Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.     

Immunoreactive growth hormone-releasing hormone in rat placenta

It has been shown that immunoreactive and biologically active GH-releasing hormone (GHRH)-like material is present in rat placenta. To investigate the role of placental GHRH, we measured it in human and rat placenta of different gestational stages, using specific RIA systems. GHRH and somatostatin contents in median eminence, pituitary GH contents, and plasma GHRH levels were also quantified in rats. Immunoreactive GHRH was detectable in rat placenta [13 days of gestation, 1.4 +/- 0.4 (+/- SD); 16 days, 1.4 +/- 0.2; 20 days, 1.7 +/- 0.4 ng/g] but not in human placenta (less than 0.06 ng/g in both full-term and mid-term placenta). GHRH concentrations in rat placenta did not change significantly during pregnancy, but total contents increased progressively in relation to placental growth. GHRH and somatostatin contents in median eminence of pregnant rats were not different from those of control female rats. In contrast, rat pituitary GH contents in pregnant rats were significantly lower than those of control female rats. Immunoreactive GHRH was not detectable in plasma of either pregnant rats or nonpregnant rats. Molecular sieve chromatography revealed two peaks of immunoreactive GHRH in rat placental extracts: a major peak eluted in the position of synthetic rat GHRH and a minor peak in the higher molecular weight region. In contrast, a single peak in the position of rat GHRH was observed in rat median eminence extracts. Detection of immunoreactive GHRH in rat placenta but not in human may suggest that the mechanism of GHRH gene expression in placenta is species specific. Failure of detection of immunoreactive GHRH in rat maternal circulation suggests that placental GHRH may not affect the maternal hypothalamic pituitary axis. Presence of high molecular weight materials of immunoreactive GHRH in rat placenta but not in median eminence suggests that posttranslational processing of the GHRH precursor molecule may be different in the two organs. Placental GHRH may have a paracrine function or may be secreted into fetal circulation and contribute to fetal growth.     

An autocrine/paracrine role for insulin-like growth factors in the regulation of human placental growth

Tissue derived from preterm (9-19 weeks gestation) and term (38-41 weeks gestation) human placentae were examined for their ability to synthesize and secrete insulin-like growth factors (IGFs) in organ culture. IGF-I was measured by a specific RIA, and IGF-II by a rat placental membrane radioreceptor assay. First, explants of placental tissue were maintained in organ culture. These explants secreted immunoreactive IGF-I (IR-IGF-I). There were no differences in the IR-IGF-I content of media conditioned by term and preterm placentae under these conditions. The similarity of this material to authentic human IGF-I was supported by parallel displacement in a specific RIA and coelution during Sephadex G-50 gel filtration. Second, monolayer cultures of fibroblasts from normal human preterm placentae (15-19 weeks gestation) were established. Confluent monolayers of these fibroblasts secreted IR-IGF-I (3-10 pg/10(5) cells X 40 h). IR-IGF-I secretion was reversibly inhibited by 5.3 microM cycloheximide, suggesting that the IR-IGF-I was the result of de novo protein synthesis. IR-IGF-I secretion was stimulated 5-fold by platelet-derived growth factor (0.6 U/ml). The response of monolayers of placental fibroblasts to IGF-I also was tested. IGF-I stimulated alpha-[3H]aminoisobutyric acid transport in these fibroblasts, with half-maximal stimulation occurring at 2-3 ng/ml. Stimulation of alpha-[3H]aminoisobutyric acid uptake by IGF-I correlated with specific binding of [125I]iodo-IGF-I. Half-maximal inhibition of [125I]iodo-IGF-I binding occurred at 2-3 ng/ml IGF-I. Placental tissue also secreted IGF-II-like activity, as measured by radioreceptor assay. Media conditioned by placental explants contained 15-20 ng/mg protein X 48 h, and media conditioned by placental fibroblasts contained 3-7 ng/10(5) cells X 40 h IGF-II determined by radioreceptor assay. These data support the hypothesis that the human placenta produces IGFs (IGF-II and/or IGF-I) that act locally to regulate placental growth.     

