Novel PLCZ1 compound heterozygous mutations indicate gene dosage effect involved in TFF after ICSI.
Reproduction
; 2024 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-39042720
ABSTRACT
Oocyte activation failure, one of the main factors of total fertilization failure (TFF) after ICSI, could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCζ), a sperm factor, was associated with Ca2+ oscillations in oocytes of mammals. To date, only a limited number of mutations in PLCZ1 (the gene encodes PLCζ) have been linked to TFF demonstrated by the observed reduction in protein levels or activity. In this study, males with normozoospermic sperm suffering TFF after ICSI and their families were recruited. Firstly, mutations in the PLCZ1 sequence were identified by Whole exome sequencing and validated by Sanger sequencing. Then the transcript and protein levels and locations of PLCZ1/PLCζ in sperms of patients were studied followed by in vitro function analysis and in silico analysis to investigate the function-structure correlation of mutations identified in PLCZ1 through Western blotting, Immunofluorescence, RT-qPCR and Molecular Simulation. Ca2+ oscillations were detected after cRNA microinjection with Mâ
¡ mouse oocyte to investigate the calcium oscillations of abnormal PLCζ. Five variants in compound heterozygosity were identified including five new mutations and three-reported mutations which were located across the main domains of PLCζ, except the EF hands domain. The transcript and protein levels were decreased among all the mutations identified in PLCZ1 at different degrees when transfected with HEK293T cells. Among these mutations, M138V and R391* of PLCζ could not trigger normal Ca2+ oscillations. In case 5, an abnormal location in the head of sperm and a higher expression of PLCζ in the sperm were found.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Reproduction
Assunto da revista:
MEDICINA REPRODUTIVA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China