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Temporal and Regional Expression of a Sperm Chemoattractant in Mice Meaghan Harmon, Department of Biology, York College Review of Literature cont’d http://www.gettingovergod.com/wp-content/uploads/2011/11/sperm_zygot.jpg Introduction Allurin was isolated from Xenopus laevis and shown to direct sperm movement toward a chemical gradient (Olson et. Al 2001). The protein sequence of allurin shows homology to the cysteine rich secretory protein (CRISP) family found in mammalian reproductive tracts (Olson et al. 2001). An allurin homolog, ubiquitin conjugating enzyme, was isolated in the mouse oviduct using amphibian allurin primers (Harrison 2010). It is believed that ubiquitin conjugating enzyme’s role in protein processing performs a similar function in the mouse oviduct that allurin performs in the oviduct of X. laevis (Harrison 2010). An allurin homolog was isolated in the mouse oviduct using amphibian allurin primers (Figure 1) (Harrison 2010). Figure 2. The sequence of events present throughout the DIG system in situ hybridization process (Roche Applied Science). Note: Human reproductive system shown for comparison. Figure 1. Allurin primers designed from the amphibian sequence were used to identify a homologous cDNA sequence in the mouse oviduct (Harrison 2010). Research Design and Methods Figure 3. Ubiquitin conjugating enzyme sequence identified by Harrison (2010). Figure 6. Comparative levels of DIG expression at various times during the estrous cycle. T=0 no allurin is present during the follicular phase. T=48 small concentrations of allurin are present as follicles are maturing prior to ovulation. T=72 highest concentrations of allurin are present after ovulation. All times measured in hours post injection stimulation (see methods). Objectives To determine: • Where in the mouse oviduct the allurin gene is expressed. • When in the mouse estrous cycle the gene is expressed. Review of Literature A small protein (allurin) isolated from the egg jelly of X. laevis directed sperm movement toward a chemical gradient. Protein concentration and amino acid sequence was measured (Olson et. al 2001). Allurin is homologous to other cysteine rich secretory proteins (CRISP) found in mammalian reproductive tract (Olson et. al 2001). Allurin is the first amphibian protein to be added to the CRISP family and it is also the first in the family to be found in the female reproductive tract (Olson et. al 2001). Allurin expression is restricted to the amphibian upper oviduct (Xiang et. al 2004). Injection of hCG increased the expression of allurin (Xiang et. al 2004) Figure 5. Expected gradient of allurin expression in the oviduct. Highest concentrations are expected near the ovary, lower concentrations are expected near the uterus. Experimental Significance In-situ hybridization using digoxygenin (DIG) will be used to detect allurin mRNA within the mouse oviduct (Figure 2). cDNA probes will be created targeting the ubiquitin conjugating enzyme sequence (Figure 3) (Harrison 2010). Identifying a sperm chemoattractant in the mammalian reproductive system and pinpointing its time of expression could possibly aid in the fight against infertility in humans. If infertility can be linked to a missing or misexpressed protein with sperm chemoattractant capabilities, a supplemental protein could be manufactured. Literature Cited Figure 4. Anticipated appearance of purple DIG staining within oviductal epithelial cells. Purple stain would indicate positive allurin expression (Lapointe et al. 2005). Expected Results Allurin expression and DIG staining (Figure 4) will appear within the epithelial cells of the mouse oviduct, particularly near the ampulla where fertilization occurs. An increasing gradient of expression is anticipated moving from the uterus towards the ampulla (Figures 5 and 6). Greater allurin expression is anticipated during the ovulatory phase vs. the follicular phase of the estrous cycle (Figure 6) such that: T=0 No/little allurin will be expressed T=48 Allurin will be expressed in small concentrations T=72 Allurin expression will peak as the ovulated egg enters the oviduct Lapointe, J., Kimmins, S., MacLaren, L., Bilodeau, J. 2005. Estrogen Selectivity Up-Regulates the Phospholipid Hydroperoxide Glutathione Peroxidase in the Oviducts. Endocrinology 146:25832592. Lee, K., Kwok, K., Chung, M., Lee, Y., Chow, J., Yeung, W. 2005. Journal of Cellular Biochemistry 95: 740-749. Olson, J., Xiang, X., Ziegert, T., et al. Allurin, a 21kDa Sperm Chemoattractant from Xenopus Egg Jelly, is related to mammalian sperm-binding proteins. 2001. PNAS 98: 11205-11210. Segi, E., Haraguchi, K., Sugimoto, Y., et al. 2003. Expression of Messenger RNA for Prostaglandin E Receptor Subtypes EP4/EP2 and Cyclooxygenase Isozymes in Mouse Periovulatory Follicles and Oviducts During Superovulation. Biology of Reproduction 68:804-811. Sun, F., Giojalas, L., Rovasio, R., Tur-Kaspa, I., Sanchez, R., Eisenbach, M. 2002. Lack of speciesspecificity in mammalian sperm chemotaxis. Developmental Biology 255:423-427. Xiang, X., Burnett, L. , Rawls, A. , Bieber, A. , Chandler, D. 2004. The sperm chemoattractant “allurin” is expressed and secreted from the Xenopus oviduct in a hormone-regulated manner. Developmental Biology 275:343-355. Acknowledgements A special thank you to Dr. Deborah Ricker for mentoring my project, and devoting so much time to me throughout the duration of my thesis experience. Dr. Wendy Boehmler for helping me to thoroughly understand the in situ hybridization process using the DIG system. Dr. Jeffery Thompson for his consultations regarding Zach’s project. Zach Harrison for sharing his project with me so that I may continue his research. http://www.sanger.ac.uk/research/projects/cellsurfacesignalling/gfx/sperm_egg.jpg