Melanocortin 4 receptor signaling in Sim1 neurons permits sexual receptivity in female mice.

Introduction: Female sexual dysfunction affects approximately 40% of women in the United States, yet few therapeutic options exist for these patients. The melanocortin system is a new treatment target for hypoactive sexual desire disorder (HSDD), but the neuronal pathways involved are unclear. Methods: In this study, the sexual behavior of female MC4R knockout mice lacking melanocortin 4 receptors (MC4Rs) was examined. The mice were then bred to express MC4Rs exclusively on Sim1 neurons (tbMC4RSim1 mice) or on oxytocin neurons (tbMC4ROxt mice) to examine the effect on sexual responsiveness. Results: MC4R knockout mice were found to approach males less and have reduced receptivity to copulation, as indicated by a low lordosis quotient. These changes were independent of body weight. Lordosis behavior was normalized in tbMC4RSim1 mice and improved in tbMC4ROxt mice. In contrast, approach behavior was unchanged in tbMC4RSim1 mice but greatly increased in tbMC4ROxt animals. The changes were independent of melanocortin-driven metabolic effects. Discussion: These results implicate MC4R signaling in Oxt neurons in appetitive behaviors and MC4R signaling in Sim1 neurons in female sexual receptivity, while suggesting melanocortin-driven sexual function does not rely on metabolic neural circuits.

Low bone mineral density in achondroplasia and hypochondroplasia.

BACKGROUND: Achondroplasia (ACH) and hypochondroplasia (HCH) are the most common form of short-limb skeletal dysplasias caused by activated fibroblast growth factor receptor 3 (FGFR3) signaling. Although decreased bone mass was reported in gain-of-function mutation in Fgfr3 mice, both disorders have never been described as osteoporotic. In the present study, we evaluated bone mineral density (BMD) in ACH and HCH patients. METHODS: We measured spinal BMD (L1-L4) in 18 ACH and four HCH patients with an average age of 19.8 ± 7.5 years (range, 10-33 years). BMD Z-score in each individual was calculated for normalizing age and gender. Correlation between body mass index (BMI) and BMD was analyzed. Moreover, BMD and Z-score were compared between ACH patients and HCH patients. RESULTS: The average BMD of ACH/HCH patients was 0.805 ± 0.141 g/cm(2) (range, 0.554-1.056 g/cm(2) ), resulting in an average Z-score of -1.1 ± 0.8 (range, -2.4 to 0.6) of the standard value. A slightly positive correlation was observed between BMI and BMD (r = 0.45; P = 0.13). There was no significant difference in BMD and Z-score between ACH and HCH patients. CONCLUSION: Spinal BMD was reduced in ACH/HCH patients, and was mildly correlated with individual BMI. We should carefully monitor BMD and examine osteoporosis-related symptoms in adolescent and adult ACH/HCH patients. © 2016 Japan Pediatric Society.

Spartan deficiency causes genomic instability and progeroid phenotypes.

Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

Estradiol dose-dependent regulation of membrane estrogen receptor-α, metabotropic glutamate receptor-1a, and their complexes in the arcuate nucleus of the hypothalamus in female rats.

