Author Institution: Department of Biological Sciences, Bowling Green State UniversityWe examined the influence of thiouraeil-induecd hypothyroidism imposed at various stages of development upon pituitary growth hormone (OH) concentration. Balb/e-derived mice were mated and fed thionracil, 0.25% in Purina Lab Chow, from the first day of pregnancy until 25 days postpartum, or for shorter periods of time (last % of pregnancy and postpartum, last j/$ of pregnancy and postpartum, or postpartum only). Using disc gel eleetrophoresis, pituitary Gil concentration of 25 day old pups exposed to thiouracil was compared with that of unexposed animals. Concentration of pituitary GH was depressed significantly in animals whose mothers were first given thiouracil at the initiation of pregnancy or \% of the way through pregnancy. Young of mothers first given thiouracil % of the way through gestation or on the first day postpartum did not have significantly lower pituitary GH concentrations than controls. The factor determining the presence or absence of thiouracil-induced depression of pituitary GH concentration may be the initiation of fetal thyroid function that occurs between days 15-17, approximately % of the way through gestation

Meserve, Lee A.

Rhodes, Audrey B.

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Copyright © 1979 Ohio Acad. Sci. ()()30-0950/79/0006-02()7/$l.f)0/0INFLUENCE OF PRE- AND POSTNATAL THIOURACILADMINISTRATION ON PITUITARY GROWTHHORMONE CONTENT IN MICE1LEE A. MESERVE and AUDREY B. RHODES, Department of Biological Sciences, BowlingGreen State University, Bowling Green, OH 43403Abstract. We examined the influence of thiouraeil-induecd hypothyroidism imposedat various stages of development upon pituitary growth hormone (OH) concentration.Balb/e-derived mice were mated and fed thionracil, 0.25% in Purina Lab Chow, fromthe first day of pregnancy until 25 days postpartum, or for shorter periods of time(last % of pregnancy and postpartum, last j/$ of pregnancy and postpartum, or post-partum only). Using disc gel eleetrophoresis, pituitary Gil concentration of 25 dayold pups exposed to thiouracil was compared with that of unexposed animals. Con-centration of pituitary GH was depressed significantly in animals whose mothers werefirst given thiouracil at the initiation of pregnancy or \% of the way through preg-nancy. Young of mothers first given thiouracil % of the way through gestation or onthe first day postpartum did not have significantly lower pituitary GH concentrationsthan controls. The factor determining the presence or absence of thiouracil-induceddepression of pituitary GH concentration may be the initiation of fetal thyroid func-tion that occurs between days 15-17, approximately % of the way through gestation.OHIO J. SCI. 79(6): 267, 1979Hypothyroidism induced around thetime of birth has long been known to re-tard skeletal development and growth inrats (Salmon 1938). Mice born of euthy-roid mothers were reported to grow fasterthan those of thyroidectomized dams(Davenport and Swingle 1927). More re-cent studies have suggested that growthis depressed because of reduced synthesisor secretion of growth hormone (GH) in-duced by the hypothyroid condition.For example, Peake et al (1973), usingradioimmunoassay, and Wilkins et al(1974) using disc gel column eleetro-phoresis have shown altered pituitaryGH concentration in pituitaries of ratsmade hypothyroid as young adults.Furthermore, pituitaries of rats thyroi-dectomized at birth are virtually devoidof GH at 25 days of age (Geel andTimiras 1970).The length of time necessary for hypo-thyroidism to alter GH status has notbeen extensively studied. Hamburgh(196S) suggested that withholding thy-1Manuscript received 16 August 1978 and inrevised form 19 February 1979 (#78-47).roid hormone from developing rats inutero does not appreciably alter matura-tion of the nervous and skeletal systems.Our study was carried out to determinethe importance of thyroid status duringdevelopment as a factor in establishingpostnatal pituitary growth hormone con-centration. Hypothyroidism was in-duced by exposing mice to thiouracil, adrug that prevents thyroid hormoneformation, during a portion of gestationand postnatally.MATERIALS AND METHODSFemale Balb/c-derived mice were mated tomales of the same strain. First day of preg-nancy was determined by presence of a vaginalplug. Pregnant females were isolated and as-signed to one of the following feeding schedules(see fig. 1):(A) Thiouracil diet from the first day ofpregnancy to 25 days postpartum (46days of thiouracil exposure).