Grantee Research Project Results
Final Report: The Mechanisms and Effects of Endocrine Disruption on Infertility in the Bonnethead Shark on Florida's Gulf Coast
EPA Grant Number: R826128Title: The Mechanisms and Effects of Endocrine Disruption on Infertility in the Bonnethead Shark on Florida's Gulf Coast
Investigators: Manire, Charles A. , Cortes, Enric , Rasmussen, L. E.L. , Gelsleichter, James
Institution: Mote Marine Laboratory , Oregon Graduate Institute of Science & Technology , National Marine Fisheries Service
EPA Project Officer: Aja, Hayley
Project Period: January 1, 1998 through December 31, 2001
Project Amount: $399,653
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Environmental Justice , Endocrine Disruptors , Human Health , Safer Chemicals
Objective:
The objectives of this research project were to: (1) determine the degree to which the infertility observed in the bonnethead shark, Sphyrna tiburo, in the Tampa Bay/Anclote River area of Florida is caused by disruption of the shark's endocrine system; (2) determine the mechanism(s) of such disruption; (3) determine if this infertility is correlated with organochlorine levels in the shark's serum and/or liver; and (4) estimate the effects that the infertility rates, as well as other possible effects of the contaminants, observed in three different geographical areas, may have on the rate of population growth of the species in the different areas.
Summary/Accomplishments (Outputs/Outcomes):
As of the end of the fourth year of this 4-year study, all samples have been collected from all three study sites, and the analysis of the samples has been completed. The RIAs to be performed by Oregon Graduate Institute, Oregon Regional Primate Center, and the University of Florida have been completed, as have the histology, immunocytochemistry, and organochlorine analyses. The development of the RIA for 1 -hydroxycorticosterone was not possible, due to an inability to produce the steroid in purified form. The vertebra analysis (of age and growth) is completed on all samples. In addition, as of March 2002, all data analyses were completed.
Efforts to isolate native vitellogenin of S. tiburo were largely unsuccessful, most likely due to low circulating levels of this protein in elasmobranch serum. However, yolk products (i.e., lipovitellin and phosvitin) have been identified in crude yolk extracts of preovulatory female S. tiburo. Selective precipitation of these extracts using ammonium sulfate yielded two major protein bands at approximately 105 and 120 kDa, which are believed to represent putative lipovitellins. These proteins were cross-reactive with antibody against striped bass vitellogenin. Polyclonal antibodies directed against have been produced commercially and used for western blot analysis of serum from immature and mature male and female S. tiburo.
The sharks collected during this study were from Anclote Key/Tampa Bay and Florida Bay. From Apalachicola Bay, sharks were collected as follows: 34 immature females, 12 mature females, 29 immature males, and 17 mature males. There are a number of mature animals that could not be collected from this area, due to different migratory patterns. Consequently, mature animals were absent from the area during at least three of the eight stages that were to be collected.
We observed that infertility rates were much higher in the Tampa Bay/Anclote River area than in the Florida Bay area. We found 17 infertile ova that were distributed in 28 percent of the potential females captured in the Tampa Bay/Anclote River area and found only two infertile ova (one each in two females) in sharks from Florida Bay. We also observed that sperm viability from mated females was lower in the Tampa Bay/Anclote River population, compared to the Florida Bay population. We further observed that sperm counts from males prior to mating and from females after mating and prior to ovulation from the Tampa Bay/Anclote River population were lower than those of the Florida Bay population.
Analysis of the steroid hormone concentrations of 17 β-estradiol (E2), progesterone (P4), testosterone (T), and dihydrotestosterone (DHT) for animals from the three different areas have yielded some interesting results. When comparing all animals, regardless of stage of pregnancy, there were no significant differences in concentrations of any of the four hormones in either mature females or mature males. However, when immature animals were compared, immature females from Florida Bay (control) had significantly higher E2 and T concentrations (P = 0.019 and P = 0.011, respectively) than immature females from Anclote Key area and from Apalachicola Bay. Immature males from Florida Bay had significantly higher P4 concentrations (P = 0.038) than immature males from Apalachicola Bay, and immature males from Anclote Key had significantly higher DHT concentrations (P = 0.002) than ones from either Florida Bay or Apalachicola Bay. When mature females were compared by stage of pregnancy between Anclote Key and Florida Bay (inadequate sample size for Apalachicola Bay), females from Florida Bay had significantly greater E2 concentrations during pre-ovulation (P = 0.021) and greater P4 concentrations during early pregnancy (P = 0.017) than ones from Anclote Key during the same stages. Analysis of corticosterone in the sharks revealed that mature males and immature females from Apalachicola Bay had significantly higher concentrations than animals from the other two sites. There were no significant differences in corticosterone concentrations in either immature males or mature females. Corticosterone most likely plays a role in reproduction, rather than in stress, in elasmobranchs (Manire, unpublished data). Analysis of 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone yielded no significant differences among the sites.
