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Biomedical subjects

L J Guillette

Publications and source records attributed to L J Guillette.

At least 19 recordsLinked to original sources
PubMed2001

Altered serum sex steroids and vitellogenin induction in walleye (Stizostedion vitreum) collected near a metropolitan sewage treatment plant.

Several recent in situ studies have reported that domestic and mixed domestic/industrial sewage effluents contain one or more natural or anthropogenic estrogenic substances. Those studies examined caged or feral fish for the presence of the egg yolk precursor protein, vitellogenin (VTG), in the blood of male fish. We have previously reported that male, feral carp (Cyprinus carpio) obtained from the effluent channel of a major sewage treatment plant (STP) exhibited depressed serum testosterone (T) concentrations, as well as detectable levels of VTG. The present study examines male and female walleye (Stizostedion vitreum), a native species with a different life history and feeding habits, collected from the same Mississippi River locations below the St. Paul metropolitan STP. All male and female walleye collected from the effluent channel contained measurable levels of VTG in their blood. Males from that location also exhibited depressed serum T concentrations and elevated serum estradiol-17beta (E2) concentrations compared with males from the Snake River reference site. Males obtained from Mississippi River Navigational Pool #2 (MRP-2), 3-20 miles downstream of the STP also exhibited reduced serum T concentrations, but showed no alterations in E2 concentrations or the presence of VTG in the serum. Females collected at the STP site had greatly elevated serum E2 concentrations, but serum T concentrations were not different from females collected in the Snake River. Our results demonstrate that the St. Paul metropolitan STP continues to release an estrogenic effluent, capable of inducing VTG production and altering normal serum sex steroid concentrations in a commercially valuable, native fish, the walleye. Additional studies will be required to determine whether these observations portend long-term population level effects.

Animals↗
PubMed2001

Masculinization of female mosquitofish in Kraft mill effluent-contaminated Fenholloway River water is associated with androgen receptor agonist activity.

Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft paper mills in Florida display masculinization of the anal fin, an androgen-dependent trait. The current investigation was designed to determine if water contaminated with pulp-mill effluent (PME) from the Fenholloway River in Florida displayed androgenic activity in vitro and to relate this activity to the reproductive status of female mosquitofish taken from this river. We tested water samples for androgenic activity from a reference site upstream of a Kraft pulp and paper mill on the Fenholloway River, from 3 sites downstream from the mill, and from another reference site on the Econfina River, also in Florida, where there is no paper mill. We also examined anal fin ray morphology in mosquitofish from these rivers for evidence of masculinization. Eighty percent of the female mosquitofish from the Fenholloway River were partially masculinized while another 10% were completely masculinized, based upon the numbers of segments in the longest anal fin ray (18.0 +/- 0.4 vs. 28.1 +/- 0.9 [p < 0.001]) in the Econfina River vs. the Fenholloway River, respectively). In a COS whole cell-binding assay, all 3 PME samples displayed affinity for human androgen receptor (hAR) (p < 0.001). In addition, PME induced androgen-dependent gene expression in CV-1 cells (cotransfected with pCMV hAR and MMTV luciferase reporter), which was inhibited by about 50% by coadministration of hydroxyflutamide (1 microM), an AR antagonist. Water samples collected upstream of the Kraft mill or from the Econfina River did not bind hAR or induce luciferase expression. When CV-1 cells were transfected with human glucocorticoid receptor (hGR) rather than hAR, PME failed to significantly induce MMTV-luciferase expression. Further evidence of the androgenicity was observed using a COS cell AR nuclear-translocalization assay. PME bound hAR and induced translocalization of AR into the nucleus. In contrast, AR remained perinuclear when treated with water from the control sites (indicating the absence of an AR ligand). Interestingly, PME also displayed "testosterone-like" immunoreactivity in a testosterone radioimmunoassay, whereas water from the reference sites did not. In summary, water collected downstream of the Kraft mill on the Fenholloway River contains unidentified androgenic substances whose presence is associated with masculinization of female mosquitofish.

