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The distribution of radiolabeled corticotropin-releasing factor in pregnant rats : an investigation of placental transfer to the FETUSESfn2
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Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 118Ð123\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII ] S9625Ð4637"87#99911Ð6
THE DISTRIBUTION OF RADIOLABELED CORTICOTROPIN! RELEASING FACTOR IN PREGNANT RATS ] AN INVESTIGATION OF PLACENTAL TRANSFER TO THE FETUSES& MICHAEL T[ WILLIAMS\$ HARRY N[ DAVIS\$ ANNE E[ MCCREA% and MICHAEL B[ HENNESSY$ $ Wright State University\ Dayton\ OH 34324 ^ % Sinclair Community College\ Dayton\ OH 34391\ U[S[A[ "Received 5 October 0886 ^ revised 2 January 0887 ^ accepted 3 January 0887# Abstract*Stress during gestation can have serious consequences on the development of the fetus[ Many of these e}ects appear to be mediated by hormones of the hypothalamic!pituitary!adrenal "HPA# axis[ Corticotropin!releasing factor "CRF#\ released by the hypothalamus during times of stress serves to activate release of pituitary hormones and is also present in low levels in rat plasma[ Moreover\ the uterus contains signi_cant quantities of CRF at implantation sites\ probably from local sources[ Therefore\ the possibility exists that CRF may cross the placenta and activate the fetal HPA axis[ However\ the ability of CRF to cross the placenta has not been demonstrated[ In the present study\ pregnant rats were administered radiolabeled CRF intraperitoneally\ and the distribution of the labeled product was determined in the fetuses and various maternal organs[ High levels of activity were observed in the pregnant female|s uterus\ adrenals\ heart and the placentae\ but only background levels of activity were detected in the maternal brain[ Very low levels of activity were observed in the fetuses\ indicating that the transfer of CRF across the placenta is greatly restricted[ These _ndings suggest that maternal CRF has little or no direct e}ect on the developing fetus during gestational stress[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] corticotropin!releasing factor\ blood!brain barrier\ prenatal stress\ placental transfer[
INTRODUCTION Stress during pregnancy\ or gestational stress\ can in~uence the anatomical\ behavioral\ and physio! logical development of the rat fetus[15\26\28 One of the more striking e}ects of gestational stress is on the sexual di}erentiation of the o}spring[ In general\ males appear and:or behave more like females00\24\26 and in some instances\ females appear and:or behave more like males[7\00\05 Probable mediators of these gestational stress e}ects are the hormones of the hypothalamic!pituitary!adrenal "HPA# axis ði[e[\ corticotropin!releasing factor "CRF#\ adrenocorticotropin "ACTH#\ b!endorphin\ corticosterone\ and possibly aldosteroneŁ\ which are released during times of stress[ Recently\ we found that peripheral administration of CRF to pregnant rats produced several e}ects that were similar to those observed following gestational stress\ speci_cally ] "0# reduced weight gain of the pregnant females ^ "1# reduced body weights of o}spring at birth ^ "2# enhanced emission of ultrasonic vocalizations on postnatal day 03^ and "3# shorter anogenital distances of the male o}spring[27\28 It is not known where CRF exerted its actions\ but several possibilities exist ] "0# CRF caused release of maternal hormones which directly a}ected o}spring development ^ "1# CRF caused release of maternal hormones which\ in turn\ activated release of hormones from the fetuses ^ "2# CRF altered the physiology of the pregnant female in other ways "e[g[\ constriction of blood vessels leading to hypoxia in the fetuses# which a}ected o}spring development ^ or "3# CRF had direct actions on fetal hormone release\ possibly through translocation of CRF across the placenta[ Any one of these possibilities alone or in combination might account for the e}ects of both administration of CRF and of stress during pregnancy[ Furthermore\ it is known that administration of either corticosterone\ ACTH\ or b!endorphin to pregnant rats\ can produce some consistent and inconsistent consequences\ compared to e}ects observed following gestational stress[2\4\8\09 Address correspondence to ] Michael B[ Hennessy\ Ph[D[\ Wright State University\ Department of Psychology\ 224 Fawcett Hall\ 2539 Col[ Glenn Hwy[\ Dayton\ OH 34324\ U[S[A[ Tel[ ] 990 826 664 1832 ^ fax ] 990 826 664 2236 ^ e!mail ] mhennessyÝdesire[wright[edu & This research was supported by grant IBN!8111966 from the National Science Foundation[ 118
