Regulation of growth hormone expression by thyrotropin-releasing hormone through the pituitary-specific transcription factor Pit-1 in chicken pituitary

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone beta- (TSHbeta) encoding genes. Pit-1 has been identified in several mammalian and avian species. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and it stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully developed a competitive RT-PCR for the detection of Pit-1 expression in the chicken pituitary, that was sensitive enough to detect picogram levels of Pit-1 mRNA. Applying this method, the effect of TRH injections on Pit-1 mRNA expression was determined in the pituitary of chick embryos and growing chicks. In both 18-day-old embryos and 10-day-old male chicks the Pit-1 mRNA expression was significantly increased following TRH injection, thereby indicating that the stimulatory effects of TRH on several pituitary hormones is mediated via its effect on Pit-1 expression. Therefore, a semi-quantitative RT-PCR method was used to detect possible changes in GH levels. TRH affected the GH mRNA levels at both developmental stages. These results, combined with the data on Pit-1 mRNA expression, indicate that Pit-1 has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH.     

The chicken pituitary-specific transcription factor PIT-1 is involved in the hypothalamic regulation of pituitary hormones

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.     

六盘山肉牛杂交改良群体Pit-1基因多态性分析

本试验旨在对宁夏六盘山区肉牛杂交改良群体垂体特异性转录因子1(Pit-1)基因的多态性进行研究,为其杂交改良提供理论基础。采用聚合酶链式反应—限制性片段长度多态性方法（PCR-RFLP）,对该群体101个个体Pit-1基因多态性进行检测。结果表明,扩增出的451 bp片段为目的基因片段,其PCR产物经限制性内切酶HinfⅠ消化后表现出多态性,共检测到AA、BB和AB 3种基因型。其中AA基因型频率6.93%（7个）;AB基因型频率34.65%（35个）;BB型基因型频率58.42%（59个）,等位基因A频率24.26%;等位基因B频率75.74%。因此,宁夏六盘山地区的肉牛杂交改良群体Pit-1基因具有多态性。     

巢湖鸭生长素基因和垂体特异性转录因子1基因的PCR-SSCP检测

以生长素基因Ghrelin和垂体特异性转录因子1 Pit-1基因为候选基因,采用PCR-SSCP和DNA测序技术检测2个候选基因在巢湖鸭群体中的单核苷酸多态性(SNPs)。结果表明,Ghrelin基因exon 3第54 bp位置有G→A碱基的点突变,在巢湖鸭群体中检测到AA、AB、BB 3种基因型,A等位基因频率为0.49,B等位基因频率为0.51;在exon 5第149位和166位发生了C→A和G→T的突变,检测到MM、MN、NN 3种基因型,M等位基因频率为0.19,N等位基因频率为0.81。Pit-1基因的2对引物的扩增片段均未检测到多态性,说明所检测的Pit-1基因外显子3和4序列比较保守。     

Immunocytochemical identification of Pit-1 containing cells in the anterior pituitary of hens

Our goal was to identify the cells expressing Pit-1 protein in chicken anterior pituitary. The anterior pituitaries were collected from laying hens after perfusion with formalin-PBS, and fixed with Bouin's fixative followed by paraffin embedding. Sections of the anterior pituitaries were immunostained for Pit-1 in the first staining sequence followed by staining for 6 types of pituitary hormones in the second sequence. Pit-1 positive nuclei were observed in the glandular cells in both the cephalic and caudal lobes. Pit-1 immunoreaction products were colocalized in the glandular cells immunopositive for growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone or prolactin. These results indicate that Pit-1 protein induction occurs in 6 types of glandular cells, suggesting that Pit-1 may regulate hormone synthesis in each glandular cell in the chicken pituitary.     

Pituitary Hormone Responses to Luteal Regression after Administration of Prostaglandin F2α-Analogue

As an experiment to elucidate the canine luteal function maintenance system, a PGF2alpha-analogue, fenprostalene (PGF-F), was administered 24 and 54 days after ovulation to induce luteal regression, and the responses of luteinizing hormone (LH) and prolactin (PRL), which are considered to support the canine corpus luteum, were investigated. The plasma progesterone (P(4)) level was rapidly decreased, by which the plasma PRL level was increased, but no change was observed in the plasma LH level. The decrease in the plasma P(4) level after PGF-F administration may have induced positive feedback to the superior system, and stimulated secretion of the pituitary hormones. These findings suggested that the canine corpus luteum is maintained by PRL, not by LH.     

