In addition to the well-characterized role of the sex steroid receptors in regulating fertility and reproduction, reproductive events are also mediated by the hypothalamic-pituitary-adrenal axis in response to an individual's environment. Glucocorticoid secretion in response to stress contributes to the well-characterized suppression of the hypothalamic-pituitary-gonadal axis through central actions in the hypothalamus and pituitary. However, both animal and in vitro studies indicate that other components of the reproductive system are also regulated by glucocorticoids. Furthermore, in the absence of stress, it appears that homeostatic glucocorticoid signaling plays a significant role in reproduction and fertility in all tissues comprising the hypothalamic-pituitary-gonadal axis. Indeed, as central regulators of the immune response, glucocorticoids are uniquely poised to integrate an individual's infectious, inflammatory, stress, nutritional, and metabolic status through glucocorticoid receptor signaling in target tissues. Endocrine signaling between tissues regulating the immune and stress response and those determining reproductive status provides an evolutionary advantage, facilitating the trade-off between reproductive investment and offspring fitness. This review focuses on the actions of glucocorticoids in tissues important for fertility and reproduction, highlighting recent studies that show glucocorticoid signaling plays a significant role throughout the hypothalamic-pituitary-gonadal axis and characterizing these effects as permissive or inhibitory in terms of facilitating reproductive success.

Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2^–/– and Cyp27b1^–/– mutant mice. Gcm2^–/– mice exhibited low serum PTH and 1,25(OH)₂D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1^–/– mice had high PTH, undetectable 1,25(OH)₂D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2^–/– mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)₂D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)₂D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1^–/– mice in the absence of 1,25(OH)₂D and in Gcm2^–/– mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)₂D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia.

Resistance of estrogen receptor positive (ERα+) breast cancers to antiestrogens is a major factor in the mortality of this disease. Although activation of ERα in the absence of ligand is hypothesized to contribute to this resistance, the potency of this mechanism in vivo is not clear. Epidemiologic studies have strongly linked prolactin (PRL) to both development of ERα+ breast cancer and resistance to endocrine therapies. Here we employed genetically modified mouse models to examine the ability of PRL and cross talk with TGFα to activate ERα, using a mutated ERα, ERα(G525L), which is refractory to endogenous estrogens. We demonstrate that PRL promotes pubertal ERα-dependent mammary ductal elongation and gene expression in the absence of estrogen, which are abrogated by the antiestrogen, ICI 182,780 (ICI). PRL and TGFα together reduce sensitivity to estrogen, and 30% of their combined stimulation of ductal proliferation is inhibited by ICI, implicating ligand-independent activation of ERα as a component of their interaction. However, PRL/TGFα-induced heterogeneous ERα+ tumors developed more rapidly in the presence of ICI and contained altered transcripts for surface markers associated with epithelial subpopulations and increased signal transducer and activator of transcription 5b expression. Together, these data support strong interactions between PRL and estrogen on multiple levels. Ligand-independent activation of ERα suggests that PRL may contribute to resistance to antiestrogen therapies. However, these studies also underscore ERα-mediated moderation of tumor phenotype. In light of the high expression of PRL receptors in ERα+ cancers, understanding the actions of PRL and cross talk with other oncogenic factors and ERα itself has important implications for therapeutic strategies.

There has been great interest in the extent of β-cell regeneration after pancreatic duct ligation (PDL) and whether α- to β-cell conversion might account for β-cell regeneration after near-complete β-cell loss. To assess these questions, we established a PDL-model in adult male rats after almost complete beta-cell depletion achieved by giving a single high dose of streptozocin (STZ) in the fasted state. Because of the resultant severe diabetes, rats were given islet cell transplants to allow long-term follow-up. Although animals were followed up to 10 months, there was no meaningful β-cell regeneration, be it through replication, neogenesis, or α- to β-cell conversion. In contrast, the acinar cell compartment underwent massive changes with first severe acinar degeneration upon PDL injury followed by the appearance of pancreatic adipocytes, and finally near-complete reappearance of acini. We conclude that β-cells and acinar cells, although originating from the same precursors during development, have very distinct regenerative potentials in our PDL model in adult rats.

The incretin hormone glucagon-like peptide-1 (GLP-1) is an important insulin secretagogue and GLP-1 analogs are used for the treatment of type 2 diabetes. GLP-1 displays antiinflammatory and surfactant-releasing effects. Thus, we hypothesize that treatment with GLP-1 analogs will improve pulmonary function in a mouse model of obstructive lung disease. Female mice were sensitized with injected ovalbumin and treated with GLP-1 receptor (GLP-1R) agonists. Exacerbation was induced with inhalations of ovalbumin and lipopolysaccharide. Lung function was evaluated with a measurement of enhanced pause in a whole-body plethysmograph. mRNA levels of GLP-1R, surfactants (SFTPs), and a number of inflammatory markers were measured. GLP-1R was highly expressed in lung tissue. Mice treated with GLP-1R agonists had a noticeably better clinical appearance than the control group. Enhanced pause increased dramatically at day 17 in all control mice, but the increase was significantly less in the groups of GLP-1R agonist-treated mice (P < .001). Survival proportions were significantly increased in GLP-1R agonist-treated mice (P < .01). SFTPB and SFTPA were down-regulated and the expression of inflammatory cytokines were increased in mice with obstructive lung disease, but levels were largely unaffected by GLP-1R agonist treatment. These results show that GLP-1R agonists have potential therapeutic potential in the treatment of obstructive pulmonary diseases, such as chronic obstructive pulmonary disease, by decreasing the severity of acute exacerbations. The mechanism of action does not seem to be the modulation of inflammation and SFTP expression.

