Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2)) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E(2). The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

Decreased expression of A-kinase anchoring protein 150 in GT1 neurons decreases neuron excitability and frequency of intrinsic gonadotropin-releasing hormone pulses.

The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca(2+) oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca(2+) oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 +/- 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 +/- 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion.

Intermolecular interactions of homologs of germ plasm components in mammalian germ cells.

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.

Decreased gonadotropin-releasing hormone neuronal activity is associated with decreased fertility and dysregulation of food intake in the female GPR-4 transgenic rat.

Expression of a cAMP-specific phosphodiesterase in GnRH neurons in the GPR-4 transgenic rat resulted in decreased LH levels and pulse frequency and diminished fertility. We have characterized changes in fertility, adiposity, and reproductive and metabolic hormones with age. Although LH levels were decreased in 3-, 6-, and 9-month-old GPR-4 females relative to wild-type (WT) controls, GPR-4 females did not become anovulatory until 6 months of age. No differences were observed in FSH, estradiol, or androstenedione levels in 3-, 6-, or 9-month-old GPR-4 and WT females. At 9 months of age, GPR-4 females had significantly increased abdominal and sc fat depot weights that were associated with increased leptin and insulin levels not observed in WT females. We tested the hypothesis that metabolic changes observed at 9 months of age were the result of dysregulation of the mechanisms controlling energy balance. Two-month-old female GPR-4 rats placed on a high-energy diet gained weight at a rate significantly greater than WT females and, after 24 d, developed the same metabolic phenotype observed in 9-month-old GRP-4 females (increased abdominal and sc fat associated with elevated leptin and insulin concentrations). Overeating did not correlate with changes in estradiol or androstenedione levels. We conclude that decreased GnRH neuronal activity is closely associated with decreased reproductive function and dysregulation of food intake.

Targeted expression of a dominant-negative fibroblast growth factor (FGF) receptor in gonadotropin-releasing hormone (GnRH) neurons reduces FGF responsiveness and the size of GnRH neuronal population.

Increasing evidence suggests that fibroblast growth factors (FGFs) are neurotrophic in GnRH neurons. However, the extent to which FGFs are involved in establishing a functional GnRH system in the whole organism has not been investigated. In this study, transgenic mice with the expression of a dominant-negative FGF receptor mutant (FGFRm) targeted to GnRH neurons were generated to examine the consequence of disrupted FGF signaling on the formation of the GnRH system. To first test the effectiveness of this strategy, GT1 cells, a GnRH neuronal cell line, were stably transfected with FGFRm. The transfected cells showed attenuated neurite outgrowth, diminished FGF-2 responsiveness in a cell survival assay, and blunted activation of the signaling pathway in response to FGF-2. Transgenic mice expressing FGFRm in a GnRH neuron-specific manner exhibited a 30% reduction in GnRH neuron number, but the anatomical distribution of GnRH neurons was unaltered. Although these mice were initially fertile, they displayed several reproductive defects, including delayed puberty, reduced litter size, and early reproductive senescence. Overall, our results are the first to show, at the level of the organism, that FGFs are one of the important components involved in the formation and maintenance of the GnRH system.

Role of cAMP signaling in the mediation of dopamine-induced stimulation of GnRH secretion via D1 dopamine receptors in GT1-7 cells.

Pharmacologically increasing cyclic adenosine monophosphate (cAMP) levels in GT1 gonadotropin-releasing hormone (GnRH) cell lines increased the secretion of GnRH. Dopamine (DA) increased the GnRH secretion in GT1 cells via a DA receptor positively coupled to adenylate cyclase. We then asked whether inhibition of the DA-induced increase in cAMP would block the stimulatory effect of DA on GnRH release. Expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to inhibit the DA-induced increase in cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad) or, for controls, with an empty Ad (Null-Ad). Infection with the PDE-Ad completely blocked the forskolin-induced stimulation of GnRH secretion and [Ca2+]i and decreased the majority of the release of cAMP into the culture medium. In contrast, although PDE-Ad infection blocked virtually all of the DA-induced increase in extracellular cAMP, the release of GnRH and the increase in [Ca2+]i were only delayed for approximately 15 min. GT1 cells express the D1 DA receptor which is positively coupled to adenylate cyclase but not the D5 DA receptor. These data suggest that the initial phase of the DA-induced secretion of GnRH is dependent on an increase in cAMP levels. However, it appears that an additional non-cAMP-regulated signaling pathway is involved in the stimulation of GnRH release via the D1 DA receptor.

