Pituitary DNA Research Articles

DNA damage and growth hormone hypersecretion in pituitary somatotroph adenomas.

Drivers of sporadic benign pituitary adenoma growth are largely unknown. Whole-exome sequencing of 159 prospectively resected pituitary adenomas showed that somatic copy number alteration (SCNA) rather than mutation is a hallmark of hormone-secreting adenomas and that SCNAs correlate with adenoma phenotype. Using single-gene SCNA pathway analysis, we observed that both cAMP and Fanconi anemia DNA damage repair pathways were affected by SCNAs in growth hormone-secreting (GH-secreting) somatotroph adenomas. As somatotroph differentiation and GH secretion are dependent on cAMP activation and we previously showed DNA damage, aneuploidy, and senescence in somatotroph adenomas, we studied links between cAMP signaling and DNA damage. Stimulation of cAMP in C57BL/6 mouse primary pituitary cultures using forskolin or a long-acting GH-releasing hormone (GHRH) analog increased GH production and DNA damage measured by H2AX phosphorylation and a comet assay. Octreotide, a somatostatin receptor ligand that targets somatotroph adenoma GH secretion in patients with acromegaly, inhibited cAMP and GH and reversed DNA damage induction. In vivo long-acting GHRH treatment also induced pituitary DNA damage in mice. We conclude that cAMP, which induces somatotroph proliferation and GH secretion, may concomitantly induce DNA damage, potentially linking hormone hypersecretion to SCNA and genome instability. These results elucidating somatotroph adenoma pathophysiology identify pathways for targeted treatment.

MON-LB082 Elevated Expression of KISSR Is Associated with Secretory Function of Functioning Gonadotroph Adenomas

Background: Gonadotroph adenomas are identified as the most common histological subtype of pituitary adenomas. But overwhelming majority of them are hormonally silent and act as non-functioning pituitary adenomas that presenting only with mass effects. The rest few named functioning gonadotroph adenomas (FGAs) can secrete biologically active gonadotropins and lead to characteristic abnormities of the hypophysial-gonadal axis function, such as isosexual precocious puberty in children, ovarian hyperstimulation and menstrual irregularity in premenopausal women and testicular enlargement in men. The mechanism remains unknown why FGAs have different secretory function from the clinical non-functioning ones which are morphologically same. Kisspeptin secreted by KISS1 neurons on hypothalamus can stimulate GnRH secretion when combines with its receptor KISS1R. Objective: To investigate whether kisspeptin and KISS1R are of effect for the secretory function of FGAs. Methods: we studied 18 clinically and histologically verified pituitary adenoma tissue samples: Group I(3 FGAs including 2 females and 1 male) ,Group II(12 NFPAs with immunohistochemistry positive for FSH and/or LH)and Group III(3 NFPAs with immunohistochemistry negative for FSH and LH). The mRNA expression levels of Kisspeptin and KISS1R were measured by RT-qPCR and the protein expression of KISS1R were determined by Western bolt. We also sequenced KISS1R gene in sera of all 3 FGAs patients and 2 female FGA stissue samples. The DNA methylation analyses of KISS1R promoter region in PA tissues were also performed. Results: We found that the KISS1R mRNA level was significantly different among three groups(p<0.05): Group I was highest,which was 1.8 times as Group II and 8.5 times as Group III. But there was no difference of Kisspeptin mRNA level among the groups. Western bolt showed that the KISS1R protein level was also significantly up-regulated in FGA group than the other two groups(p<0.05). A missense change c.1091T>A in exon 5 of KISS1R gene was found in the both female FGA pituitary adenoma DNA while no mutations of this gene were found in the serum samples of 3 FGA patients. No DNA methylation differences of KISS1R promoter region were observed among the three groups. Conclusion: We speculate that the elevated expression of KISS1R could play a role in the secretion function of FGAs. The expression of KISS1R might need to achieve to a certain threshold that could bring the secretory ability of FGAs. Sources of Research Support: The National Key Research and Development Program of China” (No. 2016YFC0901501) and CAMS Innovation Fund for Medical Science (CAMS-2016-I2M-1-002) awarded to HJZ. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Involvement of DNA methylation in regulating rat Prop1 gene expression during pituitary organogenesis.

