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The homeodomain transcription factor Six1 and its modulator, the protein phosphatase Eya1, cooperate to promote cell differentiation and survival during mouse organ development. Here, we studied the effects caused by loss of eya1 and six1 function on pituitary development in zebrafish. eya1 and six1 are co-expressed in all adenohypophyseal cells. Nevertheless, eya1 (aal, dog) mutants show lineage-specific defects, defining corticotropes, melanotropes, and gonadotropes as an Eya1-dependent lineage, which is complementary to the Pit1 lineage. Furthermore, eya1 is required for maintenance of pit1 expression, leading to subsequent loss of cognate hormone gene expression in thyrotropes and somatotropes of mutant embryos, whereas prolactin expression in lactotropes persists. In contrast to other organs, adenohypophyseal cells of eya1 mutants do not become apoptotic, and the adenohypophysis remains at rather normal size. Also, cells do not trans-differentiate, as in the case of pit1 mutants, but display morphological features characteristic for nonsecretory cells. Some of the adenohypophyseal defects of eya1 mutants are moderately enhanced in combination with antisense-mediated loss of Six1 function, which per se does not affect pituitary cell differentiation. In conclusion, this is the first report of an essential role of Eya1 during pituitary development in vertebrates. Eya1 is required for lineage-specific differentiation of adenohypophyseal cells, but not for their survival, thereby uncoupling the differentiation-promoting and anti-apoptotic effects of Eya proteins seen in other tissues.

In addition to regulating anterior pituitary function by being released from the median eminence, mammalian neurotensin (NT) may also exert an autocrine or a paracrine action within the anterior pituitary. In this study, using double immunostaining with elution restaining, we identified the specific anterior pituitary cells which express NT immunoreactivity (NT-IR) during the rat oestrous cycle. In the normal cycling rat, NT-IR was present in both gonadotrophs and thyrotrophs and displayed plastic changes along the oestrous cycle. Both the number of TSH-NT positive cells and the intensity of immunological reaction were elevated during dioestrus, and decreased through pro-oestrus and early oestrus. NT-IR was also high in both follicle stimulating hormone (FSH)- or luteinizing hormone (LH)-positive cells during early pro-oestrus, and decreased during late pro-oestrus. Treatment of intact rats with either the anti-oestrogens Tamoxifen or LY117018, or the anti-progestagen RU486 prevented the normal expression of NT-IR in thyroid-stimulating hormone (TSH)-, FSH-, and LH-positive cells during pro-oestrus. Bilateral ovariectomy induced a dramatic reduction in the number of NT-IR cells. This effect was completely prevented by treatment of ovariectomized rats with oestradiol and progesterone, and was unaffected by the concurrent administration of a GnRH antagonist. Furthermore, administration of an anti-oestrogen together with an anti-progestagen to ovariectomized-oestrogen, progesterone-treated rats, blocked the stimulatory effect of ovarian hormones on NT-IR in anterior pituitary cells. These findings demonstrate that, in female rats, NT is specifically localized in gonadotrophs or thyrotrophs. In addition, they strongly suggest that changes in circulating concentrations of ovarian steroids may control both NT synthesis in, and release from, these cells.

Glucose-6-phosphatase is generally accepted as a functional component of rough endoplasmic reticulum and has been histochemically examined in many organs. The aim of this study is to know the ultracytochemical localization of glucose-6-phosphatase in each type of hormone-producing cell constituting the anterior pituitary gland in the rat. Pituitaries of male Sprague-Dawley rats were perfused with 1.5% glutaraldehyde from the left ventricles. After buffer washing 40 microns sections were incubated in the medium of Hugon et al. for 60 min at 37 degrees C. The sections were then postfixed with 1% osmium tetroxide, embedded in epoxy resin and observed under an electron microscope. The reaction product for glucose-6-phosphatase was observed in the lumen of rough endoplasmic reticulum and nuclear envelope of all anterior pituitary cells. The enzyme activities in thyroid-stimulating hormone-producing cells and luteinizing hormone/follicle-stimulating hormone-producing cells (LH/FSH cells) were stronger than those in growth hormone-producing cells and prolactin-producing cells; adrenocorticotropic hormone-producing cells and folliculo-stellate cells presented intermediate activity. In LH/FSH cells, the activity in dilated cisternae of endoplasmic reticulum had weaker density than that in flattened cisternae. In addition, substantial reaction product was also frequently observed in the cis saccules of the Golgi apparatus. These findings suggest that glucose-6-phosphatase may play different functional roles in hormone synthesis within different types of anterior pituitary cells.

