Open AccessProcaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism1
Author(s) -
Bart Leemans,
Bart M. Gadella,
T.A.E. Stout,
Sonia Heras,
Katrien Smits,
Minerva Ferrer-Buitrago,
Eline Claes,
Björn Heindryckx,
Winnok H. De Vos,
Hilde Nelis,
Maarten Hoogewijs,
Ann Van Soom
Publication year - 2015
Publication title -
biology of reproduction
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.366
H-Index - 180
eISSN - 1529-7268
pISSN - 0006-3363
DOI - 10.1095/biolreprod.114.127423
Subject(s) - procaine , oocyte activation , oocyte , cortical granule , sperm , andrology , cytokinesis , biology , acrosome , intracytoplasmic sperm injection , staining , hyperactivation , polar body , human fertilization , ionomycin , depolymerization , microbiology and biotechnology , anatomy , chemistry , in vitro fertilisation , in vitro , embryo , cell division , biochemistry , pharmacology , genetics , cell , medicine , organic chemistry
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 6 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