PROP1 is a pituitary specific transcription factor that plays a crucial role in pituitary organogenesis. The Prop1 shows varied expression patterns that promptly emerge and then fade during the early embryonic period. However, the regulatory mechanisms governing Prop1 expression remain unclear. Here, we investigated whether Prop1 was under epigenetic regulation by DNA methylation. Bisulfite sequencing was performed on DNA obtained from the pituitary glands and livers of rats on embryonic days (E) 13.5 and E14.5, and postnatal days (P) 4 and P30. The methylation of CpG sites in seven regions from 3-kb upstream of the Prop1 transcription start site through to its second intron were examined. Certain differences in CpG-methylation levels were observed in Region-1 (–2772 b to –2355 b), Region-4 (–198 b to +286 b), Region-5 (+671 b to +990 b), and Region-6 (+1113 b to +1273 b) based on comparisons between pituitary and liver DNA on E13.5. DNA methylation in pituitary glands on E14.5, P4, and P30 was generally similar to that observed in in the pituitary gland on E13.5, whereas the anterior and intermediate lobes of the pituitary gland on P4 and P30 showed only small differences. These results indicate that Prop1 is under regulation by CpG methylation during the early period of pituitary primordium development around E13.5.

Research Advances in Pituitary Adenoma and DNA Methylation.

DNA methylation is closely related to the genesis and development of pituitary adenoma. Studies have shown that high methylation in the promoter region of potassium voltage-gated chanel,shaker related subfamily,beta member 2,O-6-methylguanine-DNA methyltransferase,echinoderm microtubule associated protein like 2 ,ras homolog family member D ,homeobox B1 ,NNAT, and P16 inhibits the expression of these genes and regulates of the proliferation of pituitary adenoma. DNA methylation is also closely related to invasive pituitary adenoma. Therefore,further study on molecular mechanism of DNA methylation of pituitary adenoma will offer a new strategy for the diagnosis and treatment of pituitary adenoma.

Germline or somatic GPR101 duplication leads to X-linked acrogigantism: a clinico-pathological and genetic study.

Non-syndromic pituitary gigantism can result from AIP mutations or the recently identified Xq26.3 microduplication causing X-linked acrogigantism (XLAG). Within Xq26.3, GPR101 is believed to be the causative gene, and the c.924G > C (p.E308D) variant in this orphan G protein-coupled receptor has been suggested to play a role in the pathogenesis of acromegaly.We studied 153 patients (58 females and 95 males) with pituitary gigantism. AIP mutation-negative cases were screened for GPR101 duplication through copy number variation droplet digital PCR and high-density aCGH. The genetic, clinical and histopathological features of XLAG patients were studied in detail. 395 peripheral blood and 193 pituitary tumor DNA samples from acromegaly patients were tested for GPR101 variants.We identified 12 patients (10 females and 2 males; 7.8 %) with XLAG. In one subject, the duplicated region only contained GPR101, but not the other three genes in found to be duplicated in the previously reported patients, defining a new smallest region of overlap of duplications. While females presented with germline mutations, the two male patients harbored the mutation in a mosaic state. Nine patients had pituitary adenomas, while three had hyperplasia. The comparison of the features of XLAG, AIP-positive and GPR101&AIP-negative patients revealed significant differences in sex distribution, age at onset, height, prolactin co-secretion and histological features. The pathological features of XLAG-related adenomas were remarkably similar. These tumors had a sinusoidal and lobular architecture. Sparsely and densely granulated somatotrophs were admixed with lactotrophs; follicle-like structures and calcifications were commonly observed. Patients with sporadic of familial acromegaly did not have an increased prevalence of the c.924G > C (p.E308D) GPR101 variant compared to public databases.In conclusion, XLAG can result from germline or somatic duplication of GPR101. Duplication of GPR101 alone is sufficient for the development of XLAG, implicating it as the causative gene within the Xq26.3 region. The pathological features of XLAG-associated pituitary adenomas are typical and, together with the clinical phenotype, should prompt genetic testing.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0328-1) contains supplementary material, which is available to authorized users.