Sexual receptivity in the female rat is dependent on dose and duration of estradiol exposure. A 2 µg dose of estradiol benzoate (EB) primes reproductive behavior circuits without facilitating lordosis. However, 50 µg EB facilitates lordosis after 48 hours. Both EB doses activate membrane estrogen receptor-α (mERα) that complexes with and signals through metabotropic glutamate receptor-1a (mGluR1a). This mERα-mGluR1a signaling activates a multisynaptic lordosis-inhibiting circuit in the arcuate nucleus (ARH) that releases ß-endorphin in the medial preoptic nucleus (MPN), activating µ-opioid receptors (MOP). MPN MOP activation is maintained, inhibiting lordosis for 48 hours by 2 µg EB, whereas 50 µg EB at 48 hours deactivates MPN MOP, facilitating lordosis. We hypothesized that 50 µg EB down-regulates ERα and mERα-mGluR1a complexes in the ARH to remove mERα-mGluR1a signaling. In experiment I, 48 hours after 2 µg or 50 µg EB, the number of ARH ERα-immunopositive cells was reduced compared with controls. In experiment II, compared with oil controls, total ARH ERα protein was decreased 48 hours after 50 µg EB, but the 2 µg dose was not. These results indicate that both EB doses reduced the total number of cells expressing ERα, but 2 µg EB may have maintained or increased ERα expressed per cell, whereas 50 µg EB appeared to reduce total ERα per cell. In experiment III, coimmunoprecipitation and Western blot revealed that total mERα and coimmunoprecipitated mERα with mGluR1a were greater 48 hours after 2 µg EB treatment vs rats receiving 50 µg EB. These results indicate 2 µg EB maintains but 50 µg EB down-regulates mERα-mGluR1a to regulate the lordosis circuit activity.

Hypochondroplasia in a child with 1620C>G (Asn540Lys) mutation in FGFR3.

Hypochondroplasia (HCP) is an autosomal dominant skeletal dysplasia characterized by short extremities, short stature and lumbar lordosis, usually exhibiting a phenotype similar to but milder than achondroplasia (ACP). Fibroblast growth factor receptor 3 gene (FGFR3) mutations in the germline are well-known causes of skeletal syndromes. FGFR3 is a negative regulator of bone growth and all mutations in FGFR3 are gain-of-function mutations that lead to skeletal dysplasias. We report a child who presented with short stature, a relatively long trunk, short legs, short arm span, radiographic evidence of HCP and mild mental retardation. Genetic analysis revealed a heterozygous 1620C>G (Asn540Lys) mutation in FGFR3. To our knowledge, ours is the first case report of HCP with a heterozygous 1620C>G (Asn540Lys) mutation in Turkey.

In vivo and in vitro evaluation of the estrogenic properties of the 17ß-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17ß-estradiol.

BACKGROUND: Buame [17ß-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods. METHODS: Buame (10, 100, 500, and 1,000 µg/kg), 17ß-estradiol (E(2)) (100 µg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E(2) was 78 (E(2) = 100) with a relative uterotrophic potency of 1.48 (E(2) = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 µg of E(2) (≈ 30 µg/kg), buame (≈ 750, 1,500, 3,000 µg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4-5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts x 100) was evaluated. RESULTS: Buame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 µg/kg) and E(2) LQmax 56 ± 8; ED50 10 ± 2 µg/kg; the relative LQ-potency was 0.51 (E(2) = 100). Buame competed with [(3)H]E(2) for the estrogen receptor (Buame RBA= 0.15 and Ki = 5.9 x 10(-7) M; E(2) RBA= 100;Ki = 6.6 x 10(-9) M). Buame increased MCF-7 cells proliferation, from 10(-11) to 10(-)9 M, its proliferative effect was 1.73-1.79 (E(2) = 3.0-3.9); its relative proliferative effect to E(2) was 33-40% (E(2) = 100%) and relative potency 10.4-10.7 (E(2) = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response. CONCLUSION: Buame is therefore an estrogen partial agonist with a weak estrogenic activity.

Activity of protein kinase C is important for 3alpha,5alpha-THP's actions at dopamine type 1-like and/or GABAA receptors in the ventral tegmental area for lordosis of rats.

