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In mouse models used to study parturition or pre-clinical therapeutic testing, measurement of uterine contractions is limited to either isometric tension or operative intrauterine pressure (IUP). The goal of this study was to: (1) develop a method for transcervical insertion of a pressure catheter to measure intrauterine contractile pressure during mouse pregnancy, (2) determine whether this method can be utilized numerous times in a single mouse pregnancy without affecting the timing of delivery or fetal outcome and (3) compare the contractile activity between mouse models of term and preterm labor (PTL). Visualization of the cervix allowed intrauterine pressure catheter (IUPC) placement into anesthetized pregnant mice (plug = day 1, delivery = day 19.5). The amplitude, frequency, duration and area under the curve (AUC) of IUP was lowest on days 16-18, increased significantly ( < 0.05) on the morning of day 19 and reached maximal levels during by the afternoon of day 19 and into the intrapartum period. An AUC threshold of 2.77 mmHg discriminated between inactive labor (day 19 am) and active labor (day 19 pm and intrapartum period). Mice examined on a single vs every experimental timepoint did not have significantly different IUP, timing of delivery, offspring number or fetal/neonatal weight. The IUP was significantly greater in LPS-treated and RU486-treated mouse models of PTL compared to time-matched vehicle control mice. Intrapartum IUP was not significantly different between term and preterm mice. We conclude that utilization of a transcervical IUPC allows sensitive assessment of uterine contractile activity and labor progression in mouse models without the need for operative approaches.
© 2018 Society for Reproduction and Fertility.
Cyclooxygenase (COX)-derived prostaglandins stimulate uterine contractions and prepare the cervix for parturition. Prior reports suggest Cox-1 knockout (KO) mice exhibit delayed parturition due to impaired luteolysis, yet the mechanism for late-onset delivery remains unclear. Here, we examined key factors for normal onset of parturition to determine whether any could account for the delayed parturition phenotype. Pregnant Cox-1KO mice did not display altered timing of embryo implantation or postimplantation growth. Although messenger RNAs of contraction-associated proteins (CAPs) were differentially expressed between Cox-1KO and wild-type (WT) myometrium, there were no differences in CAP agonist-induced intracellular calcium release, spontaneous or oxytocin (OT)-induced ex vivo uterine contractility, or in vivo uterine contractile pressure. Delayed parturition in Cox-1KO mice persisted despite exogenous OT treatment. Progesterone (P4) withdrawal, by ovariectomy or administration of the P4-antagonist RU486, diminished the delayed parturition phenotype of Cox-1KO mice. Because antepartum P4 levels do not decline in Cox-1KO females, P4-treated WT mice were examined for the effect of this hormone on in vivo uterine contractility and ex vivo cervical dilation. P4-treated WT mice had delayed parturition but normal uterine contractility. Cervical distensibility was decreased in Cox-1KO mice on the day of expected delivery and reduced in WT mice with long-term P4 treatment. Collectively, these findings show that delayed parturition in Cox-1KO mice is the result of impaired luteolysis and cervical dilation, despite the presence of strong uterine contractions.
Copyright © 2018 Endocrine Society.
Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity leads to debilitating neuroendocrine or metabolic disorders such as Cushing's syndrome (CS). Glucocorticoids control HPA axis activity through negative feedback to the pituitary gland and the central nervous system (CNS). However, the cellular mechanisms involved are poorly understood, particularly in the CNS. Here we show that, in mice, selective loss of TrkB signalling in cholecystokinin (CCK)-GABAergic neurons induces glucocorticoid resistance, resulting in increased corticotrophin-releasing hormone expression, chronic hypercortisolism, adrenocortical hyperplasia, glucose intolerance and mature-onset obesity, reminiscent of the human CS phenotype. Interestingly, obesity is not due to hyperphagia or decreased energy expenditure, but is associated with increased de novo lipogenesis in the liver. Our study therefore identifies CCK neurons as a novel and critical cellular component of the HPA axis, and demonstrates the requirement of TrkB for the transmission of glucocorticoid signalling.
Globally, an estimated 13 million preterm babies are born each year. These babies are at increased risk of infant mortality and life-long health complications. Interventions to prevent preterm birth (PTB) require an understanding of processes driving parturition. Prostaglandins (PGs) have diverse functions in parturition, including regulation of uterine contractility and tissue remodeling. Our studies on cervical remodeling in mice suggest that although local synthesis of PGs are not increased in term ripening, transcripts encoding PG-endoperoxide synthase 2 (Ptgs2) are induced in lipopolysaccharide (LPS)-mediated premature ripening. This study provides evidence for two distinct pathways of cervical ripening: one dependent on PGs derived from paracrine or endocrine sources and the other independent of PG actions. Cervical PG levels are increased in LPS-treated mice, a model of infection-mediated PTB, consistent with increases in PG synthesizing enzymes and reduction in PG-metabolizing enzymes. Administration of SC-236, a PTGS2 inhibitor, along with LPS attenuated cervical softening, consistent with the essential role of PGs in LPS-induced ripening. In contrast, during term and preterm ripening mediated by the antiprogestin, mifepristone, cervical PG levels, and expression of PG synthetic and catabolic enzymes did not change in a manner that supports a role for PGs. These findings in mice, supported by correlative studies in women, suggest PGs do not regulate all aspects of the parturition process. Additionally, it suggests a need to refocus current strategies toward developing therapies for the prevention of PTB that target early, pathway-specific processes rather than focusing on common late end point mediators of PTB.
Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox. To this end, we have developed and validated reagents for the Dre-rox system. These include an Escherichia coli-inducible expression vector based on the temperature-sensitive pSC101 plasmid, a mammalian expression vector based on the CAGGs promoter, a rox-lacZ reporter embryonic stem (ES) cell line based on targeting at the Rosa26 locus, the accompanying Rosa26-rox reporter mouse line, and a CAGGs-Dre deleter mouse line. We also show that a Dre-progesterone receptor shows good ligand-responsive induction properties. Furthermore, we show that there is no crossover recombination between Cre-rox or Dre-loxP. Hence, we add another set of efficient tools to the genomic toolbox, which will enable the development of more sophisticated mouse models for the analysis of gene function and disease.
Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.
Gonadal function plays a major role in bone homeostasis. It is widely held that the skeletal consequences of hypogonadism are solely due to a loss of sex steroids; however, increases in bone turnover begin during perimenopause before decreases in serum estradiol levels. These data and our demonstration that inhibins acutely regulate bone cell differentiation in vitro led us to test whether inhibin A (InhA) regulates bone mass in vivo. Using a transgenic model of inducible human InhA expression, InhA increased total body bone mineral density, increased bone volume, and improved biomechanical properties at the proximal tibia in intact mice and also prevented the loss of BMD and bone volume and strength associated with gonadectomy at both the spine and proximal tibia. In addition, InhA increased mineral apposition rate, double-labeled surface, and serum osteocalcin levels in vivo and osteoblastogenesis ex vivo without affecting osteoclast number or activity. Together these results demonstrate novel stimulatory effects of InhA on the skeleton in vivo. These studies provide in vivo evidence demonstrating that gonadal factors other than sex steroids play an important role in regulating bone mass and strength and, combined with our previous clinical data, suggest that gonadal InhA may be a component of the normal endocrine repertoire that regulates bone quality in both the axial and appendicular skeleton.
The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). In this disorder, cortisol is not inactivated by 11betaHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11betaHSD2 inhibition and examined its possible role in the development of hypertension. 11betaHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11betaHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11betaHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11betaHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11betaHSD2 deficiency and that 11betaHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.
Production of prostaglandins involved in renal salt and water homeostasis is modulated by regulated expression of the inducible form of cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex. Because inflammatory COX-2 is suppressed by glucocorticoids, and prostaglandin levels in the kidney are sensitive to steroids, the sensitivity of COX expression to adrenalectomy (ADX) was investigated. By 2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity increased 10-fold in the cortical thick ascending limb and macula densa. The constitutive isoform, COX-1, was unchanged. The magnitude of the changes and specificity of COX-2 immunoreactivity were validated by in situ hybridization histochemistry of COX-2 mRNA and Western blot analysis. Increased COX-2 activity (>5-fold) was documented by using a specific COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by replacement therapy with either corticosterone or deoxycorticosterone acetate. In normal rats, inhibition of glucocorticoid receptors with RU486 or mineralocorticoid receptors with spironolactone caused up-regulation of renal cortical COX-2. These results indicate that COX-2 expression in situ is tonically inhibited by adrenal steroids, and COX-2 is regulated by mineralocorticoids as well as glucocorticoids.
The effect of dexamethasone on ACTH-induced accumulation of CYP11A and CYP17 mRNAs was studied in bovine adrenocortical cells in primary culture. The cells were treated with either ACTH (1 microM) or the adenylate cyclase activator forskolin (25 microM) and/or dexamethasone (100 nM). The accumulation of CYP11A and CYP17 mRNAs was evaluated by Northern blot analysis with the use of [alpha-32P]deoxy-CTP-labeled bovine CYP11A and CYP17 cDNAs. Chloramphenicol acetyltransferase (CAT) activity was monitored in bovine adrenocortical cells transfected with recombinant plasmids containing either CYP11A or CYP17 regulatory regions coupled to the CAT reporter gene and treated with forskolin and/or dexamethasone. Dexamethasone treatment of the cells cultured in the presence of ACTH or forskolin resulted in about 50% suppression of both CYP11A and CYP17 mRNA accumulation, with a concomitant fall in cortisol secretion to about 60% of the stimulated value. The effects of dexamethasone on accumulation of CYP11A and CYP17 mRNAs and cortisol secretion were blocked by pretreatment of the cells with RU 486 (100 nM), while RU 486 had no effect on forskolin-induced accumulation of either mRNA or cortisol secretion. Dexamethasone also inhibited the forskolin-induced expression of the transfected CYP11A- or CYP17-CAT constructs in bovine adrenocortical cells. The inhibitory effect of dexamethasone was greatly reduced by cotreatment of the transfected cells with RU 486. It is concluded that dexamethasone inhibits the ACTH-induced accumulation of CYP11A and CYP17 mRNAs at a transcriptional level and that the effect of dexamethasone is mediated by the glucocorticoid receptor.