2005 Progress Report: Individual Level Indicators: Reproductive Function in Estuarine FishesEPA Grant Number: R829458C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R829458
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico
Center Director: Brouwer, Marius
Title: Individual Level Indicators: Reproductive Function in Estuarine Fishes
Investigators: Thomas, Peter , Cheek, Ann , Nunez, Scott , Rose, Kenneth A.
Institution: The University of Texas at Austin , University of Southern Mississippi
EPA Project Officer: Hiscock, Michael
Project Period: December 1, 2001 through November 30, 2005 (Extended to May 20, 2007)
Project Period Covered by this Report: December 1, 2004 through November 30, 2005
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text | Recipients Lists
Research Category: Water , Ecosystems , Ecological Indicators/Assessment/Restoration
Hypoxia is a well-defined physiological stressor and presents a new mechanism for endocrine disruption. Despite numerous studies on the physiological responses of fish to hypoxia, the effects of hypoxia on neuroendocrine mechanisms and reproductive output remain largely un known. The goal for year 4 of this project was to determine whether reproductive neuroendocrine function was impaired in croaker chronically exposed to low dissolved oxygen (DO, 1.7 ppm) level. The results of our recent laboratory experiments provide clear evidence that the central nervous system in croaker is extremely sensitive to hypoxia. Hypothalamic gonadotropin-releasing hormone (GnRH) I mRNA levels showed a marked decrease, indicating a disturbance of neuroendocrine function in hypoxia-exposed fish. Low DO exposure also resulted in a decrease of serotonin (5-HT) concentrations in the hypothalamus, where the 5-HT cell bodies con trolling GnRH neuronal activity are located. This decrease in 5-HT levels was associated with a 40 percent decline in the activity of tryptophan hydroxylase (TPH), suggesting that the decrease in 5-HT function was due to a decline in 5-HT synthesis. We recently cloned two full-length cDNAs for TPH-1 and TPH-2 from croaker hypothalamus. Initial results show that hypoxia exposure decreases the expression of TPH-1 mRNA levels, suggesting that hypoxia acts at the level of TPH gene transcription to down-regulate hypothalamic TPH and 5-HT activity and is associated with disruption of GnRH-gonadotropin (GtH) neuroendocrine function in croaker, leading to an inhibition of reproductive function. Circulating levels of progestin, 20β-S, which regulates gamete maturation, did not change significantly in the exposed fish. However, there were significant decreases in gamete functions as assessed by measurements of sperm membrane progestin receptor (mPR) in western blot analysis and oocyte maturation in an in vitro assay. Interestingly, low DO exposure also resulted in a decrease in in vitro production of 20β-S by intact ovarian follicles, indicating that reproductive endocrine function during the final stages of the ovarian cycle in croaker is extremely susceptible to disruption by hypoxic conditions. For field studies of year 4 of this project, we conducted two field samplings in mid-September and October 2005 to assess the reproductive condition of croaker in the East Bay, Florida. Measurements of DO con tent in the bay at the time of sampling did not show evidence of hypoxia, data reported from summer deployments of logging sondes showed lowered DO over an extended period previously. There was a significant decline in the number of captured fish compared to the fish collected in the previous year. The sampling had to be suspended because a major hurricane destroyed roads and other infrastructure and dramatically affected the sampling sites, making further collections logistically daunting and of limited scientific value.Fundulus grandis
The overall long-term objective of this project is to evaluate biomarkers of reproductive function in Gulf killifish (Fundulus grandis) as early warning indicators of fish population hazards due to degradation of estuarine environments, using low DO as a stressor. The major goals for the fourth year were to compare reproductive biomarker responses between estuaries and between years. Collection sites were characterized as normoxic or mildly, moderately, or severely hypoxic based on mean daily DO minimum, mean daily period with DO ≤ 2 mg/L, and frequency of diel hypoxia based on continuous recordings. Reproductive biomarkers varied in sensitivity to hypoxia: gonadosomatic index (GSI) and the male-specific androgen 11-ketotestosterone were consistently reduced by mild, moderate, or severe hypoxia. Testosterone (T) changes were sex-specific¾female T was reduced by moderate, but not severe hypoxia, while male T was reduced only by severe hypoxia. Female estradiol (E2) was reduced only by severe hypoxia. Female vitellogenin (VTG) appeared to be reduced by mild hypoxia only. This result may be misleading, since VTG has a long half-life in plasma and may be responding to stimuli not measured. These morphometric and endocrine responses are potentially useful as early warning indicators of reproductive failure, an ecologically relevant endpoint, because in the laboratory they correlated with declines in fecundity.
The objective was to compare reproductive biomarker responses between estuaries and between years.
Laboratory Studies. Several laboratory studies were conducted from March 2005 through March 2006:
An experiment (Experiment 1) to determine the effects of 1 and 2 weeks exposure to low DO concentrations on hypothalamic GnRH and 5-HT functions was completed in the middle of November. Six mature female and six mature male Atlantic croaker were put into each of four tanks (total no. fish = 48) with a recir culating seawater system (2 x control and 2 x 1.7 ppm DO). A YSI probe was used to monitor DO, pH, and temperature several times a day.
A second experiment (Experiment 2) to determine the effects of 2 weeks exposure to low DO concentrations on hypothalamic TPH expression and activity , the rate limiting enzyme in 5-HT synthesis, was begun in October and completed in November. Twelve fish (both sexes) were put into each of four tanks (total: 48 fish) with a recirculating seawater system (2 x control and 2 x 1.7 ppm DO). DO, pH, and temperature were monitored as before.
A third experiment (Experiment 3) to determine the effects of 2 and 4 weeks exposure to low DO concentrations on croaker sperm mPR expression, hypoxia-inducible factor-2α (HIF-2α) protein expression, and in vitro oocyte matura tion (germinal vesicle breakdown [GVBD]) was begun in October and completed at the end of December. Twelve fish (both sexes) were put into each of four tanks (total fish: 48) with a recircu lating seawater system (2 x control and 2 x 1.7 ppm DO). DO, pH, and temperature were moni tored as before.
A fourth experiment (Experiment 4) to determine the effects of 4 weeks exposure to low DO concentrations on plasma GtH concentrations and pituitary GnRH receptor levels was begun in October and completed at the end of December. Twelve fish (both sexes) were put into each of four tanks (total: 48 fish) with a recirculating seawater system (2 x control and 2 x 1.7 ppm DO). DO, pH, and temperature were monitored as before.
Results. The results of the laboratory studies provide clear evidence that reproductive function in Atlantic croaker is extremely sensitive to hypoxia, resulting in a dramatic decrease in several indicators of reproductive function. Hypothalamic GnRH I mRNA levels show a marked de crease (Experiment 1, Figure 1A), indicating a disturbance of neuroendocrine function in hypoxia-exposed fish. Low DO exposure also resulted in a decrease of 5-HT concentrations in the hypo thalamus (Experiment 1, Figure 1B), where the 5-HT cell bodies controlling GnRH neuronal activity are located. This decrease in 5-HT levels was associated with a 40 percent decline in the activity of TPH (Experiment 2, Figure 4A), suggesting that the decrease in 5-HT function was due to a decline in 5-HT synthesis. We recently cloned two full-length cDNAs for TPH-1 (2118 bp) and TPH-2 (1932 bp) from croaker hypothalamus (Figure 2). Initial results show that hypoxia exposure decreases the expression of TPH-1 mRNA levels (Experiment 2, Figure 4B), suggesting that hypoxia acts at the level of TPH gene transcription to down-regulate hypothalamic TPH (Experiment 2, Figure 4A) and 5-HT activity and is associated with disruption of GnRH- GtH neuroendocrine function in croaker, leading to an inhibition of reproductive function. A search of the partial sequence of the 5’ untranslated region of croaker TPH-1 indicates the presence of three hypoxia response elements (HRE) in the promoter region (Figure 3). Therefore, these results suggest that the decline of TPH gene expression observed in our study after hypoxia exposure involves activation of HIF-2α (Experiment 3, Figure 5) that interacts with a specific HRE. Circulating levels of progestin, 20β-S, which regulates gamete maturation, did not change significantly in the exposed fish (Experiment 3, Figure 6). However, there were significant decreases in gamete functions as assessed by measurements of sperm mPR in western blot analy sis (Experiment 3, Figure 7) and oocyte maturation in an in vitro assay (Experiment 3, Figure 8A). Interestingly, low DO exposure also resulted in a decrease in in vitro production of 20β-S by in tact ovarian follicles (Experiment 3, Figure 8B), indicating that the final stages of the reproductive cycle in croaker are extremely susceptible to disruption by hypoxic conditions. Pituitary GnRH receptor concentrations or mRNA levels will be assayed soon (Experiment 4). A decrease in pi tuitary GnRH receptor concentrations or mRNA levels is predicted.
Figure 1. Neuroendocrine Indicators, Hypothalamic GnRH mRNA (A) and 5-HT (B), Levels in Atlantic Croaker Chronically Exposed to Hypoxia in Laboratory Studies. N = 7-12. ** p < 0.001 compared to control.