Autophagy is cellular machinery for maintenance of β-cell function and mass. The implication of autophagy failure in β-cells on the pathophysiology of type 2 diabetes and its relation to the effect of treatment of diabetes remains elusive. Here, we found increased expression of p62 in islets of db/db mice and patients with type 2 diabetes mellitus. Treatment with exendin-4, a glucagon like peptide-1 receptor agonist, improved glucose tolerance in db/db mice without significant changes in p62 expression in β-cells. Also in β-cell-specific Atg7-deficient mice, exendin-4 efficiently improved blood glucose level and glucose tolerance mainly by enhanced insulin secretion. In addition, we found that exendin-4 reduced apoptotic cell death and increased proliferating cells in the Atg7-deficient islets, and that exendin-4 counteracted thapsigargin-induced cell death of isolated islets augmented by autophagy deficiency. Our results suggest the potential involvement of reduced autophagy in β-cell dysfunction in type 2 diabetes. Without altering the autophagic state in β-cells, exendin-4 improves glucose tolerance associated with autophagy deficiency in β-cells. This is mainly achieved through augmentation of insulin secretion. In addition, exendin-4 prevents apoptosis and increases the proliferation of β-cells associated with autophagy deficiency, also without altering the autophagic machinery in β-cells.

Diet-induced obesity is commonly associated with leptin resistance, and attenuated leptin signaling contributes to the progression of obesity. PAQR3 is a member of the progesterone and AdipoQ receptor (PAQR) family with close homology to adiponectin receptors. We hypothesized that PAQR3 is implicated in the regulation of obesity and energy homeostasis. To address this hypothesis, we fed Paqr3-deleted mice with high-fat diet (HFD), followed by analyses to evaluate obesity, hepatic steatosis, insulin resistance, metabolic rate, and leptin signaling. We found that mice with deletion of Paqr3 are resistant to HFD-induced obesity and hepatic steatosis, accompanied by improvement of insulin resistance and insulin signaling. Paqr3-deleted mice have an increased energy expenditure and physical activity. HFD-induced leptin resistance is reversed by Paqr3 ablation. Overexpression of PAQR3 reduces leptin signaling whereas deletion of Paqr3 enhances leptin signaling in the hypothalamus. In conclusion, this study reveals that PAQR3 has an important physiological function in modulating obesity, energy metabolism, and leptin signaling.

Nonalcoholic fatty liver disease (NAFLD) has been associated with glucocorticoid excess and androgen deficiency, yet in the majority of patients with steatohepatitis, circulating cortisol and androgen levels are normal. The enzyme 5α-reductase (5αR) has a critical role in androgen and glucocorticoid action. We hypothesize that 5αR has an important role in the pathogenesis of steatohepatitis through regulation of intracrine/paracrine hormone availability. Human liver samples from patients with NAFLD and normal donor tissue were used for gene expression and immunohistochemical analysis. NAFLD samples were scored using the Kleiner classification. In addition, 5αR1^–/–, 5αR2^–/–, and wild-type (WT) mice were fed normal chow or American lifestyle-induced obesity syndrome (ALIOS) diet for 6 or 12 months. Liver histology was graded and staged. Hepatic and circulating free fatty acid and triglyceride levels were quantified, and gene and protein expression was measured by real-time PCR and immunohistochemistry. 5αR1 and -2 were highly expressed in human liver, and 5αR1 protein expression increased with severity of NAFLD. 5αR1^–/– (but not 5αR2^–/–) mice fed an ALIOS diet developed greater hepatic steatosis than WT mice, and hepatic mRNA expression of genes involved in insulin signaling was decreased. Furthermore, 60% of WT mice developed focal hepatocellular lesions consistent with hepatocellular carcinoma after 12 months of the ALIOS diet, compared with 20% of 5αR2^–/– and 0% of 5αR1^–/– mice (P < .05). 5αR1 deletion accelerates the development of hepatic steatosis but may protect against the development of NAFLD-related hepatocellular neoplasia and therefore has potential as a therapeutic target.

Thrombospondin 1 (THBS1 or TSP-1) is a circulating glycoprotein highly expressed in hypertrophic visceral adipose tissues of humans and mice. High-fat diet (HFD) feeding induces the robust increase of circulating THBS1 in the early stages of HFD challenge. The loss of Thbs1 protects male mice from diet-induced weight gain and adipocyte hypertrophy. Hyperinsulinemic euglycemic clamp study has demonstrated that Thbs1-null mice are protected from HFD-induced insulin resistance. Tissue-specific glucose uptake study has revealed that the insulin-sensitive phenotype of Thbs1-null mice is mostly mediated by skeletal muscles. Further assessments of the muscle phenotype using RNA sequencing, quantitative PCR, and histological studies have demonstrated that Thbs1-null skeletal muscles are protected from the HFD-dependent induction of Col3a1 and Col6a1, coupled with a new collagen deposition. At the same time, the Thbs1-null mice display a better circadian rhythm and higher amplitude of energy expenditure with a browning phenotype in sc adipose tissues. These results suggest that THBS1, which circulates in response to a HFD, may induce insulin resistance and fibrotic tissue damage in skeletal muscles as well as the de-browning of sc adipose tissues in the early stages of a HFD challenge. Our study may shed new light on the pathogenic role played by a circulating extracellular matrix protein in the cross talk between adipose tissues and skeletal muscles during obesity progression.

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Using the retrogradely transported immunotoxin, antidopamine β-hydroxylase-saporin (DSAP), we showed previously that hindbrain catecholamine neurons innervating corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus are required for glucoprivation-induced corticosterone secretion. Here, we examine the metabolic consequences of the DSAP lesion in male rats using indirect calorimetry. Rats injected into the paraventricular nucleus of the hypothalamus with DSAP or saporin (SAP) control did not differ in energy expenditure or locomotor activity under any test condition. However, DSAP rats had a persistently higher respiratory exchange ratio (RER) than SAPs under basal conditions. Systemic 2-deoxy-D-glucose did not alter RER in DSAP rats but rapidly decreased RER in SAP controls, indicating that this DSAP lesion impairs the ability to switch rapidly from carbohydrate to fat metabolism in response to glucoprivic challenge. In SAP controls, 2-deoxy-D-glucose-induced decrease in RER was abolished by adrenalectomy but not adrenal denervation. Furthermore, dexamethasone, a synthetic glucocorticoid, decreased RER in both SAP and DSAP rats. Thus, rapid switching of metabolic substrate use during glucoprivation appears to be due to impairment of the catecholamine-mediated increase in corticosterone secretion. Sustained elevation of basal RER in DSAP rats indicates that catecholamine neurons also influence metabolic functions that conserve glucose under basal conditions.