We examined tissue architecture histologically in reproductive tissues of embryonic, immature, and mature male and female S. tiburo, to detect any abnormalities that may be present and/or associated with data on serum hormone concentrations. In virtually all cases, the primary sex organs-the testes and ovaries-were the focus of these observations. However, in embryonic and mature female S. tiburo, we also inspected structure of the oviducal gland, a specialized component of the reproductive tract that is sensitive to hormone alteration during development and, in mature females, stores spermatozoa between mating and ovulation. There was no evidence of abnormal gonad development or structure in any male or female S. tiburo, and there were no visible differences in these parameters in animals from different sites. Normal development of the oviducal gland appeared to have occurred in all female S. tiburo embryos that were examined. In adult females, oviducal structure also appeared normal and stored sperm were observed in virtually all preparations. To further investigate if stored cells were viable, we used the TUNEL reaction to detect evidence of apoptosis, or programmed cell death, in spermatozoa. Although apoptotic and non-functional sperm were detected, there was no overall difference in the degree of sperm apoptosis in animals from different sites, and some non-apoptotic and functional cells were present.
We examined immunoreactivity of the reproductive steroids testosterone/dihydrotestosterone (T/DHT), 11-ketotestosterone (11-KT), 17 β-estradiol (E2), and progesterone (P4) in all structures described above. We detected immunoreactive 11-KT in testes from mature male S. tiburo in the interstitial cells that border spermatocysts. Immunoreactive T/DHT and E2 were also observed in testes of mature male S. tiburo, yet were confined to mature spermatocysts that contained tightly packed, late-stage spermatozoa. Hormone reactivity was low or absent in all other structures. Although hormone immunoreactivity differed in testes from individual animals, there were no trends associated with the collection site.
Analysis of age and growth yielded some significant differences between the three populations. Sharks from the Florida Bay population are born smaller, grow more slowly, mature at a smaller size, reach a smaller maximum size but the same maximum age as the Tampa Bay sharks. The sharks from the Apalachicola Bay population are born larger, grow more rapidly, mature at a larger size, reach a larger maximum size, but do not reach as high a maximum age as the Tampa Bay sharks. It is not known at this point whether these are latitudinal differences or are caused by something else. A total of 536 sharks were aged, of which ten percent were discarded, due to reader disagreement. Three indices of precision were calculated: (1) the Average Percent Error (APE; 8.6 percent; (2) the Index of Precision (0.09); and (3) the Percent of Readings in Agreement within ± 1 year (88.8 percent), which increased to 97.3 percent within ± 2 years.
There was a significant linear relationship between vertebral radius and total length (mm) for sharks from each area (northwest Florida: r2 = 0.93, p < 0.001, n = 175; Anclote Key: r2 = 0.87, p < 0.001, n = 159; Florida Bay: r2 = 0.85, p < 0.001, n = 141). We used a marginal increment analysis to determine the timing of vertebral band formation. No differences among month of collection were found either in Anclote Key (single factor ANOVA; F = 0.76, p = 0.38, df = 157) or Florida Bay (single factor ANOVA; F = 0.21, p = 0.65, df = 139). There was a statistically significant difference among months in northwest Florida (single factor ANOVA; F = 4.36, p = 0.04, df = 173), but it was most likely due to a small sample size for March (n = 1).