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.     

Expression of mRNA species encoding steroidogenic enzymes in the rat ovary.

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyroid hormone regulation of TRH mRNA levels in rat paraventricular nucleus of the hypothalamus changes during ontogeny

The changing roles of the hypothalamus and pituitary in regulating thyroid hormone levels in the rat during ontogeny has not been fully elucidated. It has been reported that endogenous TRH begins to stimulate TSH secretion at 5-8 days after birth but that the pituitary responds to hypothyroidism during late gestation. To determine the onset and extent of TRH response to low thyroid hormone levels during ontogeny, normal and hypothyroid rats treated with methimazole for 7 days were sacrificed at 16 days gestation (E16), 20 days gestation (E20), 7, 21 and 56 days after birth (n = 5/study group). Plasma hormones were assayed from pregnant mothers, pups (pooled) and adults. Levels of TRH mRNA were measured in the paraventricular nuclei (PVN) by in situ hybridization histochemistry. A labeled 48-base cDNA oligonucleotide for TRH was hybridized with brain slices (n = 6/animal) in the region of the medial parvocellular division of the PVN of the hypothalamus and the signal was quantitated by digitized computer analysis. Plasma-free T4 levels decreased and plasma TSH levels increased in the animals treated with methimazole as compared to the euthyroid controls. TRH mRNA was detected in the PVN at E16 after brain slices were dipped in emulsion and granules observed by dark-field microscopy. In the euthyroid animals, TRH mRNA increased from E20 (150 +/- 9 OD units) to 7 days (222 +/- 5 OD units) and remained unchanged at 21 days (252 +/- 27 OD units) and 56 days (244 +/- 6 OD units).(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin receptor subtypes mRNA in TSH-secreting pituitary adenomas: a case showing a dramatic reduction in tumor size during short octreotide treatment

TSH-secreting adenoma is a rare pituitary adenoma, and the expression levels of the specific subtypes of somatostatin receptors (sstr) mRNAs have remained obscure. To determine the quantitative expression of the sstr1-5 mRNAs in TSH-secreting adenomas that may be related to the efficacy of treatment with a somatostatin analogue, expression of the sstr1-5 mRNAs was examined and compared in TSH-secreting adenomas and other pituitary adenomas. The pituitary adenomas were obtained at transsphenoidal surgery from 4 cases of TSH-secreting adenoma, including 1 patient showing a significant shrinkage of the tumor size after only 10 days of octreotide treatment, 2 patients without tumor size reduction and 1 patient without treatment, and 5 GH-secreting adenomas, 6 prolactinomas, 5 nonfunctioning adenomas, 4 ACTH-secreting adenomas and normal pituitaries at autopsy from 4 normal subjects. In comparison to the normal pituitary, sstr2A>sstr1>sstr5>sstr3 mRNAs were expressed in the TSH-secreting adenomas examined. No expression of sstr2B or sstr4 mRNA was observed. The expression level of sstr2 mRNA was significantly higher than those in normal pituitary, prolactinomas, ACTH-secreting and nonfunctioning pituitary adenomas. The patient with marked shrinkage of the tumor showed the highest expression of both sstr2 and sstr5 mRNAs among all the cases of pituitary adenoma. A TSH-secreting tumor without shrinkage showed a similar expression level of sstr2 mRNA. These findings demonstrated that TSH-secreting adenomas express sstr1, 2A, 3 and 5 mRNAs, predominantly sstr2A, and in addition to the expression of sstr2 mRNA, the expression level of sstr5 mRNA may be a factor affecting the tumor shrinkage by somatostatin analogues against TSH-secreting adenomas.     

