PROTEIN PROFILE OF THE OVIDUCTAL FLUID FROM CYCLIC COWS

Abstract

The present study evaluated protein profiles of the oviductal fluid (ODF), collected from indwelling catheters inserted in the isthmus and ampulla of six mature cows. ODF as well as blood samples were obtained daily from two or more consecutive estrus cycles. We determined peripheral concentrations of progesterone (P4) by RIAs and grouped ODF samples from luteal (P4 > 1.5 ng/ml) or non-luteal (P4 < 1.5 ng/ml) phases of estrous cycle, also considering the two regions of the oviduct. Luteal ODF samples were pooled only from days 12, 13 or 14 of the cycle. Proteins of the ODF were separated by 2-D SDS-PAGE and gel images digitalized by PDQuest software (BioRad). Protein quantities were given as PPM of the total integrated optical density of the spots. On average, 84 spots appeared in the 2-D gels but a single spot identified as albumin comprised 42.3% of intensity of all spots, preventing the detection of low abundance proteins. Thus, we used spin columns to deplete albumin from all samples and subjected them again to 2-D electrophoresis. After this procedure, digitalized ODF maps had, on average, 90% less albumin. Individual spots were then excised from gels, subjected to in-gel trypsin digestion and analyzed by MALDI-ToF-ToF. There were no spots unique to oviductal samples collected either in the luteal or non-luteal phase of the estrus cycle, regardless of anatomical region. However, the expression of 34 spots were at least 4 times more intense in luteal than in non-luteal ODF, including albumin, transferrin, apolipoprotein A1, cathelicidin 1 and calgranulin A. Coincidently, our previous studies detected both transferrin and apolipoprotein A1 in the cauda epididymal fluid of mature bulls. Apo A1 is part of the HDL complex known to be present in the ODF, binds to bovine seminal plasma protein A1/A2 and may participate in sperm capacitation. Cathelicidins and calgranulins are modulators of immunoreactions. In contrast, only 5 spots appeared more abundant in the non-luteal period, such as serpins and calgranulin C. Serpins modulate proteases of the plasminogen activator cascade and their activity can be regulated by heparin, a galactosaminoglycan abundant in the oviduct and involved in sperm capacitation. Other proteins identified in ODF maps included apolipoprotein A2, calgranulin B, alfa 1 acidic glycoprotein and IgGs. Many proteins were present in small quantities since they were detected only after albumin depletion. In summary, we showed that bovine oviductal fluid is an albumin-rich secretion, regardless of anatomical location or phase of the estrous cycle. Albumin is a multi-functional component, can interact with other proteins, participate in sperm capacitation and acrosome reaction and exhibit protective actions on membranes and sperm motility. Using tools of indirect immunocytochemistry and confocal microscopy, we detected that both immature and in vitro matured bovine oocytes incubated with non-luteal ODF show intense immunoreaction with antibodies against albumin, supporting the notion that this protein plays a role in spermoocyte interaction. We hypothesize that the expression of ODF proteins linked to phases of the estrous cycle result from changes in estradiol and P4 and the presence of these proteins in the oviduct fluid are required for gamete functions and post-fertilization events. (poster)