(B) Control diet during the first ]/A of preg-nancy, and thiouracil diet during thelast % of pregnancy to 25 days post-partum (39 days).(C) Control diet for the first % of preg-nancy, and thiouracil diet during thelast l/i of pregnancy to 25 days post-partum (32 days).267268 L. A. MESERVE AND A. B. RHODES Vol. 79(D) Control diet during pregnancy andthiouracil diet from day 1 to day 25postpartum (25 days).(E) Control diet from the first day of preg-nancy to 25 days postpartum (0 days).MatedThiouracil Feeding ScheduleBirth21 days 25 daysPituitaryremovalR°UFIGURE 1. Digramatic representation offeeding schedule. Bar represents time ofthiouracil incorporation in maternal diet.Groups correspond to those in table 1: A =46 days; B=39 days; C = 32 days; D = 25days; E = 0 days.The control diet consisted of Purina Lab Chowad libitum, and the test diet had 0.25% thioura-cil added to the ground Lab Chow. Each groupconsisted of 5 litters except A and C, which had4 and 6 respectively.At birth, litters were standardized to 5 pups.At 25 days of age, all pups were killed by de-capitation and body weights were determined.Thyroid and pituitary glands were rapidly ex-cised, and the weights of individual thyroidsand pooled pituitaries (5 glands per pool) wereobtained to the nearest 0.1 mg. Pooled pitui-taries were stored frozen at — 20 °C for less than30 days after which they were homogenized in40% sucrose at a concentration of 4 mg pituitarytissue per ml.Pituitary GH was separated from other pro-teins in the homogenate by the polyacrylamidedisc electrophoresis methods developed byDavis (1964) and Ornstein (1964) as modifiedby Lewis et al (1965). Electrophoresis wasdone at room temperature using a 7% acryla-mide separating gel (pH 9.5). Duplicate ortriplicate analyses were made of each ho-mogenate. After electrophoresis, gels werestained with aniline blue black (1% in 7%, aceticacid) and were destained by diffusion in 7%acetic acid.^ Pituitary GH was quantified bydensitomctric comparison of gels containingGH from pituitary homogenates to those con-taining standard rat GH (NLAMDD RatGH-3) supplied by Dr. A. F. Parlow as agentfor the NIAMDD Rat Pituitary Hormone Dis-tribution Program.Differences in group means for body and organweights and growth hormone concentrationswere determined using two-way analysis ofvariance, and Scheffe's method of multiplecomparisons was used to test for significant dif-ferences between litters within treatmentgroups (Scheffe 1959).RESULTSMultiple comparisons showed no dif-ferences between litters within treatmentgroups for any of the parameters studied.An analysis of variance, however, demon-strated a significant effect of thiouracilexposure on body and organ weights.Body weights indicated a depression ofgrowth in all thiouracil-exposed pups.This influence was most pronounced inyoung exposed to thiouracil after birthonly (25 days, table 1), but subnormalmaternal food intake may have been aconfounding unmeasured variable in thisgroup. Thyroid weight gain by allthiouracil-exposed pups (overall mean10.9 mg vs. 2.2 mg for controls) demon-strated the influence of the drug as aninducer of thyroid hypertrophy throughinhibition of hormone secretion. Pooledpituitary weights showed the influence ofinduced hypothyroidism with a lineardepression in gland mass from pups ex-posed to thiouracil for 0 days (3.6 mg)to those exposed for 46 days (2.4 mg)(table 1).Growth hormone concentration of thepituitary gland was also affected bythiouracil, showing a significant lineardepression from 25 days (28.1 Mg/mgpituitary tissue) through 46 days (11.5Mg/mg) of exposure (P<{).