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Many di}erent compounds are known to cross the placental barrier\ and do so with varying degrees of ease\0\3\01\02\07\08\17\18\39\30 whereas other compounds appear completely unable to cross[6\06 Very low circulating levels of CRF exist in rat plasma ^ these appear to be derived primarily from peripheral rather than hypothalamic sources[14 During human pregnancy\ CRF is produced by the placenta[16 However\ in the rat\ CRF was not detected in either the placenta or the amnion[29\25 Yet\ a recent study has shown that CRF is produced at implantation sites on the rat uterus[03 Although CRF is produced in close proximity to the fetal environment in the rat "i[e[\ the uterus#\ the ability of CRF to cross the placental barrier has not been reported[ The capability of other hormones of the HPA axis to cross the placental barrier has been studied[ Evidence exists that ACTH does not cross the placenta6\06 while corticosterone0\07\39 and b!endorphin do[18 Because the adrenal steroids are lypophilic\ it is possible that aldosterone may also cross the placenta\ and some data exist to suggest this may be the case\ at least from the fetal to the maternal compartment[3 To help clarify a possible site of action for CRF during gestational stress\ this study was designed to determine if CRF can cross the placental barrier in the rat[ The experiment was conducted during the third trimester "gestational day 06# which is when CRF administration was found to a}ect o}spring development27 and when gestational stress produces the clearest e}ects on sexual di}er! entiation and other parameters[7\00\05\24\26 Radiolabeled CRF was administered to pregnant females and levels of radioactivity were measured in the fetuses[ The placentae\ as well as the pregnant female|s brain\ pituitary\ adrenals\ kidneys\ and uterus\ were also examined because of known CRF receptors in these regions19\13\23 and for comparison with fetal levels of radioactivity[
EXPERIMENTAL PROCEDURES Animals and conditions Pregnant female Sprague!Dawley rats "shipped pregnant from Harlan\ Indianapolis\ IN# and their fetuses were used for this experiment[ All females were allowed free access to food and water prior to testing[ Each pregnant rat was injected IP with 4 mCi 014I!ðTyr9Ł!CRF "rat\ human ^ Peninsula Laboratories\ Inc[\ Belmont\ CA# in a volume of 9[4 ml distilled water on day 06 of gestation[ Equal numbers of females "n 4# were decapitated at intervals of 09\ 19\ or 29 min following the injection[ Tissue dissection and measurement of 014I!ðTyr9Ł!CRF Females were anesthetized by halothane inhalation and decapitated[ Immediately following decapitation\ the uterus was excised from the abdominal region[ The fetuses from each uterine horn were counted and placentae and fetuses were removed[ The amniotic sac was cleared from each fetus and the umbilical cord was snipped proximal to the fetus[ Simultaneous with the fetal extraction\ the whole brain "excluding olfactory bulbs# and the pituitary were removed from the pregnant female|s skullcase[ Each pregnant rat|s heart and adrenals were harvested after removal of the uterus[ All samples were placed\ immediately upon removal\ into an acidic solution "9[0 N HCL and 1 N Acetic acid in a 0 ] 0 concentration#[ All tissues were homogenized for 09 s and then centrifuged at 1399 rpm for 29 min[ Following centrifugation\ 099 ml of supernatant was removed and counted in a gamma counter for 0 min in no less than duplicate aliquots[ The activity of each sample was expressed in counts per min "cpm#:099 ml supernatant[ The speci_c activity of each tissue sample was calculated by subtracting the mean background level "43[4 cpm# from the total activity[ Statistics The weights of the pregnant females\ the total number of fetuses\ and the number of fetuses in each uterine horn were subjected to one!way analyses of variance "ANOVA#[ For the tissue samples\ one!way ANOVAs were used to determine if there were any changes in the speci_c activity over time[ StudentÐNewmanÐKeuls was used for post!hoc analysis of any signi_cant main e}ect of Time Point[ Any sample that did not show a signi_cant e}ect was collapsed across time points for subsequent analysis[ Dependent Student|s t!tests were performed to compare speci_c activity to background[
Distribution of 014I!ðTyr9Ł!CRF
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RESULTS ANOVAs for the weights of the pregnant females\ total number of pups in a litter\ and number of pups in the left and right uterine horns yielded no signi_cant e}ects[ Inspection of Table 0 shows that the highest levels of speci_c activity were observed in the uterus[ The levels of speci_c activity in the placentae were much lower than that found in the uterus\ especially during the _rst two time periods[ The speci_c activity levels in the fetuses were very low during the _rst 19 min "i[e[\ less than 04 cpm over background# and increased at 29 min\ although they were less than a seventh of that in the placentae\ and a fortieth of that found in the uterus at this _nal time point[ For placentae\ ANOVA revealed a signi_cant e}ect of Time Point F"1\01# 3[18\ P ³ 9[94[ Increased levels of radioactivity were observed at 29 min relative to 09 or 19 min[ The e}ect of Time Point was not signi_cant for the other samples\ although that for fetuses approached signi_cance\ P ³ 9[95[ Dependent t!tests revealed that the fetuses\ uterus\ heart\ adrenals\ and placentae "at all time points# exhibited activity above background levels[ No di}erence from background was found for brain and pituitary "Table 0#[ Because no di}erence between background activity and brain activity was found\ another set of dependent t!tests was performed using the brain as a negative tissue control[ All tissues\ with the exception of pituitary\ had higher levels of activity than did the brain[ DISCUSSION This study described the distribution of 014I!