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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology & Biomedical Sciences

Year Degree Awarded

2017

Month Degree Awarded

May

First Advisor

Pablo E. Visconti

Second Advisor

Rafael Fissore

Third Advisor

Barbara Osborne

Fourth Advisor

Thomas Zoeller

Subject Categories

Biotechnology | Other Animal Sciences

Abstract

Mammalian sperm acquire the fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with the up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+, ATP consumption and an increase in tyrosine phosphorylation. However, little is known about the function of Ca2+ and ATP during this important process. These signaling systems interact during capacitation are still not well understood.

Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Sperm incubated in zero Ca2+ in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists and by the Calcineurin inhibitor. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin.

Several other models revealed the need of Ca2+ for capacitation and fertilization. For example, sperm from Ca2+ channel CatSper1 knockout (KO) mice underwent PKA activation and an increase in tyrosine phosphorylation, but it was not able to fertilize. When CatSper KO sperm was treated with a transient exposure of Ca2+ ionophore A23187 fertilizing capacity was rescued. In the same sense, transient incubation of mouse sperm with A23187 accelerated capacitation and rescued fertilizing capacity in sperm when PKA inhibitor H-89 was used. As a result, infertile KO models linked to cAMP-dependent signaling (soluble adenylyl cyclase and K+ channel) were also rescued with A23187. In contrast, sperm from infertile mice lacking the Ca2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.

Sperm required energy for motility, and glucose is one of the main drivers. When both glucose and pyruvate are removed sperm motility decrease becoming immotile. Unusually, immotile sperm significantly recovered motility and hyperactivation when substrates are provided back (SER treatment: starve + rescue). Increased sperm hyperactivation improve fertilization rates in different sub-fertile mouse models, and also, embryo development and number offspring are improved compared with the control. Infertile model CatSper KO sperm fertilizing ability is not rescued. Still, A23187 and SER treatment combination rescue fertilization and embryo development. Suggesting that, metabolism and Ca2+ are fundamental players fertilization and embryo development.

Available for download on Tuesday, May 12, 2020

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