Development and differentiation of pituitary cells.

Classically, it was thought that the adenohypophyseal gland originated from the oral ectoderm. Its development has been the object of numerous studies over many years. However, several questions are still raised about its origin, differentiation, and commitment. The adenohypophyseal gland could originate from the anterior ridge of the neural plate. Glandular adenohypophyseal cells are committed very early in embryonic life. Interactions between adenohypophyseal presumptive territory and neighboring tissues can exist very soon, as early as at the open neural stage. The expression of a given phenotype by the committed cells seems to be controlled by a number of differentiation and/or transcription factors. In view of all these studies, performed with the use of different in vivo and in vitro models, classical concepts of the embryology of the adenohypophyseal gland need to be reevaluated. Indeed, many questions remain unanswered concerning the molecular mechanisms of known and unknown factors controlling development of the adenohypophyseal gland.

Fetal development of delta-sleep-inducing-peptide-like immunoreactivity in hypothalamus of guinea pig with special regard to the prenatal colocalization with gonadotropin-releasing-hormone-like immunoreactivity.

Delta-sleep-inducing peptide (DSIP) colocalizes within gonadotropin-releasing-hormone (GnRH)-containing neurons in adult hypothalamus and could play a role in the regulation of hypothalamic-pituitary axis in adults. To support the possibility that DSIP also participates in fetal neuroendocrine events and to demonstrate the ontogenic evidence of coexisting neuropeptides, we have performed a detailed immunocytochemical study of DSIP- and GnRH-immunoreactivity in fetal hypothalamus of guinea pig. Using indirect immunofluorescent and sequential double-immunolabeling (elution-restaining) techniques, the results indicated that DSIP immunoreactivity was initially detected at the 38th day of gestation. In contrast to the first appearance of GnRH immunoreactivity at day 28, therefore, a 10-day delay was found. Such a delay remains as yet unexplained. From its first occurrence, DSIP immunoreactivity was always labeled with GnRH, whereas some of GnRH-immunoreactive structures did not display a DSIP immunoreactivity. But with the growth of fetus, especially before and after birth, a complete overlap between DSIP and GnRH immunoreactivity was observed throughout various regions of hypothalamus. Attention was also focused on prenatal morphological development of DSIP/GnRH- and GnRH-immunolabeled neurons. Initially, labeled neurons were visualized as uni- or bipolar types. Thereafter, their smooth and irregular subtypes could be distinguished. During later fetal age, relatively mature features were evident such as the increase of multipolar and irregularly labeled neurons. Taken together, these data provide, for the first time, anatomical evidence that DSIP exists in fetal hypothalamus and that, like GnRH, it could regulate the hypothalamic-pituitary axis during ontogenesis.

Fetal development and regulation of pituitary cell types.

The ontogenesis of the pituitary gland is considered from anatomical and functional points of view. Embryogenesis of the hypothalamo-pituitary unit involving development of the hypothalamo-hypophyseal portal system is complete during early life as shown in several mammalian species. The ultrastructural characteristics of the different cell types during development are described according to observations made by using immunochemical techniques. The patterns of differentiation of the cell types are reviewed according to studies of pituitary glands from human anencephalic fetuses and encephalectomized rat fetuses as well as in vitro studies of cultured pituitary primordia in synthetic media. The maturation of the neuroendocrine mechanisms controlling the secretion of fetal hormones is also analyzed. During fetal life, the factors implicated in the regulation of pituitary hormone secretion are generally the same as in adults, but the intensity of the response of pituitary cells to their action is variable according to the species, thus reflecting an immaturity in the functioning of certain cell types.

Mapping of neuropeptide Y-like immunoreactivity in the feline hypothalamus and hypophysis.