The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11β-HSD1 inhibitor (compound C) inhibited liver 11β-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)–fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11β-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11β-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting "off-target" mechanisms. Based on the patent literature, we synthesized another 11β-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11β-HSD1 inhibitors occurs via 11β-HSD1–independent pathways, and only limited efficacy is achievable from selective 11β-HSD1 inhibition. These data challenge the concept that inhibition of 11β-HSD1 is likely to produce a "step-change" treatment for diabetes and/or obesity.

Men with prostate cancer who receive androgen deprivation therapy show profound skeletal muscle loss. We hypothesized that the androgen deficiency activates not only the ubiquitin-proteasome systems but also the autophagy and affects key aspects of the molecular cross talk between protein synthesis and degradation. Here, 2-month-old male mice were castrated and treated with either testosterone (T) propionate or vehicle for 7 days (short term) or 43 days (long term), and with and without hydroxyflutamide. Castrated mice showed rapid and profound atrophy of the levator ani muscle (high androgen responder) at short term and lesser atrophy of the triceps muscle (low androgen responder) at long term. Levator ani and triceps muscles of castrated mice showed increased level of autophagy markers and lysosome enzymatic activity; only the levator ani showed increased proteasomal enzymatic activity. The levator ani muscle of the castrated mice showed increased level and activation of forkhead box protein O3A, the inhibition of mechanistic target of rapamicyn, and the activation of tuberous sclerosis complex protein 2 and 5'-AMP-activated protein kinase. Similar results were obtained in the triceps muscle of castrated mice. T rescued the loss of muscle mass after orchiectomy and inhibited lysosome and proteasome pathways dose dependently and in a seemingly IGF-I-dependent manner. Hydroxyflutamide attenuated the effect of T in the levator ani muscle of castrated mice. In conclusion, androgen deprivation in adult mice induces muscle atrophy associated with proteasomal and lysosomal activity. T optimizes muscle protein balance by modulating the equilibrium between mechanistic target of rapamicyn and 5'-AMP-activated protein kinase pathways.

Previously we showed that bisphenol A (BPA), an environmental estrogenic endocrine disruptor, rapidly altered Ca²⁺ handling and promoted arrhythmias in female rat hearts. The underlying molecular mechanism was not known. Here we examined the cardiac-specific signaling mechanism mediating the rapid impact of low-dose BPA in female rat ventricular myocytes. We showed that protein kinase A (PKA) and Ca²⁺/CaM-dependent protein kinase II (CAMKII) signaling pathways are the two major pathways activated by BPA. Exposure to 1 nM BPA rapidly increased production of cAMP and rapidly but transiently increased the phosphorylation of the ryanodine receptors by PKA but not by CAMKII. BPA also rapidly increased the phosphorylation of phospholamban (PLN), a key regulator protein of sarcoplasmic reticulum Ca²⁺ reuptake, by CAMKII but not PKA. The increase in CAMKII phosphorylation of PLN was mediated by phospholipase C and inositol trisphosphate receptor-mediated Ca²⁺ release, likely from the endoplasmic reticulum Ca²⁺ storage. These two pathways are likely localized, impacting only their respective target proteins. The rapid impacts of BPA on ryanodine receptors and PLN phosphorylation were mediated by estrogen receptor-β but not estrogen receptor-α. BPA's rapid signaling in cardiac myocytes did not involve activation of ERK1/2. Functional analysis showed that PKA but not CAMKII activation contributed to BPA-induced sarcoplasmic reticulum Ca²⁺ leak, and both PKA and CAMKII were necessary contributors to the stimulatory effect of BPA on arrhythmogenesis. These results provide mechanistic insight into BPA's rapid proarrhythmic actions in female cardiac myocytes and contribute to the assessment of the consequence and potential cardiac toxicity of BPA exposure.

The Dickkopf family of proteins is comprised of four members (Dkk1, Dkk2, Dkk3, Dkk4) that are known to modulate Wnt/β-catenin signaling, which is activated during bone formation. Although the effects of Dkk1 on Wnt/β-catenin signaling have been well studied, little is known about the effects of Dkk4. Therefore, to evaluate the role of Dkk4 in osteoblastogenesis, we used the mouse osteoblastic cell line MC3T3-E1, in which Dkk4 expression was suppressed by small interfering RNA knockdown. Our results showed that the suppression of Dkk4 expression promoted osteoblast proliferation and differentiation and suppressed apoptosis. In colony-forming unit alkaline phosphatase assay, Dkk4 knockdown cells possessed markedly higher alkaline phosphatase activity compared with Dkk1 knockdown cells. Reduced Dkk4 expression also led to the up-regulation of β-catenin levels, β-catenin/T cell factor activity, and Wnt-target genes. In contrast, overexpression of Dkk4 in MC3T3-E1 cells led to inhibition of osteoblast differentiation. Our findings reveal that Dkk4 functions as an inhibitor of osteoblastogenesis through Wnt/β-catenin signaling, providing new insights into the relationship between Wnt/β-catenin signaling and Dkk4 in bone formation.

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shh^flx/flx) and mice expressing a Cre transgene under the control of the H⁺,K⁺-ATPase β-subunit promoter. Shh^flx/flx, the H⁺,K⁺-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair.

It has been reported that various endocrine hormones exert prominent effects on erythropoiesis. We conducted experiments to identify the mechanisms involved in the regulatory functions of adrenergic signaling on erythroid differentiation and the expression of hemoglobin genes. The reactivation of fetal hemoglobin (HbF) expression is also an important therapeutic option in patients with hemoglobin disorders. We determined that the adrenergic agonists phenylephrine (PE) and isoproterenol (ISO) can induce the production of β-hemoglobin embryonic 1 (hbbe1) mRNA and protein in adult zebrafish erythrocytes. Elevated levels of HbF mRNA and protein were also observed in human K562 cells after the adrenergic agonist treatments. In addition, elevated levels of histone acetylation were observed in both the PE- and the ISO-treated K562 cells. Additionally, our data further indicate that the induction effects of the adrenergic agonists on HbF synthesis and erythroid differentiation in K562 cells are mainly mediated by the p38 MAPK/cAMP response element binding pathway. In summary, the present study identifies the role of the adrenergic agonists PE and ISO on p38 MAPK and ERK signaling for the stimulation of HbF production and erythroid differentiation.

