Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor α and Epidermal Growth Factor Receptor mRNAs in Mice

Both estrogen and prolactin (PRL) are key hormones for the normal, preneoplastic and neoplastic growth of mammary glands (Nagasawa et al., 1986), and these hormones have been proposed to stimulate cell growth indirectly through an autocrine or paracrine mechanism by enhancing the produc- tion of peptide factors, as well as by direct effects. Transform- ing growth factor α (TGFα) participates in the growth of nor- mal and neoplastic mammary glands (Perroteau et al., 1986; Derynck et al., 1987; Liu et al., 1987; Zajchowski et al., 1988; Valverius et al., 1989; Matsui et al., 1990; Halter et al., 1992; Mizuno et al ., 1994), acting through binding to epidermal growth factor receptor (EGFR) (Todaro et al., 1980). How- ever, the information of the participation of estrogen and/or PRL on this process is rather sporadically. TGFα is suggested to be modulated by estrogen (Dickson et al., 1986; Arteaga et al., 1988; Bates et al., 1988; Manni et al., 1991). Prusheik et al. (1997) suggested that PRL inhibited EGFR signaling. In the present study, the effects of OVX and of the modu- lation of circulating PRL on expressions of TGF α and EGFR mRNAs in the mammary gland were examined in virgin mice. Perphenazine (Singtripop et al., 1991) and danazol (Singtripop et al., 1992) were used for the stimulant and the inhibitor of pituitary PRL release, respectively. The effect of ovine pro- lactin (oPRL) in this process was also examined.

Animals and treatments The mice used were SHN/Mei virgin mice maintained in our labo- ratory by strict brother x sister mating (Nagasawa et al., 1976) and Jcl: ICR virgin mice purchased from CLEA Japan (Tokyo, Japan). At 3 months of age, mice of each strain were divided into five groups. Four groups were bilaterally ovariectomized via a dorsal approach under anesthesia by pentobarbital sodium (Nembutal; Abbott Labo- ratories, North Chicago, IL, USA) and the remaining one group un- derwent a sham operation and served as the intact control. Begin- ning 15 days after the operation, the OVX group received subcutane- ous injections of saline (Otsuka Seiyaku, Tokyo, Japan; 0.1 ml, twice a day) or olive oil (Kozakai Seiyaku, Tokyo, Japan; 0.1 ml, once a day), the data of which were pooled in the Results section, since they differed little (OVX control); danazol (Sigma Chem. Co., St. Louis, MO, USA; 0.5 µg / 0.1 ml olive oil, once a day), perphenazine (Sigma; 0.05 mg / 0.1 ml saline, twice a day) or ovine PRL (AFP-10677C, NIDDK; 0.25 mg / 0.05 ml buffer, twice a day) for 3 days and on the morning of day 4. The mice undergone the sham operation were treated as was in the OVX control. All mice were killed one hour after Downloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026 Terms of Use: https://bioone.org/terms-of-use H. Nagasawa et al.526 the last injection under light ether anesthesia and blood was collected from the trunk. All procedures were carried out according to the USA NIH Guide for the Care and Use of Laboratory Animals. Throughout the experiment, mice were kept in plastic cages (18 × 30 × 13 cm) with wood shavings, maintained in an animal room, which was air-conditioned (20-22 °C and 60-70% relative humidity), artificially illuminated (14 hr of light from 5:00 AM to 7:00 PM) and ventilated (16 times / hr) and provided with commercial pellets (Lab MR-Breeder; Nihon Nosan Kogyo, Yokohama, Japan) and tap water ad libitum. Serum PRL level Blood collected was left at room temperature for 6 hr, kept in the refrigerator overnight and centrifuged at 1,000 × g for 20 min at 4°C. Serum was stored at –20 °C. The PRL level was determined by a homologous radioimmunoassay. End-bud formation in the mammary glands At autopsy, unilateral third thoracic mammary glands were pre- pared for the wholemount evaluation and were examined under 10- fold magnification. The degree of end-bud formation was rated from 1 to 7 in increments of 1 (Nagasawa et al., 1980). Northern blot analysis At autopsy, bilateral inguinal and unilateral third thoracic mam- mary glands were immediately removed, pooled and stored at –70°C. RNA was extracted from 200-250 mg of frozen tissue by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). The RNA concentration was deter- mined at 260 nm by spectrophotometer. Each 