Semester

Fall

Date of Graduation

2007

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Jianbo Yao.

Abstract

Genes specifically expressed in oocytes are important for the development of oocytes and early embryos. By analyzing ESTs from a rainbow trout oocyte cDNA library, we identified a novel EST sequence that does not show homology to any sequences in the GenBank. Analysis of tissue distribution by RT-PCR revealed that this gene was only expressed in unfertilized oocytes. Sequencing of the EST clone identified a cDNA of 3163 bp. Northern blot analysis showed the novel gene has a single transcript of 3.4 kb. Additional 5' sequence was obtained by 5' RACE, extending the novel cDNA to 3333 bp. Analysis of the full length cDNA identified an open reading frame encoding a protein of 564 amino acids. The novel protein contains a conserved oxysterol binding protein (OSBP) domain at the C terminus that is characteristic of OSBP-related proteins implicated in lipid metabolism. Therefore, we named the novel gene as Oocyte-specific Oxysterol binding protein Related-Protein of Trout (OORP-T). In situ hybridization showed that the OORP-T mRNA appears to be confined to the cytoplasm of vitellogenic oocytes. Transcription of OORP-T appears to start during pre-vitellogenesis and increases steadily, reaching its peak in the late vitellogenic stage. OORP-T transcript is abundantly present in unfertilized eggs but the level drops significantly in day 2 embryos and continues to decline in day 7 embryos after which it remains low. It is proposed that OORP-T may play an important role in the utilization of yolk derived lipid products during oocyte development and early stages of embryonic development in rainbow trout.;Maternal-zygotic transition (MZT) is the first major transition in early development leading to the activation of embryonic genome. Effective transcription machinery including transcription factors must be in place during MZT for it to occur. Therefore, measuring the transcript abundance of key transcription factors prior to and after MZT can give important clues about the roles of transcription factors during this process. In this study, we quantitatively measured mRNA abundance of 9 selected transcription factors (Figla, P300, YY1, HMGA1, HMGB1, HMGN1, ATF-1, TEAD2 and OCT-4) in unfertilized eggs and early stage embryos from day 1 through day 7 post fertilization using quantitative real time PCR. Our results demonstrate that significant amounts of mRNA for all transcription factors studied are present in unfertilized eggs and day 1 embryos, and the expression of all transcription factors reaches minimum levels in day 2 embryos. While some transcription factors remain at low levels of expression throughout late stage development, others show significant increase of expression following embryonic genome activation. The expression patterns of these transcription factors are suggestive of their roles in MZT as well as in early development in rainbow trout.;Current literature and our results on expression patterns of oocyte specific genes and transcription factors suggest global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). We hypothesized that microRNAs (miRNAs), naturally occurring 19-21bp long post-transcriptional regulators, are involved in this degradation process. We analyzed the expression pattern of dicer, an enzyme required for the processing of microRNAs. Dicer is abundantly expressed until 24 hours post-fertilization and gets down-regulated afterwards. This supports the hypothesis that dicer processes mature miRNAs during these stages and these miRNAs in turn degrade maternal mRNAs. To identify candidate microRNAs involved in this process, we constructed a miRNA library from a pool of oocytes and early stage embryos (0 hour post-fertilization through 96 hours post-fertilization). Sequencing analysis of clones showed that there are at least 15 miRNAs expressed during these stages, 4 of which are novel to rainbow trout. We carried out quantitative real-time PCR to learn more about their expression pattern. Our results show that several microRNAs are up-regulated when maternal RNAs are degraded. Stat3, a transcription factor which is involved in activating the transcription of miR-21 is also abundantly expressed in early rainbow trout embryos. Taken together, these results indicate that up-regulated microRNAs, some induced by stat3, could be responsible for degradation of maternal mRNAs in early embryos.;Identification and characterization of a novel oocyte specific gene with a conserved domain that is involved in oxysterol (a metabolite of cholesterol, a precursor molecule of all steroid hormones) metabolism is described here. Expression pattern of OORP-T follows the pattern of estrogen during oogenesis indicating its unique role in oogenesis and early embryonic development although the functions of OORP-T remain to be discovered. None the less, the OORP-T can potentially be used as a marker in selecting for high growth, better nutritional efficiency, disease resistance etc. Based on the results of studies on expression pattern of transcription factor mRNAs and microRNAs, it appears that microRNAs may be involved in maternal mRNAs degradation before EGA. The microRNAs identified and characterized here might also serve as markers for above mentioned economically important traits especially because microRNA might be regulating several target genes involved in any of the above mentioned phenotypes.

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