Changes in the presence of progesterone receptor isoforms in the oviduct magnum of newly-hatched chicks after gonadotropins treatment

The effects of Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) on progesterone receptor (PR) isoforms presence in different cell populations from the oviduct magnum of newly-hatched chicks treated in vivo on days 13, 15 and 17 of embryonic development, were analyzed by immunohistochemistry. We found that FSH promoted cytodifferentiation of the magnum's mucosa and increased PR immunoreactivity in all cell types of the oviduct magnum, whereas LH-treatment did not exert cytodifferentiation of magnum's mucosa, and PR immunoreactivity was only induced in some epithelial and stromal cells of the oviduct magnum. In all treatments the number of PR immunopositive cells incubated with the antibody PgR Ab-8 that recognizes both PR isoforms were significantly higher than the number of immunopositive cells incubated with antibody PgR Ab-6 that only recognizes PR-B. This suggests that PR-A should be the predominant isoform in the oviduct magnum of newly-hatched chicks treated with gonadotropins during embryonic development.We conclude that gonadotropins differentially regulate PR-A isoform presence in the oviduct magnum of newly-hatched chicks.     

Ontogenic variations in the content and distribution of progesterone receptor isoforms in the reproductive tract and brain of chicks

Progesterone participates in the regulation of several functions in chicks such as ovulation, gonadal differentiation, and sexual and nesting behaviors. Many progesterone actions are mediated by specific intracellular receptors (PR) which are ligand-induced transactivators. Two PR isoforms that are functionally distinct in their ability to activate genes and regulate distinct physiological processes have been described in chicks: a full length form PR-B and the N-terminally truncated one PR-A which lacks the amino-terminal 128 amino acids of PR-B. PR isoforms have been detected in several tissues of both the adult and the embryo chick such as brain, ovary and oviduct. PR isoforms expression ratio varies among progesterone target tissues and under different hormonal and environmental conditions such as those presented during avian sexual maturity and the seasons of the year. These data let us to conclude that progesterone actions in brain, ovary, and oviduct highly depend on PR isoforms expression pattern and regulation.     

Sexual dimorphism in the content of progesterone and estrogen receptors, and their cofactors in the lung of adult rats

Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.     

Sexual maturation-associated and estrogen-induced progesterone receptor expression in the bursa of Fabricius

The expression of the progesterone receptor (PR) was studied in the chicken bursa of Fabricius (BF) in both sexes from the time of hatching until the bursal involution. Steroid binding studies, immunohistochemistry, and autoradiography were used to characterize and localize the receptor. Three different polyclonal antibodies (IgG-RB, IgG-G3, and IgG-RB2) directed against the chick oviduct progesterone receptor were used for the studies. With immunohistochemistry, no receptor-positive cells were detected in the bursae of young chicks. The first receptor-positive cells were occasionally seen at the age of 10 wk in the frozen sections, not in the paraffin sections. In older female chicks, the staining became more abundant. In males, the PR was expressed only after estradiol treatment. The staining was located in the nuclei of the subepithelial and the interfollicular cells, which were probably mesenchymal in origin. The bursal epithelium and the lymphocytes were not stained. By using a combined technique of autoradiography and immunohistochemistry, we were able to demonstrate that the same cells also concentrated tritiated ORG 2058 (a specific synthetic progestin) in their nuclei. In steroid binding studies with tritiated ORG 2058, the receptor concentration after the age of 10 wk was 50 to 120 fmol/mg protein. Low-level ORG 2058 binding was also detected in young chicks of both sexes before the age of 10 wk. The progestin-binding molecule resembled the progesterone receptor of the chick oviduct in molecular size (studied with HPLC) and binding properties. The PR expression in the BF was preceded by the expression of PR in the oviduct stromal cells and by an increase in oviduct epithelial proliferation, indicating the BF is affected by factors associated with sexual maturation. It is concluded that the subepithelial and the interfollicular stromal cells in the BF, but not the epithelial or follicular cells, are estradiol-sensitive in both sexes immediately after hatching. The endogenous estrogens, however, are not able to induce PR until after the onset of sexual maturation, and only in females. This implies that estrogen and progesterone may affect the structural organization of the BF through the stromal cells, but probably not before the onset of puberty.     

