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Isolation and characterisation of oviductal proteins involved in formation of sperm oviductal reservoir in cow
Kračmerová, Jana ; Liberda, Jiří (advisor) ; Postlerová, Pavla (referee)
The oviduct plays an important role in the complex process of fertilization and in the early phase of embryo development. Isolation and characterization of some components of bovine oviduct participating in the formation of oviductal sperm reservoir was a subject of the present study. Two affinity sorbents containing immobilized components of bull seminal plasma (non-separated proteins of bull seminal plasma and their phosphorylcholine binding fraction) were prepared and used for the study of interaction of immobilized ligands with components of oviductal epithelium. Two types of preparation of oviductal membrane proteins of the isthmus region were isolated: complete membrane proteins and the apical fraction of membrane proteins. These preparations were separated using prepared affinity matrices. in both cases only an insignificant amount of proteins was found in the adsorbed fractions. Further attention has been paid to the identification of four protein zones detected after electrophoretic separation in the presence of SDS (rel. mol. wt. 15 500 - 18 500) in the fraction of apical membrane proteins. These proteins were identified after the electrophoretic separation and tryptic digestion using MS analysis as bovine histones H4, H2A typ 2-C, H2B typ 1-K a H3.3. Proteins identified as bovine...
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Detection of mannosidase in the porcine urogenital tract – study of the sperm releasing from oviductal reservoir
Maňásková, Pavla ; Ren, Š. ; Jelínková, Jitka ; Krejčová, T. ; Liberda, J.
One of the most important steps of reproduction process is the meeting of sperm with oocyte. Binding of sperm with oviductal cells maintains spermatozoa in fertile state. The beginning of sperm capacitation is associated with oocyte ovulation resulting in the sperm release from oviductal reservoir. Hormonal changes after ovulation probably induce distinct oviductal secretion leading to disruption of the sperm protein binding with oviductal saccharide moieties. Another eventuality of the sperm releasing from oviductal reservoir is a change of oviductal environment caused by components of follicular fluid transported with oocyte after ovulation. In the pig, previous studies indicate lectin-type interaction of sperm protein receptors by mannose structures on the surface of oviductal cells. Our study was focused on enzymatic activity of mannosidase and its detection in porcine oviduct (fluid and tissue) and follicular fluid during hormonal cycle. In fluid from follicles in early and late hormonal stages, we measured mannosidase activity by colorimetric methods at physiological and acidic pH. Expression of secreted mannosidase was studied by specific antibody in follicular and oviductal fluids, and oviductal tissues during hormonal cycle. Clearly increased enzymatic activity of secreted mannosidase was found as specific-species in porcine fluid from follicles in late stage of hormonal cycle. On the other hand, detection of secreted mannosidase in follicular fluid as well as in oviductal fluid did not shown any significant differences during hormonal cycle. In oviductal isthmic tissue, we detected decreased protein expression of secreted mannosidase at middle and late follicular phases. These results suggest possible role of follicular mannosidase rather than oviductal one in the sperm releasing from oviductal reservoir in the pig. The additional study of the gene expression of secreted form of mannosidase in oviductal tissue during hormonal cycle should be necessary.
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