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Home / Equine Research Bank / Biol Reprod / pH-dependent effects of procaine on equine gamete activation...
Biology of reproduction2019; 101(5); 1056-1074; doi: 10.1093/biolre/ioz131

pH-dependent effects of procaine on equine gamete activation†.

Abstract: Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway.
© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
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Publication Date: 2019-08-03 PubMed ID: 31373616PubMed Central: PMC6877780DOI: 10.1093/biolre/ioz131Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates the pH-dependent impact of the drug procaine on the activation of horse reproductive cells, revealing it can trigger important capacitative events in these cells, but can also hinder the fertilization process if administered incorrectly.

The role of procaine in reproductive cell activation

  • The study first observed that procaine, a local anesthetic, can influence equine reproduction.
  • Procaine was found to directly induce pH-dependent cytokinesis (cell division) in equine oocytes (egg cells) and hypermotility (increased movement) in stallion spermatozoa — an essential process during capacitation (preparation for fertilization).
  • However, they also found that this procaine-induced hyperactivity is halted if sperm is washed to remove procaine before being combined with the oocyte.

Investigating mechanism of procaine effect

  • To better understand how procaine brings about these effects, the researchers tested the dependency of procaine-induced hyperactivity on external calcium (Ca2+) and sodium ions (Na+) and weak base activity.
  • This was accomplished by using computer-assisted sperm analysis of stallion spermatozoa that had been washed with Percoll, a solution often used for sperm washing in in vitro fertilization.
  • The researchers found that procaine still induced hyperactivity under conditions of calcium depletion, but not when the medium was devoid of sodium.
  • Procaine and another weak base, NH4Cl, were shown to trigger an increase in the cytoplasmic pH, with an accompanying increase in sperm motility.

The relationship between procaine dosage, capacitation indicators, and fertilization

  • Fascinatingly, hyperactivity in sperm was reliably induced by 2.5-10 mM concentration of procaine, but there was a significant increase in two capacitation markers — high cytoplasmic cAMP levels and protein tyrosine phosphorylation of the sperm tail — only observed at a concentration of 10 mM.
  • While a high concentration of NH4Cl also induced oocyte cell division, the same capacitation characteristics observed with procaine were not seen.
  • Additionally, the study uncovered that cAMP levels in boar sperm were more than 10 times higher than in stallion sperm under similar capacitating conditions.
  • Finally, the research showed that equine oocytes were not fertilized by stallion sperm pre-treated with a 10 mM concentration of procaine, demonstrating that despite its stimulatory effects, procaine administration can impede subsequent fertilization.

Conclusion of the study

  • The comprehensive conclusion of this study is that procaine triggers a simultaneous increase in pH, hyperactivity, and capacitation characteristics in stallion spermatozoa, but these effects are largely independent of each other.
  • Meanwhile, the increased motility provoked by procaine is not dependent on the cAMP/protein tyrosine phosphorylation pathway familiar to capacitation, implying that procaine uses a different mechanism to incite hyperactivity.
  • Thus, while procaine does alter equine gamete activity, more investigation is required to ensure it can do so in a way that supports, rather than hinders, successful fertilization.

Cite This Article

APA
Leemans B, Stout TAE, Soom AV, Gadella BM. (2019). pH-dependent effects of procaine on equine gamete activation†. Biol Reprod, 101(5), 1056-1074. https://doi.org/10.1093/biolre/ioz131

Publication

Biology of reproduction
ISSN: 1529-7268
NlmUniqueID: 0207224
Country: United States
Language: English
Volume: 101
Issue: 5
Pages: 1056-1074

Researcher Affiliations

Leemans, Bart
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Stout, Tom A E
  • Departments of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
  • Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Soom, Ann Van
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Gadella, Bart M
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
  • Departments of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
  • Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

MeSH Terms

  • Animals
  • Calcium
  • Cytoplasm / chemistry
  • DNA
  • Embryonic Development
  • Fertilization in Vitro / veterinary
  • Horses / embryology
  • Horses / physiology
  • Hydrogen-Ion Concentration
  • Image Processing, Computer-Assisted / methods
  • Male
  • Oocytes
  • Organelles / chemistry
  • Procaine / pharmacology
  • Semen Analysis / veterinary
  • Sodium
  • Spermatozoa / drug effects

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