()b). No sta-tistically significant difference existed,however, between GH concentration ofpups exposed to thiouracil for 39 or 46days and those exposed for (), 25, or 32days (table 1). Indeed, when analyzedas single treatment periods, pups frommothers fed thiouracil before the last ^of pregnancy had a significant!}- de-pressed pituitary GH concentration (39and 46 day groups; 14.3 /xg/mg) whencompared with offspring of mothers giventhiouracil from the last }4> °f pregnancyor later (25 and 32 day groups; 26.5jig/mg). The latter animals did not dif-fer significantly from controls (0 daygroup; 30.3 ng/mg).DISCUSSIONPublished values for pituitary GHconcentration in untreated animals varysomewhat with species, strain, age, sex,and method of measurement. For ex-ample, Geel and Timiras (1970) re-ported a concentration of 17.7 ^g GH/mgpituitary tissue in 25 day old rats as as-sayed by disc gel electrophoresis, whereasOhio |. Sci. THIOURACIL AND PITUITARY GHTABLE 1Body and organ weights and pituitary growth hormone concentrationsof control and thiouracil-exposed 25 day old mice.ThiouracilExposure(days)02.1;>2: ; < >4(>BodyWeight(g)1.">.9*±0.4(2.-,)±0.;")(2.1)11.0**±0.3(30)13.2**±o.o(2r,)10.8**±0.1(20)ThyroidWeight(mg)2 . 2±0.1(2,->)8.5**±0.8(2-,)13.8**±1.2(30)10.(>**± 1 .0(2,->)9.8**±0.8(20)PituitaryWeight***(mg)3. o±0.2(•">)3 . 4±0.2(•'))3.0**±0.1(«)2.(i**±0.4(•"))2.4**±0.1(4)GH Cone.(Mg/mg tissue)30.3±2.1(ID28.1±1.5(13)24.8±2.4(12)l <>.:>**± 1 . 7( 1 4 )l l . T ) * *± 2 . 200*Mean± Standard Error.Number in parentheses indicate mice in each group.**Significantly different from 0 days (/J<0.0o).•••Pituitary weights are based on pooled samples.Walker et al (1977), using radioimmuno-assay, found about 100 ng GH/^ug pitu-itary protein in rats the same age. Thisvalue converts to approximately 12.6 MgGH/mg pituitary tissue if the pituitaryis considered to be about 12.6% proteinas reported by Treloar and Leathern(1969) in 50 day old rats. Slightly olderanimals appear to have higher pituitaryGH concentrations as measured by radio-immunoasay. Mosier et al (1977) showed150 idg GH/mg in 56 day old rats andSinha et al (1977) reported levels of60 jug/mg in female and 120 /xg/mg inmale mice 76-96 days old. The controlGH concentration found by disc gelelectrophoresis in the pituitaries of 25day old mice in the present study (30.3Mg/mg) was slightly greater than thatreported for 25 day old rats. The dif-ference observed may be the result ofusing rat GH as a standard to quantifymouse GH, or perhaps the mouse actuallyhas a slightly greater pituitary GH con-centration than the rat.Thiouracil belongs to the class of com-pounds that induce hypothyroidism bypreventing thyroid hormone synthesis(Astwood et al 1943, MacKenzie andMacKenzie 1943). Our data indicatethat the length of time mice are exposedto this drug in utero is important in de-termining pituitary GH concentration at25 days of age. One explanation for thepresence of an influence on pituitary GHin mice first exposed to thiouracil beforethe last 3^3 of gestation and the absenceof effect in mice first exposed later in de-velopment can be based on the time ofonset of fetal thyroid function. Func-tional activity of the fetal mouse thyroidgland develops between days 15 and 17of gestation (van Heyningen 1961).Since mothers of pups exposed to thioura-cil during the last }/§ of gestation firstreceive thiouracil in their diet on the 15thday of pregnancy, the fetal thyroid glandprobably has developed some minimalfunctional capability before the effect ofthiouracil becomes pronounced. There-fore, pituitary GH content may be lessdrastically altered than it would be inpups whose mothers have been fedthiouracil from day 1 or day 8 of preg-nancy. Since thiouracil can pass throughthe placenta (Hayashi and Gilling 1967),administration of the drug early in de-velopment could result in its presence inthe fetus well before the onset of thyroid270 L. A. MESERVE AND A. B. RHODES Vol. 79function, bringing about significant al-teration of pituitary GH concentration.The present study does not corroboratethe finding of Hamburgh (1968) thatprenatal thyroid restriction does notsignificantly affect the developmentalprocesses of young rodents. It may bepossible to explain the discrepancy be-tween our results and those