The Effects of Estradiol on Sperm
Transport in Female Red-Sided Garter
Snakes (Thamnophis sirtalis parietalis)
Mattie Squire ● Chris Friesen ● Dr. Robert T.
Mason
Department 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…
♀
Behavior
Morphology
Physiology
Environment
External Signals: Pheromones
Male’s Behavior
Seasonality
Internal Signals: Hormones
Male Quality
Reproduction & Fitness
I need to find
some male
gametes…
♀
Behavior
Morphology
Physiology
Environment
External Signals: Pheromones
Male Quality
Internal Signals: Hormones
Male’s Behavior
Seasonality
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
Maltaris et al. 2006
Hawk and Cooper, 1976
Estradiol in Reptiles
• Rises in E2 associated with ovulation and
vitellogenesis
Almeida-Santos et al. 2004
Edwards and Jones 2001
Girling et al. 2000
• In Red-sided garter snakes (Thamnophis sirtalis
parietalis) E2 may also serve another function—
sperm transport
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 redsided 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
Photo: Chris Friesen
•36 females collected from den
Photo: Don Powers
•Mated in arenas
•Tissue sample taken from males
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Experimental Design
Treatment
E2
Vehicle
Tamoxifen
Day
3
6
10
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
Anterior oviduct
Posterior
Oviduct
Middle
Oviduct
Posterior
Anterior
POV
MOV
Anterior
Anterior
PAO
AAO
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
Results: Two Way ANOVA
*
*
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
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
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
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, JessiAnn Michaelson
Questions?