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PLCζ disruption with complete fertilization failure in normozoospermia

  • Mercè Durban
  • Montserrat Barragán
  • Marta Colodron
  • Minerva Ferrer-Buitrago
  • Petra De Sutter
  • Björn Heindryckx
  • Valérie Vernaeve
  • Rita Vassena
Gamete Biology

Abstract

Purpose

Intracytoplasmic sperm injection (ICSI) is widely used to achieve fertilization in the presence of severe male factor, resulting in high fertilization rates. Nevertheless, 1–3 % of couples experience complete fertilization failure after ICSI. When a male factor is identified, assisted oocyte activation (AOA) can help overcome fertilization failures. The objective of this study is to describe a case of repeated complete fertilization failures after ICSI with donor oocytes, and to investigate the molecular and functional aspects of phospholipase C zeta (PLCζ) protein in the patient semen.

Methods

The patient was a normozoospermic male who had previously fathered, through natural conception, four children by a different partner. Molecular and functional analysis of sperm-specific PLCζ in the patient and control samples by means of gene sequencing, immunocytochemistry, Western blot, mouse oocyte activation test (MOAT), and mouse oocyte calcium analysis (MOCA) were used.

Results

PLCζ expression levels and distribution were significantly disrupted, although MOAT and MOCA did not indicate a decrease in activation ability.

Conclusions

Normozoospermic males can have disrupted expression and distribution of PLCζ, and reduced activation ability after ICSI in human oocytes, despite their normal activation potential in functional testing using mouse oocytes. Discrepancy among molecular and functional data might exist, as mutations in the gene sequence may not be the only cause of alteration in PLCζ protein related to activation failures.

Keywords

ICSI Normozoospermia PLCζ Assisted oocyte activation Fertilization failure 

Notes

Acknowledgments

The authors wish to thank Dr. Anna Ferrer, Dr. Elena Rebollo and Sara Casadesús for technical support.

Compliance with ethical standards

Informed consent

Informed consent was obtained from the couple involved in the study in order to analyze the semen samples and to report the case.

Ethical approval

All procedures performed were in accordance with the ethical standards of the institutional research committees and with the 1964 Helsinki declaration and its subsequent amendments.

Financial support

Financial support for this study was provided in part by a fundamental clinical research mandate and a university grant.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This study was funded in part by Fundació Privada EUGIN, a fundamental clinical research mandate from the FWO-Vlaanderen to PDS, and a Ghent University grant to BH.

Supplementary material

10815_2015_496_MOESM1_ESM.docx (15 kb)
Suppl. Table 1 (DOCX 15 kb)
10815_2015_496_MOESM2_ESM.docx (15 kb)
Suppl. Table 2 (DOCX 14 kb)
10815_2015_496_MOESM3_ESM.docx (14 kb)
Suppl. Table 3 (DOCX 13 kb)
10815_2015_496_MOESM4_ESM.docx (19 kb)
Suppl. Table 4 (DOCX 19 kb)
10815_2015_496_Fig2_ESM.gif (16 kb)
Suppl. Fig. 1

Representative patterns for the Ca2+ frequency scores (0, +, ++, +++). The frequency score is given by the number of calcium spikes recorded during 2 h post ICSI: a) +++ = >10; b) ++ = 3–10; c) + = 1–2; d) 0 = no spikes recorded (AU, arbitrary units). (GIF 15 kb)

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High resolution image (TIFF 2445 kb)
10815_2015_496_Fig3_ESM.gif (159 kb)
Suppl. Fig. 2

Western Blot results from both blots. Immunoblotting was applied to determine protein expression of endogenous PLCζ (left panel), using tubulin as a loading control (right panel). The relative intensity of PLCζ bands (bar graph) were determined by densitometry referred to tubulin in fertile (white bar) and patient sperm (black bar). Different amounts of protein extracts, 50 (a) and 100 (b) μg, were applied and figures show the whole exposed blot. Arrows indicate bands for PLCζ (apparent MW of ≈70 kDa), * indicates unspecific bands. (GIF 158 kb)

10815_2015_496_MOESM6_ESM.tif (14.9 mb)
High resolution image (TIFF 15296 kb)
10815_2015_496_Fig4_ESM.gif (235 kb)
Suppl. Fig. 3

Cellular localization of PLCζ protein in normozoospermic fertile human sperm. Representative image of PLCζ localization patterns by immunolocalization in fertile (a) and patient sperm (b). Squares represent corresponding upper (i) and lower (ii) panels in Fig. 1. Scale bars, 10 μm. (GIF 234 kb)

10815_2015_496_MOESM7_ESM.tif (3 mb)
High resolution image (TIFF 3107 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Mercè Durban
    • 1
  • Montserrat Barragán
    • 1
  • Marta Colodron
    • 1
  • Minerva Ferrer-Buitrago
    • 2
  • Petra De Sutter
    • 2
  • Björn Heindryckx
    • 2
  • Valérie Vernaeve
    • 1
  • Rita Vassena
    • 1
  1. 1.Clínica EUGINBarcelonaSpain
  2. 2.Ghent Fertility and Stem Cell Team (GFaST), Department for Reproductive MedicineGhent University HospitalGhentBelgium

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