Bovine follicular fluid and serum share a unique isoform of matrix metalloproteinase-2 that is degraded by the oviductal fluid

Whereas the mammalian fertilization environment consists of possible produc ts of the mutual interaction between oviductal and follicular fluids in add ition to both fluid components, little is known regarding the interaction. In the present study, we have demonstrated that a mutual interaction occurs , resulting in the biochemical changes of follicular fluid components. Gela tin zymographic analyses of bovine follicular fluid (bFF) showed consistent ly a distinct, gelatinolytic activity having a molecular weight of 110 kDa (GA110) in addition to other gelatinases, whereas bovine oviductal fluid (b OF) showed a lack of GA110. Surprisingly, when bFF was mixed with bOF befor e zymography, the GA110 of bFF mostly disappeared at a 1:1 (v/v) mixture, c ompletely disappeared at a 1: 10 mixture, as fast as within 30 min after mi xing. Other bFF gelatinase activities were not affected by bOF at 1:1 or 10 :1 mixtures. Addition of EDTA or phenanthroline, but not of phenylmethylsul fonyl fluoride or trypsin inhibitor, to the mixture greatly increased the g elatinolytic activity of bFF GA110. The increased activity of bFF GA110 by EDTA was again abolished by subsequent bOF treatment. Addition of aminophen ylmercuric acetate to the EDTA-treated bFF also abolished GA110; however, t his was accompanied by the disappearance of other gelatinases, except the 6 2-kDa gelatinase, the activity of which increased as the treatment continue d up to 24 h. Addition of EDTA or phenanthroline to the gelatin gel incubat ion buffer after electrophoresis abolished almost all gelatinases; of bFF, except those of 88-84 kDa, demonstrating that they were indeed gelatinases or isoforms. Bovine serum and fetal bovine serum also showed the presence o f GA110, the activity of which was increased by EDTA. However, ovarian gran ulosa cell homogenate did not exhibit GA110. Immunoblot experiments using a ntibodies against matrix metalloproteinase (MMP)-2 and MMP-9 demonstrated t hat bFF GA110 was an isoform of MMP-2, and that the 62-kDa form was an acti ve form of MMP-2. Disappearance of immunoreactive GA110 of bFF and serum by bOF was also observed. Based on these observations, we conclude that bFF a nd bovine serum share a unique isoform of MMP-2, and that bOF can specifica lly degrade the isoform, suggesting that a mutual interaction between bFF a nd bOF could occur at the time of ovulation.