The distribution of neuropeptide Y (NPY) in the cat hypothalamus and hypophysis was studied with the indirect immunofluorescence technique of Coons and co-workers (Coons, Leduc, and Connolly: J. Exp. Med. 102:49-60, 1955), which provided a detailed map of NPY-like immunoreactive neurons. The immunolabelling was detected in cell bodies, fibers, and terminallike structures widely distributed throughout the whole hypothalamus. A large population of medium-sized NPY-like immunoreactive cell bodies was localized in the area of arcuate nucleus. The number of immunoreactive cell bodies visualized was dramatically increased after intracerebroventricular injections of colchicine. Numerous immunolabelled cell bodies were also visible in the median eminence and scattered in the lateral hypothalamic area. Dense plexuses of NPY-immunoreactive fibers were observed in the arcuate nucleus, internal layer of median eminence, periventricular zone, and paraventricular nucleus. Other regions of hypothalamus displaying numerous NPY-like immunoreactive fibers included dorsal and ventrolateral hypothalamic areas. In contrast, certain hypothalamic areas were almost devoid of NPY-like immunoreactive fibers-namely, the mammillary bodies and suprachiasmatic nucleus. Finally, in neurohypophysis, bright immunofluorescent fibers were observed along the pituitary stalk and penetrating the neural lobe. These results suggest the widespread distribution of the NPY-containing neuronal systems in the cat hypothalamus and hypophysis.

Ontogeny of substance P in the digestive tract, spinal cord and hypothalamus of the human fetus.

The time of appearance and tissue concentrations of substance P-like immunoreactivity (SP-LI) were studied in 53 human fetuses aged 8-21 weeks. Detectable amounts were present at 8 weeks of gestation in available fragments of spinal cord and intestine. Thereafter, the tissue concentrations were highest in spinal cord, intermediate in hypothalamus and lowest in digestive tract. Except for a significant increase in the intestinal wall, the concentrations did not vary from the 8-14 to the 15-21 week period. At chromatography, SP-LI in extracts of spinal cord and intestine was essentially eluted in the volume of the synthetic undecapeptide. Using the indirect immunofluorescence technique, the localization of SP-LI positive structures in the digestive tract was studied in 5 fetuses aged 12-18 weeks. Scarce cell bodies were observed in the myenteric plexus. Nerve fibers were recognized in the muscular layer, in the myenteric plexus and in connective tissue of pancreas. The present results demonstrate the early appearance of SP-LI positive structures both in central nervous system and in the enteric nervous system in the human fetus. In the age range tested, SP-LI concentrations were noticeably higher in spinal cord and hypothalamus than in the digestive tract.

Ontogeny of hypothalamic luteinizing hormone-releasing hormone (GnRH) and pituitary GnRH receptors in fetal and neonatal rats.

Although it is known that LH secretion starts at 17 days of gestation in the fetal rat and that this first LH release is most likely driven by hypothalamic GnRH, an earlier role for GnRH during fetal life has been postulated with the observation that presence of GnRH is important before day 13 of gestation for the differentiation of the pituitary anlage. In order to clarify the different roles of GnRH during fetal life, we have studied the first appearance of GnRH in the fetal brain, the expression of GnRH receptors in the fetal pituitary gland, and the presence of GnRH immunoreactivity within the fetal gonadotrophs. GnRH was present in the earliest brain tissue examined (12 days of gestation). From 12-17 days, GnRH content of fetal brain remained low and then increased markedly by the end of gestation. No immunoreactive GnRH-like material could be detected in rat placental tissue throughout gestation. Binding sites for GnRH were detected as early as 12 days of gestation in fetal pituitary glands. However, binding was very low until 16 days. At 17 days, Scatchard analysis indicated the presence of high affinity, low capacity binding sites [affinity constant (Ka) = 10(10) M-1]. Intracellular presence of GnRH as seen by immunocytochemistry using ultrathin sections prepared by cryoultramicrotomy was first visible at 14 days and started to increase at 16 days. LH was first detectable in the fetal pituitary by RIA at 17 days; FSH was first detectable at 21 days, and PRL at 1 day of postnatal life. Thereafter, neonatal pituitary contents of LH, FSH, and PRL increased linearly with-time, as did the number of pituitary GnRH receptors. At 10 days of postnatal life, pituitary contents of LH and FSH were significantly higher in females than in males. In summary, hypothalamic GnRH appears early in fetal life and potentially can induce differentiation of the pituitary anlage. Conversely, the presence at 15 days of gestation of specific binding sites for GnRH and of intracellular GnRH immunoreactivity in gonadotrophs indicates that the hypophysiotropic action of GnRH clearly precedes the start of LH biosynthesis.