of Hamburghby consideration of procedural differencesor differences in experimental animals.Hamburgh used the drug propylthiouracilto induce hypothyroidism but did notfeed it until day 15 of gestation, hypo-thesizing that it was unimportant tobegin treatment before the onset of fetalthyroid function. Our study shows thatit is necessary to give the drug earlier toinfluence pituitary GH concentration.In addition, physiological differences be-tween rats and mice that may result indissimilar responses to thiouracil ex-posure are facts that cannot be ignored.It is important to note that the de-pressed pituitary GH level in thiouracil-exposed mice does not warrant conclu-sions regarding its synthesis or secretion.As mentioned by Kraicer et al (1977),hormone content of a gland is the resultof dynamic hormone synthesis, release,activation, and inactivation. An altera-tion in one component brought about byexperimental manipulation (e.g., thioura-cil exposure) indicates an altered balanceamong these 4 factors. The importanceof this alteration is exemplified by theabsence of a direct correlation betweenbody weight and pituitary GH concen-tration in the present study. Lack ofcorrelation is presumably the result ofunmeasured metabolic alterations presentin thiouracil-exposed mice. For example,in the neonatal rat Stuart et al (1976)found a paradoxical relationship betweencirculating levels of GH and somato-medin, a peptide that mediates GH ac-tion on skeletal tissues. GH levels inplasma were high, but somatomedinlevels were low during a period of rapidgrowth. These investigators hypothe-sized the importance of insulin, abundantin the circulation of neonatal rats, as agrowth factor in early life. Neither in-sulin nor somatomedin was measured inthe present study. As mentioned earlier,the pups exposed to thiouracil duringlactation only (25 days) may have hadtheir growth stunted by depressed ma-ternal food intake that probably occurredwhen thiouracil was first encountered atparturition. Besides the possible thioura-cil-induced metabolic alterations, thereappears to be an absence of direct corre-lation between pituitary GH content andgrowth in some species examined undernormal physiological conditions. Borerand Kelch (1978) have reported this tobe the case in the hamster, but this speciesis well known for endocrine responsesunlike those of other experimental ro-dents. Our present study shows that inmice exposed to thiouracil before thelast 3^3 of gestation, pituitary GH concen-trations were depressed whereas in thosefirst exposed to thiouracil during the last3^3 of gestation or later, the GH contentwas not significantly altered. The impor-tance of this finding in the overall meta-bolic scheme remains to be determined.Acknowledgments. The authors thank Dr.A. F. Parlow and the NIAMDD for the gift ofrat GH. J. Dailey, P. Froelich, D. Schulte,and Drs. P. Dailey and R. St. John are acknowl-edged for their assistance with the manuscript.Our study was supported in part by a BowlingGreen State University FRC grant.LITERATURE CITEDAstwood, E. B., J. Sullivan, A. Brissell and R.Tyslowitz 1943 Action of certain sulfona-mides and of thiourea upon the function of thethyroid gland of the rat. Endocrinology 32:210—225Borer, K.' T. and R. P. Kelch 1978 A heter-ologous radioimmunoassay method for mea-surement of hamster growth hormone.Neuroendocrinologv 25: 65-76.Davenport, C. B. and W. W. Swingle 1927Effects of operation upon the thyroid glandsof female mice on the growth of their off-spring. J. Exp. Zool. 48: 395-440.Davis, B. J. 1964 Disc electrophoresis. II.Method and application of human serumproteins. Ann. N. Y. Acad. Sci. 121: 404-427.Geel, S. E. and P. S. Timiras 1970 Influenceof growth hormone on cerebral cortical RNAmetabolism in immature hypothyroid rats.Brain Res. 22: 63-72.Hamburgh, M. 1968 An analysis of the ac-tion of thyroid hormone on developmentbased on in vivo and in vitro studies. Gen.Comp. Endocrinol. 10: 198-213.Hayashi, T .T . andB.Gilling 1967 Placentaltransfer of thiouracil. Obstet. Gynccol. 