Peter Thomas University of Texas at Austin

1. Widespread Endocrine Disruption and Reproductive Impairment in Atlantic croaker Exposed to Seasonal Hypoxia Peter Thomas University of Texas at Austin Collaborators: Dr. Md.S. Rahman J. Kummer Dr. I.Khan

2. HPG axis controlling reproduction in fish Environmental Stimuli Primary Regulators Stressors • • • Te • Catastrophic Events • • Catastrophic Events Temperature • Day Length Physical stressors eg.hypoxia • • Day Length Day Length • Captivity • • Captivity Captivity • • • • Pollution • • Pollution Pollution DayLength • • • Sense Organs Toxic chemicals Brain Water Chemistry LHRH Disease Social Interaction Pituitary Gonadotropins Sensitive stages: ●Gonadal sex differentiation ● Puberty ● Gamete production and gonadal growth ● Gamete maturation, reproductive success Testes Ovary Feedback Yolk Proteins Liver Androgens Progestins Estrogens

3. Effects of Hypoxia on Gamete Production Outline • Evidence from field studies • Pattern of endocrine disturbance in lab studies • Neuroendocrine effects of hypoxia and proposed mechanism of disruption • Conclusions

4. 87o10‘ 87o00‘ 30o30‘ N Escambia Bay H1 W E S East Bay H2 H3 H4 TR Pensacola Bay N2 N1 Santa Rosa Sound 30o20‘ Gulf of Mexico 5 km SC MS AL GA LA FL Study area Field Study:High rainfall in 2003 resulted in extensive and persistent hypoxia throughout East Bay, Florida

5. Field Study: Gonadal growth severely impaired in both sexes at low DO sites OCTOBER normoxic hypoxic Conclusion: chronic hypoxia exposure likely results in complete reproductive failure of exposed population NOVEMBER normoxic hypoxic

6. Gonadal Function • Plasma E2, T • ER conc, ERmRNA Liver Function (vitellogenesis) ER conc, ERmRNA Gamete Production -Females Plasmagonadotropin Plasma E2 steroids Oocyte Production • Gonadosomatic index (GSI) • Fecundity and developmental • stage of oocytes Plasma vitellogenin

7. Oocyte Development impaired in croaker from low DO sites in East Bay Field Study: OCTOBER PNS PNS SYS SYS TYS P 12 Pensacola Bay PB 5 4.6 ppm 3.0 ppm 1.4 ppm normoxic hypoxic  Low DO sites in September

8. Evidence for reproductive dysfunction in croaker exposed to hypoxia at field sites - 2 ● Plasma sex steroids and liver estrogen receptor mRNA levels lower in females from low DO sites STEROIDS normoxic hypoxic Conclusion: Chronic hypoxia exposure causes impairment of estrogen signaling in females normoxic ER mRNA hypoxic

9. Very few mature oocytes developed in croaker from low DO sites Field Study: Atretic Tert. yolk 2ndy yolk 1my yolk Cort. alv. Perinucl. 100 80 60 Oocyte (%) 40 20 0 Sampling sites PB5 PB5 P12 P12 P13 P13 P14 P14 Trans Bridge Bridge Trans PenBay PenBay         Low DO T N T N N N Low DO November October

10. Evidence for reproductive dysfunction in croaker exposed to hypoxia at field sites - 3 ● Plasma vitellogenin levels lower in females from low DO sites,Oct. & Nov. normoxic Conclusion: the decrease in yolk production results in very low fecundity in females exposed to hypoxia hypoxic ● Fecundity much lower in females from low DO sites normoxic normoxic hypoxic hypoxic

11. Gonadal Function • Plasma E2, T, 11 KT • ER conc, ERmRNA Gamete Production -Males Plasma Gonadotropin T, 11 KT Sperm Production • Gonadosomatic index (GSI • Number and developmental • stage of sperm

12. Androgen levels decreased in males from hypoxic sites in October Field Study: OCTOBER normoxic hypoxic

13. Field Study: Androgen levels decreased in males from hypoxic sites in November NOVEMBER normoxic testosterone hypoxic 11-ketotestosterone normoxic Conclusion: consistent pattern of impaired endocrine function in males hypoxic

14. Sperm production dramatically decreased in males from low DO sites Field Study: October November 250 normoxic normoxic 200 150 Relative sperm production 100 hypoxia hypoxia 50 0 P 13 P 14 P B5 Sampling sites P 12 P 13 P 14 P 12 P B5 Bridge Trans Trans Bridge Pensa Bay Pensa Bay