Dwarf tyrosine hydroxylase-human GH (TH-hGH) transgenic mice carrying the hGH reporter gene targeted by the TH promoter express hGH in those regions of the hypothalamus responsible for regulation of pituitary GH secretion. Central expression of the hGH gene decreases GH-releasing hormone (GHRH) and increases somatostatin, which ultimately impacts on pituitary function by reducing the overall amount of GH produced. In the present study, we sought to determine if the reduction of pituitary GH in TH-hGH mice could be attributed to a decrease in somatotrope cell numbers and/or an impairment of somatotrope function. Pituitaries from TH-hGH or wild-type (WT) male and female mice were enzymatically dispersed, counted, and immunostained for GH, PRL, TSH, and ACTH. The total number of pituitary cells recovered from TH-hGH pituitaries was approximately one-half of that from WT controls. However, the proportion of cells that stained for GH and PRL were virtually identical (males, GH-TH-hGH, 58.1 +/- 1.0% [mean +/- SEM] vs. WT, 60.7 +/- 1.0%; PRL-TH-hGH, 43.4 +/- 2.2% vs. WT, 43.1 +/- 0.7%; females, GH-TH-hGH, 47.9 +/- 2.3% vs. WT, 41.5 +/- 3.5%; PRL-TH-hGH, 43.3 +/- 3.2% vs. WT, 47.1 +/- 3.3%). In contrast, percentages of both TSH- and ACTH-containing cells were increased in TH-hGH pituitaries relative to controls (males, TSH-TH-hGH, 15.1 +/- 2.3% vs. WT, 9.6 +/- 1.5%; ACTH-TH-hGH, 24.5 +/- 2.5% vs. WT, 10.9 +/- 0.9%; females: TSH-TH-hGH, 11.3 +/- 0.7% vs. WT, 7.5 +/- 0.6%; ACTH-TH-hGH, 19.8 +/- 1.6% vs. WT, 9.3 +/- 0.8%; P < 0.05). Calculation of the absolute number of each cell type per pituitary demonstrated TH-hGH mice to have about one-half the number of GH and PRL cells, whereas TSH and ACTH cell populations were comparable with that of their WT counterparts. Immunocytochemical localization of GH cells within pituitary sections from TH-hGH mice revealed that somatotropes were confined primarily to the lateral wings of the adenohypophysis, in contrast to the heterogeneous distribution of GH-immunostained cells in WT pituitaries. To assess the functional capacity of the somatotrope populations, pituitary cells from TH-hGH and WT mice were challenged with mouse GHRH (0.01-10 nM). The quantity of GH released (as assessed by both RIA and reverse hemolytic plaque assay) under basal and stimulated conditions did not differ among TH-hGH and WT pituitary cell cultures. Similarly, GHRH induced intracellular cAMP levels were comparable. These results indicate that proliferation of pituitary somatotropes and lactotropes is much more sensitive to changes in GHRH input than is the capability of developing regulated GH secretory function.

The anterior pituitary provides a model to study the molecular mechanisms responsible for emergence of distinct cell types within an organ. Dwarf mice (Snell) that express a mutant form of the tissue-specific POU-domain transcription factor Pit-1 fail to generate three cell types, including the thyrotrope (S. Li, E. B. Crenshaw, E. J. Rawson, D. S. Simmons, L. Swanson and M. G. Rosenfeld (1990), Nature 347, 528-533). Analyses of wild-type and Pit-1-defective mice, presented here, have revealed that thyrotropes unexpectedly arise from two independent cell populations. The first population is Pit-1-independent and appears on e12 in the rostral tip of the developing gland, but phenotypically disappears by the day of birth. The second is Pit-1-dependent and arises subsequently in the caudomedial portion of the developing gland (e15.5), following the initial expression of Pit-1 in this region. The failure of caudomedial thyrotrope cells to appear in the Snell dwarf, and the observation that Pit-1 can bind to and transactivate the TSH beta promoter, apparently enhanced by its phosphorylation, suggests that Pit-1 is directly required for the appearance of this distinct population that serves as the precursors of the mature thyrotrope cell type. These data suggest that different molecular mechanisms, based on the actions of distinct transcription factors, can serve to independently generate a specific cell phenotype during mammalian organogenesis.

The expression of the alpha-subunit of the glycoprotein hormones was studied in the developing rat pituitary by in situ hybridization and immunocytochemical techniques. The alpha-subunit mRNA labelling was detected in Rathke's pouch at 12 days' gestation (E12); at E13, it was exclusively concentrated in the pars tuberalis primordium (the lateral lobes) where its intensity rapidly increased at following stages. The alpha-subunit was first immunocytochemically detectable in the pars tuberalis at E14. Labelling of the whole pars tuberalis primordium with both techniques indicated that the specific glandular cells were involved. Immunocytochemical detection of bromodeoxyuridine, injected into the pregnant rat 4 h prior to fixation, revealed a total arrest of cell divisions in the pars tuberalis between E13 and E17, which was not the case in the pars distalis or the pars intermedia at any stage. In the pars distalis, the onset of alpha-subunit expression was detected much later than in the pars tuberalis, at E16-17 by in situ hybridization and at E17-18 by immunocytology. The functional significance of the early secretory differentiation of the pars-tuberalis-specific cells remains to be established.

