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.     

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.     

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.     

Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching

In the developing pituitary gland of embryos of the annual fish Cynolebias whitei and the medaka, Oryzias latipes, prolactin cells have been identified before hatching by means of a light-microscopic immunocytochemical method with antiserum against ovine prolactin. At the time of hatching, changes in the intensity of the immunoperoxidase staining occur. Histological staining by Cleveland and Wolfe's trichrome shows differentiation of cell types in the adenohypophysis only later in ontogeny. Our results indicate that, in teleosts, differentiated prolactin cells are present before hatching and that prolactin may be involved in the endocrine control of the hatching process.     

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.     

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.     

Immunohistochemical Study of Adenohypophysial Cells During Embryonic Development in the Reptile Chalcides chalcides (Squamata, Scincidae)

The immunohistochemical avidin-biotin complex method was used to study hormone-producing cells in the adenohypophysis of the skink Chalcides chalcides during embryonic development. In Chalcides, the formation of Rathke's pouch was evident between stages 28 and 30 of embryonic development. The adenohypophysial cells begin to differentiate before the morphological development of the gland was complete. At stage 29, few corticotropic cells were present only in the dorsal face of Rathke's pouch. No other immunoreactive cell type was revealed at this stage. At stage 32, the hypophysis had developed to a great extent though it was not yet elongated in a cephalic-caudal direction. At this stage, the corticotropic cells appeared more numerous and well differentiated in the rostral pars distalis and in the pars intermedia. Melanotropic, somatotropic and gonadotropic cells appeared simultaneously, with the same distributions as in the adult skink. At stage 34, the first thyrotropic cells appeared in the pars distalis but also in the pars intermedia, whereas rare prolactin cells were observed only at stage 35 in the medial pars distalis. Between stages 36 and 38, the gland was developed in the cephalic-caudal direction and all the cell types were completely differentiated with an evident increase in the number of prolactin cells. In embryos close to birth (stages 39-40), the hypophysis and the adenohypophysial cells were already similar to those of the adult animal.     

Effect of nutritional insufficiency on cell proliferation in the matrix zones of the cerebellar anlage in mice

The work has been performed on 62 CBA mice. In the ventricular zone and in the external granular layer of the cerebellar anlage of embryos (13-17 days of the intrauterine development) mitotic index, labelled nuclei index, part of labelled mitoses have been counted. Parameters of the mitotic cycle of the matrix cells have been calculated by means of the graphic method. The proliferative pool value has been calculated. At malnutrition the cerebellar anlage structure retards in its maturation from the norm. For the matrix zones of the cerebellar anlage, higher indices of the proliferative activity are specific. At the same time, duration of the mitotic cycle of the matrix cells increases by 15-17%. It is possible, that retardation of histogenesis of the mouse cerebellar anlage, when developing under conditions of alimentary insufficiency depends on decreased rate of cell proliferation, as a result of prolonged mitotic cycle of the matrix cells.     

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.     

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.     

Development of the ductule system of the epididymis in the prenatal period of human ontogeny

In 285 human embryos, prefetuses, fetuses and newborns by means of a complex of morphological methods, development of the epididymal canalicular system has been studied. The anlage of the epididymal canalicular system is stated to appear in embryos 13.0-17.0 mm long (the 6th week of development) and is presented as an accumulation of epithelial cells and primary germ cells between reducing glomeruli and mesonephric canaliculi. The canalization process of the cellular accumulations and their transformation into real canaliculi takes place during the first half of the prefetal period (the 8th week of development). Further growth and development of the epididymal canaliculi takes place in the craniocaudal direction and by the end of the prefetal period (the 12th week) the canaliculi of the head are already formed, they have a twisted course; within the limits of the body and tail they are yet poorly differentiated and their course is nearly straight. During the fetal period of ontogenesis, further differentiation of the epididymal parts occurs and structure of their canaliculi becomes more complex. They acquire a twisted course in all the parts of the organ; by the end of the fetal period the form and structure of the epididymal canaliculi resemble those of the definitive organ.     

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.     

