Biomedical subjects

I P Callard

Publications and source records attributed to I P Callard.

At least 19 recordsLinked to original sources

PubMed2006

Laboratory exposure to 17beta-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis.

To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes-estrogen receptor (ER) and vitellogenin (VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17beta-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner-confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1-3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.

Amino Acid Sequence↗

PubMed2005

Reproductive endocrine disruption in a sentinel species (Chrysemys picta) on Cape Cod, Massachusetts.

Freshwater turtles (Chrysemys picta) were collected from two sites on Cape Cod, MA. One site (Moody Pond), adjacent to the Massachusetts Military Reservation (MMR), was considered potentially impacted by toxic agents deriving from contaminant point sources on the MMR. The second (reference) site (Washburn Pond), to the east of the MMR, was considered not impacted by these pollutants and was chosen as a control site. Plasma estradiol 17 beta and vitellogenin were significantly lower in female turtles from Moody Pond. Ovarian follicular analysis indicated a significant decrease in the >16.00-mm follicular cohort in Moody Pond female turtles compared with Washburn Pond animals. Although testicular weight was lower at the Moody Pond site, histology, plasma testosterone, and sperm number were similar to these parameters in Washburn Pond animals. The data suggest that in Moody Pond, the reproductive capacity of turtles may be negatively affected by contaminants from the MMR.

Animals↗

PubMed2001

Phylogenetic distribution of apolipoproteins A-I and E in vertebrates as determined by Western blot analysis.

A putative apolipoprotein E (apoE) has been identified in the HDL and VHDL fractions of the turtle. This observation is of particular interest considering apoE has been reported absent in the domestic hen (Hermier et al., '95; Biochim Biophys Acta: 105-118, 1995) and thus presumed absent in nonmammalian vertebrates altogether. As a result, partial amino acid sequencing of this protein was performed and revealed that one fragment shared 41% sequence identity to human apoE. Western blot analysis using antisera to apoE demonstrated cross-reactivity to a 34-kDa protein (putative apoE) in turtle plasma. Further investigation using anti-apoE antibody in Western blot analysis detected immunoreactive apoE in the plasma of lamprey, spiny dogfish, skate, and alligator, but not in flounder, newt or python; its absence in several species of birds was confirmed. Using anti-apoA-I antibody, apoA-I was detected in all vertebrate groups except a representative teleost (flounder). Apo-A-I antibody cross-reacted weakly with some putative apoE proteins (chicken, spiny dogfish and skate) and the reverse was true for anti-apoE, which cross-reacted with putative apoA-I in birds, reptiles, and elasmobranchs, confirming the molecular similarity and phylogenetic relatedness of these two proteins.

Animals↗

PubMed2001

Cadmium: tissue distribution and binding protein induction in the painted turtle, Chrysemys picta.

The freshwater painted turtle, Chrysemys picta, was used to investigate (a) the distribution of an injected dose of 109Cd in tissues over a period of 192 h (8 days) and (b) the effect of non-isotopic cadmium injection on tissue metal-binding protein levels. Cadmium is cleared from the blood with 9% remaining in the circulation at 192 h. 109Cd is found in all tissues, but is accumulated preferentially in liver, kidney, pancreas, and gastrointestinal tract. The liver is the primary site of Cd accumulation, accounting for 46.4% of the injected dose by 192 h and the highest Cd concentration (cpm/mg tissue). Steroidogenic tissues and the oviduct accumulate significant amounts of 109Cd and the isotope is present in yolk. An increase in tissue metal-binding protein level after non-isotopic CdCl2 injection is consistent with 109Cd distribution, in that metal-binding protein concentration after CdCl2 injection is highest in liver, followed by pancreas and kidney with low, but with significant levels of cadmium-binding protein in gonads and steroid target organs. We conclude that the liver is the major site of storage after a single injection of isotopic cadmium and induction of a metal-binding protein may be an adaptive response to exposure to cadmium.

