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Biomedical subjects

L J Guillette

Publications and source records attributed to L J Guillette.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1997

The reproductive cycle of the female American alligator (Alligator mississippiensis).

We examined ovarian and oviducal gross morphology and collected blood samples from wild female alligators in central Florida during most of the year. Females with vitellogenic follicles were observed throughout the year, although ovaries containing follicles larger than 15 mm were seen only during the spring and early summer (March-June). We detected a poor relationship between female body size and the number of vitellogenic follicles whereas body size was significantly correlated with clutch size. Plasma samples were analyzed for (1) estradiol-17beta (E2), testosterone (T), and corticosterone by specific radioimmunoassays; (2) vitellogenin by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and (3) total protein, phosphorus, and calcium by spectrophotometric assays. Reproductively active females showed elevated plasma concentrations of E2 during the fall (September-November) and spring (March-May) whereas non-reproductively active females exhibited basal levels with no apparent peaks. Vitellogenin was detected in the plasma during the same months that plasma E2 concentrations were elevated. Elevated plasma vitellogenin and E2 were not correlated with plasma total protein but were correlated with plasma calcium concentration. During late vitellogenesis, plasma T concentrations were elevated in reproductively active females coincident with a period of intense courtship and mating. Corticosterone plasma concentrations exhibit no significant monthly variation, nor apparent changes during various stages of reproductive activity although plasma concentrations were elevated during late gravidity. Our data suggest that female reproductive activity begins in the fall with an increase in plasma E2 concentration in September or October and vitellogenesis in October. Ovarian activity slows during winter and reactivates with the onset of spring.

Alligators and Crocodiles↗
PubMed1997

Reproductive toxins and alligator abnormalities at Lake Apopka, Florida.

The alligator population at Lake Apopka in central Florida declined dramatically between 1980 and 1987. Endocrine-disrupting chemicals and specifically DDT metabolites have been implicated in the alligators' reproductive failure. The DDT metabolite hypothesis is based largely on the observation of elevated concentrations of p,p-DDE and p,p-DDD in alligator eggs obtained from Lake Apopka in 1984 and 1985. In the following commentary, we draw attention to two nematocides that are established reproductive toxins in humans, dibromochloropropane (DBCP) and ethylene dibromide (EDB), which could also have played a role in the reproductive failure observed in alligators from Lake Apopka in the early 1980s.

Alligators and Crocodiles↗
PubMed1997

Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants.

Many environmental contaminants alter the reproduction of animals by altering the development and function of the endocrine system. The ability of environmental contaminants to alter the endocrine system of alligators was studied both in a descriptive study in which juvenile alligators from a historically contaminated lake were compared to animals from a control lake and in an experimental study in which hatchling control alligators were exposed in ovo to several endocrine-disrupting standards and two modern-use herbicides. Endocrine status was assessed by examining plasma hormone concentrations, gonadal-adrenal mesonephros (GAM) aromatase activity, and gonadal histopathology. In the descriptive study, juvenile alligators from the contaminated lake had significantly lower plasma testosterone concentrations (29.2 pg/ml compared to 51.3 pg/ml), whereas plasma 17 beta-estradiol concentrations did not vary when compared to controls. GAM aromatase activity was significantly decreased n the alligators from the contaminated lake (7.6 pmol/g/hr compared to 11.4 pmol/g/hr). In the experimental study, the endocrine-disrupting standards had the expected effects. 17 beta-Estradiol and tamoxifen caused sex reversal from male to female, with a corresponding increase in aromatase activity. Vinclozolin had no apparent effect on male or female alligators. Among the herbicides tested, atrazine induced GAM aromatase activity in male hatchling alligators that was neither characteristic of males nor females, although testicular differentiation was not altered. Exposure to 2,4-dichlorophenoxyacetic acid had no effect on the endocrine parameters that were measured. Together, these studies show that exposure to some environmental chemicals (such as atrazine) can alter steroidogenesis in alligators, but the endocrine alterations previously noted for Lake Apopka, Florida, alligators can not be fully explained by this mechanism.

Alligators and Crocodiles↗
PubMed1997

In vitro synergistic interaction of alligator and human estrogen receptors with combinations of environmental chemicals.

The effect of mixtures of environmental chemicals with hormonal activity has not been well studied. To investigate this phenomenon, the estrogen receptor (ER) from the American alligator (aER) or human (hER) was incubated with [3H]17beta-estradiol in the presence of selected environmental chemicals individually or in combination. The environmental chemicals included the insecticide chlordane, which has no estrogenic activity, and the pesticides dieldrin and toxaphene, which have very weak estrogenic activity. Chlordane, dieldrin, and toxaphene individually demonstrated no appreciable displacement of [3H]17beta-estradiol from aER and hER at the concentration tested. A combination of these chemicals inhibited the binding of [3H]17beta-estradiol by 20 to 40%. Alachlor, a chemical recently discovered to have weak estrogenic activity, also displaced [3H]17beta-estradiol more effectively in combination with dieldrin than alone. These results indicate that combinations of some environmental chemicals inhibit [3H]17beta-estradiol binding in a synergistic manner. This suggests that the ER may contain more than one site for binding environmental chemicals. The possibility that the ER binds multiple environmental chemicals adds another level of complexity to the interaction between the environment and the endocrine system.

