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?