Animals↗

PubMed2001

Caenorhabditis elegans as an environmental monitor using DNA microarray analysis.

In order to assist in the identification of possible endocrine disrupting chemicals (EDC) in groundwater, we are developing Caenorhabolitis elegans as a high throughput bioassay system in which responses to EDC may be detected by gene expression using DNA microarray analysis. As a first step we examined gene expression patterns and vitellogenin responses of this organism to vertebrate steroids, in liquid culture. Western blotting showed the expected number and size of vitellogenin translation products after estrogen exposure. At 10(-9) M, vitellogenin decreased, but at 10(-7) and 10(-5), vitellogenin was increased. Testosterone (10(-5) M) increased the synthesis of vitellogenin, but progesterone-treated cultures (10(-5) M) had less vitellogenin. Using DNA microarray analysis, we examined the pattern of gene expression after progesterone (10(-5), 10(-7), and 10(-9) M), estrogen (10(-5) M), and testosterone (10(-9) M) exposure, with special attention to the traditional biomarker genes used in environmental studies [vitellogenin, cytochrome P450 (CYP), glutathione s-transferase (GST), metallothionein (MT), and heat shock proteins (HSP)]. GST and P450 genes were affected by estrogen (10(-5) M) and progesterone (10(-5) and 10(-7) M) treatments. For vitellogenin genes, estrogen treatment (10(-5) M) caused overexpression of the vit-2 and vit-6 genes (2.68 and 3.25 times, respectively). After progesterone treatment (10(-7) M), the vit-5 and vit-6 were down-regulated and vit-1 up-regulated (3.59-fold). Concentrations of testosterone and progesterone at 10(-9) M did not influence the expression of the vit, CYP, or GST genes. Although the analysis is incomplete, and low doses and combinations of EDC need to be tested, these preliminary results indicate C. elegans may be a useful laboratory and field model for screening EDC.

Animals↗

PubMed1999

Reproductive endocrinology of female elasmobranchs: lessons from the little skate (Raja erinacea) and spiny dogfish (Squalus acanthias).

Conventional classification of reproductive modes in female elasmobranchs fails to account for the diversity in ovarian dynamics that operate during oviparous and viviparous cycles. Delineating this diversity is crucial for understanding the endocrine regulation of the manifold physiological mechanisms utilized to retain and protect eggs and developing embryos, to fuel embryogenesis, and to manage the intrauterine milieu. Oocyte development and follicular steroidogenesis overlap with egg retention and pregnancy in some species, whereas in others the follicular phase of the cycle is temporally separated from the gravid period. A luteal phase predominates the post-ovulatory period in viviparous species. In oviparous species, the luteal phase overlaps with the follicular cycle. This heterogeneity in ovulatory cycles suggests that the endocrine system evolved a transmutable system for regulating steroidogenesis and the control of the reproductive events. The reproductive biology and endocrinology of the oviparous little skate and lecithotrophic viviparous spiny dogfish are reviewed in order to derive a working hypothesis that explains the complex nature of endocrine patterns observed in species utilizing disparate reproductive modes. An understanding of the adaptations in ovarian dynamics to particular ovulatory cycles is key to developing theories about the evolution of reproductive strategies in female elasmobranchs. J. Exp. Zool. 284:557-574, 1999.

Animals↗

PubMed1998

Characterization of progesterone-binding moieties in the little skate Raja erinacea.

In this study we report evidence of a [3H]progesterone-binding moiety in the liver and oviduct of the little skate Raja erinacea. It is characterized by high affinity, low capacity and DNA-cellulose-binding activity. Furthermore Western blot analysis revealed that monoclonal antibodies against the chicken progesterone receptor (PR) subunits A and B cross-reacted with a 110-kDa band in the liver and a 80-kDa band in the oviduct. When analyzed by DEAE-Sepharose ion-exchange column chromatography, [3H]progesterone-binding molecules resolved into two peaks, one nonadherent and one adherent to the column. The liver adherent peak eluted in a linear gradient at a NaCl concentration of about 0.07 M and resolved on Western blot as a single band of a 110 kDa. The oviduct adherent peak eluted at about 0.14 M NaCl and resolved on Western blot as a single band of 80 kDa. Competition studies showed that the progesterone-binding moiety in the cytosol was specific for progesterone. On the contrary, the nuclear component is not specific for progesterone; it also binds testosterone and estradiol 17 beta in the oviduct, and progesterone, testosterone, dihydrotestosterone, estradiol 17 beta, mibolerone, and R5020 in the liver. The [3H]progesterone-binding activity was monitored in both liver and oviduct of females in different reproductive stages. Females were separated into three groups; laying, nonlaying, and immature. [3H]Progesterone-binding activity levels were higher in the liver of immature than of nonlaying skates, and it was undetectable in laying skates. [3H]Progesterone binding was higher in the oviduct of laying and nonlaying skates than of immature skates. This PR-binding moiety has many characteristics of a true receptor: high affinity, low capacity, binds to DNA, and cross-reacts with antibodies against chicken PR. However, while the cytosolic form of this progesterone-binding component was quite specific for P, nuclear extracted material was nonspecific. If these progesterone-binding components are homologous with the PR A and PR B forms of other vertebrates, as we believe, it is clear that there are species differences that probably relate to phylogenetic level and physiology of the organism.

Animals↗

PubMed1996

Radioligand and immunochemical studies of turtle oviduct progesterone and estrogen receptors: correlations with hormone treatment and oviduct contractility.

Progesterone (PR) and estrogen (ER) receptors were previously identified and characterized in the reproductive tract of the turtle, Chrysemys picta, and changes in PR levels were monitored during the seasonal cycle. To understand the hormonal regulation of PR, intact and ovariectomized animals were treated with estradiol, progesterone, and a combination of estradiol and progesterone, and high affinity PR and ER levels were determined by radioligand binding studies. Ovariectomy significantly decreased ER levels; in contrast, PR levels increased following ovariectomy. In both intact and ovariectomized animals, estradiol alone did not elevate PR levels above control; however, the PR was down-regulated by progesterone. ER levels in ovariectomized animals were not restored by any of the steroid regimens. By Western blot analysis, PR levels appeared to increase following ovariectomy, were unaffected by estradiol, and were somewhat decreased following progesterone treatment in estradiol-primed ovariectomized animals. While not quantitative, these results are supportive of radioligand binding studies. Immunocytochemical studies of oviduct PR followed the same pattern showing increased immunoreactivity following ovariectomy, no change with estradiol, and a decrease following progesterone treatment of estradiol-primed animals. Oviduct contractility was monitored as a physiological index of progesterone action. Estradiol significantly increased the amplitude of the contractions both in vivo and in vitro, whereas progesterone in combination with estradiol significantly inhibited the estrogen effect. This study suggests that estradiol alone may not be adequate for regulation of both ER and PR. While progesterone down-regulates its own receptor, it does not appear to influence the ER. These data are in contrast to mammalian and avian studies which show that estradiol increases both the ER and PR in the reproductive tract, and progesterone down-regulates both receptors.

Ammonium Sulfate↗

PubMed1995

Distribution and characterization of apolipoproteins in Chrysemys picta plasma.

Very low density lipoproteins (VLDL) (d < 1.006), low density lipoproteins (LDL) (1.006 < d < 1.063), and high density lipoproteins (HDL) (1.063 < d < 1.21) were isolated by ultracentrifugation from the plasma of female and male Chrysemys picta. The protein components of lipoproteins (apolipoproteins) were analysed by electrophoresis in denaturing polyacrylamide gradient gel. Gel filtration chromatography, electroelution from gradient gel and two dimensional electrophoresis were utilized to partially purify and characterized the main apolipoproteins. A 350 kDa Apo B-like protein was found associated with VLDL and LDL fractions. In HDL, Apo AI was the main protein component which, after purification on Sephadex G-200, showed the same electrophoretic mobility as human Apo AI (28 kDa). When analysed by two dimensional electrophoresis, Apo AI gave a pI value of 5.2. A 36 kDa apolipoprotein was purified from VLDL fraction. The purified putative Apo E showed an electrophoretic mobility slightly lower than human Apo E (34 kDa). The only sex difference observed was a band in the female bottom fraction (d > 1.21) with similar electrophoretic mobility to vitellogenin.

Animals↗

PubMed1995

Reptilian (Chrysemys picta) hepatic progesterone receptors: relationship to plasma steroids and the vitellogenic cycle.

In non-mammals, estrogen-induced yolk precursors produced by the adult female liver are the main nutritional source for development. Evidence exists that progesterone exerts counter-regulatory effects on estrogen-induced vitellogenesis, and we have used the turtle model (Chrysemys picta) to study changes in hepatic progesterone receptor during the vitellogenic cycle. Using radioligand methods, we show that high and lower affinity binding sites are present in the cytosolic but not nuclear extracts. The lower affinity site is detectable at all times of the year; the high affinity site is mainly observed during non-vitellogenic periods and does not correlate with plasma estrogen. DNA-cellulose chromatography shows that PR-A is present in spring, summer, and winter, and that PR-B is down-regulated except in animals which recently laid eggs. Western blots confirm the presence of PR in all months, but PR-A (88 kDa) is the dominant isoform. PR-B (125 kDa) is well correlated with the luteal phase, winter and fall. Immunocytochemical studies show that PR is nuclear in location, and nuclear heat shock protein 90 (hsp90) is present. Competitive binding studies of both sites reveal that progesterone is the most effective ligand for both, followed by pregnenolone, deoxycorticosterone, and R5020. RU 486 does not bind to the high affinity site but binds moderately well to the lower affinity site. This study suggests that progesterone receptor isoforms are differentially expressed and may be involved as transcriptional regulators of hepatic function outside the periods of active vitellogenesis in the turtle.

Animals↗

PubMed1995

Myometrium of the spiny dogfish Squalus acanthias: peptide and steroid regulation.

The phylogenetic age of endocrine control of viviparous reproduction in vertebrates may be estimated by examination of elasmobranch models. We have shown in pregnant Squalus acanthias that Squalus relaxin (sRLX) significantly decreased the frequency of myometrial contractions in a dose-dependent reversible manner in vitro and in vivo, without altering the intensity or duration of contractions. In contrast, neurointermediate lobe extract provoked a marked and reversible enhancement of the duration and intensity of contractions but was ineffective in altering the frequency of contractions. In steroid-primed animals, untreated and estradiol-17 beta (E2)-treated animals exhibited a decrease in the frequency of activity after injection of sRLX in vivo while pretreatment with progesterone (P4) alone or in combination with E2 fully suppressed the effects of sRLX. These results suggest that homologous sRLX slows the frequency of spontaneous uterine contractions in third-trimester sharks (stage C) in which endogenous P4 is reduced and E2 levels are rising. These data demonstrate the physiological importance of these hormones and the antiquity of reproductive tract control mechanisms.

Animals↗

PubMed1992

Regulation of ovarian steroidogenesis in vitro in the viviparous shark, Squalus acanthias.