ðTyr9Ł!CRF in pregnant female rats and their fetuses[ The capacity of CRF to cross the blood!brain barrier has been examined in rats1 and mice[04 These researchers did not _nd CRF to cross from blood to brain\ but did _nd CRF to cross from brain to blood\ at least for mice[ In the present study\ the total cpm for the brains of the pregnant females were not di}erent from background levels\ supporting Banks and Kastin|s04 _nding that CRF does not enter the brain from the periphery in the rat[ Although CRF is produced in the human placenta during pregnancy\16 there is no evidence for placental expression of CRF during either stress or nonstress periods in rats[29\25 However\ a recent study found that CRF is expressed at the implantation sites on the rat uterus[03 These researchers hypothesized that CRF may serve to mediate the in~ammation of the uterus observed during the implantation of rat blastocysts[ In the present study\ the uterus had the highest levels of radioactivity of the tissues sampled[ This suggests that a high density of CRF binding sites is present in the uterus during the later stages of rat pregnancy[ Because these levels were found during the third trimester\ activation of CRF binding sites in the uterus may serve functions unrelated to implantation[ Moreover\ because CRF can modify blood ~ow\12 the activation of CRF receptors in the uterus or placenta during times of stress could conceivably restrict or alter blood ~ow and therefore produce hypoxia in the fetuses[ Hypoxia has been suggested to underlie some of the e}ects of gestational stress[11 Levels of radioactivity in the fetuses were just a fraction of the levels observed in the placentae and uterus[ When the radioactivity of the fetuses was statistically analyzed\ the di}erence over time approached signi_cance and the total radioactivity was greater than background and brain\ suggesting that some CRF crossed the placental barrier[ However\ inspection of Table 0 shows that the speci_c activities at 09 and 19 min are at least 2 times lower than the speci_c activity at 29 min[ In humans\ a bi!exponential clearance rate for CRF has been found with a fast and a slow half! life[10\20\22 The fast half!life of CRF was reported to be in the range of 3[0 minÐ00[5 min[ Similar _ndings have been found in cynomolgus monkeys with a fast half!life of approximately 06[0 min[21 If the half!life of CRF in rat plasma is the near the range for humans and cynomolgus monkeys\ then the background levels of radioactivity in the fetuses shortly following injection\ with increasing levels thereafter\ could indicate that detected radioactivity in the fetuses was due to a metabolite of CRF crossing the placenta[ Since the heart\ adrenals\ placentae\ and uterus were radiolabeled with CRF within the _rst 09 min and showed fairly constant levels throughout 29 min\ it would further suggest that the radioactivity observed in the fetuses at 29 min was the result of metabolized parent compound\ 014I!ðTyr9Ł!CRF[ Clearly\ the passage of CRF through the placenta is greatly restricted ^ it appears that little\ and
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Table 0[ Distribution of
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I!CRF ] total counts per min "CPM# and speci_c activity "minus background$# per 099 ml supernatant\ observed for maternal brain\ heart\ adrenals\ pituitary\ and uterus\ and the placentae and all fetuses from both uterine horns at 09\ 19\ or 29 min following injection 09 min
29 min
Speci_c CPM
Total CPM
Speci_c CPM
Total CPM
Speci_c CPM
36[17200[38 193[41210[45 0074[392155[95 50[20200[52 1037[992336[02 120[09211[58 59[87200[38
−6[11200[38 049[91210[45 0029[892155[95 5[70200[52 1982[492336[02 065[59211[58 5[37200[38
49[2828[87 150[18222[23 0951[092012[44 52[49201[67 1467[212328[02 122[93215[39 54[29202[24
−3[0028[87 195[68222[23 0996[592012[44 8[99201[67 1412[712328[02 067[43215[39 09[79202[24
44[65200[94 239[79245[69 640[412063[17 60[57205[31 1145[522333[32 347[612092[66 098[22205[71
0[15200[94 175[29245[69 586[912063[17 06[07205[31 1191[022333[32 393[112092[66 43[72205[71
$ The mean background was 43[4 cpm[ The asterisk"s# next to the tissue sample represents the level of signi_cance for all time points combined[ For placentae\ there was a signi_cant e}ect of Time Point ^ therefore\ tests for increased activity were carried out separately for each time point[ P ³ 9[94[ P ³ 9[90[ P ³ 9[990[
M[ T[ Williams et al[
Brain Heart Adrenals Pituitary Uteri Placentae Fetuses
19 min
Total CPM
Distribution of 014I!ðTyr9Ł!CRF