Regulation of progesterone receptor isoforms content in human astrocytoma cell lines

Progesterone regulates several functions through the interaction with its intracellular receptor (PR) which expresses two isoforms with different functions and regulation: PR-A and PR-B. Both PR isoforms have been detected in human astrocytomas, the most common and aggressive primary brain tumours, but their regulation and function are unknown. We studied the effects of estradiol, progesterone and their receptor antagonists (ICI 182,780 and RU 486) on PR isoforms content in U373 and D54 human astrocytoma cell lines, respectively derived from grades III and IV astrocytomas, by Western blot analysis. In U373 cells we also evaluated the effects of PR-A overexpression on cell growth. We observed that in U373 cells estradiol increased the content of both PR isoforms whereas in D54 cells it had no effects. Estradiol effects were blocked by ICI 182,780. In both cell lines, PR isoforms content was down-regulated by progesterone after estradiol treatment. This effect was blocked by RU 486. We observed that overexpression of PR-A significantly diminished the increase in U373 cells number produced after progesterone treatment. Our results suggest a differential PR isoforms regulation depending on the evolution grade of human astrocytoma cells, and an inhibitory role of PR-A on progesterone effects on astrocytomas cell growth.     

Progesterone receptor isoforms in ovary of newly-hatched chick after gonadotropin treatment during embryonic development

We evaluated by immunohistochemistry the expression of progesterone receptor (PR) isoforms in different cell subpopulations of the ovary of newly-hatched chicks after a treatment with Follicle-stimulating hormone (FSH) or Luteinizing hormone (LH) administered on days 13, 15 and 17 of embryonic development. Two monoclonal antibodies that recognize either both PR isoforms or only PR-B, were used. The results indicate that FSH increased both the total number of cells and the number of PR-immunoreactive ones in all cell subpopulations of the ovary. In all cases, PR-B was the isoform regulated by FSH. In contrast, LH did not modify the number of total cells in any cell subpopulations of the ovary. Besides, LH decreased the number of PR-B immunoreactive interstitial cells, without modifying PR expression in any other cell subpopulations of the ovary. These results reveal differential effects of FSH and LH on PR-expression in cell subpopulations of the ovary of newly hatched chicks treated during embryonic development. We conclude that gonadotropins regulate PR-B isoform in the prefollicular ovary of the chick.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks

1. The behaviour of total acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of chick embryos between 11th and 21st day of development and of 8 and 180-day-old chicks is described. 2. Total acid soluble carnitine fluctuates around the same levels in the brain, liver and muscle until 18th day of development, whereas it attains a peak on that day in the heart. At hatching compared to 18th day, it suddenly increases three times in the muscle, drops not significantly in the heart and brain, but sharply in the liver (-40%). However the levels are always higher than those of the grown chick in the brain but lower in the other tissues. 3. Free carnitine levels are almost constant in all tissues during the embryonic life; if compared to adult ones, they are very much lower in the liver, heart and muscle, but higher in the brain, even in 8 day-old chick. 4. Short chain esterified, carnitine reaches a maximum on 18th day of egg incubation in the liver, brain and heart; in the muscle it stays on constant levels until this day and then rapidly increases so that at hatching it doubles the values. 5. The short chain esterified to free carnitine percentage ratio peaks in all tissues on 18th day of development, attaining figures which are well above those determined in the grown chick.     

Parental care of the Common Tern Sterna hirundo

Parental care activities of male and female Common Terns Sterna hirundo were recorded over two breeding seasons. Males and females exhibited distinct parental roles throughout a breeding bout. Courtship feeding by males was extensive prior to and during egg-laying, but declined with the onset of incubation. Females performed significantly more incubation behaviour than males although both sexes spent equal time attending at the nest site. During the chick stage, females spent significantly more time on the territory than did males. Chick feeding was largely the responsibility of the male; males fed chicks at a rate approximately three times higher than that of females. In addition, whereas females showed no trend in the size of fish delivered to chicks relative to chick age, the size of fish delivered by males increased with chick age. Courtship feeding activities and extensive chick feeding contributions by male Common Terns appear to outweigh parental contributions by females, contrary to predictions for a monogamous species.     

Notes on the behavioural ecology of the Magnificent Frigatebird Fregata magnificens

The Magnificent Frigatebird population breeding on Man-o-War Cay, Belize in 1980 laid their single eggs between mid-December and early April, and had a mean hatching date of mid- to late April. There were no differences between the sexes in the percent of time spent incubating eggs or brooding small chicks (males 48%, females 52%). However, males were not observed to feed larger (75–100 d) chicks. All 81 feedings of juvenile (approximately 1 year old) frigatebirds observed during 126 hr of observations were by females, and the majority of these feedings occurred at midday (46%) when approximately equal numbers of females and juveniles frequented the study area. Our data confirm Diamond's (1972, 1973) reports of unequal roles in chick rearing and post-fledging care between male and female F. magnificens , and support the hypothesis that males and successful females may breed at one- and two-year intervals, respectively.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Immunoreactive insulin and insulin-like activity of the blood in ontogenesis of the hen]