Embryology and cytodifferentiation of the pituitary gland in the lizard Anolis carolinensis

Anolis embryos have limb buds at the time eggs are laid and require about 39 days to complete development at 28°C. Rathke's pouch is present at five days, and the subdivisions of the adenohypophysis are differentiated by ten days after oviposition. The cells of the rostral half of the pars distalis (PD) are derived from the anterior face of Rathke's pouch; cells of the caudal half from the posterior face. Lateral lobe cells differentiate on the lateral margins of the developing caudal PD, and knob-like outgrowths of this tissue attach to the walls of the diencephalon to form the pars tuberalis (PT). Subsequently, the cells of the PT lose their connection with the PD and become a pair of flattened oblong plaques. They reach maximal size in midincubation, and are gradually invaded by nervous elements and incorporated into the walls of the hypothalamus. Electron micrographs demonstrate that the embryonic PT is secretory. Ultrastructurally the pars intermedia (PI) and PD are composed of parenchymous secretory cells in a framework of stellate cells. Stellate cells surround the lumen of Rathke's pouch and are connected laterally by complex junctions that exclude the secretory cells from the luminal surface. They extend in sheet-like processes among the secretory cells to the outer margin of the gland where they form a partial sheath within the basal lamina around the secretory tissue. As development proceeds, the lumen becomes subdivided and the resulting reduced lumina are recognizable as the forerunners of the follicles of the adult adenohypophysis. The cells of the PI are differentiated into secretory or stellate cells halfway through incubation. At this time only half of the cells of the PD can be so classified. Four of the five granulated cell types described in the adult are recognizable by mid-incubation; the fifth cell type (prolactin cell) becomes distinguishable within ten days thereafter, and at hatching appears to be actively synthesizing secretory products.     

On the specificity of staining by Alcian blue in the study of human,foetal adenohypophysis

Summary The specificity of Alcian blue staining has been investigated on a material comprising the adenohypophysis from 26 human foetuses.Two distinct types of alcianophilic cells were observed. The first cell type appears about 28 mm CRL forming small groups at the epithelial-mesenchymal junction and beneath the anlage of the fibrous capsule. Besides its alcianophilic property the cell shows a pronounced maltase-resistant PAS-positivity. The alcianophilia is due to carboxylgroups — probably of proteins as no sialic acid could be detected. By the performic acid — Alcian blue method the cell seems to show a high content of SH and S-S groups.The second type of cell appears about 70 mm CRL and is mostly located singly. It shows a pronounced alcianophilia — probably due to sulphate groups and to some extent to carboxyl groups.The role of the alcianophilic properties of the cells in typing definite cells of the foetal adenohypophysis was discussed. — Finally the pitfalls caused by different fixatives and by the use of the general polychrome staining methods for the typing of pituitary parenchymal cells were discussed.This work was supported by grants from the Association for the Aid of Crippled Children, New York, and from the Danish State Research Foundation, Copenhagen.     

Mapping and developmental expression analysis of the WD-repeat gene Preb

We have isolated from mouse a novel WD-motif-containing gene designated Preb. This gene encodes a predicted protein of 416 amino acids and has significant homology with other members of the WD-motif gene superfamily that play a role in cell fate determination. Preb maps to the proximal end of chromosome 5 in mouse, near the Hmx1 homeobox gene. Preb is detectable in early stage embryos in the peripheral nervous system, developing liver, and surface ectoderm. Later, Preb is expressed in the anterior portion of Rathke's pouch, which gives rise to the anterior pituitary, the organ responsible for the production of prolactin and other hormones. In midgestation embryos, the most extensive expression of Preb is observed in the perichondrium of the craniofacial, axial, and appendicular skeleton. The expression pattern of Preb in murine embryos suggests a potential role in the specification of multiple cell types, in particular, the fetal skeleton.     

Morphometric analysis of the autophagic and crinophagic lysosomal systems in mammotropes throughout the estrous cycle of the rat

Summary The crinophagic and autophagic lysosomal systems were studied in mammotropes (prolactin secreting cells) of the adenohypophysis throughout the estrous cycle of the rat. By means of morphometric analysis, it was found that the volume of secondary autophagic lysosomes was usually greater than that of the crinophagic type. Although the volumes of both secondary autophagic and crinophagic lysosomes were minimal throughout proestrus and diestrus 2, the autophagic lysosomal volume per mammotrope was elevated during the estrous period. The volume of secondary crinophagic lysosomes per mammotrope increased during late estrus and remained elevated throughout early diestrus 1. Furthermore, there was an inverse relationship between the volume of mature secretory granules per cell and of the crinophagic system. These data suggest a role for lysosomes in the regulation of synthesis and secretion of prolactin by the adenohypophysis of the rat.Supported by grant HD 11571 from the National Institutes of Health