2000 Progress Report: The Mechanisms and Effects of Endocrine Disruption on Infertility in the Bonnethead Shark on Florida's Gulf CoastEPA Grant Number: R826128
Title: 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.
Current Investigators: Manire, Charles A. , Cortes, Enric , Gelsleichter, James , Rasmussen, L. E.L.
Institution: Mote Marine Laboratory , National Marine Fisheries Service , Oregon Graduate Institute of Science & Technology
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1998 through December 31, 2001
Project Period Covered by this Report: January 1, 1999 through December 31, 2000
Project Amount: $399,653
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
The objectives of this study are 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.
As of the end of the third year of this 4-year study, all samples that can be collected have been collected from all three study sites, and a majority of the analysis of the samples is completed. Most of the RIAs to be performed by Oregon Graduate Institute and Oregon Regional Primate Center have been completed and most of the histology and immunocytochemistry have been completed. The organochlorine analysis has been completed except that PCB determinations should be finished within the next 2 months. The RIAs to be run at University of Florida are underway and should be completed in the next 2 months. These were intentionally delayed to await the completion of the development of the RIA for 1 -hydroxycorticosterone, but it now appears that the development of that test may not be possible. The vertebra analysis (of age and growth) is completed on all samples. We fully expect to complete the entire project within the next year.
Efforts to isolate native vitellogenin of S. tiburo have been largely unsuccessful, 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, which was provided by Dr. Charles Rice of Clemson University. Continued efforts to isolate this protein are underway and polyclonal antibodies directed against it will be produced commercially and used for western blot and ELISA analysis of serum from immature and mature male and female S. tiburo.
Of the sharks to be collected during this study, all of the animals from Anclote River/Tampa Bay and Florida Bay were collected. 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. This means that mature animals are absent from the area during at least three of the eight stages to be collected.
The sample analysis phase of this study is well underway. We observed during our field collections 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 decreased in the Tampa Bay/Anclote River population as 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 ?-estradiol (E2), progesterone (P4), testosterone (T), and dihydrotestosterone (DHT) for animals from the three different areas has 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 is likely to play a role in reproduction, rather than in stress, in elasmobranchs (Manire, unpublished data).
Tissue architecture was examined histologically in reproductive tissues of embryonic, immature, and mature male and female Sphyrna 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 nonfunctional sperm were detected, there was no overall difference in the degree of sperm apoptosis in animals from different sites and at least some non-apoptotic and functional cells were present. Immunoreactivity of the reproductive steroids testosterone/dihydrotestosterone (T/DHT), 11-ketotestosterone (11-KT), 17 ?-estradiol (E2), and progesterone (P4) was examined in all structures described above. Immunoreactive 11-KT was detected in testes from mature male S. tiburo in the interstitial cells that border spermatocysts. Immunoreactive T/DHT and E2 also were 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 site of collection.
Analysis of age and growth have 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 10 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). Marginal increment analysis was used 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 probably due to a small sample size for March (n = 1).
Estimates of reproductive parameters were determined 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, a significant difference in embryo length was determined 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).