Acetamides↗
PubMed1996

Synergistic activation of estrogen receptor with combinations of environmental chemicals.

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

Binding, Competitive↗
PubMed1996

Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment.

The development of the male reproductive ducts and external genitalia in vertebrates is dependent on elevated androgen concentrations during embryonic development and the period of postnatal growth. We have observed that a population of juvenile alligators living on Lake Apopka exhibit significantly smaller penis size (24% average decrease) and lower plasma concentrations of testosterone (70% lower concentrations) when compared to animals of similar size on Lake Woodruff. In addition to smaller phalli, no relationship exists between plasma testosterone concentrations and penile size in males from Lake Apopka, whereas a positive relationship exists for males from Lake Woodruff. The alligators on Lake Apopka are known to have elevated concentrations of the antiandrogenic DDT breakdown product p.p'-DDE stored in their fat. We suggest a number of hypotheses that could explain the modification in the phenotype of the juvenile male living in Lake Apopka. These modifications in phenotype include a smaller penis size, lower plasma androgen concentrations, and lack of responsiveness of the penis to the plasma androgens present.

Alligators and Crocodiles↗
PubMed1996

Plasma insulin-like growth factor-I concentration during the reproductive cycle of the American alligator (Alligator mississippiensis).

Insulin-like growth factor I (IGF-I) is involved in the growth, reproduction, and development of vertebrates. In this study, we report plasma IGF-I concentrations throughout the reproductive cycle of the American alligator (Alligator mississippiensis) and consider these concentrations with respect to several reproductive steroid hormones and ions involved in reptilian reproduction. Mean plasma IGF-I concentrations were significantly different among reproductive stages. Maximal plasma concentrations of IGF-I were observed throughout gravidity. Plasma IGF-I concentration was not correlated with plasma concentrations of estradiol-17 beta, calcium, phosphorus, albumin, total protein, or body size, but was correlated with plasma progesterone concentration. We hypothesize that the elevated concentrations of IGF-I in gravid female alligators are due to the synthesis of this hormone for incorporation into eggs as well as residual IGF-I that could function in the stimulation of uterine secretory products.

Alligators and Crocodiles↗
PubMed1996

Plasma corticosterone concentrations associated with acute captivity stress in wild loggerhead sea turtles (Caretta caretta).

Plasma corticosterone concentrations were measured in wild loggerhead sea turtles (Caretta caretta) in response to acute captivity (capture, serial bleeding, and restraint up to 6 hr). In general, concentrations of corticosterone dramatically increased 1 hr after capture, peaked at 3 hr, and decreased by 6 hr. Initial corticosterone concentrations were significantly lower in animals captured by tangle net than in those captured by trawl and were thought to more closely represent baseline levels. Significant effects of season and size class on corticosterone concentrations were found for turtles captured by trawl. Corticosterone concentrations of small turtles captured in summer were higher than those of large turtles captured in the same season and of all turtles captured during winter. In winter, corticosterone concentrations for small turtles were higher than those for large turtles at 3 hr after capture. Large turtles captured during winter experienced the slowest rate of increase in plasma corticosterone and a decline at 3 hr after capture. Although cloacal temperatures were significantly higher in summer samples, corticosterone concentrations of large turtles did not differ between seasons until 1 hr after capture. In addition, several large turtles during summer did not experience an increase in corticosterone concentrations 1 hr after capture. It is possible that the lower corticosterone response of large turtles captured during summer may be associated with reproductive condition.

Animals↗
PubMed1996

Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen.

We have used the yeast estrogen (YES) consisting of the human estrogen receptor and a reporter containing two estrogen response elements linked to the lacZ gene to evaluate the interaction between ovarian, phyto-, and synthetic estrogens with extracellular binding proteins. YES was incubated with charcoal-stripped human serum, human sex hormone-binding globulin, or human alpha-fetoprotein in the presence of concentrations of various estrogens that induced a 100% estrogenic response, as measured by beta-galactosidase activity. The activity of estradiol and coumestrol, a phytoestrogen, was reduced 75% with physiological levels of serum, sex hormone-binding globulin, or alpha-fetoprotein. The beta-galactosidase activity of genistein, another phytoestrogen, also decreased with extracellular proteins but to a lower extent than estradiol. In contrast, the activity of the synthetic estrogens diethylstilbestrol, kepone, and p,'p-DDD was only minimally reduced with extracellular proteins. These results indicate a potential fundamental difference in the interaction of estrogens from diverse sources with extracellular binding proteins. This suggests that the capacity for various estrogens to induce estrogen-associated responses is in part regulated by their affinity for extracellular bindings proteins.

