2002 Progress Report: Individual Level Indicators: Reproductive Function in Estuarine Fishes

EPA 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
Current 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, 2001 through November 30, 2002
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Water , Ecosystems , Ecological Indicators/Assessment/Restoration

Objective:

The overall objective of this research project is to evaluate biomarkers of reproductive function in two estuarine fish species, Atlantic croaker and Fundulus grandis, as early warning indicators of fish population hazards due to the degradation of estuarine environments. One focus of the CEER-GOM program is to investigate the effects of hypoxia, which often occurs in estuaries along the Gulf of Mexico. Currently, little is known about the effects of exposure to moderate hypoxia on reproductive and endocrine functions in estuarine fishes.

One objective of Year 1 of the project was to determine whether reproductive function was impaired in croaker chronically exposed to low dissolved oxygen (DO) levels in a controlled laboratory study. A low DO exposure system was designed that maintained DO levels at 2.5 ppm and 3.5 ppm, 35 percent and 50 percent of normoxic levels, respectively, as well as maintaining water quality. Gonadal growth in males, but not in females, was significantly impaired in croaker exposed to 2.5 ppm for 6 weeks. After 10 weeks of exposure, sperm motility and plasma androgen levels were significantly decreased in males exposed to both of the low DO treatments. An initial spawning trial suggested that fertilization success also is impaired after chronic exposure to low DO. Overall, the patterns of reproductive and endocrine disturbances after exposure to low DO appeared to differ from those typically observed in fish exposed to other environmental stressors such as pollutants. These findings suggest that the mechanisms of reproductive interference by low DO may be specific to this stressor.

Another objective for Year 1 was to develop additional indicators for subsequent evaluation in field studies during the remainder of the project. The genes for two key steroidogenic enzymes involved in gonadal development at puberty, aromatase (estrogen synthesis) and 11ß-hydroxylase (androgen synthesis), have been cloned and almost completely sequenced. Other indicators in croaker and F. grandis have undergone further development and initial evaluation in preliminary field studies in Corpus Christi Bay (CCB), Galveston Bay, TX, and Terrebonne Bay, LA. Site differences in several reproductive indicators such as gonadal growth were observed in both species, which were associated with moderate environmental degradation due to low DO or chemical contamination. These initial field trials suggest that some of the reproductive endpoints selected are potentially useful as individual-level indicators of fish population hazards, whereas others appear less promising. In conclusion, no major problems were encountered and all of the major objectives proposed for Year 1 of the project were accomplished.

Progress Summary:

To test the effect of moderate hypoxia on reproduction in Atlantic croaker, a simple, high-volume (500-gallon), moderate hypoxia exposure system was developed to maintain DO levels at 2.5 ppm or 3.5 ppm without the need for removing oxygen by gassing with nitrogen. DO concentrations in the croaker tanks declined to target levels within 1 day upon lowering the rate of aeration. Water quality was maintained by increasing the biological filtration capacity. One-year-old croakers were exposed to 2.5 ppm, 3.5 ppm, or 7.0 ppm (control) DO for 6 and 10 weeks (3 tanks/treatment) during the period of gonadal recrudescence. Gonadal growth was significantly impaired in males exposed to the lowest DO levels for 6 weeks, whereas it was unaltered in females. These preliminary results suggest that gonadal function in male croaker may be more susceptible to low DO than in females at this stage of the reproductive cycle. Interestingly, there was no evidence that the impairment of testicular growth was due to endocrine disturbances. Plasma levels of 11-ketotestosterone (11 KT) (males), testosterone (T) and vitellogenin (VTG), or estradiol-17ß (EZ) (females), were unaltered after low DO exposure for 6 weeks. Testicular growth also was decreased after an additional 4 weeks exposure to low DO concentrations and this was accompanied by significant decreases in plasma androgen levels and in somatic growth.

