Immunohistochemical detection of prolactin in the hypophysis of the chick and chick embryo

The appearance and localization of immunoreactive prolactin in the adenohypophysis of chick embryos and chickens was studied by antisera to the bovine prolactin. Immunoreactive prolactin was found in the chick embryos from the 15th day of development on using the methods of indirect immunofluorescence and of unlabelled antibodies with a complex PAP. In the chick embryos and in chickens during the first 10 days of life, the prolactin-containing cells were distributed, mainly, in the cephalic part of adenohypophysis; in the chickens, scarce cells were also found in the caudal part. These results suggest that in the domestic fowl the immunoreactive prolactin, similar by immunochemical specificity with the mammalian prolactin, is a late appearing marker of the adenohypophysis differentiation.     

Immunochemical identification of the growth hormone and prolactin in the hypophyseal polypeptide spectrum of chickens

Forty-eight fractions of polypeptides including 39 fractions with a molecular weight of 14-95 kD were identified in chick adenohypophysis by sodium dodecyl sulphate electrophoresis in 10-20% gradient polyacrylamide gel slabs. The immunochemical identification of the polypeptides was performed with the aid of the electroblotting of proteins and antisera to human STH, to bovine prolactin, and to the tissue-specific antigen A-1 of chick adenohypophysis. Antisera to human STH and to antigen A-1 reacted with the same major polypeptide fraction, m.w. 26 kD, characteristic of the caudal lobe of the adenohypophysis. Immunoreactive prolactin was present in chick adenohypophysis in the form of a polypeptide fraction with a molecular weight of 25 kD and in the form of two minor fractions of polypeptides with molecular weights of 27 and 28 kD. The data obtained indicate the identity of the adenohypophyseal tissue-specific antigen A-1 to chick STH.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.     

Immunocytological study on the differentiation of chick and quail adenohypophysis epithelial rudiments, grafted or cultivated in vitro: evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11--13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-beta-(1--24)ACTH, anti-alpha-(17--39)-ACTH, anti-alpha-endorphin, anti-beta-endorphin and anti-beta-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to beta-(1--24)-ACTH, alpha-(17--39)-ACTH, and beta-LPH, as did adenohypophysis from quail embryos of corresponding age (9--10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

The determination of the cytodifferentiation of the adenohypophysis in embryonic development

This is a review of the literature and author's own data on determination of various cell types of adenohypophysis during embryonic development. Recent studies using techniques of organ culture and immunohistochemistry have established the time of determination of glandular cells of adenohypophysis. It has been shown in rat embryos that the direction of differentiation of all major cell types of adenohypophysis is programmed late during the development of the epithelial anlage of this organ. Similar data as concerns somatotropic and prolactin cells have been obtained on chick embryos. Chick embryos possess regional type of determination of prolactin and somatotropin-containing cells in the anlage in correspondence with their location in definitive adenohypophysis.     

Tissue specific antigen of the caudal lobe of the chicken adenohypophysis]

The tissue-specific water-soluble antigen of the chick pituitary gland was revealed by immunochemical analysis. The content of this antigen was found to be predominant in the caudal lobe of the adenohypophysis. The antigen was found from the 13th day of embryogenesis by immunoelectrophoresis and immunofluorescence. Two kinds of the antigen (with high and low relative electrophoretic mobility) were discovered in the chick adenohypophysis. A conclusion was drawn that the adenohypophysis cells differed by the differentiation level.     

