Glucocorticoids,thyroid hormones,and iodothyronine deiodinases in embryonic saltwater crocodiles

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.     

Control of the development of the pulmonary surfactant system in the saltwater crocodile,Crocodylus porosus

Pulmonary surfactant is a mixture of lipids and proteins that controls the surface tension of the fluid lining the inner lung. Its composition is conserved among the vertebrates. Here we hypothesize that the in ovo administration of glucocorticoids and thyroid hormones during late incubation will accelerate surfactant development in the saltwater crocodile, Crocodylus porosus. We also hypothesize that the increased maturation of the type II cells in response to hormone pretreatment will result in enhanced responsiveness of the cells to surfactant secretagogues. We sampled embryos at days 60, 68, and 75 of incubation and after hatching. We administered dexamethasone (Dex), 3,5,3'-triiodothyronine (T(3)), or a combination of both hormones (Dex + T(3)), 48 and 24 h before each prehatching time point. Lavage analysis indicated that the maturation of the phospholipids (PL) in the lungs of embryonic crocodiles occurs rapidly. Only T(3) and Dex + T(3) increased total PL in lavage at embryonic day 60, but Dex, T(3), and Dex + T(3) increased PL at day 75. The saturation of the PLs was increased by T(3) and Dex + T(3) at day 68. Swimming exercise did not increase the amount or alter the saturation of the surfactant PLs. Pretreatment of embryos with Dex, T(3), or Dex + T(3) changed the secretion profiles of the isolated type II cells. Dex + T(3) increased the response of the cells to agonists at days 60 and 68. Therefore, glucocorticoids and thyroid hormones regulate surfactant maturation in the crocodile.     

Embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in chickens: effects of dose and embryonic stage on hatchability and growth

Chicken embryos (Gallus domesticus) were injected with 0, 8, 20 or 50 ng tetrachlorodibenzo-p-dioxin (TCDD) per egg at embryonic day (ED) 4, 8 or 12 to investigate the effects of differential periods of sensitivity to TCDD exposure. At hatch, all chicks were weighed, sexed and examined macroscopically to identify possible malformations. Liver, bursa, heart and spleen masses were recorded from a number of chicks. The remaining chicks were raised until 6 weeks of age and body and organ masses, plasma concentrations of thyroid hormones, triglycerides and glucose were measured. Dose and stage during embryonic development at which injection was performed affected hatchability. Fifty nanogram of TCDD was highly toxic for 4-day-old chicken embryos. TCDD was less toxic for chicken embryos of 8- and especially 12-days old. One-day-old chick and organ weights were not different between TCDD doses at all injection days. However, injection performed at ED4 or ED8 with 20 and 50 ng, respectively, significantly depressed post-hatch body mass gain. Moreover, body mass gain in males was more depressed than in females. The delayed growth in TCDD treated chickens was accompanied by changes in T(3)/T(4) ratio that at some ages were significantly higher compared to control animals. No pronounced changes in plasma triglycerides or glucose concentrations during postnatal life were observed. Absolute and relative organ masses of 6-week-old chickens showed no remarkable changes.     

Effect of the sex-linked dwarf gene on thyrotrophic and somatotrophic axes in the chick embryo

Plasma concentrations of thyroxine (T4), triiodothyronine (T3), reversed triiodothyronine (rT3), and insulin-like growth factors I and II (IGF-I, IGF-II) together with peripheral 5'-monodeiodination activity were measured in both normal and sex-linked dwarf embryos between day 14 of incubation and day 1 posthatch. Plasma T4 levels increased gradually during embryonic development while T3 concentrations remained low until day 20, when a sharp increase was observed. rT3 levels also increased from day 14 and dropped on day 20 when T3 levels started to increase. 5'-monodeiodination activity was high on day 14 of incubation, decreased thereafter, and showed an increase at the time of air sac penetration together with increased T3 levels. At this stage, differences between normal and dwarf embryos were observed; the latter had lower nonsignificant 5'-Monodeiodination activity and lower (P less than 0.01) plasma T3 levels. Plasma IGF-II levels were high during the whole embryonic period studied. Dwarf embryos had lower (P less than 0.05) IGF-II levels at the time of hatching. IGF-I levels were high on days 14 and 16, declined afterwards, and started to increase again around hatching. With the exception of T3 and IGF-II levels, introduction of the dwarf gene did not cause major changes in the hormonal parameters studied. This may explain the identical body weight at hatching.     

