Mattie Squire ● Chris Friesen ● Dr. Robert T. Mason

1. 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

2. Reproduction & Fitness Fitness- an individual’s relative ability to survive, reproduce, and propagate its genes into future generations

3. Reproduction & Fitness

4. Reproduction & Fitness

5. Reproduction & Fitness

6. Reproduction & Fitness I need to find some male gametes… ♀

7. Reproduction & Fitness I need to find some male gametes… ♀ Behavior Morphology Physiology

8. 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

9. Reproduction & Fitness I need to find some male gametes… ♀ Behavior Morphology Physiology Environment External Signals: Pheromones Male’s Behavior Internal Signals: Hormones Male Quality Seasonality

10. 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

11. Estradiol and Sperm Transport • Administration of exogenous E2 has been shown to improve sperm transport and sperm retention: Hawk and Cooper, 1976 Maltaris et al. 2006 Hawk and Cooper, 1975

12. 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 Edwards and Jones 2001 Almeida-Santos et al. 2004 Girling et al. 2000

13. 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

14. 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

15. 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

16. 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.

17. 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.

19. Migrate to group den sites; hibernate Nov-April Emerge in spring to mate Spend summers feeding in marshes

20. Experimental Design Photo: Chris Friesen 36 females collected from den Mated in arenas Tissue sample taken from males Photo: Don Powers

21. Experimental Design Treatment E2 Vehicle Tamoxifen

22. Experimental Design Treatment E2 Vehicle Tamoxifen

23. Experimental Design Treatment E2 Vehicle Tamoxifen

24. Experimental Design Treatment E2 Vehicle Tamoxifen

25. Experimental Design Treatment E2 Vehicle Tamoxifen

26. Experimental Design Treatment E2 Vehicle Tamoxifen 3 Day 6 10 Sperm transport Sperm transport Sperm transport > > Predictions:

27. Experimental Design • Collection of oviducts and sperm • Left oviduct maintained for histology Redrawn from Halpert et al. 1982

28. 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 Middle Oviduct MOV Posterior Oviduct POV Posterior Anterior PAO Anterior Anterior AAO

29. 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

30. Results: Three Way ANOVA: Treatment E2 Tamoxifen Vehicle F statistic: 1.079 P =0.385

31. 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

32. 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

33. 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

34. Results: Two Way ANOVA POV = Posterior Oviduct MOV = Middle Oviduct PAO = Posterior Infundibulum AAO = Anterior Infundibulum

35. 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

36. 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

37. 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

38. Questions?