Potential involvement of EGF-like growth factors and phosphodiesterases in initiation of equine oocyte maturation.

Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when log transformed. Epiregulin and amphiregulin mRNA increased (P<0.01) from controls by 4 h after hCG administration, with amphiregulin increasing (P<0.01) by 2 h after hCG administration. Epiregulin and amphiregulin mRNA levels remained elevated (P<0.01) at 6h after hCG. These results indicate that EGF-like growth factors are likely paracrine mediators of the LH signal in the horse.

Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila.

Over the course of a 4-day period of metamorphosis, the Drosophila larval nervous system is remodeled to prepare for adult-specific behaviors. One example is the reorganization of peripheral nerves in the abdomen, where five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. The studies will help to establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1144-1160, 2017.

Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.

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.

Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback.

Kisspeptins are encoded by the gene KiSS-1 and regulate gonadotrophin-releasing hormone (GnRH) and gonadotrophin secretion in various species, including humans. Here, we quantify gene expression of KiSS-1 in the arcuate nucleus (ARC) across the ovine oestrous cycle and demonstrate an increase in the caudal division of the ARC during the preovulatory period. These data strongly suggest that kisspeptins are involved in the generation of the preovulatory GnRH and luteinising hormone surge.

The tripartite basal enhancer of the gonadotropin-releasing hormone (GnRH) receptor gene promoter regulates cell-specific expression through a novel GnRH receptor activating sequence.

The molecular mechanisms regulating restricted expression of GnRH receptor and gonadotropin subunit genes to gonadotrope cells have been the focus of intense interest. Using deletion and mutational analysis we have identified a tripartite enhancer that regulates cell-specific expression of the GnRH receptor gene in the gonadotrope-derived alphaT3-1 cell line. Individual elements of this enhancer include binding sites for steroidogenic factor-1; activator protein 1 (AP-1); and a novel element referred to as the GnRH receptor activating sequence (GRAS). Mutation of each element alone results in loss of approximately 60% of promoter activity. Combinatorial mutations of any two elements decreases promoter activity by approximately 80%. Finally, mutation of all three elements reduces promoter activity to a level not different from promoterless vector. Using 2-bp mutations, we have defined the functional requirements for transcriptional activation by GRAS. The core motif of GRAS is at -391 to -380 bp relative to the start site of translation and has the sequence 5'-CTAGTCACAACA-3'. Three copies of GRAS or GRAS with a 2-bp mutation (muGRAS) were cloned into a luciferase expression vector immediately upstream of the thymidine kinase minimal promoter (TK) and tested for expression in alphaT3-1 cells. When compared with TK promoter alone, activity of 3xGRAS-TKLUC was increased by more than 5-fold while activity of 3xmuGRAS-TKLUC was unchanged. When 3xGRAS-TKLUC was transfected into a variety of nongo-nadotrope cell lines, it did not increase activity of the TK promoter. We propose that basal activity of the GnRH receptor gene is regulated by a tripartite enhancer, and the key component of this enhancer is an element, GRAS, that activates transcription in a cell-specific fashion.

Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors.

We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element.

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.

Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer.

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.

Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor.

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.

Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.

A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.

Is gonadotrope expression of the gonadotropin releasing hormone receptor gene mediated by autocrine/paracrine stimulation of an activin response element?

Expression of the FSHbeta subunit and GnRH receptor (GnRHR) genes in gonadotropes is stimulated by activin. We sought to identify the cis-acting element(s) in the murine GnRHR gene promoter which confer activin responsiveness. We established that 600 bp of 5'flanking sequence from the murine GnRHR gene were sufficient to confer activin responsiveness in the gonadotrope-derived alphaT3-1 cell line. Since alphaT3-1 cells, like gonadotropes, secrete activin, we examined the ability of follistatin, an activin binding protein, to block the activin response. Increasing concentrations of follistatin from 0 to 100 ng/ml resulted in a dose dependent decrease in activity of the -600 promoter. Contained within this region are three elements important for expression in alphaT3-1 cells: a Steroidogenic Factor-1 binding site (SF-1), an Activator Protein-1(AP-1) element, and an element termed the GnRH receptor activating sequence or GRAS. A block mutation of GRAS inhibited the ability of the promoter to respond to follistatin. A more refined analysis using a series of two-bp mutations which scan GRAS and flanking sequence revealed exact convergence of GRAS with activin/follistatin responsiveness. Finally, a construct consisting of 3 copies of GRAS placed upstream of a heterologous minimal promoter (3xGRAS-PRL-LUC) was responsive to both activin stimulation and follistatin inhibition in alphaT3-1 cells. Thus, autocrine/paracrine stimulation of gonadotropes by activin illustrates a unique mechanism for cell-specific gene expression.

Regulation of gonadotropin-releasing hormone (GnRH) receptor messenger ribonucleic acid and GnRH receptors during the early preovulatory period in the ewe.

