (EE) was evaluated and scored from 1 to 4. Mares were classified in the following experimental groups: G 15 (n = 4) F1 ≤ 15 mm, EE ≤ 2.5, CL > 27 mm; G 20 (n = 8) F1 20 to 26 mm, EE 2.5 to 3, CL 17 to 26 mm; G 30 (n = 4) F1 30 to 38 mm, EE > 3, CL 40 mm, EE > 3, CL < 16 mm. Plasma progesterone concentrations were assayed by chemilluminescence. After slaughter, the FF of F1 and F2 was aspirated and submitted to 2D-PAGE for protein separation and identification by mass spectrometry. For statistical analysis a one-way analysis of variance (GLM procedure of SAS) was performed to evaluate the relative optical density of each protein spot as the dependent factor and the experimental groups, F1 and F2 and their interactions as independent variables. From the 20 mares studied, four constituted G15, eight G20, four G30 and four G40. Plasma progesterone concentrations varied from 8.1 to 12.7 in G15, 6.7 to 12.6 in G20, 0.6 to 1.3 in G30 and 0.6 to 0.7 in G40. A total of 43 spots was observed in gels (38 from F1 and 35 from F2). Nine spots presenting significant differences between treatments were submitted to mass spectrometry. Albumin, apolipoprotein A-I, gelsolin, serotransferrin and alpha-1-antiproteinase 2 were detected in the fluid of F1 and differed in abundance (P ˂ 0.05) between the experimental groups. POM121 and ZP3 fusion protein (POMZP3) differed (P = 0.02) in abundance since deviation (G20), and alpha-1-antiproteinase 2 showed interaction (P = 0.05) between F1 and F2. The majority of the proteins identified in FF are present in blood plasma. It was not possible to correlate a specific protein with a particular stage of follicular development. However, serotransferrin and alpha-1-antiproteinase 2 had greater abundance during dominance and apolipoprotein A-1 and gelsolin during the pre-ovulatory stage. POMZP3 showed higher abundance in the dominant follicle compared to the subordinate one.