Mattie Squire ● Chris Friesen ● Dr. Robert T. Mason
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Transcript of Mattie Squire ● Chris Friesen ● Dr. Robert T. Mason
The Effects of Estradiol on Sperm Transport in Female Red-Sided Garter Snakes (Thamnophis sirtalis parietalis)
Mattie Squire ● Chris Friesen ● Dr. Robert T. MasonDepartment of Zoology ● Oregon State University
Reproduction & Fitness
Fitness- an individual’s relative ability to survive, reproduce, and propagate its genes into future generations
Reproduction & Fitness
Reproduction & Fitness
Reproduction & Fitness
Reproduction & Fitness
I need to find some
male gametes…
♀
Reproduction & Fitness
I need to find some
male gametes…
♀
Behavior Morphology Physiology
Reproduction & Fitness
I need to find some
male gametes…
♀
MorphologyBehavior Physiology
Male Quality
Male’s BehaviorExternal Signals: PheromonesEnvironment
Seasonality Internal Signals: Hormones
Reproduction & Fitness
I need to find some
male gametes…
♀
MorphologyBehavior Physiology
Male Quality
Male’s BehaviorExternal Signals: PheromonesEnvironment
SeasonalityInternal Signals: Hormones
Hormones and Reproduction: Estradiol
• Hormone- chemical substance that acts as an internal signal within the body
• Estradiol (E2) is an important steroid hormone in vertebrate females– Primarily secreted from the ovaries– Has behavioral and physiological effects on
both females and males
Estradiol and Sperm Transport
• Administration of exogenous E2 has been shown to improve sperm transport and sperm retention:
Hawk and Cooper, 1975 Hawk and Cooper, 1976 Maltaris et al. 2006
Estradiol in Reptiles
• Rises in E2 associated with ovulation and vitellogenesis
• In Red-sided garter snakes (Thamnophis sirtalis parietalis) E2 may also serve another function—sperm transport
Almeida-Santos et al. 2004Girling et al. 2000
Edwards and Jones 2001
Estradiol and T. s. parietalis
• Hormonal assays show a peak in E2 levels – Starts 6 hours
after mating– Peaks at 24 hours
• E2 peak not necessary to start vitellogenesis or ovulation Redrawn from Whittier et al. 1987
Sperm Transport and T. s. parietalis
• Females store sperm– Mate ~6 weeks before ovulation need to
store sperm– Sperm stored in sperm storage tubules within
anterior end of oviduct– Long term storage also possible
Redrawn from Halpert et al. 1982
Sperm Transport and T. s. parietalis
• Sperm transport increases 24-48 hrs after copulation
• Sperm transport between 2 and 40 days?
From Halpert et al. 1982
Question:
Does the peak in E2 seen in female red-sided garter snakes play a role in aiding
sperm transport after mating?
Hypothesis:
E2 improves sperm transport in the oviducts of female red-sided garter snakes.
Predictions
• Administering an additional dose of E2 will increase sperm transport in the garter snake oviduct.
• Also, injection of Tamoxifen, an estrogen antagonist will decrease sperm transport in the garter snake oviduct.
• Migrate to group den sites; hibernate Nov-April
• Emerge in spring to mate
• Spend summers feeding in marshes
Experimental Design
•36 females collected from den
•Mated in arenas
•Tissue sample taken from males
Photo: Chris Friesen
Photo: Don Powers
Experimental Design
TreatmentE2 Vehicle Tamoxifen
Experimental Design
TreatmentE2 Vehicle Tamoxifen
Experimental Design
TreatmentE2 Vehicle Tamoxifen
Experimental Design
TreatmentE2 Vehicle Tamoxifen
Experimental Design
TreatmentE2 Vehicle Tamoxifen
Experimental Design
TreatmentD
ay
3
6
10
E2 TamoxifenVehicle
Predictions:Sperm
transport Sperm
transport Sperm
transport > >
Experimental Design
• Collection of oviducts and sperm– Left oviduct maintained for histology
Redrawn from Halpert et al. 1982
Experimental Design
• Collection of oviducts and sperm– Left oviduct maintained for histology– Right oviduct sectioned and sections placed into
sperm washing medium for sperm collection for counting and competitive PCR
Posterior Oviduct
POV
Middle Oviduct
MOV
Posterior
Anterior
PAO
Anterior
Anterior
AAO
Anterior oviduct
Methods: Sperm Scoring
• 1 µl sample removed to be visualized with light microscopy– Checked for presence of sperm– Counting chamber used to assess sperm
numbers/score
• Scoring:– 0: No sperm– 1: 1-10 sperm– 2: 11-50 sperm– 3: >50 sperm
Results: Three Way ANOVA: Treatment
E2
Tamoxifen
Vehicle
F statistic: 1.079
P = 0.385
E2
Tamoxifen
Vehicle
F statistic: 1.079
P = 0.385
Results: Two Way ANOVA
*
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
*
Holm-Sidak multiple comparisons
N = 12
P < 0.012
Bars – standard error
Results: Two Way ANOVA
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
*
Holm-Sidak multiple comparisons
N = 12
P < 0.001
MOV > POV, PAO, AAO
Bars – standard error
Results: Two Way ANOVA
**
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
Holm-Sidak multiple comparisons
N = 12
P < 1.00
Bars – standard error
Results: Two Way ANOVA
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
POV = Posterior Oviduct
MOV = Middle Oviduct
PAO = Posterior Infundibulum
AAO = Anterior Infundibulum
Conclusions Thus Far…
• Treatment with E2 or an E2 antagonist had no significant effect on sperm transport– Can still test whether or
not treatment was effective and look at histological results
• However, we were able to elucidate sperm transport for time periods between 2 and 40 days
What’s Next?
• Competitive PCR– Finding a way to extract enough DNA from
samples– Constructing a standard curve to quantify
relative sperm numbers– Running PCR and genotyping for sperm
samples from the females’ oviducts
• Histology
Acknowledgements
• Funding:– Howard Hughes Medical Institute– Offiyai funds
• People:– Dr. Robert T. Mason– Dr. Kevin Ahern– Chris Friesen– Mason Lab: Ben Burke, Emily Uhrig,
Anna Vigeland, King Yabut– Help in the field: Dr. Deborah
Lutterschimdt, Dr. Don Powers, Jessi-Ann Michaelson
Questions?