妊娠期山羊腺垂体中催乳素促卵泡素和促黄体素的分布特点

为了阐明妊娠期垂体中催乳素(PRL)、促卵泡素(FSH),促黄体素(LH)的分布和三者之间协调关系.本试验采用免疫组化SP法检测了PRL、FSH,LH三种激素在妊娠各期山羊的腺垂体细胞中免疫反应产物的分布特点.结果显示,在山羊妊娠过程中,腺垂体中PRL阳性细胞多存在于远侧部,数量逐渐增多,胞质呈强阳性;FSH和LH阳性细胞分布在远侧部和结节部,FSH胞质内颗粒着色深浅不一,LH多核膜着色;相对表达量都逐渐升高,而升高程度不同,表现为妊娠后期LH表达量急剧升高,FSH则变化较小,除了FSH的相对表达量在妊娠前期和中期之间无显著差异(P>0.05)外,3种激素的相对表达量在妊娠前期、中期和后期3个时期之间都存在显著差异(P<0.05),妊娠前、中期FSH的相对表达量均高于PRL和LH,妊娠后期LH的表达量最高.FSH次之,PRL最低.提示PRL,FSH和LH三种激素相互协同以促进胚胎着床并维持妊娠,而且在妊娠早期PRL起主要作用,之后协同FSH和LH维持妊娠.     

High level expression of Prop-1 gene in gonadotropic cell lines

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.     

猪垂体细胞单层培养

本研究建立了猪垂体前叶细胞单层培养模型 ,并进行了免疫细胞化学研究。结果显示 ,用 0 2 5 %胶原酶 ,0 0 5 %透明质酸酶和 0 0 1%DNA酶消化垂体前叶 2次 ,每个垂体可获 5× 10 8个细胞 ,细胞活率在 90 %以上。用含 10 %小牛血清的McCoy’s 5A培养液培养 2 0h ,细胞贴壁 ,4 0h左右形成单层 ,形成单层后换无血清培养液仍维持生长。抗LH和FSH免疫细胞化学染色表明 ,大多数LH阳性细胞同时也是FSH阳性细胞。在单层细胞中 ,LH和FSH阳性细胞占细胞总数的比例不足 10 %。本培养模型可用于体外研究垂体细胞的功能及调控     

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH_1-24 expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.     

Effects of dopamine, norepinephrine and serotonin on secretion of luteinizing hormone, follicle-stimulating hormone and prolactin in ovariectomized, pituitary stalk-transected ewes

Two experiments were conducted in ovariectomized, pituitary stalk-transected ewes to determine if dopamine (DA), norepinephrine (NE) or serotonin (5-HT) alter secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL). In experiment 1, ewes were infused (iv) with saline (control), DA (66 micrograms/kg/min), NE (6.6 micrograms/kg/min) or 5-HT (6.6 micrograms/kg/min). Treatments did not alter pulse frequency, but 5-HT increased (P less than .05) amplitude of pulses of LH and mean concentrations of LH, DA and NE were without effect on basal secretion of LH. DA but not NE or 5-HT decreased (P less than .05) the release of LH in response to gonadotropin hormone-releasing hormone (GnRH, 25 micrograms, im). Concentrations of FSH were not affected by treatments. Secretion of PRL was reduced (P less than .05) by treatment with DA and NE but not 5-HT. Each amine reduced (P less than .05) the release of PRL in response to thyrotropin-releasing hormone (TRH; 3 micrograms, im). In experiment 2, ewes were given DA at doses of 0, 0.66, 6.6 or 66.0 micrograms/kg/min, iv. No dose altered basal LH, but each dose reduced (P less than .05) basal and TRH-induced release of PRL. Key findings from these studies include direct pituitary action for: (1) 5-HT enhanced basal secretion of LH, (2) suppression of GnRH-induced secretion of LH by DA. (3) DA and NE inhibition of PRL secretion, and (4) DA, NE and 5-HT inhibition of release of PRL in response to TRH.     

Bovine Posterior Pituitary Extract Stimulates Prolactin Release from the Anterior Pituitary Gland In Vitro and In Vivo in Cattle

