Secretoneurin (SN) is a 31-42 amino acid neuropeptide, derived from the proteolytic processing of the precursor protein secretogranin-II (Scg2). In zebrafish, SNa and SNb are respectively 34 and 31 amino acids long, deriving from selective processing of the distinct Scg2a and Scg2b precursors. Our lab recently reported that frameshift mutations in Scg2 leads to reduces sexual behavior and disrupted spawning. This defect was partially rescued by injection of SNa. In my work, we determined the distribution of SNa in relation to other known reproductive hormones in zebrafish brain and pituitary by double immunofluorescent staining. SNa-immunoreactivity (ir) was observed in neuronal cell bodies in the ventral telencephalon, preoptic area (POA) and hypothalamus. Neuronal fibers staining for SNa projecting from the magnocellular POA passed through the pituitary stalk and terminated largely in the neurointermediate lobe (NIL). The SNa-ir fibers were less abundant but clearly present in the pars distalis. Moreover, SNa colocalized with isotocin in cell bodies in the POA and fibers in the NIL. Using the lhb-RFP x fshb-eGFP transgenic zebrafish line, we observed SNa-ir near gonadotroph cell bodies but not in them. Peptidomic analysis uncovered shorter processed fragments of both in SNa and SNb in whole brain and pituitary. We performed mass spectrometry to determine natural periovulatory variations and studied their potential bioactivities. Both SNa1-34 and SNa1-14 in the brain varied during the ovulatory cycle, while SNb-related peptides were relatively stable. The levels of SNa1-34 in brain peaked coincident with increased Gnrh3 at the time of the luteinizing hormone (Lh) surge. The levels of SNa1-14 in brain and ovaries peaked at the time of ovulation. To investigate the potential bioactivity of SNa1-34 and SNa1-14, we performed intraperitoneal injections and analyzed the expression numerous reproductive genes. The results suggested that SNa1-34 could induce ovulation by stimulating time-dependent expression of gnrh3 in brain, cga and lhb in pituitary and npr in ovaries. In contrast, SNa1-14 exhibited far fewer effects, but stimulated the expression of gnrh2 but suppressed gnrhr2, so its natural biological function remains unknown. After a single injection of SNa1-34 in females isolated from males, 61% (11/18) zebrafish ovulated. This compares favorably with the effects of the Lh analog human chorionic gonadotropin, inducing ovulation in 72% (13/18) of females. Natural variations in levels of SN in relation to other well-known neuropeptides and biological activity data in the zebrafish model support the hypothesis that SNa is a new stimulatory reproductive hormone. The SN peptides are conserved in evolution so what we uncover in fish may help us speculate on its importance in other vertebrates.

ZNF292 is a complex protein of ≈3000 amino acids with 16 zinc finger DNA binding domains. First discovered as a Growth Hormone (GH) transcription activator, ZNF292 was then found to play a role in the initiation of certain cancers, in specific autism spectrum disorder types, and to produce high levels of unique circular RNAs (circRNAs). In the rat, we have found that ZNF292 circRNAs are present in multiple related structures containing differing combinations of exons 1-7, thus increasing in sizes like “babushka” or nested Russian dolls. To begin to understand the functions of these ZNF292 circRNAs, we assayed expression in four rat MtT cell lines that model stages of pituitary somatotroph development. These cell lines have specific GH levels: MtT/E are empty: MtT/Se have estrogen stimulated GH expression; MtT/SM somatomammotrophs have both GH and PRL; and MtT/S somatotrophs produce only GH. Rat GH3 somatomammotroph cells were also assayed for comparison. Thus, the defined stages of pituitary development in these cells are an excellent model for evaluating linear or circular ZNF292 RNA changes. All cell lines were grown according to standard conditions, then harvested and total RNA was extracted (RNeasy kit, Qiagen) and used in qRT-PCR assays where both RT and PCR were performed in each well (Luna kit, NEB). Primers were designed in MacVector: RPL19 as a normalization control, and GH, linear, and circular ZNF292 primers targeted selected exons. Standards for quantitation used purified total rat RNA (ThermoFisher). After qRT-PCR, all data was normalized to the amount of RPL19 in each sample, then average RNA levels calculated for each cell line using Excel and GraphPad Prizm. We found that GH RNA rose from 0% in MtT/E and MtT/Se cells to 157% in MtT/SM and 275% in MtT/S cells. Linear ZNF292 was increased to 219% in MtT/Se cells, then declined to 110% in MtT/S and 37% in MtT/SM cells. Circular ZNF292 was expressed in MtT/E cells at 26%, increased to 184% in MtT/Se cells and fell to ≈100% in MtT/SM and MtT/S cells. GH3 cell expression was similar in pattern to MtT/SM cells but reduced in relative abundance. Statistical analysis with two-way ANOVA indicated significant differences. First, these data confirmed that GH RNA levels correlate with stages of somatotroph differentiation in these cell lines. Second, ZNF292 linear RNA expression increases prior to the onset of GH expression, agreeing with the function of linear ZNF292 in initiating GH transcription. Third, ZNF292 circRNAs have a developmental pattern of expression that is higher in somatomammotroph cells. Expression of other RNAs central to pituitary development will be used to evaluate whether ZNF292 circRNA levels specifically relate to GH status or another criterion of cellular differentiation.

No description available.

Controversy exists concerning the possible involvement of serotonin in the pituitary-adrenocortical response to stress. In the present research, a variety of physiological and pharmacological manipulations were used in male rats to study the role of this neurotransmitter in the adrenocortical response to insulin- induced hypoglycemia. First, the effect of insulin stress on hypothalamic 5-HT metabolism was examined, and an increased turnover was found as determined by an enhanced accumulation of 5-HT following monoamine oxidase inhibition. The corticos terone response to insulin was potentiated by prior administration of L-tryptophan, and blocked by pretreatment with valine, an amino acid that competes with tryptophan for transport across the blood-brain barrier. Treatment with the 5-HT receptor blocker methysergide, or serotonin depletion by intraventricular injection of 5 , 7-dihydroxy tryptamine significantly attenuated the insulin- induced rise in circulating corticosterone.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

Computed tomography (CT) is becoming more available as a diagnostic tool in the evaluation of the equine skull and brain. Objectives of this study were: 1) refine a CT protocol for evaluating the equine pituitary gland, 2.) define the CT anatomy of the pituitary region, 3.) determine a set of normal values for the pituitary dimensions (length, width, height, volume and weight), 4.) refine CT techniques for measuring pituitary size. Horses were scanned using 10x10mm, 10x5mm, 4x4mm and 4x2mm slice thickness and interval combinations. The pituitary glands were removed immediately after CT and gross measurements were performed. CT measurements were compared with gross pituitary measurements using analysis of variance (ANOVA) in a randomized block design. Accuracy percentages were also calculated using gross measurements as the known value. Mean dimensions of the histologically normal pituitary glands were: length 21.07mm, width 21.62mm, height 9.78mm and volume 2.66cm³. The weights ranged from 1.7g to 3.4g with a mean of 2.6g. Computed tomographic measurement analysis demonstrated that the 10mm slices were the most accurate way to estimate the length of the gland. The 4mm slices yielded the highest accuracy values for width, height and volume of the pituitary gland. The volume was underestimated by all interval and slice thickness combinations performed by CT. No evidence of an overlap effect was identified for any of the dimensions. Our findings indicated that contrast-enhanced CT is an accurate technique for estimating pituitary linear dimensions. Three-dimensional CT volumetry may not be an accurate method for estimating pituitary volume. / Master of Science

Improvements in turkey reproductive efficiency is a very desirable goal for the turkey industry. The ability to maintain turkey hens in the egg-laying (LAY) stage and produce one additional egg per hen a year is estimated to save the turkey industry approximately $1.5 million dollars per year. Overall protein expression generated by tissues of the hypothalamic-hypophyseal complex, namely the anterior pituitary, of the mature turkey hen have a profound impact on reproductive cycling (Scanes, 2000). One of the key physiological factors produced by the anterior pituitary and shown to play a significant role in the regulation of egg laying is the protein prolactin (Prl). The objectives for this study are to examine the overall protein expression patterns from turkey hen pituitary tissue during the nonphotostimulated (NPS), photostimulated (PS), and egg laying (LAY) stages. Attempts to isolate transcription factors that regulate the expression of Prl using an affinity chromatography technique or southwestern screening of a bacteriophage expression library were not successful. A global analysis of protein expression, using two-dimensional polyacrylamide gels (2D gels), was conducted using whole cell, cytoplasmic and nuclear protein extracts from pituitary tissue collected during the NPS, PS and LAY reproductive stages. Approximately 1,046 proteins ranging in pI from 4.6-8.2 and molecular weights between 100 kDa-6kDa were resolved. Protein expression patterns were replicated and verified using pituitaries harvested from NPS, PS and LAY stage turkey hens from another laboratory. Proteins showing considerable changes (563 proteins increased in expression and 98 proteins decreased in expression from the NPS to the LAY stage) in their expression between the reproductive stages were grouped in analysis sets for future identification. These proteins may prove to be important to the reproductive cycling of the turkey hen and warrant future investigation. The results of this study contribute to the overall understanding of the role that the pituitary, as a critical part of the hypothalamic-hypophyseal complex, plays in turkey hen reproductive cycling. / Master of Science

Wong Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 73-85). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Abbreviations --- p.viii / Chapter 1 --- Introduction --- p.1 / Overview --- p.1 / Chapter a. --- Steroidogenesis --- p.1 / Chapter i --- Ectopic steroidogenesis --- p.2 / Chapter ii --- Steroidogenesis in the pituitary --- p.5 / Chapter iii --- Proteins and enzymes involved in steroidogenesis and their distribution in brain and other sites --- p.6 / Chapter iv --- Regulation of adrenal steroidogenesis --- p.11 / Chapter b. --- Regulation of pituitary ACTH secretion --- p.13 / Chapter i --- Hypothalamic regulation --- p.13 / Chapter ii --- Paracrine regulation --- p.14 / Chapter iii --- Feedback regulation --- p.15 / Chapter c. --- Aims of the study --- p.15 / Chapter 2 --- Materials and methods --- p.18 / Chapter a. --- Materials --- p.18 / Chapter b. --- In vivo experiments --- p.25 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.25 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.25 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.26 / Chapter c. --- In vitro experiments --- p.26 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.28 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.28 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.29 / Chapter d. --- Statistical analysis --- p.29 / Chapter 3 --- Results --- p.30 / Chapter a. --- In vivo experiments --- p.30 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.30 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.38 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.47 / Chapter b. --- In vitro experiments --- p.51 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.51 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.55 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.57 / Chapter 4 --- Discussion --- p.58 / Further studies --- p.71 / References --- p.73 / Appendix 1 --- p.86

Preliminary analyses of three kinds of promoter activity showed that a-actin gene promoter was chosen to initiate the hormone transcription for the first consideration. We have fused the cDNAs encoding the intact somatolactins in frame to a zebrafish a-actin gene promoter to generate transgenic zebrafish lines co-injected with a GFP protein driven by the same promoter. The transgenic zebrafish were selected from GFP expression and confirmed by genomic PCR and Southern blot analysis, then maintained as transgenic founders. Measurement of the transgenes' expressions and the expressions of marker genes in different pathways by using real-time PCR provided a general understanding of SLs' actions. The data obtained indicated that the over-expressing of SLalpha and SLbeta in vivo significantly enhance the transcriptions of the insulin-like growth factors, IGF1 (5.46-fold and 6.77-fold), IGF2a (4.38-fold and 4.35-fold) and IGF2b (2.83-fold and 3.94-fold), but down-regulated IGF3 (a novel member found specifically in gonad) in larvae. However, the stimulation by administration of recombinant proteins (SLalpha and SLbeta) only showed a slight induction of the mRNA levels of IGFs (IGF1, IGF2a and IGF2b) on ZFL cells in vitro. / Somatolactin (SL) is a novel member of pituitary polypeptide hormone found only in fish; it shares significant structural homology with prolactin and growth hormone. Since somatolactin receptor (SLR) was first defined as GHR1 and orthologous to the growth hormone receptor GHR2, SL and GH may share similar actions in growth and development. Recently, two SLs have been identified as SLalpha and SLbeta with similar structures, freshwater fish have these two isoforms found in the same species and only one isoform (SLalpha) is found in marine species. The two isoforms of SL may have different functions and physiological actions. To investigate the roles of SLs on vertebrate development and embryogenesis, we generated transgenic fish models with "all zebrafish" elements in origin to study the physiological functions of SLs in zebrafish. / The ectopic expression of somatolactins also results in up-regulating gene expression of insulin, leptin, sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FAS), as well as the expression of vitellogenin and melanocyte-stimulating hormone (MSH) levels while causing reduction of catalase (CAT) and glutathione S-transferase (GST) levels in larvae. The results here represent the similar function between SLalpha and SLbeta and reveal more details in fish of the endocrinology system involvement in growth development, glucose synthesis, lipid metabolism, reproduction, pigmentation and antioxidant defense system through the actions of SLs. / Three different gene promoters of zebrafish have been isolated to initiate the ectopic expression of somatolactins in vivo, which including a constitutional beta-actin gene promoter, a liver specific transferrin gene promoter and a zinc ion inducible metallothionein (MT) gene promoter. The promoter activities were tested in fish cell-line by using luciferase reporter assay. In MT gene promoter, two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5'-flanking region (1,514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn2+ and the Zn 2+ induction was dose-dependent. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn2+ or Cd2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein. / Wan, Guohui. / Adviser: Chan King Ming. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.