30:736-740.Kraicer, J., G. Beraud and D. W. Lywood1977 Pars intermedia ACTH and MSH con-tent: effect of adrenalectomy, gonadectomyand a ncurotropic (noise) stress. Ncuro-endocrinology 23: 3.12-367.Ohio J. Sci. THIOURACIL AND PITUITARY GH 271Lewis, U. J., E. V. Chcever and W. P. Vandcr-laan 19(35 Studies on the growth hormoneof normal and dwarf mice. Endocrinology76: 210-215.MacKenzic, C. G. and J. B. MacKenzic 1943Effect of sulfonamides and thiourea on thethyroid gland and basal metabolism. Endo-crinology 32: 185-209.Mosier, H. D., Jr., L. C. Dearden, R. A. Jansonsand R. R. Hill 1977 Growth hormone,somatomedin and cartilage sulfation in fail-ure of catch-up growth after propylthiouracil-induced hypothyroidism in the rat. Endo-crinology 100: 1644-1651.Ornstein, L. 1964 Disc clectrophoreis. 1.Background and theory. Ann. N. Y. Acad.Sci. 121:321-349.Peakc, G. T., C. A. Birge and W. H. Daughaday1973 Alterations of radioimmunoassayablcgrowth hormone and prolactin during hypo-thryoidism. Endocrinology 92: 487-493.Salmon, N. T. 1938 The effect on the growthrate of thyro-parathyroidectomy in newbornrats and of subsequent administration ofthyroid, parathyroid and anterior hypophy-sis. Endocrinology 23: 446-457.Scheffe, H. 1959 The Analysis of Variance.John Wiley and Sons, New York. 477 pp.Sinha, Y. N., C. B. Salocks, M. A. Wickes andW. P. Vanderlaan 1977 Scrum and pituitaryconcentrations of prolactin and growthhormone in mice during a twenty-four hourperiod. Endocrinology 100: 786-791.Stuart, M. C , L. Lazarus, S. S. Moore and G.A. Smythc 1976 Somatomedin productionin the neonatal rat. Hormone Me tab. Res.8: 442-445.Treloar, O. L. and J. H. Leathern 1969 Hy-pophyseal nucleic acids: castration and thio-uracil. Proc. Soc. Exp. Biol. Med. 132:968-970.van Henyngcn, H. E. 1961 The initiation ofthyroid function in the mouse. Endocrinol-ogy 69: 720-727.Walker, P. J., H. Dussault, G. Alvarado-Urbina and A. Dupont 1977 The develop-ment of the hypothalamo-pituitary axis inthe neonatal rat: hypothalamic somato-statin and pituitary and serum growth hor-mone concentrations. Endocrinology 101:782-787.Wilkins, J. N., S. E. Mayer and P. Vanderlaan1974 Effects of hypothyroidism and 2,4dinitrophenol on growth hormone synthesis.Endocrinology 95: 1259-1267.Copyright © 1979 Ohio Acad. Sci. 0030-0950/79/0006-0271/11.00/0BRIEF NOTEELEMENTAL ANALYSIS OF BIOLOGICAL MATERIAL INTHE FRESH-FROZEN STATE1WAYNE B. PARRISH, Department of ZoologyDONALD G. BRONN, JAMES R. WRIGHT JR. and JOHN P. MINTON, Department of Sur-gery, The Ohio State University, Columbus, OH 43210OHIO J. SCI. 79(6): 271, 1979The acceptance of x-ray probe analysisof biological materials depends upon themethods used for the preparation of speci-mens. Fixation and embedding create aproblem since many embedding mediacontain high levels of electrolytes (Mizu-hira 1976). Other fixatives such as glu-taraldehyde urea resin, which do not con-tain electrolytes, contribute to the loss oflabile elements. In an effort to preventthis loss during the dehydration, embed-ding, and sectioning process, additivessuch as oxalates have been incorporatedinto the fixative (Poddsky et al 1970),but Coleman (1976) has reported that1Note received 28 July 1978 and in revisedform 4 January 1979 (#78-42).this method causes redistribution of ele-ments into the intercellular spaces. Inorder to more accurately detect the ele-mental distribution in biological materi-als by x-ray probe analysis, a number ofmethods for tissue preparation have beenexplored. Critical point drying offerssuperior preservation of surface structureand morphology for electron microscopy,but this technique promotes the redis-tribution of the normal intracellular ele-ments and therefore is not useful forx-ray probe studies (Warner and Cole-man 1975). Cryogenic techniques havebeen utilized for the preparation of speci-mens for x-ray probe analysis. Rapidfreezing not only preserves morphologicalstructure but also the normal distribution

Influence of Pre- and Postnatal Thiouracil Administration on Pituitary Growth Hormone Content in Mice

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Influence of Pre- and Postnatal Thiouracil Administration on Pituitary Growth Hormone Content in Mice

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