pH-dependent effects of procaine on equine gamete activation

• Published in: Biology of reproduction 2019-11, Vol.101 (5), p.1056-1074
• Main Authors: Leemans, Bart, Stout, Tom A.E, Van Soom, Ann, Gadella, Bart M
• Format: Article
• Language: English
• Subjects: Ammonium salts; Beef cattle; cAMP; Cyclic adenosine monophosphate; Cytokines; cytokinesis; equids; Fertility; Germ cells; Horses; hyperactivated motility; Motility; pH; Phenols (Class of compounds); Phosphorylation; Physiological aspects; Procaine; Proteins; RESEARCH ARTICLE; Sodium chloride; Sperm; Tyrosine
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1093/biolre/ioz131

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. Summary Sentence Procaine induces cAMP dependent tail-associated protein tyrosine phosphorylation in stallion spermatozoa at 10 mM as a result of its weak base activity, whereas hypermotility is induced over a much broader concentration range namely, 2.5–10 mM.