Prolonging the lifespan of bovine follicles is known to result in reduced fertility after ovulation and insemination. In this study, the effect of prolonged development of follicles on oocyte viability was examined. In Expt 1, six cows in the aspirated-prolonged-follicle group received a vaginal progesterone releasing device on day 4 of the oestrous cycle (day 1 = ovulation) and prostaglandin F2 alpha (40 mg) on day 6. Ultrasound-guided follicular aspiration was performed on day 13. Six cows in the aspirated-growing-follicle group received prostaglandin F2 alpha on day 6, and follicular aspiration on day 7. In Expt 2, all cows were stimulated with 36 mg FSH-P to develop multiple large follicles for study. Three cows in the prolonged-multiple-follicle group received the same treatment as did cows in Expt 1, but were ovariectomized on day 13. Three cows in the growing-multiple-follicle group received prostaglandin F2 alpha on day 6 and were ovariectomized on day 7. Oocytes recovered in both experiments were stained to reveal the stage of nuclear development. All oocytes from aspirated-prolonged and prolonged-multiple follicles showed expanded cumulus cells and condensed chromatin dispersed in the ooplasm, with possible germinal vesicle breakdown. Oocytes from aspirated-growing and growing-multiple follicles showed compact cumulus cells and an intact germinal vesicle. Plasma concentrations of oestradiol in both growing follicle groups increased until oocyte recovery. Oestradiol in the aspirated-prolonged-follicle group increased after luteolysis on day 6 and remained high until follicular aspiration. In contrast, in the FSH-stimulated prolonged-multiple-follicle group, oestradiol fell to trace amounts on day 8 and remained low. Oestradiol concentrations in follicular fluid were consistent with plasma concentrations for all groups. Bovine oocytes from prolonged dominant follicles undergo premature maturation in vivo, which perhaps accounts for the poor fertility observed in other studies when oestrous synchronization with progestins prolongs follicle lifespan.
Sperm binding to oviductal epithelium produces a reservoir in vivo that may serve to maintain sperm fertility and provide sperm for fertilization when ovulation occurs. Previously, it was determined that bull sperm binding could be blocked by fucoidan and its component fucose; furthermore, treatment of epithelium with fucosidase prevented binding. The present study was conducted to further characterize binding. Because fucose would probably be present on the epithelium as part of oligosaccharide moieties of glycoproteins and/or glycolipids, competitive binding inhibition activity was tested for fucose in five linkages commonly found in oligosaccharides. Binding inhibition was assayed by determining the concentration of motile, frozen/thawed sperm bound to fresh epithelial explants in the presence of test inhibitors. Initially, 5 monosaccharides were tested at 30 mM (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose), and only fucose significantly reduced sperm binding compared to vehicle control (p = 0.03). Of the oligosaccharides tested (lacto-N-fucopentaose I, 3-fucosyllactose, Lewis-X, Lewis-a, and GlcNAcbeta1-4[Fucalpha1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.
In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean +/- SD:167 +/- 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by capacitating washed sperm in medium containing 10 microg/ml heparin (50 +/- 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 +/- 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.
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