Maternal dietary restriction during the periconceptional period results in an increase in adrenal growth and in the cortisol stress response in the offspring. The intraadrenal mechanisms that result in the programming of these changes are not clear. Activation of the IGF and the signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways regulate adrenal growth. We have used an embryo transfer model in sheep to investigate the impact of exposure to either dietary restriction in normal or obese mothers or to maternal obesity during the periconceptional period on adrenal growth and function in the offspring. We assessed the adrenal abundance of key signaling molecules in the IGF-I and Janus kinase/STAT/SOCS pathways including IGF-I receptor, IGF-II receptor, Akt, mammalian target of rapamycin, ribosomal protein S6, eukaryotic translation initiation factor 4E-binding protein 1, eukaryotic translation initiation factor 4E, STAT1, STAT3, STAT5, SOCS1, and SOCS3 in female and male postnatal lambs. Maternal dietary restriction in the periconceptional period resulted in the hypertrophy of the adrenocortical cells in the zona fasciculata-reticularis and an up-regulation in STAT1, phospho-STAT1, and phospho-STAT3 (Ser727) abundance and a down-regulation in IGF-I receptor, Akt, and phospho-Akt abundance in the adrenal cortex of the postnatal lamb. These studies highlight that weight loss around the time of conception, independent of the starting maternal body weight, results in the activation of the adrenal Janus kinase/STAT pathway and adrenocortical hypertrophy. Thus, signals of adversity around the time of conception have a long-term impact on the mechanisms that regulate adrenocortical growth.

Cardiovascular diseases, the most common leading death diseases, occur more in men than women of the same ages. Increasing evidence shows that urocortin (Ucn1), an autocrine or paracrine pro-inflammatory factor, can be regulated by sex hormones. The purpose of the study is to investigate the role of Ucn1 in gender disparity in a sodium laurate–induced vasculitis model. Rats exhibited visible signs of vasculitis on the 14th day after sodium laurate injection. Inflammatory states of the rat femoral artery were observed by histological examination. Significant gender disparity, with the symptoms much grosser in males than females, was seen. In males, the serum levels of Ucn1, prostaglandin estradiol, and soluble intercellular adhesion molecule-1 and the expressions of Ucn1, cyclooxygenase-2, and intercellular adhesion molecule-1 in femoral artery were higher than those in females. Orchidectomy significantly ameliorated the symptoms of vasculitis accompanied with a decrease in the plasma Ucn1 level. However, estradiol supplement after orchidectomy failed to improve the inflammatory states further. In females, ovariectomy and/or dihydrotestosterone supplement significantly increased Ucn1 level and exacerbated symptoms of vasculitis. Furthermore, ip administration of rabbit antiserum to Ucn1 almost abolished the gender differences in vasculitis. These results demonstrated that vasculitis of this model is androgen-responsive and hormonal manipulation by surgical orchidectomy could substantially attenuate the symptoms of vasculitis. Moreover, Ucn1 is a contributory factor to the gender disparity in vasculitis and dihydrotestosterone-promoted Ucn1 secretion exacerbated the development of vasculitis.

The development and maintenance of the zones of the adrenal cortex and their steroidal output are extremely important in the control of gluconeogenesis, the stress response, and blood volume. Sonic Hedgehog (Shh) is expressed in the adrenal cortex and signals to capsular cells, which can respond by migrating into the cortex and converting into a steroidogenic phenotype. Delta-like homologue 1 (Dlk1), a member of the Notch/Delta/Serrate family of epidermal growth factor-like repeat-containing proteins, has a well-established role in inhibiting adipocyte differentiation. We demonstrate that Shh and Dlk1 are coexpressed in the outer undifferentiated zone of the male rat adrenal and that Dlk1 signals to the adrenal capsule, activating glioma-associated oncogene homolog 1 transcription in a β1 integrin- and Erk1/2-dependent fashion. Moreover, Shh and Dlk1 expression inversely correlates with the size of the zona glomerulosa in rats after manipulation of the renin-angiotensin system, suggesting a role in the homeostatic maintenance of the gland.

Several adipokines are known to influence skeletal metabolism. Fasting-induced adipose factor (FIAF) is an adipokine that gives rise to 2 further peptides in vivo, the N-terminal coiled-coil domain (FIAF_CCD) and C-terminal fibrinogen-like domain (FIAF_FLD). The skeletal action of these peptides is still uncertain. Our results show that FIAF_CCD is a potent inhibitor of osteoclastogenesis and function, as seen in mouse bone marrow and RAW_264.7 cell cultures, and in a resorption assay using isolated primary mature osteoclasts. The inhibitory effects at 500 ng/mL were approximately 90%, 50% and 90%, respectively, in these assays. FIAF_CCD also stimulated osteoblast mitogenesis by approximately 30% at this concentration. In comparison, FIAF_FLD was only active in decreasing osteoblast mitogenesis, and intact FIAF had no effect in any of these assays. In murine bone marrow cultures, FIAF_CCD reduced the expression of macrophage colony-stimulating factor (M-CSF), nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP), and to lesser extent suppressed the expression of connective tissue growth factor (CTGF). FIAF_CCD also decreased expression of M-CSF and CTGF in stromal/osteoblastic ST2 cells. Its effect on receptor activator of nuclear factor B (RANKL) and osteoprotegerin expression in bone marrow was not consistent with its inhibitory action on osteoclastogenesis, but it decreased RANKL expression in ST2 cells. In RAW_264.7 cell cultures, FIAF_CCD significantly reduced the expression of NFATc1 and DC-STAMP. In conclusion, FIAF_CCD inhibits osteoclast differentiation and function in vitro and decreases expression of genes encoding key osteoclastogenic factors such as M-CSF, CTGF, NFATc1, and DC-STAMP. FIAF_CCD's action on osteoclasts may be independent of the RANKL/osteoprotegerin pathway. These results suggest a novel mechanism by which adipose tissue may regulate bone resorption and skeletal health.

