Wood, Amanda J.

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University of Puget SoundSound IdeasSummer Research2010Zebrafish (Danio rerio) oocyte maturation anddevelopmentAmanda J. WoodUniversity of Puget SoundFollow this and additional works at: http://soundideas.pugetsound.edu/summer_researchThis Presentation is brought to you for free and open access by Sound Ideas. It has been accepted for inclusion in Summer Research by an authorizedadministrator of Sound Ideas. For more information, please contact soundideas@pugetsound.edu.Recommended CitationWood, Amanda J., "Zebrafish (Danio rerio) oocyte maturation and development" (2010). Summer Research. Paper 60.http://soundideas.pugetsound.edu/summer_research/60Zebrafish (Danio rerio) oocyte maturation and development   Mandi Wood Advisor: Dr. Alyce DeMarais University of Puget Sound Biology Department                      Figure 2. Agarose gel of Bub-1 RT-PCR products. Densitometry reveals that cells treated with Nocadozole expressed Bub-1 significantly more than the control. (p=0.0295, 95% confidence interval 166.26, 1192.06, n=6) Figure 3. Fluorescent microscope image (20x) of follicle cell and oocyte in egg culture stained with Hoescht 33342.      •We have asked whether prematurational oocytes respond to a signaling cascade destabilizing treatment by upregulating the expression of genes found in the spindle assembly checkpoint (specifically, Bub-1) •We have also asked whether it be possible to detect chromosomal or nuclear morphological data from the follicular cells themselves.    •During development, the oocyte has signalling mechanisms that help promote the fidelity of chromosome segregation during meiosis.  •The spindle assembly checkpoint will halt the cell cycle if chromosomal abnormalities are present, in order to promote repair mechanisms (Tunquist and Maller, 2003) •The signalling cascade is  to be triggered by abnormalities in the spindle apparatus            •Follicle cells provide nutrients and transmit signals to the developing ovum for growth and maturation  •Studies of checkpoint proteins in oocytes should be corresponded with studies of follicle cells  Figure 1. The current molecular model for mitotic checkpoint activation.(Tunquist and Maller, 2003) Background   •The oocyte mitotic process is sensitive to cellular condition and treatments •The oocyte responds to microtubule destabilization by up-regulating checkpoint protein Bub-1  •Follicular nuclei can be successfully visualized from oocyte culture using the newly-developed protocol  The University of  Puget Sound Biology Department Murdock Charitable Trust Dr. Alyce DeMarais Dr. Leslie Saucedo Dr. Mark Martin  •Connect the variation in oocyte condition with that of the follicle cells. •Immunofluorescent studies of follicle cells in conjunction with the checkpoint proteins •More gene expression assays should be performed with other genes in the pathway Conclusions Present Study   •PART I: expression of Bub-1 under altered oocyte conditions •Oocytes  were treated with microtubule destabilizing drug, Nocadozole (Ikegami et al., 1997). •Total RNA was extracted from treated oocytes •Bub-1 gene transcripts were amplified by RT-PCR, with EF1-α sequences also amplified as a loading control. •Densitometry analysis was performed on the resulting PCR products using ImageJ to determine statistical power  PART II: developing a protocol for visualization of follicular nuclei •Oocytes were fixed for 2 hours using a new “fixing solution” (100mM HEPES, 50mM EGTA, 5mM MgSO4, 0.4 M Dextrose, 0.2% Triton X-100, 32% Formaldehyde, and 1x PBS in water) and washed in PBTriton 0.1%  •Nuclear material was stained with Hoechst 33342  •Follicle cell nuclei were visualized on a fluorescent microscope at 20x magnification.     •The Bub-1 sequence is up-regulated in oocytes treated with Nocadozole.           •Follicle cell nuclei are visible under fluorescent microscope        Materials and Methods Results Future Research Acknowledgements References   1. Tunquist, B.J. and Maller, J.L. 2003. Under  arrest: Cytostatic factor (CSF)-mediated  metaphase arrest in vertebrate eggs. Genes  Dev. 17: 683-710.  2. Ikegami, Richard; Zhang, Jianshe; Rivera- Bennetts, Alma K.; Yager, Thomas D.;  1997. Activation of  the metaphase  checkpoint. Zygote. 5: (November) 329-350.   

Zebrafish (Danio rerio) oocyte maturation and development

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Zebrafish (Danio rerio) oocyte maturation and development

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