Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We (7)], it is difficult to devise protocols for chronic administration of exogenous LH that mimic endogenous pulse patterns of LH. To circumvent this limitation, we report a transgenic mouse model in which elevated hormone levels are maintained chronically, without requiring multiple injections,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. supraphysiologic dosing, or dampening of the hypothalamicpituitary-gonadal axis.We utilized a two-pronged approach to achieve elevated levels of serum LH. (i) Increased secretion of hormone from the pituitary was achieved by expression of an LH(3 subunit transgene, targeted to the pituitary by a previously characterized bovine a-subunit promoter (8,9). (ii) The LH,B transgene was modified to encode a peptide extension that we proposed would slow the elimination of LH from the serum. This peptide is normally found at the C terminus of the 3 subunit of human chorionic gonadotropin (hCG) (hCG,B) and is referred to as the C-terminal peptide (CTP) (10). The CTP is thought to be a major determinant of the serum t,'2 of hCG and has been shown to increase the til2 of FSH 2-to 3-fold when fused to its (3 subunit (11). Accordingly, we constructed a transgene with the coding region of bovine (b) LH,B fused in-frame to the coding region of CTP (bLH3-CTP). MATERIALS AND METHODSConstruction of the bLH3-CTP Transgene. The bLH,B-CTP minigene was engineered by a two-step PCR. (i) A short fragment containing the C terminus of bLH,B (30 bp) fused in-frame to the CTP (last 87 bp of the hCGj3 gene) was generated. (ii) This fragment was lengthened to contain the entire bLHf3 cDNA fused in-frame to the CTP. This fusion gene was utilized for transfection experiments but was modified to contain the first intron of bLH3 for the transgene construct. The resulting insert was ligated into a BSK-plasmid already containing the bovine a-subunit promoter (positions -315 to +45) and the simian virus 40 late polyadenylylation signal. Transgenic mice were generated by microinjecting the bLH3-CTP insert into fertilized oocytes as described (12). Mice were genotyped by PCR analysis of tail DNA using primers specific to the a-subunit promoter and (3-subunit gene.Analysis of t,,2. Recombinant bLH and bLH-CTP heterodimers were generated by stably cotransfecting CHO cells with viral expression vectors containing the bovine a-subunit gene and either the bLH( or bLH,3-CTP genes as described (13). Serum-free medium was collected and ammonium sulfate-precipitated or concentrated with an Amicon ultrafiltration cell. Female rats were pretreated with 50 ,tg of antide (Sigma) in 20% (vol/vol) propylene glycol, injected subcutaneously 2 h prior to hormone injections t...

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.

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Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

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