{"paper_id":"fa7dc3f3-c9e1-4a13-bff5-10cd45c79a39","body_text":"Effects of Ovariectomy and Prolactin on Mammary Gland\nExpressions of Transforming Growth Factor α and\nEpidermal Growth Factor Receptor mRNAs in Mice\nAuthors: Nagasawa, Hiroshi, Ikezaki, Yoshie, and Yamamoto,\nKazutoshi\nSource: Zoological Science, 15(4) : 525-529\nPublished By: Zoological Society of Japan\nURL: https://doi.org/10.2108/0289-\n0003(1998)15[525:EOOAPO]2.0.CO;2\nThe BioOne Digital Library (https://bioone.org/) provides worldwide distribution for more than 580 journals\nand eBooks from BioOne’s community of over 150 nonprofit societies, research institutions, and university\npresses in the biological, ecological, and environmental sciences. The BioOne Digital Library encompasses\nthe flagship aggregation BioOne Complete (https://bioone.org/subscribe), the BioOne Complete Archive\n(https://bioone.org/archive), and the BioOne eBooks program offerings ESA eBook Collection\n(https://bioone.org/esa-ebooks) and CSIRO Publishing BioSelect Collection (https://bioone.org/csiro-\nebooks).\nYour use of this PDF, the BioOne Digital Library, and all posted and associated content indicates your\nacceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use.\nUsage of BioOne Digital Library content is strictly limited to personal, educational, and non-commmercial\nuse. Commercial inquiries or rights and permissions requests should be directed to the individual publisher\nas copyright holder.\nBioOne is an innovative nonprofit that sees sustainable scholarly publishing as an inherently collaborative enterprise\nconnecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common\ngoal of maximizing access to critical research.\nDownloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026\nTerms of Use: https://bioone.org/terms-of-use\n\nZOOLOGICAL SCIENCE 15: 525–529 (1998) © 1998 Zoological Society of Japan\n* Corresponding author: Tel. +81-44-934-7073;\nFAX. +81-44-934-7073.\nEffects of Ovariectomy and Prolactin on Mammary Gland\nExpressions of Transforming Growth Factor ααααα and\nEpidermal Growth Factor Receptor mRNAs in Mice\nHiroshi Nagasawa1*, Yoshie Ikezaki1 and Kazutoshi Yamamoto2\n1Experimental Animal Research Laboratory, Meiji University,\nTama-ku, Kawasaki 214-8571, Japan\n2Department of Biology, School of Education, Waseda University,\nShinjuku-ku, Tokyo 169-8050, Japan\nABSTRACT—Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice\nat 3 months of age received subcutaneous injections of danazol (0.5 µg / 0.1 ml olive oil, once a day),\nperphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice\na day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased\nby danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression\nof both transforming growth factor α (TGFα) mRNA and epidermal growth factor receptor (EGFR) mRNA in\nthe mammary gland was increased by danazol. However, TGF α mRNA expression was decreased by\nperphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings\nsuggest that the manifestation of the effect of TGFα on mammary gland is rather suppressed by PRL, while\nmammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGF α on the\nmammary gland growth. OVX resulted in a significant decrease of TGFα mRNA expression in the mammary\ngland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was\nobserved in ICR mice; however, the response to hormonal modulation is generally less susceptible than\nSHN mice.\nINTRODUCTION\nBoth estrogen and prolactin (PRL) are key hormones for\nthe normal, preneoplastic and neoplastic growth of mammary\nglands (Nagasawa \net al., 1986), and these hormones have\nbeen proposed to stimulate cell growth indirectly through an\nautocrine or paracrine mechanism by enhancing the produc-\ntion of peptide factors, as well as by direct effects. Transform-\ning growth factor α (TGFα) participates in the growth of nor-\nmal and neoplastic mammary glands (Perroteau \net al., 1986;\nDerynck et al., 1987; Liu et al., 1987; Zajchowski et al., 1988;\nValverius et al., 1989; Matsui et al., 1990; Halter et al., 1992;\nMizuno et al ., 1994), acting through binding to epidermal\ngrowth factor receptor (EGFR) (Todaro et al., 1980). How-\never, the information of the participation of estrogen and/or\nPRL on this process is rather sporadically. TGFα is suggested\nto be modulated by estrogen (Dickson \net al., 1986; Arteaga et\nal., 1988; Bates et al., 1988; Manni et al., 1991). Prusheik et\nal. (1997) suggested that PRL inhibited EGFR signaling.