The first appearance of adenohypophysial cells in the developing rainbow trout embryo was studied by immunocytochemistry. Antibodies generated against the beta subunits of coho salmon gonadotropins (GTH I and GTH II), the beta subunit of human thyroid stimulating hormone (TSH), chum salmon prolactin (PRL), chum salmon growth hormone (GH), and synthetic alpha-MSH were used as immunocytochemical probes. The pituitary anlage was first recognized at developmental stage 21 (18 days postfertilization). At this stage, immunoreactive (ir) PRL and alpha-MSH cells were identified in the rostral and caudal regions of the pituitary, respectively. Cells containing ir-GH and ir-adrenocorticotropin (ACTH) appeared at stage 28 (35 days postfertilization). At the last stage before hatching (stage 29, 42 days postfertilization) ir-TSH cells were identified. Ir-GTH I cells were first observed in the pituitary at stage 32 (15 days after hatching), when mitosis of gonadal germ cells also was observed. At stage 35 (35 days after hatching), meiotic figures were first observed in a few germ cells of some fish indicating that gonadal sex differentiation probably begins at about this time. Cells containing ir-GTH II were not apparent in the pituitary at any stage of embryonic or larval development (up to stage 35) and were not present in pituitaries of trout at 6 months of age when the ovaries were in the perinucleolus stage and when only spermatogonia, but no spermatocytes were present in the testis. These observations suggest that GTH I, but not GTH II, may regulate initial gonadal growth and development in the embryonic and larval rainbow trout.(ABSTRACT TRUNCATED AT 250 WORDS)

The pituitary glycoprotein hormone, TSH, is synthesized and secreted by thyrotrophs in the anterior pituitary. Similarly to other pituitary glycoproteins, LH and FSH, it consists of two non-covalent subunits, alpha and beta. The alpha-subunit is common to all three hormones, while the beta-subunit is unique for each hormone. Both subunits are synthesized as separate peptides from distinct mRNA. T3 is the most important negative regulator of the TSH gene. The T3-T3 receptor complex binds to the specific promoter region, T3 response element (TRE), of its target genes and regulates the transcriptional activities. Although TREs have been studied in many genes, the TSH beta-TRE has not yet been fully identified. The mechanism how T3 suppresses TSH beta gene remains undetermined. Another important TSH gene regulator, TRH, is considered to stimulate the transcriptional activity by binding to the different promoter region of TSH beta gene. The interaction between TRH-response element and Pit-1/GHF-1, the pituitary specific factor, -response element is interesting. TRH affects TSH glycosylation, which is important to the biological activity. TSH in central hypothyroid patients is known to have a lower ratio of biological per immunological activity. Recently, in cases with congenital isolated TSH deficiency, a point mutation has been identified in the CAGY region of TSH beta gene, which is critical for dimer formation between alpha- and TSH beta-subunits.

Hypophysial prolactin (PRL) and thyrotropin (TSH) cells of Rana perezi were examined after treatment in vitro with synthetic thyrotropin-releasing hormone (TRH). Ultrastructural morphometry applied to PRL and TSH immunoidentified cells estimated the volume density (Vv) of the secretory granules (SG), rough endoplasmic reticulum (ER), and Golgi complex (GC) as well as the numerical density (Nv) of the granules. Hemipituitaries were cultured in a superfusion system with or without 100 ng TRH/ml for 4, 14, or 24 hr. PRL cells showed significant degranulation (42%) of medium size SG after 14 hr of stimulation, whereas the biosynthetic machinery (ER and GC) was significantly developed after 4 hr of TRH culture (increase of Vv of GC, 1.5-fold, and ER, 1.3-fold, in comparison to the control). Most of these changes remained after 24 hr of TRH treatment. Compared with controls, TRH-treated TSH cells differed only after 24 hr when SG showed degranulation (40%), mainly of the medium size ones, and Vv of GC (1.6-fold) and ER (1.3-fold) increased. These results suggest that TRH acts directly on amphibian PRL and TSH cells stimulating hormonal synthesis and release. The time courses of responses to TRH differed in that PRL cells gave an immediate response while TSH cells gave a more delayed reaction.