Androgens↗
PubMed2001

Alterations in sexually dimorphic biotransformation of testosterone in juvenile American alligators (alligator mississippiensis) from contaminated lakes.

The goal of this study was to determine whether hepatic biotransformation of testosterone is normally sexually dimorphic in juvenile alligators and whether living in a contaminated environment affects hepatic dimorphism. Lake Woodruff served as our reference site. Moonshine Bay, located on the west side of Lake Okeechobee, served as an intermediate site. Lake Apopka, the Belle Glade area located at the south end of Lake Okeechobee, and Water Conservation Area 3A, in the northern Everglades, served as our contaminated sites (all lakes are in Florida). Normal testosterone hydroxylase activity exhibited sexually dimorphic patterns of expression, with reference animals from Lake Woodruff exhibiting a female:male ratio of 1.44. This pattern was perturbed in all of the intermediate and contaminated sites investigated. Normal testosterone oxido-reductase activity exhibited sexually dimorphic expression (Lake Woodruff female:male ratio of 1.45). This pattern was altered in all contaminated sites investigated. UDP-Glucuronosyltransferase activity exhibited no sexually dimorphic pattern in animals collected from our reference site, with Lake Woodruff animals exhibiting a female:male ratio of 1.06. This pattern was perturbed in animals from Water Conservation Area 3A, which exhibited a female:male ratio of 0.65. Sulfotransferase activity demonstrated no sexual dimorphism at any of the sites investigated, although elevated activity was observed in males from the Lake Okeechobee watershed compared to those from Lake Woodruff. These data demonstrate different patterns of hepatic androgen biotransformation in animals living in contaminated environments.

Alligators and Crocodiles↗
PubMed2001

Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants.

Wildlife and human populations are affected by contaminants in natural settings. This problem has been a growing concern over the last decade with the realization that various environmental chemicals can alter the development and functioning of endocrine organs, cells and target tissues. Documented disruptions or alterations in reproductive activity, morphology or physiology in wildlife populations have been correlated with contaminant-induced modifications in endocrine system functioning. Alterations of the endocrine system are complex, and not limited to a particular organ or molecular mechanism. For instance, contaminants have been shown to (1) act as hormone receptor agonists or antagonists, (2) alter hormone production at its endocrine source, (3) alter the release of stimulatory or inhibitory hormones from the pituitary or hypothalamus, (4) alter hepatic enzymatic biotransformation of hormones, and (5) alter the concentration or functioning of serum-binding proteins, altering free hormone concentrations in the serum. This review focuses on two of these alterations, altered hormone synthesis and hepatic biotransformation, as a number of recent studies indicate that these actions are important components of endocrine disruption in developing organisms. The possible role of contaminants in altering sex determination mechanisms is also examined.

Alligators and Crocodiles↗
PubMed2001

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

Animals↗
PubMed2000

Ultrastructure of the uterus in an ovariectomized gecko (Hemidactylus turcicus) after administration of exogenous estradiol.

The uterus of an oviparous gecko, Hemidactylus turcicus, was analysed after ovariectomized females underwent a period of treatment (up to 14 days) with exogenous estradiol. Analysis focused on the uterine mucosa, which is made up of an epithelial layer and an underlying lamina propria containing the shell glands. These tissues are thought to be responsible for secretion of the eggshell components and were thus chosen for analysis using transmission electron microscopy. In ovariectomized females, the epithelial layer was low and cuboidal with minimal/no differentiation or secretory activity. Treatment with exogenous estradiol resulted in a significant increase in cell height associated with gradual differentiation of the epithelium. Development of non-ciliated cells included production of secretory granules (low electron density) at the apical cell surface. The shell glands showed less obvious changes over the course of treatment. Shell glands contained two cell types: dark cells with darkly staining nuclei and organelles, and light cells with very indistinct nuclei and organelles, except for prominent rough endoplasmic reticulum and free ribosomes. This study provides results consistent with published light microscopy studies for other reptiles and additionally provides ultrastructural details of reptilian uterine development not previously available.