\nIn the present study, the effects of OVX and of the modu-\nlation of circulating PRL on expressions of TGF α and EGFR\nmRNAs in the mammary gland were examined in virgin mice.\nPerphenazine (Singtripop et al., 1991) and danazol (Singtripop\net al., 1992) were used for the stimulant and the inhibitor of\npituitary PRL release, respectively. The effect of ovine pro-\nlactin (oPRL) in this process was also examined.\nMATERIALS AND METHODS\nAnimals and treatments\nThe mice used were SHN/Mei virgin mice maintained in our labo-\nratory by strict brother x sister mating (Nagasawa et al., 1976) and\nJcl: ICR virgin mice purchased from CLEA Japan (Tokyo, Japan). At\n3 months of age, mice of each strain were divided into five groups.\nFour groups were bilaterally ovariectomized via a dorsal approach\nunder anesthesia by pentobarbital sodium (Nembutal; Abbott Labo-\nratories, North Chicago, IL, USA) and the remaining one group un-\nderwent a sham operation and served as the intact control. Begin-\nning 15 days after the operation, the OVX group received subcutane-\nous injections of saline (Otsuka Seiyaku, Tokyo, Japan; 0.1 ml, twice\na day) or olive oil (Kozakai Seiyaku, Tokyo, Japan; 0.1 ml, once a\nday), the data of which were pooled in the Results section, since they\ndiffered little (OVX control); danazol (Sigma Chem. Co., St. Louis,\nMO, USA; 0.5 µg / 0.1 ml olive oil, once a day), perphenazine (Sigma;\n0.05 mg / 0.1 ml saline, twice a day) or ovine PRL (AFP-10677C,\nNIDDK; 0.25 mg / 0.05 ml buffer, twice a day) for 3 days and on the\nmorning of day 4. The mice undergone the sham operation were\ntreated as was in the OVX control. All mice were killed one hour after\nDownloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026\nTerms of Use: https://bioone.org/terms-of-use\n\nH. Nagasawa et al.526\nthe last injection under light ether anesthesia and blood was collected\nfrom the trunk.\nAll procedures were carried out according to the USA NIH Guide\nfor the Care and Use of Laboratory Animals.\nThroughout the experiment, mice were kept in plastic cages (18\n× 30 × 13 cm) with wood shavings, maintained in an animal room,\nwhich was air-conditioned (20-22 °C and 60-70% relative humidity),\nartificially illuminated (14 hr of light from 5:00 AM to 7:00 PM) and\nventilated (16 times / hr) and provided with commercial pellets (Lab\nMR-Breeder; Nihon Nosan Kogyo, Yokohama, Japan) and tap water\nad libitum.\nSerum PRL level\nBlood collected was left at room temperature for 6 hr, kept in the\nrefrigerator overnight and centrifuged at 1,000 × g for 20 min at 4°C.\nSerum was stored at –20 °C. The PRL level was determined by a\nhomologous radioimmunoassay.\nEnd-bud formation in the mammary glands\nAt autopsy, unilateral third thoracic mammary glands were pre-\npared for the wholemount evaluation and were examined under 10-\nfold magnification. The degree of end-bud formation was rated from 1\nto 7 in increments of 1 (Nagasawa et al., 1980).\nNorthern blot analysis\nAt autopsy, bilateral inguinal and unilateral third thoracic mam-\nmary glands were immediately removed, pooled and stored at –70°C.