RESUMO
Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca(2+)]c in sperm to down-regulate them, and thus prevent elevated levels of NO, known to induce asthenozoospermia via oxidative stress. Our studies point to the potential underlying cause of infertility in PMCA4's absence, and suggest that inactivating mutations of PMCA4 could lead to asthenozoospermia and human infertility. Screening for these mutations may serve both diagnostic and therapeutic purposes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Óxido Nítrico Sintase/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas In Vitro , Masculino , Óxido Nítrico Sintase/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ligação Proteica , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Structural cardiac defects occur in at least 1 twin in about 75% of conjoined twins with thoracic level fusion. Outcomes after surgical separation of thoracic level conjoined twins have been favorable when the hearts have been separate. However, even in this situation, the outlook is poor for an individual twin with an important cardiac defect. Arterial anastomosis between twin circulations is an important additional consideration, with poor outcomes for perfusion recipient twins. Surgical separation is contraindicated when ventricular level cardiac fusion exists. Cardiac assessment is a key component of prenatal counseling.