Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus.

Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep. Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118-122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139-142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a 35S-labeled ovine CRF type I (CRF1) receptor complementary RNA, to localize and quantify CRF1 receptor mRNA in POMC-hybridizing cells. Binding of [125I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography. The hybridization signal for the CRF1 receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF1 receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF1 receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF1 receptor hybridization signal was significantly greater in PVN-Lx (90+/-7%; mean +/- SEM), compared with SHAM (67+/-6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF1 receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89+/-8%) and SHAM (87+/-9%). Binding of [125I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140+/-19 mean arbitrary densitometry U +/- SEM), compared with SHAM (73+/-23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [125I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [125I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [125I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [125I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF1 receptor expression in the late-gestation sheep fetus.     

切除甲状腺后胎羊垂体前叶细胞的免疫组化观察

目的 观察切除甲状腺以后胎羊脑垂体中促甲状腺激素（TSH）、促生长激素（GH）和催乳素（LTH）细胞的变化。方法 实验用怀有双胎的母绵羊2只,分别在妊娠中期对其中1只胎羊行甲状腺切除术,另1只胎羊作为正常对照。术后绵羊继续妊娠,待足月后行剖腹产取胎羊。然后用免疫复合物染色（PAP）方法观察胎羊脑垂体细胞。结果 发现去甲状腺胎羊（去甲胎羊）垂体前叶出现肥大的去甲状腺细胞（去甲细胞）,并有两种类型,有的去甲细胞胞浆染色浅,胞浆内有空泡,有的去甲细胞胞浆染色深,无空泡。而在正常对照的胎羊则不出现上述类型细胞。GH细胞大多数较正常组的GH细胞直径小。结论 切除甲状腺以后的胎羊其脑垂体前叶细胞的形态有一定的改变。     

Characteristics and developmental changes of corticotrophin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland.

The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120-130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65-70, 85-88, 100-110, 125-130 and at term (approximately 145 days). Binding assays were carried out using 125I-labelled Tyr-ovine CRH (125I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 degrees C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (Kd congruent to 1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65-70 to a maximum at days 125-130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation.     

Hypothalamic, pituitary and testicular function during sexual maturation of the male rat.

Hypothalamic content of gonadotrophin-releasing hormone (GnRH), serum LH and FSH, capacity of the testis to synthesize testosterone in vitro, and testicular 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase and 17 beta-hydroxysteroid dehydrogenase were measured in groups of rats at approximately 5 day intervals from birth to day 64 and at days 74 and 89. The capacity of the testes to synthesize testosterone in vitro was measured in the presence of a saturating dose of rat LH. Gonadotrophin-releasing hormone increased steadily from 0-17 ng per hypothalamus at birth to a maximum of 7 ng at day 52 and then remained constant. LH concentrations were highly variable and often exceeded adult values between days 10 and 32. After day 32 a steady rise was observed which reached adult values between days 37 and 42. FSH concentrations markedly increased from 255 ng/ml observed at birth and day 10 to a peak value of 1000 ng/ml at day 32. Subsequently there was a steady decline in FSH values until day 74 when the concentration returned to values found at birth. 5-ene-3 beta-Hydroxysteroid dehydrogenase-isomerase activity exhibited a rapid increase between days 12 and 19 followed by an even greater rate of increase between days 19 and 32 when adult levels were attained. 17 beta-Hydroxysteroid dehydrogenase activity was very low between birth and day 22. Enzyme activity began to increase at day 22 with a rapid increase in activity observed between days 37 and 58. The increase in capacity to synthesize testosterone closely followed the increase in 17 beta-hydroxysteroid dehydrogenase activity. The study demonstrates that during sexual maturation in the male rat, changes in serum LH and FSH do not reflect changes in hypothalamic GnRH. The appearance of Leydig cells as monitored by 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity precedes by approximately 20 days the increase in testicular capacity to synthesize testosterone in vitro. The latter coincides with the increase in 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that 17 beta-hydroxysteroid dehydrogenase is a limiting factor in the ability of the testis to respond to LH stimulation.     