[The co-localization of monoamines and enkephalins in the central nervous system]. / Colocalisation de monoamines et d'enképhalines dans le système nerveux central.

After examination of some particular methodological problems concerning the visualization of monoaminergic and "enkephalin containing" central neurones, the colocalisation of enkephalins in certains monoaminergic neurones is reported. Thus, in rat and cate raphe nuclei many serotoninergic cell bodies display enkephalin-like immunoreactivity (ELI). In the cat locus coeruleus complex most catecholaminergic neurones contain ELI. Further, the synthesis in situ of enkephalins is strongly suggested by the fact that synenkephalin-like immunoreactivity is also detected in these monoaminergic neurones. Synenkephalin is the N terminal part to the bovine medullosurrenal pro-enkephalin. Monoamine-enkephalin colocalisation has been shown in peripheral neurones and adrenal medulla. The possible functionnal significance of the coexistence of monoamine-enkephalins in neurones is discussed. In particular, some hypothetical levels of interaction are considered.

Ontogeny of somatostatin in the human gastro-intestinal tract, endocrine pancreas and hypothalamus.

The appearance, time and distribution of somatostatin in the pancreas, gastro-intestinal tract and hypothalamus were studied comparatively in human foetuses aged 6--32 weeks, by immuno-cytochemistry and radioimmunoassay. Somatostatin was detected by both methods in all segments including the colon. The first cells were observed, and somatostatin was present in measurable amounts at 8 weeks in pancreas, duodenum and intestine, while the peptide was detected at 12 weeks in antrum and colon, at 14 weeks in fundus, and at 16 weeks in hypothalamus. Subsequently, the largest cell population was located in the pancreas, where peptide concentration and age were positively correlated (P less than 0.01, r = 64). From 15 to 21 weeks of age, the mean somatostatin concentration in pancreas (12.4 +/- 1.84 ng/mg) was clearly higher than in hypothalamus (0.05 +/- 0.02 ng/mg) or in any segment of the gut, where values ranged from 0.36 +/- 0.06 (fundus) to 4.74 +/- 0.83 ng/mg (duodenum). The early appearance time of somatostatin, and its specific distribution with preferential location in the pancreas, suggest that the peptide may play a major role for the development of the foetal digestive tract, and that it may be involved in the regulation of other endocrine secretions, especially in the pancreas.

[Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]. / Influence de l'hypothalamus sur la différenciation des cellules présentant une immunoréactivité de type ACTH, beta-LPH (beta-lipotropine), alpha- et beta-endorphines dans les ébauches antéhypophysaires de foetus de rat en culture organotypique.

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.

Immunocytological evidence for oxytocin neurons in the human fetal hypothalamus.

The use of antibodies against oxytocin or neurophysin enabled the detection by immunocytochemistry of oxytocin-neurophysin neurons in the hypothalamus in the human fetus. The perikarya of these neurons are located in the paraventricular and supraoptic nuclei. Immunoreactive neurons occure in the median eminence. The neurophysin immunoreactive neurons were more numerous than the oxytocin immunoreactive neurons. The specificity of the immunocytological reaction was controlled. The first oxytocin-neurophysin neurons are seen as early as the 14th week of gestation.

Immunofluorescence study of LH-RH producing cells in the human fetal hypothalamus.

The use of antibodies to synthetic LH-RH has enabled the detection by immunofluorescence of hypothalamic LH-RH producing cells in the human fetus. The perikarya of these cells are located in the pericommissural and peroptic regions, in the lamina terminalis and in the premamillary region. Reactive axons occur in the median eminence. The first LH-RH producing cells are seen as early as nine weeks of gestation. The specificity of immunocytological reaction has been controlled.