Figure 2. Schematic Representation of Full Length cDNAs of Croaker TPH-1 and TPH-2 and the Proximal Region of Croaker TPH Promoters (In Box)
Figure 3. Croaker TPH-1 Promoter (a) Nucleotide Partial Sequence of the 5’-Flanking Region. Putative cis-regulatory elements (activating protein-1, AP-1), CAAT enhancer binding protein (C/EBP) are underlined. (b) Proximal promoter region contains three HREs.
Figure 4. Hypothalamic TPH Activity (A), and TPH-1 and TPH-2 mRNA (B) Levels in Atlantic Croaker Chronically Exposed for 2 Weeks to Hypoxia in a Laboratory Study. N = 5-8. * p < 0.05 compared to control.
Figure 5. Western Immunoblots of HIF-2α in the Ovary of Atlantic Croaker Chronically Exposed to Hypoxia for 2 and 4 Weeks. C = Control, H = Hypoxia (1.7 ppm).
Figure 6. Effect of Hypoxia on Plasma 20b-S (A), and Hypoxia + LHRHa on Plasma 20b-S Levels of Female Atlantic Croaker Chronically Exposed for 4 Weeks to Hypoxia in a Laboratory Study
Figure 7. Western Immunoblot of mPR in the Sperm of Atlantic Croaker Chronically Exposed to Hypoxia for 4 Weeks
Figure 8. Effects of HCG and Progestin 20b-S on GVBD (A), and In Vitro Production of 20b-S by Intact Follicles of Atlantic Croaker Oocytes in Response to HCG (B). Fish chronically exposed for 4 weeks to hypoxia in a laboratory study. N = 12. * p > 0.05, ** p < 0.01, and ** p > 0.001 compared to control.
Field Studies. Two field samplings were conducted in September and October 2005.
September Sampling. A field study was conducted in mid-September (9/20) 2005 to assess the condition of Atlantic croaker in Pensacola Bay. Although measurements of DO content in the bay at the time of sampling did not show evidence of hypoxia, data reported from summer deployments of logging sondes showed lowered D O over an ex tended period previously.
There was no evidence of hypoxia at 3-Mile Bridge, and just beyond the Garcon Point Bridge (PB5). Sites PB5, Bridge, and Bay were sampled, but none of the approximately 30 fish that were captured showed any evidence of maturity. PB5 (Temp. 30.15EC bottom/29.87EC top, Sal. 21.60/14.96 mg/cm, DO 4.57/5.28 mg/L, pH 7.84/7.96) produced 30 fish in four trawls with an average GSI of 0.6 for the females and 0.5 for the males. The Bridge site (Temp. 29.95/31.36EC, Sal. 28.65/17.74 mg/cm, DO 5.35/7.20 mg/L, pH 8.04/8.06) produced 29 croaker in two trawls with an average GSI of 0.8 for the females and 0.5 for the males. The Bay site (Temp. 30.40/31.50EC, Sal. 32.79/18.67 mg/cm, DO 4.95/6.65 mg/L, pH 7.95/8.13) produced 27 fish in two trawls with an average GSI of 0.8 for the females and 0.1 for the males. It is concluded from the collections that the fish at all these sites were immature. The second day H urricane Rita, centered in the Gulf, made full speed with a course projected to ward Texas. With evacuation notices going up in our hometowns, we called off the expedition and headed home.
October Sampling. A field study was conducted in mid-October (10/9-10/10) 2005 to assess the condition of Atlantic croaker in Pensacola Bay. Although measurement of DO content in the bay did not show evidence of hypoxia, data reported from summer deployments of logging sondes showed lowered D O throughout that period. The first day of sampling was conducted in the East Bay area of Pensacola Bay. Sites P12, P13, and P14 were assessed for D O and croaker. There was no evidence of hypoxia at any of these sites. The few fish that we were able to capture were significantly smaller than the size at maturity, measuring no more than 12 cm and displaying complete lack of gonadal development. Also, only a few shrimp and other organisms were present in the trawls. Three trawls were conducted at P13 (Temp. 24.94 bottom/24.34EC top, Sal. 23.25/23.03 mg/cm, DO 5.80/7.76 mg/L, pH 7.94/8.02), resulting in no fish. Likewise, three trawls at P12 (Temp. 25.15/23.95, Sal. 24.18/19.23, DO 6.00/6.82, pH 7.93/8.00) also produced no fish. The last site in East Bay was P14 (Temp. 27.26EC/24.38EC, Sal. 30.15/21.68 mg/cm, DO 3.36/7.15 mg/L, pH 7.95/8.07), and again produced no fish after two trawls. This sampling in East Bay was cut short by the ship’s winch braking, preventing further sampling in East Bay. Overall, there was a significant decline in the number of captured fish (zero fish/site) compared to the fish collected in 2003, and those that were captured were consistently smaller, suggesting that growth was impaired in this cohort.
