Involvement of calcium-dependent protein kinases and phosphatases in sperm capacitation-associated events

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

Changes in Protein O-GlcNAcylation During Mouse Epididymal Sperm Maturation

After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process

Image_1_Changes in Protein O-GlcNAcylation During Mouse Epididymal Sperm Maturation.JPEG

<p>After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.</p

Death Induced by Survival gene Elimination (DISE) correlates with neurotoxicity in Alzheimer’s disease and aging

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA-induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to Aβ42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of “SuperAgers”, humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to Aβ42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and Aβ42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration