Differentiating ability of the pars intermedia in hypothalectomized frog tadpoles.

It is generally accepted that hypothalectomy of frog tadpoles at the open neurula stage results in failure of the pars intermedia to develop. A pale body color is assumed to be evidence that the hypothalamus was completely removed. The present study, however, shows that hypothalectomized Rana japonica can develop into either albino, as already reported, or darkly pigmented tadpoles. In order to determine the extent to which the intermediate lobe can develop in these hypothalectomized tadpoles, their adenohypophyses were examined immunohistochemically by using anti-alphaMSH (melanocyte-stimulating hormone). In all the dark-colored larvae a pars intermedia had formed, though its size was very small. In the pale-colored tadpoles, on the other hand, the pars intermedia frequently failed to differentiate, but it was observed in 4 of 13 hypothalectomized larvae. In view of other investigators' data showing the complete absence of ACTH (adrenocorticotropin) cells in hypothalectomized tadpoles, hypophyses were also stained with anti-ACTH. Immunoreactive ACTH cells were detected in hypothalectomized tadpoles irrespective of the body pigmentation, although their incidence was lower than in normal controls. These data indicate that contact between the infundibulum and adenohypophysis is not absolutely essential for differentiation of MSH and ACTH cells in the frog.

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain.

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Corticosteroids stimulate the differentiation of growth hormone cells but suppress that of prolactin cells in the fetal rat pituitary.

An organ culture study was carried out to examine the effects of adrenal corticosteroids on the development of growth hormone (GH) cells and prolactin (PRL) cells. The adenohypophysial primordia were separated from 13.5-day-old fetal rats and maintained in vitro for 8 days with or without cortisol. Immunohistochemical examination of these explants showed that cortisol stimulated the differentiation of GH cells but suppressed that of PRL cells in a dose-dependent manner. In the absence of cortisol there were more PRL cells. Corticosterone had a similar effect on the developing adenohypophysis. When the pituitary primordia of Day 16.5 were cultured for 5 days and studied by the in situ hybridization technique, the expression of GH and PRL mRNA was found to be parallel with the immunoreactivity of the respective hormones. These data are discussed in relation to the normal development of GH and PRL cells in the fetal rat adenohypophysis.

Possible involvement of folliculo-stellate cells in the differentiation of muscle fibers during monolayer culture of pituitary cells.

A major objective of the present study was to examine the possibility that non-granular folliculo-stellate (FS) cells in the rat anterior pituitary are involved in the myogenesis that occurs during pituitary cell culture. Enzymatically dissociated anterior pituitary cells were fractionated by use of the Percoll gradient method. The proportion of FS cells was 5.8% on average before cell fractionation. After employing the Percoll gradient procedure, FS cells were enriched to a ratio of 12.2%. Three of five cell fractions were separately cultured, and the incidence of striated muscle fibers was quantitatively investigated. There was a good correlation between the numbers of muscle fibers and the proportions of FS cells in the fractions obtained from the Percoll gradient. These results suggest that FS cells are the cells that transform into striated muscles in pituitary monolayer cultures.

An immunohistochemical study of an anomaly of the fetal rat: adenohypophysis developing without contact with the brain.

During an experiment on developing rats, we encountered an abnormal rat fetus whose adenohypophysis was not in contact with the brain. This fetus was found and killed on the 21st day of gestation when all types of adenohypophysial hormone-producing cells are already immunohistochemically detectable in the normal gland. When compared with its normal counterpart, this brain-detached adenohypophysis was characterized by: 1) its remarkably small volume, less than 10 percent of the size of the normal gland; 2) the lack of a pars intermedia; 3) far fewer numbers of corticotropes, somatotropes and thyrotropes; and 4) the absence of prolactin cells. These results are consistent with our previous in vitro data which showed that contact between the adenohypophysial primordium and diencephalon is indispensable for the proliferation and differentiation of the adenohypophysial hormone-producing cells. The absence of the pars intermedia indicates that the formation of this part of the hypophysis is dependent on the brain in rats, as already shown for frogs.

A gene trap strategy for identifying the gene expressed in the embryonic nervous system.

An efficient gene trap strategy was devised for identifying the genes that are expressed in the mouse developing nervous system. Mouse embryonic stem (ES) cell lines that carried independent integrations of a gene trap vector, pSneolN/acZA, were allowed to differentiate in a suspension culture system. To select cells containing neurons, astrocytes or neuron-glia precursors, cell lines were immunohistochemically examined with antibodies against neuron-specific proteins (neurofilament protein 150 kD and microtubule associated protein 2), glial fibrillary acidic protein or nestin. Three cell clones (GT3-8, 11 and 12) were immunoreactive to either of the antibodies employed and at the same time positive for beta-galactosidase activity. When chimeric embryos were generated by the use of the above 3 cell lines, some cells in their nervous system showed X-gal staining. Thus the major advantage of the present gene trap method lies in its prescreening step of manipulated ES cells prior to generation of chimeric animals. This method holds promise as a useful tool for investigating the genes involved in the development of the nervous system.

Immunohistochemical study of the cytogenesis of prolactin and growth hormone cells in the anterior pituitary gland of the fetal rat.

The objectives of this study were to determine the first developmental stage when immunoreactive prolactin (PRL) cells appear in the fetal rat anterior pituitary, and then to compare their cytogenesis with that of growth hormone (GH) cells. Immunoreactive PRL cells first appeared on Day 18.5 of gestation, the same stage when GH cells were found to differentiate in the pituitary. During the fetal period, PRL cells were concentrated mainly in the anterior half of the pituitary, whereas GH cells were found predominantly in its posterior half. When estimated as the total sum of immunoreactive areas, the proportion of PRL cells remained low during fetal life in contrast to a marked increase in the GH immunoreactive area. The flip-flop (mirror) section technique revealed that, in the fetal rat, adenohypophysis PRL and GH are contained in different cells. The present study thus indicates that GH and PRL accumulation occurs in independent cells at least in early developmental stages in the rat.

The occurrence and developmental origin of epithelial cysts in the rat and mouse adenohypophysis.

The adenohypophysis occasionally contains cysts of epithelial nature. The present study describes the incidence and immunohistochemical characteristics of these epithelial cysts in the adenohypophysis of rats at different ages and in young mice. Epithelial cysts were found in about 10% of the partes distales irrespective of age and animal species. Their incidence in the pars tuberalis was higher: 22% and 58% in young rats and mice, respectively. Immunohistochemically, cells composing these cysts failed to contain S-100 protein. Although cysts found in the pars tuberalis frequently possessed immunoreactive luteinizing hormone-producing cells, most cysts in the pars distalis were immunonegative when stained with antisera to several different adenohypophysial hormones. Examination of fetal rat hypophysis has shown a close topographical relationship between cysts and the pharyngeal duct. This fact, together with the frequent occurrence of cysts in the ventro-medial region of the pars distalis in neonatal and young animals, indicates that these cysts are probably derived from a part of oral epithelium that is otherwise destined to degenerate at the time when Rathke's pouch closes.

Cellular proliferation in the anterior pituitary of the rat during the postnatal period.

Cellular proliferation in the anterior pituitary of 2-, 8-, 15- and 30-day-old rats was examined by injection of bromodeoxyuridine 1 h before autopsy. Bromodeoxyuridine incorporated into DNA was detected immunohistochemically by use of a monoclonal antibody. The highest rate of cell proliferation was found in 2-day-old animals; it decreased thereafter during the postnatal period. Possible toxic effects of colchicine on cellular proliferation were examined. Colchicine treatment (10 mg/kg in 8- and 30-day-old animals) significantly decreased the number of bromodeoxyuridine-labelled cells/mm2 in 8-day-old rats. Some sections were doubly immunostained for bromodeoxyuridine and various pituitary hormones. The proportion of doubly-immunostained cells to all proliferating cells was generally low, ranging from 23% at 2 days to 32% at 30 days of age.

Failure of luteinizing hormone-releasing hormone (LHRH) to affect the differentiation of LH cells in the rat hypophysial primordium in serum-free culture.

The aims of this study were to investigate the differentiating capacity of adenohypophysial LH cells in a serum-free culture medium and to test whether cytogenesis is affected by synthetic LHRH. The adenohypophysial primordia of fetal rats were isolated on days 11.5 and 12.5 of gestation and cultured without serum for 10 and 9 days, respectively, in synthetic Medium 199 or alpha MEM. Immunohistochemical examination using the PAP method revealed that most culture explants, apart from a few degenerate ones, contained LH cells. In comparison with Medium 199, which has been widely used as a culture medium for hypophysial explants, alpha MEM gave far better results and the primordia cultured in this medium showed better tissue growth and contained a greater number of LH cells. Administration of synthetic LHRH (10 ng/ml) on the first day of culturing had no effect on the number of LH cells, no matter whether or not the culture medium was supplemented with insulin, transferrin or thyroxine. These results suggest that at the early developmental stage LHRH is not essential for the differentiation and/or proliferation of LH cells.

Correlative study on sex differences in pituitary luteinizing hormone content and the number of immunoreactive luteinizing hormone cells in perinatal rats.