Androgens delay human fetal lung maturation in vitro

The mature lung produces pulmonary surfactant, a lipid-protein complex that prevents lung alveoli from becoming atelectatic. The prenatal surge of surfactant production in preparation for birth occurs between 28-34 weeks gestation and is triggered by glucocorticoids, which stimulate surfactant synthesis by alveolar epithelial cells through a series of biochemical steps mediated by mesenchymal-epithelial interactions. In contrast to this, when explanted midgestation human fetal lung tissue is maintained in serum-free medium, there is a spontaneous increase (40%) in de novo saturated phosphatidylcholine (SPC) synthesis on the fifth day in culture. Addition of dexamethasone (DEX; 1 x 10(-8) M) to the culture medium causes the increased synthesis in SPC to occur earlier (day 4) and to a greater extent (87%). Addition of an equimolar concentration of dihydrotestosterone (DHT) to the medium delays both the spontaneous and DEX-stimulated increases in SPC synthesis by 24 h. Weak fetal adrenal androgens are also able to block both spontaneous and DEX-stimulated SPC synthesis. Addition of DHT to the explant cultures at daily intervals reveals that inhibition of the DEX effect occurs within the first 24 h after exposure. The antiandrogen flutamide neutralizes the effect of DHT, indicating that it acts through the androgen receptor to block the glucocorticoid. Therefore, the human fetal adrenal cortex may time lung development through both inhibitory and stimulatory mechanisms.     

Granules and Golgi vesicles with differential reactivity to ACTH antiserum in the corticotroph of the rat anterior pituitary.

Slices of unembedded rat anterior pituitaries, fixed with a periodate-lysine-paraformaldehyde (PLP) fixative, were incubated with guinea pig antiserum to ACTH and stained with a peroxidase-conjugated IgG fraction of anti-guinea pig gamma-globulin serum from rabbits. The fine structure of the stained cells was identical to that of the ACTH-secreting cell, as described by Siperstein and coworkers. Immunoreactive granules were mainly located at the periphery of the cell. Numerous granules of the inner cytoplasm and also the Golgi complex were nonreactive to the antiserum. The differential labeling for granules and Golgi apparatus peptide.     

Characterization of neurophysin in the anterior pituitary gland of sheep

Portions of sheep anterior pituitary lobe tissue were extracted under acid conditions and assayed for the two neurophysins (oN-III and oN-I/II) by radioimmunoassay. In all tissues examined, oN-III and oN-I/II immunoreactivities were detected. Using a combination of isoelectric focusing and polyacrylamide gel electrophoresis, the neurophysins of the anterior pituitary gland behaved like oN-III and oN-I/II. oN-III of the anterior pituitary was purified by affinity chromatography and high-performance liquid chromatography. This corticotroph oN possessed an amino acid analysis similar to that of oN-III and an N-terminal amino acid sequence, including residues 1-24, identical to that of authentic oN-III. These findings support the work of others who have identified neurohypophysial hormones in the anterior pituitary gland.     

Hypothalamic hypothyroidism caused by lesions in rat paraventricular nuclei alters the carbohydrate structure of secreted thyrotropin

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.     

Pro-opiomelanocortin mRNA levels fall in the fetal sheep pituitary before birth

In order to clarify the corticotrophic capacity of the fetal sheep anterior pituitary in late gestation, we have measured the relative levels of messenger RNA for the ACTH precursor molecule pro-opiomelanocortin (POMC) in individual fetal sheep anterior pituitaries collected between 100 and 144 days of gestation. The mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitary glands collected between 100 and 135 days (1.35 +/- 0.15) was significantly greater than the mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitaries collected between 141 and 144 days (0.81 +/- 0.09). Northern blot analysis showed that a single band of RNA hybridized with the human POMC cDNA probe in adult and fetal sheep pituitaries. Our results do not contradict the hypothesis that an increase in basal ACTH concentrations after 140 days of gestation could reflect a change in the post-translational processing of POMC in the fetal sheep anterior pituitary.     

Hypothalamic paraventricular unit activity during labour in the rat

The spike activity of 191 antidromically identified paraventricular units was recorded during labour and the oestrous cycle of rats under urethane anaesthesia. Accelerated bursts of neuronal activity, as reported to occur during suckling, were not observed and changes in firing rate were not correlated with individual uterine contractions. There was, however, an increase in the magnitude of the mean spontaneous firing rate of antidromically identified neurosecretory cells during labour, as compared with all stages of the oestrous cycle. This increase was mainly caused by (1) a marked reduction (greater than 25%) in the percentage of totally silent cells and (2) an increase in the magnitude of the mean firing rate of the phasically firing units (1.58-3.44 spikes/s). It was concluded from these increases in spike activity that the tonic release of neurohypophysial hormones is stimulated during labour, and that this may contribute to the induction and course of labour.     