Differentiation of pituitary cells in culture

Gonadotrophs were first detected at 18 days of gestation in normal rat fetus. Encephalectomy performed at 16 days of gestation did not modify the normal aspect of cells at term. In adenohypophysial primordia explanted from 13 days of gestation differentiated gonadotrophs were detected after culture (8 days) in medium containing insulin (minimal dose required: 0.5 microgram/ml) and transferrin (5 micrograms/ml). In contrast, in primordia explanted at 11 and 12 days of gestation, GnRH 10(-9) to 10(-12) M was required for the first 24 hours of culture to induce differentiation of cells which was obtained in synergy with insulin and transferrin. On the other hand, fetal hypothalamic GnRH and pituitary GnRH receptors were observed from 12 days of gestation which can explain the observations made on primordia explanted at 13 days. Lactotrophs first appeared at term in normal rat fetus. In vitro, differentiation of lactotrophs was not observed in primordia explanted from 13 days in the presence of insulin and transferrin alone, but it was induced by GnRH (10(-9) M) for the 24 hours of culture in the same medium. The action of GnRH was mediated through glycoproteins and specifically isolated alpha subunit. Indeed, purified LH alpha-subunit added in medium instead of GnRH induced differentiation of lactotrophs from 10(-9) M with an increase in the number of cells related to the dose of hormone. Differentiation of the two cell types is very linked in these culture conditions. Gonadotroph differentiation is regulated by an hypothalamic endocrine secretion whereas lactotroph differentiation is more dependent on a paracrine secretion.     

Identification of human luteinizing hormone, follicle-stimulating hormone, luteinizing hormone beta-subunit and gonadotrophin alpha-subunit in foetal and adult pituitary glands.

Specific radioimmunoassays were used to assess the content of LH, FSH, the gonadotrophin alpha-subunit and the LH beta-subunit in four adult, 19 normal foetal pituitary glands (9-5--32 weeks of gestation) and a pituitary extract from an anencephalic foetus (36 weeks). The hormones and subunits were further identified by column chromatography on Sephadex G-100. All pituitary glands contained free alpha-subunit and intact LH but the alpha-subunit:LH ratio was significantly higher in the early foetal pituitaries (9-5--16 weeks) than in the four adult pituitaries. Only small or undetectable amounts of LH beta-subunit and 'undetectable' FSH were found in these early foetal pituitaries (9-5--11-5 weeks). The concentration of intact hormones or subunits in the pituitaries showed no significant sex difference in any of the groups. In contrast to these results, only alpha-subunit was detectable in the pituitary of the anencephalic foetus. For 14 early foetuses (age of gestation 10--16 weeks) the serum levels of LH-HCG, FSH, and alpha-subunit in the circulation were significantly higher than in 26 foetuses at term (37--41 weeks). On the basis of these results a theory for the development of the gonadotrophin secretion from the foetal pituitary gland is outlined.     

Changes in thyroxine monodeiodination in rat liver, kidney and placenta during pregnancy

Monodeiodination of thyroxine (T4) was studied in the liver, kidney and placenta of pregnant rats. Age matched female non-pregnant and pregnant Sprague-Dawley rats on the 7th, 14th, 17th and 21st days of gestation were used. The 800 X g supernatants of tissue homogenates (protein 1 mg/tube) were incubated with 1 microgram of stable T4 in the presence of 5 mM dithiothreitol (DTT) at 37 degrees C for 60 min at pH 7.5. Net triiodothyronine (T3) generation from T4 in rat liver homogenates on the 7th day of gestation was significantly lower than that in the non-pregnant rat. Thereafter it increased, but values on the 14th, 17th and 21st days of gestation were not significantly different from those obtained in the non-pregnant rat. Net renal T3 generation from T4 on the 14th day was significantly lower than that in the non-pregnant rat. It was increased thereafter and the values at the 17th and 21st days of gestation were not significantly different from those in the non-pregnant rat. Net reverse T3 (rT3) generation from T4 in the placenta rose from the 14th to the 17th day and then dropped by the 21st day and the value at the 17th day was significantly higher than those at the 14th and 21st days of gestation. These results indicate that 1) both T4 outer-ring monodeiodination in the pregnant rat liver and kidney, and T4 inner-ring monodeiodination in the placenta show significant variation with the progress of gestation; 2) the time course of the T4 outer-ring monodeiodination in pregnant rat liver and kidney is completely different from T4 inner-ring monodeiodination in the placenta.     

