Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity in the female tract in a process known as capacitation. Initially, capacitation was defined using fertilization as end-point. However, a variety of evidences suggest that the functional changes occurring in the sperm during capacitation are not one event, but a combination of sequential and concomitant processes. Some of these processes occur as soon as the sperm are released from the epididymis, others are slower and are activated only after sperm incubation for a certain period of time in conditions that support the sperm ability to fertilize the egg. These slow events are associated with changes in the motility pattern (e.g. hyperactivation) and with the acquisition of the sperm capacity to undergo an agonist-stimulated acrosome reaction (AR). Although both, fast and slow events are regulated by HCO3- , activation of Adcy10, the atypical soluble adenylyl cyclase (also known as sAC) and the subsequent activation of a cAMP-dependent pathway, slower events are limited by the release of cholesterol from the sperm plasma membrane. Using the mouse as an experimental model, we have shown that these last events are associated with a protein kinase A (PKA)-dependent increase in protein tyrosine (tyr) phosphorylation. We have also demonstrated that the increase in tyr phosphorylation as well as capacitation was regulated by the presence of cholesterol acceptors such as Bovine Serum Albumin (BSA) in the capacitation media. During the first cycle of this proposal we have discovered that although necessary, the classical linear pathway involving HCO3-/SAC/PKA is not sufficient to elicit phosphorylation of downstream targets. On top of PKA activation, another pathway leading to downregulation of ser/thr phosphatases is also necessary. This second pathway is essential to modulate the PKA pathway and for the activation of other PKA- independent phosphorylation pathways needed for sperm capacitation. The hypotheses underlying this proposal postulate that capacitation results from the combined action of two pathways, one regulated by cholesterol acceptors (e.g. BSA) inducing a cSrc family kinase (SFK)-induced downregulation of ser/thr phosphatase(s) and the other regulated by HCO3- and Ca2+ and mediated by the activation of sAC and PKA. The objective of this proposal is to understand how these two pathways are integrated during capacitation.

Public Health Relevance

Difficulties in earlier efforts to fertilize mammalian eggs in vitro were due mainly to a lack of comprehension of sperm physiology. This proposal is aimed to understand the molecular basis of sperm capacitation with emphasis in the regulation of signaling pathways involving protein phosphorylation in sperm. Accomplishment of these goals will lead to novel insights in clinical diagnosis of male infertility and provide tools for evaluating pharmacological contraceptive targets.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD044044-08
Application #
8388781
Study Section
Special Emphasis Panel (ZRG1-EMNR-E (02))
Program Officer
Moss, Stuart B
Project Start
2002-08-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
8
Fiscal Year
2013
Total Cost
$320,560
Indirect Cost
$118,897
Name
University of Massachusetts Amherst
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Navarrete, Felipe A; Alvau, Antonio; Lee, Hoi Chang et al. (2016) Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models. Sci Rep 6:33589
Ramos-Espiritu, Lavoisier; Kleinboelting, Silke; Navarrete, Felipe A et al. (2016) Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase. Nat Chem Biol 12:838-44
Stival, Cintia; Puga Molina, Lis del C; Paudel, Bidur et al. (2016) Sperm Capacitation and Acrosome Reaction in Mammalian Sperm. Adv Anat Embryol Cell Biol 220:93-106
Alvau, Antonio; Battistone, Maria Agustina; Gervasi, Maria Gracia et al. (2016) The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm. Development 143:2325-33
Navarrete, Felipe A; García-Vázquez, Francisco A; Alvau, Antonio et al. (2015) Biphasic role of calcium in mouse sperm capacitation signaling pathways. J Cell Physiol 230:1758-69
Wallingford, Mary C; Filkins, Rachel; Adams, Danielle et al. (2015) Identification of a novel isoform of the leukemia-associated MLLT1 (ENL/LTG19) protein. Gene Expr Patterns 17:11-5
Romarowski, Ana; Battistone, María A; La Spina, Florenza A et al. (2015) PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis. Dev Biol 405:237-49
Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug et al. (2015) Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biol Reprod 92:121
Buffone, Mariano G; Wertheimer, Eva V; Visconti, Pablo E et al. (2014) Central role of soluble adenylyl cyclase and cAMP in sperm physiology. Biochim Biophys Acta 1842:2610-20
Battistone, M A; Alvau, A; Salicioni, A M et al. (2014) Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation. Mol Hum Reprod 20:1054-66

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