Developmental and molecular analysis of sperm production in Drosophila
Lead Research Organisation:
Cardiff University
Department Name: School of Biosciences
Abstract
This project links developmental biology and determination of cell fate, through transcriptional controls that interpret that cell fate decision and control differentiation to evolution and optimisation of reproductive function.
Males from the Drosophila obscura group of flies make two types of sperm. Eusperm are long, and are capable of fertilising eggs, parasperm are short and protect eusperm from spermicide in the female reproductive tract.
The mechanisms underlying both the specification and differential morphogenesis of eusperm vs parasperm is not known. Many testis-specifically expressed genes identified as single copy genes in other Drosophila species (eg melanogaster) are duplicated in D. pseudoobscura. Most genes required for spermatid differentiation are transcribed in spermatocytes, we hypothesise that the spermatocytes destined to differentiate into eusperm have a different transcript profile from those that differentiate into parasperm. This would include both differential expression of single copy genes, and expression of different paralogues of multicopy genes. We have already found differential presence of mRNA from one paralogous gene pair in spermatids. Two testis-specific transcriptional regulatory complexes have been identified in D. melanogaster. Several subunits of these complexes have duplicated in the obscura group, so we propose that there are distinct versions of the transcriptional complexes, one to promote the eusperm transcript profile, the other to promote the parasperm profile. To test this hypothesis, we are conducting RNAseq on isolated individual cysts or individual spermatocytes. This will identify whether there are two distinct populations of spermatocytes, and which transcripts are differentially expressed.
You will analyse the RNAseq data sets and identify candidate genes for determining the differentiation into the two sperm morphs. You will apply bioinformatics tools to analyse evolution of both coding sequences and regulatory sequences of co-regulated genes. Q-RT-PCR on isolated cysts (early spermatocytes to spermatids) will validate and extend the analysis. You will experimentally test the roles of candidate genes you identified, in the broader context of understanding how two sperm morphs are generated. The methods applied will include primary culture of spermatogenic cysts; male fertility tests and sperm function assays; using CRISPR to knock out specific regulators and cytology and molecular analyses to determine the effects in mutant testes; generation of transgenic flies - a) expressing GFP-fusion proteins for cytology and ChIP; b) with reporter constructs to identify functional transcriptional elements; c) to over express specific genes; d) to employ clonal analysis to label progeny from single stem cells.
Males from the Drosophila obscura group of flies make two types of sperm. Eusperm are long, and are capable of fertilising eggs, parasperm are short and protect eusperm from spermicide in the female reproductive tract.
The mechanisms underlying both the specification and differential morphogenesis of eusperm vs parasperm is not known. Many testis-specifically expressed genes identified as single copy genes in other Drosophila species (eg melanogaster) are duplicated in D. pseudoobscura. Most genes required for spermatid differentiation are transcribed in spermatocytes, we hypothesise that the spermatocytes destined to differentiate into eusperm have a different transcript profile from those that differentiate into parasperm. This would include both differential expression of single copy genes, and expression of different paralogues of multicopy genes. We have already found differential presence of mRNA from one paralogous gene pair in spermatids. Two testis-specific transcriptional regulatory complexes have been identified in D. melanogaster. Several subunits of these complexes have duplicated in the obscura group, so we propose that there are distinct versions of the transcriptional complexes, one to promote the eusperm transcript profile, the other to promote the parasperm profile. To test this hypothesis, we are conducting RNAseq on isolated individual cysts or individual spermatocytes. This will identify whether there are two distinct populations of spermatocytes, and which transcripts are differentially expressed.
You will analyse the RNAseq data sets and identify candidate genes for determining the differentiation into the two sperm morphs. You will apply bioinformatics tools to analyse evolution of both coding sequences and regulatory sequences of co-regulated genes. Q-RT-PCR on isolated cysts (early spermatocytes to spermatids) will validate and extend the analysis. You will experimentally test the roles of candidate genes you identified, in the broader context of understanding how two sperm morphs are generated. The methods applied will include primary culture of spermatogenic cysts; male fertility tests and sperm function assays; using CRISPR to knock out specific regulators and cytology and molecular analyses to determine the effects in mutant testes; generation of transgenic flies - a) expressing GFP-fusion proteins for cytology and ChIP; b) with reporter constructs to identify functional transcriptional elements; c) to over express specific genes; d) to employ clonal analysis to label progeny from single stem cells.
People |
ORCID iD |
Helen White-Cooper (Primary Supervisor) | |
Fiona Messer (Student) |
Description | The morphology of the testes and organisation of developing sperm within the testes of Drosophila pseudoobscura were previously not understood. We have mapped the stages of sperm development within the testes of this species, including establishing a new model for the structure of the region containing the germline stem cell (niche). We have identified the organisation different sperm morphs within the testes, allowing for improved analysis of gene expression and localisation patterns in subsequent experiments. We have analysed differential gene expression between different sperm types in Drosophila pseudoobscura, identifying and analysing key genes which control the gene expression profiles of the different sperm types. |
Exploitation Route | Development of molecular biology techniques and mutant lines which may be used by other research teams to study aspects of D. pseudoobscura biology, or to aid in the use of D. pseudoobscura as a model system. |
Sectors | Agriculture, Food and Drink,Healthcare,Other |
Title | Kmg-GFP Tagged D pseudoobscura lines |
Description | Tagged the D pseudoobscura kumgang protein to allow tracking of Kmg protein in situ. |
Type Of Material | Biological samples |
Year Produced | 2021 |
Provided To Others? | No |
Impact | . |
Description | International Partnership Funding Award |
Organisation | The Hospital for Sick Children (SickKids) |
Country | Canada |
Sector | Hospitals |
PI Contribution | The work carried out as part of this award formed the basis for a successful bid for funding distributed by Cardiff University as part of the BBSRC International Partnership Funding, the purpose of which is to maximise, strengthen existing, and develop new international partnerships within the BBSRC remit. Experimental work carried out by us as part of this collaboration is building on work completed as part of previous collaborations with the Brill lab at The Hospital for Sick Kids/University of Toronto, to examine post-meiotic gene expression in Drosophila testes. |
Collaborator Contribution | The Brill lab are experts in RNA regulation during spermatogenesis. They are contributing mutant fly lines which are of interest in this project and are visiting our lab in April 2023 to carry out staining and imaging. |
Impact | Visit by Brill lab to White-Cooper lab for imaging and analysis |
Start Year | 2022 |
Description | Presentation at South West Fly Meeting (Bristol) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Presentation of PhD work at regional meeting of Fly researchers. Engaged with academics, PDRAs and postgraduate students. Introduced the topic and model system to researchers who were otherwise unware of it and gained valuable feedback and discussion with other researchers. |
Year(s) Of Engagement Activity | 2023 |