Studies have been made on immunoreactive insulin (IRI) and insulin-like activity (ILA) in the blood serum of chick embryos (from the 10th day of incubation), chicks and adult hens up to 1 year old. It was shown that IRI content of embryonic blood is relatively low and remains approximately constant during incubation. During postnatal ontogenesis, the level of IRI increases, the increase being most significant at the 1st day after hatching and between the 2nd and the 5th months. With respect to IRI level, 5-month chicks are similar to adult hens. Being assayed by the method of isolated epididymal rat fat, ILA was not found in the blood serum of chick embryos. It was observed in all test samples only from the 30th day after hatching. It is suggested that at this period of postnatal life, some factors are formed in the blood which increase ILA without changes of the insulin content of the blood. After the 30th day, no evident shifts were observed in ILA, although it reached maximum in adult hens. By absolute values, ILA of the blood in chicks was several times higher than the corresponding levels of IRI.     

Experimental evidence for the relationship between food supply, parental effort and chick survival in the Lesser Black-backed Gull Larus fuscus

We studied breeding success, chick growth, parental effort and chick behaviour in two groups of Lesser Black-backed Gulls Larus fuscus whose chicks were provided with additional food until 7 days after hatching or until fledging. These data were compared with those from control pairs which we studied simultaneously to test the hypotheses that food was in short supply during the chick stage at the colony site and that in such circumstances the behaviour of adults and young is mainly responsible for the low success. Pairs whose chicks were fed with additional food until fledging showed a higher fledging success than control pairs (intermediate for pairs of first experimental group). During the first week after hatching, experimental adults of both groups were present together at the territory for longer than control pairs. In adult females of experimental pairs, the length of feeding trips was shorter than in females of control pairs, whilst the rate of chick feeding was more frequent in the experimental broods. After the chicks were 7 days old, differences were significant only for the experimental pairs whose chicks were provided with additional food until fledging. Chicks fed until fledging showed a higher daily mass and wing-length increments and reached a higher fledging mass at an earlier age than both control chicks and chicks which were provided with additional food until day 7. Starvation occurred only in control chicks and in chicks of the first experimental group after we had stopped providing food. When food was in short supply, fledging success of gulls was adversely affected as a result of both starvation (because of the lower feeding rates of chicks) and a higher predation rate (arising from changes in behaviour of both adults and chicks).     

Changes in plasma testosterone levels during the peri-hatching period in the chicken.

Blood was obtained by heart puncture from 19-day-old Black Sex link chicken embryos and from Black Sex link chickens at 1.5, 6, or 24 h post-hatching. Plasma testosterone was determined by gas chromatography-mass spectrometry associated with stable isotope dilution. At 19 days the plasma of male and female chick embryos contains measurable amounts of testosterone and levels do not differ between sexes. After hatching plasma testosterone gradually declines from pre-hatch concentrations in males and females, but in all the post-hatch ages studied, plasma testosterone was significantly higher in male than in female chicks. These results indicate that in male chickens, contrary to mammals at birth, there is no surge in plasma testosterone at hatching.     

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A + PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24 h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A + B antisense (PR A + B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A + B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A + B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.     

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO₂ strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.     

Brood sex ratio in the Kentish plover

How and why do the mating opportunities of males and femalesdiffer in natural population of animals? Previously we showedthat females have higher mating opportunities than males inthe Kentish plover Charadrius alexandrinus. Both parents incubatethe eggs, and males provide more brood care than females; thusit is not obvious why the females find new mates sooner thanthe males. In this study we investigated whether the sex-biasedmating opportunities stem from biased offspring sex ratios.We determined the sex of newly hatched, precocial chicks usingCHD gene markers. Among fully sexed broods, 0.461 ± 0.024(SE) of chicks (454 chicks in 158 broods) were male, and thissex ratio was not significantly different from unity. The proportionof males at hatching decreased significantly over the breedingseason, which occurred consistently in all 3 years of the study.Large chicks were more likely to be males than females. Neitherparental age nor body size of male and female parents was relatedto brood sex ratio. We also sexed a number of chicks that werecaught after they left their nest (range of estimated ages 0–17days) and found that the proportion of males increased withbrood age. This relationship remained highly significant whencontrolling statistically for hatching date. As brood size decreaseddue to mortality after the chicks left their nest, these resultssuggest that the mortality of daughters was higher than thatof the sons shortly after hatching. Taken together, our resultsshow that the female-biased mating opportunities in the Kentishplover are not due to biased brood sex ratio at hatching but,at least in part, are due to female-biased chick mortality soonafter hatching.     

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.