Of in Utero Meconium Passage

J Lakshmanan and MG Ross Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Torrance, CA, USA

have been attributed as risk factors responsible for meconium To use sheep and rat models and demonstrate that stressors activate fetal passage as well as the potential meconium aspiration in utero. glucocorticoid (GC) system, corticotrophin-releasing factor (CRF) system Despite these risk factors, presently very little is known of the and cholinergic neurotransmitter system (ChNS) leading to propulsive cascade of events that leads to meconium passage in the colonic motility and in utero meconium passage. Immunohistochemical newborn immediately after birth or to the mechanisms studies (IHS) were performed to localize GC-Receptors, CRF-receptors and contributing ‘premature’ meconium passage in utero. There key molecules of ChNS in sheep fetal distal colon. CRF expression in are no small or large animal models that could be extrapolated placenta and enteric endocrine cells in fetal rat system were examined and to humans. We review here recent studies gathered in our the effects of acute hypoxia on in utero meconium passage was tested. IHS laboratory in rat and sheep models directed toward understanding confirmed localization and gestation dependent changes in GC-Rs, CRF-Rs the cellular and molecular basis of stress-induced in utero and cholinergic markers in sheep fetal colon. Rat placenta and enteric meconium passage. endocrine cells express CRF and gastrointestinal tract express CRF-Rs. Hypoxia is a potent inducer of meconium passage in term fetal rats. Stress is a risk factor for in utero meconium passage and laboratory animal models can be used to develop pharmacotherapy to prevent stress-induced in utero Stress and gut motility: lessons learned from meconium passage. laboratory animals Journal of Perinatology (2008) 28, S8–S13; doi:10.1038/jp.2008.144 Although impact of emotional stress on gastric and colonic motor activity has long been known in humans only during the past two decades, much attention has been paid to understand the neural circuitry and hormonal effectors that mediate effects of stress on Introduction gut functions in animal models (see review7,8). The rat is a widely Newborn meconium passage, a developmentally programmed used animal model for stress-related alteration on gut motor event, normally occurs within the first 24–48 h after birth, functions, although limited investigation has also been performed although little is known of the regulatory process. Clinically, a in mouse models.9 Inhibition of gastric emptying, gastric secretion, delay in newborn meconium passage has been observed in infants gastric contraction and stimulation of colonic motility and born with Hirschprung disease, a defect involving the absence of defecation have now been well established in adult rats as the intrinsic ganglia in colon.1 In contrast, we have no similar hallmarks of neurovisceral motor responses to stressors of anatomical or neurochemical explanations for the meconium psychological, physical, chemical and immunological origin.10–13 staining observed in 7–22% of all term deliveries.2 Greater than Members of stress hormone family, more specifically 90% of cases of clinically observed meconium-stained amniotic corticotrophin-releasing factor (CRF) and urocortin, have been fluid are noted in fetuses at or following 37 weeks gestation, being shown to mimic multifaceted acute responses to stress.8 Both uncommon in preterm deliveries.3 Passage of meconium occurs hormones mediate their actions through CRF-receptor subtype 1 most often in deliveries beyond 40 weeks of gestation.4 An increased (CRF-R1) and CRF-receptor subtype 2 (CRF-R2). Both receptors incidence of meconium passage in amniotic fluid is noted in the are encoded by distinct genes and both are G protein-coupled presence of fetomaternal stress factors such as hypoxia and receptors linked to multiple intracellular signaling pathways.14 infection, independent of fetal maturation.5,6 On the basis of the Of the two CRF receptor subtypes, CRF-R1 stimulates colonic clinical observations, fetal stress and gastrointestinal maturation motility and defecation, whereas CRF-R2 inhibits gastric emptying and gastric contraction at times of stress.15 Anatomical, Correspondence: Dr MG Ross, Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, 1000 W. Carson Street, Box 3, Torrance, CA 90502, USA. physiological, pharmacological and biochemical studies have E-mail: [email protected] provided unambiguous evidence that the CRF system Mechanism of in utero meconium passage J Lakshmanan and MG Ross S9