Animals↗
PubMed2000

Oogenesis and ovarian histology of the American alligator Alligator mississippiensis.

Although folliculogenesis and oogenesis have been observed in numerous reptiles, these phenomena have not been described in detail in a crocodilian. Oogenesis and histological features of the adult ovary of Alligator mississippiensis are described. Using a complex process, the ovary develops telolecithal oocytes that attain a diameter of 38.8 +/- 2.4 mm. The morphology of yolk platelets shows gradual changes throughout the oogenic process. Initially, yolk platelets are seen surrounded by a vesicle. As vitellogenesis advances, the vesicles contain numerous yolk spheres, with slowly growing platelets. The yolk spheres continue to increase in size and number within the vacuoles. Differences in the animal and vegetal poles are seen based on the morphology and size of the yolk platelets. The ovary of A. mississippiensis shows a well-developed system of lacunae and bundles of smooth muscle around the follicles in all stages of development. Several features seen in the ovary of A. mississippiensis are similar to those observed in birds. In particular, the morphology of the yolk platelets, especially during the middle and late vitellogenic stages, and the presence of a ovarian system of lacunae and smooth muscle. These similarities in the reproductive biology of crocodilians and birds contribute to current studies of the evolution of archosaurian reproduction.

Alligators and Crocodiles↗
PubMed2000

Metals and metalloids in tissues of American alligators in three Florida lakes.

Concentrations of metals and selenium were examined in tissues of American alligators (Alligator mississippiensis) from three lakes in central Florida, in one of which alligators have exhibited reproductive or developmental defects. Our overall objective was to determine whether the levels of metals were sufficiently high to confound the association between chlorinated hydrocarbons, which are elevated in eggs and juvenile plasma, and reproductive impairment. The concentrations of all metals were relatively low compared to those reported for alligators from elsewhere in Florida and the southeastern United States, suggesting that reproductive impairment is not due to metals and that metals pose no health risk to the alligators. We also wanted to determine whether skin, biopsied tail muscle, or tail tip tissue, all easily collected from live alligators, could be used as surrogate measures of internal tissue loads. Concentrations of arsenic, cadmium, chromium, lead, manganese, mercury, and selenium in liver were highly correlated with at least one of the three biopsied tissues. Only tin showed no significant positive correlation. No single tissue gave a high prediction of liver levels for all metals, although skin gave the highest correlation for mercury, and tail muscle gave the best overall correlation for lead and cadmium.

Alligators and Crocodiles↗
PubMed2000

Contaminant-induced endocrine disruption in wildlife.

Environmental contaminants have posed a threat to the health of wildlife since the onset of the industrial age. Over the last four decades, much concern has focused on the lethal, carcinogenic and/or extreme teratogenic manifestations of environmental pollution. During the last decade, evidence suggests that the disruption of normal endocrine signalling by environmental contaminants is a mechanism that must also be examined. Man-made chemicals, known as xenochemicals, released into the environment can act as hormone agonists or antagonists and thereby disrupt hormone synthesis, action and metabolism, that is, act as endocrine-disrupting contaminants. Our recent studies show that reptiles living in contaminated environments exhibit: (1) population declines due to the lethal and reproductive effects of the contaminants on embryos, juveniles or adults; (2) developmental abnormalities of embryos, including subtle effects in the reproductive system of alligators; and (3) abnormalities of the endocrine system. Numerous studies now demonstrate that any environmental pollutant that disrupts the normal steroid milieu of the developing embryo will have significant lifelong consequences on sex determination and on the organization and function of the reproductive and endocrine systems.

Alligators and Crocodiles↗
PubMed2000

Organochlorine pesticides as endocrine disruptors in wildlife.