Quantitative in-situ hybridization histochemistry studies on growth hormone (GH) gene expression in acromegalic somatotrophs: effects of somatostatin, GH-releasing factor and cortisol

We have examined the effects of human GH-releasing factor (1-44) (GRF), cortisol and somatostatin-(1-14) on GH gene expression in solid tissue and dispersed cells from human pituitary adenomas using quantitative in-situ hybridization histochemistry. Sections cut from tissue obtained at hypophysectomy from three acromegalic patients were hybridized to probes directed against mature alpha-subunit, GH, prolactin, pro-opiomelanocortin, TSH beta-subunit and LH beta-subunit mRNA. Only one biopsy contained GH mRNA in isolation. A second was found to coexhibit GH, prolactin and alpha-subunit mRNA, and a third was found to contain prolactin, TSH beta-subunit, alpha-subunit and LH beta-subunit mRNA, with GH mRNA below the limit of specific detection, indicating that the sample was composed of normal rather than adenomatous pituitary tissue. GH mRNA in individual dispersed cells derived from the latter declined to barely detectable levels over 287 h, both in cultures containing GRF (10 ng/ml) or GRF (10 ng/ml) plus somatostatin (10 ng/ml) and in controls, but increased fourfold in cultures containing GRF (10 ng/ml) plus cortisol (0.5 mumol/l). GH mRNA remained unchanged in both adenoma samples over 138 and 450 h, irrespective of the addition of GRF or GRF plus hydrocortisone. In these samples, somatostatin plus GRF had no consistent effect. These studies confirm that quantitative in-situ hybridization histochemistry can be used to investigate hormone gene regulation in small samples of human tissue and should enable us to define more clearly the level at which abnormal gene regulation occurs.     

Rat placental mRNA directs the synthesis of a polypeptide immunologically related to alpha-subunit of glycoprotein hormones

In order to investigate the existence of a chorionic gonadotropin (CG) in the rat, placental mRNA was prepared from either the foetal disc or the maternal site of implantation in pregnant rats and translated in a wheat-germ cell-free translation system in the presence of 35S-labeled methionine and cysteine. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the radioactive material immunoprecipitated using antiserum specific to native rat alpha-subunit allowed us to isolate in translation products from both sources (foetal disc and maternal site of implantation) a single polypeptide of 17 kDa having the electrophoretic properties of the rat pituitary alpha-precursor. This polypeptide was absent in media derived from translation of mRNA extracted from uterine horn of non-pregnant female rats, and was approximately 10 times more abundant when RNA was derived from the maternal part (about 0.25% of cpm in total proteins) rather than the foetal part (0.026%) of the placenta. Immunoprecipitation was prevented in the presence of an excess of rat (but not ovine) alpha-subunit. Antisera directed against denatured bovine alpha-subunit, which cross-react with the rat pituitary alpha-precursor, did not bind the placental peptide. These results suggest that this rat placental polypeptide and the rat alpha-subunit of pituitary glycoprotein hormones have important differences in their primary structure, but share discrete structurally and/or conformationally related regions in their polypeptide chains. The possibility that these partial homologies account for gonadotropin-like activity of a presumed rat CG remains to be ascertained.     

Neurohypophysial peptides in the human fetus: presence in pituitary extracts of immunoreactive arginine-vasotocin

A specific and sensitive radioimmunoassay was used to demonstrate the presence of arginine-vasotocin (AVT) in human fetal pituitary extracts. The mean pituitary contents of AVT in extracts of 70-101, 102-136 and 137-172 days gestation were 0.32, 0.8 and 8.1 ng/gland respectively. Combined high-pressure liquid chromatography (HPLC) and radioimmunoassay of pooled extracts at these gestational ages confirmed the presence of AVT. Radioimmunoassay of neonatal pituitary extracts (240-280 days) did not conclusively show AVT to be present, as the material assayed did not show parallelism with dilution. However, combined HPLC and radioimmunoassay of pooled extracts at this gestational age did demonstrate the presence of an immunoreactive AVT peak. Radioimmunoassay analysis of this peak indicated that a relatively small amount of AVT (at least 0.5 ng/gland) was present in the human pituitary gland at term.     