(CRF, urocortins, CRF-R1 and CRF-R2) is the key player of novel hypoxic stress paradigm to demonstrate that hypoxic stress at gastrointestinal motility disturbance during times of stress.7,16 term will provoke in utero meconium passage.21 The experimental design includes exposure of term pregnant rats (term ¼ 22 days) Hypothesis: in utero meconium passage is a fetal at 22 days of gestation to decreasing concentration of oxygen in a neurovisceral motor response to stress stepwise manner as illustrated in Figure 2a. Fetuses removed from uterine horn at the end of hypoxic exposure were alive and We hypothesize that in utero meconium passage is a neurovisceral exhibited thick meconium staining as shown in Figure 2b, whereas motor response to fetal stress and that the CRF system plays a control fetuses did not exhibit meconium. critical role in the stimulation of cholinergically mediated fetal Examination of fetal and maternal plasma revealed a marked colonic propulsive motor functions that trigger meconium passage. elevation in CRF levels,21 suggesting that the peripheral CRF An integrated model proposed for fetal in utero meconium passage pathway may mediate the stimulation of fetal colonic motility and is shown in Figure 1. The salient points of our hypothesis are as meconium passage. As acute hypoxia-induced meconium passage follows: fetal–maternal stress leads to increased fetal plasma 17 has a rapid onset, the mechanisms underlying the peripheral glucocorticoids, which stimulate the synthesis of placental CRF. pathway most likely involve the activation (directly and neurally) Stress-induced increase in glucocorticoids are also speculated to of fetal adrenal epinephrine and norepinephrine secretion as well upregulate the CRF-R1 density18 on the myenteric neurons as well 19 as sympathetic nervous system norepinephrine release, which in as the expression of peripheral choline acetyltransferase, the turn stimulate placental CRF exoctyosis. Recent studies in our enzyme that catalyzes the synthesis of acetylcholine, the principle laboratory indicate that rat placenta, similar to human placenta, is neurotransmitter that regulates the motor activity of a site of CRF synthesis.22,23 In addition, enteric endocrine cells gastrointestinal system. In conditions of stress, increased maternal expressing CRF in term rat fetal gut could also function as a and/or fetal sympathetic tone will lead to increased circulating potential local source, as speculated in humans.24,25 Increased levels of catecholamines, which are known stimulators of placental 20 peripheral CRF in the fetus stimulates colonic motility through CRF release. Thus, stress-induced activation of the CRF–CRF-R1 myenteric CRF-R1 and rapid meconium passage (Figure 3a). system at the level of myenteric neurons will evoke release of The central CRF pathway involves activation of CRF neurons acetylcholine, increased colonic smooth muscle motility and in paraventricular brain nuclei directly innervating colonic in utero meconium passage. segments, as has been demonstrated in adult rats (Figure 3b). Thus, acute hypoxic stress-induced in utero meconium passage involves release of stored CRF protein pool from peripheral and/or Stress is a potent stimulator of in utero meconium central tissues. Ongoing studies in our laboratory also support the passage in term fetal rats expression of CRF-R1 in smooth muscle and enteric neuronal Although clinical signs of stress due to hypoxia are frequently circuitry in term rat fetal gastrointestinal tract,26 lending strong observed in human neonates born with meconium staining, there anatomical support for our hypothesis that intrauterine stress may is no direct evidence to support the fact that hypoxic stress is a risk impact gut motility through CRF-R1 in a manner analogous to factor for in utero meconium passage. We recently developed a stress-induced stimulation of colonic motility and defecation in adult rats.15

Lessons learned from sheep model in support of stress-induced in utero meconium passage Sheep are used in our laboratory as a large animal model for understanding the molecular mechanism of in utero meconium passage.27–29 The larger size of the gut and longer gestational age permit in vivo studies exploring gestation- and hormone-dependent changes in gastrointestinal motility, and in vitro organ bath studies examining smooth muscle contractility. The large fetal size permits withdrawal of blood in sufﬁcient quantities for evaluating circulating hormone and cytokine levels. In the following section, we summarize some of our investigations Figure 1 An integrated model for stress-induced in utero meconium passage. An interconnected pathway involving glucocorticoids, CRF and cholinergic relevant to hormonal regulation of sheep fetal distal colonic neurocircuitry systems stimulate colonic motility leading to in utero meconium motility as well as recent progress made on the ontogeny of passage (detailed in the text). glucocorticoid receptor system, CRF system and cholinergic

Journal of Perinatology Mechanism of in utero meconium passage J Lakshmanan and MG Ross S10

Oxygen content in chamber 25

% Oxygen 10

0 3 6 8 12 15 18 21 24 27 30 33 36 39 42 Time (min) Figure 2 Hypoxic stress paradigm in term fetal rat with meconium passage. Oxygen content in hypoxic chamber is reduced in a stepwise manner from 21 to 8% as indicated in the ﬁgure (left panel). A photograph of a fetal rat with meconium passage (arrows; right panel).

