Progress 01/01/00 to 12/31/00
Outputs
This report covers the final 9 months of this project, plus an additional 3 months of no-cost extension. During this time, the fourth and last aim was completed; that is, to characterize and purify the bovine sperm lectin. This "lectin" binds sperm to the epithelial lining of the oviduct and thereby creates a reservoir of sperm. The reservoir serves to provide sperm in the appropriate condition for fertilization at the time the oocyte enters the oviduct. It is also thought to prevent polyspermic fertilization and subsequent embryonic death, by preventing too many sperm from reaching the oocyte at once. Lectins are carbohydrate-binding proteins. Purification of the sperm lectin was carried out using affinity chromatography to pull out proteins from various extracts of sperm. An affinity matrix was created by binding Lewis-a oligosaccharide, conjugated to biotinylated polyacrylamide, to avidin immobilized on agarose. We had already demonstrated that bull sperm bind to
oviductal epithelium via fucose, particularly in Lewis-a trisaccharide. Of the various extracts tested, only a KCl extract left a protein specifically bound to the affinity column. The protein could be eluted with fucose and EDTA. EDTA was used to elute the protein because we had previously demonstrated that sperm binding to epithelium and fucose is calcium-dependent. Two-dimensional gel electrophoresis revealed that this protein was a single species, with a molecular mass of about 16.5 kilodaltons and a pI of 5.8. It was determined that the protein is not glycosylated. We had previously demonstrated that capacitated sperm lose the ability to bind to oviductal epithelium and also to fucose. This is likely the mechanism that allows sperm to be released from the reservoir in order to fertilize oocytes. We found that significantly less of the protein could be recovered by Lewis-a affinity chromatography from capacitated sperm than from uncapacitated sperm. We also found that capacitated
sperm would regain the ability to bind fucose when they were treated with the purified protein. These data indicated that we had indeed isolated the sperm lectin. Because we had found that seminal plasma blocks sperm binding to fucose, we examined whether the protein was present in seminal plasma. A protein of identical molecular mass and pI was obtained using the same affinity purification procedure. The protein purified from sperm and seminal plasma was submitted for N terminal amino acid sequencing. The results indicated that the protein is PDC 109 (also known as BSP A2), which is produced in the seminal vesicles and is the most abundant protein in bovine seminal plasma.
Impacts
The impact of this work is to suggest means of improving the success of artificial insemination, particularly in the dairy industry. This information would be especially beneficial for bringing into practice the use of sexed semen to produce dairy cows, because it offers a means of reducing the number of sperm needed for insemination.
Publications
- Revah I, Gadella BM, Flesch FM, Colenbrander B, Suarez SS (2000) The physiological state of bull sperm affects fucose and mannose binding properties. Biol Reprod, 62:1010-1015.
- Suarez SS (2001) Carbohydrate-mediated formation of the oviductal sperm reservoir in mammals. Cells Tissues Organs 168:105-112.
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Progress 01/01/99 to 12/31/99
Outputs
Aim 1: Determine the specificity of the bull sperm lectin. Various polysaccharides and glycoproteins were tested for the capacity to competitively inhibit sperm binding to explants of bovine oviductal epithelium. Fucoidan was the only substance that blocked sperm binding. Its component, fucose, blocked binding in dose-dependent fashion. Pretreatment of epithelium with fucosidase reduced binding. Sections of oviducts were probed with the fucose lectins UEA-I and LTL to confirm that fucose is present throughout the oviduct in high density. It was concluded that bull sperm bind to epithelium via a molecule that contains fucose. Competitive binding inhibition activity was tested for fucose in five linkages commonly found in glycoproteins. Fucose in an alpha 1-4 linkage to N-acetyl glucosamine, as in the trisaccharide Lewis-a, was the most efficient blocker of sperm binding. Calcium dependency of binding was examined to determine whether the lectin might belong to the
family of C-lectins. Removal-less than 50 nM-of calcium ions from the medium prevented sperm binding to explants and to Lewis-a. Aim 2: Localize the lectin on the sperm. Using fucose conjugated to fluorescein-labeled albumin (fuc-BSA-FITC) and Lewis-a conjugated to fluorescein-labeled polyacrylamide, the lectin was localized to the plasma membrane overlying the acrosome. This is the region responsible for attaching sperm to oviductal epithelium. Aim 3: Determine whether the lectin is lost or modified during sperm capacitation. Capacitation of sperm in vitro reduced binding to oviductal explants. Capacitation was assayed by the lysophosphatidyl choline induction of acrosome reactions, chlortetracycline labeling, and in vitro fertilization. Fuc-BSA-FITC was used with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and excluded from analysis. Controls were BSA-FITC conjugated with mannose (man-BSA-FITC) and
BSA-FITC. Labeling of live sperm with fuc-BSA-FITC was greatest for sperm that had been washed in TALP medium. Labeling was lower in unwashed sperm and in washed sperm with seminal plasma added back. Labeling was also reduced by capacitating sperm. BSA-FITC labeling was barely detectable. Man-BSA-FITC produced little labeling of washed sperm; however, intense labeling appeared over the acrosomal region of capacitated sperm. The flow cytometry data were verified by direct observation using epifluorescence microscopy. It was concluded that removal of seminal plasma exposes fucose binding sites, which are then lost during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose binding sites appear, which could serve to bind sperm to the zona pellucida. Aim 4: Characterize and purify the bovine sperm lectin. A purification scheme was developed based on the discovery that the lectin binds Lewis-a and binding is calcium dependent. Methods for extracting
proteins from sperm were tested and a Lewis-a affinity column was constructed. A KCl extract of sperm left a 16.5 kDa protein on the column which was eluted with buffer containing EGTA and fucose.
Impacts
The aim of this project was to determine how a reservoir of sperm is created in the bovine oviduct. This reservoir serves to protect sperm and prevent polyspermic fertilization. Our characterization of the mechanism involved in creating the reservoir has led us to suggest a means of improving fertility rate during artificial insemination. This would be especially useful for sexed semen, where economics require that a minimal number of sperm be used for insemination.
Publications
- Revah I, Gadella BM, Flesch FM, Colenbrander B, Suarez SS (1999) The physiological state of bull sperm affects fucose and mannose binding properties. Biol Reprod, accepted.
- Suarez SS (1998) Gamete transport. In: Garbers D, Hardy D (eds) Fertilization, Academic Press, in press.
- Suarez SS (1999) Carbohydrate-mediated formation of the oviductal sperm reservoir in mammals In: Topfer-Petersen E (ed) Glycobiology of Fertilization, in press.
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