Novel cDNA sequences of aryl hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments

Reproductive changes have been observed in painted turtles from a site with known contamination located on Cape Cod, MA, USA. We hypothesize that these changes are caused by exposure to endocrine-disrupting compounds and that genes involved in reproduction are affected. The aryl hydrocarbon receptor (AHR) is an orphan receptor that is activated by environmental contaminants. AHR mRNA was measured in turtles exposed to soil collected from a contaminated site. Adult turtles were trapped from the study site (Moody Pond, MP) or a reference site and exposed to laboratory environments containing soil from either site. The red-eared slider was used to assess neonatal exposure to soil and water from the sites. The environmental exposures occurred over a 13-month period. Juveniles showed an age-dependent increase in brain AHR1. Juvenile turtles exposed to the MP environment had elevated gonadal AHR1. Adult turtles exposed to the MP environment showed significantly decreased brain AHR2. The painted turtle AHR is the first complete reptile AHR cDNA sequence. Phylogenetic analysis of the painted turtle AHR showed that it clusters with other AHR2s. Partial AHR1 and partial AHR2 cDNA sequences were cloned from the red-eared slider. MEME analysis identified 18 motifs in the turtle AHRs, showing high conservation between motifs that overlapped functional regions in both AHR isoforms.     

Reproductive deficits in male freshwater turtle Chrysemys picta from Cape Cod, Massachusetts

Contaminated groundwater plumes have formed on the Massachusetts Military Reservation (MMR), a Superfund site on Cape Cod, Massachusetts, as a result of chemical waste disposal. The plumes are of concern to the local people who rely on groundwater as a drinking water source. We used the freshwater turtle as a sentinel species to monitor the reproductive effects of exposure and, by inference, the potential for impact on human health. Our observations of male Chrysemys picta field-trapped from Moody Pond (an impacted site) and Washburn Pond (a reference site) on Cape Cod extended and supported prior observations of reproductive deficits. Morphometric comparison of precloacal length (PCL), which is a sexually dimorphic trait in the turtle, showed that Moody Pond males had a significantly longer PCL than Washburn Pond males. Moody Pond turtles showed reduced testicular weight, which was associated with significantly smaller seminiferous tubule diameter. Epididymal sperm counts were also markedly reduced in Moody Pond animals compared to Washburn Pond animals. Testicular histology and gonial proliferation, as determined by PCNA, were similar in both male populations, while the Moody Pond males had significantly higher germ cell apoptosis than the animals in Washburn Pond. These results suggest that a low-level mixture of xenobiotic contaminants impairs the reproductive functions of turtles exposed to the impacted site but not to the reference site environment.     

Morphogenesis of placental membranes in the viviparous,placentotrophic lizard Chalcides chalcides (Squamata: Scincidae)

In the scincid lizard Chalcides chalcides, females ovulate small ova and supply most of the nutrients for development by placental means. The yolk is enveloped precocially by extraembryonic ectoderm and endoderm during the gastrula stage, establishing a simple bilaminar yolk sac placenta. The shell membrane begins to degenerate at this time, resulting in apposition of extraembryonic and maternal tissues. A true chorioplacenta has developed by the early pharyngula stage, as has a choriovitelline placenta and the first stages of an omphaloplacenta. Although the choriovitelline membrane disappears rapidly, the omphaloplacenta spreads to occupy the entire abembryonic pole. The yolk cleft is not confluent with the exocoelom, and no omphalallantoic placenta develops. By the limb-bud stage, an allantoplacenta has been established, with a mesometrial placentome composed of interdigitating ridges of chorioallantois and uterine mucosa. The discovery of five distinct placental arrangements in this species, three of which are transitory and two of which have not previously been recorded in reptiles, emphasizes the need for accounts that specify ontogenetic stages and the precise identity and composition of squamate placental membranes. Contrary to previous interpretations, the pattern of extraembryonic membrane development in C. chalcides is evolutionarily conservative, despite the presence of a reduced yolk mass and cytological specializations for nutrient transfer. Our observations indicate that substantial placentotrophy can evolve in squamates without major modifications of morphogenetic patterns. J Morphol 232:35–55, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of the testicular binding site for iodinated rat FSH in the turtle, Chrysemys picta

