RESUMO
Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.
Assuntos
Infertilidade Masculina , Camundongos Knockout , Motilidade dos Espermatozoides , Animais , Masculino , Motilidade dos Espermatozoides/genética , Camundongos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Dineínas/metabolismo , Dineínas/genética , Espermatozoides/metabolismo , Humanos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Astenozoospermia/patologia , Capacitação Espermática/genética , Camundongos Endogâmicos C57BL , Sistemas CRISPR-CasRESUMO
The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.