Sampling on the second day was conducted in areas relating to lower Escambia Bay, a transitional site between the Bridge, East Bay, and the 3-Mile Bridge. The fish collected from these sites were more mature, although their abundance was low here. The Bridge site (Temp. 26.74EC bottom/25.12EC top, Sal. 32.10/22.66 mg/cm, DO 6.91/7.80 mg/L, pH 8.09/8.10) produced six fish after three trawls, with an average GSI of 4.1 for the females and 2.9 for the one male. No fish were collected during two trawls at the transitional site (Temp. 26.87/24.78EC, Sal. 31.24/22.27 mg/cm, DO 6.18/7.94 mg/L, pH 8.14/8.15). The Escambia Bay site (Temp. 27.38/24.85EC, Sal. 32.83/21.89 mg/cm, DO 4.42/7.02 mg/L, pH 8.08/8.16) produced eight fish in two trawls, with an average GSI of 3.7 for the females and 0.9 for the two males. Thus, although some mature fish were found in these areas, fish abun dance was very low. This is contrast to the 2003 sampling expedition in which the fish were plentiful and of larger size. Here, too, there were very few shrimp and other organisms in the trawls.
It is concluded from the field sampling that the prolonged hypoxia during the summer was the likely cause of the very low abundance of estuarine organisms collected in East Bay in the fall and also an apparent decrease in the growth and gonadal development of Atlantic croaker. However, the occurrence of a major hurricane, Rita, prior to the October sampling period, likely had a major impact both on the environmental conditions and on the movement of the biota in East Bay and the adjoining bays, so it is not possible to draw any firm conclusions from the results obtained at this sampling time.
F. grandis were collected from marsh creeks in Pensacola Bay, F lorida, and Weeks Bay, A labama, during July 2004 and 2005. Fish were captured at the same phase of the semilunar spawning cycle. Twenty males and 20 females were collected from each site (n = 2) in each estuary (n = 2). Condition factor, GSI , sex steroid hormone concentrations, and VTG concentrations were compared between sites within each estuary. Site effects were assessed using a general linear model with forward stepwise regression. Site was a categorical factor and size, lunar day, and time of day sampled were entered as biologically relevant covariates. Period of trap deployment and holding time prior to sampling were entered as potential methodological confounders.
In both years, DO cycles differed between sites within each estuary (Table 1). In Pensacola Bay, the Garcon Point marsh creek site was normoxic to mildly hypoxic during the 2004 collecting period and normoxic during the 2005 collecting period. The Indian Bayou marsh creek had similar daily DO minima to the marsh creek at Garcon Point, but the daily period of DO ≤ 2 mg/L was longer in Indian Bayou (2005 data only, no continuous recordings in 2004). In Weeks Bay, the mouth site was mildly hypoxic during the 2004 collecting period and normoxic during the 2005 period. The marsh creek site was severely hypoxic in 2004 (mean daily DOmin 0.9 ± 0.7 mg/L over 18 days, average duration of daily period ≤ 2 mg/L 3.5 hr) and mildly hypoxic in 2005 (mean daily DOmin 2.6 ± 1.1 mg/L, average duration daily period ≤ 2 mg/L 0.6 ± 0.9 hr).
Table 1. DO Cycle Amplitude, Daily Hypoxic Period Duration, and Hypoxic Episode Frequency in Marsh Sites Where F. grandis Were Collected in July 2004 and 2005. GP, Garcon Point marsh creek; IB, Indian Bayou. Period of sonde deployment includes days with ≥ 70 of a possible 96 recording periods in 24 hours. §Minimum (or maximum) recorded during point measurements taken on 7/30/04 14:00 – 20:00 and 7/31/04 6:00 – 9:30 and 18:30 – 19:30. na – unknown because no continuous recordings were made.