Many xenobiotic compounds introduced into the environment by human activity have been shown to adversely affect wildlife. The ubiquitous distribution of many contaminants and the documented, nonlethal, multigenerational effects on the reproductive, endocrine, and immune systems have lead to concerns that wildlife worldwide are affected. The reproductive disorders reported to date in wildlife include reduced fertility, reduced hatchability, reduced viability of offspring, impaired hormone secretion or activity and modified reproductive anatomy. The endocrine system exhibits an organizing effect on the developing embryo. Thus, a disruption of the normal hormonal signals can permanently modify the organization and future function of the reproductive system. An understanding of the developmental consequences of endocrine disruption in wildlife can lead to new indicators of exposure to endocrine disrupting contaminants. Thus, wildlife serve as important sentinels of ecosystem health, including human public health. An ecoepidemiological approach examining wildlife reproductive abnormalities combined with traditional toxicological studies identifying causation can provide the best model for predicting ecosystem concerns due to environmental contamination.

Animals↗
PubMed1999

Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida lakes.

Neonatal and juvenile alligators from contaminated Lake Apopka in central Florida exhibit abnormal plasma sex steroid concentrations as well as morphological abnormalities of the gonad and phallus. This study addresses whether similar abnormalities occur in juvenile alligators inhabiting six other lakes in Florida. For analysis, animals were partitioned into two subsets, animals 40-79 cm total length (1-3 years old) and juveniles 80-130 cm total length (3-7 years old). Plasma testosterone (T) concentrations were lower in small males from lakes Apopka, Griffin, and Jessup than from Lake Woodruff National Wildlife Refuge (NWR). Similar differences were observed in the larger juveniles, with males from lakes Jessup, Apopka, and Okeechobee having lower plasma T concentrations than Lake Woodruff males. Plasma estradiol-17beta (E(2)) concentrations were significantly elevated in larger juvenile males from Lake Apopka compared to Lake Woodruff NWR. When compared to small juvenile females from Lake Woodruff NWR, females from lakes Griffin, Apopka, Orange, and Okeechobee had elevated plasma E(2) concentrations. Phallus size was significantly smaller in males from lakes Griffin and Apopka when compared to males from Lake Woodruff NWR. An association existed between body size and phallus size on all lakes except Lake Apopka and between phallus size and plasma T concentration on all lakes except lakes Apopka and Orange. Multiple regression analysis, with body size and plasma T concentration as independent covariables, explained the majority of the variation in phallus size on all lakes. These data suggest that the differences in sex steroids and phallus size observed in alligators from Lake Apopka are not limited to that lake, nor to one with a history of a major pesticide spill. Further work examining the relationship of sex steroids and phallus size with specific biotic and abiotic factors, such as antiandrogenic or estrogenic contaminants, is needed.

Alligators and Crocodiles↗
PubMed1999

Serum concentrations of various environmental contaminants and their relationship to sex steroid concentrations and phallus size in juvenile American alligators.

Recent studies have reported a number of abnormalities in the hatchling and juvenile alligators of Lake Apopka, FL (USA). These abnormalities include modifications of plasma concentrations of sex steroids in males and females as well as abnormalities in gonadal morphology, gonadal enzyme activity, and steroidogenesis. Embryonic exposure to environmental contaminants in the eggs has been hypothesized to be the causal agent for these changes. However, posthatchling exposure can also contribute to changes in reproductive and endocrine functioning. We have detected serum concentrations of 16 of 18 organochlorine pesticides or metabolites (OCs) and 23 of 28 congener-specific polychlorinated biphenyls (PCBs) examined in juvenile alligators from Lake Apopka, Orange Lake, and Lake Woodruff National Wildlife Refuge. Lake Apopka juveniles had significantly elevated serum concentrations of p,p'-DDE, dieldrin, endrin, mirex, oxychlordane, SigmaDDTs, and SigmaPCBs compared to juveniles from the other lakes. Further, we observed no correlations between serum contaminant concentrations and sex steroid concentrations (estradiol-17beta and testosterone). However, serum testosterone was significantly lower in males from Lake Apopka and Orange Lake compared to Lake Woodruff NWR. We did not observe relationships between phallus size or other body parameters and serum contaminant levels. Phallus size was smaller in males from Lake Apopka even after adjustment for body size. We suggest that the observations previously reported for juvenile alligators-and observed again in this study-are apparently not associated with the current serum levels of the environmental contaminants we measured, but could be due to exposures during embryonic development to these or other pollutants. Future studies must determine if a causal relationship exists between the contaminants found in alligator eggs and abnormalities observed in the hatchlings and persisting in juveniles.