Hypophysectomy of the fetal lamb leads to a fall in the plasma concentration of insulin-like growth factor I (IGF-I), but not IGF-II

The role of the pituitary gland in the regulation of the plasma concentrations of insulin-like growth factors (IGFs) in the late gestation sheep fetus has been examined. Singleton sheep fetuses were either hypophysectomized or sham-operated between days 110-120 of gestation. Blood samples were then collected via carotid cannulae at least three times weekly for the remainder of gestation. In some hypophysectomized fetuses T4 was administered (100 g/day) to overcome the hypothyroidism caused by hypophysectomy. Blood samples were also obtained from lambs during the perinatal period, neonatal lambs within 1-10 days after birth, and pregnant and nonpregnant adult ewes. All plasma samples were subjected to Sephadex G-50 gel filtration under acidic conditions (pH 2.3) to eliminate IGF-binding protein activity. The fractions containing the free IGF peptides were collected and assayed for IGF-I by heterologous RIA, and IGF-II by a homologous RRA. Plasma concentrations of IGF-I and IGF-II did not change with advancing gestational age in any fetal group and were not affected by the prolonged gestation that results from hypophysectomy. The mean plasma IGF-I and IGF-II concentrations in the sham fetuses were 112 +/- 8 and 1340 +/- 112 ng/ml, respectively. Hypophysectomy without thyroid hormone replacement resulted in a significant decrease in plasma IGF-I concentrations to 50 +/- 5 ng/ml, whereas IGF-II concentrations were not affected (1096 +/- 124 ng/ml). IGF-I concentrations in the hypophysectomized fetuses that received T4 were significantly increased (67 +/- 6.0 ng/ml) compared to those in the hypophysectomized fetuses that did not receive T4. The IGF-II concentrations in the hypophysectomized fetuses that received T4 were similar to those in the sham-operated fetuses (1120 +/- 112 ng/ml). At term IGF-I concentrations were increased (180 +/- 21 ng/ml) and IGF-II concentrations were decreased (264 +/- 25 ng/ml) compared to fetal values. Plasma IGF-I concentrations in the prepubertal lamb were similar to the fetal values. Pregnancy in the adult ewe was associated with a significant increase in IGF-II, but had no effect on IGF-I plasma concentrations. These data show that circulating IGF-I concentrations in the fetal lamb are under some pituitary and thyroid control, whereas IGF-II concentrations are independently of pituitary or thyroid status. We confirm, using a homologous assay, that fetal IGF-II concentrations are high and then decrease at term. These data also support the concept that a pregnancy-related factor may regulate plasma IGF-II concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

STUDIES OF TWO THYROTROPHIN-SECRETING PITUITARY ADENOMAS: EVIDENCE FOR DOPAMINE RECEPTOR DEFICIENCY

Of 22 previously reported patients with TSH-secreting pituitary adenomas challenged with dopamine agonists, 18 showed no decrease in serum TSH. There have been few in-vitro studies of these rare tumours so the mechanism of the dopaminergic resistance has remained obscure. We describe two further patients with thyrotrophinomas; the first was thyrotoxic (T3 6.1 nmol/l, TSH 7 mU/l) and the second was diagnosed after radioiodine for presumed Graves' disease. The second patient had an alpha-subunit: TSH molar ratio less than unity (0.27). In-vivo TSH responses to TRH, bromocriptine and domperidone were compared with those of the resected tumour cells in vitro, the latter studied using a continuous perifusion system. Dopamine receptors were sought in membranes from each tumour using a radioreceptor assay employing 3H-spiperone. Patient 1 showed significant increases in serum TSH (7 to 13 mU/l) and alpha-subunit (18.7 to 385 ng/ml) after 200 micrograms TRH (i.v.) but patient 2 showed no such increases (TSH: 69 to 72 mU/l, alpha-subunit: 4.9 to 5.2 ng/ml). Neither patient showed a change in serum TSH following bromocriptine 2.5 mg (orally) or domperidone 10 mg (i.v.), though serum PRL responded normally. Serum TSH from patient 1 was of apparently normal molecular size but increased bioactivity (B/I ratio 3.8) and that from patient 2 was of increased molecular size but reduced bioactivity (B/I ratio 0.1). Tumour cells from each patient immunostained for TSH beta and alpha-subunit, and secreted TSH in vitro. The first showed dose-dependent TSH release after TRH (1-100 ng/ml) which could not be inhibited by dopamine (5 mumol/l) but the second was unresponsive to TRH in vitro. Neither tumour showed inhibition of TSH release by dopamine (5 mumol/l) or bromocriptine (0.01-10 nmol/l) and neither contained membrane-bound dopamine receptors. The results suggest that the dopaminergic resistance typical of most TSH-secreting pituitary adenomas may be due to altered or absent membrane-bound dopamine receptors.     

