J.M. Félix

Ontogenesis of proopiomelanocortin gene expression and regulation in the rat pituitary intermediate lobe

During ontogenesis, proopiomelanocortin (POMC) mRNA appears in the pituitary intermediate lobe (IL) at embryonic day 16 (E16), rather later than in the hypothalamic arcuate nucleus (E13) or the pituitary anterior lobe (E15). POMC mRNA onset in the IL correlates with the appearance of POMC-derived peptides detected by immunocytochemistry (ICC), indicating that there is probably no time lag between POMC mRNA translation. Subsequently, while the IL lobular organization developed progressively, the number of in situ hybridization- (ISH) and ICC-positive cells increased until after birth. During postnatal development, coinciding with innervation of the IL, the POMC mRNA level in the lobe, measured by quantitative ISH, increased about 4-fold to reach the adult value at weaning. The effects of acute or chronic postnatal treatment with a dopamine antagonist (haloperidol) or a dopamine agonist (bromocriptine) show that the physiological dopaminergic inhibitory control of POMC gene expression operates as early as postnatal day 5. The subsequent increase in mRNA levels despite the inhibitory innervation raises the question of the existence of some unknown positive regulation active during postnatal development.

Expression of the α-subunit of glycoprotein hormones in the pars tuberalis-specific glandular cells in rat, mouse and guinea-pig

Abstract.The nature of the hormone(s) secreted by the pars tuberalis (PT) is still unknown. This pituitary lobe is mainly formed by specific glandular cells that differ in their ultrastructural features from the other adenohypophysial cell types. Data from the literature indicate the presence of thyroid-stimulating hormone immunoreactivity in the PT-specific cells of the rat and the Djungarian hamster but not of other species, including the mouse and guinea-pig. The PT also encloses variable numbers of pars distalis cells, essentially gonadotrophs that are mainly dispersed in its caudal area. We studied the expression of the glycoprotein hormone α-subunit in the PT of the rat, mouse and guinea-pig by in situ hybridization and immunocytochemistry. In situ hybridization, using an oligonucleotide probe complementary to rat cDNA sequence 196–237 revealed the expression of the α-subunit gene throughout the PT of the rat and the mouse; in the guinea-pig, the probe labelled no pituitary cells. Light- and electron-microscopic immunocytochemistry demonstrated α-subunit immunoreactivity in the secretory granules of the PT-specific cells in the three species examined. These cells did not react with a specific antibody against the β-subunit of luteinizing hormone, an antibody that labelled scattered gonadotrophs. The present data suggest that hormone(s) produced by the PT-specific glandular cells are, at least partly, related to glycoprotein hormones.

Pituitary adenylate cyclase-activating polypeptide transduces through cAMP/PKA and PKC pathways and stimulates proopiomelanocortin gene transcription in mouse melanotropes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were characterized and their function investigated in mouse pituitary neurointermediate lobe explants. We show that mouse neurointermediate lobes can be maintained for up to 1 month in defined medium. After 8 days in culture, these explants are devoid of any of the original tyrosine hydroxylase or glutamate decarboxylase immunoreactive fibers, which in situ innervate the melanotropes. Under these culture conditions, no mitotic activity is detectable in melanotropes and these cells remain sensitive to physiological regulation such as dopamine and corticotropin-releasing hormone. Using in situ hybridization and polymerase chain reaction, we show that in situ and in neurointermediate lobe explants, melanotropes express PACAP receptor type I isoforms that transduce through the cAMP and inositol phosphate pathways. In neurointermediate lobe explants, PACAP 27 and PACAP 38 (10(-8) M) stimulate cAMP accumulation whereas PACAP 38 but not PACAP 27 stimulates inositol phosphate breakdown. However, both ligands are potent stimulators of proopiomelanocortin (POMC)-derived peptides exocytosis and POMC gene transcription. In addition, stimulation of POMC gene transcription is mediated both by cAMP and by inositol phosphate pathways. Taken together, our data suggest that PACAP is a major regulator of melanotrope functions.

Expression of neural cell adhesion molecules, NCAMs, and their polysialylated forms, PSA-NCAMs, in the developing rat pituitary gland

Neural cell adhesion molecules (NCAMs) can undergo post-translational modifications, such as the addition of polysialic acid chains, thus generating PSANCAMs, which are expressed mainly during development. Since polysialylation considerably modifies NCAM adhesivity, expression of NCAMs and PSANCAMs has been investigated in the developing hypophysis by immunohistochemistry. At embryonic day 13 (E13), an antibody against NCAM outlined all cellular profiles in the entire Rathkes pouch; this labelling persisted until adulthood. NCAM expression increased in all lobes during development and concerned all pituitary cell types. In contrast, at E13, PSA-NCAMs were only detected in the neural lobe, solely constituted of pituicytes at this stage, and the tuberal lobe, the only lobe expressing hormonal mRNA at the same stage. PSA-NCAMs expression increased in the neural lobe at E17 with the arrival of the neurosecretory fibres and persisted into adulthood. In the anterior lobe, PSA-NCAMs appeared at E15 where their distribution was similar to that of the differentiating corticotrophic cells; at subsequent stages, their expression extended to the whole anterior lobe. Only two cell types, corticotrophic and somatotrophic cells, remained labelled in the adult gland. In the intermediate lobe, melanotrophic cells never expressed PSA-NCAMs but these were expressed on folliculo-stellate cells at birth, preceding the onset of innervation. These results suggest that NCAMs and PSA-NCAMs play a role in pituitary histogenesis, cell differentiation and neurointermediate lobe innervation.