Cell types in the adenohypophysis of the Japanese quail and effects of injection of luteinizing hormone-releasing hormone

The cells of the adenohypophysis of the Japanese quail were studied by both light and electron microscopy after exposure to long photoperiods or injection of lutenizing hormone-releasing hormone (LRH). Six cell types were identified in the adenohypophysis by examining alternate thick and thin sections by light and electron microscopy. In the cephalic lobe, there are four types of glandular cells. They are the prolactin cells, ACTH cells, TSH cells, and gonadotropic cells (FSH?). In the caudal lobe, there are two types of cells, STH cells and gonadotropic cells (LH?). After exposure to long daily photoperiods, gonadotropic cells in both lobes were strongly activated. They became larger and accumulated many granules. ACTH cells became vacuolated; granules were sparse. Synthetic LRH injection (10 mug/0.2 ml/day) for 10 days to the non-photostimulated quail stimulated certain numbers of the gonadotropic cells in the both lobes, although the response of the cells was less than that induced by photostimulation. No response was seen in the other cell types.     

Adenohypophysis regulates cell proliferation in the gonads of the developing chick embryo

The aim of the present study was to evaluate the effect of hypophysectomy on cell proliferation in the left ovary and the left testis of 8- to 14-day-old chick embryos. Hypophysectomy was performed by the partial decapitation technique. At 44-46 h of incubation, chick embryo heads were sectioned at the mesencephalic level and the prosencephalic region removed. Embryos were further incubated until 8-14 days of development. Cell division was evaluated by bromodeoxyuridine (BrdU) incorporation and by counting the total number of somatic and germ cells in the gonads. The ovary displayed an exponential increase in the number of somatic and germ cells and a higher rate of BrdU incorporation compared to the testis. BrdU incorporation was reduced in the ovary of hypophysectomized embryos at 9-14 days of incubation, while in the testis, the reduction was significant at 14 days of development. Changes in the total number of somatic and germ cells further suggest that the absence of hypophysis affects the growth of the ovary earlier than the growth of the testis. Reduction in the number of somatic and germ cells after hypophysectomy in the ovary was reversed by a hypophyseal graft on the chorioallantoic membrane. The adenohypophysis regulates, probably through gonadotropic hormones, proliferation of somatic and germ cells in the gonads during chick embryo development.     

The distribution of competence for adenohypophysis development in the ectoderm of chick embryos

The ability of various zones of the cephalic and trunk ectoderm to differentiate into adenohypophysis after the contact with the bottom of the prosencephalon was studied in tissue culture of chick embryos as the stage of 10-13 somites. Stomodeal presumptive lens ectoderm and lateral cephalic ectoderms were shown to be competent for development into adenohypophysis. In all cases adenohypophyseal cords were formed in the zones of ectoderm contact with the brain. The cords contained antigens A-2, A-3 specific for chicken adenohypophysis as well as ACTH and beta-lopotropin. Trunk ectoderm proved to be incapable to differentiate into adenohypophysis.     

Immunohistochemical study of the differentiation of the cephalic lobe of the adenohypophysis in chick embryos]

It was shown by means of indirect immunofluorescence that ACTH appeared in the cephalic lobe of the chick embryo pituitary beginning from the 8th day of the development. A new tissue-specific antigen (A3) is revealed, which is localized in the cephalic lobe of adenohypophysis and becomes manifest at the 7th day of embryogenesis.Quantitative analysis of ACTH and A3 localization in the cells of 11-day chick embryo adenohypophyses allows a conclusion that A3 is localized in corticotropic cells.     

Rate of gluconeogenesis in the hepatocytes of the developing chick embryo

The rate of glucose formation from lactate was determined in the hepatocytes isolated from the liver of developing chick embryos and one day old chickens. The maximum rate of gluconeogenesis is observed in the hepatocytes of 16-17 day old chick embryos. A sharp fall in the rate of gluconeogenesis is noted in the hepatocytes of the embryos before hatching. It decreases still further in one day old chickens. The patterns of the gluconeogenesis rate are similar both for endogenous precursors and lactate.     