Alcohol and tobacco consumption alter hypothalamic pituitary adrenal axis DNA methylation

Alcohol and cigarette consumption have profound effects on genome wide DNA methylation and are common, often cryptic, comorbid features of many psychiatric disorders. This cryptic consumption is a possible impediment to understanding the biology of certain psychiatric disorders because if the effects of substance use are not taken into account, their presence may confound efforts to identify effects of other behavioral disorders. Since the hypothalamic pituitary adrenal (HPA) axis is known to be dysregulated in these disorders, we examined the potential for confounding effects of alcohol and cigarette consumption by examining their effects on peripheral DNA methylation at two key HPA axis genes, NR3C1 and FKBP5.We found that the influence of alcohol and smoke exposure is more prominent at the FKBP5 gene than the NR3C1 gene. Furthermore, in both genes, loci that were consistently significantly associated with smoking and alcohol consumption demethylated with increasing exposure.We conclude that epigenetic studies of complex disorders involving the HPA axis need to carefully control for the effects of substance use in order to minimize the possibility of type I and type II errors.

ISOLATION OF Β-ACTIN PROMOTER DERIVED FROM WALKING CATFISH (CLARIAS BATRACHUS)

Fish growth improvement as economic traits can be solved through fish transgenic production. Growth hormone gene is inserted into transgenic vector construction to over-express fish growth. The promoter as a part of the expression vector has an important role in its regulation. The use of promoter which is derived from mammalian or virus (such as CMV/ cytomegalovirus) in the expression vector, in specific goal as food material, has customer resistant rather than a promoter which is derived from in- sibling species. Beside of it, transgene expression level when using in-sibling promoter showed higher than using mammalian or viral promoter. The β-actin promoter is screened from walking catfish pituitary genome DNA using primers: pBA-cy-F (5’- GTGWGTGACGCYGGACCAAATC-3’) as forward primer and pBA-cy-R (5’- CCATRTCRTCCCAGTTGGTSACAAT-3’) as reverse primer, produced an amplicon of 1,7 kb in length. Sequence analysis using TF BindTM indicated transcription factor elements: TATA box, CCAAT box, enhancer (CAAT), and CarGG (CAAATGG) motif. This result showed that promoter which is obtained from this research is useful in construction of all catfish growth hormone vector expression catfish transgenic production. Keywords: β-actin promoter, growth hormone, expression vector, walking catfish (Clarias batrachus)

Means, Motive, and Opportunity: SDH Mutations Are Suspects in Pituitary Tumors

These are heady times in endocrinology, with the application of new techniques revolutionizing our understanding of the genetic pathophysiology of many syndromes of endocrine deficiency and excess. It seems that not a month goes by without a new genetic discovery, and last year’s teaching slides are already looking out of date. This ferocious pace of progress is uncovering pathways and potential targets for treatment in many rare conditions. Some discoveries are entirely new genetic causes, whereas others are known genes in new disease settings. This situation is analogous to the work of criminologists using the selfsame genetic techniques to link unsolved “cold” cases to known culprits via studies of archived evidence. In the medical setting, similar good clinical detective work is leading to known genetic culprits in other neuroendocrine tumors being hauled in for interrogation in connection with their links to pituitary tumors. In this issue of the JCEM, Dwight et al (1) describe the occurrence of a pituitary adenoma in the setting of a familial succinate dehydrogenase subunit A (SDHA) gene mutation associated with paraganglioma in a first-degree relative. This finding expands the field of the tumor features associated with succinate dehydrogenase subunit (SDHx) mutations into the pituitary, joining the first report from the Stratakis group (2) that definitively linked a succinate dehydrogenase subunit D (SDHD) germline mutation to acromegaly. Also, a case from Spain associated with a succinate dehydrogenase subunit C (SDHC) germline mutation was reported, although without pituitary tumoral DNA confirmation (3). Data from the literature back to 1952 that were reviewed by Xekouki and Stratakis (4) have included 29 instances of coexisting pituitary adenomas and pheochromocytomas/paragangliomas. Some modern cases that were negative for existing causative gene mutations appear to be persuasive for further investigation of SDHx status (4). Indeed, the clinical impact of these mitochondrial protein abnormalities already encompasses a remarkably wide range of tumors from renal cell cancers to sporadic and syndromic gastrointestinal stromal tumors (Carney triad and Carney-Stratakis syndrome) and beyond (5–9). The addition of pituitary adenomas to the expanding phenotype related to inheritable pheochromocytoma/paraganglioma syndromes is intriguing and represents both an opportunity and a challenge. An important area for future study will be to define whether SDHx mutation-associated pituitary adenomas have particular features that differentiate them from the general sporadic population. Clinically relevant pituitary adenomas occur is about 1 in 1000 people in the general population, and prolactinomas account for up to twothirds of these (10). Pituitary adenomas that occur in recognized genetic or familial settings account for about 5% of cases in our experience (11). Most occur in the setting of familial isolated pituitary adenoma—either with or without a germline aryl hydrocarbon receptor interacting protein (AIP) gene mutation—and multiple endocrine neoplasia (MEN) 1 (11–13). Rarer syndromes like that of Carney complex due to protein kinase A regulatory subunit 1A (PRKAR1A) mutations or MEN4 due to cyclindependent kinase inhibitor 1B (CDKN1B) gene mutations also feature pituitary adenomas as part of the described clinical spectrum (14, 15). Large collaborative studies of pituitary adenomas that occur against these known genetic backgrounds have revealed features related to more aggressive behavior, which are useful pointers when determining which patients to offer genetic testing to. AIP

