GnRH signaling, the gonadotrope and endocrine control of fertility.

Mammalian reproductive cycles are controlled by an intricate interplay between the hypothalamus, pituitary and gonads. Central to the function of this axis is the ability of the pituitary gonadotrope to appropriately respond to stimulation by gonadotropin-releasing hormone (GnRH). This review focuses on the role of cell signaling and in particular, mitogen-activated protein kinase (MAPK) activities regulated by GnRH that are necessary for normal fertility. Recently, new mouse models making use of conditional gene deletion have shed new light on the relationships between GnRH signaling and fertility in both male and female mice. Within the reproductive axis, GnRH signaling is initiated through discrete membrane compartments in which the receptor resides leading to the activation of the extracellular signal-regulated kinases (ERKs 1/2). As defined by gonadotrope-derived cellular models, the ERKs appear to play a central role in the regulation of a cohort of immediate early genes that regulate the expression of late genes that, in part, define the differentiated character of the gonadotrope. Recent data would suggest that in vivo, conditional, pituitary-specific disruption of ERK signaling by GnRH leads to a gender-specific perturbation of fertility. Double ERK knockout in the anterior pituitary leads to female infertility due to LH biosynthesis deficiency and a failure in ovulation. In contrast, male mice are modestly LH deficient; however, this does not have an appreciable impact on fertility.

Evaluation of a fibrillin 2 gene haplotype associated with hip dysplasia and incipient osteoarthritis in dogs.

OBJECTIVE: To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS: 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS: The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

Analysis of the calcium-dependent regulation of proline-rich tyrosine kinase 2 by gonadotropin-releasing hormone.

Calcium influx through L-type voltage-gated calcium channels (VGCC) is required for ERK activation induced by GnRH in pituitary gonadotropes. The current studies investigate VGCC-sensitive catalytic activities that may lie upstream of ERKs within the GnRH signaling network. Ion exchange fractionation of alphaT3-1 cell lysates subjected to anti-phosphotyrosine Western blot analysis revealed a nifedipine-sensitive activity that colocalized with proline-rich tyrosine kinase (Pyk) 2 immunoreactivity. Phosphorylated Pyk2 was present in alphaT3-1 cells after GnRH agonist administration for a time course that lasted up to 4 h. Pyk2 phosphorylation was also evident in gonadotropes in vivo after administration of a bolus of GnRH. Knockdown of Pyk2 using specific small interfering RNAs revealed that Pyk2 contributed to modulation of GnRH-induced ERK but not c-Jun N-terminal kinase activation. Using pharmacological approaches, calmodulin (Cam) was also demonstrated to be required for the phosphorylation of Pyk2. Pyk2 was shown to bind specifically to a Cam agarose affinity column in a calcium-dependent manner, suggesting Cam and Pyk2 are capable of forming a complex. Specific mutation of a putative Cam binding motif within the catalytic domain of Pyk2 blocked association with Cam and uncoupled Pyk2's ability to activate ERK-dependent gene transcription. Thus, GnRH induces Pyk2 tyrosine phosphorylation dependent upon calcium flux within gonadotropes. Furthermore, association of Pyk2 and Cam may be required to mediate the effects of calcium on Pyk2 phosphorylation and subsequent activation of ERKs by GnRH.

Signaling complexes associated with the type I gonadotropin-releasing hormone (GnRH) receptor: colocalization of extracellularly regulated kinase 2 and GnRH receptor within membrane rafts.

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.

Transcript profiling of immediate early genes reveals a unique role for activating transcription factor 3 in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone.

Recent studies profiling immediate early gene responses to GnRH in the LbetaT2 gonadotrope cell model revealed increased expression of numerous genes including activating transcription factor (ATF) 3. The present studies demonstrate similar results with GnRH administration in vivo in ovariectomized mice. In this model, ATF3 mRNA was markedly up-regulated at 20, 40, and 60 min after in vivo administration of a GnRH analog. In alphaT3-1 gonadotrope cells, ATF3 mRNA and protein were induced by GnRH in a manner consistent with in vivo observations. Pharmacological studies implicated a combined role for the activities of protein kinase C isozymes, ERK and c-Jun N-terminal kinase, in modulating ATF3 expression. The role of ATF3 was further investigated in the activation of the human glycoprotein hormone alpha-subunit gene promoter. GnRH induced the alpha-subunit promoter-luciferase reporter approximately 16-fold, and this induction was completely abolished with mutations in the dual cAMP response elements (CREs) or the combined inhibition of GnRH-induced ERK and c-Jun N-terminal kinase. GnRH induced recruitment of ATF3, c-Jun, and c-Fos to the dual CREs. Overexpression and specific knockdown of ATF3 by small inhibitory RNA implicate a functional role for ATF3 in mediating activation of the alpha-subunit gene promoter. These studies provide clear evidence that ATF3 is a key immediate early gene induced by GnRH administration in vivo and in the alphaT3-1 gonadotrope cell model. These studies support the conclusion that the dual CREs of the human alpha-subunit promoter are the target of GnRH-induced MAPK regulation through ATF3.

Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin.

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.

A role for CCAAT/enhancer-binding protein beta in the basal regulation of the distal-less 3 gene promoter in placental cells.

The homeodomain protein Distal-less 3 (Dlx3) is essential for normal placental development in mice. Dlx3-null mice die by embryonic day 10.0 due to placental failure. The aim of our studies was to examine the transcriptional regulation and expression of Dlx3 in choriocarcinoma cell lines and primary trophoblasts from human placenta. A Dlx3 promoter fragment coupled to a luciferase reporter gene was sufficient to increase luciferase activity more than 11-fold over a luciferase control vector in choriocarcinoma cells, but not in a heterologous gonadotrope cell line. A 5' deletion series of the Dlx3 promoter revealed that a 13-nucleotide CCAAT box-containing element was required for basal expression in choriocarcinoma cell lines. Mutation of the CCAAT box within the context of the full-length promoter resulted in reduced basal activation of the Dlx3 reporter gene, suggesting that the CCAAT box was required for full basal expression. Western blot analysis revealed that Dlx3, CCAAT/enhancer-binding protein alpha (C/EBP alpha), and C/EBP beta were present in choriocarcinoma cells and isolated trophoblasts from term human placentas. Electrophoretic mobility shift assays revealed the formation of a specific complex between choriocarcinoma cell nuclear extracts and the Dlx3 CCAAT box sequence. Competition and antibody electrophoretic mobility shift assays revealed that CCAAT/enhancer-binding protein beta (C/EBP beta) binds the Dlx3 CCAAT box sequence. Overexpression of C/EBP beta was sufficient to increase basal expression of a Dlx3 reporter gene in a dose-dependent manner. These studies provide the first insight into the mechanism(s) of Dlx3 gene expression in placental cells and suggest a role for C/EBP beta in the basal regulation of the Dlx3 gene.

Power of a Labrador Retriever-Greyhound pedigree for linkage analysis of hip dysplasia and osteoarthritis.

OBJECTIVE: To estimate the number of dogs required to find linkage to heritable traits of hip dysplasia in dogs from an experimental pedigree. ANIMALS: 147 Labrador Retrievers, Greyhounds, and their crossbreed offspring. PROCEDURE: Labrador Retrievers with hip dysplasia were crossed with unaffected Greyhounds. Age at detection of femoral capital ossification, distraction index (DI), hip joint dorsolateral subluxation (DLS) score, and hip joint osteoarthritis (OA) were recorded. Power to find linkage of a single marker to a quantitative trait locus (QTL) controlling 100% of the variation in a dysplastic trait in the backcross dogs was determined. RESULTS: For the DI at the observed effect size, recombination fraction of 0.05, and heterozygosity of 0.75, 35 dogs in the backcross of the F1 to the Greyhound generation would yield linkage at a power of 0.8. For the DLS score, 35 dogs in the backcross to the Labrador Retriever generation would be required for linkage at the same power. For OSS, 45 dogs in the backcross to the founding Labrador Retrievers would yield linkage at the same power. Fewer dogs were projected to be necessary to find linkage to hip OA. Testing for linkage to the DLS at 4 loci simultaneously, each controlling 25% of the phenotypic variation, yielded an overall power of 0.7 CONCLUSIONS AND CLINICAL SIGNIFICANCE: Based on this conservative single-marker estimate, this pedigree has the requisite power to find microsatellites linked to susceptibility loci for hip dysplasia and hip OA by breeding a reasonable number of backcross dogs.

Evaluation of multiple radiographic predictors of cartilage lesions in the hip joints of eight-month-old dogs.

OBJECTIVE: To determine the radiographic methods that best predict the development of osteoarthritis in the hip joints of a cohort of dogs with hip dysplasia and unaffected dogs. ANIMALS: 205 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbred dogs. PROCEDURE: Pelvic radiography was performed when the dogs were 8 months old. Ventrodorsal extended-hip, distraction, and dorsolateral subluxation (DLS) radiographs were obtained. An Orthopedic Foundation for Animals-like hip score, distraction index, dorsolateral subluxation score, and Norberg angle were derived from examination of radiographs. Osteoarthritis was diagnosed at the time of necropsy in dogs > or = 8 months of age on the basis of detection of articular cartilage lesions. Multiple logistic regression was used to determine the radiographic technique or techniques that best predicted development of osteoarthritis. RESULTS: A combination of 2 radiographic methods was better than any single method in predicting a cartilage lesion or a normal joint, but adding a third radiographic method did not improve that prediction. A combination of the DLS score and Norberg angle best predicted osteoarthritis of the hip joint or an unaffected hip joint. All models that excluded the DLS score were inferior to those that included it. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of the DLS score and Norberg angle was the best predictor of radiographic measures in 8-month-old dogs to determine whether a dog would have normal or osteoarthritic hip joints.