We investigated the steroid biosynthetic capabilities of ovarian granulosa and thecal elements of the viviparous dogfish, Squalus acanthias. In this report we present evidence that granulosa cells secrete quantitatively important amounts of progesterone (P), testosterone (T), and estradiol-17 beta (E), while theca has a more limited capacity to synthesize T and E. Ovarian granulosa cells were obtained from animals at each stage of gestation. After collagenase dispersion, an aliquot of 250,000 cells was incubated at 18 degrees C in basal medium, containing Eagle's salts, glutamine, penicillin, streptomycin and adjusted with 136 mM sodium chloride and 350 mM urea. After a 4 hour incubation, the content of P, T, and E in medium was determined by radioimmunoassay. P was not detectable at any time, while E was present throughout the cycle, being maximal when gestation is three quarters complete (Stage C). T gradually increased from Stage B toward late pregnancy. In Stage C granulosa cells, E production increased in the presence of graded doses of T substrate. Also, a homologous pituitary extract (1/25 equivalents) and the calcium ionophore A23187 stimulated production of all 3 steroids. Using radioisotopes, granulosa cells showed a wide range of synthetic capacities. In Stage C thecal tissue, E production also increased in the presence of graded doses of T substrate, while pituitary extract only increased T. When granulosa and theca were recombined, in the presence of pituitary extract, P levels decreased with a corresponding increase in T, when compared to granulosa alone. These data suggest a possible interaction between granulosa and theca for steroid biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Identification of vitellogenin in the little skate (Raja erinacea).

1. Vitellogenin was isolated from mature female skates by selective precipitation with MgCl2/EDTA followed by chromatography on DEAE-cellulose columns. 2. A single monomer of approximately 205 kDa was identified on 6.0% SDS-PAGE gels. 3. In addition, isolation of yolk proteins with ammonium sulfate yielded proteins of 94 and 38 kDa (putative phosvitins) and putative lipovitellins of ca 105, 91 and 67 kDa. 4. In vivo phosphate incorporation in female and male skates implanted with estradiol indicated that vitellogenin was phosphorylated. 5. Total protein phosphate incorporation was significantly higher in females than male skates. 6. In male skates treated with estradiol, phosphate incorporation increased from 2 days after implantation to a maximum at approximately 11 days after implantation. 7. Determination of the rate of disappearance of 32P-labeled protein suggests a half-life of ca 200 hr in normal female skate plasma.

Animals↗

PubMed1992

Putative apolipoprotein B-100 in the freshwater turtle Chrysemys picta: effects of estrogen and progesterone.

1. The isolation and purification of a putative apolipoprotein B-100 in the plasma of the freshwater turtle Chrysemys picta is described. 2. The protein was purified through differential ultracentrifugation and subsequent Sepharose 6B column chromatography. 3. The molecular weight of the protein determined by electrophoresis was approximately 350 kDa. 4. An antibody to chicken apolipoprotein B-100 specifically recognizes this 350 kDa protein in Western blots, suggesting its identity with apolipoprotein B-100. 5. An antibody to the putative Chrysemys apolipoprotein B-100-like protein was developed and used in an ELISA to quantitate protein levels in plasma. 6. Acute estrogen treatment increased levels of apolipoprotein B-100 (7.64 +/- 0.79 mg/ml plasma) over that of control animals (5.07 +/- 1.74 mg/ml plasma). 7. In contrast, chronic estrogen treatment reduced apolipoprotein B-100 significantly to 2.94 +/- 0.53 mg/ml plasma (P < 0.05).

Animals↗

PubMed1991

Characterization of a specific estrogen receptor in the oviduct of the little skate, Raja erinacea.

In this study we report the first estrogen receptor to be characterized in an oviparous elasmobranch. The skate receptor has high affinity for estradiol (Kd = 0.7 nM), binds both estradiol and the synthetic estrogen DES, and exists in low quantities (50-100 fmol/g oviduct). The receptor displays rapid binding kinetics with half-times of 5 min at 22 degrees and 77 min at 4 degrees. DEAE-Sepharose chromatography reveals one receptor moiety which elutes between 0.13 and 0.14 M KCl. By sucrose gradient ultracentrifugation sedimentation coefficients of 3.6 S under high-salt (0.5 M KCl) and 6.0 S under low salt (0.01 M KCl) conditions were obtained. Using Sephadex G200 gel filtration chromatography, a Stokes radius (Rs) of 2.8 nm and an estimated molecular weight of 43 kDa were calculated. DNA-cellulose elution profiles reveal that the receptor elutes as one peak between 0.34 and 0.36 M NaCl (as compared to 0.20-0.22 M NaCl in mammals and birds and 0.55 M for dogfish). Although some differences are noted between the elasmobranch ER and those of other vertebrates (e.g., dissociation kinetics, DNA affinity), in general it can be said that the skate ER is a "classical" ER in most respects. It is suggested that this steroid receptor has played a key role in the reproductive tract functions of nutrient provision, embryo protection, and as a conduit to the external environment since the earliest chordate era, approximately 400 million years ago.