We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating polypeptide (PACAP-38; 0.1-100 nmol/l) caused an increase in the release of GH, ACTH, LH and alpha-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1.3-fold (P less than 0.05), ACTH by 1.9-fold (P less than 0.05), LH by 3.5-fold (P less than 0.001) and alpha-subunit by 2.0-fold (P less than 0.005) and the accumulation of intracellular cyclic AMP by greater than 2-fold (P less than 0.001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and alpha-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and alpha-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and alpha-subunit release (10 nmol/l) added together with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 0.1 mumol/l) had no greater effect on LH and alpha-subunit release than TPA alone over a 4 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin beta subunit (TSH beta) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSH beta mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSH beta mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its medication by estradiol.

Immunocharacteristics of the pituitary pars distalis cell types of the musk shrew, Suncus murinus, were studied by the unlabeled antibody enzyme technique, using peroxidase-antiperoxidase or avidin-biotin-peroxidase complex. The thyrotropin (TSH)-, gonadotropin (GTH)-, corticotropin (ACTH)-, prolactin (PRL)-, and growth hormone (GH)-secreting cells of the PD were identified on the basis of their immunoreactivity with different heterologous antisera. The TSH cells showed specific immunoreactivity with antisera against human (h) TSH beta and rat (r) TSH beta. Cells showing immunoreactivity with the antisera against hLH beta and ovine (o) LH beta were designated as GTH cells as no immunoreactivity was observed with antisera against hFSH beta and oFSH beta. The ACTH cells as well as the cells of the pars intermedia were revealed by anti-ACTH1-24 and anti-ACTH1-10 sera. Whereas the PRL cells were recognized by their immunoreactivity with antisera against hPRL and oPRL, the GH cells were identified with anti-hGH, anti-oGH, and anti-bovine (b) GH sera. TSH and GTH, TSH and ACTH, GTH and ACTH, ACTH and GH, ACTH and PRL, and GH and PRL cells were visualized in the same section using the dual immunoperoxidase technique. Comparison of the immunohistochemically identified cells with those described histochemically reveals several discrepancies, which expose the limitations of the latter techniques identifying adenohypophysial cells.

Immunocytochemistry and in situ hybridization were used to demonstrate beta thyroid-stimulating hormone (TSH) immunoreactivity and beta TSH messenger RNA (mRNA) in the same tissue section of rat pituitary. Sections (10 micronsP of 4% paraformaldehyde-fixed pituitaries from surgically thyroidectomized and intact male rats were first hybridized with a 32P-labelled complementary RNA beta TSH probe and were then immunostained for beta TSH. In both sets of animals beta TSH mRNA and beta TSH immunoreactivity were simultaneously localized to many thyrotrophs, although there was considerable heterogeneity in mRNA labelling intensity between individual cells. In hypothyroid rats more cells were positive with both probe and antiserum, and the intensity of mRNA labelling in most quantified by direct apposition of hybridized sections to autoradiography film, and the image grey levels were measured using an image analyser to convert them into equivalent amounts of radioactivity. In hypothyroid rats this amount was 22 times greater than in controls (control 4.77 +/- 0.84 attomoles mm-2; hypothyroid 107.06 +/- 14.2 attomoles mm-2; mean +/- SEM p less than 0.001). In situ hybridization has been used in combination with immunocytochemistry, to demonstrate beta TSH and mRNA and immunoreactivity at a cellular level in rats with different thyroid status.

Cytoplasmic annulate lamellae have been observed in frog (Rana ridibunda) adenohypophysis pars distalis from normal spring animals and from others which were submitted to experimental conditions inducing selective activation of different cell types. Cell activation, because of either the normal active period in the frog cycle or the experimental treatments, seems to be correlated with the occurrence annulate lamellae. These annulate lamellae consist of a succession of two relatively parallel membranes interrupted periodically by discontinuities similar to nuclear pores. Sometimes they have been observed connected to endoplasmic reticulum.

In order to compare the functional state of the anterior pituitary of spontaneously hypertensive rats (SHR) with that of nonmotensive Wistar Kyoto rats (WKR), the anterior pituitary was examined by morphometry and autoradiography at the level of electron microscopy. The relative number and the relative volume of thyrotrophs in the anterior pituitary were significantly greater in SHR compared with age-matched WKR at 0, 7, 30-33 days and 10 months of age, while the relative number of somatotrophs in SHR was significantly smaller at 1 and 10 months of age. Electron microscope autoradiographic analysis of uptake of 3H-lysine by thyrotrophs of both strains at the age of approximately one month showed that 3H-lysine was incorporated into protein and transported finally to secretory granules which migrated to near the cell membrane to be discharged. Silver grains were significantly more numerous over the thyrotrophs of SHR than over those of WKR at 30 min, 1 h, and 4 h after the injection of 3H-lysine. The present study has ascertained morphologically than a congenital hypersynthesis of TSH by the anterior pituitary occurs in SHR.