Histopathological and biochemical changes in rat thyroid following acute exposure to hexavalent chromium.

Chromium in hexavalent form is highly toxic and a known carcinogen, although its effects on thyroid structure and function are relatively unexplored. Workers in an industrial environment can be, at times, exposed to this form of chromium. The present study was, therefore, designed using laboratory rats as a model system to investigate the effect on thyroid structure and function following two acute intraperitoneal doses of 30 mg/kg b.w. potassium dichromate administered within 48 hours. The results showed that hypothalamic chromium concentration increased (p<0.05) while thyroid chromium concentration decreased (p<0.01).The excretion of chromium in urine increased (p<0.05). The treated thyroid sections revealed hyperplasia. Follicles were disorganized, clustered and collapsed, while some of them were fused. Interfollicular spaces widened. Morphometrical analysis showed significantly (p<0.001) increased number of follicles whereas the follicular size significantly decreased (p<0.001). Nuclei were regressed (p<0.001); nuclear shapes were irregular; round, oval and shrunken. The membrane on the apical as well as the basal lamina side showed disruption. Colloid retraction within the follicles was noticeable in some sections stained with Periodic acid Schiff (PAS). Serum free tetra-iodothyronine (FT4) and free tri-iodothyronine (FT3) levels decreased (p<0.01 and p<0.001, respectively), while serum thyroid stimulating hormone (TSH) concentration increased (p<0.01). Ultrastructural analysis showed disrupted basal laminae of the follicles, regressed nuclei and disrupted cell organelles. Acridine orange stained thyroid cells demonstrated excessive dead cells, whereas DNA fragmentation assay demonstrated percent decrease of hypothalamic, pituitary and thyroidal total DNA.

Pituitary tumours: findings from whole genome analyses

Pituitary tumours are common intracranial neoplasms that cause significant morbidity through mass effects and/or the inappropriate secretion of pituitary hormones. Despite a considerable literature detailing potential pathogenic changes in these tumours, their aetiology remains largely unresolved. Recent studies have employed genome-wide profiling towards the identification of novel genes and pathways that are inappropriately expressed or regulated in this tumour type. The techniques employed vary in their complexity and interpretation; however, many of the findings from these types of studies have identified novel genes with potential and, in some cases, proven roles in pituitary tumorigenesis. These studies include comparative genomic hybridization, whole genome-wide allelotyping and methodologies for identification of novel genes associated with epigenetic silencing. In addition, differential display methodologies have been instrumental in the identification of transcripts inappropriately expressed including, pituitary tumour transforming gene, growth arrest and DNA damage-inducible protein (GADD)45gamma and a maternal expressed gene 3 isoform, which in some cases have proven roles in pituitary tumorigenesis. Although few studies of whole genome transcript analysis, as determined by microarray or gene-chip technologies, are reported, these studies of human pituitary, in some cases combined with proteomics, are yielding useful data. In addition, these types of investigation have been applied to several animal models of pituitary tumorigenesis, and in these cases novel genes are highlighted as showing significant change. The identification of the initiating events responsible for the transformation of a normal pituitary cell into one with unrestrained proliferative capacity has so far eluded us. No doubt, these new technologies allowing an essentially unbiased genome-wide analysis, perhaps in combination with animal models that display a preceding hyperplasia, will allow us to identify genes critical to tumour evolution and progression.