1.To correlate the morphological observations with the known gonadotropic activity of FSH in the turtle testis, studies of the binding of iodinated FSH were conducted. 2. These demonstrated the presence of gonadotropin-binding sites of high affinity (apparent Kd = 10(-10) M) for [125I]rFSH in turtle testicular membrane preparations. 3. Although these sites did not bind iodinated human LH or avian LH, these hormones, as well as PMSG and FSH, were effective competitive inhibitors of the binding of the radioligand. 4. Binding of the radioligand to the testis was influenced by duration of incubation and temperature. 5. Binding activity was lost after incubation with proteolytic enzymes (trypsin, pronase) but not with DNAase, lipase, collagenase and neuraminidase. 6. The binding exhibited target organ specificity (no binding observed in brain, epididymis, lung, muscle and pancreas). 7. In addition, the number of binding sites varied according to the stage spermatogenesis, being highest when the tubules contained spermatocytes and spermatids, intermediate when the tubules consisted to Sertoli cells and spermatogonia and lowest st spermiation.     

Luteinizing hormone secretion from the quail pituitary in vitro

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune function in aged mice. II. B-cell function.

A loss of B-cell function in old mice was demonstrated by measuring the in vitro response of lymphoid cells to the B-cell polyclonal activator, LPS (lipopolysaccharide), and the in vivo response to the thymus-independent antigen, pneumococcal polysaccharide type III (SIII). The reduced mitogenic reactivity of lymphoid cells from old compared with young mice could not be explained by a shift in kinetics of the responding cells. When LPS cultures were carried out in the presence of colchicine, fewer cells from old mice were found to respond to the mitogenic signal. The total number of B cells assessed by labelling with either anti-immunoglobulin serum or antigen-antibody complexes was not decreased in old animals. Taken together, these results are consistent with a qualitative rather than a quantitative loss of B-cell function with age. They did not, however, exclude the possibility of depletion of an LPS-reactive sub-population of B cells. Since the number of LPS-reactive cells could not be determined directly, the antibody response of old mice to SIII was investigated. The decreased level of antibody production by old mice to SIII was not due to a shift in kinetics of the responding cells. Extracellular influences were excluded by showing that the reduced responsiveness of old spleen cells persisted after adoptive transfer into young irradiated recipients. Furthermore, pretreatment of cells from old mice with anti-Thy.1 serum and complement before transfer did not enhance their antibody-forming potential. The loss of B-cell activity with age could not, therefore, be explained in terms of an increase in T-cell-dependent suppressive effects. Support for an intrinsic defect in the B cell itself came from the demonstration of similar numbers of SIII-binding cells in normal spleens from old and young mice. Following immunisation, a shift toward low intensity binding cells was observed in spleens from both old and young mice. This shift was, however, less pronounced in the case of old cells, which is consistent with an age-related decline in transformation potential of antibody-forming-cell precursors. The conclusion was, therefore, reached that the reduction with age in B-cell as well as T-cell function is due to a qualitative rather than a quantitative defect in lymphocytes themselves.     

The Freshwater Mussel (Elliptio complanata) as a Sentinel Species: Vitellogenin and Steroid Receptors

Freshwater mussels, Elliptio complanata were collected froma reference and pollutant-impacted pond on Cape Cod, MA. Glutathione-S-transferase(GST) activity was measured in gill, hepatopancreas and foot.In addition, content of seven heavy metals were measured inwhole bodies. GST activity was significantly elevated in hepatopancreasand foot, as was whole body cadmium level in animals from thecontaminated site suggesting that these animals have been exposedto organic and inorganic contaminants. Sodium dodecyl acrylamidegel electrophoresis (SDS-PAGE) analysis showed putative vitellogeninswith molecular weight 180 and 205 kDa bands only in the ovary.In non-denatured gel electrophoresis ovarian extracts revealedtwo higher molecular weight bands at 550 and 700 kDa, whichwere reproductive stage specific. Western blotting of SDS-PAGEand non-denatured gels using the anti-scallop yolk-protein antibodyconfirmed the presence of cross-reacting bands of the same molecularweights in the ovary but not other tissues. Although severalexperiments involving steroid hormone exposure were done, nosignificant changes in vitellogenin protein levels were observed.However, using an anti-human ERß antibody, ERßpositive bands were observed both in female foot, and the ovary.No cross reactivity with the antibody was observed in hepatopancreas.Additional studies are required to resolve questions of vitellogeninregulation and the role of (xeno)estrogens in bivalve molluscs.