In the ventral tegmental area, progestogens facilitate sexual receptivity of rodents via actions at dopamine type 1-like and/or gamma-aminobutyric acid type A receptors and activation of downstream signal transduction molecules. In the present study, we investigated whether effects of progesterone's metabolite, 3alpha,5alpha-THP, to enhance lordosis via actions at these receptors in the ventral tegmental area requires phospholipase C-dependent protein kinase C. The objective of this study was to test the hypothesis that: if progestogens' actions through dopamine type 1-like and/or gamma-aminobutyric acid type A receptors in the ventral tegmental area for lordosis require protein kinase C, then inhibiting protein kinase C in the ventral tegmental area should reduce 3alpha,5alpha-THP-facilitated lordosis and its enhancement by dopamine type 1-like or gamma-aminobutyric acid type A receptor agonists. Ovariectomized, estradiol (E(2); 10 microg s.c. at h 0)-primed rats were tested for their baseline lordosis responses and then received a series of three infusions to the ventral tegmental area: first, bisindolylmaleimide (75 nM/side) or vehicle; second, SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle; third, 3alpha,5alpha-THP (100, 200 ng/side) or vehicle. Rats were pre-tested for lordosis and motor behavior and then tested for lordosis after each infusion and 10 and 60 min after the last infusion. Rats were tested for motor behavior following their last lordosis test. As has been previously demonstrated, 3alpha,5alpha-THP infusions to the ventral tegmental area increased lordosis and effects were further enhanced by infusions of SKF38393 and muscimol. Infusions of bisindolylmaleimide to the ventral tegmental area attenuated 3alpha,5alpha-THP-, SKF38393-, and/or muscimol-facilitated lordosis. Effects on lordosis were not solely due to changes in general motor behavior. Thus, 3alpha,5alpha-THP's actions in the ventral tegmental area through membrane receptors may require activity of protein kinase C.

In the ventral tegmental area, G-proteins mediate progesterone's actions at dopamine type 1 receptors for lordosis of rats and hamsters.

RATIONALE: Previous reports indicate that the ventral tegmental area (VTA) and/or progesterone (P) can modulate the reinforcing effects of drugs of abuse, food, and sexual behavior. OBJECTIVES: We investigated if, in the VTA, P's membrane-mediated actions for lordosis involve dopamine type 1 receptors (D(1)). Also, whether P's actions at D(1) for lordosis are mediated by typical G-protein coupled mechanisms was examined. METHODS: In Exps 1 and 2, rats received estradiol (E(2) 10 microg) at h 0 and infusions of the D(1) antagonist SCH23390 (100 ng), the D(1) agonist SKF38393 (100 ng), or vehicle, to the VTA, at h 44. Thirty minutes later, rats received systemic P (0, 50, 100, or 200 microg). Subjects were tested for lordosis and motor behavior 2.5 h later. In Exps 3 and 4, E(2)+P (rats 0 or 100 microg; hamsters 200 microg)-primed animals were pretested for lordosis and motor behavior at h 47.5 and infused with SKF38393 (100 ng) or vehicle to the VTA. Thirty minutes later, subjects were retested and infused with the G-protein inhibitor guanosine 5'-O-(2-Thiodiphosphate) (GDP-beta-S; 50 microM) or vehicle. Post-testing occurred 30 min later. RESULTS: Pretreatment with SCH23390-reduced and SKF38393-enhanced P's actions, in the VTA, for lordosis of E(2)-primed rats and hamsters. As well, D(1)-mediated increases in P-facilitated lordosis of rats and hamsters were inhibited by GDP-beta-S. Changes in lordosis were independent of large alterations in motor behavior. CONCLUSIONS: In the VTA, P has actions for modulating reinforcing behaviors, such as lordosis, at D(1) that are G-protein-mediated.

Mechanisms underlying sexual and affiliative behaviors of mice: relation to generalized CNS arousal.

The field of social neuroscience has grown dramatically in recent years and certain social responses have become amenable to mechanistic investigations. Toward that end, there has been remarkable progress in determining mechanisms for a simple sexual behavior, lordosis behavior. This work has proven that specific hormone-dependent biochemical reactions in specific parts of the mammalian brain regulate a biologically important behavior. On one hand, this sex behavior depends on underlying mechanisms of CNS arousal. On the other hand, it serves as a prototypical social behavior. The same sex hormones and the genes that encode their receptors as are involved in lordosis, also affect social recognition. Here we review evidence for a micronet of genes promoting social recognition in mice and discuss their biological roles.