20 µg of RNA from each sample was applied to the nylon membrane (Immobilon TM; Millipore, Bedford, MA, USA) with a Bio-dot manifold (Bio-rad, Her- cules, CA, USA) and then the RNA was fixed to membrane using UV crosslinking. The transforming growth factor α (TGFα) and epidermal growth factor receptor (EGFR) complementary DNA (cDNA) fluolescein-labelled probes were labelled using the Gene Images ran- dom prime labelling module kit (Amersham, Buckinghamshire, En- gland). Northern blot analysis was performed by hybridization with this fluolescein-labelled cDNA probes for 16 hr at 65°C and was tested by using the Gene Images CDP-Star detection module kit (Amersham). The expression intensities of TGF α mRNA and EGFR mRNA were analyzed using Multi-Analyst (Bio-rad). The mouse TGFα and EGFR cDNA probes used were synthe- sized from the mammary gland of the mouse by reverse transcriptase- polymerase chain reaction (RT-PCR). The PCR reaction for the probes of both TGFα and EGFR was performed for 40 cycles (1 cycle=94°C for 1 min, 55°C for 1 min, 72°C for 2 min) in a program temperature control system (Astec, Fukuoka, Japan). The sequence of the prim- ers used for RT-PCR are shown in Table 1 (Avivi et al., 1991; Snedeker et al., 1991; Vaughan et al., 1992). The other conditions were the same as detailed previously (Harigaya et al., 1994; Tsunoda et al., 1997). In each assay, samples from all 5 groups were determined si- multaneously and the values were expressed in terms of the percent- ages against that of the intact control. Statistics The statistical significance of differences in each parameter among groups were evaluated by Duncan’s multiple range test. Table 1. Sequence of primers used in RT-PCR and size of predicted products amplified Primer Primer location Sequence Size (bp) of predicted product TGFα sence 21–41 5'-GGACAGCTCGCTCTGCTAGCG-3' 347 antisense 267–350 5'-TGGATCAGCACACAGGTG-3' EGFR sense 234–253 5'-GGAGGAAAAGAAAGTCTGCC-3' 304 antisense 537–518 5'-CCCATAGTTGGACAGGATGG-3' Fig. 1. Serum prolactin level in each group (mean ± SEM). Num- bers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine; oPRL, ovine prolactin. a–c Values with different superscripts differ significantly at P < 0.05 or 0.01. Downloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026 Terms of Use: https://bioone.org/terms-of-use OVX or PRL on TGFα and EGFR 527

Serum PRL level (Fig. 1) The serum PRL level was significantly higher in the perphenazine-treated group than in the intact and OVX-con- trol groups in both SHN and ICR mice. Moreover, in SHN mice, the serum PRL level of the danazol-treated group was signifi- cantly lower than those of the intact and OVX-control groups. The level of the danazol-treated group of ICR was also lower than that of the intact-control group, but the difference was not significant. Little difference was seen among the other groups in the serum PRL level in both SHN and ICR mice. End-bud formation in mammary gland (Fig. 2) In SHN mice, mammary rating as an index of end-bud formation was decreased significantly by OVX itself compared to the intact control group. The rating was further lower in the danazol-treated group than in the others. Little difference in the rating was seen among groups of ICR mice. Expression of TGFααααα mRNA and EGFR mRNA in the mam- mary gland In the first experiment, the northern blot analysis with agalose gel electrophoresis was performed to detect the mRNA size, but the band could not be detected. Then, the dot-blot analysis was carried out to determine the quantities of the mRNA. The results of transforming growth factor α (TGFα) mRNA are shown in Fig. 3. In SHN mice, TGF α mRNA expression was significantly lower in the OVX-control group and perphenazine-treated group than in the intact control group. TGFα mRNA expression of the ovine prolactin-treated group was also lower than that of the intact control group, while the difference was not significant. In ICR mice, the perphenazine- treated group and oPRL-treated group tended to be lower than the intact control group in the TGFα mRNA expression. As presented in Fig. 4, epidermal growth factor receptor (EGFR) mRNA expression was apparently higher in the danazol-treated group than in the intact control group in both SHN and ICR, while the difference in SHN was not significant owing to the large variation.

