Bovine Follicular Fluid and Serum Share a Unique Isoform of Matrix Metalloproteinase-2 That Is Degraded by the Oviductal Fluid1

Abstract

Whereas the mammalian fertilization environment consists of possible products of the mutual interaction between oviductal and follicular fluids in addition 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. Gelatin zymographic analyses of bovine follicular fluid (bFF) showed consistently a distinct, gelatinolytic activity having a molecular weight of 110 kDa (GA110) in addition to other gelatinases, whereas bovine oviductal fluid (bOF) showed a lack of GA110. Surprisingly, when bFF was mixed with bOF before zymography, the GA110 of bFF mostly disappeared at a 1:1 (v/v) mixture, completely disappeared at a 1:10 mixture, as fast as within 30 min after mixing. Other bFF gelatinase activities were not affected by bOF at 1:1 or 10:1 mixtures. Addition of EDTA or phenanthroline, but not of phenylmethylsulfonyl fluoride or trypsin inhibitor, to the mixture greatly increased the gelatinolytic activity of bFF GA110. The increased activity of bFF GA110 by EDTA was again abolished by subsequent bOF treatment. Addition of aminophenylmercuric acetate to the EDTA-treated bFF also abolished GA110; however, this was accompanied by the disappearance of other gelatinases, except the 62-kDa gelatinase, the activity of which increased as the treatment continued up to 24 h. Addition of EDTA or phenanthroline to the gelatin gel incubation 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 of GA110, the activity of which was increased by EDTA. However, ovarian granulosa cell homogenate did not exhibit GA110. Immunoblot experiments using antibodies against matrix metalloproteinase (MMP)-2 and MMP-9 demonstrated that bFF GA110 was an isoform of MMP-2, and that the 62-kDa form was an active 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 and bovine serum share a unique isoform of MMP-2, and that bOF can specifically degrade the isoform, suggesting that a mutual interaction between bFF and bOF could occur at the time of ovulation.

Dynamic in vivo changes in the activities of gelatinases, matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases (TIMPs) in buffalo (Bubalus bubalis) uterine luminal fluid during estrous cycle and early pregnancy

“…Gelatin zymography revealed that some of the gelatinase bands are common to serum/follicular fluid and ULF, some are uterus‐specific, and others are pregnancy‐specific. The 84 and 60 kDa gelatinase bands of serum and follicular fluid detected in the present study are similar to the 84–88 and 62 kDa gelatinases reported in bovine follicular fluid (Kim et al, 2001). In the absence of CaCl 2 , the activity of 60 kDa gelatinase band increased with the progress of the estrous cycle with highest activity in follicular stage ULF, whereas in the presence of CaCl 2 , the activity of this band decreased with the progress of the estrous cycle with highest activity in early luteal stage ULF (Figs.…”

Detection of the Matrix Metalloproteinases MMP‐2 and MMP‐9 and Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐2 in Llama (Lama glama) Oviduct

“…In agreement with our findings, Kim et al. () detected two gelatinase bands of 97 and 62 kDa in bovine OF. The authors suggested that based on its molecular weight, the 62 kDa form could correspond to MMP‐2.…”

“…; Kim et al. , ). MMP and TIMP oviductal functions remain elusive although it is well known that this system is involved in several aspects of the ovarian and uterine function (Curry and Osteen ).…”

Elevated non-esterified fatty acid concentrations hamper bovine oviductal epithelial cell physiology in three different in vitro culture systems

“…Non-esterified fatty acids are suggested to be introduced in the oviductal lumen via two pathways: (1) a rather small fraction through the ovulation of a NEFAcontaining follicle [55] and (2) a larger fraction through the natural formation of the OLF, which is essentially an ultrafiltrate from the blood [41]. Preliminary data from our laboratory have suggested a close correlation between serum NEFA concentrations and these in the OLF, making the effects of alternating unidirectional NEFA exposure but also bidirectional exposure physiologically relevant to study.…”