Expression of oestrogen receptor alpha during development of the skeleton in mice fetuses: immunohistochemical study.

Sequential pattern of ossification and expression of oestrogen receptor alpha (ERalpha) during development of the skeleton in male and female mice fetuses was investigated. Twenty-seven mice fetuses of gestational age between 14.5 and 18.5 days post coitum (p.c.) were examined by haematoxylin-eosin and toluidine blue staining to determine the ossification. The presence of ERalpha was detected by immunostaining using ERalpha-specific antibodies. Ossification centres were determined in fetuses of 14.5 days p.c. of both sexes in the base of skull, ribs and front limbs, while in the mandible ossification was observed only in female fetuses at that age. ERalpha was found in all investigated tissues in which the occurrence of ossification centres was determined. ERalpha was first detected in some tissues involved in ossification at 14.5 days p.c. in fetuses of both genders. There were some minor gender differences in the pattern of ERalpha expression. ERalpha was localized in the metatarsal chondrogenic condensations at 14.5 days p.c. and in phalangeal chondrocytes at 17.5 and 18.5 days p.c. only in females. ERalpha-positive osteogenic cells at 14.5 days p.c. in the mandible were seen only in females. At 16.5 days p.c. male but not female fetuses expressed ERalpha in the vertebrae. Our findings support the view that ERalpha protein is found in the tissues that undergo bone formation and that ERalpha expression in these tissues shows only minor gender differences in mice fetuses.

The long isoform of the rat thyrotropin-releasing hormone receptor down-regulates Gq proteins.

A cDNA encoding the long isoform of the rat thyrotropin releasing hormone (TRH) receptor was expressed stably in HEK-293 cells. Polymerase chain reaction analysis confirmed expression of mRNA encoding only the long and not the short isoform. Activation of this receptor with TRH caused a large stimulation in production of inositol phosphates but did not produce either activation of basal or inhibition of forskolin-amplified adenylyl cyclase activity. Sustained exposure of these transfected cells to TRH resulted in a substantial reduction in cellular levels of Gq alpha-like immunoreactivity from some 12 to 5 pmol/mg of membrane protein without significant alterations in cellular levels of the alpha subunits of Gs, Gi1, Gi2, Gi3, or Go. Equivalent experiments in GH3 cells also indicated a marked down-regulation of Gq alpha/G11 alpha. Dose-response curves indicated that 20 nM TRH produced half-maximal down-regulation of cellular Gq-like immunoreactivity in the transfected HEK-293 cells and that half-maximal loss was produced within 3-4 h. Separation of the transfected HEK-293 cell membranes in SDS-polyacrylamide gel electrophoresis conditions able to resolve individual members of the Gq family of G-proteins demonstrated the presence of two related G-proteins. Both of the expressed Gq-like G-proteins were observed to be down-regulated in parallel by TRH. The similarity of dose-response curves and time-courses for loss of the two G-proteins indicates that the long isoform of the rat TRH receptor does not functionally select between these two transducer proteins. In GH3 cells both Gq alpha and G11 alpha, which are expressed at similar levels, were observed to be down-regulated equivalently by treatment with TRH.

Calcium waves and dynamics visualized by confocal microscopy in Xenopus oocytes expressing cloned TRH receptors.

Laser scanning confocal microscopy was used to analyse changes in free cytosolic calcium ([Ca2+]i) in Xenopus laevis oocytes expressing the cloned rat TRH receptor in response to TRH. In oocytes expressing TRH receptors, TRH invariably evoked a dose-dependent, biphasic calcium response. This response consisted of an initial transient planar wave of calcium propagating just below the surface of the membrane followed by a slower, secondary calcium phase. The TRH antagonist, chlordiazepoxide, markedly inhibited this calcium wave. The origins of calcium involved in this biphasic response were investigated using a variety of intra- and extra-cellular calcium antagonists. The intracellular calcium antagonists thapsigargin and TMB-8 reduced the initial and to a lesser extent the secondary phase of the planar calcium wave. In contrast, EGTA and the calcium channel blocker nifedipine produced a profound inhibition of the secondary phase while the initial phase was only slightly reduced. These results indicate that the release of intracellular calcium is predominantly responsible for the initial phase of the calcium wave while the influx of extracellular calcium is mainly involved in the secondary phase. Qualitative changes in the patterns of calcium release induced by TRH were observed following pretreatment with intracellular calcium antagonists. Following pretreatment with these compounds, TRH induced spiral or regenerative calcium waves. Addition of EGTA to the extracellular medium did not alter these responses confirming the importance of intracellular calcium in the generation of these spiral calcium waves. This study demonstrates the nature and multiplicity of regulating mechanisms of [Ca2+]i following activation of TRH receptors expressed in Xenopus oocytes.