a b

Figure 3 Hypothesized mechanisms for hypoxia-induced in utero meconium passage. Acute hypoxia-induced in utero meconium passage could be mediated either by (a) placental CRF release or (b) central hypothalamic paraventricular nucleus CRF. In both circumstances, CRF directly stimulates CRF-R1 localized on cholinergic myenteric neurons.

synaptic circuitry system in fetal distal colonic segments. In suggesting that glucocorticoids regulate cholinergic contractile addition, we discuss our recent advances in support of knowledge of maturation through ‘genomic’ actions. The findings obtained CRF signaling system in the prevention of in utero meconium following glucocorticoid administration has provided partial passage before term. support for our hypothesis that stress-mediated glucocorticoid release may stimulate colonic motility and meconium passage. Sheep fetal colon is a glucocorticoid target organ We also examined the in vivo effect of bethanechol on colonic As the incidence of meconium-stained amniotic fluid increases muscular contractile and electromyogram activity in the sheep with increase in gestational ages and fetal stress, we hypothesized fetus.28 We noticed increased strain gauze activity in response to that fetal colon could be a glucocorticoid target organ with fetal bethanechol infusion in transverse but not in distal colon at 0.9 colonic motility function regulated by glucocorticoids.27 We gestation (135 days gestation, term ¼ 150 days). This finding examined in vivo effects of betamethasone (a synthetic suggested the presence of a local inhibitor system for glucocorticoid) in the presence and absence of thyroxine on cholinergic-dependent contractility in the distal colon before term, in vitro colonic smooth muscle cholinergic contractility.27 Muscle perhaps serving as an intrinsic mechanism to prevent premature strips prepared from sheep fetuses that received intramuscular meconium passage. injection of betamethasone alone or in combination with thyroxine exhibited greater contractile responsiveness to bethanechol Localization and gestation-dependent changes in glucocorticoid (a cholinergic agonist) than muscle strips prepared from receptor expression in sheep fetal distal colon vehicle-treated fetuses. These glucocorticoid-specific changes were To validate our hypothesis that sheep fetal colon is glucocorticoid noticed 48 h after intramuscular injections of betamethasone, target organ, we undertook studies to evaluate the cellular

Journal of Perinatology Mechanism of in utero meconium passage J Lakshmanan and MG Ross S11 localization and gestation-dependent changes in glucocorticoid endogenous CRF leading to increased motor activity and receptors in sheep fetuses at very preterm (118–120 days), preterm meconium passage. (130–132 days), near term (140–142 days) and term (146–147 days) gestation.30 Glucocorticoid receptor antibody elicited positive Cellular localization of CRF-binding protein in sheep staining in smooth muscle layers as well as in myenteric and distal colon submucosal neurons at all stages of development with maximal Corticotrophin-releasing factor-binding protein is a protein known 33 expression noticed at near term, suggesting the profound changes to compete with CRF-R1 and CRF-R2 for CRF and urocortin. Our in nuclear transcription machineries begin to occur at near term. immunohistochemical investigation revealed expression of 34 We also noticed a robust change in fetal plasma cortisol levels at CRF-binding protein in the smooth muscle-enteric unit. We term, suggesting that the expression of glucocorticoid receptors observed a marked decrease in smooth muscle expression of could be turned ‘off’ by glucocorticoids at term, or immediately CRF-binding protein and a simultaneous increase in myenteric after birth, as circulating levels of glucocorticoids reach basal levels neuronal somas at term, indicating that this protein is a critical within 24 h after birth in many species including humans. regulator of CRF and urocortin functions at the level of smooth muscle-enteric unit in sheep fetal distal colon. Evidence for prereceptor metabolism of glucocorticoids in fetal sheep distal colon CRF signaling system may play a role in preventing Glucocorticoid actions at the cellular level are controlled by 11-beta in utero meconium passage before term hydroxysteroid hydrogenase (11bHSD) type 1 and type 2, enzymes We performed in vitro study of sheep fetal colonic contractility to 29 that regulate the interconversion of active glucocorticoid and their assess the significance of CRF receptor system. In these studies, inactive 11-keto metabolites. We recently examined the presence of colonic smooth muscle strips prepared from preterm sheep fetal 11 bHSD-1 and 11 bHSD-2 in sheep fetal colonic enteric nervous distal colonic segments were subjected to cholinergically dependent system as evidence that these enzymes provide a novel means for contractility in an organ bath system following pre-incubation with regulation of glucocorticoid-mediated ‘genomic’ activities.31 The CRF or urocortin. Both neuropeptides significantly decreased the antibodies to 11bHSD-1 and 11 bHSD-2 elicited positive cholinergically stimulated contractility. Immunohistochemical immunostaining of both myenteric and submucosal ganglia at all studies confirmed the expression of muscarinic receptor subtype gestation ages. The percentage of myenteric ganglia expressing 3 and CRF-R2 in preterm sheep fetal colon. Additional studies 11bHSD-1 and 11bHSD-2 were maximal at term. The percentage of revealed positive immunostaining for CRF and urocortin in smooth submucosal ganglia expressing 11bHSD-1 was maximal at term, muscle and enteric neurons, indicating the local availability of whereas the expression of 11bHSD-2 remained constant throughout neuropeptides to inhibit smooth muscle motility during the time of 29 gestation. We speculate that increasing levels of 11bHSD-1, and thus stress. All in all, stress hormones (CRF and urocortin) and stress increased active glucocorticoid levels, at term could contribute to hormone receptors (CRF-R1 and CRF-R2) appear to function in glucocorticoid-dependent colonic contractile maturation. parallel or series pathways, and CRF-R2 plays a central role in preventing stress-induced in utero meconium passage before term.

