Identification of a Mammalian Homolog to Amphibian Allurin , a Sperm Chemoattractant. Muscle. Kidney. Uterus. Oviduct. M.W. Ovary. M.W. Oviduct. 500. 400. 300. 200. 100. 500. Product of Interest. 400. 300. 200. 100.
Identification of a Mammalian Homolog to Amphibian Allurin,
a Sperm Chemoattractant
Product of Interest
Zachary Harrison, Deborah D. Ricker, Ph.D., and Jeffrey P. Thompson, Ph.D.
Figure 2: Mouse sperm attracted to an ovum.
Department of Biological Sciences, York College of Pennsylvania
Figure 1: Ciliated epithelium (CE) lining the amphibian oviduct depositing allurin (red stain) on the jelly layer (J1) of an egg (Olson et al. 2001).
Pilot study completed in BIO356
RT-PCR revealed a 300bp product exclusively in the mouse oviduct (Figures 3 & 4).
Ovary, Uterus, Oviduct,
Muscle, and Kidney
Dissected; RNA isolated
Product of Interest
RT-PCR using amphibian primers (Olson et al., 2001)
Figure 3: Agarose gel of RT-PCR samples showing the product of interest in the mouse oviduct at 300 base pairs. No products were observed in the other tissue samples. Molecular weight standard in base pairs is also displayed (MW).
Figure 4: Oviduct PCR product from an optimized annealing temperature. This PCR product was used for ligation into pDrive Cloning Vector. Molecular weight standard in base pairs is also displayed (MW).
Gel Electrophoresis of
The 300bp product was successfully cloned into a pDrive vector (Figure 5) and used to transform E. coli (Figure 6).
of Extracted Band
Ligation of PCR product into
pDrive cloning vector
Figure 5: pDrive cloning vector used to transform E. coli. Purified PCR product was ligated at the ‘U’ sites of the plasmid and successful transformations interrupt the Placpathway.
Transformation of E. coli
Figure 7: Electropherogram of the isolated nucleotide sequence. Sequence of interest was from point 41 thru 278. Other nucleotides are a part of the cloning vector.
Cloning Vector Purification
Figure 6: Transformed E. coli on IPTG, X-gal,andkanamycin agar plates. Successfully transformed colonies (+) are white in color whereas untransformed colonies are blue (-).
The vector was purified from colonies and sequencing revealed homology to ubiquitin conjugating enzyme (Figure 7 & 8).
Xiang, X., Burnett, L. et al. 2004. The sperm chemoattractant “allurin” is expressed and secreted from the Xenopus oviduct in a hormone regulated manner. Developmental Biology [serial online] 275: 343-355. Available from: Science Direct.
Olson, J. H., Xiang, X., et al. 2001. Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins. Proceedings of the National Academy of Sciences [serial online] 98:11205-11210. Available from: EBSCO Host.
Howard, R., Sharma, P., Hajjar, C., et al. 2007. Ubiquitin conjugating enzymes participate in polyglutamine protein agregation. BMC Cell Biology 8: 32.
Baarends, W., Roest, H., & Grootegoed, J. 1999. The ubiquitin system in gametogenesis. Molecular and Cellular Endrocrinology 151:5-16.
Shibata, K., Itoh, M., Aizawa, K, Sumiharu, N., et al. 2000. RIKEN Integrated Sequence Analysis(RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer. Genome Res. 10:1757-1771.
Figure 8: BLAST comparison of isolated sequence (query) to mouse genome (sbjct), ubiquitin conjugating enzyme. Horizontal lines between the two sequences confirm nucleotide homology.
H1 : A sequence homologous to amphibian allurinwill be isolated in the mouse (Musmusculus) oviduct using the amphibian allurinprimers.
H0: No homologous sequences will be observed in any mouse tissue samples using the amphibian allurin primers.
I would like to thank Dr. Ricker and Dr. Thompson for their ongoing support of this project and overall guidance.