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

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

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

Field Study: High rainfall in 2003 resulted in extensive and persistent hypoxia throughout East Bay, Florida LA Study area FL AL SC MS GA 5 km 30 o 20‘ 87 o 00‘ 87 o 10‘ Pensacola Bay Gulf of Mexico Escambia Bay Santa Rosa Sound East Bay N S W E 30 o 30‘ H4 N2 N1 H3 H2 H1 TR

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

Gamete Production -Females Gonadal Function Plasma E 2, T ER conc, ERmRNA Oocyte Production 1.Gonadosomatic index (GSI) 2.Fecundity and developmental stage of oocytes Plasma gonadotropin Plasma E 2 Liver Function (vitellogenesis) ER conc, ERmRNA steroids Plasma vitellogenin

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

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

100 P12 P13P14 PB5 Trans Bridge PenBay P12P13 P14 PB5 Trans Bridge PenBay Perinucl.Cort. alv.1my yolk2ndy yolkTert. yolkAtretic Oocyte (%) October November Sampling sites Very few mature oocytes developed in croaker from low DO sites T N NLow DO T N N Low DO Field Study:

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

Gamete Production -Males Gonadal Function Plasma E 2, T, 11 KT ER conc, ERmRNA Sperm Production 1.Gonadosomatic index (GSI 2.Number and developmental stage of sperm Plasma Gonadotropin T, 11 KT

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

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

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

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

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

ab c SP SC SP SC d Control2.7 ppm 1.7 ppm Testicular development and sperm production impaired in low DO groups

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

perinuclear atretic Control2.7 ppm1.7 ppm Oocyte (%) Conclusion: Production of mature vitellogenic oocytes (tertiary yolk stage) decreased in low DO groups Intermediate stages tertiary yolk 1 Evidence for reproductive dysfunction in croaker exposed to hypoxia: Laboratory study - 1

ab cd SYS TYS AO TYS PNS Control2.7 ppm 1.7 ppm Ovarian and oocyte development impaired in low DO groups

Gonadal Function Oocyte Production GtH vitellogenin Liver function E2E2 Evidence for reproductive dysfunction in croaker exposed to hypoxia: Laboratory study 2 Evidence for reproductive dysfunction in croaker exposed to hypoxia: Laboratory study - 2 Conclusion: endocrine function impaired in both sexes after hypoxia exposure; Conclusion: endocrine function impaired in both sexes after hypoxia exposure; Is this effect caused by a decrease in GtH secretion?

Gonadotropin response to LHRHa decreased in low DO groups 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

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

Therefore test whether hypoxia causes a decrease in 5-HT and GnRH mRNAlevels in the POAH GnRH 5-HT GtH O2O2 Hypothesis: 5-HT (serotonin) GnRH mRNA Stim. Conclusion: decrease in GtH secretion likely due to decline in GnRH function which in turn is associated with decreased 5-HT activity

5-HTP bypasses TPH and increases substrate availability Neuroendocrine and Reproductive Functions Approach: use serotonin precursor- restore 5-HT levels Question: Does hypoxia cause reproductive impairment by decreasing hypothalamic serotonergic activity?

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 Hypoxia

hypoxia ? ? mRNA Effects of hypoxia on 5-HT, GnRH and LH ● 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?

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

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

Ovarian HIF-2α mRNA expression increased after exposure to low DO in preliminary analysis 18S HIF-2  18S HIF-2  Control2.7 ppm1.7 ppm Lab. experimentField studies P12P13PensaBay 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

Real time PCR field study