Gamete quality in males was significantly impaired in both low DO groups. The percent motile sperm in the control, 3.5 ppm, and 2.5 ppm DO groups was 85.5, 69.1, and 71.3, respectively. The finding that in vitro progestin hormonal treatment increased the percent motile sperm in the low DO treatment groups to values similar to controls suggests a possible deficit in the secretion of this hormone in males. Plasma levels of the progestin will be measured in the remaining male samples to explore this possibility. Although this is one of the first reports of impaired reproduction in male fish after exposure to low DO, there is extensive evidence of hypoxia-induced testicular dysfunction in mammals. Hypoxia-inducible factor 1alpha, a key gene indicating hypoxia exposure in mammals, is being considered for possible inclusion as an indicator of low DO exposure in fish testes. One preliminary in vitro experiment suggested that gamete function also was impaired in females exposed to low DO compared to controls. However, this study needs to be repeated because bacterial contamination was observed in some of the treatment wells. Finally, a pilot-spawning study was conducted with the remaining individuals in each treatment group. Fertilization success was 52.7 percent, 25 percent, and 17.6 percent in the control, and 3.5 ppm and 2.5 ppm DO groups, respectively. A full-scale spawning trial is planned to confirm these preliminary results. It is concluded from these studies that low DO can impair a variety of reproductive functions in Atlantic croaker with potential adverse population consequences.

Large 1,100-bp fragments of croaker aromatase and 11ß-hydroxylase mRNA were successfully isolated using degenerate primers in the polymerase chain reaction (PCR). Manual sequencing of these amplicons and National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis confirmed homology to other known forms of aromatase and 11ß-hydroxylase. Five-inch and 3-inch rapid amplification of cDNA ends (RACE) reactions has yielded more than 88 percent of the coding region for croaker aromatase. We currently are optimizing 5-inch RACE reactions to isolate the remaining aromatase sequence (approximately 130-300 bp). As few as 300 bp may remain to be isolated from the 5-inch and 3-inch ends of 11ß-hydroxylase and recent RACE reactions have yielded products of this size. These sequences have been cloned and currently are being characterized. The deduced amino acid sequence of croaker aromatase is 77, 70, 61, and 59 percent identical to medaka, rainbow trout, zebrafish, and channel catfish aromatase, respectively. The croaker 11ß-hydroxylase is 72 percent identical to eel and 78 percent identical to two forms of rainbow trout 11ß-hydroxylase. The sequence of the aromatase amplicon was used to develop a quantitative PCR (qPCR) assay to measure steady-state croaker aromatase mRNA levels. A similar assay to measure 11ß-hydroxylase mRNA levels is being developed. When the entire aromatase and 11ß-hydroxylase coding regions have been obtained, croaker aromatase/pCMV5 and 11ß-hydroxylase/pCMV5 and expression constructs will be created to confirm their aromatase and 11ß-hydroxylase enzymatic activities in cultured green monkey kidney (COS) cells. Isolation of the remaining croaker aromatase and 11ß-hydroxylase sequences and the creation of the pCMV5 expression constructs should be completed by the end of March 2003.

Adult Atlantic croaker (20-25 fish/site) were collected from four relatively clean sites and three moderately degraded (contaminants) in CCB and three degraded sites (contaminants) in upper Galveston Bay along the Houston Ship Channel (HSC) by trawling during the period of gonadal recrudescence in October (severe flooding prevented sampling in September). DO levels were above 5 ppm at all sites at the time of fish collection. Croaker sampled from most sites in CCB on October 10-16, were at a midpoint of gonadal recrudescence. However, gonadal growth was impaired in fish collected from two of the moderately degraded sites. Mean plasma VTG levels in females at each site were positively correlated with mean size of the ovaries, suggesting that it has the potential as a biochemical indicator of ovarian function at this stage of the reproductive cycle. No clear evidence of feminization of males, shown by elevated levels of VTG in male plasma, was found at any of these sites. Sampling of croaker from HSC had to be delayed until October 23, due to medical leave for the Captain. Although the mean fish size was similar to that of fish collected from CCB, the fish collected in HSC were at an earlier stage of gonadal recrudescence. Multiple collections at several stages of gonadal recrudescence would be required to determine whether gonadal growth was impaired in the entire cohort from HSC as a result of the extended period of low salinity in Fall 2002, or whether the lower mean gonadal somatic index (GSI) was the result of sampling stragglers after the mature individuals had migrated down the estuary. Gonadal weights were lowest in individuals collected at the confluence of the HSC and San Jacinto River. VTG levels were slightly elevated in the plasma of males collected from all three degraded sites in comparison to those in males from CCB. Moreover, the sex ratio was markedly skewed to females among the individuals collected from these three sites. A full-scale sampling program, involving multiple collections during the reproductive cycle, will be conducted during subsequent years to follow up on these initial findings.

