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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.
© 2015 by the Society for the Study of Reproduction, Inc.
Publication Date: 2015-06-17 PubMed ID: 26085521DOI: 10.1095/biolreprod.114.127423Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research investigated the effect of procaine on horse oocytes, finding that while it did induce cell division, it also led to irregularities in cell development, with these cells failing to progress beyond a certain stage.
Understanding the Research
- In the experiment, the researchers observed how the drug procaine impacted equine (horse) gametes (the reproductive cells interest, particularly oocytes (eggs)). There was already an understanding that procaine aided in vitro fertilization because it causes hyperactivation in sperm, but this study aimed to investigate its impact on oocytes.
- Whilst procaine was previously thought to assist stallion sperm in penetrating the equine oocytes and thus aid fertilisation, the findings revealed that the drug actually induces cytokinesis (cell division) independently of the sperm, in the oocytes.
- Following exposure to procaine, a significant percentage of oocytes divided, regardless of the presence of sperm. This cytokinesis occurred regardless of the absence of the second polar body extrusion, a process that would typically occur during normal cell division.
Abnormal Cell Development with Procaine
- Although procaine induces cell division, the research revealed that the subsequent oocytes were not developing normally. They did not progress beyond the stage of having between 8 to 16 cells, and the formed daughter cells either lacked nuclei (the control center of a cell) or contained condensed, irregular DNA fragments.
- In a control group, sperm were injected directly into the oocyte (a method known as intracytoplasmic sperm injection, ICSI), which resulted in normal development of blastocysts (early-stage embryos).
- Oocyte activation (turning the egg into a zygote capable of developing into an embryo) normally induces calcium oscillations and cortical granule reaction, but these were not observed in the procaine-treated oocytes.
What Procaine Does to Oocytes
- Rather than activating the oocyte in the same way as ICSI or ionomycin treatment, procaine initiated an alkalinization of the ooplasm (the cytoplasm of an egg cell).
- This change in pH was followed by the depolymerization of F-actin, a protein involved in cell structure and division. This stage resembled the preparations for cell division apparent in zygotes (the initial cell formed when two gamete cells are joined by means of sexual reproduction) that were fertilized via ICSI.
Conclusions
- The researchers conclude that while procaine does trigger cell division in equine oocytes, it also leads to aberrant chromatin condensation (irregular packaging of DNA) and cell division. These abnormalities likely explain why embryos resulting from oocytes exposed to procaine fail to develop beyond the 8 to 16-cell stage.
Cite This Article
APA
Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A.
(2015).
Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism.
Biol Reprod, 93(1), 23.
https://doi.org/10.1095/biolreprod.114.127423 Publication
Researcher Affiliations
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- 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.
- 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.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- Department for Reproductive Medicine, Ghent University Hospital, Ghent University, Ghent, Belgium.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- Department for Reproductive Medicine, Ghent University Hospital, Ghent University, Ghent, Belgium.
- 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.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
- Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium Ann.VanSoom@ugent.be.
MeSH Terms
- Animals
- Chromatin / metabolism
- Cytokinesis / drug effects
- Female
- Fertilization in Vitro / veterinary
- Horses
- Hydrogen-Ion Concentration
- Male
- Oocytes / drug effects
- Oocytes / metabolism
- Procaine / pharmacology
- Sperm Injections, Intracytoplasmic / veterinary
- Sperm-Ovum Interactions / drug effects
- Spermatozoa / drug effects
- Spermatozoa / physiology
Citations
This article has been cited 8 times.- Fakhar-I-Adil M, Angel-Velez D, Araftpoor E, Amin QA, Hedia M, Bühler M, Gevaert K, Menten B, Van Soom A, Chuva de Sousa Lopes SM, Stoop D, De Roo C, Smits K, Heindryckx B. Biphasic CAPA-IVM Improves Equine Oocyte Quality and Subsequent Embryo Development Without Inducing Genetic Aberrations. Int J Mol Sci 2025 Jun 8;26(12).
- Leemans B, Gadella BM, Marchand JHEAM, Van Soom A, Stout TAE. Induction of in vivo-like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells†. Biol Reprod 2024 Sep 14;111(3):580-599.
- Felix MR, Turner RM, Dobbie T, Hinrichs K. Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†. Biol Reprod 2022 Dec 10;107(6):1551-1564.
- Leemans B, Bromfield EG, Stout TAE, Vos M, Van Der Ham H, Van Beek R, Van Soom A, Gadella BM, Henning H. Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells†. Biol Reprod 2022 Apr 26;106(4):710-729.
- Cardona Barberán A, Boel A, Vanden Meerschaut F, Stoop D, Heindryckx B. Diagnosis and Treatment of Male Infertility-Related Fertilization Failure. J Clin Med 2020 Dec 1;9(12).
- Leemans B, Stout TAE, Soom AV, Gadella BM. pH-dependent effects of procaine on equine gamete activation†. Biol Reprod 2019 Nov 21;101(5):1056-1074.
- Romero-Aguirregomezcorta J, Sugrue E, Martínez-Fresneda L, Newport D, Fair S. Hyperactivated stallion spermatozoa fail to exhibit a rheotaxis-like behaviour, unlike other species. Sci Rep 2018 Nov 15;8(1):16897.
- Ferraz MAMM, Henning HHW, Stout TAE, Vos PLAM, Gadella BM. Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production. Ann Biomed Eng 2017 Jul;45(7):1731-1744.
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