We determined estimates of reproductive parameters using lengths of fertile females, litter size, and lengths of near-term embryos. A significant difference in maternal size was revealed among areas, with the largest females occurring in northwest Florida (single factor ANOVA, F = 57.06, p = 6.0 x 10-13, df = 2). No difference in litter size was found among areas (single factor ANOVA, F = 0.03, p = 0.97, df = 2). However, we noted a significant difference in embryo length among the three areas, with the smallest embryos occurring in Florida Bay (single factor ANOVA, F = 16.56, p = 2.03 x 10-7, df = 2). In addition, a significant linear relationship between maternal length and embryo length was found for northwest Florida and Florida Bay (northwest Florida: r2 = 0.15, p = 5.7 x 10-7, n = 159; Anclote Key: r2 = 0.02, p = 0.43, n = 37; Florida Bay: r2 = 0.58, p = 1.0 x 10-3, n = 15).
Journal Articles on this Report : 8 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 8 publications in selected types | All 8 journal articles |
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Bethea DM, Hale L, Carlson JK, Cortes E, Manire CA, Gelsleichter J. Geographic and ontogenetic variation in the diet and daily ration of the bonnethead shark, Sphyrna tiburo, from the eastern Gulf of Mexico. Marine Biology 2007;152(5):1009-1020 |
R826128 (Final) |
not available |
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Chapman DD, Prodohl PA, Gelsleichter J, Manire CA, Shivji MS. Predominance of genetic monogamy by females in a hammerhead shark, Sphyrna tiburo: implications for shark conservation. Molecular Ecology 2004;13(7):1965-1974 |
R826128 (Final) |
not available |
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Gelsleichter J, Steinetz BG, Manire CA, Ange C. Serum relaxin concentrations and reproduction in male bonnethead sharks, Sphyrna tiburo. General and Comparative Endocrinology 2003;132(1):27-34. |
R826128 (Final) |
not available |
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Gelsleichter J, Manire CA, Szabo NJ, Cortes E, Carlson J, Lombardi-Carlson L. Organochlorine concentrations in bonnethead sharks (Sphyrna tiburo) from four Florida estuaries. Archives of Environmental Contamination and Toxicology 2005;48(4):474-483 |
R826128 (Final) |
not available |
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Manire CA, Rasmussen LE, Gelsleichter J, Hess DL. Maternal serum and yolk hormone concentrations in the placental viviparous bonnethead shark, Sphyrna tiburo. General and Comparative Endocrinology 2004;136(2):241-247 |
R826128 (Final) |
not available |
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Mccomb DM, Gelsleichter J, Manire CA, Brinn R, Brown CL. Comparative thyroid hormone concentration in maternal serum and yolk of the bonnethead shark (Sphyrna tiburo) from two sites along the coast of Florida. General and Comparative Endocrinology 2005;144(2):167-173 |
R826128 (Final) |
not available |
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Nichols S, Gelsleichter J, Manire CA, Cailliet GM. Calcitonin-like immunoreactivity in serum and tissues of the bonnethead shark, Sphyrna tiburo. Journal of Experimental Zoology Part A-Comparative Experimental Biology 2003;298A(2):150-161. |
R826128 (Final) |
not available |
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Song J, Kinney KA. Effect of vapor-phase bioreactor operation on biomass accumulation, distribution, and activity: linking biofilm properties to bioreactor performance. Biotechnology and Bioengineering 2000;68(5):508-516. |
R826128 (Final) R826168 (1999) R826168 (Final) |
Exit |
Supplemental Keywords:
marine environment, estuarine habitat, risk assessment, elasmobranchs, organochlorines, histology, reproduction, hormones, infertility., RFA, Health, Scientific Discipline, Environmental Exposure & Risk, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Limnology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Environmental Chemistry, wildlife, Endocrine Disruptors - Environmental Exposure & Risk, Ecological Effects - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Southeast, Children's Health, Histology, Endocrine Disruptors - Human Health, Ecological Indicators, EPA Region, ecological exposure, ecological effects, risk assessment, region 4, Anclote River, Gulf Coast, Florida Bay, elasmobranchs, estuarine habitat, fish, infertility, bonnethead shark, organochlorine compounds, Tampa Bay, ecological impacts, Apalachicola Bay, reproduction, reproductive processes, reproductive health, Florida Gulf Coast, FloridaProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.