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possibly no\ CRF crosses to a}ect the fetuses[ CRF is a 30 amino acid peptide[ b!Endorphin is a 20 amino acid peptide\ and appears to cross through the placental barrier[18 ACTH\ a 28 amino acid peptide\ is unable to cross the placental barrier[6\06 Therefore\ CRF may not be able to cross the placenta because of its molecular size[ CRF receptors are located in a variety of tissues in the periphery\ such as the pituitary\ heart\ adrenal\ gonads\ liver\ spleen\ and kidney[19\13\23 A seemingly paradoxical e}ect was found in this study[ The pituitary\ which is outside of the blood!brain barrier\ did not have levels of activity di}erent than background[ The likely explanation is that the injection procedure to administer the 014 I!ðTyr9Ł!CRF resulted in a stress response which released endogenous CRF from the hypo! thalamus[ Receptors for CRF in the pituitary have been found to be saturable and to have a high a.nity for CRF[5 Therefore\ endogenous CRF may have saturated the receptor sites and no radioactivity was detected[ The present _ndings indicate that the e}ects of CRF administration to pregnant rats in our earlier study27 do not appear to have been the result of a direct in~uence of exogenous CRF on the fetuses[ Rather\ they appear to have been due to an in~uence of either other maternal hormones secreted in response to CRF that then crossed the placenta\ or another CRF!induced change in maternal physiology "e[g[\ altered uterine blood ~ow leading to fetal hypoxia#[ More generally\ similar e}ects of gestational stress may be mediated by these mechanisms[ REFERENCES 0[ Arishima\ K[\ Nakama\ S[\ Morikawa\ Y[\ Hashimoto\ Y[\ Eguchi\ Y[\ Changes in placental permeability to cortico! sterone and estradiol!06b toward the end of gestation in the rat[ Experientia\ 0866\ 23\ 151Ð152[ 1[ Banks\ W[ A[ and Kastin\ A[ J[\ Peptides and the blood!brain barrier ] Lipophilicity as a predictor of permeability[ Brain Res[ Bull[\ 0874\ 04\ 176Ð181[ 2[ Chapman\ R[ H[ and Stern\ J[ M[\ Failure of severe maternal stress or ACTH during pregnancy to a}ect emotionality of male rat o}spring ] Implications of litter e}ects for prenatal studies[ Dev[ Pyschobiol[\ 0868\ 01\ 144Ð156[ 3[ Churchill\ S[\ Bengele\ H[ H[\ Melby\ J[ C[ and Alexander\ E[ A[\ Role of aldosterone in sodium retention of pregnancy in the rat[ Am[ J[ Physiol[\ 0870\ 139\ R064ÐR070[ 4[ Dahlof\ L[\ Hard\ E[ and Larsson\ K[\ Sexual di}erentiation of o}spring of mothers treated with cortisone during pregnancy[ Physiol[ Behav[\ 0867\ 10\ 562Ð563[ 5[ De Souza\ E[ B[\ Corticotropin!releasing factor receptors ] Physiology\ pharmacology\ biochemistry and role in central nervous system and immune disorders[ Psychoneuroendocrinolo`y\ 0884\ 19\ 678Ð708[ 6[ Dupouy\ J[\ Chatelain\ A[ and Allaume\ P[\ Absence of transplacental passage of ACTH in the rat ] direct experimental proof[ Biol[ Neonate\ 0879\ 26\ 85Ð091[ 7[ Herrenkohl\ L[ R[\ Prenatal stress reduces fertility and fecundity in female o}spring[ Science\ 0868\ 195\ 0986Ð0988[ 8[ Holson\ R[ R[\ Gough\ B[\ Sullivan\ P[\ Badger\ T[ and Sheehan\ D[ M[\ Prenatal dexamethasone or stress but not ACTH or corticosterone alter sexual behavior in male rats[ Neurotoxicol[ Teratol[\ 0884\ 06\ 282Ð390[ 09[ Kashon\ M[ L[\ Ward\ O[ B[\ Grisham\ W[ and Ward\ I[ L[\ Prenatal b!endorphin can moderate some aspects of sexual di}erentiation in rats[ Behav[ Neurosci[\ 0881\ 095\ 444Ð451[ 00[ Kinsley\ C[ H[ and Bridges\ R[ S[\ Prenatal stress and maternal behavior in intact virgin rats ] response latencies are decreased in males and increased in females[ Horm[ Behav[\ 0877\ 11\ 65Ð78[ 01[ Koren\ Z[\ Pfeifer\ Y[ and Sulman\ F[ G[\ Distribution and placental transfer of C03!serotonin in pregnant rats[ Am[ J[ Obstet[ Gynecol[\ 0855\ 84\ 189Ð184[ 02[ Lasuncion\ M[ A[\ Lorenzo\ J[\ Palacin\ M[ and Herrera\ E[\ Maternal factors modulating nutrient transfer to fetus[ Biol[ Neonate\ 0876\ 40\ 75Ð82[ 03[ Makrigiannakis\ A[\ Margioris\ A[ N[\ Le Goascogne\ C[\ Zoumakis\ E[\ Nikas\ G[\ Stournaras\ C[\ Psychoyos\ A[ and Gravanis\ A[\ Corticotropin!releasing hormone "CRH# is expressed at the implantation sites of early pregnant rat uterus[ Life Sci[\ 0884\ 46\ 0758Ð0764[ 04[ Martins\ J[ M[\ Kastin\ A[ J[ and Banks\ W[ A[\ Unidirectional speci_c and modulated brain to blood transport of corticotropin!releasing hormone[ Neuroendocrinolo`y\ 0885\ 52\ 227Ð237[ 05[ Miley\ W[ M[\ Blustein\ J[ and Kennedy\ K[\ Prenatal stimulation and postnatal testosterone a}ects infanticide in female rats[ Physiol[ Behav[\ 0871\ 17\ 516Ð518[ 06[ Milkovic\ S[ and Milkovic\ K[\ Reactiveness of fetal pituitary to stressful stimuli[ Does the maternal ACTH cross the placenta< Proc[ Soc[ Exp[ Biol[ Med[\ 0850\ 096\ 36Ð38[ 07[ Milkovic\ K[\ Paunovic\ J[\ Kniewald\ Z[ and Milkovic\ S[\ Maintenance of the plasma corticosterone concentration of adrenalectomized rat by the fetal adrenal glands[ Endocrinolo`y\ 0862\ 82\ 004Ð007[ 08[ Morgan\ C[ D[\ Sandler\ M[ and Panigel\ M[\ Placental transfer of catecholamines in vitro and in vivo[ Am[ J[ Obstet[ Gynecol[\ 0861\ 001\ 0957Ð0964[ 19[ Nappi\ R[ E[ and Rivest\ S[\ Stress!induced genetic expression of a selective corticotropin!releasing factor!receptor subtype within the rat ovaries ] An e}ect dependent on the ovulatory cycle[ Biol[ Reprod[\ 0884\ 42\ 0306Ð0317[ 10[ Nicholson\ W[ E[\ DeCherney\ G[ S[\ Jackson\ R[ V[\ Rowan\ C[\ Uderman\ H[\ Alexander\ A[ N[\ Rivier\ J[\ Vale\ W[ and Orth\ D[ N[\ Plasma distribution\ disappearance half!time\ metabolic clearance rate\ and degradation of synthetic ovine corticotropin!releasing factor in man[ J[ Clin[ Endocrinol[ Metab[\ 0872\ 46\ 0152Ð0158[ 11[ Ohkawa\ T[\ Takeshita\ S[\ Murase\ T[\ Kambegawa\ A[\ Okinaga\ S[ and Arai\ K[\ Ontogeny of the response of the hypothalamo!pituitary!adrenal axis to maternal immobilization stress in rats[ Endocrinol Japon\ 0880\ 27\ 076Ð083[
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12[ Overton\ J[ M[ and Fisher\ L[ A[\ Di}erentiated hemodynamic responses to central versus peripheral administration of corticotropin!releasing factor in conscious rats[ J[ Auton[ Nerv[ Syst[\ 0880\ 24\ 32Ð41[ 13[ Owens\ M[ J[ and Nemero}\ C[ B[\ Physiology and pharmacology of corticotropin!releasing factor[ Pharmacol[ Rev[\ 0880\ 32\ 314Ð362[ 14[ Plotsky\ P[ M[\ Otto\ S[\ Toyama\ T[ and Sutton\ S[\ Lack of correlation between immunoreactive corticotropin!releasing factor concentration pro_les in hypophysial!portal and peripheral plasma[ J[ Neuroendocrinol[\ 0889\ 1\ 54Ð58[ 15[ Pollard\ I[ and Dyer\ S[ L[\ E}ects of stress administration during pregnancy on the development of fetal testes and their subsequent function in the adult rat[ J[ Endocrinol[\ 0874\ 096\ 1330Ð1334[ 16[ Riley\ S[ C[ and Challis\ J[ R[ G[\ Corticotropin!releasing hormone production by the placenta and fetal membranes[ Placenta\ 0880\ 01\ 094Ð008[ 17[ Rosso\ P[\ Changes in the transfer of nutrients across the placenta during normal gestation in the rat[ Am[ J[ Obstet[ Gynecol[\ 0864\ 011\ 650Ð655[ 18[ Sandman\ C[ A[ and Kastin\ A[ J[\ The in~uence of fragments of the LPH chains on learning\ memory and attention in animals and man[ Pharmacol[ Ther[\ 0870\ 02\ 28Ð59[ 29[ Sasaki\ A[\ Shinkawa\ O[ and Yoshinaga\ K[\ Immunoreactive corticotropin!releasing hormone in amniotic ~uid[ Am[ J[ Obstet[ Gynecol[\ 0889\ 051\ 083Ð087[ 20[ Schulte\ H[ M[\ Chrousos\ G[ P[\ Booth\ J[ D[\ Old_eld\ E[ H[\ Gold\ P[ W[\ Cutler\ G[ B[ Jr[ and Loriaux\ D[ L[\ Corticotropin!releasing factor ] Pharmocokinetics in man[ J[ Clin[ Endocrinol[ Metab[\ 0873\ 47\ 081Ð085[ 21[ Schulte\ H[ M[\ Chrousos\ G[ P[\ Gold\ P[ W[\ Old_eld\ E[ H[\ Phillips\ J[ M[\ Munson\ P[ J[\ Cutler\ G[ B[ Jr[ and Loriaux\ D[ L[\ Metabolic clearance rate and plasma half!life of radioiodinated corticotropin releasing factor in a primate[ J[ Clin[ Endocrinol[ Metab[\ 0871\ 44\ 0912Ð0914[ 22[ Schurmeyer\ T[ H[\ Averginos\ P[ C[\ Gold\ P[ W[\ Gallucci\ W[ T[\ Tomai\ T[ P[\ Cutler\ G[ B[ Jr[\ Loriaux\ D[ L[ and Chrousos\ G[ P[\ Human corticotropin!releasing factor in man ] Pharmacokinetic properties and dose!response of plasma adrenocorticotropin and cortisol secretion[ J[ Clin[ Endocrinol[ Metab[\ 0873\ 48\ 0092Ð0097[ 23[ Stenzel\ P[\ Kesterson\ R[\ Yeung\ W[\ Cone\ R[ D[\ Rittenberg\ M[ B[ and Stenzel!Poore\ M[ P[\ Identi_cation of a novel murine receptor for corticotropin!releasing hormone expressed in the heart[ Mol[ Endocrinol[\ 0884\ 8\ 526Ð534[ 24[ Takahashi\ L[ K[\ Haglin\ C[ and Kalin\ N[ H[\ Prenatal stress potentiates stress!induced behavior and reduces the propensity to play in juvenile rats[ Physiol[ Behav[\ 0881\ 40\ 208Ð212[ 25[ Takeshita\ S[\ Studies on the in~uence of acute maternal stress on the fetal endocrine system in late gestation of rats[ Acta Obstet[ Gynaecol[ Japon\ 0889\ 31\ 0560Ð0566[ 26[ Ward\ I[ L[\ Prenatal stress feminizes and demasculinizes the behavior of males[ Science\ 0861\ 064\ 71Ð73[ 27[ Williams\ M[ T[\ Hennessy\ M[ B[ and Davis\ H[ N[\ CRF administered to pregnant rats alters o}spring behavior and morphology[ Pharmacol[ Biochem[ Behav[\ 0884\ 41\ 050Ð056[ 28[ Williams\ M[ T[\ Hennessy\ M[ B[ and Davis\ H[ N[\ Stress during pregnancy alters rat o}spring morphology and ultrasonic vocalizations[ Physiol[ Behav[\ 0887\ 52\ 226Ð232[ 39[ Zarrow\ M[ X[\ Philpott\ J[ E[ and Denenberg\ V[ H[\ Passage of 03C!3!corticosterone from the rat mother to the foetus and neonate[ Nature\ 0869\ 115\ 0947Ð0948[ 30[ Zuspan\ F[ P[\ Whaley\ W[ H[\ Nelson\ G[ H[ and Ahlquist\ R[ P[\ Placental transfer of epinephrine[ I[ Maternal!fetal metabolic alterations of glucose and nonesteri_ed fatty acids[ Am[ J[ Obstet[ Gynecol[\ 0855\ 84\ 173Ð178[
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 118Ð123\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII ] S9625Ð4637"87#99911Ð6
THE DISTRIBUTION OF RADIOLABELED CORTICOTROPIN! RELEASING FACTOR IN PREGNANT RATS ] AN INVESTIGATION OF PLACENTAL TRANSFER TO THE FETUSES& MICHAEL T[ WILLIAMS\$ HARRY N[ DAVIS\$ ANNE E[ MCCREA% and MICHAEL B[ HENNESSY$ $ Wright State University\ Dayton\ OH 34324 ^ % Sinclair Community College\ Dayton\ OH 34391\ U[S[A[ "Received 5 October 0886 ^ revised 2 January 0887 ^ accepted 3 January 0887# Abstract*Stress during gestation can have serious consequences on the development of the fetus[ Many of these e}ects appear to be mediated by hormones of the hypothalamic!pituitary!adrenal "HPA# axis[ Corticotropin!releasing factor "CRF#\ released by the hypothalamus during times of stress serves to activate release of pituitary hormones and is also present in low levels in rat plasma[ Moreover\ the uterus contains signi_cant quantities of CRF at implantation sites\ probably from local sources[ Therefore\ the possibility exists that CRF may cross the placenta and activate the fetal HPA axis[ However\ the ability of CRF to cross the placenta has not been demonstrated[ In the present study\ pregnant rats were administered radiolabeled CRF intraperitoneally\ and the distribution of the labeled product was determined in the fetuses and various maternal organs[ High levels of activity were observed in the pregnant female|s uterus\ adrenals\ heart and the placentae\ but only background levels of activity were detected in the maternal brain[ Very low levels of activity were observed in the fetuses\ indicating that the transfer of CRF across the placenta is greatly restricted[ These _ndings suggest that maternal CRF has little or no direct e}ect on the developing fetus during gestational stress[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] corticotropin!releasing factor\ blood!brain barrier\ prenatal stress\ placental transfer[
INTRODUCTION Stress during pregnancy\ or gestational stress\ can in~uence the anatomical\ behavioral\ and physio! logical development of the rat fetus[15\26\28 One of the more striking e}ects of gestational stress is on the sexual di}erentiation of the o}spring[ In general\ males appear and:or behave more like females00\24\26 and in some instances\ females appear and:or behave more like males[7\00\05 Probable mediators of these gestational stress e}ects are the hormones of the hypothalamic!pituitary!adrenal "HPA# axis ði[e[\ corticotropin!releasing factor "CRF#\ adrenocorticotropin "ACTH#\ b!endorphin\ corticosterone\ and possibly aldosteroneŁ\ which are released during times of stress[ Recently\ we found that peripheral administration of CRF to pregnant rats produced several e}ects that were similar to those observed following gestational stress\ speci_cally ] "0# reduced weight gain of the pregnant females ^ "1# reduced body weights of o}spring at birth ^ "2# enhanced emission of ultrasonic vocalizations on postnatal day 03^ and "3# shorter anogenital distances of the male o}spring[27\28 It is not known where CRF exerted its actions\ but several possibilities exist ] "0# CRF caused release of maternal hormones which directly a}ected o}spring development ^ "1# CRF caused release of maternal hormones which\ in turn\ activated release of hormones from the fetuses ^ "2# CRF altered the physiology of the pregnant female in other ways "e[g[\ constriction of blood vessels leading to hypoxia in the fetuses# which a}ected o}spring development ^ or "3# CRF had direct actions on fetal hormone release\ possibly through translocation of CRF across the placenta[ Any one of these possibilities alone or in combination might account for the e}ects of both administration of CRF and of stress during pregnancy[ Furthermore\ it is known that administration of either corticosterone\ ACTH\ or b!endorphin to pregnant rats\ can produce some consistent and inconsistent consequences\ compared to e}ects observed following gestational stress[2\4\8\09 Address correspondence to ] Michael B[ Hennessy\ Ph[D[\ Wright State University\ Department of Psychology\ 224 Fawcett Hall\ 2539 Col[ Glenn Hwy[\ Dayton\ OH 34324\ U[S[A[ Tel[ ] 990 826 664 1832 ^ fax ] 990 826 664 2236 ^ e!mail ] mhennessyÝdesire[wright[edu & This research was supported by grant IBN!8111966 from the National Science Foundation[ 118
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Many di}erent compounds are known to cross the placental barrier\ and do so with varying degrees of ease\0\3\01\02\07\08\17\18\39\30 whereas other compounds appear completely unable to cross[6\06 Very low circulating levels of CRF exist in rat plasma ^ these appear to be derived primarily from peripheral rather than hypothalamic sources[14 During human pregnancy\ CRF is produced by the placenta[16 However\ in the rat\ CRF was not detected in either the placenta or the amnion[29\25 Yet\ a recent study has shown that CRF is produced at implantation sites on the rat uterus[03 Although CRF is produced in close proximity to the fetal environment in the rat "i[e[\ the uterus#\ the ability of CRF to cross the placental barrier has not been reported[ The capability of other hormones of the HPA axis to cross the placental barrier has been studied[ Evidence exists that ACTH does not cross the placenta6\06 while corticosterone0\07\39 and b!endorphin do[18 Because the adrenal steroids are lypophilic\ it is possible that aldosterone may also cross the placenta\ and some data exist to suggest this may be the case\ at least from the fetal to the maternal compartment[3 To help clarify a possible site of action for CRF during gestational stress\ this study was designed to determine if CRF can cross the placental barrier in the rat[ The experiment was conducted during the third trimester "gestational day 06# which is when CRF administration was found to a}ect o}spring development27 and when gestational stress produces the clearest e}ects on sexual di}er! entiation and other parameters[7\00\05\24\26 Radiolabeled CRF was administered to pregnant females and levels of radioactivity were measured in the fetuses[ The placentae\ as well as the pregnant female|s brain\ pituitary\ adrenals\ kidneys\ and uterus\ were also examined because of known CRF receptors in these regions19\13\23 and for comparison with fetal levels of radioactivity[
