No description available.

No description available.

No description available.

No description available.

Chicken spermatozoa undergo a post-testicular maturation process similar to that reported for mammals. Association of secreted epididymal proteins and glycoproteins with the sperm surface appears to be complete at the anterior ductus deferens. However, antigenic change to, and glycosylation and deglycosylation of seminal plasma and sperm surface-associated proteins, selective loss of proteins from the sperm surface, as well as secretion of some seminal plasma proteins and selective absorption of others by epithelial cells lining the excurrent ducts, continues in the ductus deferens and ampulla. Hypertonic treatment or neuraminidase treatment of spermatozoa without apparent loss of sperm integrity in-vitro, reduced the ability of spermatozoa to gain access to the uterovaginal insemination. This indicates a clear role for sperm surface-associated proteains and sperm surfarce sialic acid in vaginal sperm transport. Sperm surface-associated proteins were also extracted by glycerol in a temperature-dependent and concentration-dependent manner. This was accompanied at room temperature by a glycerol concentration-dependent reduction in sperm motility. These effects may be implicated in the contraceptive action of glycerol in the chicken vagina. Spermatozoa from several avian and mammalian species entered quail uterovaginal junction SSTs in-vitro, and turkey spermatozoa were found in chicken uterovaginal junction SSTs following uterovaginal junction but not intravaginal insemination, thus showing that the SSTs are not selective, and identifying the vagina as a major site of oviducal sperm selection. Spermatozoa washed from the vagina following intravaginal insemination had immunoglobulin bound to their surface, which was shown to be associated with cell death. Spermatozoa recovered from the anterior oviduct however, were generally devoid of bound immunoglobulin. Furthermore, sperm access to the newly ovulated egg in-vivo following incubation with vaginal mucosa in-vitro did not differ significantly from that of control spermatozoa despite fewer viable spermatozoa inseminated, suggesting a true 'selection' of spermatozoa in the vagina. Ovarian pocket fluid, postulated to be the milieu in which fertilisation takes place in-vivo, altered sperm surface antigenicity and reduced sperm motility in-vitro, suggesting that spermatozoa may naturally undergo oviduct-induced changes prior to fertilisation. Sperm-egg interaction appears specific and receptor-mediated, as in mammals. Spermatozoa showed no preference for the animal pole of the egg, and heterologous gamete combinations indicated limited, order-dependent rather than species-dependent specificity. Presenting carbohydrate residues on the sperm surface and sperm surface antigenicity showed a lack of change proportional to species divergence, although the vagina clearly presents the main barrier to interspecies fertilisation within the order Galliformes.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 63-70).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 116-132).

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Because diagnostic tests for highly pathogenic avian influenza (HPAI) viruses require the use of replication-competent viruses in a biosafety level 3 containment, numerous studies have looked at ways to develop alternative tests. Lentiviral particles pseudotyped with H5 hemagglutinin (HA), the surface glycoprotein of influenza virus, have been described as useful and safe tools for research and serological surveillance on the HPAI viruses. However, not all H5 HA give rise to efficient H5 pseudotyped lentiviral particles (H5pp) production. HA from A/Cambodia/408008/05 H5N1 (H5Cam) and HA from A/Anhui/1/05 H5N1 (H5Anh) exhibit a dramatic difference in their ability to pseudotype lentiviral particles. H5Cam gives the highest H5pp production among all HAs tested, whereas the lowest has been observed with H5Anh. The objective of this study was to investigate the molecular determinants that govern efficient H5pp production. Based on the amino acid differences between H5Cam and H5Anh, H5Anh mutants were generated by site-directed mutagenesis. Strikingly, a single amino acid change, A134V, in the 130-loop receptor-binding domain of HA, significantly increased H5pp production with H5Anh. The finding that valine 134 is crucial for H5pp production was confirmed by reciprocal H5Cam and H5Anh mutants, which displayed either a dramatic decrease or increase in H5pp production, respectively. Influenza virus and H5pp bud at the plasma membrane, therefore changes in HA cell surface expression could affect the production of H5pp. Thus, cell surface expressions of H5Cam and H5Anh were compared by flow cytometry. Intriguingly, H5Cam displayed a higher plasma membrane expression than H5Anh, suggesting that transport is important for H5pp production. Introduction of V134A mutation in H5Cam reduced its surface expression to that of H5Anh; by contrast, H5Anh mutant harboring A134V mutation largely restored its expression. Next the effect of A134V mutation on the binding of HA to sialic acid receptors was investigated. A cell-based Enzyme-linked Immunosorbent Assay was developed to measure binding of wild-type and mutated HA. Soluble recombinant proteins were produced by mammalian cells stably transfected with HA gene ectodomain and were mostly trimeric as indicated by discontinuous native gel electrophoresis. Interestingly, H5Anh proteins exhibited a stronger binding to MDCK cells than H5Cam proteins, and introduction of A134V mutation in H5Anh proteins reduced the binding. By contrast, as predicted, the reciprocal V134A mutation induced a major increase in binding to cellular receptors. It is likely that stronger binding of H5Anh to sialic acids could hinder the release of H5pp. Consistent with this notion, the ability of H5Anh to generate H5pp was significantly increased in a sialylation deficient Lec2 cell, a CHO mutant cell line. In conclusion, H5Cam allows efficient H5pp production whereas H5Anh does not. With several lines of evidence, it is likely that the behavior of H5Anh can be explained by a stronger binding to sialic acid receptors that is dependent on a single amino acid residue at position 134. Since A134V is a naturally occurring mutation observed occasionally in human host, these results may have implications for the understanding of human host adaptations of H5N1 viruses. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy