Elucidation of the causes of reproductive dysfunction in Penaeid prawns: a functional genomic approach

Mr Philip Brady (2009). Elucidation of the causes of reproductive dysfunction in Penaeid prawns: a functional genomic approach PhD Thesis, school of agriculture and food sciences, The University of Queensland.

       
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Author Mr Philip Brady
Thesis Title Elucidation of the causes of reproductive dysfunction in Penaeid prawns: a functional genomic approach
School, Centre or Institute school of agriculture and food sciences
Institution The University of Queensland
Publication date 2009-04
Thesis type PhD Thesis
Supervisor Richard Williams
Wayne Knibb
Abigail Elizur
Total pages 393, i dont want a printed copy if it can only have uq phd thesis on the spine.
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Abstract The black tiger prawn Penaeus monodon is an internationally significant aquaculture species. However, as with many species of penaeids, reliance on wild-caught broodstock, due to compromised fecundity in captive-reared animals, remains a major constraint to industry development. The physiological factors inhibiting ovarian maturation* and spawning in captive-reared animals are unknown. Knowledge of the cellular and associated molecular events occurring during ovarian maturation in penaeids is also fragmentary. Identification and functional characterisation of genes involved in ovarian maturation could therefore be fundamental knowledge required to improve reproductive success in captive-reared animals. In this study, we employed the first application of microarray technology to gene expression profiling in the ovaries and other key tissues associated with reproduction among wild-caught and captive-reared P. monodon (Chapters 2, 3 and 4). To this end, we created three tissue specific P. monodon EST libraries, and using these libraries, developed a reproduction targeted 2000 spot custom oligonucleotide microarray. Microarray technology offers the unprecedented ability to study the simultaneous expression profiles of thousands of genes from a range of tissues and provides a platform for gaining a greater understanding of gene function, interaction, and regulation with respect to penaeid reproduction. Using microarray analysis, it was possible to identify 55 transcripts as differentially expressed in the ovaries of wild-caught (and captive-reared) animals during ovarian maturation. 53 transcripts were also identified as differentially expressed in the ovary between wild-caught and captive-reared animals (Chapter 2). Key transcripts# identified as up-regulated at vitellogenic stages in the ovary include, as expected, vitellogenin (Vg) – the major egg yolk protein precursor, P4HB – a subunit of the MTP complex which, consistent with its expression profile, may play a key role in Vg assembly, and p8 – an architectural transcription factor which may be involved in initiating expression of large numbers of genes involved in ovarian maturation. Key transcripts identified as differentially expressed in the ovary between wild-caught and captive-reared animals include Vg and pmLSD – a member of the PAT-family (Perilipin, Adipophilin, TIP47) of lipid droplet associated proteins, the latter not previously described in crustaceans. Both transcripts displayed reduced expression levels in the ovaries of captive-reared compared with wild-caught animals, suggestive of reduced vitellogenic activity and lipid accumulation in captive-reared animals. Several further transcripts displayed differences in expression profiles between wild-caught and captive-reared animals, together suggestive of further possible differences in processes associated with cellular proliferation and differentiation and other energetic and metabolic processes in the ovaries between the two sources. Ovarian expression profiles of several key known and novel transcripts were further characterised by RNA in situ hybridisation (ISH) (Chapter 2). In Chapter 2, we also explored gene expression profiles in the whole cephalothorax of wild-caught animals during ovarian maturation. The cephalothorax contains further target organs involved in reproduction including the hepatopancreas, the mandibular organ and the Y-organ. Key transcripts displaying expression profiles of interest included βGBP-HDL – the bi-functional non-sex-specific high density lipoprotein and pattern recognition protein, and SCP2/SCPx – the sterol carrier protein/oxoacyl-CoA thiolase. Both transcripts were up-regulated at vitellogenic stages, reflecting the increased mobilisation of lipids towards ovarian maturation. The expression profiles of βGBP-HDL and SCP2/SCPx have not been previously described in relation to ovarian maturation in crustaceans. Also in Chapter 2, we examined histological differences in the ovary between wild-caught and captive-reared animals. We identified several key differences in developing oocytes between the two sources, including reduced lipid accumulation in the oocytes of captive-reared animals. This finding was consistent with the reduced expression levels observed for transcripts encoding Vg and pmLSD (both involved in lipid accumulation) in ovaries of captive-reared compared with wild-caught animals. In Chapter 3, we further examined gene expression profiles in the ovary and cephalothorax among wild-caught and captive-reared animals using heterologous (cross-species) screening of an Australian Blue swimmer crab Portunis pelagicus 5000 spot cDNA microarray. These results complemented and extended gene expression profiles obtained in Chapter 2. Many studies have previously utilised heterologous screening of existing microarrays to examine gene expression in closely related species for which few genomic resources are available. However, this represents one of the most divergent studies in evolutionary terms using heterologous microarray screening reported to date. As the crab microarray was initially developed for identification of genes involved in moult-cycle regulation in portunid crabs, we first evaluated the utility of the heterologus technique by comparing gene expression profiles obtained for both P. pelagicus and Fenneropenaeus merguiensis (banana prawn) between key moult stages. We identified a low, but significant correlation between expression profiles obtained for the two species and identified several transcripts involved in cuticle formation that are commonly differentially expressed between the two species. This provided a partial ‘validation’ to the use of the heterologous technique. In subsequent P. monodon ovarian maturation stage-specific hybridisations, using the P. pelagicus array, we identified several key transcripts displaying expression profiles of interest. Of particular interest, transcripts encoding farnasoic acid methyl transferase (FAMeT) were up-regulated in the whole cephalothorax of animals with mature ovaries. The promotory effect of methyl farnesoate (MF) on reproduction in crustaceans, together with the role of FAMeT in the synthesis of MF, suggests a key role for FAMeT in regulation of ovarian maturation in crustaceans. This is the first report of the differential expression of FAMeT in relation to ovarian maturation in crustaceans. In crustaceans, vitellogenesis (yolk formation) is negatively regulated by neuropeptides expressed in the neuroscretory centres of the eyestalk. Eyestalk ablation (removal of one or both eyestalks) remains the only method widely utilised by industry to promote ovarian maturation in penaeids. The effect from ablation is attributed to removal of the medulla terminalis X-organ-sinus gland complex (MTXO-SG), the source of the gonad inhibiting hormone (GIH). However, ablation also causes myriad detrimental effects including high mortality. The need clearly exists for development of novel, benign, more physiological methods for manipulation of reproduction. Accordingly, in Chapter 4, to examine MTXO-SG function, we utilised oligonucleotide microarray analysis to examine ovarian maturation stage-specific differential gene expression in the eyestalk in both wild-caught and captive-reared animals. Key transcripts displaying expression profiles of interest included transcripts encoding arrestin – regulators of G-protein coupled receptor (GPCR) activity, that were found to be up-regulated at pre-vitellogenic stages. As the GPCRs include receptors for the biogenic amines, which have been demonstrated to modulate release of the eyestalk neuropeptides, this observation highlights the possibility that further functional characterisation of the various hormone receptors in the eyestalk, including their modes of regulation and probable coupling to excitation-release mechanisms in the neurosecretory cells of the MTXO-SG, may offer new opportunities for manipulation of eyestalk neuropeptide levels. Transcripts encoding known/previously identified eyestalk neuropeptides could not be detected using microarray analysis in this study. This result is attributed to low relative expression levels of these transcripts. Intense deposition of lipid occurs during oocyte development. Lipid accumulating in crustacean oocytes provides fuel for the biosynthetic processes of oogenesis and vitellogenesis and is later used by developing larvae. Given the reduced levels of lipid accumulation observed in oocytes of captive-reared compared with wild-caught animals (Chapter 2), together with the observed reduced expression of transcripts encoding pmLSD – a novel member of the PAT-family of lipid storage droplet associated proteins (Chapter 2), we chose to further investigate pmLSD gene and protein expression among wild-caught and captive-reared animals. In Chapter 5, we report the full length pmLSD cDNA sequence, generated using the 5’ and 3’ rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence of the 368-residue pmLSD peptide shares similar identity with other invertebrate and vertebrate PAT-family proteins. Results of ISH revealed pmLSD transcripts as restricted to pre-vitellogenic oocytes regardless of ovarian maturation stage. We subsequently developed a polyclonal anti-pmLSD antibody. Results of western blot and immunohistochemical analyses together revealed expression of pmLSD protein exclusively in oocytes at vitellogenic and cortical rod stages, localised to the surface of lipid droplets, and at higher levels in wild-caught compared with captive-reared animals. We suggest that low levels of pmLSD plays a key role in the reduced lipid accumulation observed in oocytes of captive-reared animals. We also provide evidence of further PAT-family proteins in P. monodon displaying tissue specific expression profiles. These incremental, yet important gains in understanding of key cellular and molecular events associated with peneaid ovarian maturation contribute to identification of the possible causes of reduced reproductive potential in captive-reared animals. * Throughout this thesis, the term ovarian maturation is used to describe all the stages of ovarian development and maturation. This convention is widely adopted in the literature and avoids repetition of the lengthy term ovarian development and maturation. # Throughout this thesis, the term key transcripts is used to refer to transcripts which display significant homology (through BLAST) to genes previously identified as playing key roles in the process of ovariation maturation and other related metabolic processes.
Keyword Penaeus monodon
oocyte
Ovarian maturation
Gene Expression
Dna Microarray

 
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Created: Fri, 04 Mar 2011, 13:39:36 EST by Mr Philip Brady on behalf of Library - Information Access Service