Analysis In Vitro of Capacity of Fetal Fat Pad to Support Mammary Gland Embryogenesis

Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo , without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis.
³H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.     

Granulated ‘marginal cell layer’ in the rat anterior pituitary gland

A granulated ‘marginal layer cell’ was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5–20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the ‘invading’ residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by ‘marginal layer cells’ had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the ‘marginal cell layer’ and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

The origin of yolk in the oocytes of Nereis virens (Annelida,Polychaeta)

Summary Comparative studies have been made of development of the adenohypophysis using the Rathke's pouch (RP)-derived model system. Rathke's pouch with associated mesenchyme and ventral hypothalamus, was microsurgically isolated from 15-day fetal rats and placed in mild trypsin solution. Three variations of donor tissue were isolated and transplanted beneath the kidney capsule of adult hosts: A) pure pouch epithelium; B) pouch epithelium plus mesenchyme; and C) pouch epithelium with mesenchyme and ventral hypothalamus. After 30 days the grafts were isolated and processed for light and electron microscopy. Cell types were characterized by immunostaining as well as by morphological criteria. In group A well differentiated mammotrophs dominated the grafts, many of which were hypertrophied with widely dilated endoplasmic reticulum and Golgi saccules. Mammotrophs, frequently with mitotic figures, were distributed evenly throughout the grafts. Somatotrophs and gonadotrophs were neither abundant nor well differentiated in group A, but were both abundant and more extensively differentiated in groups B and C. Both somatotrophs and gonadotrophs were typically localized at margins of the graft adjacent to connective tissue spaces. Well differentiated mammotrophs were present in groups B and C although there were fewer hypertrophied mammotrophs than in group A; and immunoreaction to prolactin was weaker than in group A.Tumor-like features found in all three groups included some loss of tissue integrity and large, vascular lakes unlined by endothelium.These findings suggest that differentiation of mammotrophs may be inhibited in part by mesenchyme associated with Rathke's pouch, since in the absence thereof these cells become hyperplastic. Conversely, differentiation of somatotrophs and gonadotrophs appears more dependent on these mesenchymal elements for normal development.     

大鼠实验性腺胃癌形成过程中脑垂体远侧部ACTH细胞的免疫组织化学观察

用断乳雄性 Wistar大鼠 15只 ,分为实验性胃癌组和盐水对照组。实验性胃癌组大鼠用 MNNG灌胃 ,流式细胞仪测定有异倍体 (10 0 % )后 ,断头处死动物 ,快速取出脑垂体。用免疫组织化学、图像分析和形态计量方法观察大鼠实验性腺胃癌发生过程中脑垂体远侧部 ACTH细胞的变化。结果表明 ,实验性胃癌组大鼠脑垂体远侧部 ACTH阳性细胞的面数密度增大 ,反应增强 ,与对照组相比有显著性差异 (P<0 .0 1,P<0 .0 5 )。结果提示 ,在大鼠实验性腺胃癌发生过程中 ,脑垂体远侧部 ACTH细胞可能参与了机体抗肿瘤的内分泌调节。     

The hypothalamo-hypophysial vascular relationship in Indian fresh-water goby, Glossogobius giuris (Ham.).920u.

The hypothalamo-hypophysial vascular relationship and intra-hypophysial vasculatisation have been described in order to understand the regulatory mechanism of hypothalamic control over the functions of the pituitary gland. In Glossogobius giuris, the disposition of the blood vessels in the head region is on typical teleostean pattern with certain modifications. The nucleus preopticus is supplied through the nucleus preopticus artery, a small blood vessel arising from the anterior branch of the posterior cerebral artery, whereas the pituitary gland receives blood through a pair of hypophysial arteries. The blood from the pituitary is drained off by the pituitary veins whch pour their blood into the supra-orbital sinus. The anterior cerebral vein after taking the blood from anterior part of the brain including the hypothalamus and the nucleus preopticus joins with the supra-orbital sinus. The hypothalamo-hypophysial portal system is absent in this fish. The saccus vasculosus receives blood from the posterior cerebral artery through a small blood vessel and is collected by a prominent saccus vasculosus vein which pours blood into the supra-orbital sinus before it joins the infra-orbital sinus to form the heat vein. There seems to be no physological connection between the saccus vasculosus and pituitary gland. The highly vascularised neurohypophysis interdigitate with the pars intermedia and extends upto the proximal pars distalis. The blood vessels are restricted to the neurohypophysial extensions only. However, in the rostral pars distalis the blood vessels are present but the neurohypophysis does not extend to this part. The blood capillaries enter the rostral pars distalis from the capillary network on the surface of pituitary gland along with the connected tissue covering of the pituitary. The neurohypophysis shows a greater vascularisation in comparison to that of the other glandular part of the pituitary gland. In the present study of Glossogobius giuris, though an extensive ramification of neurohypophysis occurs with the pars intermedia and the proximal pars distalis, the neurosecretory axons do not innervate the endocrine cells of the pituitary gland and the blood vessels are found restricted to the neurohypophysial extensions except that of the rostral pars distalis. The neuro-vascular way of hypothalamic control over the functions of the pituitary gland seems to be justified as the neurosecretory fibres have been found associated with the blood vessels.     