Hypothalamic hypopituitarism after pituitary apoplexy in acromegaly.

Pituitary apoplexy in acromegaly is an uncommon event having been recorded approximately 30 times in the English literature.This report records two additional cases that included growth hormone measurements and an assessment of pituitary function. The apoplectic event developed spontaneously in one, and in the other it developed within two weeks of completing a course of radiotherapy to the pituitary gland. Autocure of the acromegaly was apparent. Basal levels of growth hormone were in the normal range but failed to change with provocative stimuli. Luteinizing hormone and follicle-stimulating hormone titers, although detectable, were inappropriately low for the degree of hypogonadism. Pituitary insufficiency was associated with a significant thyroid-stimulating hormone response to protirelin in one patient tested. It is suggested that these experiments of nature lend credence to the proposal that the hypothalamus may play a critical role in the perpetuation of growth hormone hypersecretion in some patients with acromegaly.     

Effect of fetal adrenalectomy on messenger ribonucleic acid for proopiomelanocortin in the anterior pituitary and for corticotropin-releasing hormone in the paraventricular nucleus of the ovine fetus

In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at approximately 122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothalamus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [adrenalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 +/- 2.5 ng/ml) than in ADX fetuses (mean +/- SD, 1.97 +/- 0.9 ng/ml; P less than 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hybridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of approximately 1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at approximately 1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P less than 0.025) in ADX fetuses (236 +/- 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P less than 0.05; mean +/- SEM, 179 +/- 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proopiomelanocortin processing in the anterior pituitary of the ovine fetus after lesion of the hypothalamic paraventricular nucleus

The hypothalamic-pituitary-adrenocortical axis plays an essential role in the maturation of fetal organs and, in sheep, birth. Lesioning the paraventricular nucleus (PVN) in fetal sheep prevents adrenocortical maturation and parturition without altering plasma immunoreactive ACTH concentrations. The purpose of this study was to determine the effect of PVN lesion on anterior pituitary processing of proopiomelanocortin (POMC) to ACTH, plasma concentrations of ACTH and ACTH precursors (POMC; 22-kDa proACTH), and expression of subtilisin-like prohormone convertase 3 (SPC3) in corticotropes in fetal sheep. PVN lesion did not affect anterior pituitary POMC and 22-kDa proACTH levels, whereas ACTH was significantly affected. The ACTH precursor (POMC plus 22-kDa proACTH) to ACTH ratio in the anterior pituitary was significantly increased after PVN lesion. Post-PVN lesion, fetal plasma ACTH(1-39), was below the limit of detection, whereas ACTH precursors (POMC plus 22-kDa proACTH) were not affected. In the inferior region of the anterior pituitary, 40-50% of corticotropes had detectable SPC3 hybridization signal, and PVN lesion did not change the extent of colocalization of POMC and SPC3, or SPC3 mRNA levels within corticotropes. Neither the percent of corticotropes in the superior region containing SPC3 hybridization (7-12%) or hybridization signal strength was altered in response to PVN lesion. In conclusion, the fetal PVN is necessary for sustaining adequate anterior pituitary processing of POMC to ACTH and ACTH release needed for maturing the adrenal cortex in the sheep fetus.     

Periconceptional undernutrition in sheep accelerates maturation of the fetal hypothalamic-pituitary-adrenal axis in late gestation

We investigated the effects of moderate maternal periconceptional undernutrition from 60 d before to 30 d after mating on fetal hypothalamic-pituitary-adrenal axis function in late gestation. Ewes were sampled regularly during the period of undernutrition for circulating hormone levels. Vascular catheters were inserted into ewes and their singleton fetuses at 112 d gestation (term, 145 d), and fetal ACTH(1-24) and metyrapone challenge tests were performed at 127 and 128 d. Postmortems were performed at 132 d. Fetuses of undernourished ewes (UN, n = 12) had elevated baseline cortisol concentrations (P < 0.05), compared with fetuses of ad libitum-fed ewes (n = 10). There were no differences between groups in fetal responses to ACTH challenge, but only UN fetuses demonstrated ACTH and 11-deoxycortisol responses to metyrapone (P < 0.05). UN fetuses had increased mRNA levels for proopiomelanocortin and prohormone convertase-1, but not -2, in the pars intermedia of the pituitary gland (P < 0.05). Glucocorticoid receptor mRNA levels were not different between groups in pituitary or hypothalamus. Maternal cortisol and ACTH levels during undernutrition were profoundly suppressed (P < 0.001), rather than elevated, in UN ewes. Furthermore, the normal pregnancy rise in maternal serum progesterone concentrations was delayed in undernourished mothers. These data demonstrate that events around the time of conception have profound effects on fetal hypothalamic-pituitary-adrenal development in late gestation and that factors other than fetal exposure to excess glucocorticoids may be important.     

Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary.

Galanin is a 29-amino acid neuropeptide widely distributed in the central nervous system. Galanin-like immunoreactivity (galanin-LI) was investigated in 23 nontumorous human adenohypophyses and 27 pituitary adenomas. Galanin-LI was demonstrated by immunohistochemistry on formalin-fixed paraffin-embedded sections, using the avidin-biotin-peroxidase complex method. All 23 nontumorous pituitaries showed the presence of galanin-positive cells which were similar to the corticotrophs in shape, size, histological features, and distribution. Immunoreactivity based on the study of mirror sections was found to be present in the cytoplasm of corticotrophs, indicating that galanin-LI colocalizes with ACTH. Crooke's cells and basophilic cells spreading to the posterior lobe (basophil invasion) were also positive for galanin. No galanin-LI was seen in lactotrophs of the human pituitaries, including 4 estrogen-treated men and 4 pregnant women. Varying numbers of tumor cells in 13 of 18 corticotroph adenomas were positive for galanin. Galanin-LI and ACTH were coexpressed in the cytoplasm of the same adenoma cells. In 19 pituitary adenomas composed of cells other than corticotrophs, no galanin-LI was noted. This study is the first report providing evidence of the presence of galanin-LI in corticotrophs of the human pituitary.     

Adrenocorticotrophic hormone in the anterior and neurointermediate lobes of the fetal rat pituitary gland

The concentration of ACTH in the pars distalis and pars intermedia of the fetal rat hypophysis from days 17-21 of pregnancy was measured with a specific radioimmunoassay and a bioassay using isolated adrenal cells from adult rats. In both lobes of the pituitary gland, a significant correlation was observed between immunoreactive and bioreactive values, expressed as pg equivalents synthetic human 1-39 ACTH per microgram protein. In the pars distalis, ACTH concentrations increased steadily from days 17-20 and then remained unchanged to term. At this time they were tenfold higher than on day 17. In the neurointermediate lobe, ACTH was detected only from day 18; the concentration of ACTH increasing between days 18 and 19. At each of the stages of pregnancy examined, the concentration of ACTH in the pars distalis was greater than that in the pars intermedia. These data have demonstrated that ACTH is present in both anterior and neurointermediate lobes of the fetal rat hypophysis, that the functional differentiation of the pars distalis takes place earlier than that of the pars intermedia, and that the concentrations of corticotrophin in the pars distalis and in the pars intermedia have different patterns of development as gestation progresses.     

Analysis of the appearance of fenestrations in the blood vessels of the fetal sheep pituitary

Using electron microscopy, the appearance of fenestrations within blood vessels of the fetal sheep pituitary gland was studied at 40, 80 and 122 days of gestation (term = 150 days). While fenestrations were extremely rare at 40 days of gestation, blood vessels contained characteristic regions of narrow endothelium, termed attenuations. There was an increase in the frequency of fenestrations with increasing gestational age, while there was a decrease in the number of attenuations, to the extent that the latter were not observed at 122 days of gestation. In order to examine the 'leakiness' of vessels with relatively few fenestrations, the fate of intracardially injected horseradish peroxidase (HRP) was studied in 50- and 51-day fetuses. Flocculant HRP reaction product was observed in the lumen of pituitary blood vessels, within endothelial cells, as well as within perivascular spaces and intercellular spaces between pituitary glandular cells. HRP was also observed in internalised vesicles within pituitary cells. These results indicate that even though fenestrations are scarce at early gestational ages, blood-borne substances can leave the portal network to interact with pituitary cells. The previously reported patency of the portal vascular system in the immature fetus is supported by our observations.