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1^G93A mice throughout the course of ALS disease. Male wild-type and hSOD1^G93A mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1^G93A mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1^G93A mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

Recent studies have revealed the presence and regulation of aromatase at the vertebrate synapse, and identified a critical role played by presynaptic estradiol synthesis in the electrophysiological response to auditory and other social cues. However, if and how synaptic aromatization affects behavior remains to be directly tested. We have exploited 3 characteristics of the zebra finch hippocampus (HP) to test the role of synaptocrine estradiol provision on spatial memory function. Although the zebra finch HP contains abundant aromatase transcripts and enzyme activity, immunocytochemical studies reveal widespread pre- and postsynaptic, but sparse to undetectable somal, localization of this enzyme. Further, the superficial location of the avian HP makes possible the more exclusive manipulation of its neurochemical characteristics without perturbation of the neuropil and the resultant induction of astroglial aromatase. Last, as in other vertebrates, the HP is critical for spatial memory performance in this species. Here we report that local inhibition of hippocampal aromatization impairs spatial memory performance in an ecologically valid food-finding task. Local aromatase inhibition also resulted in lower levels of estradiol in the HP, but not in adjacent brain areas, and was achieved without the induction of astroglial aromatase. The observed decrement in acquisition and subsequent memory performance as a consequence of lowered aromatization was similar to that achieved by lesioning this locus. Thus, hippocampal aromatization, much of which is achieved at the synapse in this species, is critical for spatial memory performance.

Recent in vitro studies highlighted G protein-coupled receptor kinase (GRK)2 and β-arrestins as important players in driving somatostatin receptor (SSTR) desensitization and trafficking. Our aim was to characterize GRK2 and β-arrestins expression in different pituitary adenomas and to investigate their potential role in the response to somatostatin analog (SSA) treatment in GH-secreting adenomas (GHomas). We evaluated mRNA expression of multiple SSTRs, GRK2, β-arrestin 1, and β-arrestin 2 in 41 pituitary adenomas (31 GHomas, 6 nonfunctioning [NFPAs], and 4 prolactinomas [PRLomas]). Within the GHomas group, mRNA data were correlated with the in vivo response to an acute octreotide test and with the GH-lowering effect of SSA in cultured primary cells. β-Arrestin 1 expression was low in all 3 adenoma histotypes. However, its expression was significantly lower in GHomas and PRLomas, compared with NFPAs (P < .01). GRK2 expression was higher in PRLomas and NFPAs compared with GHomas (P < .05). In the GHoma group, GRK2 expression was inversely correlated to β-arrestin 1 (P < .05) and positively correlated to β-arrestin 2 (P < .0001). SSA treatment did not affect GRK2 and β-arrestin expression in GHomas or in cultured rat pituitary tumor GH3 cells. Noteworthy, β-arrestin 1 was significantly lower (P < .05) in tumors responsive to octreotide treatment in vitro, whereas GRK2 and SSTR subtype 2 were significantly higher (P < .05). Likewise, β-arrestin 1 levels were inversely correlated with the in vivo response to acute octreotide test (P = .001), whereas GRK2 and SSTR subtype 2 expression were positively correlated (P < .05). In conclusion, for the first time, we characterized GRK2, β-arrestin 1, and β-arrestin 2 expression in a representative number of pituitary adenomas. β-Arrestin 1 and GRK2 seem to have a role in modulating GH secretion during SSA treatment.

We have previously demonstrated that the cleavage product of the full-length GnRH, GnRH-(1–5), is biologically active, binds G protein-coupled receptor 173 (GPR173), and inhibits the migration of cells in the immortalized GnRH-secreting GN11 cell. In this study, we attempted to characterize the GnRH-(1–5) intracellular signaling mechanism. To determine whether the signaling pathway mediating GnRH-(1–5) regulation of migration involves a G protein-dependent mechanism, cells were treated with a generic G protein antagonist in the presence and absence of GnRH-(1–5), and a wound-healing assay was conducted to measure migration. G Protein antagonist 2 treatment abolished the GnRH-(1–5) inhibition of migration, indicating that the mechanism of GnRH-(1–5) is G protein coupled. To identify the potential Gα-subunit recruited by GnRH-(1–5) binding GPR173, we measured the second messengers cAMP and inositol triphosphate levels. GnRH-(1–5) treatment did not alter cAMP levels relative to cells treated with vehicle or forskolin, suggesting that GnRH-(1–5) does not couple to the Gαs or Gαi subunits. Similarly, inositol triphosphate levels remained unchanged with GnRH-(1–5) treatment, indicating a mechanism not mediated by the Gαq/11 subunit. Therefore, we also examined whether GnRH-(1–5) activating GPR173 deviated from the canonical G protein-coupled receptor signaling pathway by coupling to β-arrestin 1/2 to regulate migration. Our coimmunoprecipitation studies indicate that GnRH-(1–5) induces the rapid interaction between GPR173 and β-arrestin 2 in GN11 cells. Furthermore, we demonstrate that this association recruits phosphatase and tensin homolog to mediate the downstream action of GnRH-(1–5). These findings suggest that the GnRH-(1–5) mechanism deviates from the canonical G protein-coupled receptor pathway to regulate cell migration in immortalized GnRH neurons.