Implication for melatonin and its receptor in the spinal deformities of hereditary Lordoscoliotic Rabbits.

STUDY DESIGN: The association between melatonin system and the spontaneous development of the spinal deformities in the Hereditary Lordoscoliotic Rabbit, the natural animal model for idiopathic scoliosis, was studied. OBJECTIVES: To examine the implication for melatonin and its receptor in the spinal deformities of the natural animal model, the Hereditary Lordoscoliotic Rabbit. SUMMARY OF BACKGROUND DATA: We previously reported radiologic and histologic studies investigating the etiology of spinal deformities in a breed of Japanese White Rabbit, the Hereditary Lordoscoliotic Rabbit. These animals develop thoracic lordoscoliosis during growth and as such can be used as a model for human idiopathic scoliosis. Although previous studies in chickens have established that pinealectomy produces scoliosis, the cause of the condition is yet to be fully elucidated. METHODS: Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were measured by radioimmunoassay and compared with those of Japanese White Rabbits (controls). The expression of melatonin receptor in the rabbit was detected by homology cloning to access the number of the melatonin receptor mRNA in the rabbit spinal cord by quantitative reverse-transcribed polymerase chain reaction. RESULTS: Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were significant higher than those of controls in each period until 20 weeks. We detected the expression of melatonin receptor mRNA in rabbit spinal cord. However, no significant quantitative differences were found in the level of expression of melatonin mRNA in the spinal cord between Hereditary Lordoscoliotic Rabbits and controls. CONCLUSIONS: In relation to the present study, we suggest that causes of spinal deformities in the Hereditary Lordoscoliotic Rabbit may be the result of the contribution of melatonin receptors as well as that of altered serum melatonin levels in the Hereditary Lordoscoliotic Rabbit. Further studies will be required to investigate the expression of melatonin receptor in other tissues of the Hereditary Lordoscoliotic Rabbit as well as to delineate the role of melatonin in the pathogenesis of idiopathic scoliosis.

Evaluation of spinal curvatures after a recent osteoporotic vertebral fracture.

OBJECTIVES: To evaluate spinal curvature changes over a 3-year period in postmenopausal women who had had an osteoporotic vertebral fracture within the last 3 months. METHODS: Thoracic kyphosis and lumbar lordosis were measured using a curviscope at baseline and after 1, 3, 6, 12, and 36 months. Anteroposterior and lateral radiographs of the thoracolumbar spine were obtained after 1 and 3 years. RESULTS: Sixty-one patients were included. At baseline, a significant increase in thoracic curvature was found in the subgroup with thoracic fractures as compared to the subgroups with thoracolumbar or lumbar fractures (64 degrees +/- 9 degrees, 56 degrees +/- 10, and 56 degrees +/- 13, respectively; P < 0.05). No lumbar curvature differences were found. Thoracic curvature was significantly correlated with age (r = -0.48, P < 0.001) and with the vertebral deformity index (r = 0.6, P < 0.001). A significant increase in thoracic curvature was apparent 3 months into the study; after 3 years, the increase was 5.6 degrees +/- 0.7 (P < 0.01). A moderate increase in lumbar curvature was found after 3 years (P < 0.01). Five of 13 patients and five of 10 patients had at least one incident fracture after 1 and 3 years, respectively. Mean thoracic curvature was greater among the patients with than without incident fractures after 1 and 3 years, although the difference was not statistically significant. CONCLUSION: Thoracic compression fractures significantly increase thoracic kyphosis as compared to dorsolumbar and lumbar fractures. Thoracic kyphosis worsens overtime in patients with prevalent vertebral fractures. These data invite an evaluation of techniques capable of providing early correction of alignment disorders, such as widespread use of bracing or kyphoplasty.

Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.

The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.