Fish condition factor was equivalent between sites within each estuary, suggesting that F. gran-dis may be able to maintain somatic growth under a wide range of DO conditions. Male 11-ketotestosterone (11KT) and female GSI were significantly lower in F. grandis collected from Indian Bayou and Weeks Bay creek —marsh sites where diel hypoxia was of longer duration and occurred more frequently than at Garcon Point or Weeks Bay mouth (Figure 9). T, the precursor to both E2 and 11KT, was lower in males collected in Weeks Bay creek in July 2004, the site and sampling period with the most severe hypoxia¾the lowest mean daily DOmin, the longest mean daily period with DO ≤ 2.0 mg/L, and the most frequent hypoxia. T was significantly lower in females collected from an Indian Bayou creek with moderate hypoxia (Figure 9). In females, higher frequency, longer hypoxic periods, such as the conditions occurring at Weeks Bay creek in July 2004, had no effect on T but suppressed E2 (Figure 9). Circulating VTG concentration might be expected to change in concert with female GSI, since yolk uptake increases oocyte weight. However, VTG supply and uptake did not seem to be in phase: GSI was significantly reduced even when circulating VTG was unchanged (Figure 9). This result should be considered with caution, since plasma VTG has a long half-life in fish, 14 days in flounder (Platichthyes flesus) and sheepshead minnow (Cypri nodon variegatus), and hence may be responding to stimuli not measured.
Figure 9. Reproductive Biomarkers in F. grandis. WB, Weeks Bay; PB, Pensacola Bay. Based on continuous DO recordings at collection sites (Table 1), WBS (Weeks Bay shoreline = WBM) was normoxic in July 2005 and WBC (Weeks Bay creek) was mildly hypoxic. GP (Garcon Point) was normoxic in July 2005 and IB (Indian Bayou) was moderately hypoxic. WBS in July 2004 was mildly hypoxic, while WBC was severely hypoxic. GP in July 2004 was mildly hypoxic, but no continuous recordings were made at IB. * p < 0.05, ** p < 0.01, *** p < 0.001. GSI, gonadosomatic index; T, testosterone; KT, 11-ketotestosterone; E2, estradiol-17β; VTG, vitellogenin.
Conclusions. All the major objectives of the project have been completed. Reproductive function in F. grandis was shown to be sensitive to hypoxia in the field. Comparing field sites with differing severity of diel hypoxia demonstrated variation in stressor sensitivity of indicators: GSI and 11KT de crease in response to even mild hypoxia. T in females is reduced by moderate hypoxia, but in males is only reduced by severe hypoxia. Female E2 is reduced by severe hypoxia. The re sponse of VTG is equivocal¾in one case (WB 05), it was reduced by mild, but not by moderate or severe hypoxia. In another (PB 04), the degree of hypoxia was unknown, but responses of other indicators suggest moderate hypoxia and VTG was reduced.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other subproject views:||All 24 publications||5 publications in selected types||All 4 journal articles|
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||Martinez ML, Landry C, Boehm R, Manning S, Cheek AO, Rees BB. Effects of long-term hypoxia on enzymes of carbohydrate metabolism in the Gulf killifish, Fundulus grandis. Journal of Experimental Biology 2006;209(19):3851-3861.||
||Thomas P, Rahman MS, Kummer JA, Lawson S. Reproductive endocrine dysfunction in Atlantic croaker exposed to hypoxia. Marine Environmental Research 2006;62(Suppl 1):S249-S252.||
Supplemental Keywords:RFA, Health, Scientific Discipline, Geographic Area, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Ecosystem/Assessment/Indicators, Endocrine Disruptors - Environmental Exposure & Risk, Aquatic Ecosystem, endocrine disruptors, Aquatic Ecosystems, Environmental Monitoring, Ecological Monitoring, Ecology and Ecosystems, Biology, Endocrine Disruptors - Human Health, Gulf of Mexico, Ecological Indicators, monitoring, ecoindicator, ecological exposure, molecular ecology, estuaries, estuarine integrity, ecosystem assessment, biomarkers, fish, endocrine disrupting chemicals, ecological assessment, estuarine ecoindicator, fish reproduction, animal models, environmental indicators, environmental stress, reproductive processes, water quality, nutrient fluxes
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R829458 EAGLES - Consortium for Estuarine Ecoindicator Research for the Gulf of Mexico
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829458C001 Remote Sensing of Water Quality
R829458C002 Microbial Biofilms as Indicators of Estuarine Ecosystem Condition
R829458C003 Individual Level Indicators: Molecular Indicators of Dissolved Oxygen Stress in Crustaceans
R829458C004 Data Management and Analysis
R829458C005 Individual Level Indicators: Reproductive Function in Estuarine Fishes
R829458C006 Collaborative Efforts Between CEER-GOM and U.S. Environmental Protection Agency (EPA)-Gulf Ecology Division (GED)
R829458C007 GIS and Terrestrial Remote Sensing
R829458C008 Macrobenthic Process Indicators of Estuarine Condition for the Northern Gulf of Mexico
R829458C009 Modeling and Integration