Adult↗
PubMed1996

Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant.

Endocrine disrupting chemicals can potentially alter the reproductive physiology of fishes. To test this hypothesis, serum was collected from common carp (Cyprinus carpio) at five riverine locations in Minnesota. Male fish collected from an effluent channel below the St. Paul metropolitan sewage treatment plant had significantly elevated serum egg protein (vitellogenin) concentrations and significantly decreased serum testosterone concentrations compared to male carp collected from the St. Croix River, classified as a National Wild and Scenic River. Carp collected from the Minnesota River, which receives significant agricultural runoff, also exhibited depressed serum testosterone concentrations, but no serum vitellogenin was apparent. These data suggest that North American rivers are receiving estrogenic chemicals that are biologically active, as has been reported in Great Britain.

Animals↗
PubMed1996

Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator.

Reports of reproductive abnormalities in the American alligator from Lake Apopka, Florida, have been linked to a spill of DDT and other pesticides suspected of having hormonelike activity. To determine whether environmental chemicals had the potential to function as exogenous hormones in the American alligator, we examined the ability of chemicals to bind the estrogen receptor (aER) and progesterone receptor (aPR) in a protein extract prepared from the oviduct of the alligator. In competition binding assays with [3H]17 beta-estradiol, some DDT metabolites showed inhibition of [3H]17 beta-estradiol binding to aER. A combination of DDTs demonstrated an additive decrease in [3H]17 beta-estradiol binding to aER. Modern-use chemicals such as alachlor, trans-nonachlor, endosulfan, and atrazine also competed with [3H]17 beta-estradiol for binding to the aER. To test the effect of chemicals identified in alligator eggs from Lake Apopka on [3H]17 beta-estradiol binding, we mixed these chemicals at concentrations measured in eggs in the competition binding assay. 2,2-bis(4-chlorophenyl)-N-(methoxymethyl)acetamide (p,p'-DDD) and trans-nonachlor, both found in Lake Apopka, interacted with aER, whereas others such as chlordane and toxaphene did not. Surprisingly, combinations of these chemicals decreased [3H]17 beta-estradiol binding in a greater than additive manner. To assess the ability of chemicals to interact with aPR, we performed commpetition binding assays with the synthetic progestin [3H]R5020. Most of the chemicals tested did not reduce [3H]R5020 binding to aPR, whereas endosulfan, alachlor, and kepone inhibited binding. These results provide the first evidence that environmental chemicals bind the aER and aPR from the American alligator, supporting the hypothesis that the reported reproductive abnormalities may be related to the modulation of endocrine-related responses. The findings that combinations of chemicals demonstrated a greater than additive interaction with the aER and some chemicals bind to the aPR in the competition binding assay are novel. This suggests that interactions of these chemicals with the endocrine system are complex.

Alligators and Crocodiles↗
PubMed1996

A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens.

Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol. To test this hypothesis, the yeast estrogen screen (YES) was created by expressing human estrogen receptor (hER) and two estrogen response elements (ERE) linked to the lacZ gene. The beta-galactosidase activity of the YES system was significantly increased after treatment with 17 beta-estradiol or the xenoestrogens diethylstilbestrol (DES), o,p'-DDT, and OP but not with vehicle, antiestrogen ICI 164,384, dexamethasone, or testosterone. To determine whether serum proteins affected the bioavailability of natural estrogens compared to xenoestrogens, albumin, sex hormone binding globulin (SHBG), or charcoal-stripped serum were added to the YES system and beta-galactosidase activity assayed. Albumin and SHBG decreased beta-galactosidase activity in the presence of estradiol to a greater extent than DES, o,p'-DDT, and OP. Human and alligator charcoal-stripped serum were also effective at selectively reducing beta-galactosidase activity in the presence of estradiol compared to xenoestrogens. Human serum was more effective than alligator serum in reducing beta-galactosidase activity in the presence of xenoestrogens, indicating that serum may serve as a biomarker for sensitivity to xenoestrogens. Selective binding of 17 beta-estradiol by proteins in serum indicates that certain xenoestrogens may exert greater estrogenicity than originally predicted. The estrogenic potency of a compound involves its binding affinity, bioavailability in serum, and persistence in the environment. Our data demonstrate the utility of the YES system for identifying and characterizing environmental estrogens.

Albumins↗
PubMed1996

Male reproductive health and environmental xenoestrogens.

Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood. Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.

Animals↗