EXPERIMENTAL PROCEDURES Animals and conditions Pregnant female Sprague!Dawley rats "shipped pregnant from Harlan\ Indianapolis\ IN# and their fetuses were used for this experiment[ All females were allowed free access to food and water prior to testing[ Each pregnant rat was injected IP with 4 mCi 014I!ðTyr9Ł!CRF "rat\ human ^ Peninsula Laboratories\ Inc[\ Belmont\ CA# in a volume of 9[4 ml distilled water on day 06 of gestation[ Equal numbers of females "n 4# were decapitated at intervals of 09\ 19\ or 29 min following the injection[ Tissue dissection and measurement of 014I!ðTyr9Ł!CRF Females were anesthetized by halothane inhalation and decapitated[ Immediately following decapitation\ the uterus was excised from the abdominal region[ The fetuses from each uterine horn were counted and placentae and fetuses were removed[ The amniotic sac was cleared from each fetus and the umbilical cord was snipped proximal to the fetus[ Simultaneous with the fetal extraction\ the whole brain "excluding olfactory bulbs# and the pituitary were removed from the pregnant female|s skullcase[ Each pregnant rat|s heart and adrenals were harvested after removal of the uterus[ All samples were placed\ immediately upon removal\ into an acidic solution "9[0 N HCL and 1 N Acetic acid in a 0 ] 0 concentration#[ All tissues were homogenized for 09 s and then centrifuged at 1399 rpm for 29 min[ Following centrifugation\ 099 ml of supernatant was removed and counted in a gamma counter for 0 min in no less than duplicate aliquots[ The activity of each sample was expressed in counts per min "cpm#:099 ml supernatant[ The speci_c activity of each tissue sample was calculated by subtracting the mean background level "43[4 cpm# from the total activity[ Statistics The weights of the pregnant females\ the total number of fetuses\ and the number of fetuses in each uterine horn were subjected to one!way analyses of variance "ANOVA#[ For the tissue samples\ one!way ANOVAs were used to determine if there were any changes in the speci_c activity over time[ StudentÐNewmanÐKeuls was used for post!hoc analysis of any signi_cant main e}ect of Time Point[ Any sample that did not show a signi_cant e}ect was collapsed across time points for subsequent analysis[ Dependent Student|s t!tests were performed to compare speci_c activity to background[
Distribution of 014I!ðTyr9Ł!CRF
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RESULTS ANOVAs for the weights of the pregnant females\ total number of pups in a litter\ and number of pups in the left and right uterine horns yielded no signi_cant e}ects[ Inspection of Table 0 shows that the highest levels of speci_c activity were observed in the uterus[ The levels of speci_c activity in the placentae were much lower than that found in the uterus\ especially during the _rst two time periods[ The speci_c activity levels in the fetuses were very low during the _rst 19 min "i[e[\ less than 04 cpm over background# and increased at 29 min\ although they were less than a seventh of that in the placentae\ and a fortieth of that found in the uterus at this _nal time point[ For placentae\ ANOVA revealed a signi_cant e}ect of Time Point F"1\01# 3[18\ P ³ 9[94[ Increased levels of radioactivity were observed at 29 min relative to 09 or 19 min[ The e}ect of Time Point was not signi_cant for the other samples\ although that for fetuses approached signi_cance\ P ³ 9[95[ Dependent t!tests revealed that the fetuses\ uterus\ heart\ adrenals\ and placentae "at all time points# exhibited activity above background levels[ No di}erence from background was found for brain and pituitary "Table 0#[ Because no di}erence between background activity and brain activity was found\ another set of dependent t!tests was performed using the brain as a negative tissue control[ All tissues\ with the exception of pituitary\ had higher levels of activity than did the brain[ DISCUSSION This study described the distribution of 014I!ðTyr9Ł!CRF in pregnant female rats and their fetuses[ The capacity of CRF to cross the blood!brain barrier has been examined in rats1 and mice[04 These researchers did not _nd CRF to cross from blood to brain\ but did _nd CRF to cross from brain to blood\ at least for mice[ In the present study\ the total cpm for the brains of the pregnant females were not di}erent from background levels\ supporting Banks and Kastin|s04 _nding that CRF does not enter the brain from the periphery in the rat[ Although CRF is produced in the human placenta during pregnancy\16 there is no evidence for placental expression of CRF during either stress or nonstress periods in rats[29\25 However\ a recent study found that CRF is expressed at the implantation sites on the rat uterus[03 These researchers hypothesized that CRF may serve to mediate the in~ammation of the uterus observed during the implantation of rat blastocysts[ In the present study\ the uterus had the highest levels of radioactivity of the tissues sampled[ This suggests that a high density of