The WNT pathway has been well characterized in embryogenesis and tumorigenesis. In humans, specific polymorphisms in the T cell-specific transcription factor 7 and the WNT coreceptor, low-density lipoprotein receptor-related protein-6 (LRP-6), both prominent components of this pathway, correlate with a higher incidence of type 2 diabetes, suggesting that the WNT pathway might be involved in the control of adult glucose homeostasis. We previously demonstrated that glycogen-synthase-kinase-3β (GSK-3β), the key enzyme of the WNT pathway, is increased in the hypothalamus during obesity and exacerbates high-fat diet-induced weight gain as well as glucose intolerance. These data suggest that WNT action in the hypothalamus might be required for normal glucose homeostasis. Here we characterized whether WNT signaling in general is altered in the hypothalamus of adult obese mice relative to controls. First we identified expression of multiple components of this pathway in the murine arcuate nucleus by in situ hybridization. In this region mRNA of ligands and target genes of the WNT pathway were down-regulated in obese and glucose-intolerant leptin-deficient mice. Similarly, the number of cells immunoreactive for the phosphorylated (active) form of the WNT-coreceptor LRP-6 was also decreased in leptin-deficient mice. Leptin treatment normalized expression of the WNT-target genes Axin-2 and Cylin-D1 and increased the number of phospho-LRP-6-immunoreactive cells reaching levels of lean controls. Leptin also increased the levels of phosphorylated (inactive) GSK-3β in the arcuate nucleus, and this effect was colocalized to neuropeptide Y neurons, suggesting that inactivation of GSK-3β may contribute to the neuroendocrine control of energy homeostasis. Taken together our findings identify hypothalamic WNT signaling as an important novel pathway that integrates peripheral information of the body's energy status encoded by leptin.

Little is known about the functions of chromosome Y (chrY) genes beyond their effects on sex and reproduction. In hearts, postpubertal testosterone affects the size of cells and the expression of genes differently in male C57BL/6J than in their C57.Y^A counterparts, where the original chrY has been substituted with that from A/J mice. We further compared the 2 strains to better understand how chrY polymorphisms may affect cardiac properties, the latter being sexually dimorphic but unrelated to sex and reproduction. Genomic regions showing occupancy with androgen receptors (ARs) were identified in adult male hearts from both strains by chromatin immunoprecipitation. AR chromatin immunoprecipitation peaks (showing significant enrichment for consensus AR binding sites) were mostly strain specific. Measurements of anogenital distances in male pups showed that the biologic effects of perinatal androgens were greater in C57BL/6J than in C57.Y^A. Although perinatal endocrine manipulations showed that these differences contributed to the strain-specific differences in the response of adult cardiac cells to testosterone, the amounts of androgens produced by fetal testes were not different in each strain. Nonetheless, chrY polymorphisms associated in newborn pups' hearts with strain-specific differences in genomic regions showing either AR occupancy, accessible chromatin sites, or trimethylation of histone H3 Lysine 4 marks, as well as with differential expression of 2 chrY-encoded histone demethylases. In conclusion, the effects of chrY on adult cardiac phenotypes appeared to result from an interaction of this chromosome with the organizational programming effects exerted by the neonatal testosterone surge and show several characteristics of being mediated by an epigenetic remodeling of chromatin.

Nesfatin-1, a satiety-inducing peptide identified in hypothalamic regions that regulate energy balance, is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant. We investigated its production by human cardiomyocytes and its effects on glucose uptake, in the main cardiac glucose transporter GLUT-4 and in intracellular signaling. Quantitative RT-PCR, Western blots, confocal immunofluorescence microscopy, and ELISA of human and murine cardiomyocytes and/or cardiac tissue showed that cardiomyocytes can synthesize and secrete nesfatin-1. Confocal microscopy of cultured cardiomyocytes after GLUT-4 labeling showed that nesfatin-1 mobilizes this glucose transporter to cell peripherals. The rate of 2-deoxy-d-[³H]glucose incorporation demonstrated that nesfatin-1 induces glucose uptake by HL-1 cells and cultured cardiomyocytes. Nesfatin-1 induced dose- and time-dependent increases in the phosphorylation of ERK1/2, AKT, and AS160. In murine and human cardiac tissue, nesfatin-1 levels varied with diet and coronary health. In conclusion, human and murine cardiomyocytes can synthesize and secrete nesfatin-1, which is able to induce glucose uptake and the mobilization of the glucose transporter GLUT-4 in these cells. Nesfatin-1 cardiac levels are regulated by diet and coronary health.

Hyperaldosteronism is linked to the development and progression of several different cardiovascular diseases. Angiotensin (Ang) II increases aldosterone secretion and adrenal blood flow. Ang II peptide fragments are produced by various peptidases, and these Angs have diverse and vital physiologic roles. Due to the uncharacteristic vasorelaxation of adrenal arteries by Ang II, we tested the hypothesis that Ang II metabolism contributes to its relaxant activity in adrenal arteries. Metabolism of Angs by bovine adrenal cortical arteries and isolated bovine adrenal vascular cells was measured by liquid chromatography-mass spectrometry. The primary Ang metabolites of adrenal arteries are Ang III and Ang (1–7), with Ang IV produced to a lesser extent. Bovine microvascular endothelial cells produced a similar metabolic profile to adrenal arteries, whereas bovine adrenal artery smooth muscle cells exhibited less metabolism. In preconstricted adrenal arteries, Ang II caused relaxation in picomolar concentrations and constrictions at 10nM. Ang-converting enzyme 2 inhibition augmented this relaxation response, whereas aminopeptidase inhibition did not. Ang III was equipotent to Ang II in relaxing adrenal arteries. Ang IV did not cause relaxation. Nitric oxide synthase inhibition enhanced Ang II-induced constriction of adrenal arteries. Aminopeptidase inhibition increased the concentration range for Ang II-induced constriction of adrenal arteries. Ang III and Ang IV did not change the basal tone but caused constriction of adrenal arteries with nitric oxide synthase inhibition. These data indicate that Ang II metabolism modulates the vascular effects of Ang II in the adrenal vasculature.

A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in "extrapituitary" tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been "humanized" with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL^–) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology.

Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa^–/– (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P < .02) and tended to take longer for all subsequent parturition intervals (9 days; P < .07). Heterozygous males sired fewer pups per litter (P < .03) and after the first litter took 10 days longer (P < .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P < .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P < .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P < .02), Bcl2:Bax (P < .02), Neurog3 (P < .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P < .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility.