Prolactin-producing Cells in Clupea harengus membras,Polypterus palmas and Calamoichthys calabaricus Studied by Immuno-histochemical Methods

The prolactin-producing cells in Clupea harengus membras, Polypterus palmas and Calamoichthys calabaricus have been localized by fluores-ceinlabelled antibodies to ovine prolactin. Pituitary sections were cut in a cryostat and both direct and indirect protein tracing methods have been used. As an additional method the paraffin embedding technique was carried out with Polypterus palmas. The prolactin-producing cells in Clupea harengus membras were localized to the follicle epithelium in the rostral pars distalis. In Polypterus palmas and Calamoichthys calabaricus the prolactin producing cells were scattered all over the pars distalis. A possible relationship between the erythrosinophilic prolactin cells and the epithelium cells in the buccohypophyseal canal is discussed.     

Early Determination of Developmental Fate in Presumptive Intestinal Endoderm of the Chicken Embryo

The endodermal epithelia of esophagus, proventriculus and gizzard of 6-day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract.
The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6-day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland-like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization.
These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into intestinal epithelium.     

Immunohistochemical evidence of progestin target cells in the pituitary gland of ovariectomized rat

Progestin (P) target cells were identified in the pituitary gland of gonadectomized female rats which had been primed with estrogen (E). P staining was localized using the immunohistochemical avidin-biotin-peroxidase (ABP) complex method. Dark brown precipitates were primarily found over the cytoplasm of cells in the pars distalis, but not in the pars intermedia nor in the pars nervosa. The majority of P-sensitive cells in the pars distalis were identical with luteotrophs, a few being lactotrophs. These observations suggest a role of P in the regulation of production and secretion of gonadotrophins in the pituitary glands of female rats.     

Biology of mammary fat pad in fetal mouse: capacity to support development of various fetal epithelia in vivo

Epithelia from the lobular part of submandibular salivary gland, glandular stomach, intestine and colon of 14-day C3H/HeN fetuses, and from pituitary gland and pancreas of 12-day fetuses were recombined with 14-day mammary fat pad precursor tissue and syngrafted under the kidney capsule. The normal organogenetic development typical of the epithelium occurred. The same epithelia taken from earlier stage fetuses did not develop normally. Thus, 14-day fetal mouse mammary fat pad precursor tissue has the capacity to support normal organogenesis of various fetal epithelia of developmentally advanced stages. This supportive capacity is decreased in the fat pad precursor tissue of 17- to 18-day fetal mice and is entirely lost postnatally.     

Organogenesis of the pituitary, adrenal, and lung at birth in the wallaby, Macropus rufogriseus

The ultrastructure of the pituitary, the adrenal, and the lung was examined in the newborn wallaby, Macropus rufogriseus. Tissue from six wallaby neonates (less than 8 hr of age), two near-term fetuses (26 days after removal of suckling pouch young [RPY]), and a two-day-old pouch young was examined; and tissue levels of cortisol in the adrenal glands of five neonates and a near-term fetus (26 days) were measured by radioimmunoassay. At birth the adenohypophysis comprised the bulk of the pituitary gland. The pars distalis was well vascularized and many cells contained electron-dense, membrane-bound granules. The adrenal glands lacked specific zones but comprised two distinct populations of cells. The cytoplasm of one cell type contained electron-dense, membrane-bound granules, similar to those observed inside catecholamine-secreting cells of the adrenal medulla; the other cell type possessed large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. These features are characteristic of cells which are actively synthesizing steroid hormones. The concentration of cortisol was 0.58 ng/adrenal in the wallaby at birth. The fetal lungs near term were at the glandular stage of development, and epithelial differentiation of type I and type II pneumocytes was imminent although attenuation was not evident. The canalicular neonatal lung did not contain true alveoli, but type II pneumocytes contained osmiophilic lamellar inclusions of surfactant. The fetal pituitary and adrenal are functional at birth and are thus capable of initiating parturition and of influencing lung maturation in the fetus.     