F. grandis were sampled in July, September, and November from one hypoxic site (5-29 percent DO saturation in early morning), one moderately hypoxic site (50 percent DO at noon), and one normoxic (>90 percent DO at noon) site in Terrebonne Bay, LA. Salinity differed among sites (hypoxic: 10 ppt; moderately hypoxic: ~5 ppt; normoxic: 23 ppt). Sampling efforts focused on F. grandis because only one site had large numbers of Cyprinodon variegatus. In November, all sites were normoxic (>70 percent DO), and a polyaromatic hydrocarbon contaminated site at Port Fourchon, LA, on the eastern side of the Terrebonne/Timbalier Bay system also was sampled. DO was normoxic and F. grandis were abundant. The physiological variables measured in F. grandis were GSI, steroid hormone levels, and VTG. In addition, percent hematocrit was measured as an indicator of exposure to oxygen stress. GSI values showed that the spawning season is ongoing in July, but has ended by September. In July, male and female GSI were considerably higher at the normoxic site than at the moderately hypoxic site. In November, when DO levels were high at all sites, we found that percent hematocrit did not vary between sites. Initially, we planned to assess the steroid function by measuring gonadal steroidogenesis. This approach proved to be impractical due to contamination and sample processing time. Instead, plasma steroid levels (SL) were measured because approximately 20-500 µL of plasma can be obtained from F. grandis >55 mm SL, sufficient for measurement of steroid levels in individual samples. Validation of E2, T, and 11KT enzyme-linked immunosorbent assays (ELISAs) for F. grandis plasma showed that 3 µL of plasma is sufficient for analysis of T in males and females and E2 in females. Approximately 20 µL are required for 11KT analysis in males. Currently, F. grandis VTG is being purified for ELISA development. In July, when GSIs were high, almost no females had ovulated eggs at the times sampled. Ovulation is a discrete event that occurs before dawn on the full moon, so it is unlikely that the early morning sampling scheme and midday and afternoon processing could capture this event. Because of the difficulty in reliably obtaining ovulating females in the field, estimations of female fecundity should be made in laboratory studies. A ranked male secondary sex character index was not developed because males showed little variation in intensity of coloration. Spermatocrit was not measured, but will be measured in all future collections by expressing 5 µL of semen into a microcapillary tube and smearing it on a slide for later sperm counting.

Significant progress has been made with all the Year 1 objectives . Low DO exposure systems have been developed and a preliminary low DO experiment has indicated several reproductive impairments that have the potential as ecological indicators (Objective 1). The two key steroidogenic genes, aromatase and 11ß-hydroxylase, have been cloned and are almost completely sequenced (Objective 2). In addition, field sites and collecting vessels have been located and pilot field collections have been performed. Moreover, initial evaluations of the variability of reproductive parameters and their ease of measurement in the field and their assay in the laboratory have been performed (Objective 3). Therefore, the project is on track and the necessary preliminary studies are completed.

Future Activities:

The finding that in vitro progestin hormonal treatment increased the percent motile sperm in the low DO treatment groups to values similar to controls suggests a possible deficit in the secretion of this hormone in males. Plasma levels of the progestin will be measured in the remaining male samples to explore this possibility.

The sequence of the aromatase amplicon was used to develop a quantitative PCR (qPCR) assay to measure steady-state croaker aromatase mRNA levels. We continue the development of a similar assay to measure 11ß-hydroxylase mRNA levels. When the entire aromatase and 11ß-hydroxylase coding regions have been obtained, croaker aromatase/pCMV5 and 11ß-hydroxylase/pCMV5 and expression constructs will be created to confirm their aromatase and 11ß-hydroxylase enzymatic activities in cultured green monkey kidney (COS) cells. Isolation of the remaining croaker aromatase and 11ß-hydroxylase sequences and the creation of the pCMV5 expression constructs should be completed by the end of March 2003.

A full-scale sampling program, involving multiple collections during the reproductive cycle, will be conducted during subsequent years to follow up on these initial findings. In addition, spermatocrit, which was not measured in Year 1, will be measured in all future collections by expressing 5 mL of semen into a microcapillary tube and smearing it on a slide for later sperm counting.

Journal Articles:

No journal articles submitted with this report: View all 24 publications for this subproject

Supplemental Keywords:

dissolved oxygen, DO, polymerase chain reaction, PCR, Corpus Christi Bay, CCB, rapid amplification of cDNA ends, RACE, population, community, ecosystem, watersheds, estuary, Gulf of Mexico, nutrients, hypoxia, innovative technology, ecoindicators, biomarkers, water quality, remote sensing, geographic information system, GIS, integrated assessment, risk assessment, fisheries, conservation, restoration., 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
  • 2003 Progress Report
  • 2004 Progress Report
  • 2005 Progress Report
  • 2006
  • Final

  • 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