Effects of thyroxine on mitochondrial morphology and uptake of 35S in embryonic chicken livers

Ultrastructural analyses of reactions of mitochondria in hepatocytes of chicken embryos to low levels of exogenous thyroxine (T4) reveal that such reactions (overall swelling accompanied by disruption of crest geometry) first take place at about 10 days of incubation, T4 having been administered on the 6th day. Physically altered mitochondria may be seen after 11-12 days of incubation but are no longer evident by 13 days. Correlated with the initial evidence of T4 effects on mitochondria at 10 days of incubation is a spurt in hepatocyte proliferation. The time correlation observed between T4 induced mitochondrial changes in morphology and abrupt increases in rates of cell proliferation, suggests that liver nuclear receptors for thyroxine are unavailable prior to 9-10 days of incubation. Golgi complexes within the hepatocytes appear to be especially active in the production of electron-opaque vesicles from at least the 8th day of incubation to 11-12 days. Uptake of 35S (probably into chondroitin sulphates) was found to be fifteen times greater on the 8th day of incubation than at 15 days. This correlates with the period of heightened activity of the Golgi complex. In livers exposed to T4 on the 6th day, uptake of 35S was higher on the 9th and 10th days of incubation as compared to controls.     

Effects of glucocorticoids on circulating concentrations of thyroxine (T4) and triiodothyronine (T3) and on peripheral monodeiodination in pre- and post-hatching chickens

The administration of either glucocorticoids (dexamethasone or corticosterone) or adrenocorticotropic hormone (ACTH) to chicken embryos was followed by increase in the circulating concentration of triiodothyronine (T3), the T3 to thyroxine (T4) ratio and the activity of liver T4-5' monodeiodinase. No consistent changes in plasma concentrations of T4 or GH were observed. In post-hatching chicks, corticosterone and dexamethasone depressed the circulating concentrations of both T4 and T3. Iopanoc acid, an inhibitor of liver T4-5' monodeiodinase, elevated plasma concentrations of T4 and depressed those of T3 in both chicken embryos and young chicks. It is suggested that glucocorticoids affect circulating concentrations of T4 and T3 both by affecting the activity of the liver T4-5' monodeiodinase and by influencing the hypothalamo-pituitary axis.     

Effect of cyclical cold stress during embryonic development on aspects of physiological responses and HSP70 gene expression of chicks

The objective of the present study was to evaluate the effects of cyclical lower incubation temperature at different embryonic ages on the hatchability, body and organs weights, thyroid hormones, and liver HSP70 gene expression of newly hatched chicks. In a completely randomized design, fertile eggs of a broiler breeder (34 weeks of age) were assigned to three treatment groups with six replicates and 145 eggs per each. The treatment groups were as: control group (C) that eggs were incubated at 37.6 °C during the whole incubation period; incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 12 to 14 (T1); and incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 15 to 17 (T2). No significant difference was found among treatments for hatchability (P>0.05). There were no differences (P>0.05) among treatments for body weight and liver weight, while heart weight of chicks in T1 and T2 groups were significantly higher than the control group (P<0.05). There were no differences (P>0.05) among treatments for the levels of thyroid hormones, however, the levels of both hormones tended to increase in chicks exposed to cold stress (T1 and T2). Chicks in T2 group had higher liver HSP70 gene expression compared with those in T1 and the control group (P<0.05). Cold stress in both incubation periods had no significant effect on the plasma levels of aspartate aminotransferase and alanine aminotransferase. Treatments had no effect on the plasma levels of glucose, cholesterol and triglyceride. The results of this study suggest that cyclical lower incubation temperatures (36 °C) at the embryonic age from day 15–17 could induce the liver HSP70 gene expression, without negative effects on the hatchability and body weight of hatched chicks.     