Prolactin Cells: Study of the Pituitary Gland of the Roach (Leuciscus rutilus) by Immuno-histochemical Methods

Fluorescein-labelled antibodies against ovine prolactin have been used to localize the prolactin-producing cells in the pituitary gland of the roach, Leuciscus rutilus. Two methods of protein tracing have been used: the direct and the indirect. In the direct method gammaglobulin fractions of rabbit-antisera were conjugated with fluorescein-isothiocyanate. Frozen sections of the pituitary gland were treated with conjugate. Cells with a content of prolactin gave off green fluorescence. Conjugate made from normal rabbit-serum failed to give this result. Blocking-experiments were also used to control the conjugate fluorescence. In the indirect method sheep-anti-rabbit-gammaglobulin was labelled with fluorescein. First the sections of the hypophysis were treated with antiserum from rabbits. After careful rinsing the sections were exposed to the dyemarked sheep-anti-rabbit-gammaglobulin. For orientation in the pituitary gland histological staining was carried out in parallel to that by fluorescence. The investigation has shown that the erythrosinophilic cells in the rostral pars distalis contained prolactin. Since no other cells in the gland were fluorescent, it can be concluded that the eta-cells are the prolactin cells.     

Pituitary gland of the garter snake (Thamnophis): Morphology and immunocytochemistry

The three-dimensional gross morphology of the pituitary gland of the garter snake (Thamnophis sirtalis) is presented. Hormone-producing cells of the pars distalis were localized immunocytochemically. Corticotropes and lactotropes occur in the anterior two-thirds of the gland; corticotropes are especially numerous in the area of the pars distalis nearest the median eminence, and lactotropes are most abundant medially. Somatotropes are restricted to the posterior one-third of the pars distalis. Gonadotropes and thyrotropes are scattered throughout the pars distalis and in favorable sections form a network of cells enclosing clusters of peptide-secreting cells.     

Histology of the pituitary gland of the grey mullet, Mugil cephalus L.

The present communication deals with a histological study of the pituitary gland of the teleost fish Mugil cephalus , found in the estuarine waters of Cochin area. Six different cell types were identified in the pituitary gland on the basis of their grouping, distribution and staining properties. The prolactin and the TSH cells (thyroid stimulating hormone producing cells or thyrotrops) were identified in the rostral pars distalis and the ACTH cells (adrenocorticotropic hormone producing cells or corticotrops) in the interphase between the neurohypophysis and the rostral pars distalis. The STH cells (somatotropic hormone producing cells or somatotrops) and the gonadotropic cells were distinguished in the proximal pars distalis and the MSH cells (melanin stimulating hormone producing cells or melanotrops) in the pars intermedia.     

A histological study of the hypophysial-gonadal system during sexual maturation and spawning in the milkfish, Chanos chanos (Forskal)

The pituitary gland of the milkfish, Chanos chanos , was studied at different stages of sexual maturation and spawning. Consecutive median sagittal sections were treated with a range of stains to demonstrate the different cell types and regions. The milkfish pituitary consists of a neural component, the neurohypophysis, and an epithelial component, the adenohypophysis, which in turn consists of three regions: the rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). However, unlike most teleosts, the pituitary gland of the milkfish is encased in a bony chamber, has dorsal and ventral lobes and extends anteriorly from its point of origin at the base of the brain. PAS (+) basophils are found in all regions of the adenohypophysis, but mostly in the proximal pars distalis. These cells undergo hypertrophy and hyperplasia during sexual maturation, shrinkage and degranulation during spawning.     

Presence of immunoreactive alpha-endorphin in rat pituitary gland.

Utilizing radioimmunoassay for α-endorphin, we attempted to demonstrate immunoreactive α-endorphin in acid extracts of pars distalis and combined pars intermedia and pars nervosa of the rat pituitary gland after chromatography on Sephadex G-25. β-Lipotropin, β-endorphin and γ-endorphin were not converted into α-endorphin during the extraction and gel chromatographic procedures. Concentrations of immunoreactive α-endorphin determined after gel chromatography of extracts from pars distalis and combined pars intermedia and pars nervosa were 1.1±0.6 and 130±17 ng/mg wet tissue (mean±SE), respectively. Serial dilution of these extracts gave parallel lines to the standard curve of synthetic α-endorphin, but not to that of γ-endorphin or δ-endorphin. These results suggest the existence of immunoreactive α-endorphin indistinguishable in molecular size from synthetic α-endorphin in the rat pituitary gland.     

