Adoyo, Pius Aoko; (1994) Isolation and characterisation of genes coding for candidate sperm antigens for contraceptive vaccines. Doctoral thesis (Ph.D.), University College London (United Kingdom).

Text Isolation_and_characterisation.pdf Download (9MB)

Abstract

Specific protein components of the mammalian sperm acrosome are potential antigens for the development of an immunocontraceptive vaccine. In order to identify such acrosomal components, a series of specific antisera has been generated by immunising rabbits with purified acrosomal membrane fraction prepared from hamster epididymal spermatozoa. Initial characterisation of the antisera by immunocytochemistry was performed on formaldehyde fixed, frozen sections of hamster and baboon testis and epididymis. Only post-meiotic germ cells in the testis were recognised by immune sera and staining was limited to the acrosomal region of spermatozoa. To analyze proteins recognised by these sera, baboon testis extract was resolved on 10% SDS-PAGE. Western-blot analysis indicated that rabbit antisera reacted to particular protein bands in the extracts even after pre-absorption of anti-coliform antibodies from each polyclonal serum. Diluted rabbit polyclonal antisera designated R7 and R10, inhibited significantly sperm-zona binding and fertilisation in a hamster in vitro fertilisation (IVF) assay. A combination of R7 and R10 antiserum was used as a probe for screening a human testis ?gt11 expression cDNA library. This screen resulted in the selection of over 70 clones. Cloned cDNA inserts were isolated by amplification through the use of the polymerase chain reaction. Cloned inserts were characterised by restriction enzyme digestion and oligonucleotide probing techniques, ?-galactosidase fusion proteins expressed by these clones were tested for their effect on fertilisation in a hamster IVF. One clone (HA5-2), showed significant blocking of fertilisation and marked reduction of sperm-zona binding. Two other clones (HA6-2 and HB4-1), showed marked reduction of fertilisation and considerably lower levels of sperm binding. Sequence data from the 1.75 kb cloned HB4-1 insert revealed it to consist of two domains. The first 1132 nucleotides (nt) with >96% homology to human testis specific lactate dehydrogenase (LDH-C4) gene followed by the second downstream sequence 34 nt away from the end of LDH-C4 gene with >71% homology to Chlamydomonas caltractin gene. The findings suggest that these clones encode functional molecules involved in sperm-zona binding, and hence the latter are possible candidates for contraceptive vaccine development.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item