ERK signaling, but not c-Raf, is required for gonadotropin-releasing hormone (GnRH)-induced regulation of Nur77 in pituitary gonadotropes.

Stimulation of pituitary gonadotropes by hypothalamic GnRH leads to the rapid expression of several immediate early genes that play key roles in orchestrating the response of the gonadotrope to hypothalamic stimuli. Elucidation of the signaling mechanisms that couple the GnRH receptor to this immediate early gene repertoire is critical for understanding the molecular basis of GnRH action. Here we identify signaling mechanisms that underlie regulation of the orphan nuclear receptor Nur77 as a GnRH-responsive immediate early gene in αT3-1 cells and mouse gonadotropes in culture. Using a variety of approaches, we show that GnRH-induced transcriptional upregulation of Nur77 in αT3-1 cells is dependent on calcium, protein kinase C (PKC), and ERK signaling. Transcriptional activity of Nur77 within the gonadotrope is regulated posttranslationally by GnRH signaling via PKC but not ERK activity. Surprisingly, neither activation of the ERK pathway nor the transcriptional response of Nur77 to GnRH requires the activity of c-Raf kinase. In corroboration of these results, Nur77 responsiveness to GnRH was maintained in gonadotropes from mice with pituitary-targeted ablation of c-Raf kinase. In contrast, gonadotropes from mice with pituitary deficiency of ERK signaling failed to up-regulate Nur77 after GnRH stimulation. These results further clarify the role of ERK and PKC signaling in regulation of the GnRH-induced immediate early gene program as well as GnRH-induced transcription-stimulating activity of Nur77 in the gonadotrope and shed new light on the complex functional organization of this signaling pathway in the pituitary gonadotrope.

Membrane rafts and GnRH receptor signaling.

The binding of hypothalamic gonadotropin-releasing hormone (GnRH) to the pituitary GnRH receptor (GnRHR) is essential for reproductive function by stimulating the synthesis and secretion of gonadotropic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Engagement of the GnRHR by GnRH initiates a complex series of signaling events that include the activation of various mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinase (ERK). GnRHR signaling is thought to initiate within specialized microdomains in the plasma membrane termed membrane rafts. These microdomains are enriched in sphingolipid and cholesterol and are believed to be highly dynamic organizing centers for receptors and their cognate signaling molecules associated with the plasma membrane. Within this review we discuss the composition and role of membrane rafts in cell signaling and examine evidence that the mammalian type I GnRHR is constitutively and exclusively localized to these membrane microdomains in various experimental models. We conclude that membrane raft composition and organization potentially underlie the functional ability of GnRH to elicit the assembly of multi-protein signaling complexes necessary for downstream signaling to the ERK pathway that ultimately is critical for controlling fertility.

ERK signaling in the pituitary is required for female but not male fertility.

Males and females require different patterns of pituitary gonadotropin secretion for fertility. The mechanisms underlying these gender-specific profiles of pituitary hormone production are unknown; however, they are fundamental to understanding the sexually dimorphic control of reproductive function at the molecular level. Several studies suggest that ERK1 and -2 are essential modulators of hypothalamic GnRH-mediated regulation of pituitary gonadotropin production and fertility. To test this hypothesis, we generated mice with a pituitary-specific depletion of ERK1 and 2 and examined a range of physiological parameters including fertility. We find that ERK signaling is required in females for ovulation and fertility, whereas male reproductive function is unaffected by this signaling deficiency. The effects of ERK pathway ablation on LH biosynthesis underlie this gender-specific phenotype, and the molecular mechanism involves a requirement for ERK-dependent up-regulation of the transcription factor Egr1, which is necessary for LHbeta expression. Together, these findings represent a significant advance in elucidating the molecular basis of gender-specific regulation of the hypothalamic-pituitary-gonadal axis and sexually dimorphic control of fertility.

IL-10 regulates movement of intestinally derived CD4+ T cells to the liver.

