Developmental and molecular analysis of sperm in Drosophila pseudoobscura

Messer, Fiona Rachel ORCID: https://orcid.org/0000-0002-9377-750X 2022. Developmental and molecular analysis of sperm in Drosophila pseudoobscura. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Drosophila pseudoobscura produce three distinct sperm morphs: a long fertilising morph, the eusperm, and short and medium non-fertilising morphs, parasperm 1 and 2. Parasperm protect the eusperm from female-derived spermicides in the female reproductive tract. Drosophila spermatogenesis follows a well-characterised pattern of differentiation, mitosis, meiosis, elongation and individualisation. The majority of transcription of genes whose products are required during meiosis and post-meiosis occurs during the pre-meiotic primary spermatocyte stage. Prior to this work, little was known regarding the specific molecular and developmental processes required for the production of multiple sperm morphs in D. pseudoobscura. I hypothesised that transcriptional variation would be present between sub-sets of primary spermatocyte cysts, which would contribute to development of the sperm morphs. RNA-seq analysis of single spermatocyte cysts showed transcriptional differences between sub-sets of cysts, prior to the onset of meiosis. Over 1000 genes were identified as differentially expressed between primary spermatocyte cysts. RNA-seq analysis of post-meiotic spermatid cysts suggested that transcriptional differences between cyst types are also present during elongation and individualisation, identifying around 1400 genes. Analysis of cyst RNA-seq data, and subsequent validation by in situ hybridisation, revealed differentially expressed genes with potential functions in transcription, spermatogenesis and spermiogenesis, notably components of the testis meiotic arrest complex (tMAC) and the tMAC regulator kumgang (kmg). A Kmg-GFP fusion revealed that the Kmg protein is also differentially expressed in D. pseudoobscura spermatocytes and may contribute to morph differentiation. I have used immunofluorescence to characterise the structure of the hub and apical proliferation centre in D. pseudoobscura testes, and propose an updated model of hub structure in this species. I have also developed D. pseudoobscura lines expressing endogenous cas9, and describe the results of validation experiments. In this work, I have identified genetic components contributing to the development of the multiple sperm morphs in D. pseudoobscura.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Funders:	BBSRC SWBio
Date of First Compliant Deposit:	7 July 2022
Date of Acceptance:	7 July 2022
Last Modified:	10 Nov 2022 11:35
URI:	https://orca.cardiff.ac.uk/id/eprint/151104

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