Estradiol stimulation of pituitary primordia transplanted to the kidney capsule

Summary The purpose of this study was to define better the influence of hormones on the normal and pathological development of the anterior pituitary using Rathke's pouch (RP)-derived model system. RP from either 12- or 15-day fetuses were microsurgically isolated and transplanted beneath the kidney capsule (KC) of intact adult hosts. Eighteen days later the hosts were hypophysectomized. Ten-12 days after hypophysectomy hosts were injected daily with either 1.0 g estradiol benzoate (EB) or 0.1 cc corn oil until necropsy at 80 days. Both 12- and 15-day RP differentiated into large, pars distalis tissues consisting of a variety of granulated and agranulated cell types, as well as large, secretory cysts. Cytodifferentiation was consistently most advanced in 15-day RP-derived grafts. Evidence of secretory granule synthesis, but not exocytosis, was apparent in granulated cells in oil-treated controls and EB-treated 12-day RP-derived grafts. Immunostaining and electron microscopy demonstrated hypertrophied somatotrophs and mammotrophs with numerous profiles of exocytosis in EB-treated 15-day RP-derived grafts. Mammotrophs and somatotrophs were infrequent and not well differentiated in 12-day RP-derived grafts whether EB- or oil-treated, nor in oil-treated 15-day RP-derived grafts. Radioimmunoassay demonstrated highest levels of plasma PRL in EB-treated 15-day RP-derived grafts. Implant invasiveness was noted only in EB-treated 12-day RP-derived grafts when basal laminae were disrupted or absent, and graft cells mixed with connective tissue elements.Results indicate that the Cytodifferentiation of pars distalis cell types derived from KC transplanted RP can be maintained to 80 days. Mammotrophs are especially well differentiated and responsive to EB treatment during this development. However, continued maintenance of a differentiated state by mammotrophs and somatotrophs appears to require the presence of host pituitary and/or end organ hormones as evidenced by the lack of maintenance in oil-treated controls. Furthermore, the loss of well defined tissue boundaries between host and graft tissues of EB-treated 12 day RP animals suggests tumorigenic transformation.Supported in part by Grant No. R01-CA-21426 from the National Cancer Institute, DHEW; California Division ACS Special Grant No. 851; HEW-NIH Grant CA-14089, Los Angeles County/USC Cancer Center; and GRS 53-5104-5283The authors gratefully acknowledge the technical assistance of Vivian Valentin, Alicia Thompson, Linda Melsek, Carolyn Tallent, and Lindsay Gilpin.     

Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme

Persistence of the capacity for embryogenic morphogenesis in adult mammary epithelium was demonstrated by allowing it to interact with grafted embryonic mesenchyme in vivo. When 14-day embryonic mammary or salivary mesenchyme was transplanted in the mammary gland of syngeneic young adult virgin mice, organogenetic development of the mammary epithelial cells occurred responding to closely attached mesenchyme. An early change, within 2–4 days, that was observed equally in both types of the mesenchymes was proliferation of mammary epithelial cells in multiple layers resembling rudimental architecture. Subsequently, ductal branching occurred from the rudimental architecture by mesenchyme-dependent branching pattern, of mammary gland type with mammary mesenchyme and of salivary gland-like type with salivary mesenchyme. This developmental response did not require hormones secreted from ovaries since it was observed similarly in ovariectomized mice. The mammary epithelium at the lactating stage did not show such a potential to the transplanted salivary mesenchyme.     

Granulated 'marginal cell layer' in the rat anterior pituitary gland

A granulated 'marginal layer cell' was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5-20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the 'invading' residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by 'marginal layer cells' had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the 'marginal cell layer' and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

Appearence of LH-Immunoreactive Cells in the Rathke's Pouch of the Chicken Embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions

Summary Immunofluorescent ACTH cells are present in the developing chick pituitary gland from the 9th day of incubation.Rathke pouch grafts from 4–5 day or 5.5 and 6.5 day-old chicks, grafted into chick chorioallantoic membrane and grown for 12 days, gave rise to tinctorially normal pituitary glands in both cases.The early grafts were of pouch epithelium alone, separated from mesenchyme by trypsinization. The later grafts were surrounded by their attached mesenchyme, from which they are virtually inseparable.In 17 out of 18 of the 4.5 day grafts no immunofluorescent ACTH cells developed. (In the 18th case a few feebly stained single cells). In 16 out of 30 of the 5.5 and 6.5 day grafts ACTH cells were present in normal numbers.Of the 3 hypotheses put forward to explain these findings only one appears valid. This is that the ACTH cells are contributed directly by the mesectoderm (neural crest) surrounding the 5.5 and 6.5 day pituitary primordia.