ABSTRACT: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta.
Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations.
Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.
Human Reproduction 08/2009; 24(10):2417-28. · 4.47 Impact Factor
ABSTRACT: Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa.
A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro.
After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region.
PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.
Human Reproduction 11/2008; 23(11):2513-22. · 4.47 Impact Factor
ABSTRACT: The study of gene function in testis and sperm has been greatly assisted by creation of transgenic mice by injection of a transgene into the fertilised egg. However this approach is costly and laborious and is not applicable to other species of importance for the study of sperm function, such as the hamster. We have investigated alternative ways of expressing transgenes in mouse and hamster testis and sperm by in vivo gene transfer. DNA expression constructs were introduced into the testis by injection of DNA followed by electroporation, or by injection of a lentiviral vector. Expression of fluorescent proteins was assessed by fluorescence microscopy. In vivo gene transfer by electroporation led to expression of a fluorescent reporter protein and a fluorescently tagged version of sperm protein phospholipase C zeta in hamster and mouse testis and epididymal sperm. In vivo gene transfer by lentiviral infection led to high level expression of a fluorescent reporter protein in male germ cells. In conclusion, in vivo gene transfer offers a novel way to study gene function in testis and sperm and may also have potential as a way of creating transgenic versions of important model organisms such as the hamster.
Society of Reproduction and Fertility supplement 02/2007; 65:469-74.