Diagnosis of genetic defects through parallel assessment of PLCζ and CAPZA3 in infertile men with history of failed oocyte activation.
OBJECTIVE
Phospholipase C ζ (PLCζ) is considered as a nominee for sperm associated oocyte activating factors and is located back-to-back with CAPZA3, an actin-capping protein controlling actin polymerization during spermiogenesis. They contain a common bidirectional promoter. The objective of this study was to identify individuals with parallel low expression of PLCζ and CAPZA3 mRNA, in hope of detecting genetic defects in this bidirectional promoter.


METHODS
Semen samples were collected from 24 fertile and 59 infertile individuals with total failed, low and high fertilization rate post intra-cytoplasmic sperm injection (ICSI), as well as globozoospermic individuals. Expression of PLCζ and CAPZA3 were assessed by Real time PCR. In addition, PLCζ was assessed by Western blot.


RESULTS
Significant correlations between PLCζ with CAPZA3 and also between these two genes with fertilization were observed. Individuals with low fertilization presented significantly lower expression of these two genes. Low expression of PLCζ was also verified by Western analysis. Sequence analysis of bidirectional promoter of these two genes in an individual with parallel low expression of both PLCζ and CAPZA3, revealed a mutation within the CAPZA3 predicted promoter, known as human regulatory factor X4 which is a testis-specific dimeric DNA-binding protein. In the opposite stand, in the same location, the mutation appears to be outside but in the vicinity of PLCζ, in a binding region predicate by Genomatix.


CONCLUSIONS
Parallel assessment of CAPZA3 with PLCζ at mRNA level in individuals with inability to induce oocyte activation may help researcher to identify genetic defects associated with failed fertilization.
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Soudabeh
S
Javadian-Elyaderani
Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran.
Kamran
K
Ghaedi
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Marziyeh
M
Tavalaee
Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Farzaneh
F
Rabiee
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Mohammad Reza
MR
Deemeh
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Isfahan Fertility and Infertility Center, Isfahan, Iran.
Mohammad Hossein
MH
Nasr-Esfahani
CAPZA3; Failed fertilization; ICSI; Mutation; PLCζ; Promoter