Differential regulation of gonadotropins and glycoprotein hormone alpha-subunit by IGF-I in anterior pituitary cells from male rats

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites alpha-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhlGF-I; 2 microg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p < 0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only alpha-subunit release but also its mRNA levels. This study provides the first observation of alpha-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and 1-subunits is also shown.     

Glycoprotein hormone alpha-subunit production in somatotroph adenomas with and without Gs alpha mutations.

Activating mutations of the Gs alpha subunit have been identified in a subset of somatotroph adenomas. The mutant form of the Gs alpha subunit causes persistent activation of adenylyl cyclase and consequently results in high intracellular levels of cAMP. Because cAMP is known to stimulate the synthesis of the glycoprotein hormone (GPH) alpha-subunit as well as GH, we examined somatotroph tumors with and without Gs alpha mutations for GPH alpha-subunit production. GPH alpha-subunit production was assessed in vivo by measuring serum hormone levels and in vitro by analyzing hormone secretion by cultured pituitary tumor cells. DNA was extracted from the pituitary tumors of 26 acromegalic patients. The Gs alpha gene was amplified by the polymerase chain reaction and screened for mutations at codons 201 and 227 using oligonucleotide specific hybridization. Nine of the 26 tumors (35%) had point mutations at Arg 201. Seven of these tumors contained a CGT to TGT mutation (Arg to Cys) and 2 contained a CGT to CAT mutation (Arg to His). No mutations were detected at codon 227. There were no significant differences in age, sex distribution, tumor size, or serum levels of GH or insulin-like growth factor-1 between the groups of patients with or was Gs alpha mutations. The mean serum level of the free GPH alpha-subunit was 1.9-fold higher in the group with Gs alpha mutations (0.48 +/- 0.37 micrograms/L) than in patients without mutations (0.25 +/- 0.17) (P less than 0.05). In pituitary tumor cell culture, 75% of somatotroph tumors with Gs alpha mutations secreted free GPH alpha-subunit into the media compared with 45% of tumors without Gs alpha mutations. The amount of GPH alpha-subunit secretion was 12-fold greater in the group of tumors containing the Gs alpha mutation (P less than 0.05). Immunocytochemical detection of the free GPH alpha-subunit was similar in the two groups of patients with 75% positive for the GPH alpha-subunit in tumors with Gs alpha mutations and 67% positive in tumors without mutations (P = 0.69). We conclude that GPH alpha-subunit production occurs in somatotroph tumors with and without Gs alpha mutations. The increased levels of GPH alpha-subunit secretion in vivo and in vitro suggest that the Gs alpha mutation may increase the amount of preexisting GPH alpha-subunit biosynthesis in the tumors, perhaps via activation of the cAMP pathway.     

