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Author ORCID Identifier

https://orcid.org/0000-0003-2030-360X

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology & Biomedical Sciences

Year Degree Awarded

2020

Month Degree Awarded

May

First Advisor

Pablo E. Visconti

Second Advisor

Jesse Mager

Third Advisor

Michele Markstein

Fourth Advisor

Ana Maria Salicioni

Subject Categories

Cell Biology | Molecular Biology

Abstract

ABSTRACT

To acquire fertilizing ability, mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation1,2. At the molecular level, capacitation is associated with a fast bicarbonate (HCO3-)-dependent activation of a unique type of soluble adenyl cyclase (sAC) and a consequent increase in cyclic AMP (cAMP) levels and PKA activation3. Activation of a cAMP/PKA pathway results in the phosphorylation of PKA substrates, which in turn initiates activation of several signaling cascades ultimately leading to an increase in phosphorylation on tyrosine residues (P-Tyr) of sperm axonemal proteins4,5. Increase in P-Tyr has been associated to sperm hyperactivation, an asymmetric and whip-like motion of the sperm tail necessary for fertilization4,6.

Although the increase in protein phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites7–9. As a part of the work on Chapter 2, we found that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is highly phosphorylated in capacitated mouse sperm. Phylogenetic analysis showed that Rsph6a gene contains six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a. Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.

Besides HCO3-, the role of Ca2+ in capacitation pathway is indispensable. Ca2+ regulates cAMP/PKA pathway through direct stimulation of sAC3. Ca2+ also binds to another binding partner calmodulin (CaM) and regulates activity of multiple enzymes such as phosphodiesterases (PDEs), protein phosphatase 2B (PP2B, also known as calcineurin), and to protein kinases; Ca2+/CaM-dependent kinase II (CaMKII) and Ca2+/CaM-dependent kinase IV (CaMKIV)10. The role of Ca2+ and Ca2+/CaM in sperm capacitation was demonstrated by genetic and pharmacological approaches. Mice lacking CatSper, a sperm specific Ca2+ channel, failed to hyperactive and displayed aberrant/premature P-Tyr of sperm axonemal proteins, and consequently were infertile11–14. Consistently, inhibition of CaM inhibitors also inhibited onset of sperm hyperactivation15. Nonetheless, the role of downstream targets of Ca2+/CaM in capacitation process is not well understood.

cAMP controls several signaling events during sperm capacitation and its levels during this process are quite dynamic. Depending on the need to fine tune PKA signaling, its levels are regulated by constant synthesis and degradation events. Such dynamics in cAMP levels can be explained by crosstalk between cAMP and Ca2+ signaling pathways. On one hand, Ca2+ positively promotes cAMP synthesis by direct stimulation of sAC3,16; on the other hand, Ca2+ binds to calmodulin (CaM), which activates a phosphodiesterase (PDE) and promotes cAMP degradation17–19. Besides PDE, PKA has been shown to control cAMP synthesis in a negative feedback loop by direct or indirect phosphorylation of sAC20. However, there is no clear understanding as to how this feedback loop is regulated, and how it fine-tunes PKA signaling during capacitation. As a part of this work on Chapter 3, we showed that calcineurin, a Ser/Thr phosphatase, is involved in this process. Inhibition of calcineurin by calcineurin inhibitors such as Cyclosporin A (CsA) and FK506 negatively PKA substrate phosphorylation. By measuring PKA and sAC activity in vitro in the presence of calcineurin inhibitors, we demonstrated that inhibition of PKA substrate phosphorylation is not due to off-target effect of the pharmacological drugs. Furthermore, we hypothesized that calcineurin being a Ser/Thr phosphatase, is able to activate sAC and thus regulates cAMP levels during sperm capacitation. Consistently, calcineurin inhibitors significantly reduced intracellular cAMP levels of the sperm incubated under capacitating conditions, which suggested a mechanistic role of calcineurin in a PKA feedback loop. Moreover, built on the rationale of the significance of Ca2+/CaM signaling, as a part of our work on Chapter 4, we characterized the presence and localization of Ca2+ signaling molecules involved in mouse and human sperm. In addition, we provided evidence that CaMKII is regulated during capacitation and is associated to cAMP/PKA pathway. Taken together, our data provide evidence for the role of novel signaling molecules in sperm capacitation. Understanding of the underlying mechanisms how these molecules regulate sperm capacitation will provide new insights into the development of novel contraceptive methods for men.

Available for download on Saturday, May 08, 2021

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