Sex differences in both pituitary luteinizing hormone (LH) content and the number of LH cells were correlatively studied in perinatal male and female rats. In the fetal pituitaries of late gestation, no sex difference was observed. On the day of birth, LH content and LH cell numbers were significantly greater in female than in male rats. Both of the two sex differences became more pronounced during the 1st and 4th postnatal days. Hormone synthesis and proliferation of pituitary LH cells are probably suppressed by testicular steroids in perinatal male rats.

A comparative in vitro study on LHRH responsiveness of LH cells of the pars tuberalis and pars distalis.

The aim of the present study was to test whether the luteinizing-hormone (LH) cells in the pars tuberalis (PT) of the rat and mouse respond to LH-releasing hormone (LHRH) as do those of the pars distalis. A part of the basal hypothalamus containing the pituitary stalk, median eminence and the pars tuberalis (H-PT), was dissected out and incubated in vitro. The LH-secreting capacity of the PT was investigated after removal of the "pituitary body" (i.e., partes distalis, intermedia and nervosa). First, some rat and mouse H-PT tissues were treated with synthetic LHRH (100 ng/ml), while others were incubated without LHRH. After 24 h of incubation, variable amounts of LH release were detected in the medium. This LH discharge, however, was not LHRH-dependent but proportional to the number of PT LH cells that were immunohistochemically detected in each incubated tissue. Since there was marked individual variation in the number of LH cells in the PT, the LH levels in the incubation medium were next compared before and after LHRH treatment using the same H-PT of the rat. An effect of LHRH could not clearly be shown in this experiment. Finally, the cytological response of the PT to LHRH was investigated by incubating both the H-PT and pituitary body connected to the intact pituitary stalk. Immunohistochemical examination of LHRH-treated tissues after 24 h revealed that, in females of both rats and mice, hormone depletion occurred in LH cells of the pars distalis but not in those of the PT.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in pituitary LH storage and release in LHRH-stimulated pubertal rats. A correlative immunohistochemical and radioimmunoassay study.

This study investigates the relationship between pituitary LHRH responsiveness and the depletion of LH in pubertal rats. The anterior pituitaries of 7-week-old rats of both sexes were stimulated for a maximum of 24 h with either a continuous, or pulsatile exposure to LHRH in vitro. Immunohistochemical examination revealed that most LH-cells in females became depleted of immunoreactive material, regardless of the mode of LHRH administration. In contrast, the majority of LH-cells in the male gland retained a strong immunostaining intensity. Radioimmunoassay showed that the initial pituitary LH content was significantly lower in the female rats (P less than 0.001), but, even so, they released a higher percentage of stored LH in response to LHRH stimulation in vitro. A similar result was also obtained after a single injection of LHRH in vivo. Thus, the lower LH content and higher LHRH responsiveness of the female pituitary explain why LHRH treatment induced a pronounced LH depletion in this sex. These results are discussed in relation to available data on heightened LH secretion in maturing female rats.

An immunohistochemical study on the mouse adenohypophysis with reference to the spatial relationship between GH cells and other types of hormone-producing cells.

The objective of the present immunohistochemical study was to determine whether the close spatial relationship between hormone-producing cells as described in rats also exists in the mouse adenohypophysis. In both immature and mature mice, GH cells were the only cell type that had a round shape throughout the pars distalis. All other types of secretory cells had angular or irregular shapes and were closely apposed to round GH cells. Thus, between GH and ACTH cells the same intimate relationship pertains as in rats. Unlike in rats, however, the juxtaposition of LH and Prl cells was observed only occasionally in mature female mice. The salient features of the mouse adenohypophysis were that most LH cells closely surrounded GH cells. These findings show that the cytoarchitectural interrelationship between adenohypophysial cells of mice differs from that of rats.

A correlative radioimmunoassay and immunohistochemical study of LHRH-induced LH depletion in the anterior pituitary of male and female neonatal rats in vitro.

After an exposure of 24 h to synthetic LHRH (100 ng/ml) in vitro, the anterior pituitaries of 4-day-old rats show a notable loss of immunoreactive material in most LH cells in males, but not in females. When radioimmunoassayed without incubation, the pituitary LH content of 4-day-old female rats is 2.8 times higher than that of males of the same age. LHRH treatment stimulates a higher rate of LH discharge in females than in males, but if LH release is expressed as a percentage of the initial pituitary LH content, there is no apparent difference. In both sexes, more than 70% of the initially stored LH is discharged into the medium after 24 h of LHRH stimulation. In males, this discharge produces a pronounced depletion, but in females, the pituitary still contains 78.2% of the initial LH content despite the large amount of hormone released. From these results, it is concluded that in newborn rats the LH synthetic rate in females is higher than that in males. This high synthetic activity, together with the large store of LH, may explain why prolonged LHRH treatment fails to cause LH depletion in females. At 4 days of age LHRH had no stimulatory effect on pituitary synthesis of LH in either sex.

Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro. II. Differentiation of LH cells.

The objective of this in-vitro study was to examine whether the diencephalic floor or the mesenchyme is involved in differentiation of LH cells in the developing rat adenohypophysis. Overall growth of the adenohypophysial tissue was retarded when the adenohypophysial primordium was cultivated after enzymatic removal of the diencephalic floor on days 11.5 and 12.5 of gestation. This malgrowth was more marked when the brain was separated on day 11.5; most explants retained a simple cystiform structure that consisted of a few layers of undifferentiated cells. Removal of the brain also caused a highly significant decrease (P less than 0.001) in the number of immunoreactive LH cells, if it was performed on day 11.5 but not day 12.5. Mesenchyme had little effect on the adenohypophysial growth or the number of immunopositive cells. Cultivation of the adenohypophysial primordium with the diencephalic floor resulted in the appearance of many immunoreactive LH cells. The number of LH cells significantly decreased, however, when the co-cultivated brain completely surrounded the adenohypophysial tissue. These results indicate that in 11.5-day-old fetal rats the diencephalic floor is indispensable for the initial proliferation of adenohypophysial primordial cells and for the early determinating process of LH cells. Once determined, the development of LH cells may proceed without the surrounding tissues. The cytodifferentiation seems to be rather inhibited when in contact with the brain. The significance of the intimate spatial relationship between developing LH cells and the surrounding mesenchyme is also discussed.

A quantitative electron microscopic study on the frequency of exocytosis in the anterior pituitary of perinatal rats.

Granule exocytosis was quantitatively investigated in the perinatal rat anterior pituitary at the electron microscopic level. Both the number of cells in the process of exocytosis and the number of extruded granules per cell profile found in a standardized area of section were counted. The first distinct figure of exocytosis was detected in the anterior pituitary of fetal rats on day 18.5 of gestation, although occasional cells on day 17.5 had structures resembling granule extrusion. The frequency of cells showing granule discharge was very low on day 18.5 of gestation, but it sharply increased on day 19.5; a similar level was maintained up to day 21.5 of gestation. While the number of exocytosed granules per cell profile was almost unchanged during the fetal and neonatal period up to day 3 after birth. The frequency of cells undergoing exocytosis decreased near the time of birth, after which it transiently increased and dropped again to a minimum at 12 h after delivery. During days 1 to 3 of postnatal life, cells in the process of exocytosis were less frequent compared to fetuses between day 19.5 and 21.5. Both the number of cells undergoing exocytosis and the number of discharged granules per cell profile first exceeded the fetal values on the 6th postnatal day and were remarkably augmented between days 9-20 of the neonatal period. These data are discussed in relation to the hormone secreting activity of the anterior pituitary gland of perinatal rats.

Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro. I. Differentiation of adrenocorticotropes.

This study investigates the role of the developing diencephalic floor or mesenchymal tissue in the differentiation of ACTH-producing cells. The adenohypophysial primordia of fetal rats on days 12.5 and 13.5 of gestation were treated with collagenase; some primordia were allowed to retain an association with the brain and mesenchyme, but in others the brain and/or mesenchyme were removed. These different combinations of tissues were cultured and examined by immunohistochemical techniques using antisera against pACTH and synthetic alpha-MSH. Removal of mesenchyme alone had little effect on the development of ACTH cells as compared to primordia maintained with brain and mesenchyme. In contrast, removal of the brain with or without mesenchyme on day 12.5 resulted in a marked decrease of ACTH cells accompanied by a mal-growth of adenohypophysial tissue. Such changes were slight when the brain was separated from day 13.5 primordia. Immunoreactive alpha-MSH cells were sparse or absent in all cases. These results suggest that in fetal rats the developing diencephalic floor is essential for differentiation of ACTH cells before day 13.5 of gestation whereas mesenchyme has no apparent effect.

Immunohistochemical study of the responsiveness of LH cells of fetal rats to synthetic LHRH in vitro.

Fetal rat pituitaries on days 17-19 of gestation were maintained in serum-free Medium 199 for 24 h in the presence of 1, 10 and 100 ng/ml of synthetic LHRH. Immunohistochemical examination of such stimulated tissue reveals a complete depletion of immunoreactive material in most of the LH cells, irrespective of the LHRH concentrations tested, though some cells remain weakly immunopositive in the pituitaries of later developmental stages. Once discharge has occurred, there is little reaccumulation of secretory material in LH cells during prolonged incubation for 48 h in LHRH-free medium containing 10% calf serum. The LHRH treatment causes no immunohistochemical change in TSH cells. It is concluded that in fetal rats recently differentiated LH cells can release the secretory product if they are stimulated by hypothalamic LHRH.