15. Testicular Development impaired in Croaker from low DO sites in East Bay during October Field Study: SC SP SC SP SP Pensacola Bay P 13 PB 5 Low DO Low DO normoxic Sampling sites

16. Confirm reproductive effects due to hypoxia in controlled laboratory studies ● Expose croaker to 3 DO levels: 7.0, 2.7 and 1.7ppm in simple low DO exposure system for 10 week periods during gametogenesis

17. Testicular development and sperm production impaired in low DO groups SP Control 2.7 ppm SC SP SC a b 1.7 ppm 1.7 ppm SP c d

18. GtH Lab Study: Sperm production and endocrine function decreased in low DO groups GtH Testis function T,11KT Sperm Production

19. Evidence for reproductive dysfunction in croaker exposed to hypoxia: Laboratory study -1 Intermediate stages tertiary yolk perinuclear atretic 100 80 Conclusion: Production of mature vitellogenic oocytes (tertiary yolk stage) decreased in low DO groups 60 Oocyte (%) 40 20 0 Control 2.7 ppm 1.7 ppm

20. Ovarian and oocyte development impaired in low DO groups Control 2.7 ppm SYS TYS PNS a b 1.7 ppm 1.7 ppm TYS AO c d

21. Gonadal Function E2 Liver function GtH vitellogenin Oocyte Production Evidence for reproductive dysfunction in croaker exposed to hypoxia: Laboratory study - 2 Conclusion: endocrine function impaired in both sexes after hypoxia exposure;Is this effect caused by a decrease in GtH secretion?

22. Gonadotropin response to LHRHa decreased in low DO groups Conclusion: neuroendocrine system controlling GtH (LH) secretion is a site of impairment on H.P.G. axis after hypoxia exposure. --- mechanism unclear

23. Neuroendocrine Control of Gonadotropin Secretion in Teleosts inhibitory some species but not croaker stimulatory Inhibitory on GnRH melatonin steroids both positive and negative feedback-stage dependent

24. Therefore test whether hypoxia causes a decrease in 5-HT and GnRH mRNAlevels in the POAH Hypothesis: 5-HT O2 Stim. GnRH GtH GnRH mRNA 5-HT (serotonin) Conclusion: decrease in GtH secretion likely due to decline in GnRH function which in turn is associated with decreased 5-HT activity

25. Question: Does hypoxia cause reproductive impairment by decreasing hypothalamic serotonergicactivity? Approach:useserotonin precursor- restore 5-HT levels 5-HTP bypassesTPH and increases substrate availability Neuroendocrine and Reproductive Functions

26. Hypoxia Hypoxia Injection of 5-HTP to restore 5-HT levels in POAH Conclusion: Decline in GnRH mRNA and neuroendocrine function after hypoxia exposure probably due to decrease in serotonergic function in POAH

27. Effects of hypoxia on 5-HT, GnRH and LH hypoxia ● Tryptophan hydroxylase- rate limiting enzyme for 5-HT synthesis ● Monoamine oxidase- catabolizes 5-HT to 5-HIAA ? What is the cause of the decline in 5–HT? ? mRNA

28. Hypoxic mechanism: Determine whether turnover or synthesis of 5-HT altered Ratio of metabolite/5-HT unchanged, therefore turnover not increased TPH activity decreased Conclusion: 5-HT decrease due to decreased synthesis, not turnover, via decrease in TPH activity

29. Conclusions • Negligible oocyte and sperm production in fish from hypoxic field sites • Hypoxia severely disrupts gametogenesis in both male and female croaker by impairing endocrine function. • Hypoxia caused similar impairment in the lab and field studies • Several endocrine and morphometric biomarkers potentially useful for detection of reproductive dysfunction in field samples • Hypoxia disrupts neuroendocrine function by inhibiting the activity of tryptophan hydroxylase, the enzyme for 5-HT synthesis

30. Ovarian HIF-2α mRNA expression increased after exposure to low DO in preliminary analysis Lab. experiment Field studies Control 2.7 ppm 1.7 ppm P12 P13 PensaBay HIF-2a HIF-2a 18S 18S hypoxic normoxic Conclusion: first evidence for activation of a molecular marker of hypoxia in a vertebrate exposed to low DO in the natural environment – potentially specific biomarker of hypoxia exposure

31. Real time PCR field study