Regulation of the alpha and thyrotropin beta-subunit messenger ribonucleic acids by thyroid hormones

We studied the regulation of mRNAs encoding the alpha- and beta-subunits of TSH by thyroid hormones (T4 and T3) in mouse thyrotropic tumors and pituitary glands. Hypothyroid male (LAF1) mice bearing thyrotropic tumor (TtT97) were injected daily with T4 for 0, 1, 5, 12, or 33 days. After day 33, plasma levels of TSH and free (unassociated) TSH beta-subunit were reduced to less than 1% of control levels, whereas free alpha-subunit was reduced to 6% of control levels. Steady state levels of subunit mRNAs in extracts of the thyrotropic tissues were measured by blot hybridization analyses using mouse subunit-specific cloned cDNAs. Treatment of mice with T4 caused a rapid decline in the levels of tumor mRNAs for both alpha and TSH beta; after day 1, alpha and TSH beta mRNA levels decreased to 35% and 10% of control values, respectively. Levels of TSH beta mRNA were undetectable after 5 days of T4 treatment, whereas levels of alpha-subunit mRNA remained at 30-35% of control levels even after day 33. In a separate experiment, TSH beta mRNA decreased to 42% of the control level (P less than 0.05), whereas alpha-subunit mRNA remained at 64% of the control level (P = NS) 4 h after a single injection of T4. Finally, T3 also caused a rapid decrease in the levels of both subunit mRNAs in the anterior pituitary glands of hypothyroid mice, but the effect was more complete on TSH beta mRNA levels. We conclude that thyroid hormones have rapid suppressive effects on the levels of mRNAs encoding the subunits of mouse TSH in the thyrotrope. The suppressive effects of thyroid hormones occur more rapidly and are greater for TSH beta than alpha-subunit mRNAs. The parallel changes observed in the subunit mRNA levels and the plasma subunit protein levels in animals treated with thyroid hormones suggest that the changes in the plasma levels of TSH and subunits may reflect effects of thyroid hormones on TSH gene expression in addition to effects on secretion.     

Effects of orchidectomy on gonadotropin and inhibin subunit messenger ribonucleic acids in the pituitary of the rhesus monkey (Macaca mulatta).

Our research programs required the preparation of hypophysectomized and orchidectomized rhesus monkeys. This afforded us the possibility to characterize and compare levels of the gonadotropin and inhibin subunit mRNAs in pituitaries from intact and castrate monkeys. Eighteen adult male monkeys, four of which had been bilaterally orchidectomized 5-9 months previously, were used in this study. Plasma concentrations of LH and FSH were, respectively, 188.5 +/- 5.3 and 246.8 +/- 25.2 ng/ml in the castrate monkeys and 25.8 +/- 4.5 and 4.1 +/- 1.1 ng/ml (mean +/- SEM) in the intact animals. Total pituitary RNA was hybridized to cDNA probes for cynomolgus monkey gonadotropin subunits (FSH beta, LH beta, and the common alpha-subunit) and for human inhibin subunits (alpha, beta B, and beta A) by Northern blot analysis, and mRNA levels were normalized by subsequent hybridization to cyclophilin. Each of the gonadotropin subunit probes hybridized to a single RNA species with the approximate sizes of 1.6 kilobases (kb; FSH beta), 0.7 kb (LH beta), and 0.8 kb (alpha). Levels of LH beta and alpha-subunit mRNAs in pituitaries from castrate monkeys were about 5- and 2-fold higher, respectively, than those in pituitaries from intact monkeys. FSH beta mRNA, on the other hand, was elevated about 27-fold in castrate monkeys [mean +/- SEM, 3176 +/- 408 cpm bound (n = 4 castrate) and 116 +/- 30 cpm bound (n = 8 intact]). Inhibin beta B-subunit mRNA was present in the monkey pituitary as a doublet of about 5 kb, and it was approximately twice as abundant in intact pituitaries as in castrate pituitaries. Hybridizations involving inhibin beta A cDNA revealed a faint band in the region expected for monkey beta A mRNA (6.5 kb) in three of six RNA samples from intact monkeys and a 0.3- to 0.4-kb mRNA species. mRNA encoding the inhibin alpha-subunit was undetectable by Northern blot hybridization. These results indicate that the postpubertal testis imposes an inhibition on the expression of the genes encoding FSH beta, LH beta, and glycoprotein hormone alpha-subunit and that this suppression of the FSH beta gene in the monkey is much greater than that in the rat. In addition, the monkey pituitary may be a source of activin, which may act locally to modulate FSH gene expression and secretion.