The appearance and relative distribution of immunoreactive gonadotropic and thyrotropic cells of the anterior pituitary of normal rat fetuses were studied by immunocytology with anti-rat luteinizing hormone serum, anti-porcine luteinizing hormone beta serum, anti-bovine thyrotropic hormone serum after saturation with bovine luteinizing hormone. The thyrotropic cells appeared at 17 days of gestation. They were preferentially localized in the dorsal part of the anterior lobe. The gonadotropic cells localized in the ventral part of the same lobe, appeared 1 day later. The number of immunoreactive cells of the two populations increased until the end of gestation. In the pituitaries of 21-day-old rat fetuses encephalectomized at 16 days of gestation, the same number of gonadotropic and thyrotropic cells was observed as in control fetuses at the same age. Gonadotropic cells were also observed in rat adenohypophysial primordia explanted at 14 days of gestation and maintained in organ culture for 7 days. Thyrotropic cells were stained when the primordia were explanted at 15 days of gestation and cultured for 6 days. No immunoreactive gonadotropic or thyrotropic cells were detected when explantation was performed before 14 days of gestation. These results clarify the problem of the role of hypothalamus on the differentiation of these two cell types.

An influence of thyrotrophin-releasing hormone (TRH) on TSH heterogeneity in close association with de-novo biosynthesis was studied in rat anterior pituitary glands. Hemipituitary glands from adult male rats were incubated in Krebs-Henseleit-glucose media containing [3H]glucosamine and [14C]alanine for 3 and 6 h in the presence or absence of 10 ng TRH per ml. Fractions of TSH in the pituitary extracts were obtained using affinity chromatography coupled with an anti-rat TSH globulin. These TSH fractions were analysed by isoelectric focusing. The control pituitary glands were composed of four component peaks (isoelectric point (pI) 8.7, 7.8, 5.3 and 2.5) of [3H]glucosamine and [14C]alanine incorporated into TSH, and the amounts of radioactivity of these components were increased with the incubation time. Of these peaks, radioactive components of pI 8.7 coincided with the non-radioactive TSH components measured by radioimmunoassay. Addition of TRH increased incorporation of [14C]alanine into TSH in each of the components to a greater extent than that of [3H]glucosamine. In addition, new components with pI 7.2, 6.5 and 6.2, each component corresponding to each unlabelled TSH component, were demonstrated in the presence of TRH. Because addition of TRH did not change the amounts of [14C]alanine-labelled TSH in the media, the newly formed components were assumed to be connected with protein synthesis occurring in the anterior pituitary gland, which may be specific substances in response to TRH administration. These results indicate that TRH principally elicits an increase in protein synthesis in TSH at the anterior pituitary level, resulting in an alteration of TSH heterogeneity.

The effects of in vivo TRH administered either as bolus or continuous doses on TSH secretion, synthesis, and glycosylation were studied in normal and hypothyroid rats. Nine-week-old normal or 3-week postthyroidectomy rats were administered bolus doses of saline or TRH (0.5 mg/kg) twice daily or continuous saline or TRH (1 mg/kg/day) via an osmotic pump. After 5 days, pituitaries were removed and incubated with [35S]methionine (MET) and [3H]glucosamine (GLCN), with or without 10(-8) M TRH, for 6 and 24 h. Samples were precipitated with anti-TSH beta sera and then analyzed by gel electrophoresis. In normal rats, plasma TSH, T4 and T3 increased with continuous in vivo TRH but not with bolus TRH; in hypothyroid rats, plasma TSH, T4 and T3 were not altered by continuous or bolus doses of TRH. Additionally, in normal rats, continuous in vivo TRH increased incorporation of MET in secreted TSH (477 vs. 212 X 10(3) dpm/mg DNA; P less than 0.05) and intrapituitary TSH (5035 vs. 2124 X 10(3) dpm/mg DNA; P less than 0.05), and GLCN in secreted TSH (148 vs. 50 dpm/mg DNA; P less than 0.05) and intrapituitary TSH (2344 vs. 744 X 10(3) dpm/mg DNA; P less than 0.05). In contrast, in hypothyroid animals, continuous in vivo TRH did not alter MET or GLCN incorporation in TSH. Bolus TRH did not alter secreted or intrapituitary MET or GLCN incorporation into TSH in the normal rat. However, bolus TRH in the intrapituitary MET or GLCN incorporation into TSH in the normal rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells from the rabbit pars distalis and pars intermedia have been cultured in several ways. In clump cultures the glandular adenohypophysial cells of the primary clump remain identifiable for long periods. Both acidophil and mucoid cells can be maintained in this way for over 50 days. During this time two different modes of granule extrusion are observed, one being found only in acidophil cells and the other being exclusive to mucoid cells. From this it is concluded that specific release of secretory products from these cells can occur in the absence of hypothalmic control. A special form of collagenous fibre is produced in clump cultures. Monolayer cell cultures are derived either from isolated adenohypophysial cells or from secondarily aggregated clumps, but in either case the predominant cells are fibroblasts, which persist for over 50 days (as isolated cultures) and for over 90 days (as spreads from clumps). Rounded cells can be recognized as a separate type in isolated cell culture.

