Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596826
Title: Characterisation of gene expression during spermatogenesis in mammalian species
Author: Bowgen, C.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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Abstract:
In studies carried out previously, transcripts that are expressed in the testis were isolated from a human adult testis cDNA library. The characterisation of these cDNA clones was carried out by a number of methods. In order to determine the sizes of the transcripts and the tissues in which they are expressed, the clones were analysed by Northern blotting using human multiple tissue blots. It was seen that out of the 38 clones analysed, four were exclusively expressed in the testis and a further 17 were preferentially expressed in the testis, suggesting that they may have a role in testis function. Two clones produced multiple transcripts in the testis. This may be the result of post-transcriptional processing of the mRNA molecules in the testis. Southern analysis of the testis-specific and testis-elevated clones demonstrated the conservation of the human cDNA clones with other mammalian species. There was greater conservation between human and pig DNA compared with human and rodent DNA. This allowed the human clones to be used as probes for mRNA in situ hybridization studies using sections of pig testis. The expression patterns of the transcripts was determined at the cellular level by mRNA in situ hybridization. It was seen that all of the clones that produced a signal were expressed in the primary spermatocytes, when RNA synthesis is maximal. There was also germ cell-specific patterns of expression, indicating that the gene products are required at different stages of the spermatogenic cycle. The spermatogenic cells from pig testis were separated into germ cell enriched populations by centrifugal elutriation, and RNA was extracted from each cell fraction and used to prepare cDNA. Amplification of the cDNA with gene-specific primers showed in which germ cells the gene is expressed. The sensitive technique of RT-PCR provides information on low-level transcripts that do not produce a signal in mRNA in situ hybridization studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.596826  DOI: Not available
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