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. In this study, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae. By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally derived CD4+ cells increased in the livers of IL-10 KO mice, and Ab-mediated blockade of MAdCAM-1 inhibited the accumulation of CD4+alpha(4)beta(7)+ cells in the liver. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of wild-type donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.

Quantitative trait loci for hip dysplasia in a cross-breed canine pedigree.

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p<0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p<0.01), 16, 20, 22, 25, 29 (p<0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the cross-breed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

Use of recombinant tissue-plasminogen activator in a dog with chylothorax secondary to catheter-associated thrombosis of the cranial vena cava.

A 4-year-old, castrated male Maltese developed cranial vena caval thrombosis and chylothorax following central venous catheterization for treatment of postoperative sepsis. Vena caval thrombolysis was attempted using recombinant human tissue-plasminogen activator (t-PA). Thrombolytic therapy led to an acute reduction in the size of the caval thrombus and was followed by prompt resolution of the chylothorax. Hemorrhage at the entry sites of a jugular catheter and esophagostomy tube placed at the time of treatment was a dose-limiting complication of t-PA therapy in this dog.

Colloid osmotic pressure after hemorrhage and replenishment with Oxyglobin Solution, hetastarch, or whole blood in pregnant sheep.

OBJECTIVE: To compare plasma colloid osmotic pressure (COP) of both maternal and fetal blood, before and after hemorrhage, and replenishment with Oxyglobin Solution (Biopure Corporation, Cambridge, MA, USA), hetastarch or whole blood in pregnant ewes. STUDY DESIGN: Prospective, randomized study. ANIMALS: A total of 17 adult Rambouillet ewes at 131 (128-133) [median (minimum, maximum)] days gestation, weighing 56 (46, 63) kg. METHODS: Ewes and fetuses were chronically instrumented with catheters in a maternal jugular vein, maternal carotid artery and fetal femoral artery. Twenty milliliters per kilograms of blood were removed from each ewe over 1 hour. The ewes were then given 20 mL kg(-1) of either Oxyglobin Solution (n = 5), hetastarch (n = 6), or autologous whole blood (n = 6) IV. Maternal plasma COP was measured before hemorrhage, after hemorrhage, after replenishment, and 1 and 2 hours later. Fetal plasma COP was measured after maternal hemorrhage and 2 hours after maternal volume replenishment. RESULTS: Median COP of all ewes before hemorrhage was 20 (16, 24) mm Hg and after hemorrhage (p < 0.05), decreased to 16 (11, 19) mm Hg. After volume replenishment, the COP of the Oxyglobin Solution group was 22 (21, 25) mm Hg, the autologous whole blood group was 17 (16, 22) mm Hg and the hetastarch group was 20 (17, 21) mm Hg. The COP of the Oxyglobin Solution group was significantly greater (p < 0.05) than the COP of the hetastarch group immediately and 60 minutes after volume replenishment, and greater (p < 0.05) than that of the autologous whole blood group at 60 minutes after volume replenishment. The COP of all the fetuses after maternal hemorrhage was 16 (12, 19) mm Hg and at 120 minutes after maternal volume replenishment was 15 (11, 18) mm Hg. There were no differences in COP between or within any of the fetal groups. CONCLUSIONS: When used to treat blood loss, Oxyglobin Solution increases plasma COP more than an equal volume of hetastarch in the first hour following administration. Maternal administration of Oxyglobin Solution did not alter fetal COP. CLINICAL RELEVANCE: Oxyglobin Solution is a more potent colloid than hetastarch. Oxyglobin Solution did not appear to translocate fluid from the fetal to maternal circulation.

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK.

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.

The extent and distribution of linkage disequilibrium in a multi-hierarchic outbred canine pedigree.

A canine integrated linkage-radiation map has been recently constructed by using microsatellite markers. This map, with a good coverage of the canine genome, allows for a genome-wide search for the extent and distribution of linkage disequilibrium derived from linkage and evolutionary forces. In this study, we genotyped an outbred pedigree between Labrador retriever and Greyhound breeds with a set of microsatellite markers (240) from the canine linkage map. Linkage disequilibrium was measured between all syntenic and nonsyntenic marker pairs. Analysis of syntenic pairs revealed a significant correlation (-0.229, P < 0.001) between linkage disequilibrium and genetic distance (log transformed). Significant linkage disequilibria were observed more frequently between syntenic pairs spaced <40 cM than those paced >40 cM. There is a clear trend for linkage disequilibrium to decline with marker distance. From our results, a genome-wide screen with markers at low to moderate density (1-2 per 10 cM) should take full advantage of linkage disequilibrium for quantitative trait locus mapping in dogs. This study supports the appropriateness of linkage disequilibrium analysis to detect and map quantitative trait loci underlying complex traits in dogs.