Endometriosis is a chronic inflammatory disease of reproductive age women leading to chronic pelvic pain and infertility. Current antiestrogen therapies are temporizing measures, and endometriosis often recurs. Potential nonestrogenic or nonsteroidal targets are needed for treating endometriosis. Peroxisome proliferator-activated receptor (PPAR), a nuclear receptor, is activated by thiazolidinediones (TZDs). In experimental endometriosis, TZDs inhibit growth of endometriosis. Clinical data suggest potential use of TZDs for treating pain and fertility concurrently in endometriosis patients. Study objectives were to 1) determine the effects of PPAR action on growth and survival of human endometriotic epithelial and stromal cells and 2) identify the underlying molecular links between PPAR activation and cell cycle regulation, apoptosis, estrogen biosynthesis, and prostaglandin E2 biosynthesis and signaling in human endometriotic epithelial and stromal cells. Results indicate that activation of PPAR by TZD ciglitazone 1) inhibits growth of endometriotic epithelial cells 12Z up to 35% and growth of endometriotic stromal cells 22B up to 70% through altered cell cycle regulation and intrinsic apoptosis, 2) decreases expression of PGE2 receptors (EP)2 and EP4 mRNAs in 12Z and 22B cells, and 3) inhibits expression and function of P450 aromatase mRNA and protein and estrone production in 12Z and 22B cells through EP2 and EP4 in a stromal-epithelial cell-specific manner. Collectively, these results indicate that PGE2 receptors EP2 and EP4 mediate actions of PPAR by incorporating multiple cell signaling pathways. Activation of PPAR combined with inhibition of EP2 and EP4 may emerge as novel nonsteroidal therapeutic targets for endometriosis-associated pain and infertility, if clinically proven safe and efficacious.

Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17β and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.

LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.

Women with polycystic ovary syndrome (PCOS) and hyperandrogenism have altered hormone levels and suffer from ovarian dysfunction leading to subfertility. We have attempted to generate a model of hyperandrogenism by feeding mice chow supplemented with dehydroepiandrosterone (DHEA), an androgen precursor that is often elevated in women with PCOS. Treated mice had polycystic ovaries, low ovulation rates, disrupted estrous cycles, and altered hormone levels. Because DHEA is an inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway, we tested the hypothesis that oocytes from DHEA-exposed mice would have metabolic disruptions. Citrate levels, glucose-6-phosphate dehydrogenase activity, and lipid content in denuded oocytes from these mice were significantly lower than controls, suggesting abnormal tricarboxylic acid and pentose phosphate pathway metabolism. The lipid and citrate effects were reversible by supplementation with nicotinic acid, a precursor for reduced nicotinamide adenine dinucleotide phosphate. These findings suggest that elevations in systemic DHEA can have a negative impact on oocyte metabolism and may contribute to poor pregnancy outcomes in women with hyperandrogenism and PCOS.

Knockdown of the transcription factors GATA4 and GATA6 in granulosa cells (GCs) impairs folliculogenesis and induces infertility. To investigate the pathways and genes regulated by these factors, we performed microarray analyses on wild-type GCs or GCs lacking GATA4, GATA6, or GATA4/6 (G4^gcko, G6^gcko, and G4/6^gcko) after in vivo treatment with equine chorionic gonadotropin. GATA4 deletion affected a greater number of genes than GATA6, which correlates with the subfertility observed in G4^gcko mice and the normal reproductive function found in G6^gcko animals. An even greater number of genes were affected by the deletion of both factors. Moreover, the expression of FSH receptor, LH receptor, inhibin α and β, versican, pregnancy-associated plasma protein A, and the regulatory unit 2b of protein kinase A, which are known to be crucial for ovarian function, was greatly affected in double GATA4 and GATA6 knockouts when compared with single GATA-deficient animals. This suggests that GATA4 and GATA6 functionally compensate for each other in the regulation of key ovarian genes. Functional enrichment revealed that ovulation, growth, intracellular signaling, extracellular structure organization, gonadotropin and growth factor actions, and steroidogenesis were significantly regulated in G4/6^gcko mice. The results of this analysis were confirmed using quantitative polymerase chain reaction, immunohistochemical, and biological assays. Treatment of GCs with cAMP/IGF-I, to bypass FSH and IGF-I signaling defects, revealed that most of the affected genes are direct targets of GATA4/6. The diversity of pathways affected by the knockdown of GATA underscores the important role of these factors in the regulation of GC function.

Oocyte-derived paracrine factors (ODPFs) and estrogens are both essential for the development and function of ovarian follicles in mammals. Cooperation of these two factors was assessed in vitro using intact cumulus-oocyte complexes, cumulus cells cultured after the removal of oocytes [oocytectomized (OOX) cumulus cells], and OOX cumulus cells cocultured with denuded oocytes, all in the presence or absence of 17β-estradiol (E2). Effects on the cumulus cell transcriptome were assessed by microarray analysis. There was no significant difference between the cumulus cell transcriptomes of either OOX cumulus cells cocultured with oocytes or intact cumulus-oocyte complexes. Therefore, oocyte-mediated regulation of the cumulus cell transcriptome is mediated primarily by ODPFs and not by gap junctional communication between oocytes and cumulus cells. Gene ontology analysis revealed that both ODPFs and E2 strongly affected the biological processes associated with cell proliferation in cumulus cells. E2 had limited effects on ODPF-regulated biological processes. However, in sharp contrast, ODPFs significantly affected biological processes regulated by E2 in cumulus cells. For example, only in the presence of ODPFs did E2 significantly promote the biological processes related to phosphorylation-mediated signal transduction in cumulus cells, such as the signaling pathways of epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor. Therefore, ODPFs and E2 cooperate to regulate the cumulus cell transcriptome and, in general, oocytes modulate the effects of estrogens on cumulus cell function.

A successful postpartum involution permits the postnatal uterus to rapidly regain its prepregnancy function and size to ultimately facilitate an ensuing blastocyst implantation. This study investigates the molecular mechanisms that govern the initiation of the involution process by examining the signaling events that occur as the uterus transitions from the pregnant to postnatal state. Using mouse and baboon uteri, we found a remarkable cross-species conservation at the signal transduction level as the pregnant uterus initiates and progresses through the involution process. This study originated with the observation of elevated levels of caspase-3 activation in both the laboring mouse and baboon uterus, which we found to be apoptotic in nature as evidenced by the concurrent appearance of cleaved poly(ADP-ribose) polymerase. We previously defined a nonapoptotic and potential tocolytic role for uterine caspase-3 during pregnancy regulated by increased antiapoptotic signaling mediated by myeloid cell leukemia sequence 1 and X-linked inhibitor of apoptosis. In contrast, this study determined that diminished antiapoptotic signaling in the postpartum uterus allowed for both endometrial apoptotic and myometrial autophagic episodes, which we speculate are responsible for the rapid reduction in size of the postpartum uterus. Using our human telomerase immortalized myometrial cell line and the Simian virus-40 immortalized endometrial cell line (12Z), we demonstrated that the withdrawal of antiapoptotic signaling was also an upstream event for both the autophagic and apoptotic processes in the human uterine myocyte and endometrial epithelial cell.

Children with monocarboxylate transporter 8 (MCT8) deficiency lose weight, even when adequately nourished. Changes in serum markers of thyroid hormone (TH) action compatible with thyrotoxicosis suggested that this might be due to T₃ excess in peripheral tissues. Mct8-deficient mice (Mct8KO) replicate the human thyroid phenotype and are thus suitable for metabolic studies so far unavailable in humans. In the current work, compared with wild-type (Wt) mice, Mct8KO mice were leaner due to reduced fat mass. They tended to use more carbohydrates and fewer lipids during the dark phase. Mct8KO mice had increased total energy expenditure (TEE) and food and water intake, with normal total activity, indicating hypermetabolism. To determine whether this is due to the high serum T₃, we studied mice deficient in both Mct8 and deiodinase 1 (Mct8D1KO) with serum T₃ similar to Wt mice and Wt mice given L-T₃ to raise their serum T₃ to the level of Mct8KO mice. Contrary to Mct8KO, Mct8D1KO mice had similar fat mass, TEE, and food intake as their D1KO littermates, whereas T₃-treated Wt mice showed increased food intake and TEE, similar to Mct8KO mice. In skeletal muscle, Mct8KO mice had increased T₃ content and TH action and increased glucose metabolism, which improved in Mct8D1KO mice. These studies indicate that the high serum T₃ in MCT8 deficiency increases the TEE and fails to maintain weight despite adequate calorie intake. This is mediated by tissues that are not predominantly MCT8 dependent for TH transport, including skeletal muscle. Normalizing serum T₃ level by deleting deiodinase 1 corrects body composition and the metabolic alterations caused by the MCT8 deficiency.

The great apes include, in addition to Homo, the genera Pongo (orangutans), Gorilla (gorillas), and Pan, the latter comprising two species, P. troglodytes (chimpanzees) and P. paniscus (bonobos). Adult-onset hypothyroidism was previously reported in 4 individual nonhuman great apes. However, there is scarce information on normal serum thyroid hormone levels and virtually no data for thyroid autoantibodies in these animals. Therefore, we examined thyroid hormone levels and TSH in all nonhuman great ape genera including adults, adolescents, and infants. Because hypothyroidism in humans is commonly the end result of thyroid autoimmunity, we also tested healthy and hypothyroid nonhuman great apes for antibodies to thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSH receptor (TSHR). We established a thyroid hormone and TSH database in orangutans, gorillas, chimpanzees, and bonobos (447 individuals). The most striking differences are the greatly reduced free-T₄ and free-T₃ levels in orangutans and gorillas vs chimpanzees and bonobos, and conversely, elevated TSH levels in gorillas vs Pan species. Antibodies to Tg and TPO were detected in only 2.6% of adult animals vs approximately 10% in humans. No animals with Tg, TPO, or TSHR antibodies exhibited thyroid dysfunction. Conversely, hypothyroid nonhuman great apes lacked thyroid autoantibodies. Moreover, thyroid histology in necropsy tissues was similar in euthyroid and hypothyroid individuals, and lymphocytic infiltration was absent in 2 hypothyroid animals. In conclusion, free T₄ and free T₃ are lower in orangutans and gorillas vs chimpanzees and bonobos, the closest living human relatives. Moreover, thyroid autoantibodies are rare and hypothyroidism is unrelated to thyroid autoimmunity in nonhuman great apes.

Rapid actions of T₃ on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T₃ on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T₃. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T₃ dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T₃ chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T₃ is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-β subunit, designated TSH-βv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-βv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-β, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Goitrogenesis in Graves' disease (GD) has been attributed to anti-TSH receptor antibody stimulation. Recently, a role for adenosine A2a receptor (A2aR) in goiter formation was reported in the thyroglobulin-A2aR transgenic mice. However, it is unclear whether A2aR is expressed in the thyroid and whether it is associated with the pathogenesis of goiter in GD. Here, we confirmed the expression of A2aR in FRTL-5 cells, primary normal human thyrocytes (both sexes were used without regard to sex), and thyroid tissue (both sexes were used without regard to sex) by PCR, Western blotting, immunohistochemistry, and immunofluorescence. After treatments with A2aR-specific agonist 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine or GD IgG, the mRNA and protein levels of vascular endothelial growth factor (VEGF), a growth factor related to goitrogenesis, were evaluated along with upstream signaling pathways. A2aR activation and GD IgG promoted the expression of VEGF in thyrocytes, which was accompanied by the activation of cAMP/protein kinase A/phosphorylated-cAMP-response element-binding protein, peroxisome proliferator-activated receptor coactivator-1α, and hypoxia-inducible factor-1α. The changes induced by GD IgG were partially abrogated by A2aR small interfering RNA and an A2aR antagonist. These results were supported by data on the goiter samples from the thyrotropin receptor adenovirus-induced GD mouse model (female). These data demonstrate that GD IgG could up-regulate the VEGF expression through A2aR, indicating a potential mechanism for goitrogenesis in GD.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-μL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.