Cells binding anti-bovine TASH beta serum were found exclusively in the rostral lobe of the adenohypophysis of the drake using the peroxidase-antiperoxidase complex unlabelled antibody method. The specificity of the binding of the anti-serum to TSH cells was established by relating the morphology and relative abundance of immunochemically stained cells to the TSH content of the adenohypophysis after experimentally altering the activity of the pituitary-thyroid axis. The TSH activity of the adenohypophysis was assessed indirectly, by the weight of the thyroid glands, and directly, by bioassay. As determined by bioassay, the TSH content of the rostral lobe of the adenohypophysis was much greater than that of the caudal lobe. Compared with control drakes, immunochemically stained cells in birds fed a goitrogen, methimazole, seemed to be enlarged and were closer together, while the stained cells in drakes injected with thyroxine were shrunken and less intensely stained. The TSH content of the adenohypophysis was increased in drakes fed methimazole. Castration did not alter the TSH content of the adenohypophysis or change the morphology of immunochemically stained cells. These observations suggest that in the drake: 1) anti-bovine TSH beta serum binds specifically to TSH cells; 2) the TSH cells occur in the rostral and not in the caudal lobe of the adenohypophysis; and 3) the activity of TSH cells is not inhibited by the feedback effects of gonadal steroids.

The effects of hypothalamic hypothyroidism vs. primary hypothyroidism on TSH carbohydrate structure were studied in the rat. Adult male rats with bilateral paraventricular nuclear lesions (n = 10), sham lesions (n = 10), and thyroidectomies (n = 6) were studied 2 weeks postoperatively and compared to normal animals without surgery (n = 6). Pituitaries were incubated in medium containing [3H]glucosamine for 24 h. TSH was immunoprecipitated from medium and pituitary sonicates using anti-TSH beta serum, digested with pronase to obtain TSH glycopeptides, desalted, then analyzed by Concanavalin-A (Con-A) chromatography. Compared to sham controls, hypothalamus-lesioned animals contained a greater proportion of secreted TSH glycopeptides that bound weakly to Con-A, indicating a shift from bisecting and/or multiantennary structures in control animals to biantennary and/or truncated hybrid forms in hypothalamus-lesioned animals. In contrast, thyroidectomized animals, compared to normal and lesioned animals, contained a greater proportion of secreted TSH glycopeptides that did not bind to Con-A, indicating a shift from biantennary and/or truncated hybrid forms to bisecting and/or multiantennary forms. The characteristics of the carbohydrate chains on secreted TSH differed markedly in hypothalamic vs. primary hypothyroidism despite equally low thyroid hormone levels in vivo. Thus, in addition to regulating TSH secretion, hypothalamic hormones alter TSH carbohydrate structure, which may affect its bioactivity and MCR.

Actinomycin D and cycloheximide were used to clarify the tight relationship between protein synthesis and heterogeneous carbohydrate synthesis of rat pituitary thyroid-stimulating hormone (TSH) under thyrotropin-releasing hormone (TRH) stimulation in vitro. Rat anterior pituitaries were incubated with [3H]glucosamine in the presence of TRH, cycloheximide and actinomycin D at 37 degrees C for 3 and 6 h. The TSH fraction of pituitary homogenates was obtained using affinity chromatography coupled with anti-TSH globulin, and was analyzed by isoelectric focusing. Anterior pituitary in the presence of TRH showed heterogeneous components of [3H]glucosamine-labeled TSH with 6 different isoelectric points (components I-VI). Radioactivities of the components increased with the incubation period. Cycloheximide changed these heterogeneous components. Actinomycin D also caused a striking change in the heterogeneity of pituitary [3H]glucosamine-labeled TSH: components I and II decreased and components III and IV increased as compared to the control group after a 6-hour incubation. The present study implies that actinomycin D affects the TRH-induced heterogeneous components of pituitary TSH glycosylation. The data indicate that messenger RNA is essential for the normal processing of carbohydrate synthesis of pituitary TSH.

