Anna Ferrer-Vaquer1, Montserrat Barragan1, Thomas Freour1,2,3, Valérie Vernaeve1, Rita Vassena4. 1. Clínica EUGIN, Travessera de les Corts 322, 08029, Barcelona, Spain. 2. Service de médecine et biologie de la reproduction, CHU de Nantes, Nantes, France. 3. INSERM UMR 1064, Nantes, France. 4. Clínica EUGIN, Travessera de les Corts 322, 08029, Barcelona, Spain. rvassena@eugin.es.
Abstract
PURPOSE: Sperm-borne PLCζ protein induces Ca(2+) oscillations in the oocyte and is believed to play a major role during oocyte activation. However, its implication in fertilization failure following ICSI is still debated. We analyzed PLCζ gene sequence, protein expression level, and localization in both patients with previous failed fertilization by ICSI and sperm donors with proven fertility in order to assess the association of PLCζ with both sperm characteristics and ability to fertilize. METHODS: Semen from 15 patients and 13 sperm donors with proven fertility was included in the study. Analysis of the PLCζ gene sequence, protein expression through Western blot, and protein localization by immunofluorescence were performed. RESULTS: Two patients with total fertilization failure presented mutations in heterozygosis in the PLCζ gene. Comparison with donor sample sequences displayed comparable SNP allele frequency. Distribution pattern of PLCζ did not vary significantly between donor and patient samples. Levels of PLCζ protein in sperm cells showed an interindividual variability both in patient and donor samples. Several SNPs previously reported in infertile patients were also present in fertile men. CONCLUSION: Failed fertilization occurs even when levels and distribution of PLCζ protein are within normal range. PLCζ seems to be a necessary but not sufficient factor in determining the molecular pathway involved in oocyte activation.
PURPOSE: Sperm-borne PLCζ protein induces Ca(2+) oscillations in the oocyte and is believed to play a major role during oocyte activation. However, its implication in fertilization failure following ICSI is still debated. We analyzed PLCζ gene sequence, protein expression level, and localization in both patients with previous failed fertilization by ICSI and sperm donors with proven fertility in order to assess the association of PLCζ with both sperm characteristics and ability to fertilize. METHODS: Semen from 15 patients and 13 sperm donors with proven fertility was included in the study. Analysis of the PLCζ gene sequence, protein expression through Western blot, and protein localization by immunofluorescence were performed. RESULTS: Two patients with total fertilization failure presented mutations in heterozygosis in the PLCζ gene. Comparison with donor sample sequences displayed comparable SNP allele frequency. Distribution pattern of PLCζ did not vary significantly between donor and patient samples. Levels of PLCζ protein in sperm cells showed an interindividual variability both in patient and donor samples. Several SNPs previously reported in infertilepatients were also present in fertile men. CONCLUSION: Failed fertilization occurs even when levels and distribution of PLCζ protein are within normal range. PLCζ seems to be a necessary but not sufficient factor in determining the molecular pathway involved in oocyte activation.
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