Alligators and Crocodiles↗
PubMed1999

A comparison of the reproductive physiology of largemouth bass, Micropterus salmoides, collected from the Escambia and Blackwater Rivers in Florida.

Largemouth bass (LMB), Micropterus salmoides, were taken from the Escambia River (contaminated site) and the Blackwater River (reference site) near Pensacola, Florida. The Escambia River collection occurred downstream of the effluent from two identified point sources of pollution. These point sources included a coal-fired electric power plant and a chemical company. Conversely, the Blackwater River's headwaters and most of its length flow within a state park. Although there is some development on the lower part of the Blackwater River, fish were collected in the more pristine upper regions. Fish were captured by electroshocking and were maintained in aerated coolers. Physical measurements were obtained, blood was taken, and liver and gonads were removed. LMB plasma was assayed for the concentration of 17ss-estradiol (E2) and testosterone using validated radioimmunoassays. The presence of vitellogenin was determined by gel electrophoresis (SDS-PAGE) and Western blotting using a monoclonal antibody validated for largemouth bass vitellogenin. No differences in plasma concentrations of E2 or testosterone were observed in females from the two sites. Similarly, males exhibited no difference in plasma E2. However, plasma testosterone was lower in the males from the contaminated site, as compared to the reference site. Vitellogenic males occurred only at the contaminated site. Additionally, liver mass was proportionately higher in males from the contaminated site, as compared to males from the reference site. These data suggest that reproductive steroid levels may have been altered by increased hepatic enzyme activity, and the presence of vitellogenic males indicates that an exogenous source of estrogen was present in the Escambia River.

Analysis of Variance↗
PubMed1999

Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis.

The objectives of this study were to identify, characterize and examine differences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippiensis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state. After capture, alligators from lakes in north central Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of the tract dissected out and partitioned for explant culture. Analysis of the biosynthetic activity indicated regional variations within the tract, differences among reproductive groups and region by status interactions. When oviductal regions were considered regardless of reproductive status, the greatest incorporation of [3H]Leu into secreted nondialysable macromolecules was by the anterior and posterior infundibulum and oviductal tube compared with the transition zone and the uterus. When status was included, the biosynthetic activity of the anterior and posterior portion of the tract in non-reproductive alligators was not different, whereas that of the posterior region of the reproductive group (vitellogenic, gravid, post-clutch) was significantly lower than that of the anterior region. This finding indicates that regulation of protein synthesis and secretion by the non-reproductive alligator tract is different from that in the tract of the reproductive group. Explant-conditioned media were analysed by one-dimensional and two-dimensional SDS-PAGE and fluorography. Sixteen major proteins in culture media were identified as de novo synthesized, by relative molecular weight, by isoelectric point and by differences in distribution determined for reproductive status and oviductal region. Six proteins were examined by N-terminal amino acid microsequence analysis. On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M(r) 55,000, basic), found in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen. Four proteins (aP4-aP7) were sequenced and shown to be significantly related to immunoglobulin heavy chains from several species. This study demonstrated that a large number of proteins are synthesized de novo and released by the female alligator reproductive tract and that there are biosynthetic activity differences by reproductive status and region. Six proteins have been identified, several of which may be incorporated into alligator egg albumen and some of which appear to be different from proteins found in the egg albumens of other species.