Metabolic response to cooling temperatures in chicken embryos and hatchlings after cold incubation

We asked to what extent cold exposure during embryonic growth, and the accompanying hypometabolism, may interfere with the normal development of thermogenesis. White Leghorn chicken eggs were incubated in control conditions (38 degrees C) or at 36 or 35 degrees C. Embryos incubated at a lower temperature (34 degrees C) failed to hatch. The cold-incubated embryos had lower oxygen consumption (VO2) and body weight (W) throughout incubation, and hatching was delayed by about, respectively, 1 and 2 days. The W-VO2 relationship of the cold-incubated embryos was as in controls, indicating that cold-induced hypometabolism was at the expense of the growth, not the maintenance, component of VO2. At embryonic day E11, the metabolic response to changes in ambient temperature (T) over the 30-39 degrees C range was typically poikilothermic, with Q10 = 1.8-1.9, and similar among all sets of embryos. Toward the end of incubation (E20), the thermogenic responses of the cold-incubated embryos were significantly lower than in controls. This difference occurred also in the few-hour old hatchlings (H1), even though, at this time, W was similar among groups. Exposure to cold during only the last 3 days of incubation (from E18 to H1), i.e. during the developmental onset of the endothermic mechanisms, did not lower the thermogenic capacity of the hatchlings. In conclusion, sustained cold-induced hypometabolism throughout incubation blunted the rate of embryonic growth and the development of thermogenesis. This latter phenomenon could be an example of epigenetic regulation, i.e. of environmental factors exerting a long-term effect on gene expression.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Changes in sex steroid levels in yolks of the leghorn chicken,Gallus domesticus,during embryonic development

Yolk steroid hormones have been documented to have growth and behavior effects on hatchlings in several avian species. The purpose of these investigations was to determine initial levels of androstenedione (A), dihydrotestosterone (DHT), estradiol (E(2)), and testosterone (T), and document any changes in those hormones during the course of embryonic development in the Leghorn chicken, Gallus domesticus. Eggs were collected, labeled for hen of origin and egg sequence, incubated at 37.8 degrees C, and sacrificed at predetermined times during development. The embryos were staged, the yolk material collected, homogenized and hormones extracted. A, DHT, E(2), and T were separated via column chromatography and hormone levels determined using radioimmunoassays (RIAs). Results indicate a significant decrease in A and T during embryonic development, similar to that reported by our laboratory for the alligator, with A levels being significantly greater initially than levels of all other hormones. Changes in DHT mirrored changes in T levels. Chicken E(2) yolk content dynamics differ from those we have measured in both the turtle and the alligator. After an initial decline, E(2) in the yolks of chicken eggs undergoes a significant increase at the end of development, between embryonic stages 40 and 45 (days 14 and 20 of development). As the increase is much larger than could be accounted for by hormones present in the yolk material, this may represent early embryonic production of steroid hormones by the developing gonads.     

ONTOGENY OF CHICKEN HEMOGLOBIN II. CHROMATOGRAPHIC STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

Some properties of the carbonmonoxyhemoglobin (HbCO) from chicken embryos of ages 5, 10 and 15 days of incubation, from 1-day posthatching and from adult chickens have been investigated by chromatography on carboxymethylcellulose (CM-cellulose) column and by starch gel electrophoresis.
Chromatogram of the hemoglobin (Hb) from 5-day chicken embryos has shown that it consists of at least 6 components. Starch gel electrophoresis of each isolated component from the column in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown later that there are 3–4 embryonic type Hb components in 5-day embryos.
Chromatogram of the hemoglobin from adult chickens has shown that it consists of at least 4 components, but the examination of each isolated component from the column by electrophoresis in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown that there are 4–6 adult type Hb components in adults.
In ontogenic process, embryonic Hb type is detectable in embryos up to 15 days of incubation. Fetal Hb type, which is not detectable in adult chickens, can be first found in 10-day embryos.     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

Immunoglobulin-containing cells in chick embryo urogenital tissues: a new site for early B lineage cells in endothermic vertebrates.

We have employed histological and immunofluorescent staining procedures in order to characterize the distribution of mu + B lineage cells in tissue sections prepared from developing chicken embryo urogenital tissues (UGTs) between 14 and 21 days of incubation. B lineage cells were observed in tissue sections prepared from developing UGTs, especially the mesonephros and its associated tissue, throughout the sample period. The highest densities of mu + B lineage cells were observed in tissue sections prepared from 18 day embryos. The mu + UGT cells were distributed singly and in clusters in subcapsular regions and within the peritubular interstitium of the mesonephros. These observations (1) are consistent with those which suggest nonbursal site(s) for origin of cells in B lineage, (2) may help account for the varying effects of embryonic caudectomy performed between the second and third days of incubation and surgical bursectomy performed close to hatching, (3) may help provide new insights on the effects of sex hormones on B cell development, and (4) suggest fundamental ontogenetic and phylogenetic similarities between developing vertebrate immune systems.     

