title: Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism.
abstract: Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Leemans
authors.affiliation: Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Bart M
authors.initials: BM
authors.lastname: Gadella
authors.affiliation: Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Deparment of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Tom A E
authors.initials: TA
authors.lastname: Stout
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Sonia
authors.initials: S
authors.lastname: Heras
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Smits
authors.affiliation: Department for Reproductive Medicine, Ghent University Hospital, Ghent University, Ghent, Belgium.
authors.forename: Minerva
authors.initials: M
authors.lastname: Ferrer-Buitrago
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Eline
authors.initials: E
authors.lastname: Claes
authors.affiliation: Department for Reproductive Medicine, Ghent University Hospital, Ghent University, Ghent, Belgium.
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
authors.affiliation: Department Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp University, Antwerp, Belgium Department Molecular Biotechnology, Cell Systems and Cellular Imaging, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
authors.forename: Winnok H
authors.initials: WH
authors.lastname: De Vos
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Hilde
authors.initials: H
authors.lastname: Nelis
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Hoogewijs
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium Ann.VanSoom@ugent.be.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism; D048749:Cytokinesis / Q000187:drug effects*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008297:Male; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D011343:Procaine / Q000494:pharmacology*; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D002843:Chromatin; D011343:Procaine
keywords: cytokinesis; donkeys; equids (horses; in vitro fertilization (IVF); pH; procaine; zebras)