AbstractConsiderable progress has been made in mammalian species since the advent of in vitro sperm-egg interaction systems. Although in vitro systems for studying sperm-egg interaction in chickens have been developed, they have not been characterised and are limited by the availability of material as only one fertilised egg and a few follicles can be obtained per bird and the bird has to be sacrificed in order to obtain them. Therefore, inner perivitelline layer (IPVL) separated from laid chicken eggs was investigated as a readily available material for studying spermatozoa-egg interaction in vitro. This layer was found to have a similar response to hydrolysis by spermatozoa as IPVL from ovulated and follicular ova in terms of the number of points of hydrolysis produced in a 5 min assay at 40°C. Initiation of hydrolysis of the layer was found to have occurred within 2.5 min, with only the size of the holes, not the frequency, increasing with increased incubation times. The number of points of hydrolysis was found to be positively correlated with the concentration of spermatozoa in the assay. IPVL taken from the same laid egg was found to have a similar response to hydrolysis by spermatozoa, but greater variability was found when fragments of IPVL were taken from different laid eggs. Up to twenty replicate pieces of IPVL can be obtained from a single laid egg, which could be used as an 'experimental unit', providing a plentiful source of material for studying sperm-1 PVL interaction in vitro.
The in vitro sperm-IPVL assay, utilising IPVL from laid eggs, was found to be a suitable assay for assessing sperm quality, and therefore potentially, the fertilizing ability of spermatozoa. In fresh ejaculates of semen, individual males were found to have a different and characteristic ability to hydrolyse the layer and the results were linearly correlated with standard sperm quality assays. The number of holes formed in the IPVL in vitro also correlated linearly with the number of holes formed in the IPVL of eggs fertilized in vivo, in inseminated hens; and logarithmically with the percentage of fertile eggs laid. The sperm-IPVL assay was also able to detect more damaged spermatozoa in chicken and turkey semen, which had been stored at 5°C, under both aerobic and anaerobic conditions, than did other tests of sperm quality, which are known to overestimate the fertilizing ability of stored avian semen. The sperm-IPVL assay is therefore likely to be highly discriminatory against compromised spermatozoa and the fact that the test is 'low-tech', inexpensive and simple to perform should ensure its ready application to avian fertility systems.
Chicken spermatozoa were able to hydrolyse the IPVL separated from laid eggs of several avian species. The heterologous gamete combinations showed order dependent, but not species dependent specificity at the level of sperm-IPVL interaction. Mammalian spermatozoa were unable to hydrolyse the IPVL from laid chicken eggs.
Co-incubation of freshly ovulated ova and chicken spermatozoa resulted in preferential hydrolysis of the IPVL overlying the germinal disc (GD) region of the ovum. However, isolated fragments from the same ova incubated with spermatozoa under the same conditions resulted in an increase in IPVL-holes to numbers comparable to the GD region. Furthermore, when fragments of IPVL with the yolk still attached were incubated with spermatozoa, hydrolysis of the layer was severely inhibited. This suggests that rather than being attracted to the IPVL overlying the GD region of the ovum, spermatozoa are inhibited from hydrolysing the IPVL at areas away from this region by the presence of yolk material.
Fluoroscein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) was able to reliably detect acrosome-reacted chicken spermatozoa. PNA was found to bind to the exposed acrosomes of acrosome-reacted, but not acrosome-intact spermatozoa. At 40°C chicken spermatozoa co-incubated with IPVL required extracellular Ca2+ for induction of the acrosome reaction (AR). However, at 30°C, the IPVL was the sole requirement for acrosomal exocytosis. FITC-PNA was also able to detect acrosome-reacted turkey and quail spermatozoa.
Carbohydrates have been shown to play a crucial role in sperm-IPVL interaction in chickens. Removal of N-linked glycans from the IPVL severely inhibited the ability of spermatozoa to hydrolyse the layer. Wheat germ agglutinin (WGA) completely inhibited hydrolysis of the IPVL by spermatozoa, and the addition of D-N-Acetylglucosamine (D-GIcNAc) to the sperm-IPVL assay caused a drastic reduction in the number of IPVL-holes. Therefore, Nlinked glycans, possibly with terminal D-GIcNAc residues appear to be involved in sperm-IPVL interaction in chickens. Furthermore, isolated IPVLglycans were able to induce the AR in chicken spermatozoa.
|Date of Award||Dec 1999|
|Sponsors||Biotechnology and Biological Sciences Research Council|
Sperm-egg interaction in birds: assays and mechanisms
Robertson, L. (Author). Dec 1999
Student thesis: Doctoral Thesis