title: An alkaline follicular fluid fraction induces capacitation and limited release of oviduct epithelium-bound stallion sperm.
abstract: Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca(2+)-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca(2+) in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70-120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca(2+), a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca(2+) and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium.
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands baleeman.leemans@ugent.be.
authors.forename: Bart
authors.initials: B
authors.lastname: Leemans
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Bart M
authors.initials: BM
authors.lastname: Gadella
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Tom A E
authors.initials: TA
authors.lastname: Stout
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Hilde
authors.initials: H
authors.lastname: Nelis
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Hoogewijs
authors.affiliation: Department of ReproductionObstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, BelgiumDepartments of Farm Animal HealthBiochemistry and Cell BiologyEquine SciencesFaculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D020101:Acrosome Reaction; D000818:Animals; D002118:Calcium / Q000378:metabolism; D061207:Calcium Ionophores / Q000494:pharmacology; D002448:Cell Adhesion; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D015571:Follicular Fluid / Q000378:metabolism*; D006736:Horses / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D010057:Oviducts / Q000378:metabolism*; D010766:Phosphorylation; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D046509:Tissue Culture Techniques; D014443:Tyrosine / Q000378:metabolism
chemical_list: D061207:Calcium Ionophores; D014443:Tyrosine; D002118:Calcium