Alligators and Crocodiles↗
PubMed1998

Reptiles as models of contaminant-induced endocrine disruption.

Historically, reptiles have been used as bioindicators of environmental contaminants and, currently, reptiles have the potential to elucidate the mechanisms of a newly described group of environmental contaminants--endocrine disrupters. Reptiles are particularly good models for studying endocrine altering compounds due to the fact that different species of reptiles have varying modes of gender determination (genotypic sex determination or temperature-dependent sex determination) and parity modes (oviparity or viviparity). This review focuses both on laboratory and field studies of contaminant-induced endocrine alterations in reptiles. Laboratory studies of oviparous reptiles with temperature-dependent sex determination reveal that embryonic exposure to natural hormones and many man-made chemicals (including the ubiquitous PCBs and common herbicides) can permanently alter the functioning of the reproductive system. It is hypothesized that similar permanent, organizational changes occur in wild reptiles exposed to endocrine-disrupting contaminants.

Animals↗
PubMed1998

Oviducal structure in four species of gekkonid lizard differing in parity mode and eggshell structure.

Oviducal structure was analysed in vitellogenic females from four species of gekkonid lizard exhibiting variation in parity mode and eggshell structure: Hemidactylus turcicus (oviparous) which produces a hard, calcareous eggshell; Saltuarius wyberba (oviparous) which produces a soft, parchment-like eggshell; and Hoplodactylus maculatus and Hoplodactylus duvaucelii (both viviparous). Oviducts were analysed by light, scanning electron and transmission electron microscopy. The uterus exhibited differences among species that were directly attributable to parity mode. H. turcicus and S. wyberba (oviparous) had numerous uterine shell glands; H. maculatus and H. duvaucelii (viviparous) had very few. The uterus also exhibited differences between the two oviparous species (H. turcicus and S. wyberba) which may be related to the type of eggshell produced. Variations were noted in the staining properties of the uterine glandular and epithelial cells. The structure of the infundibulum, uterine tube, isthmus and vagina also differed among species, but differences could not be directly related to parity mode or eggshell structure. Instead, the differences may be related to how prepared the oviduct is for ovulation in individuals analysed from the different species. This study confirms, in the Gekkonidae, aspects of oviducal structure that have been associated with parity mode in other squamate taxa.

Animals↗
PubMed1997

Identification and characterization of estrogen and progesterone receptors from the oviduct of the American alligator (Alligator mississippiensis).

A protein extract prepared from the egg shell fiber-secreting region of the oviduct of Alligator mississippiensis was assayed for the presence of receptors for estrogen and progesterone. In the presence of [3H]-estradiol-17 beta, the extract contained an estrogen-binding activity which reached equilibrium at 25 degrees C in 1 h. Scatchard analysis demonstrated that a single estrogen-binding activity was present in the extract with a Kd of 0.5 nM for [3H]estradiol-17 beta. A steroid specificity competition assay showed the estrogen binding activity strongly recognized estradiol-17 beta and diethylstilbestrol (DES) and weakly interacted with estrone, estriol, estradiol-17 alpha, and dihydrotestosterone (DHT). The estrogen binding activity did not recognize testosterone, dexamethasone or progesterone. The extract exhibited a DNA-binding activity that recognized an estrogen response element in a gel mobility shift assay. We have also identified a high affinity binding activity in the extract that specifically recognized the synthetic progestin R5020 with a Kd of 0.9 nM. This binding activity recognized 17 alpha-estradiol, dexamethasone, testosterone, and estriol. This activity did not recognize DHT, DES, or estradiol-17 beta. These data suggest the presence of estrogen and progesterone receptors in the oviduct of the alligator.

Alligators and Crocodiles↗