Dopamine D2 receptor mRNA in the pituitary during the oestrous cycle, pregnancy and lactation in the rat.

We have used the technique of quantitative in situ hybridization in order to study the changes in the levels of expression of D2 receptor mRNA in the anterior pituitary gland of female rats at different stages of the reproductive cycle. Plasma prolactin levels in the same animals were determined by radioimmunoassay. Rats in the prooestrous, oestrous, dioestrous 1, dioestrous 2 phases of the oestrous cycle and in pregnant, lactating, ovariectomised and ovariectomised animals treated with diethylstilbestrol (DES) have been examined. Our results show that expression of D2 receptor mRNA in the anterior pituitary gland varies during the oestrous cycle, with the lowest expression measured during oestrus. Expression levels increased during dioestrus 1, reaching the highest values in dioestrus 2 and declining again in prooestrus. Expression of D2 receptor mRNA was reduced during pregnancy when compared to lactating animals. In ovariectomised animals, the level of D2 receptor mRNA was similar to that observed in intact animals during oestrus. Ovariectomised animals treated with DES showed dramatically increased prolactin levels, while D2 receptor mRNA remained low. Prolactin secretion might be controlled not only by variations in the release and plasma concentrations of dopamine itself, but also by modulation of D2 receptor expression in pituitary cells. Our results suggest that the variations in D2 receptor density in the anterior pituitary cells at different physiological states are, at least to some extent, regulated at the level of gene expression.

Distribution of thyrotrophin-releasing hormone receptor messenger RNA in rat pituitary and brain.

The distribution sites of messenger RNA encoding for the thyrotrophin-releasing hormone receptor have been studied in rat pituitary and brain. A specific 35S-labelled riboprobe generated from a rat thyrotrophin-releasing hormone receptor complementary DNA clone was used to perform in situ hybridization experiments on brain and pituitary sections. A positive hybridization signal was found in the anterior lobe of the pituitary gland, the intermediate and posterior lobes were negative. Hybridization was also detected in different areas of the brain. These areas include distinct regions in the olfactory system, septal area, amygdaloid complex, cerebral cortex, hypothalamus, hippocampus, basal ganglia and the motor nuclei of cranial nerves in brainstem. This study has shown for the first time the exact site of thyrotrophin-releasing hormone receptor expression in the central nervous system. These results correlate well with regions thought to possess thyrotrophin-releasing hormone recognition sites.

Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland.

We have isolated the TRH receptor (TRH-R) from a rat anterior pituitary cDNA library, determined its sequence and examined its functional characteristics. Expression studies were carried out in Xenopus oocytes and in COS-7 cells. Microinjection of sense RNA transcripts into Xenopus oocytes showed electrophysiological responses of between 800 and 1000 nA under voltage-clamp conditions. COS-7 cells were transiently transfected with the cDNA clone under the control of a cytomegalovirus promoter and inositol phosphate (IP) measurements carried out. In TRH-R transfected cells, TRH (100 nM) produced an approximately twofold increase in total IP production. In-situ hybridization in the rat anterior pituitary revealed a heterogeneous distribution of label, a characteristic pattern of TRH-R expression. The rat 3.3 kb insert coded for a protein of 411 amino acids compared with 393 for the mouse protein. Over its length, the rat TRH-R protein showed considerable homology with that of the mouse, except for a deletion of 232 bp in the 3'-coding region. This deletion did not appear to affect the functional characteristics of the receptor, as shown by electrophysiological studies with Xenopus oocytes and by transfection of the cDNA into COS-7 cells. The sequence given for the 3'-untranslated region is 1.5 kb longer than that reported for the mouse receptor, and extends to the poly(A) tail.

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland.

A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus oocytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor.