Gonadotropin-releasing hormone and its receptor in normal and malignant cells.

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells.

1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence. 2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis. 3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression. 4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.

Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells.

To dissect the functional architecture of the rat gonadotropin releasing hormone (GnRH) gene promoter and its regulation by estrogen, gene transfer studies were performed in a placental cell line. 5'-Deletional constructs demonstrated that cis-acting elements important for rGnRH promoter activity in placenta were contained within a region of the proximal promoter between -73 and -16 bp upstream of the transcription initiation site. In addition, an inhibitory region was found from -903 to -424. Deletion of sequences to -424 which removed the inhibitory region, produced a promoter fragment which exhibited a 50% inhibition of GnRH promoter activity in the presence of estrogen (E) and cotransfected estrogen receptor. Negative regulation by E was retained in constructs deleted to -73 bp. We conclude that sequences important for placental cell expression and estrogen regulation of rGnRH lie within the region from -73 to -16. In addition, potential repressor sequences active in placental cells are present between -903 to 424.

Structure of the rat gonadotropin releasing hormone (rGnRH) gene promoter and functional analysis in hypothalamic cells.

The gonadotropin releasing hormone (GnRH) gene encodes a protein which plays a critical role in mammalian reproductive physiology. Its expression is predominantly restricted to the hypothalamus although it has also been described in the placenta. To begin to determine the promoter elements important for tissue specific expression and to examine the mechanisms of developmental and hormonal regulation of the rat GnRH (rGnRH) gene, we cloned the rGnRH gene from a rat liver genomic DNA library. The nucleotide sequence of greater than 3 kb of 5'-flanking region was determined. The transcriptional initiation site in rat hypothalamic tissue and a mouse hypothalamic cell line were mapped by primer extension analysis and found to be different. In addition, transient transfection studies demonstrated that multiple regions of the distal promoter are important for tissue specific and basal promoter activity in hypothalamic cells. Furthermore, in these cells a potent activation region resides between -3026 and -1031 bp and suppressor region between -1031 and -903 bp upstream of the transcriptional start site. We conclude that different portions of the 5'-flanking region, which are activating and suppressing in nature, are critical for hypothalamic expression of the rGnRH gene.

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe.

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.

Intravenous administration of pulsatile gonadotropin-releasing hormone in hypothalamic amenorrhea: effects of dosage.

Eighteen women with well characterized hypothalamic amenorrhea underwent 30 cycles of pulsatile GnRH treatment in an effort to examine the role of GnRH dosage in pituitary and ovarian responses. GnRH was administered iv at 2 doses (25 and 100 ng/kg bolus) at a physiological range of frequencies (90 and 60 min) in the follicular phase of the induced cycles. After demonstration of ovulation by ultrasound and clinical parameters, the frequency of GnRH administration was progressively slowed from every 60 min to every 90 min and then to every 240 min to mimic the slowing of endogenous LH secretion that occurs during the luteal phase in normal women. The results of these induced cycles were compared to those of 62 ovulatory cycles from normal women. Overall clinical and biochemical results revealed the following. Patients receiving doses of 25 ng/kg GnRH successfully ovulated only 80% of the time, with recruitment of a single dominant follicle. Two of 5 patients became pregnant. Peak estradiol levels were significantly lower than normal [261 +/- 33 (+/- SE) vs. 342 +/- 11 pg/ml, respectively; P less than 0.02]. Integrated luteal phase progesterone production was also significantly reduced in the 25 ng/kg group compared to normal (78 +/- 17 vs. 145 +/- 8 ng/ml/entire luteal phase, respectively; P less than 0.02). All women receiving bolus doses of 100 ng/kg GnRH ovulated; maturation of multiple follicles occurred in 5 of 20 cycles, and 6 of 7 women conceived. Peak estradiol values were significantly higher than those in either normal women or the 25 ng/kg group (478 +/- 48 pg/ml; P less than 0.02 for both), with integrated luteal phase progesterone levels significantly higher than those in patients receiving the 25 ng/kg dose (196 +/- 25 ng/ml/luteal phase; P less than 0.02). This study demonstrates that ovulation and fertility can be achieved with a physiological frequency regimen of pulsatile GnRH administration using bolus doses of both 25 and 100 ng/kg in women with hypothalamic amenorrhea; the 25 ng/kg dose of GnRH may represent a threshold of stimulation of the pituitary-ovarian axis and recreates cycles with an inadequate luteal phase; and a 100 ng/kg dose of GnRH may well cause a supraphysiological stimulation of the pituitary-gonadal axis.

Preparation for barium enema: comparison of a commercial formula diet and low-residue, clear-liquid diets in inpatients.

The barium enema examination is the most frequently done initial diagnostic evaluation for disease of the lower gastrointestinal tract. Adequate preparation of the colon is essential because inadequate preparation necessitates repetition, with patient discomfort, decreased caloric intake, and increased cost. A formula diet (Ensure) has been reported as superior to the usual low-residue, clear-liquid diet for bowel preparation. We evaluated the feasibility of using this formula diet in a general medical inpatient population. We found no significant difference in the quality of preparation (72% and 76% optimal or adequate preparation, respectively) for our inpatient population. The formula diet has several theoretical advantages, including a greater number of calories, lower cost, and better patient acceptance.