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.     

Tissue-specific antigen of chick adenohypophysis appearing at early stages of histotypic differentiation]

The tissue-specific water-soluble antigen characterized by alpha1-globulin electrophoretic mobility was revealed in chick adenohypophysis. The antigen was shown to appear in embryogenesis at early stages of histotypical differentiation of the adenohypophysis by indirect immunofluorescence. The first cells with specific fluorescence were found simultaneously in the cephalic and caudal lobes of 6-day embryo adenohypophysis. The bright patterned fluorescence was observed in all cellular cords of the adenohypophysis by the 8--10th day of the development. This antigen may be used as a common marker for pituitary cell differentiation.     

Immunohistochemical study of the appearance of somatotropic hormone in the adenohypophysis of chick embryos

The differentiation of somatotropocytes was studied in the Leghorn chick embryos during 11 to 18 days of incubation and in chickens during the first week of life using the immunohistochemical method of nonlabelled antibodies with PAP complex and antiserum against human somatotropic hormone (STH). Unlike in humans, the fixation of pituitaries in the Carnoy mixture is optimal for STH to be immunohistochemically estimated in the chickens and chick embryos. STH was found in adenohypophysis from 12-13 days of development. Besides predominant localization of somatotropocytes in the adenohypophysis caudal lobe, individual STH-positive cells are also present in the cephalic lobe of chickens and chick embryos. The results obtained suggest a relatively late appearance of STH during histotypical development of adenohypophysis in chick embryos.     

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.     

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.     

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.     

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.     

Synthesis of a lens-specific antigen (delta-crystallin) in rudimentary chicken adenohypophysis

The synthesis of two lens-specific proteins, delta- and beta-crystallins, by adenohypophyseal anlage of 4-day chick embryos was studied by the immunofluorescence technique in conjunction with autoradiography. Isolated anlages were incubated for 16 hours in a culture medium containing 14c-leucine. The synthesis was determined with the use of an unlabelled carrier, extract of chick lens, as well as of antisera against delta- and beta-crystallins. 14C-Leucine incorporation was found to occur only in delta-crystalline precipitation line rather than in beta-crystallin line. This evidence attests to the synthesis of delta-crystalline by the chick embryo adenohypophyseal anlage. The results are in agreement with the previously obtained immunohistochemical data on delta-crystalline localization in cells of the developing adenohypophysis.     

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.     

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.     

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.     

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.     

Chicken Growth-Associated Protein GAP-43 Is Tightly Bound to the Actin-Rich Neuronal Membrane Skeleton

We have identified the chicken equivalent of growth-associated protein GAP-43 in a detergent-resistant membrane skeleton from cultures of chick neurones and embryonic chick brain. Antisera to the membrane skeleton protein, the 3D5 antigen, precipitate the translation product of chick GAP-43 cDNA, and the 3D5 antigen is also detected by antisera against synthetic peptides from the known amino acid sequence of rat GAP-43. The chick protein and the rat GAP-43 are biochemically similar proteins that both serve as major targets of phosphorylation by endogenous protein kinase C. The detergent-resistant complex in which GAP-43 is found also contains actin (approximately 5% of the total protein) and a neurone-specific cell surface glycoprotein. We suggest that the membrane skeleton of neurones may be a primary site of action of GAP-43.     

Isolation and antigen production of herpes simplex virus in the chorioallantoic membrane of duck embryos

Summary Isolation of herpes simplex virus and production of CF antigen was tried in chorioallantoic membranes of embryonated duck eggs. Duck CAM is twice as sensitive as chick CAM to natural virus and has almost the same susceptibility as suckling mice after intracerebral inoculation. Herpes simplex virus can be more rapidly adapted to duck eggs than chicken eggs. The first few subcultures result in higher virus titers if the virus is grown in duck CAM. No difference of virus titer is found, however, if the same sample of egg-adapted virus is titrated in duck and chick membranes. Herpes reactions are in the first few passages more progressive on duck CAM than on chick membranes and show continued proliferation between 3–5 days after inoculation. Herpes reactions on chick membranes tend to decrease after the 3rd day of incubation. Intranuclear inclusions of the Lipschütz type are always found in large numbers in reactions of the first passage on duck membranes, even when they are scanty in parallel membranes of chick eggs. CF antigens of herpes simplex virus were prepared from selected duck chorioallantoic membranes harvested 5 days after inoculation. CF antigens of herpes simplex virus prepared from duck eggs are reproducible and show high antigen titers averaging about 10 times those of chick egg preparations.     

Spatiotemporal distribution of 1P1 antigen expression in the plexiform layers of developing chick retina

Changes in the distribution of 1P1-antigen in the developing chick retina have been examined by indirect immunofiuorescence staining technique using the novel monoclonal antibody (MAb) 1P1. Expression of the 1P1 antigen was found to be regulated in radial as well as in tangential dimension of the retina, being preferentially or exclusively located in the inner and outer plexiform layers of the neural retina depending on the stages of development. With the onset of the formation of the inner plexiform layer 1P1 antigen becomes expressed in the retina. With progressing differentiation of the inner plexiform layer 1P1 immunofiuorescence revealed 2 subbands at E9 and 6 subbands at E18. At postnatal stages (after P3) immunoreactivity was reduced in an inside-outside sequence leading to the complete absence of the 1P1 antigen in adulthood. 1P1 antigen expression in the outer plexiform layer was also subject to developmental regulation. The spatio-temporal pattern of 1P1 antigen expression was correlated with the time course of histological differentiation of chick retina, namely the synapse rich plexiform layers. Whether the 1P1 antigen was functionally involved in dendrite extension and synapse formation was discussed.     

Diurnal rhythm of the reproductive activity of cells and the functional morphology of the adenohypophysis of rats (histological and the autoradiographic study)]

The adenohypophysis of intact mongrel male rats was studied histologically and autoradiographically. The diurnal periodicity was revealed and the successive spikes of indices of the DNA synthesis, mitotic process and secretory activity of the adenohypophysis cells were established. The maximum amount of the dividing cells was found about 12 o'clock p. m.-at the period of the least secretory and synthetizing activity of the gland.     

Role of the thyroid in the involution of the mesonephric Malpighian corpuscles in the chick embryo

The mesonephros of the chick embryo normally begins to regress during the second half of embryonic life. Experimental methods, such as adenohypophysis grafting, hypophysectomy or use of antithyroid drugs, which stimulate or depress the thyroid function of the embryo, modified accordingly the regressive processes occurring in the mesonephric Malpighian corpuscles, particularly at the level of the glomerular basement laminae. These results as well as the known sensitivity of the mesonephros to thyroxine and the concordance between the steps of embryonic thyroid development and the mesonephric modifications show that the thyroid normally plays a major determining role in this phenomenon.     

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.     

Expression and Electrophysiological Function of Actin in Chick Cerebellar Neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a ¹²⁵I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.