2001 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 , 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, 2000 through December 31, 2001
Project Amount: $399,653
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
Objective:The objectives of this research project are to: (1) determine the degree to which the infertility of the bonnethead shark, Sphyrna S. tiburo, 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 contaminants may have on the rate of population growth of the species in different areas. This study examined bonnethead sharks at three locations off Florida?s Gulf Coast, in the Tampa Bay/Anclote River and Florida Bay areas.
As of the end of the fourth year of this study, all samples that can be collected have been collected from all three study sites, and analysis of the samples is complete. The radioammunoassays (RIAs) performed by Oregon Graduate Institute, Oregon Regional Primate Center, and the University of Florida have been completed, as well as the histology, immunocytochemistry, and organochlorine analyses. 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. As of March 2002, all data analyses have been 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, provided by Dr. Charles Rice of Clemson University. Polyclonal antibodies directed against striped bass vitellegenin have been produced commercially and used for western blot analysis of serum from immature and mature male and female S. tiburo.
During our field collections, 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 distributed in 28 percent of the females captured in the Tampa Bay/Anclote River area. We found only two infertile ova (one each in two females) in sharks from the Florida Bay. We also observed that sperm viability from mated females was lower in the Tampa Bay/Anclote River population as compared to the Florida Bay population. 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 areas 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 the Florida Bay (controls) had significantly higher E2 and T concentrations (P = 0.019 and P = 0.011, respectively) than immature females from the Anclote River area and from the Apalachicola Bay. Immature males from the Florida Bay had significantly higher P4 concentrations (P = 0.038) than immature males from Apalachicola Bay, and immature males from the Anclote River had significantly higher DHT concentrations (P = 0.002) than ones from either the Florida Bay or the Apalachicola Bay. When mature females were compared by stage of pregnancy between the Anclote River and the Florida Bay (inadequate sample size for Apalachicola Bay), females from the 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 the Anclote River during the same stages. Analysis of corticosterone in the sharks revealed that mature males and immature females from the 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). Analysis of 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone yielded no significant differences among the sites.
Tissue architecture was examined 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. In mature females, it 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. In all female S. tiburo embryos that were examined, normal development of the oviducal gland appeared to have occurred. In adult females, oviducal structure also appeared normal, and stored sperm were observed in virtually all preparations. We used the terminal deoxnucleotidyl transfer (TdT)-mediated dUTP digoxigenin nick end labeling (TUNEL) reaction to detect evidence of apoptosis, or programmed cell death, in spermatozoa to investigate if stored cells were viable. 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 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) were 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 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 site of collection.
Analysis of age and growth have yielded 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 reach the same maximum age as the Tampa Bay sharks. 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 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 (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 River: 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 the Anclote River (single factor analysis of the variance (ANOVA); F = 0.76, p = 0.38, df = 157) or the 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 the 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 the 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).