Download X. laevis - York College of Pennsylvania

Temporal and Regional Expression of a
Sperm Chemoattractant in Mice
Meaghan Harmon, Department of Biology, York College
Review of Literature cont’d
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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.
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