Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.Mark A. Baker, Louise Hetherington, Heath Ecroyd, Shaun D. Roman, and R. John Aitke

Tyrosine phosphorylation of HSP-90 during mammalian sperm capacitation

© 2003 by the Society for the Study of ReproductionThe process of sperm capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation. Whereas phosphotyrosine expression is an essential prerequisite for fertilization, the proteins that are phosphorylated during capacitation have not yet been identified. In the present study, we observed that a major target of this signaling pathway is the molecular chaperone protein, heat shock protein (HSP)-86, a member of the HSP-90 family of HSPs. We used cross-immunoprecipitation experiments to confirm the tyrosine phosphorylation of HSP-86, a process that is not inhibited by the ansamycin antibiotic, geldanamycin. The general significance of these findings was confirmed by studies in which HSP- 90 was also found to be tyrosine phosphorylated in human and rat spermatozoa when incubated under conditions that support capacitation. To our knowledge, these results represent the first report of a protein that undergoes tyrosine phosphorylation during mouse sperm capacitation and the first study implicating molecular chaperones in the processes by which mammalian spermatozoa gain the ability to fertilize the oocyte.Heath Ecroyd, Russell C. Jones, and R. John Aitke

Endogenous redox activity in mouse spermatozoa and Its role in regulating the tyrosine phosphorylation events associated with sperm capacitation

© 2003 by the Society for the Study of Reproduction, Inc.We investigated the role of endogenous redox activity in regulating the signal transduction pathway leading to tyrosine phosphorylation in mouse spermatozoa. Endogenous redox activity was monitored using a luminol-peroxidase chemiluminescent probe. Chemiluminescence increased in spermatozoa that were actively undergoing cAMP-mediated tyrosine phosphorylation events associated with capacitation and was inhibited in a dose-dependent manner by addition of catalase or diphenylene iodonium, both of which also inhibited tyrosine phosphorylation within the cell at points downstream of cAMP. Excluding bicarbonate from the incubation medium reduced the redox activity of sperm by 80–90% and dramatically reduced tyrosine phosphorylation. This study provides the first evidence that tyrosine phosphorylation associated with capacitation in mouse spermatozoa is redox regulated by a flavinoid-containing enzyme involving mediation by hydrogen peroxide. Bicarbonate regulated the redox activity of mouse spermatozoa, and this regulation may contribute to the impact of this anion on tyrosine phosphorylation during capacitation of mouse spermatozoa.Heath W. Ecroyd, Russell C. Jones, and R. John Aitke

Compartmentalization of Prion Isoforms Within the Reproductive Tract of the Ram

© 2004 by the Society for the Study of Reproduction, Inc.Cellular prion protein (PrpC) is a glycoprotein usually associated with membranes via its glycosylphosphatidylinositol (GPI) anchor. The trans-conformational form of this protein (PrpSC) is the suggested agent responsible for transmissible neurodegenerative spongiform encephalopathies. This protein has been shown on sperm and in the reproductive fluids of males. Antibodies directed against the C-terminal sequence near the GPI-anchor site, an N-terminal sequence, and against the whole protein showed that the Prp isoforms were compartmentalized within the reproductive tract of the ram. Immunoblotting with the three antibodies showed that the complete protein and both N- and C-terminally truncated and glycosylated isoforms are present within cauda epididymal fluid and seminal plasma. Moreover, we demonstrate that in these fluids, the PrpC isoforms are both in a soluble state as well as associated with small membranous vesicles (epididymosomes). We also report that only one major glycosylated 25 kDa C-terminally truncated PrpC isoform is associated with sperm from the testis, cauda epididymis, and semen, and this form is also present in the sperm cytoplasmic droplets that are released during maturation. In sperm, this C-terminal truncated form was found to be associated with membrane lipid rafts present in the mature sperm, suggesting a role for it in the terminal stages of sperm maturation.Heath Ecroyd, Pierre Sarradin, Jean-Louis Dacheux, and Jean-Luc Gatt

The development of signal transduction pathways during epididymal maturation is calcium dependent

Heath Ecroyd, Kelly L. Asquith, Russell C. Jones and R. John Aitkenhttp://www.elsevier.com/wps/find/journaldescription.cws_home/622816/description#descriptio