Ethanol Induces Hyperprolactinemia by Increasing Prolactin Release and Lactotrope Growth in Female Rats

Background Alcohol drinking is known to cause hyperprolactinemia in both humans and laboratory animals. The mechanism by which alcoholism causes hyperprolactinemia is not known. This study investigated whether increased pituitary production of prolactin, which leads to alcohol‐induced hyperprolactinemia, results from an increase in cell number and/or cell production of prolactin in the pituitary.Methods The effects of ethanol on lactotropes were determined in vivo using female rats as an animal model and in vitro using primary cultures of mixed rat anterior pituitary cells and enriched lactotropes. In vivo experiments involved administration of ethanol for 2 and 4 weeks using a liquid diet containing 8.7% ethanol (v/v), which provides 37% of the calories in cyclic, ovariectomized, and estradiol‐17β–treated ovariectomized Fischer‐344 rats. The control group was pair‐fed an isocaloric diet minus the ethanol or fed a normal diet ad libitum. These animals were used to determine ethanol's effects on plasma prolactin levels, pituitary wet weights, pituitary total protein levels, and the number of mitotic lactotropes. In vitro studies determined ethanol's effects in the presence and absence of estradiol on prolactin release and lactotropic cell proliferation. Prolactin levels in plasma and media samples were measured using radioimmunoassay. Mitotic lactotropes were determined using bromodeoxyuridine incorporation assay.Results Ethanol treatment increased in a time‐dependent manner the plasma levels of prolactin in cyclic, ovariectomized, and estradiol‐treated ovariectomized rats. Ethanol treatment also increased pituitary wet weight and/or pituitary total protein levels and DNA synthesis in lactotropes. Determination of ethanol's action on lactotropic cell proliferation and hormone secretion in vitro using primary cultures of mixed pituitary cells revealed that ethanol stimulated both basal and estradiol‐induced prolactin secretion and lactotropic cell proliferation. When ethanol's actions were studied in isolated lactotropes, ethanol alone or in combination with estradiol stimulated prolactin secretion but failed to increase lactotropic cell proliferation.Conclusions These results suggest that ethanol causes hyperprolactinemia by elevating prolactin release from lactotropes and by increasing the number of lactotropes in the anterior pituitary gland. The mitotic action of ethanol requires cell–cell communication between lactotropes and other pituitary cells. Furthermore, ethanol's mode of action on prolactin release and lactotrope growth is similar to that observed for estradiol.

Ethanol induces hyperprolactinemia by increasing prolactin release and lactotrope growth in female rats.