Pulsatile luteinizing hormone and follicle-stimulating hormone secretion and gonadotropin subunit mRNA levels in the ovariectomized GPR-4 transgenic rat.

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the alpha-glycoprotein hormone subunit (alpha-GSU), LH-beta and FSH-beta subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the alpha-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-beta subunit by 38% and the FSH-beta subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the alpha- and beta-subunit mRNA levels.

Lowering cyclic adenosine-3',5'-monophosphate (cAMP) levels by expression of a cAMP-specific phosphodiesterase decreases intrinsic pulsatile gonadotropin-releasing hormone secretion from GT1 cells.

Pulsatile GnRH secretion is an intrinsic property of GnRH neurons. Since increases in cAMP levels increase excitability and GnRH secretion in the GT1-1 GnRH cell line, we asked whether cAMP levels play a role in timing excitability and intrinsic pulsatile GnRH secretion. The expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to lower cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad), or for controls with an empty Ad (Null-Ad) or an Ad expressing green fluorescent protein (GFP-Ad). Infection with the PDE-Ad significantly inhibited forskolin-induced increases in cAMP production, GnRH secretion, and Ca2+ oscillations. Infection of GT1-1 cells with the PDE-Ad vs. GFP-Ad or Null-Ad controls significantly decreased spontaneous Ca2+ oscillations and inhibited the frequency of GnRH pulses. These data support the hypothesis that the level of cAMP in GT1 neurons is a component of the biological clock timing neuron excitability and pulsatile GnRH secretion. Genetically targeted expression of PDE4D1 represents a powerful approach to study the role of cAMP levels in specific populations of neurons in transgenic animals.

The antiangiogenic factor 16K human prolactin induces caspase-dependent apoptosis by a mechanism that requires activation of nuclear factor-kappaB.

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats.

Experiments in the GT1 gonadotropin-releasing hormone (GnRH) cell line have shown that the cAMP signaling pathway plays a central role in regulating the excitability of the cells. Lowering cAMP levels by expressing the constitutively active cAMP-specific phosphodiesterase PDE4D1 in GT1 cells inhibited spontaneous Ca2+ oscillations and intrinsic pulsatile GnRH secretion. To address the role of cAMP levels in endogenous GnRH neurons, we genetically targeted expression of PDE4D1 (P) to GnRH neurons in transgenic rats (R) by using the GnRH gene promoterenhancer regions (G). Three lines of transgenic rats, GPR-2, -4, and -5, were established. In situ hybridization and RT-PCR studies demonstrated that transgene expression was specifically targeted to GnRH neurons. Decreased fertility was observed in female but not in male rats from all three lines. The mean luteinizing hormone (LH) levels in ovariectomized rats were significantly reduced in the GPR-4 and -5 lines but not in the GPR-2 line. In castrated male and female GPR-4 rats, the LH pulse frequency was dramatically reduced. Six of twelve GPR-4 females studied did not ovulate and had polycystic ovaries. The remaining six females ovulated, but the magnitude of the preovulatory LH surge was inhibited by 63%. These findings support the hypothesis that cAMP signaling may play a central role in regulating excitability of GnRH neurons in vivo. The GPR-4 line of transgenic rats provides a genetic model for the understanding of the role of pulsatile gonadotropin release in follicular development.

Localization of olfactory cyclic nucleotide-gated channels in rat gonadotropin-releasing hormone neurons.

The GT1 GnRH cell lines express all three subunits of the cyclic nucleotide-gated (CNG) channels (CNG2, 4.3 and 5) expressed in olfactory neurons. We investigated using in situ hybridization and double immunofluorescence whether endogenous GnRH neurons in therat also express CNG channel subunits. Sections from male and female adult rats were hybridized with a digoxigenin-labeled riboprobe made to regions of rat GnRH, CNG2, CNG4.3 or CNG5 cDNAs. In both sexes, 70-80% of GnRH neurons contained mRNAs for CNG2, CNG4.3 and CNG5. Similarly, double immunofluorescence staining for GnRH and CNG2, CNG4.3 or CNG5 confirmed that 70-80% of GnRH perikarya contained all three CNG subunit proteins. Moreover, the distribution of the immunostaining of CNG subunits in the external layer of the median eminence overlapped with GnRH with the presence of functional cAMP-gated cation channels. The presence of CNG channel subunits in the median eminence supports the notion that coordination of the excitability of the scattered GnRH perikarya may occur at the level of the nerve terminals.