Estradiol increases the number of GnRH receptors in the ewe. Although results from studies conducted in vitro indicate that progesterone may have a negative influence on the number of ovine GnRH receptors, this effect of progesterone has not been documented in vivo. To explore the regulation of GnRH receptors at the level of gene expression, a partial complementary DNA (cDNA) encoding ovine GnRH receptor was isolated using reverse transcription and polymerase chain reaction methodology. This partial cDNA (701 basepairs) was used to isolate a full-length cDNA encoding GnRH receptor from an ovine pituitary cDNA library. Northern blot analysis of RNA from ovine pituitary glands using the partial cDNA as a molecular probe revealed four messenger RNA (mRNA) transcripts at 5.6, 3.8, 2.1, and 1.3 kilobases. In some samples, a fifth transcript at 0.8 kilobases was also evident. GnRH receptor mRNA was not detected in ovine brain, heart, kidney, adrenal, or liver tissues. To examine the regulation of GnRH receptor mRNA and GnRH receptors during the early preovulatory period, relationships among steady state concentrations of GnRH receptor mRNA, numbers of GnRH receptors, and circulating concentrations of progesterone and estradiol during luteolysis were characterized. We hypothesized that during luteolysis, decreased concentrations of progesterone would be associated with increased concentrations of GnRH receptor mRNA and increased numbers of GnRH receptors. On day 11 or 12 of the estrous cycle, luteolysis was induced in 14 ewes by treatment with prostaglandin F2 alpha (PGF2 alpha). Four ewes were treated with saline (saline controls). Anterior pituitary tissue was collected 4 h (n = 4), 12 h (n = 5), and 24 h (n = 5) after treatment with PGF2 alpha or 24 h after treatment with saline and from four untreated ewes on day 11 or 12 of the estrous cycle (untreated controls). Twelve hours after treatment with PGF2 alpha, circulating concentrations of progesterone had decreased (P < 0.05) to 46% of the control values; however, concentrations of estradiol were not different from those in control ewes. Concentrations of GnRH receptor mRNA increased 2-fold during luteolysis and were higher than control values 12 h after PGF2 alpha treatment (P < 0.05). This increase in GnRH receptor mRNA was not accompanied by an increase in the number of GnRH receptors. Twenty-four hours after treatment with PGF2 alpha, concentrations of progesterone in PGF2 alpha-treated ewes had decreased (P < 0.05) to 15% of control values, whereas concentrations of estradiol had increased (P < 0.05) to 321% of control values.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion.

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.

Expression of a murine gonadotropin-releasing hormone receptor-luciferase fusion gene in transgenic mice is diminished by immunoneutralization of gonadotropin-releasing hormone.

A line of transgenic mice harboring a fusion gene consisting of 1900 bp of proximal 5'-flanking region from the murine GnRH receptor gene linked to the complementary DNA encoding luciferase was established to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for identifying the molecular mechanisms underlying hormonal regulation of this gene. Of 10 tissues screened, luciferase was detected predominantly in pituitary gland, but also in brain and testis. To assess hormonal regulation, luciferase activity was measured in intact males and ovariectomized females treated with an anti-GnRH serum alone, and in combination with testosterone or 17beta-estradiol. No effect of steroid treatment on transgene expression was detected. However, immunoneutralization of GnRH resulted in decreased serum LH concentrations and suppressed pituitary expression of luciferase. Furthermore, the effects of GnRH antiserum could be prevented by the administration of a noncross-reactive GnRH agonist. Thus, 1900 bp of 5'-flanking DNA from the murine GnRH receptor gene are sufficient to target luciferase expression in transgenic mice to established sites of GnRH receptor gene expression. Furthermore, we suggest that GnRH regulation of GnRH receptor gene expression is mediated by regulatory elements residing within 1900 bp of the 5'-flanking region.

Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice.

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization.

We have isolated four lambda clones, which, in their aggregate, contain the entire coding sequence of the ovine gene encoding the gonadotropin-releasing hormone (GnRH) receptor (GnRHR). Like its human and murine counterparts, ovine GnRHR exists as a single-copy gene and is comprised of three exons and two introns. Furthermore, the locations of all exon-intron boundaries are perfectly conserved among the human, ovine and murine genes. The most striking difference among these genes is the location of the transcription start points (tsp) and, thus, the length of 5' untranslated region (UTR). This variation in size of the 5' UTR between the murine, human and ovine genes raises the possibility that different mechanisms have evolved for cell-specific expression of this gene. Isolation of the ovine GnRHR and its associated 5' flanking region is the essential first step in defining the molecular mechanisms underlying cell-specific and hormonal regulation of its expression in ruminants.

Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

Education, smoking and non-cardiovascular mortality: findings in three Chicago epidemiological studies.

The interrelationships among education, smoking, and non-cardiovascular (non-CVD) mortality were examined in middle-aged white males from the Chicago Peoples Gas Company Study (PG), the Chicago Western Electric Company Study (WE), and the Chicago Heart Association Detection Project in Industry (CHA). In each study, college graduates had the lowest prevalence of current smokers and the highest prevalence of former smokers. The associations between education and smoking were strongest in CHA, a study with baseline measurements 10-14 years after those of PG and WE and 3-8 years after the US Surgeon General's report on smoking and health in 1964. In PG and WE, the relative risks of non-CVD death for those who did not attend college compared to those who did were 1.50 and 1.38 (95% limits, 1.04 to 2.18 and 0.95 to 2.02). In CHA, the relative risk for those who did not graduate from college compared to those who did was 1.55 (1.17, 2.05). Differences in baseline cigarette smoking could account for only 23-29% of these increased risks. Because the associations between education and non-CVD mortality may have been confounded by changes in smoking status over the course of follow-up in these studies, non-CVD deaths were subdivided into those from causes related to smoking and causes not related to smoking. For smoking-related causes, the relative risk of death for those who did not attend/graduate from college was 1.95 (0.96, 3.95) in WE, 2.13 (1.18, 3.87) in PG, and 2.34 (1.47, 3.84) in CHA, while the relative risks for causes not related to smoking were 1.17, 1.12 and 1.16, respectively. These findings suggest that education is related inversely to non-CVD mortality primarily through smoking and smoking-related causes of death. With smoking becoming increasingly a habit of the less well-educated, these findings underscore the need for smoking prevention and cessation programmes targeted at the lower end of the socioeconomic scale.