It has been reported that the posterior pituitary (PP) gland contains a potent, unknown prolactin (PRL)-releasing factor (PRF) in rats. PRFs are assumed to be produced in neurones located within the hypothalamus, and to be peptidergic in nature. However, little is known about PRFs in domestic animals. To characterize the PRF in the PP of domestic animals, the present study examined the PRL-releasing activity of an acidic extract from bovine PP (bPP) in vitro and in vivo in cattle. First, the PRL-releasing effect of bPP extract was compared with that of PRL-releasing peptide (PrRP), and thyrotropin-releasing hormone (TRH) from cultured bovine anterior pituitary cells. The extract significantly increased PRL concentrations in the culture medium, at doses of 0.002 and 0.02 eq./ml (one eq. is the PP extract from one animal), compared with the control (p < 0.05). PrRP failed to stimulate the release of PRL. TRH significantly increased PRL concentrations in the culture medium, at doses from 10(-9) to 10(-7) M, compared with the control (p < 0.05). The rate of increase in the PRL concentration, by 0.02 eq./ml bPP extract, was significantly greater than that in TRH (p < 0.05). Secondly, plasma PRL responses to the intravenous (i.v.) injection of bPP extract (0.5 eq./head), PrRP [3.59 mug/kg body weight (BW)], TRH (1 mug/kg BW), and a dopamine receptor antagonist (sulpiride, 0.1 mg/kg BW), were examined in calves. PrRP failed to stimulate PRL release; however, plasma PRL increased immediately following the injection of bPP extract, TRH and sulpiride. The PRL-releasing effect of i.v. injections of TRH and sulpiride was more potent than that of bPP extract. Finally, plasma PRL responses to the intra-hypothalamic injection of bPP extract were examined in calves. The intra-hypothalamic infusion (arcuate nucleus) of 0.0625 eq./head of bPP extract strongly stimulated PRL release in calves (p < 0.05). The present results show that PP contains a physiologically potent PRF in cattle.     

Correlations between presence of spontaneous lesions of the pituitary (adenohypophysis) and plasma prolactin concentration in aged Wistar rats.

The predictive value of elevated plasma prolactin concentrations for the presence of spontaneous pituitary lesions was studied in 40 male and 38 female Wistar (Cpb:WU) rats, all 30 months old. The pituitaries were examined light microscopically and stained for prolactin using immunohistochemical methods. Plasma prolactin concentrations were measured by radioimmunoassay. Pituitary lesions were classified on the basis of their morphology in hematoxylin and eosin-stained sections as foci of hypertrophic or hyperplastic cells and hemorrhagic, pleomorphic, or spongiocytic adenomas; no carcinomas were found. There were significantly (P = 0.001) more female than male rats with pituitary adenomas (58% females, 33% males) or without any pituitary lesions (21% females, 5% males); however, there were less female (21%) than male rats (63%) with foci of hyperplastic and/or hypertrophic cells but no adenomas in the pituitary (P = 0.001). Elevation of plasma prolactin concentration above the upper 99th percentile value in age-matched rats without lesions was predictive, but not conclusively, of the presence of pituitary hemorrhagic adenomas in both sexes. It was, however, not predictive of the presence of foci of hypertrophic or hyperplastic cells. Elevation of plasma prolactin concentration above 10 ng/ml in male and 60 ng/ml in female rats was conclusive for the presence of hemorrhagic adenomas. Using multivariate analysis, significant positive correlations (P less than 0.01) were found between plasma prolactin concentration and presence and size of hemorrhagic adenomas and their prolactin staining intensity (correlation coefficients between 0.392 and 0.652). Foci of hyperplastic cells stained positively for prolactin, whereas hypertrophic cell foci and pleomorphic and spongiocytic adenomas did not stain for prolactin. There were no correlations (coefficients of less than +/- 0.189) between plasma prolactin concentration and the presence of hypertrophic or hyperplastic cell foci and pleomorphic or spongiocytic adenomas in the pituitary. The morphologic criteria developed to distinguish spontaneous hypertrophic, hyperplastic, and neoplastic lesions of the rat pituitary corresponded well with their prolactin immunoreactivity and/or ability to elevate plasma prolactin concentration. These criteria constitute a biologically meaningful classification system for these rat pituitary lesions.     

Histopathological and immunohistochemical features of proliferative lesions in the pituitary pars distalis of rats

Pituitary proliferative lesions derived from the endocrine cells of the pars distalis are frequently encountered and adenomas/carcinomas are a common cause of death in standard 2-year carcinogenicity studies using various rat strains, especially Sprague-Dawley. This report describes the immunohistochemical characteristics of pituitary tumors derived from the pars distalis in rats. Prolactin (PRL)-containing tumors are the most common, with PRL/growth hormone (GH) dual positive tumor masses (PRL/GH co-positive tumor masses) being more prevalent than only PRL-positive tumor masses (PRL single-positive tumor masses). GH-containing tumors are relatively numerous and many of these are also PRL/GH co-positive tumor masses. TSH-containing tumors are common in females. PRL-containing tumors have been shown to increase the incidence of hyperlactation in males and mammary adenomas/adenocarcinomas in females, suggesting that these masses are functional tumors.