The present results show that a decrease of the circulat- ing PRL by danazol induced an apparent elevation of both transforming growth factor α (TGFα) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary glands of SHN mice. Furthermore, TGF α mRNA expression in the mammary gland was decreased by perphenazine associated with an increase in the PRL level. These findings indicate that PRL acts suppressively on the manifestation of effects of TGFα on the mammary glands. On the other hand, mammary end- bud formation was lower in the danazol group than in the group treated with perphenazine or oPRL. The findings would indi- cate that the stimulating effects of TGF α on the mammary glands would need the simultaneous participation of PRL; in other words, the role of PRL on mammary gland growth is dominant to that of TGFα. TGFα expression in the mammary gland was decreased by OVX compared to the intact control group despite of little difference in serum PRL level. This confirmed the previous reports that TGFα is modulated by estrogen (Dickson et al., 1986; Arteaga et al., 1988; Bates et al., 1988; Manni et al., 1991). The differences among groups in the parameters exam- ined except EGFR mRNA were smaller in ICR than in SHN. This would reflect that mammary gland of ICR is less suscep- Fig. 2. Mammary rating as an index of end-bud formation in each group (mean ± SEM). Numbers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine; oPRL, ovine prolactin. a–c Values with different superscripts differ significantly at P < 0.05 or 0.01. Downloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026 Terms of Use: https://bioone.org/terms-of-use H. Nagasawa et al.528 Fig. 4. Northern blot analysis of EGFR mRNA expression in the mammary glands in each group. Relative band intensity, which is expressed as a percentage against the intact control (mean ± SEM). Numbers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine; oPRL, ovine prolactin. a–b Values with different superscripts differ significantly at P < 0.05. Fig. 3. Northern blot analysis of TGFα mRNA expression in the mammary glands in each group. Relative band intensity, which is expressed as a percentage against the intact control (mean ± SEM). Numbers of estimates are in parentheses. OVX, ovariectomy; D, danazol; Per, perphenazine; oPRL, ovine prolactin. a–c Values with different superscripts differ significantly at P < 0.05 or 0.01. tible to OVX and the modulation of circulating PRL than that of SHN. Incidentally, in mice, high mammary gland suscepti- bility to mammotropic hormones is a characteristic of high mammary tumor strains (Nagasawa and Yanai, 1978). ACKNOWLEDGMENTS We thank Dr. T. Harigaya, Professor of Functional Anatomy of this University for his invaluable advice and continued interest, Pro- fessor A. F. Parlow, Pituitary Hormones & Antisera Center, Harbor UCLA-Medical Center, Torrance, CA, for the radioimmunoassay kit for mouse PRL and Professor K. Wakabayashi, Gunma University, for goat anti-rabbit IgG serum. The ovine PRL (AFP-10677C) was Downloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026 Terms of Use: https://bioone.org/terms-of-use OVX or PRL on TGFα and EGFR 529 donated by the National Hormone and Pituitary Program, NIDDK, Bethesda, MD, USA, which is also acknowledged.

Arteaga CL, Coronado E, Osborne CK (1988) Blockade of the epi- dermal growth factor receptor inhibits transforming growth factor α-induced but not estrogen-induced growth of hormone-depen- dent human breast cancer. Mol Endocrinol 2: 1064–1069 Avivi A, Lax I, Ullrich A, Schlessinger J, Givol D, Morse B (1991) Comparison of EGF receptor sequences as a guide to study the ligand binding site. Oncogene 6: 673–676 Bates SE, Davidson NE, Valverius EM, Freter CE, Dickson RB, Tam JP, Kudlow JE, Lippman ME, Salomon DS (1988) Expression of transforming growth factor α and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its pos- sible functional significance. Mol Endocrinol 2: 543–555 Chomczynski P, Sacchi N (1987) Single-step method of RNA isola- tion by acid guanidinium thiocyanate-phenol-chloroform extrac- tion. Anal Biochem 162: 156–159 Derynck R, Goeddel DV, Ullrich A, Guttermam JU, Williams RD, Bringman TS, Berger WH (1987) Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epider- mal growth factor receptor by human tumors. Cancer Res 47: 