Technical Reference #1658

1658.

Reduced Amounts and Abnormal Forms of Phospholipase C Zeta (PLCz) in Spermatozoa From Infertile Men E. Heytens; J. Parrington; K. Coward; C. Young; S. Lambrecht; S.-Y. Yoon; R.A. Fissore; R. Hamer; C.M. Deane; M. Ruas; P. Grasa; R. Soleimani; C.A. Cuvelier; J. Gerris; M. Dhont; D. Deforce; L. Leybaert; and P. De Sutter;, Ghent University, Human Reproduction, 24(1658), (2009)
Link To Paper

Abstract:
background: In mammals oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta(PLCz). However it still remains to be conclusively shown that PLCz is the endogenous agent of oocyte activation. Some types o

Keywords:
PLCz / infertility / sperm / globozoospermia / oocyte activation

Materials & Methods:
Calcium imaging in these experiments was carried out as described previously (Yoon and Fissore 2007). In brief oocytes loaded with Fura 2- acetoxymethyl ester (Fura 2-AM; Molecular Probes Eugene OR USA) were subjected to microinjection and transferred within 20 min into TL-HEPES microdrops placed on a glass-bottom monitoring dish (MatTek Corporation Ashland MA USA) covered with mineral oil. Oocytes were monitored simultaneously using a 20 objective on an inverted microscope (Nikon Corp. Tokyo Japan) outfitted for fluorescence measurements and with a temperature-controlled stage (20/20 Technology Inc Wilmington NC USA). The excitation wavelength was alternated between 340 and 380 nm by a filter wheel (Ludl Electronic Products Hawthorne NY USA) and fluorescence ratios were taken every 20 or 30 s. After passing through a 510 nm barrier filter the emitted light was collected by a cool SNAP ES digital camera (Roper Scientific Tucson AZ USA). Images were analysed by SimplePCI software (Compix Imaging Inc. Cranberry PA USA). Calcium values are reported as the ratio of 340/380 nm fluorescence in the whole oocyte obtained every 20–30 s.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
sperm