Co-culture of hypothalamic neurons and melanotrope cells: a model to study synaptogenesis between central neurons and endocrine cells

As a first step towards elucidating mechanisms involved in neuroendocrine synaptogenesis, we developed a model of co-culture based on hypothalamic-intermediate pituitary interactions. Dissociated hypothalamic neurons from fetal rats at embryonic day 15 were cultured in a defined medium together with melanotrope cells of the pituitary intermediate lobe from neonatal rats. In these co-cultures, establishment of synaptic contacts between GABAergic or dopaminergic neurons and an endocrine target cell the melanotrope cell, was studied by morphofunctional approaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and alpha-melanocyte-stimulating hormone, we demonstrated morphological contacts between GABAergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synapsin I, we showed a modification of synapsin I immunoreactivity from diffuse to punctate distribution correlated with the establishment of contacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrophysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic currents, which occurred either spontaneously and/or could be evoked chemically by 50 mM KCl or 100 microM kainate. The proportion of the melanotrope cells receiving functional synaptic inputs increased until 10 days in culture, a stage at which virtually all melanotrope cells in contact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approaches.

L1 and laminin : their expression during rathypophysis ontogenesis and in adult neurohemal areas

L1 is a murine multidomain glycoprotein implicated in cell aggregation, fasciculation,neurite outgrowth and synaptogenesis. Laminin, a trimeric polypeptide, is implicated in neuronalsurvival, growth cone guidance, neurite outgrowth and cell differentiation. Laminin can alsointeract with the cell adhesion molecule L1. Their expressions were investigated from embryonicday 15 (E15) to adult in the rat hypophysis, and in adult neurohemal zones.

Ontogeny of glucocorticoid and D2 receptors in the rat pituitary : an in situ hybridization study

The expression of glucocorticoid and D2 dopamine receptors (GR and D2R) during rat pituitary ontogenesis was studied by in situ hybridization (ISH). On early stages, E13-E14, a weak specific signal for GR mRNA was obvious in the whole Rathkes pouch (RP) whereas subsequently, from E17-E18, strong labelling was restricted to the anterior lobe (AL) and the neural lobe (NL). At the same time, D2R mRNAs appeared in the intermediate lobe (IL) and the long isoform of the D2R (D2R 444) was detectable with specific probes. On the postnatal stages, until adult, GR mRNA, if present, was always undetectable in the IL using the conventional ISH technique. These data indicate a possible early regulation of POMC gene expression by glucocorticoid in corticotrophic cells of the AL and by dopamine in the melanotrophic cells of the IL. The possibility of a negative regulation of GR mRNA by dopamine (DA) in the IL as soon as E17 is discussed.

Activation of protein kinase C differentially regulates corticotropin-releasing factor-stimulated peptide secretion and cyclic AMP formation of intermediate and anterior pituitary cells in culture

The present study was aimed at investigating the effect of protein kinase C (PKC) activation on CRF receptor function of proopiomelanocortin (POMC) cells in culture. Incubation of tissues with the phorbol ester PMA selectively potentiated corticotropin-releasing factor (CRF)-stimulated ACTH secretion and cyclic AMP formation of anterior pituitary (AP) cells, while, in sharp contrast, it failed to similarly affect intermediate pituitary (IP) cells and AtT-20 corticotrophs exposed to CRF. Unexpectedly, however, long-term treatment of cultures with PMA, which depletes cell stores of PKC, resulted in a similar dramatic attenuation of stimulated peptide release from both corticotrophs and melanotrophs, while being without significant effect on cyclic AMP production. Exposure of cells to PMA did not change either basal or CRF-enhanced levels of POMC mRNA. We conclude that activation of PKC fails to synergize with CRF-mediated signalling in IP and AtT-20 cells, although optimal CRF receptor expression requires the presence of a functional kinase C pathway, thus suggesting cross-talks between both messenger systems.

Expression of the c-ets 1 gene in the hypothalamus and pituitary during rat development

The Ets gene family codes for transcription factors containing a conserved DNA binding domain: the Ets-binding domain. The proto-oncogene c-ets1 is highly expressed in lymphoid organs and in developing mesodermal-originating structures. We studied c-ets1 gene expression in the developing rat hypothalamo-hypophyseal system, using in situ hybridization on paraformaldehyde-fixed frozen sections. At embryonic day 12 (E12) and E13, cells synthesizing c-ets1mRNA are found in the neural tube where they form small, heavily labeled strand-like and punctate structures; positive mesenchymatous cells, corresponding to the surface capillary network, surround the brain and hypophysis. C-ets1mRNA is synthesized from E14 in the neural pituitary and E15 in the adenohypophysis, during angiogenesis; no c-ets1mRNA is detected in the avascular intermediate pituitary at any stage. Strand-like c-ets1mRNA labeling is intense from E14 to E21 in the diencephalon. This labeling is also detected during perinatal stages in the hypothalamic magnocellular nuclei, one of the most richly vascularized brain areas. In the rat hypothalamo-hypophyseal system, c-ets1 gene expression is maximal during fetal and perinatal stages and progressively decreases thereafter until adulthood. The spatio-temporal correlation observed between c-ets1 gene expression and blood vessel formation in the rat hypothalamus and pituitary suggests a role for c-ets1 in angiogenesis in this system.

Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium

SummaryOrganotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 35S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2–3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against α-melanocyte-stimulating hormone (αMSH), γ3 and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of αMSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the αMSH content of the explants and αMSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in αMSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.