Animals↗

PubMed1991

Vitellogenin--homologs of mammalian apolipoproteins?

1. To determine if a relationship exists between vertebrate vitellogenins and mammalian plasma proteins the EMBL and NBRF computer databases were searched with two partial amino acid sequences from Xenopus laevis and Gallus gallus vitellogenin. 2. A significant relationship was found between vitellogenin and human apolipoprotein B-100 genes, and confirmed using homology-determination programs. 3. Further analysis shows that unique multiple proline consensus regions found in apolipoprotein B-100 are significantly similar to proline dominant regions in vitellogenin. 4. This work suggests that these proteins are functionally and structurally related and should be categorized as a functional group of hepatic lipid transport and metabolism proteins.

Amino Acid Sequence↗

PubMed1991

The role of steroids in reproduction in female elasmobranchs and reptiles.

Adequate evidence exists to suggest the importance of temporal changes in steroid hormone ratios in the normal reproductive/vitellogenin cycle in oviparous and viviparous elasmobranchs and reptiles. In oviparous species, where the cycle is relatively short, secretion of gonadal hormones is synchronous; thus inhibitory actions of progesterone (P) on hepatic or reproductive tract functions would be offset by stimulatory actions of estradiol (E), resulting in appropriate vitellogenin secretion and reproductive tract development. In viviparous species, temporal asynchrony of E and P secretion occurs, and the actions of the individual hormones can be more easily dissected out. Thus, during gestation, where P is the dominant hormone, antagonistic or stimulatory actions of E may be prevented, and the inhibitory action of P on vitellogenesis dominant. Hence vitellogenesis is limited to the follicular phase and eggs are retained. Although the elasmobranch and reptilian species discussed here do not form a continuum through phylogenesis, but rather are extant forms of a particular line of evolution, it is possible to extrapolate from these observations to the probable endocrine interactions in a species as viviparity evolves from oviparity. The theoretical intermediate stage would involve; (a) egg retention, (b) extension of the luteal phase and increased P secretion and (c) resulting in E/P asynchrony and potential expression of "independent" P action, egg retention and yolk suppression.

Animals↗

PubMed1990

Vitellogenesis in reptiles as a model for mammalian sex-differentiated hepatic protein synthesis.

The stimulation of yolk protein synthesis by estrogen is a characteristic of female non-mammalian vertebrates; in mammals, or their reptilian ancestors, however, vitellogenesis has been suppressed as a corollary of the evolution of viviparity. It is our hypothesis that progesterone has a dual role in this phylogenetic trend: a) to inhibit myometrial contraction and thus set the stage for internal development of embryos and associated placentation and b) to inhibit yolk protein synthesis in a coordinate manner as placentation became an efficient direct supply of nutrients to the fetus. Despite the absence of vitellogenesis per se in eutherian mammals, significant sex-differentiated hepatic protein-lipid synthetic functions remain, which are under complex hormonal control. We have presented evidence that in the reptiles, the central vertebrate group from which the ancestors of modern mammals evolved, the control of yolk protein synthesis is also complex, involving both pituitary hormones (GH, PRL, and LH) and ovarian steroids (estradiol, testosterone, and progesterone). Adequate evidence exists to suggest that mammalian hepatic lipoprotein synthesis and its regulatory elements are phylogenetically derived from their reptilian ancestors and may be better understood in this context. This is of particular relevance to cardiovascular disease in which there is a clear sex bias yet for which no coordinated research program exists which takes into account the relevant phylogenetic history. We believe that reptilian, and possibly avian, models could be used to great advantage to probe the endocrine components of cardiovascular disease.

Animals↗