Alcohol drinking is known to cause hyperprolactinemia in both humans and laboratory animals. The mechanism by which alcoholism causes hyperprolactinemia is not known. This study investigated whether increased pituitary production of prolactin, which leads to alcohol-induced hyperprolactinemia, results from an increase in cell number and/or cell production of prolactin in the pituitary. The effects of ethanol on lactotropes were determined in vivo using female rats as an animal model and in vitro using primary cultures of mixed rat anterior pituitary cells and enriched lactotropes. In vivo experiments involved administration of ethanol for 2 and 4 weeks using a liquid diet containing 8.7% ethanol (v/v), which provides 37% of the calories in cyclic, ovariectomized, and estradiol-17beta-treated ovariectomized Fischer-344 rats. The control group was pair-fed an isocaloric diet minus the ethanol or fed a normal diet ad libitum. These animals were used to determine ethanol's effects on plasma prolactin levels, pituitary wet weights, pituitary total protein levels, and the number of mitotic lactotropes. In vitro studies determined ethanol's effects in the presence and absence of estradiol on prolactin release and lactotropic cell proliferation. Prolactin levels in plasma and media samples were measured using radioimmunoassay. Mitotic lactotropes were determined using bromodeoxyuridine incorporation assay. Ethanol treatment increased in a time-dependent manner the plasma levels of prolactin in cyclic, ovariectomized, and estradiol-treated ovariectomized rats. Ethanol treatment also increased pituitary wet weight and/or pituitary total protein levels and DNA synthesis in lactotropes. Determination of ethanol's action on lactotropic cell proliferation and hormone secretion in vitro using primary cultures of mixed pituitary cells revealed that ethanol stimulated both basal and estradiol-induced prolactin secretion and lactotropic cell proliferation. When ethanol's actions were studied in isolated lactotropes, ethanol alone or in combination with estradiol stimulated prolactin secretion but failed to increase lactotropic cell proliferation. These results suggest that ethanol causes hyperprolactinemia by elevating prolactin release from lactotropes and by increasing the number of lactotropes in the anterior pituitary gland. The mitotic action of ethanol requires cell-cell communication between lactotropes and other pituitary cells. Furthermore, ethanol's mode of action on prolactin release and lactotrope growth is similar to that observed for estradiol.

Ethanol Induces Hyperprolactinemia by Increasing Prolactin Release and Lactotrope Growth in Female Rats

Background Alcohol drinking is known to cause hyperprolactinemia in both humans and laboratory animals. The mechanism by which alcoholism causes hyperprolactinemia is not known. This study investigated whether increased pituitary production of prolactin, which leads to alcohol-induced hyperprolactinemia, results from an increase in cell number and/or cell production of prolactin in the pituitary. Methods The effects of ethanol on lactotropes were determined in vivo using female rats as an animal model and in vitro using primary cultures of mixed rat anterior pituitary cells and enriched lactotropes. In vivo experiments involved administration of ethanol for 2 and 4 weeks using a liquid diet containing 8.7% ethanol (v/v), which provides 37% of the calories in cyclic, ovariectomized, and estradiol-17β–treated ovariectomized Fischer-344 rats. The control group was pair-fed an isocaloric diet minus the ethanol or fed a normal diet ad libitum. These animals were used to determine ethanol's effects on plasma prolactin levels, pituitary wet weights, pituitary total protein levels, and the number of mitotic lactotropes. In vitro studies determined ethanol's effects in the presence and absence of estradiol on prolactin release and lactotropic cell proliferation. Prolactin levels in plasma and media samples were measured using radioimmunoassay. Mitotic lactotropes were determined using bromodeoxyuridine incorporation assay. Results Ethanol treatment increased in a time-dependent manner the plasma levels of prolactin in cyclic, ovariectomized, and estradiol-treated ovariectomized rats. Ethanol treatment also increased pituitary wet weight and/or pituitary total protein levels and DNA synthesis in lactotropes. Determination of ethanol's action on lactotropic cell proliferation and hormone secretion in vitro using primary cultures of mixed pituitary cells revealed that ethanol stimulated both basal and estradiol-induced prolactin secretion and lactotropic cell proliferation. When ethanol's actions were studied in isolated lactotropes, ethanol alone or in combination with estradiol stimulated prolactin secretion but failed to increase lactotropic cell proliferation. Conclusions These results suggest that ethanol causes hyperprolactinemia by elevating prolactin release from lactotropes and by increasing the number of lactotropes in the anterior pituitary gland. The mitotic action of ethanol requires cell–cell communication between lactotropes and other pituitary cells. Furthermore, ethanol's mode of action on prolactin release and lactotrope growth is similar to that observed for estradiol.

Different functions of QTL for estrogen-dependent tumor growth of the rat pituitary.