707–712 Dickson RB, McManaway ME, Lippman ME (1986) Estrogen-induced factors of breast cancer cells partially replace estrogen to pro- mote tumor growth. Science 232: 1540–1543 Halter SA, Dempsey P, Matsui Y, Stokes MK, Deal RG, Hogan BLM, Coffey RJ (1992) Distinctive patterns of hyperplasia in transgenic mice with mouse mammary tumor virus transforming growth fac- tor-α. Amer J Pathol 140: 1131–1146 Harigaya T, Tsunoda S, Mizuno M, Nagasawa H (1994) Different gene expression of mouse transforming growth factor α between preg- nant mammary glands and mammary tumors in C3H/He mice. Zool Sci 11: 625–627 Liu SC, Sanfilippo B, Perroteau I, Derynck R, Salomon DS, Kidwall WR (1987) Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production. Mol Endocrinol 1: 683–692 Manni A, Wright C, Buck H (1991) Growth factor involvement in the multihormonal regulation of MCF-7 breast cancer cell growth in soft agar. Breast Cancer Res Treat 20: 43–52 Matsui Y, Halter SA, Holt JT, Hogan BLM, Coffey RJ (1990) Develop- ment of mammary hyperplasia and neoplasia in MMTV-TGF α transgenic mice. Cell 61: 1147–1155 Mizuno M, Yamamoto K, Sakamoto S, Mori T, Harigaya T, Nagasawa H (1994) Normal and neoplastic mammary gland growth in MMTV/ TGFα mice. In Vivo 8: 263–270 Nagasawa H, Yanai R, Taniguchi H, Tokuzen R, Nakahara W (1976) Two-way selection of a stock of Swiss albino mice for mammary tumorigenesis: Establishment of two new strains (SHN and SLN). J Natl Cancer Inst 57: 425–430 Nagasawa H, Yanai R (1978) Normal and abnormal growth of the mammary gland. In “Physiology of Mammary Glands” Ed by Yokoyama A, Mizuno H, Nagasawa H, Jpn Sci Soc Press/Univ Park Press, Tokyo/Baltimore, pp 122–159 Nagasawa H, Yanai R, Nakajima Y, Namiki H, Kikuyama S, Shiota K (1980) Inhibitory effects of potassinm thiocyanate on normal and neoplastic mammary development in female mice. Europ J Can- cer 16: 473–480 Nagasawa H, Ohta K, Nakajima K, Noguchi Y, Miura K, Niki K, Namiki H (1986) Interrelationship between pituitary and ovarian hormones in normal and neoplastic growth of mammary glands of mice. Ann NY Acad Sci 464: 301–315 Perroteau I, Salomon D, DeBortoli M, Kidwell W, Hazarika P, Pardue R, Dedman J, Tam J (1986) Immunological detection and quantitation of alpha transforming growth factors in human breast carcinoma cells. Breast Cancer Res Treat 7: 201–210 Prusheik K, Ladd AN, Sheffield LG (1997) Effects of epidermal growth factor (EGF) and prolactin on EGF receptor cytoskeletal asso- ciation in mammary epithelial cells. Proc Soc Exp Biol Med 215: 393–398 Singtripop T, Mori T, Park MK, Sakamoto S, Kawashima S (1991) Development of uterine adenomyosis after treatment with dopam- ine antagonists in mice. Life Sci 49: 201–206 Singtripop T, Mori T, Sakamoto S, Sassa S, Park MK, Kawashima S (1992) Suppression of the development of uterine adenomyosis by danazol treatment in mice. Life Sci 51: 1119–1125 Snedeker SM, Brown CF, DiAugustine RP (1991) Expression and functional properties of transforming growth factor a and epider- mal growth factor during mouse mammary gland ductal morpho- genesis. Proc Natl Acad Sci USA 88: 276–280 Todaro GJ, Fryling C, DeLarco JE (1980) Transforming growth fac- tors produced by certain human tumor cells: Polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci USA 77: 5258–5262 Tsunoda S, Ikezaki Y, Nagasawa H (1997) Transforming growth fac- tor-α mRNA and epidermal growth factor receptor mRNA expres- sion in normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials. Zool Sci 14: 803– 807 Valverius EM, Bates SE, Stampfer MR, Clark R, McCormick F, Salomon DS, Lippman ME, Dickson RB (1989) Transforming growth factor alpha production and epidermal growth factor re- ceptor expression in normal and oncogene transformed human mammary epithelial cells. Mol Endocrinol 3: 203–214 Vaughan TJ, Pascall JC, Brown KD (1992) Nucleotide sequence and tissue distribution of mouse transforming growth factor-α. Biochim Biophys Acta 1132: 322–324 Zajchowski D, Band V, Pauzie N, Tager A, Stampfer M, Sager R (1988) Expression of growth factors and oncogenes in normal and tu- mor-derived human mammary epithelial cells. Cancer Res 48: 7041–7047 (Received March 16, 1998 / Accepted April 14, 1998) Downloaded From: https://bioone.org/journals/Zoological-Science on 08 Sep 2026 Terms of Use: https://bioone.org/terms-of-use