- Title
- Large-scale proteomic screen and signal transduction analysis used to study embryonic gonad and germ cell development
- Creator
- Ewen, Katherine
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2010
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- In the fields of sex determination, embryonic gonad development and germ cell differentiation, much effort has been placed in performing large-scale screens anchored in RNA and DNA technologies. Whilst these technologies have identified new candidate genes, they are unable to provide expression data for functionally active gene products. Recent improvements in the sensitivity of proteomic screening technologies have made analysis of the embryonic gonadal proteome experimentally feasible. Thus, major aims of my PhD were to generate a data set of gonadal proteins expressed at the time of sex determination, and to identify differentially expressed proteins that potentially regulate early events in gonadogenesis, germ cell development and sex differentiation. To detect and identify gonadal proteins, I used two-dimensional nano-flow liquid chromatography and tandem mass spectrometry. I identified 1037 proteins which primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. Over 300 proteins were not identified in a similar transcriptomic study. Over 60 proteins were identified with potential links to human disorders of sexual development (DSDs). I identified four proteins up-regulated in the ovary and three proteins up-regulated in the testis at a critical time point in sex determination. I also identified five proteins increasing, and three proteins decreasing, in expression during early testis development. Two of these temporally-regulated proteins are associated with human DSDs. The majority of these expression differences have not been detected at the transcript level. These data provide novel targets potentially controlling events in gonadogenesis, sex determination and germ cell differentiation. Recently, the field of germ cell sex differentiation was revolutionised with the discovery that retinoic acid (RA) initiates meiosis in female embryonic germ cells, and that the RA-degrading enzyme CYP26B1 inhibits meiosis in male germ cells, which consequently cease mitotic division till after birth. How these somatic environmental factors regulate the transition from mitosis to meiosis or mitotic arrest remains unanswered. p38 MAP kinase (MAPK) signalling initiates mitotic arrest in other differentiating cell types. Thus, a specific aim of my PhD was to investigate the role of p38 MAPK in controlling male germ cell differentiation. To address this aim experimentally, I analysed expression of p38 MAPK in embryonic gonads and found it to be activated in differentiating male germ cells. I then blocked p38 MAPK signalling and found that male germ cells expressed pluripotency and meiosis-associated proteins in a similar pattern to their female counterparts, and that testes exhibited more meiotic germ cells. These data suggest that p38 MAPK signalling contributes to meiosis inhibition in testicular germ cells, and potentially directs them towards quiescence. The studies outlined in this thesis have identified new candidates potentially regulating early events in gonadogenesis, sex determination and germ cell differentiation. Further analysis of these proteins and signalling pathways may provide valuable insights into these important events which are essential not only for sexual development and reproductive competence, but also for fertilisation, genetic recombination and ultimately species evolution.
- Subject
- gonad; germ cell; proteomic; p38 MAPK
- Identifier
- http://hdl.handle.net/1959.13/807499
- Identifier
- uon:7423
- Rights
- Copyright 2010 Katherine Ewen
- Language
- eng
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| View Details Download | ATTACHMENT02 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download |