Estrogen treatment to rats of the Fischer 344 (F344) strain induces growth of pituitary tumors that exhibit accelerated cell proliferation, breakdown of basement membrane, and formation of hemorrhagic lakes. Estrogen-dependent pituitary growth is due to variation in a group of quantitative trait loci (QTL), called Edpm for estrogen-dependent pituitary mass, that we previously identified in an F(2) intercross of F344 and the tumor-resistant Brown Norway strain. We previously identified 5 QTL, and microsatellite markers developed since our earlier work have allowed us to scan new chromosomal regions, resulting in two new QTL for estrogen-dependent pituitary mass: Edpm9-2 and a possible QTL on the X Chromosome (Chr). Here we report evidence that these QTL differ from each other in how they affect growth. To examine the effect of the Edpm QTL on biochemical components of tumor growth, we tested their effects in 138 progeny of a backcross to the F344 strain which were given a 10-week chronic estrogen treatment. Hemoglobin/DNA ratio (a measure of blood volume relative to cell number) and total pituitary DNA (a measure of cell number) correlated only weakly, and very large pituitaries were observed which had a low hemoglobin/DNA ratio resembling a normal gland. Through QTL mapping, we found that Edpm2-1, Edpm3, Edpm5, and Edpm9-2 all had significant effects on pituitary mass, but Edpm2-1 and Edpm9-2 primarily affected DNA content, Edpm5 primarily affected hemoglobin/DNA ratio, and Edpm3 affected all traits equally.

Cyclical expression of egr-1/NGFI-A in the rat anterior pituitary: a molecular signal for ovulation?

Molecular genetic studies of egr-1/NGFI-A have recently established a key role for this immediate early gene in anterior pituitary development, and particularly in transcriptional regulation of the LH beta-subunit gene. These studies, using null mutant mice, addressed the developmental aspects of gonadotrophin gene regulation by egr-1 and, because of the limitations of this approach, did not address the role of egr-1 in adult reproductive physiology. Using the rat, we have now investigated the expression of egr-1 during the female reproductive cycle, and have found evidence of cyclical changes in anterior pituitary egr-1 expression, at mRNA, protein, and DNA binding activity levels. Specifically, we have shown that egr-1 mRNA is significantly elevated during proestrous, and conversely suppressed on the subsequent day of oestrous. We have also demonstrated significantly raised levels of an approximately 70 kDa Egr-1 immunoreactive protein band during the night of proestrous, together with markedly raised levels of a Egr-1 consensus sequence DNA binding complex. These studies are indicative of both direct egr-1 gene regulation by oestrogen, and also regulation by GnRH. Finally, we have sequenced previously uncharacterized regions of the egr-1 gene and found evidence of a potential oestrogen response element. Our findings are consistent with the hypothesis that Egr-1 forms at least part of the molecular signal for ovulation in the pituitary.

Genetic basis of congenital hypothyroidism: abnormalities in the TSHbeta gene, the PIT1 gene, and the NIS gene.

We have elucidated the molecular pathology of three types of congenital hypothyroidism. Thyrotropin (TSH) is the major regulator of thyroid function. In cases of isolated congenital TSH deficiency, we found that they are caused by a missense mutation in the conserved CAGYC region of the TSHbeta gene. Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), TSHbeta genes, and the PIT1 gene itself. In cases of combined deficiency of GH, PRL, and TSH, we found that they are caused by abnormalities in the PIT1 gene, either recessively or dominantly. Sodium dependent iodide symporter (NIS) actively transports iodide into the thyroid cells to produce thyroid hormones. In cases of iodide transport defect, we elucidated that a missense mutation in the transmembrane region of the NIS gene caused them.

Gender-specific induction of pituitary RNA by estrogen and its modification by thyroid hormone.

Estrogen and thyroid hormones play important roles in the regulation of pituitary function. We presently show that pituitary weight and total cellular RNA levels were significantly decreased by ovariectomy in female rats and were significantly increased by castration in males, without alterations in pituitary DNA levels as compared to intact animals. Treatment with a single dose of estrogen produced a significant increase in pituitary RNA in ovariectomized females but not castrated males. This effect was more obvious following multiple doses of estrogen, and was blocked by pretreatment with cycloheximide, or surprisingly by concomitant administration of triiodothyronine (T3). Analysis of estrogen response element (ERE) binding activity in pituitary nuclear protein extracts revealed that estrogen produced a rapid induction of a slow mobility complex of ERE binding in ovariectomized females much greater than in castrated males. Thus, estrogen-induced increases in pituitary total RNA levels are dependent on new protein synthesis, are gender-specific, are inhibited by T3, and may be mediated via specific estrogen-induced changes in protein-DNA interactions.

Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.

Human pituitary adenomas express endogenous inhibin subunit and follistatin messenger ribonucleic acids.

Specific factors involved in the pathogenesis of tumors that stimulate clonal human pituitary adenoma cell proliferation remain unknown. An important question regarding the pathogenesis of human pituitary tumors is whether they synthesize autocrine regulatory factors that regulate both hormone biosynthesis and neoplastic growth. Activin and inhibin are both comprised of inhibin subunits and have diverse regulatory roles as growth and differentiation factors in normal and neoplastic tissue. Activin stimulates FSH beta messenger ribonucleic acid (mRNA) biosynthesis and FSH secretion, and these effects are down-regulated in normal gonadotrophs by the endogenous glycoprotein follistatin. In addition to its effects on gonadotrophs, activin modulates hormone secretion by somatotroph and corticotroph cell lines. It is not known whether human neoplastic pituitary tissue synthesizes inhibin subunits or follistatin or whether their expression is cell type specific. We investigated whether alpha-, beta A-, and beta B-inhibin subunit and follistatin mRNAs could be detected in 27 human pituitary adenomas [clinically nonfunctioning (n = 11), somatotroph (n = 5), corticotroph (n = 5), and lactotroph (n = 6)] using reverse transcriptase-polymerase chain reaction techniques. Twenty-six of the tumors contained mRNAs encoding one or more inhibin subunits. beta B-Inhibin mRNA was the most prevalent (81% of tumors), followed by beta A-inhibin (59% of tumors) and alpha-inhibin (52% of tumors). Endogenous alpha-, beta A-, and beta B-inhibin subunit mRNA synthesis was also examined in normal human pituitary and testicular complementary DNA libraries, and all subunit mRNAs were detected. In contrast to the widespread expression of inhibin subunits in pituitary tumors, follistatin mRNA was detected in a subset of nonfunctioning tumors (54%) as well as in control normal human pituitary and testicular complementary DNA libraries. Tumor-specific follistatin biosynthesis was not observed in other pituitary tumor subtypes. These data are the first to demonstrate that 1) endogenous inhibin subunits are synthesized in human pituitary adenomas of all known secretory phenotypes as well as normal pituitary tissue; and 2) follistatin gene expression in pituitary adenomas is specific to clinically nonfunctioning or gonadotropin subunit-producing tumors. The characterization of inhibin subunit and follistatin biosynthesis by human pituitary tumors will be important in investigating their potential roles in regulating both tumor phenotype and cell proliferation.

Biochemical parameters in the anterior pituitary during the course of tumorigenesis induced by diethylstilbestrol treatment

Treatment of F 344 rats with diethylstilbestrol (DES) for 1–2 months induces a prolactin (PRL)-secreting pituitary adenoma. After 8 weeks of DES treatment, we have shown that the ratio of regulatory subunits of the cAMP-dependent protein kinase (RI/RII) increased in the tumors. Presently we report the variations in RI/RII ratio, pituitary weight, DNA content, serum PRL, nuclear estrogen receptor (E 2R) and of ornithine decarboxylase (ODC) activity from the time of DES pellet implantation until 8 weeks. Pituitary weight, DNA content and serum PRL rose significantly at 4 weeks with a maximum at 6–8 weeks, and significantly correlated with each other. E 2R and ODC activity increased from week 1 onwards, with a maximum at 2 weeks and decreased at 8 weeks. Both variables showed a positive correlation but neither E 2R nor ODC activity correlated with pituitary weight, DNA or serum PRL. Values for RI remained stable with time, but RII decreased progressively. The RI/RII ratio was maintained around unity between 1–4 weeks, increasing to 1.6–2 thereafter. This ratio positively correlated with pituitary weight and DNA. It is suggested that during tumor induction by estrogen in a sensitive strain of rats, growth signals with different time-courses become activated. Increases in pituitary weight and DNA content, indicators of mammotroph hypertrophy and hyperplasia, were preceeded by early rises in E 2R and ODC activity. Increases in the RI/RII ratio accompanied the adenomatous change, suggesting their role in cell transformation after 6 weeks of estrogen exposure.