AN EVALUATION OF OVARIAN STATUS AND VITELLOGENIN FOR AMERICAN ALLIGATORS FROM CONTAMINATED LAKES IN FLORIDA.

Eileen K. Monck¹, Jon J. Wiebe², Maria S. Sepúlveda¹, Janet S. Buckland¹, Denise A. Gross², Christopher J. Borgert^1,3, Timothy S. Gross^1,2

¹University of Florida, College of Veterinary Medicine, Gainesville, FL 32610,
²USGS/Florida Integrated Science Center, Gainesville, FL 32653,
³Applied Pharmacology and Toxicology, Inc., Alachua, FL 32615

Presented at the The Society of Environmental Toxicology and Chemistry (SETAC) meeting in November 2002.

ABSTRACT

Organochlorine pesticides (OCPs) in Florida lakes have been associated with decreased egg hatchability /quality and increased developmental/ embryonic mortality in the American Alligator (Alligator mississippiensis). Although levels in yolk and offspring do not correlate with hatchability/ survivability, OCPs may decrease egg/embryo quality by altering maternal reproductive and ovarian function. To test this hypothesis, ovarian follicles and vitellogenin (Vtg) proteins were compared in 20 female alligators (10 of which were late follicular stage) collected during the peak follicular season from both Lake Griffin, FL and Rockefeller Wildlife Refuge, LA (reference site).  Lake Griffin animals had two distinct follicular populations: 10-20cm and 21-35cm, with approximately 50 mature, pre-ovulatory follicles.  Rockefeller animals had predominantly one population of follicles (16-25cm) with approximately 40 mature, pre-ovulatory follicles.  SDS-PAGE of plasma revealed Vtg protein bands unique to follicular females at ~250, 300 & 350kD, which are similar to published molecular weights for Vtg or Vtg metabolites from other species. Two additional Vtg protein bands, ~150 & 230kD, were also identified and both were more pronounced in Lake Griffin females. Follicular contents had similar protein profiles and showed similar site differences. These data suggest that there may be differences in the post-translational processing of Vtg in animals from Lake Griffin.
 

INTRODUCTION

Florida Lakes (specifically those located in the Ocklawaha River Basin) have been the subject of environmental concern for the past few decades. In the 1980's, the alligator population in Lake Apopka (located at the head of the Ocklawaha River) experienced a noticeable decline which was associated with the presence of organochlorine pesticides (OCPs). OCPs were widely used in the United States from the early 1940s – 1960s to control mosquitoes and agricultural pests but have since been removed from the markets due to effects on wildlife, persistence in the environment, and ability to biomagnify.  Reports of reproductive effects associated with OCPs, such as decreased egg viability / quality and increased embryonic mortality, but the mechanisms are not clearly understood.  Lake Griffin is one of several lakes (located upstream of Lake Apopka in the Ocklawaha River basin) exhibiting elevated OCP concentrations in alligator egg yolks and decreased egg viability, and is therefore a suitable study site to investigate this relationship.

 There are several potential aspects of egg quality which could potentially explain reduced egg viability that has been noted in these alligator populations.  They range from nutritional contents such as vitamins, minerals, fatty acids and proteins to egg shell quality such as thickness and porosity.  Vitellogenin (Vtg), an egg yolk precursor protein is synthesized in the liver of oviparous animals, circulates through the bloodstream and subsequently deposits as metabolites in egg yolk as a nutrient source. Vtg has been identified as a biomarker of OCP exposure in several oviparous species (ie. fish and birds).  Therefore this study was designed to investigate Vtg as a potential biomarker for OCP exposure in adult female alligators as well as being a possible mechanism for decreased egg quality. A concurrent aim of this study was to develop an overall assessment of the pre-ovulatory condition of the reproductive tracts (including the follicular development) of these impacted animals.
 

Ocklawaha River Basin, Florida

Fig. 1: Map of the impacted study area.  Arrow indicates lake sampled.

Fig. 2: Map of the control study area. Arrow indicates Rockefeller State Wildlife Refuge, LA.

OBJECTIVE

Characterize plasma Vtg and reproductive tract characteristics of adult female American Alligators (Alligator mississippiensis) during the pre-ovulatory season from two sites; an OCP contaminated site in Florida (Lake Griffin) and a control site in Louisiana (Rockefeller State Wildlife Refuge).
 

SPECIFIC AIMS

• Reproductive Tract Characterization
• Purpose: Develop a quantitative/qualitative assessment protocol for comparing reproductive tracts of pre-ovulatory female alligators between sites.
• Current study: Impacted site – Lake Griffin, FL. Control site - Rockefeller Refuge. 
• Rationale: Previous studies in our laboratory have shown the reproductive tract in impacted animals (alligators as well as other species) to be quantitatively as well as qualitatively different from those from control sites.
   
•  Molecular analysis of Vitellogenin
• Purpose: Develop a reliable reproductive biomarker in the American Alligator as an indicator of potential effects of various toxic chemicals in the environment.
• Current study: Vitellogenin (Vtg)
• Rationale: Vtg is a protein developed in the female liver which is subsequently processed and deposited in egg yolk as a nutrient source. There is evidence in other species suggesting this protein may be altered in animals from contaminated sites.
   