The thyrotropic activity of the hypophysis (TTH synthesis and secretion) was studied in rats at various age periods. The work was conducted on mongrel male rats of four age groups: 1 1/2-2, 8-10, 18-24, and 28-32-months old. Being relatively low in the sexually immature animals, the thyrotropic activity of the hypophysis considerably intensified in adult rats and diminished with the ageing of the organism. A reduction of the TTH synthesis and secretion in old animals, testified to by the hormone content in the hypophysis and the blood, was apparently connected with the intensification of the T4-deiodating capacity of the hypophysis tissue.

A cytological and optical microscopic study of the anterior lobe of hypohysis in the Wistar adult rat 10, 30 or 60 minutes after intravenous injection of 50 mug of synthetic TRH discloses modifications of thyrotropic cells and prolactin cells. It's possible to recognize a quick degranulation of the prolactin cells (with a maximum at the 10th minute) and a slower degranulation of the thyrotropic cells (perceptible at the 10th minute and very pronounced at the 60th minute). The somatrotopic cells did not show any modification.

The cells that produce luteinizing hormone (LH) in the adenohypophysis of the Japanese quail were identified immunohistochemically using anti-chicken LH serum and horseradish peroxidase-labeled goat anti-rabbit gamma globulin serum. The LH cells are localized in the caudal lobe of the pars distalis. They are elongate in shape shape and are polarized toward the sinusoids, especially in their active states. Alterations in size of LH cells are directly related to changes in circulating LH levels as induced by castration or photostimulation. The LH cells identified immunohistochemically were only stained by alcian blue with periodic acid-Schiff (PAS), alcian blue and orange G. PAS-positive gonadotropic cells in the cephalic lobe were stained immunohistochemically only slightly if at all using anti-chicken LH serum and consequently may be FSH producing cells. In the cephalic lobe another type of basophilic cell was stained with alcian blue. These cells were also stained immunohistochemically with anti-chicken LH serum. These cells may possibly be identified as TSH cells due to the characteristics of the anti-chicken LH serum used in this study which cross react with LH and TSH but only slightly with FSH, and also on the basis of previous light and electron microscopic studies.

To investigate whether TRH regulates TSH production through a pre- or posttranslational mechanism, we determined the pituitary levels of mRNAs for alpha-subunit and TSH beta in male Sprague-Dawley rats given TRH in the presence or absence of thyroid hormones, with or without hypothalamic influence. In normal rats, serum TSH increased 6-fold after a single sc injection of TRH (7 micrograms/kg BW), but the levels of mRNA for both TSH subunits did not differ from the control values. Infusion of TRH, achieved by osmotic minipumps that were implanted sc, increased serum TSH for 3 days. Conversely, the pituitary content of TSH dropped to and remained 35% of that in the controls. In these normal rats, throughout the TRH infusion, the pituitary levels of mRNA for the TSH subunits did not differ from those in the controls. Thyroidectomy increased, by 27 and 75 times, the normal levels of mRNAs for alpha and TSH beta, respectively. TRH, given either as a single injection or a 3-day infusion, did not further elevate these levels. We then studied thyroidectomized animals whose pituitaries were transplanted under their renal capsules. These pituitaries responded to TRH infusion by releasing TSH. T4 injection inhibited this response significantly, but not completely. In spite of this evidence of normal responsiveness to TRH, infusion of TRH for a week did not increase the level of mRNAs for either TSH subunit in transplanted pituitaries. We conclude that in the presence or absence of thyroid hormones, with or without concurrent hypothalamic influence, TRH did not affect rat pituitary level of mRNA for either TSH subunit despite persistent high levels of serum TSH. Therefore, TRH does not regulate TSH production through a pretranslational mechanism. Although a translational regulation cannot be completely excluded, the present data, in conjunction with previous findings, support the hypothesis that TRH regulates TSH production primarily by stimulating both posttranslational carbohydrate processing and secretion of this hormone.

The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells. Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted. The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.