\nRNA was extracted from 200-250 mg of frozen tissue by the acid\nguanidinium thiocyanate-phenol-chloroform extraction method\n(Chomczynski and Sacchi, 1987). The RNA concentration was deter-\nmined at 260 nm by spectrophotometer. Each 20 µg of RNA from\neach sample was applied to the nylon membrane (Immobilon\nTM;\nMillipore, Bedford, MA, USA) with a Bio-dot manifold (Bio-rad, Her-\ncules, CA, USA) and then the RNA was fixed to membrane using UV\ncrosslinking. The transforming growth factor α (TGFα) and epidermal\ngrowth factor receptor (EGFR) complementary DNA (cDNA)\nfluolescein-labelled probes were labelled using the Gene Images ran-\ndom prime labelling module kit (Amersham, Buckinghamshire, En-\ngland). Northern blot analysis was performed by hybridization with\nthis fluolescein-labelled cDNA probes for 16 hr at 65°C and was tested\nby using the Gene Images CDP-Star detection module kit (Amersham).\nThe expression intensities of TGF α mRNA and EGFR mRNA were\nanalyzed using Multi-Analyst (Bio-rad).\nThe mouse TGFα and EGFR cDNA probes used were synthe-\nsized from the mammary gland of the mouse by reverse transcriptase-\npolymerase chain reaction (RT-PCR). The PCR reaction for the probes\nof both TGFα and EGFR was performed for 40 cycles (1 cycle=94°C\nfor 1 min, 55°C for 1 min, 72°C for 2 min) in a program temperature\ncontrol system (Astec, Fukuoka, Japan). The sequence of the prim-\ners used for RT-PCR are shown in Table 1 (Avivi \net al., 1991; Snedeker\net al., 1991; Vaughan et al., 1992). The other conditions were the\nsame as detailed previously (Harigaya et al., 1994; Tsunoda et al.,\n1997).\nIn each assay, samples from all 5 groups were determined si-\nmultaneously and the values were expressed in terms of the percent-\nages against that of the intact control.\nStatistics\nThe statistical significance of differences in each parameter\namong groups were evaluated by Duncan’s multiple range test.\nTable 1. Sequence of primers used in RT-PCR and size of predicted products amplified\nPrimer Primer location Sequence Size (bp) of predicted product\nTGFα sence 21–41 5'-GGACAGCTCGCTCTGCTAGCG-3' 347\nantisense 267–350 5'-TGGATCAGCACACAGGTG-3'\nEGFR sense 234–253 5'-GGAGGAAAAGAAAGTCTGCC-3' 304\nantisense 537–518 5'-CCCATAGTTGGACAGGATGG-3'\nFig. 1. Serum prolactin level in each group (mean ± SEM). Num-\nbers of estimates are in parentheses. OVX, ovariectomy; D, danazol;\nPer, perphenazine; oPRL, ovine prolactin. a–c Values with different\nsuperscripts differ significantly at P < 0.05 or 0.01.\nDownloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026\nTerms of Use: https://bioone.org/terms-of-use\n\nOVX or PRL on TGFα and EGFR 527\nRESULTS\nSerum PRL level (Fig. 1)\nThe serum PRL level was significantly higher in the\nperphenazine-treated group than in the intact and OVX-con-\ntrol groups in both SHN and ICR mice. Moreover, in SHN mice,\nthe serum PRL level of the danazol-treated group was signifi-\ncantly lower than those of the intact and OVX-control groups.\nThe level of the danazol-treated group of ICR was also lower\nthan that of the intact-control group, but the difference was\nnot significant. Little difference was seen among the other\ngroups in the serum PRL level in both SHN and ICR mice.\nEnd-bud formation in mammary gland (Fig. 2)\nIn SHN mice, mammary rating as an index of end-bud\nformation was decreased significantly by OVX itself compared\nto the intact control group. The rating was further lower in the\ndanazol-treated group than in the others.\nLittle difference in the rating was seen among groups of\nICR mice.\nExpression of TGFααααα mRNA and EGFR mRNA in the mam-\nmary gland\nIn the first experiment, the northern blot analysis with\nagalose gel electrophoresis was performed to detect the\nmRNA size, but the band could not be detected. Then, the\ndot-blot analysis was carried out to determine the quantities\nof the mRNA.\nThe results of transforming growth factor α (TGFα) mRNA\nare shown in Fig. 3. In SHN mice, TGF α mRNA expression\nwas significantly lower in the OVX-control group and\nperphenazine-treated group than in the intact control group.\nTGFα mRNA expression of the ovine prolactin-treated group\nwas also lower than that of the intact control group, while the\ndifference was not significant. In ICR mice, the perphenazine-\ntreated group and oPRL-treated group tended to be lower than\nthe intact control group in the TGFα mRNA expression.\nAs presented in Fig. 4, epidermal growth factor receptor\n(EGFR) mRNA expression was apparently higher in the\ndanazol-treated group than in the intact control group in both\nSHN and ICR, while the difference in SHN was not significant\nowing to the large variation.\nDISCUSSION\nThe present results show that a decrease of the circulat-\ning PRL by danazol induced an apparent elevation of both\ntransforming growth factor α (TGFα) mRNA and epidermal\ngrowth factor receptor (EGFR) mRNA in the mammary glands\nof SHN mice. Furthermore, TGF α mRNA expression in the\nmammary gland was decreased by perphenazine associated\nwith an increase in the PRL level. These findings indicate that\nPRL acts suppressively on the manifestation of effects of TGFα\non the mammary glands. On the other hand, mammary end-\nbud formation was lower in the danazol group than in the group\ntreated with perphenazine or oPRL. The findings would indi-\ncate that the stimulating effects of TGF α on the mammary\nglands would need the simultaneous participation of PRL; in\nother words, the role of PRL on mammary gland growth is\ndominant to that of TGFα.\nTGFα expression in the mammary gland was decreased\nby OVX compared to the intact control group despite of little\ndifference in serum PRL level. This confirmed the previous\nreports that TGFα is modulated by estrogen (Dickson \net al.,\n1986; Arteaga et al., 1988; Bates et al., 1988; Manni et al.,\n1991).\nThe differences among groups in the parameters exam-\nined except EGFR mRNA were smaller in ICR than in SHN.\nThis would reflect that mammary gland of ICR is less suscep-\nFig. 2. Mammary rating as an index of end-bud formation in each\ngroup (mean ± SEM). Numbers of estimates are in parentheses. OVX,\novariectomy; D, danazol; Per, perphenazine; oPRL, ovine prolactin.\na–c Values with different superscripts differ significantly at P < 0.05 or\n0.01.\nDownloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026\nTerms of Use: https://bioone.org/terms-of-use\n\nH. Nagasawa et al.528\nFig. 4. Northern blot analysis of EGFR mRNA expression in the mammary glands in each group. Relative band intensity, which is expressed as\na percentage against the intact control (mean ± SEM). Numbers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine;\noPRL, ovine prolactin. a–b Values with different superscripts differ significantly at P < 0.05.\nFig. 3. Northern blot analysis of TGFα mRNA expression in the mammary glands in each group. Relative band intensity, which is expressed as\na percentage against the intact control (mean ± SEM). Numbers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine;\noPRL, ovine prolactin. a–c Values with different superscripts differ significantly at P < 0.05 or 0.01.\ntible to OVX and the modulation of circulating PRL than that\nof SHN. Incidentally, in mice, high mammary gland suscepti-\nbility to mammotropic hormones is a characteristic of high\nmammary tumor strains (Nagasawa and Yanai, 1978).\nACKNOWLEDGMENTS\nWe thank Dr. T. Harigaya, Professor of Functional Anatomy of\nthis University for his invaluable advice and continued interest, Pro-\nfessor A. F. Parlow, Pituitary Hormones & Antisera Center, Harbor\nUCLA-Medical Center, Torrance, CA, for the radioimmunoassay kit\nfor mouse PRL and Professor K. Wakabayashi, Gunma University,\nfor goat anti-rabbit IgG serum. The ovine PRL (AFP-10677C) was\nDownloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026\nTerms of Use: https://bioone.org/terms-of-use\n\nOVX or PRL on TGFα and EGFR 529\ndonated by the National Hormone and Pituitary Program, NIDDK,\nBethesda, MD, USA, which is also acknowledged.\nREFERENCES\nArteaga CL, Coronado E, Osborne CK (1988) Blockade of the epi-\ndermal growth factor receptor inhibits transforming growth factor\nα-induced but not estrogen-induced growth of hormone-depen-\ndent human breast cancer. Mol Endocrinol 2: 1064–1069\nAvivi A, Lax I, Ullrich A, Schlessinger J, Givol D, Morse B (1991)\nComparison of EGF receptor sequences as a guide to study the\nligand binding site. 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