CRF system Cholinergic circuitry system Identification and gestation-dependent changes in CRF Maturational changes in sheep fetal colonic cholinergic circuitry system in sheep fetal distal colon system parallels plasma glucocorticoid surge We recently undertook a systematic investigation to obtain We utilized antibodies specific to vesicular acetylcholine anatomical support that CRF system is an integral component of transporter, a high-affinity choline transporter, and peripheral distal colonic contractility apparatus.32 Our findings can be choline acetyl transferase as markers for cholinergic synaptic summarized as follows: CRF-R1- and CRF-R2-specific antibodies circuitry system to examine the maturation of cholinergic system strongly immunostained longitudinal and circular smooth muscles in distal colon of sheep fetuses from very preterm to term in addition to muscularis mucosa in colonic segments at all gestation.35 The percentage of myenteric ganglia expressing gestational ages. A marked decrease in CRF-R2 immunostaining cholinergic markers in term fetuses were significantly higher than occurred in smooth muscle layers close to term. CRF-R1 antibody those observed in preterm. The marked increase observed in immunostained neuronal somas both in myenteric and cholinergic markers in myenteric ganglia in fetal distal colon at submucosal ganglia, and the percentage of ganglia expressing term is possibly mediated by effects of endogenous glucocorticoid CRF-R1 were maximal at term gestation. On the basis of the levels, which peak at term. We speculate that glucocorticoid is the reciprocal changes in CRF-R1 and CRF-R2 expression in the physiological hormone that regulates enteric cholinergic neural smooth muscle-enteric unit, we speculate that CRF-R1 could be synaptic circuitry system similar to their actions in central nervous hyper-responsive to stressors that stimulate the release of system regions.

