Association of Cumulus Oocyte Complexes with the intrafollicular levels of a blood protein in B. bubalis.

The protein pattern of the follicular fluid (FF) and the ultrastructure of the inner cumulus–oocyte complex (COC) has been analysed in single antral follicles (n = 146) of buffalo B. Bubalis ovaries. The protein population of FF was fractionated by SDS-PAGE; the resulting pattern was Coomassie stained and processed for densitometry. Comparartive analysis of sera and autologous FFs showed a marked difference in the level (measured as the percentage of total proteins) of one 21 kDa polypeptide band, called ‘L’. Concentration of L, which was mainly higher in the serum (2.05 ± 1.5%) than in the surrounding FF(0.98 ± 0.94%), fluctuated widely in fluids from the in the same ovary. On gel filtration of FF and SDS-PAGE of ther factions collected, the L polypeptide was found and eluted together with a 36 kDa polypeptide, called ‘H’, with an exclusion volume lower than that of albumin. The levels of both polypeptides in the eluted fractions were measured by gel densitometry and the same ratio H/L was found (2:1). These data suggest that H and L are subunits of a complex high-molecular-weight protein. The presence of L levels in male sera comparable to those detected in females indicates that this putative protein does not originate in the ovary but is transported from the blood. Moreover, a correlation between the increase in the percentage of Lf (calculated as %L in FF/%L in serum) and atresia was observed. COCs (n = 86) obtained during the collection of the single FF samples were processed for transmission electron microscopy. The ultrastructure of each COC was compared with the SDS-PAGE data of the associated FF. Healthy COCs were found to be related to very low levels of Lf (between 0 and 14% of those measured in serum). COCs with an early atretic ultrastructure undetectable at the dissection microscope, were associated with FFs having Lf levels between 24% and 60%; advanced atresia was associated with Lf values up to 70%. Finally, the acrosome reaction of buffalo precapacitated spermatozoa in vitro was monitored by adding one volume of FF with high (FF +; Lf = 80%) or undetectable (FF - ) values of Lf to the sperm suspension. It was found that 96.2% of spermatozoa treated with FF - showed a normal acrosome reaction, without any change in their motility pattern; spermatozoa treated with FF +, in contrast, exhibited poor motility, and only 66.4% of them underwent the acrosome reaction. The ionophore A23187, in a control experiment, resulted in lower percentages of acrosome reactions (38%). In conclusion, our results suggest that enhanced influx of L from serum into the follicle antrum causes an increase in the polypeptide level in FF. This might be due to a loss of selectivity in the blood-follicle barrier of early atretic follicles. The use of the L polypeptide as a biochemical marker for identifying the earlier stage of follicular atresia is proposed