MATERIALS & METHODS

Animals were captured, euthanized, and necropsied according to IACUC guidelines.

• Captures were coordinated such that animals from each site would be at equivalent points in their reproductive seasons.
• Rockefeller animals were captured March 24-26^th.
• Lake Griffin animals were captured April 5^th.
• These time points were confirmed to be equivalent when eggs were collected and staged later in the season.
• Rockefeller embryos were staged at day 12 on June 29^th.
• Lake Griffin embryos were staged at day 12 on July 1^st.
• Total OCP Analysis: Tissues were weighed, homogenized, and extracted into ethyl acetate.  Sample clean-up includes use of C18 and NH2 SPE (solid phase extraction) cartridges before analysis by GC-MS.

Hormone Analysis was performed by standard RIA method to detect plasma estradiol.

Anatomical evaluations were performed using the following:

• Digital calipers for follicle and oviductal measurements and a centimeter tape measure for body measurements.
• A gram scale for repro-tract weights and a kilogram scale for body weights.
• Weights, measurements and photographic documentation were supervised by the same personnel at each site for consistency.

SDS-PAGE Analysis

• A surfactant buffer (containing protease inhibitors) was applied to samples to liberate and denature proteins. Samples were kept on ice during extraction and snap-frozen for storage to minimize degradation.
• Plasma samples were clarified by spinning at 1000K for 5 minutes (to remove RBC's and WBC's). 10x buffer was added to give a 1x final concentration.
• Follicular contents were homogenized in the surfactant buffer (1x concentration).
• 20ug total protein (quantitated by Bronsted-Lowery technique) was resolved on 5% acrylamide +/or 4-20% gradient denaturing gels.

Statistics

• Statistics were run for mean, SEM, equality of variance, and ANOVA or T-tests using the Minitab statistical package.

RESULTS & DISCUSSION

Total OCP analysis

• Animals from Lake Griffin had a higher OCP burden in fat and follicular tissue than those from Rockefeller during the pre-ovulatory season.
• Egg yolk collected from Lake Griffin in the years 2000 through 2002 also exhibited a higher OCP burden than those collected in Rockefeller.

Hormone analysis

• There was no significant difference between sites for plasma estradiol levels.

Anatomical evaluations

• Lake Griffin animals were significantly larger and had larger reproductive tract weights and measurements (before and after normalizing to anatomical weight).
• Lake Griffin animals had significantly larger follicles overall which represented a larger total number of follicles.  These data are strengthened by comparing to the fecundity data (table 2) from previous years at both sites.
• Lake Griffin animals have larger clutch sizes composed of relatively larger eggs.

SDS-PAGE Analysis

• Plasma analysis was accurate in predicting vitellogenesis.
• Plasma analysis revealed some qualitative site differences in the high MW Vtg and Vtg metabolite bands (150-350 kD),^1-4 but not in the lower MW range of other Vtg metabolites (data not shown).
• Follicular fluid analysis however indicated there were qualitative site differences in the lower MW Vtg metabolites as well as other proteins in that range (35-100 kD)⁵ unique to follicular fluid.
   

CONCLUSIONS

The chosen set of indices proved to be appropriate for a qualitative/quantitative assessment of reproductive status and as an indicator of exposure to this environmental xenobiotic.

• There was an increased estrogenic response in animals from Lake Griffin which may be due to OCP exposure.
• greater number of larger follicles.
• overall repro-tract measurements are greater.
• larger eggs and overall larger clutch sizes.
• Note: This did not include a significantly higher level of circulating estrogen.

SDS-PAGE analysis of plasma has proven to be a useful tool for prediction of vitellogenesis.

Results indicate Vtg and Vtg metabolites may be used as biomarkers of exposure to these environmental xenobiotics in alligators.

• Preliminary analysis of plasma and follicular fluid protein profiles suggests qualitative differences in the post-translational processing of Vtg in animals from Lake Griffin.
   

REFERENCES

1. B. Allner et al.  The Science of the total Environment 1995; 233:21-31.
2. F. Brion et al. J. Chromatog. B 2000; 737:3-12
3. S.A. Heppell et al.  Environmental Health Perspectives 1995; 103, Supp.(7):9-15.
4. M.A. Brown et al. Comp-Biochem-Physiol-B-Biochem-Mol-Biol. 1997; 117(2):159-68
5. N. Hiramatsu, et al. Comp-Biochem-Physiol-A-Mol-&-Integ-Physiol. 2002; 131(2):429-441.
    

ACKNOWLEDGEMENTS

• NIEHS, Superfund Project: "Chlorinated pesticides and developmental mortality in wildlife".
• Partially funded by a grant from The American Chemical Council to Timothy S. Gross and Christopher J. Borgert.
• University of Florida, Department of Physiological Sciences, College of Veterinary Medicine.
• USGS/Florida Integrated Science Center.
• Florida Fish and Wildlife Conservation Commission.
• Ruth Elsey and staff - Rockefeller State Wildlife Refuge, LA.