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Localization of inhibitory and stimulatory muscarinic Disclosure receptor subtypes in sheep fetal distal colon: implication Jayaraman Lakshmanan has received grant funding from NIH R03. Michael Ross for meconium passage has received grant funding from the March of Dimes. Acetylcholine mediates gastrointestinal motility through muscarinic receptors. To date, five muscarinic receptors (M1–M5) have been cloned, with M3 confirmed as the prominent mediator of smooth muscle contraction. Of the other subtypes, M1, M2 and M4 have References been shown to function as potent autoinhibitor (that is, inhibitory 1 Momoh JT. Hirschsprung’s disease: problems of diagnosis and treatment. Ann Trop presynaptic muscarinic receptor) of Ach release from myenteric Paediatr 1982; 2: 31–35. plexus. We examined the maturation and topographical 2 Wiswell TE, Tuggle JM, Turner BS. Meconium aspiration syndrome: have we made a distribution of muscarinic receptor subtypes in distal colon from difference? [see comments]. Pediatrics 1990; 85: 715–721. 36 3 Ahanya SN, Lakshmanan J, Morgan BL, Ross MG. Meconium passage in utero: very preterm to term. M1–M5 antibodies elicited positive staining mechanisms, consequences, and management. Obstet Gynecol Surv 2005; 60: in colonic sections at all gestational ages. M4 is expressed most 45–56. abundantly in circular and longitudinal smooth muscle layers, 4 Usher RH, Boyd ME, McLean FH, Kramer MS. Assessment of fetal risk in postdate whereas M3 is expressed in rather low levels. Positive pregnancies. Am J Obstet Gynecol 1988; 158: 259–264. immunostaining for both M5 and M1 in smooth muscle layers are 5 Miller FC, Read JA. Intrapartum assessment of the postdate fetus. Am J Obstet Gynecol 1981; 141: 516–520. similar, but their immunostaining intensity was significantly lower 6 Richey SD, Ramin SM, Bawdon RE, Roberts SW, Dax J, Roberts J et al. Markers of compared with M3, M4 and M2. The percentage of neurons acute and chronic asphyxia in infants with meconium-stained amniotic fluid. expressing inhibitory autoreceptors (M1, M2 and M4) in the enteric Am J Obstet Gynecol 1995; 172: 1212–1215. ganglia was markedly higher in distal colonic sections of preterm 7 Tache Y, Bonaz B. Corticotropin-releasing factor receptors and stress-related alterations and near-term fetuses, with expression declining rapidly and of gut motor function. J Clin Invest 2007; 117: 33–40. markedly at term. Our results indicate that before term, muscarinic 8 Tache Y, Martinez V, Million M, Rivier J. Corticotropin-releasing factor and the brain- gut motor response to stress. Can J Gastroenterol 1999; 13(Suppl A): 18A–25A. autoreceptors may inhibit presynaptic release of Ach and 9 Martinez V, Wang L, Rivier J, Grigoriadis D, Tache Y. Central CRF, urocortins and consequently limit the cholinergically mediated smooth muscle stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors contractility. This could represent a physiological mechanism delays gastric transit in mice. J Physiol 2004; 556: 221–234. analogous to CRF-R2 functions in preventing stress-induced 10 Million M, Wang L, Martinez V, Tache Y. Differential Fos expression in the in utero meconium passage before term. The rapid decline of paraventricular nucleus of the hypothalamus, sacral parasympathetic nucleus and colonic motor response to water avoidance stress in Fischer and Lewis rats. Brain Res muscarinic inhibitory autoreceptors noticed at term most 2000; 877: 345–353. likely relieves the colonic contractile machinery from 11 Williams CL, Villar RG, Peterson JM, Burks TF. Stress-induced changes in intestinal auto-inhibition and will increase efficacy of cholinergic synaptic transit in the rat: a model for irritable bowel syndrome. Gastroenterology 1988; 94: transmission, facilitating ready responsiveness of colonic contractile 611–621. machinery to Ach. 12 Miampamba M, Sharkey KA. c-Fos expression in the myenteric plexus, spinal cord and In summary, we have established for the first time a rat model brainstem following injection of formalin in the rat colonic wall. J Auton Nerv Syst 1999; 77: 140–151. for stress-induced in utero meconium passage. We have further 13 la Fleur SE, Wick EC, Idumalla PS, Grady EF, Bhargava A. Role of peripheral obtained anatomical support for the expression of key molecules of corticotropin-releasing factor and urocortin II in intestinal inflammation and motility glucocorticoid system, CRF system and cholinergic circuitry system in terminal ileum. Proc Natl Acad Sci USA 2005; 102: 7647–7652. in sheep fetal distal colon. All three systems seem positioned 14 Hillhouse EW, Grammatopoulos DK. The molecular mechanisms underlying the in distal colon readily to respond to stress. Our studies unexpectedly regulation of the biological activity of corticotropin-releasing hormone receptors: implications for physiology and pathophysiology. Endocr Rev 2006; 27: 260–286. revealed the presence of potential mechanisms to prevent the 15 Maillot C, Million M, Wei JY, Gauthier A, Tache Y. Peripheral corticotropin-releasing in utero meconium passage before term, including the factor and stress-stimulated colonic motor activity involve type 1 receptor in rats. expression of CRF-R2. Future studies should improve our Gastroenterology 2000; 119: 1569–1579. understanding of the mechanisms of in utero meconium passage 16 Grundy D, Al-Chaer ED, Aziz Q, Collins SM, Ke M, Tache Y et al. Fundamentals of and possibly to develop pharmacotherapy to prevent stress-induced neurogastroenterology: basic science. Gastroenterology 2006; 130: 1391–1411. in utero meconium passage and consequences of meconium 17 Jones SA, Brooks AN, Challis JR. Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 1989; aspiration. 68: 825–830. 18 Bruder ED, Jacobson L, Raff H. Plasma leptin and ghrelin in the neonatal rat: interaction of dexamethasone and hypoxia. J Endocrinol 2005; 185: 477–484. 19 Takahashi LK, Goh CS. Glucocorticoid facilitation of cholinergic development in the rat Acknowledgments hippocampus. Neurosci 1998; 83: 1145–1153. 20 Coukos G, Monzani A, Saletti C, Petraglia F. [Noradrenaline and interleukin-1 This research was supported by grants from the March of Dimes Foundation stimulate CRF secretion from human placental cells in culture]. Medicina (Firenze) (MGR) and the National Institutes of Health (JL). 1988; 8: 441–442.