CRF binding sites is present in the uterus during the later stages of rat pregnancy[ Because these levels were found during the third trimester\ activation of CRF binding sites in the uterus may serve functions unrelated to implantation[ Moreover\ because CRF can modify blood ~ow\12 the activation of CRF receptors in the uterus or placenta during times of stress could conceivably restrict or alter blood ~ow and therefore produce hypoxia in the fetuses[ Hypoxia has been suggested to underlie some of the e}ects of gestational stress[11 Levels of radioactivity in the fetuses were just a fraction of the levels observed in the placentae and uterus[ When the radioactivity of the fetuses was statistically analyzed\ the di}erence over time approached signi_cance and the total radioactivity was greater than background and brain\ suggesting that some CRF crossed the placental barrier[ However\ inspection of Table 0 shows that the speci_c activities at 09 and 19 min are at least 2 times lower than the speci_c activity at 29 min[ In humans\ a bi!exponential clearance rate for CRF has been found with a fast and a slow half! life[10\20\22 The fast half!life of CRF was reported to be in the range of 3[0 minÐ00[5 min[ Similar _ndings have been found in cynomolgus monkeys with a fast half!life of approximately 06[0 min[21 If the half!life of CRF in rat plasma is the near the range for humans and cynomolgus monkeys\ then the background levels of radioactivity in the fetuses shortly following injection\ with increasing levels thereafter\ could indicate that detected radioactivity in the fetuses was due to a metabolite of CRF crossing the placenta[ Since the heart\ adrenals\ placentae\ and uterus were radiolabeled with CRF within the _rst 09 min and showed fairly constant levels throughout 29 min\ it would further suggest that the radioactivity observed in the fetuses at 29 min was the result of metabolized parent compound\ 014I!ðTyr9Ł!CRF[ Clearly\ the passage of CRF through the placenta is greatly restricted ^ it appears that little\ and
121
Table 0[ Distribution of
014
I!CRF ] total counts per min "CPM# and speci_c activity "minus background$# per 099 ml supernatant\ observed for maternal brain\ heart\ adrenals\ pituitary\ and uterus\ and the placentae and all fetuses from both uterine horns at 09\ 19\ or 29 min following injection 09 min
29 min
Speci_c CPM
Total CPM
Speci_c CPM
Total CPM
Speci_c CPM
36[17200[38 193[41210[45 0074[392155[95 50[20200[52 1037[992336[02 120[09211[58 59[87200[38
−6[11200[38 049[91210[45 0029[892155[95 5[70200[52 1982[492336[02 065[59211[58 5[37200[38
49[2828[87 150[18222[23 0951[092012[44 52[49201[67 1467[212328[02 122[93215[39 54[29202[24
−3[0028[87 195[68222[23 0996[592012[44 8[99201[67 1412[712328[02 067[43215[39 09[79202[24
44[65200[94 239[79245[69 640[412063[17 60[57205[31 1145[522333[32 347[612092[66 098[22205[71
0[15200[94 175[29245[69 586[912063[17 06[07205[31 1191[022333[32 393[112092[66 43[72205[71
$ The mean background was 43[4 cpm[ The asterisk"s# next to the tissue sample represents the level of signi_cance for all time points combined[ For placentae\ there was a signi_cant e}ect of Time Point ^ therefore\ tests for increased activity were carried out separately for each time point[ P ³ 9[94[ P ³ 9[90[ P ³ 9[990[
M[ T[ Williams et al[
Brain Heart Adrenals Pituitary Uteri Placentae Fetuses
19 min
Total CPM
Distribution of 014I!ðTyr9Ł!CRF
122
possibly no\ CRF crosses to a}ect the fetuses[ CRF is a 30 amino acid peptide[ b!Endorphin is a 20 amino acid peptide\ and appears to cross through the placental barrier[18 ACTH\ a 28 amino acid peptide\ is unable to cross the placental barrier[6\06 Therefore\ CRF may not be able to cross the placenta because of its molecular size[ CRF receptors are located in a variety of tissues in the periphery\ such as the pituitary\ heart\ adrenal\ gonads\ liver\ spleen\ and kidney[19\13\23 A seemingly paradoxical e}ect was found in this study[ The pituitary\ which is outside of the blood!brain barrier\ did not have levels of activity di}erent than background[ The likely explanation is that the injection procedure to administer the 014 I!ðTyr9Ł!CRF resulted in a stress response which released endogenous CRF from the hypo! thalamus[ Receptors for CRF in the pituitary have been found to be saturable and to have a high a.nity for CRF[5 Therefore\ endogenous CRF may have saturated the receptor sites and no radioactivity was detected[ The present _ndings indicate that the e}ects of CRF administration to pregnant rats in our earlier study27 do not appear to have been the result of a direct in~uence of exogenous CRF on the fetuses[ Rather\ they appear to have been due to an in~uence of either other maternal hormones secreted in response to CRF that then crossed the placenta\ or another CRF!induced change in maternal physiology "e[g[\ altered uterine blood ~ow leading to fetal hypoxia#[ More generally\ similar e}ects of gestational stress may be mediated by these mechanisms[ REFERENCES 0[ Arishima\ K[\ Nakama\ S[\ Morikawa\ Y[\ Hashimoto\ Y[\ Eguchi\ Y[\ Changes in placental permeability to cortico! 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