Comparison of techniques for the detection of avian infectious bronchitis virus as a contaminant of vaccines

Techniques for detecting various levels of both field and vaccine strains of infectious bronchitis virus in a deliberately contaminated Newcastle disease vaccine were compared using chick embryos, chick kidney cells, chick tracheal organ cultures and chickens with a view to determining the most appropriate method for screening vaccines for freedom from IBV contamination. Techniques examined included detection of abnormalities and deaths in embryos, cytopathic effect in chick kidney cells, ciliostasis in chick tracheal organ cultures and clinical signs and virus isolation in chickens as well as the fluorescent antibody test, negative contrast electron microscopy and serology where appropriate. Results showed that the techniques capable of detecting both strains of infectious bronchitis virus were, in order of sensitivity, the fluorescent antibody test on allantoic cells from infected embryos, ciliostasis and direct electron microscopy of allantoic fluid. One surprising feature was the poor results obtained using chickens. Some detection was achieved with tracheal virus isolation and tracheal organ cultures prepared from inoculated birds and to a lesser extent with histology and clinical signs, but no technique detected the field strain.     

Somatotropic cell differentiation in an organ culture of the chick embryo adenohypophysis

Morphogenetic potencies of the adenohypophysis tissue from 4.5 to 11-day old chicken embryos used for the differentiation of somatotropic cells were investigated by methods of organ tissue culture. STG-cells were detected in cultures by immunofluorescence using an antiserum to human STG. In vitro studies of organ cultures revealed differentiation of STG-cells when adenohypophysis tissue was cultured from the 5.5th, 7th, 9th and 11th day of development in the absence of the diencephalon. Differentiation of STG-cells occurred predominantly in embryo caudal lobe transplants after chorion-allantois culturing of Rathke's pocket fragments from 4.5-, 5.0- and 5.5-day old embryos. The data obtained suggest that at late stages of Rathke's pocket development differentiation of STG-cells is preprogrammed and that determined precursors of these cells are located in the caudal lobe of the germ.     

草鱼垂体STH细胞组织化学和超微结构研究

本文分别对草鱼性成熟前、后垂体ＳＴＨ细胞进行了组织化学和超微结构研究。垂体ＳＴＨ细胞多位于中腺垂体中部和背部,为嗜酸性细胞,用ＰＭＢ（ＰＡＳ－ＭＢ）和ＡＰＧ（ＡＢ－ＰＡＳ－ＯＧ）两种组织化学方法染色,对橙黄Ｇ阳性,对ＰＡＳ、ＡＢ阴性;电镜下电子密度较高,内质网绕核呈环形,分泌颗粒多而小。     

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.     

Insulin binding by receptors of the plasma membranes of the liver in hens in ontogenesis

Specific binding of 125I-insulin to the liver plasma membranes was studied in the chick embryos from the 10th day of incubation on, in chickens and adult fowl. The level of binding was the same in all cases although the insulin concentration of blood increases during ontogenesis, the number of receptors and their affinity to the hormone remaining constant. The data on insulin-receptor interactions in the liver have been compared with the earlier results of the authors obtained on the chick skeletal muscle and erythrocytes.     

Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.     

Action of the testosterone treated chicken testis on the regression of the Müllerian duct]

After administration of testosterone propionate to male chick embryos and chickens, their testis have an activity, on the retrogression of mullerian ducts, much more important than that observed in testis of normal subjects of the same age, activity measured by grafting testis fragments in undifferentiated female chick embryos. The results suggest that testosterone gives such an effect by inhibiting pituitary gonadotropins, peculiarly FSH.     

Changes in somatotropic and lactotropic functions of the adenohypophysis of rats with acute alloxan diabetes

Increased growth hormone and prolactin contents of the rat adenohypophysis during the development of experimental diabetes were found by colorimetric studies of stained electrophoregrams. 4 to 5 days after alloxan administration the levels of somatotropic hormone (STH) and prolactin were higher in comparison to those in intact animals by 58% and 43%, respectively. Experiments on the primary cell culture using the precursor 14C-L-leucine revealed an enhanced secretion of somatotropic hormone and prolactin by cells of the rats with alloxan diabetes. A possible role of the adenohypophyseal changes in the development of experimental diabetes is discussed.