To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.
Post-translational modification of intracellular proteins with O -linked N -acetylglucosamine ( O -GlcNAc) catalysed by O -GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT ( Dm OGT) is encoded by super sex combs ( sxc ), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O -GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and Dm OGT informed the rational design of Dm OGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.
Significance Unlike in birds and mammals, cells in most other organisms live at temperatures that correspond closely to those of the environment. Change in ambient temperature is a tremendous challenge for these cells. How ectothermic cells can adjust to the complex effects of temperature change is not understood in detail. Here we demonstrate that ambient temperature correlates with the level of a particular posttranslational modification of cytoplasmic and nuclear proteins in cells of different ectotherms (fruit fly, roundworm, zebrafish). Moreover, high levels of this protein modification ( O- GlcNAc) are shown to be important for successful development at high temperatures. Concerted temperature-dependent O- GlcNAcylation of a wide range of cellular proteins might therefore support the myriad adjustments required in response to temperature change.
During mammalian fertilization, spermatozoa must undergo capacitation and the acrosome reaction. These processes of sperm function are critically associated with various molecular events and one such process is protein tyrosine phosphorylation (PYP). This event is downstream of increases in intracellular Ca2+ and activities of HCO3- activated adenylate cyclase, cAMP-dependent-protein kinase-A and reactive oxygen species. Though, PYP is known to be mediated by tyrosine kinases and phosphatases, only a few of them have been identified and characterized in spermatozoa. Since most identified tyrosine kinases are soluble proteins from somatic cells, it is believed that distinct mechanisms could exist in spermatozoa for PYP. Such sperm-specific protein tyrosine kinases/ phosphatases still remain to be thoroughly characterized in most species, including hamsters. Nevertheless, a few tyrosine phosphorylated sperm proteins have been identified in hamsters and in other mammals as well. There is very limited information available on our understanding of the molecular and ultrastructural localization, as well as the characteristics of tyrosine phosphorylated proteins. Functionally, how sperm motility is regulated by PYP is also poorly understood. Knowledge of tyrosine phoshorylated proteins and how they regulate sperm function is of immense significance in our understanding of male (in)fertility and clinical management of fertility; especially, in the light of studies that implicate the hypo-tyrosine phosphorylated state of sperm proteins with asthenozoospermic condition in humans. This article provides a comprehensive review on PYP and its regulation by kinases and phosphatases.
O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O-N-acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development.
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