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

6. I need to find some male gametes… ♀

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

8. Reproduction & Fitness I need to find some male gametes… ♀ MorphologyBehaviorPhysiology Male Quality Male’s BehaviorExternal Signals: PheromonesEnvironment SeasonalityInternal Signals: Hormones

9. Reproduction & Fitness I need to find some male gametes… ♀ MorphologyBehaviorPhysiology Male Quality Male’s BehaviorExternal Signals: PheromonesEnvironment Seasonality Internal Signals: Hormones

10. Hormones and Reproduction: Estradiol Hormone- chemical substance that acts as an internal signal within the body Estradiol (E 2 ) 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 E 2 has been shown to improve sperm transport and sperm retention: Hawk and Cooper, 1975 Hawk and Cooper, 1976 Maltaris et al. 2006

12. Estradiol in Reptiles Rises in E 2 associated with ovulation and vitellogenesis In Red-sided garter snakes (Thamnophis sirtalis parietalis) E 2 may also serve another function— sperm transport Almeida-Santos et al. 2004 Girling et al. 2000 Edwards and Jones 2001

13. Estradiol and T. s. parietalis Hormonal assays show a peak in E 2 levels –Starts 6 hours after mating –Peaks at 24 hours E 2 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 E 2 seen in female red- sided garter snakes play a role in aiding sperm transport after mating? Hypothesis: E 2 improves sperm transport in the oviducts of female red-sided garter snakes.

17. Predictions Administering an additional dose of E 2 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 36 females collected from den Mated in arenas Tissue sample taken from males Photo: Chris Friesen Photo: Don Powers

21. Experimental Design Treatment E2E2 VehicleTamoxifen

22. Experimental Design Treatment E2E2 VehicleTamoxifen

23. Experimental Design Treatment E2E2 VehicleTamoxifen

24. Experimental Design Treatment E2E2 VehicleTamoxifen

25. Experimental Design Treatment E2E2 VehicleTamoxifen

26. Experimental Design Treatment Day 3 6 10 E2E2 TamoxifenVehicle Predictions: Sperm transport >>

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 Posterior Oviduct POV Middle Oviduct MOV Posterior Anterior PAO Anterior AAO Anterior oviduct

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 E 2 Tamoxifen Vehicle F statistic: 1.079 P = 0.385 E 2 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 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 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 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 POV = Posterior Oviduct MOV = Middle Oviduct PAO = Posterior Infundibulum AAO = Anterior Infundibulum

35. Conclusions Thus Far… Treatment with E 2 or an E 2 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?