Gene activation of alcohol dehydrogenase in Japanese quail and chicken-quail hybrid embryos

No preferential activation of the maternally derived alcohol dehydrogenase (ADH) allele was found in any of the chicken male x Japanese quail female hybrids examined. ADH activity in the liver was, in fact, found to exist in two different cathodal zonal regions on starch gel electropherograms; the zone II bands appeared at day 5 of incubation in the quail embryo (day 6 in the hybrid embryo) and the zone I bands appeared in 9-day quail embryos (10-day hybrid embryos). By day 13 of incubation, only the faster-migrating zone I bands could be detected in both quail and hybrid embryos.     

Alcohol dehydrogenase activity in the developing chick embryo

Before day 9 of incubation, chick embryos contain no measurable alcohol dehydrogenase (ADH) activity. Following day 9 of incubation, chick embryo liver ADH activity increases as a linear function of liver mass. A single dose of ethanol given at the start of incubation is cleared only slowly prior to day 9 of incubation but is completely cleared by day 13. Chick embryo liver ADH has two detectable isozymes throughout development. The percentage contribution of each isozyme to total ADH activity does not change significantly during development. The Km apparent of chick liver ADH is significantly increased shortly after hatching relative to the Km apparent of embryonic ADH. Ethanol exposure during incubation has no effect on the development of ADH activity or isozyme distribution.     

Ontogenetic development of L-gulonolactone oxidase activity in several vertebrates

Activity of L-gulonolactone oxidase (EC 1.1.3.8) in livers of fetal Rattus norvegicus and Mus musculus was detectable on the 18th day of gestation, increased rapidly to maxima at 15 and 5 days postpartum for the two species, respectively, and thereafter declined to adult levels. L-Gulonolactone oxidase was not detectable in liver or kidney of fetal guinea pigs at any stage of development. Near-term fetal snowshoe hares had higher activities of liver L-gulonolactone oxidase than observed in a large sample of adults. L-Gulonolactone oxidase was detectable in chicken (Gallus gallus) embryos by the sixth day of incubation, increased rapidly in the kidney with no discontinuity at hatching, reached a maximum at about the 35th day from the beginning of incubation, and then declined to adult levels. Barn swallow (Hirundo rustica) embryos appeared to synthesize little if any L-ascorbic acid; nestlings had considerably higher levels of L-gulonolactone oxidase than adults. Tadpoles of three species of frogs had appreciable levels of L-gulonolactone oxidase activity.     

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

IgG Fc receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.     

Avian hepatic T-3 production by two pathways of 5'-monodeiodination: effects of fasting and patterns during development

Two forms of iodothyronine 5'-monodeiodinase (5'-D) were studied in liver homogenates from adult and developing quail. The influence of fasting in adults and corticosterone treatment in embryonic quail on 5'-D also were examined. Liver homogenates were assayed for 5'-D activity in the presence of abundant substrate (T4) and cofactor (dithiothreitol; DTT). Generation of T3 during a 15 min incubation at 37 degrees C was assessed by an ethanol-based RIA. In adults, both Type I [the fraction of activity inhibited by propylthiouracil (PTU)] and a putative Type II (the PTU-insensitive fraction) were present in liver homogenates. Type II activity typically comprised about 30% of Total activity. Type I activity first appeared on day 15 of the 16.5 day incubation period, increased 20-fold to peak at hatching, then gradually declined to reach adult levels by 21 days of age. Type II activity was present at all developmental stages and was highest during the perinatal period. Corticosterone treatment in vivo on day 13 of development induced increases in both Type I and Type II activities in liver homogenates 24- and 48-h after treatment. This study demonstrates that in avian liver a putative Type II 5'-D activity (generally considered to be lacking in mammalian liver) is present and may be important in the maintenance of minimal concentrations of tissue T3 during fasting. Both types of 5'-D contribute to the developmental pattern of serum T3 concentrations. Type II comprises a large proportion of total activity during late embryonic life; Type I becomes predominant at the beginning of the perinatal period.(ABSTRACT TRUNCATED AT 250 WORDS)