Stuart P. Bliss

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.

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.

Quantitative trait loci for hip dysplasia in a crossbreed 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 crossbreed 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.

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.

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.

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 Pyk2s 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.

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+α4β7+ 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.

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, 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.