A sensitive and specific radioimmunoassay has been developed for rat prolactin (rPRL), employing the double antibody solid phase technique for the separation of free and antibody-bound [125I]rPRL. The anti-serum was raised in rabbits and showed no cross-reaction with rat growth hormone (rGH), follicle stimulating hormone (rFSH), luteinizing hormone (rLH) and thyrotrophin (rTSH). The immunosorbent (sheep anti-rabbit IgG bound to cellulose) showed a surprisingly high binding of [125I]rPRL, but not of the other iodinated anterior pituitary hormones. Addition of serum to the incubation mixtures prevented the binding between [125I]rPRL and the immunosorbent. Three different clonal strains of pituitary cells have been examined for production of rPRL, rLH and rTSH, both in the basal state as well as after treatment with thyrotrophin releasing hormone (TRH) and gonadotrophin releasing hormone (LH/FSH-RH). Monolayer cultures of two of the cell strains produced and secreted rPRL spontaneously, and they showed a 2-fold increase in rPRL production after treatment with TRH (3-10(-7) mol/1). The third cell strain did not produce rPRL spontaneously, or after treatment with TRH. None of these cell strains could be stimulated to produce rTSH by treatment with TRH. Treatment of the same three cell strains with LH/FSH-RH (1.2-10(-6) mol/1) failed to induce production of rLH, and there were no changes in production of rPRL. Prostaglandins E1 and E2 (3-10(-8) mol/1) and oestradiol-17beta (10(-7)-10(-10) mol/1), however, stimulated the production of rPRL. The effects of TRH and prostaglandins E1 and E2 were observed within 24 h of treatment, while the first effect of oestradiol-17beta was seen after 3 days. These results suggest that the stimulatory effect of oestradiol-17beta on rPRL production differs from that of TRH and prostaglandins.

Ten patients (8 women, 2 men) with the "empty sella syndrome" were studied to evaluate the pituitary reserve of human thyrotropin (hTSH) and prolactin (hPRL). None of the patients had signs or symptoms of hypopituitarism or primary hypothyroidism. All patients had normal baseline thyroid function tests except for 2 patients with mild elevations in total triiodothyronine as measured by competitive protein displacement assay (T3D). Eight of ten patients had normal hTSH responses to thyrotropin releasing hormone (TRH), whereas the 2 patients with elevated T3D had blunted TRH responses. In the 4 patients studied, the metabolic clearance (MCR) and production rates (PR) of hTSH were normal. In 9 of 10 patients normal baseline serum hPRL levels were detected, and each responded to TRH. In one case serum hPRL was undetectable and failed to respond to TRH. The assessment of other anterior pituitary function revealed few minor abnormalities. In summary, like other anterior pituitary hormones, the pituitary reserve in "empty sella syndrome" of hTSH and hPRL is usually normal. When abnormalities do occur, they are attributable to other co-existent endocrine pathology.

The fetal porcine pituitary was investigated by means of ultrastructural immunocytochemistry (1) to identify the first cells synthesizing the adenohypophyseal hormones, (2) to follow their differentiation during fetal development, and (3) to compare their ultrastructural characteristics with those of mature adult cells. The first ACTH-cells, which produced and stored ACTH, beta-LPH, beta-MSH, and alpha- and beta-endorphin in the same granules, were very numerous at day 34 and displayed a uniform morphology. At day 50 and thereafter, until the end of gestation, the ACTH-cells differed in their appearance probably reflecting various stages of differentiation of one cell type. The GH-cells gained rapidly ultrastructural features comparable to those of mature GH-cells. In contrast, in the case of PRL-cells, which appeared only at the end of the gestation period as immature elements containing very small secretory granules, the morphological maturation seemed to take place only after birth. The first cells synthesizing the glycoprotein hormones (LH alpha, LH beta, FSH and TSH) displayed ultrastructural features of immature cells. At day 50, their ultrastructural organization started to show a different pattern. At the end of gestation, the TSH-cells and the gonadotropic cells displayed the ultrastructural features of mature cells.

The ultrastructure of each adenohypophysial secretory cell type was examined in pituitaries of adult female mollies (Poecilia latipinna) after various periods in vitro, and with varied medium osmotic pressure (OP), Na+, and Ca2+ concentrations. Prolactin (PRL) cells were markedly activated by 18 hr, and after 7 or 14 days were almost totally degranulated, with massive arrays of Golgi and RER. Reduction in OP, but not Na+ or Ca2+, caused an additional activation of PRL cells after periods of 18 hr or longer. Corticotroph (ACTH) cells became noticeably activated by 4 hr, and were possibly affected by OP, but not Na+ or Ca2+. Growth hormone (GH) cells were activated by 6 hr, and after 18 hr were quite degranulated with extensive arrays of RER. OP had no effect on GH cells before 3 days, when reduced OP appeared to cause an additional activation, with the appearance of large irregular secretory granule (SG)-like inclusions. Na+ and Ca2+ again had no effect. Gonadotrophic (GtH) cells appeared to be little affected by in vitro incubation; however, the very active cells from vitellogenic fish underwent a reduction in dilated RER after prolonged culture. Thyrotrophic (TSH) cells gradually became activated in vitro, but the response again varied with the sexual condition of the fish. Neither GtH nor TSH cells were affected by OP, Na+, or Ca2+. The findings are discussed in relation to hypothalamic control, via releasing/inhibiting factors, of adenohypophysial cell activity.