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21 Lakshmanan J, Ahanya SN, Rehan V, Oyachi N, Ross MG. Elevated plasma 29 Lakshmanan J, Oyachi N, Ahanya SA, Liu G, Mazdak M, Ross MG. corticotrophin release factor levels and in utero meconium passage. Pediatr Res 2007; Corticotropin-releasing factor inhibition of sheep fetal colonic contractility: mechanisms 61: 176–179. to prevent meconium passage in utero. Am J Obstet Gynecol 2007; 196: 357. 22 Lakshmanan J, Salido E, Amidi F, Amidi E, Raj R, Ross MG. Rat placenta expresses 30 Lakshmanan J, Oyachi N, Liu GL, Choi GY, Ross MG. Fetal colonic enteric nervous corticotrophin releasing factor protein and mRNA. Reproductive Sciences 2007; system is a site of glucocorticoid-induced gastrointestinal maturation. Reprod Sci 2007; 14(1 Suppl): 175A. 14(1 Suppl): 287A. 23 Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA. 31 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Evidence for pre-receptor metabolism of Characterization and gestational regulation of corticotropin-releasing hormone glucocorticoids in ovinee fetal distal colonic enteric nervous system. Reprod Sci 2007; messenger RNA in human placenta. J Clin Invest 1988; 82: 287–292. 14(1 Suppl): 288A. 24 Lakshmanan J, Richard JD, Liu GL, Ross MG. Corticotrophin releasing factor is a fetal 32 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Localization and gestation-dependent gut hormone. Reprod Sci 2007; 14(1 Suppl): 251A. pattern of CRF-receptos (R1, R2) expression in ovine fetal distal colon. Reprod Sci 25 Kawahito Y, Sano H, Kawata M, Yuri K, Mukai S, Yamamura Y et al. Local secretion of 2007; 14(1 Suppl): 258A. corticotropin-releasing hormone by enterochromafﬁn cells in human colon. 33 Seasholtz AF, Valverde RA, Denver RJ. Corticotropin-releasing hormone-binding Gastroenterology 1994; 106: 859–865. protein: biochemistry and function from ﬁshes to mammals. J Endocrinol 2002; 175: 26 Richard JD, Lakshmanan J, John TA, Ross MG. Rat fetal gastrointestinal tract is a target 89–97. organ for corticotrophin-releasing factor family neuropeptides. Am J Obstet Gyn 2006; 34 Lakshmanan J, Liu GL, Oyachi N, Ross MG. Cellular localization of corticotrophin 195(6 Suppl): S214. releasing factor binding protein (CRF-BP) in fetal ovine distal colon: a possible local 27 Ross B, Bradley K, Nijland MJ, Polk DH, Ross MG. Increased fetal colonic muscle inhibitor of stress-induced colonic motility. Reprod Sci 2007; 14(1 Suppl): 288A. contractility following glucocorticoid and thyroxine therapy: implications for 35 Lakshmanan J, Lips KS, Liu GL, Ross MG. Maturational changes in ovine fetal colonic meconium passage. J Matern Fetal Med 1997; 6: 129–133. cholinergic circuitry parallels plasma glucocorticoid surge. Reprod Sci 2007; 28 Acosta R, Oyachi N, Lee JJ, Lakshmanan J, Atkinson JB, Ross MG. 14(1 Suppl): 249A. Mechanisms of meconium passage: cholinergic stimulation of 36 Lakshmanan J, Liu GL, Ross MG. Localization of inhibitory and stimulatory muscarinic electromechanical coordination in the fetal colon. J Soc Gynecol Investig receptor subtypes in ovine fetal distal colon: implications for meconium passage. 2005; 12: 169–173. Reprod Sci 2007; 14(1 Suppl): 168A.

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