IN VITRO Volume 13, No. 8, 1977 All rights reserved 9
EFFECTS OF PERINATAL ESTROGEN ON MOUSE MAMMARY RESPONSE TO CORTICOIDS IN VITRO M. R. WARNER 1ANDR. L. WARNER 2
SUMMARY
Effects of three adrenal corticoids on in vitro mammary differentiation were compared in neonatally estrogenized (E) and uninjected control (N) B A L B / c Crgl female mice. E mice were injected with 10/~g of 17/3-estradiol on each of the first 5 days of life. At 4 weeks of age, all mice were pretreated with 1/~g 17/3-estradiol and 1 mg progesterone for 9 consecutive days. Groups of 10 or more entire mammary glands then were organ-cultured for 5 days at 37~ on chemically defined medium in 95% 02/5% CO2 with various combinations of hormones: mammotropin (M), somatotropin (S), insulin (I), thyroxine (T); and corticosterone (B), aldosterone (A), or cortisol (F). Differentiation followed a quantitative pattern of M S I T < B M S I T < A M S I T < F M S I T for both E and N tissues. E tissues formed more alveoli and more lobules in vitro than N tissues with each of the corticoids tested. These findings may have pathologic significance.
Key words: perinatal; differentiation; hormones; mammary; in vitro. INTRODUCTION
with a daily dose of 10/~g of 17/~-estradiol in 0.02 ml aqueous microsuspension subcutaneously into the intrascapular region for 5 consecutive days, starting within 24 hr after birth. Warner (7) has shown that this dose of estradiol to newborn mice results in altered growth of mammary glands. Pretreatment. Beginning at 4 weeks of age, both the perinatally estradiol-injected group and control mice were injected daily for 9 days with 1 /~g of 17/3-estradiol and 1 mg of progesterone in 0.2 ml aqueous microsuspension subcutaneously in the intrascapular region (8k Experimental groups. M a m m a r y glands were compared in order to determine the effect of neonatal administration of estradiol on in vivo and in vitro hormone responsiveness of mammary tissues. Pretreated mice were killed 24 hr after the last hormone injection. Several mammary glands from each mouse were fixed immediately in 15% formalin to determine the effect of pretreatment MATERIALS AND METHODS on morphology. The remaining glands were Animals. Newborn and immature B A L B / c grown in organ culture with various hormone supCrgl female mice were studied. Mice were housed, plements. Each experiment was repeated at least five to a cage, with their mothers. With the excep- once. tion of uninjected controls, all mice were injected Hormone source. Hormones used as pretreatment and in culture media were as follows: 17/3'To whom reprint requests should be sent at the De- estradiol (Schering Corp., Bloomfield, N.J.); partment of Cell Biology, Baylor College of Medicine, cortisol and corticosterone (Sigma Chemical Co., Houston, Texas 77030. 2Department of Neurobiology and Anatomy, Univer- St. Louis, Mo.); progesterone and thyroxine sosity of Texas Medical School, Houston, Texas 77025. dium (Nutritional Biochemical Corp., Cleveland, 477 Exposure of mice to 17/3-estradiol early in life (E mice) induces an increased susceptibility of their reproductive tissues (ovary, uterus, vagina, cervix and mammary glands) to tumors in later life (1-3L Increased tumor incidence in one of these organ systems, the mammary gland, could result from alterations in tissue level sensitivity to hormones. Indeed, we have described a decrease in estrogen and progesterone pretreatment requirement in E mice, together with an enhanced response to several combinations of steroid and protein hormones (4k Nandi and Bern (5) have outlined in vivo mammary responses to corticoids, and Rivera (6) has provided similar data for in vitro studies. The following experiments were performed to see how tissues of neonatally estradiolinjected mice respond in vitro to major corticoids in terms of these established norms.
478
WARNER AND WARNER
Ohio,; d-aldosterone (Ciba Pharmaceutical Co., Summit, N.J.); mammotrophin {ovine) and somatotrophin {bovine) (Hormone Research Laboratory, University of California, Berkeley}; and insulin (Calbiochem, Los Angeles, Calif.). Culture method. Whole mammary glands were explanted according to the method described by Ichinose and Nandi (8). Culture dishes were incubated in polystyrene chambers in a water-jacketed incubator at 37~ An atmosphere of 95% 02/5% CO2, saturated with water vapor, was provided. Gas flow was adjusted to maintain a pH of about 7.4 in the medium. At day 2.5 to 3 of incubation, medium was removed and replaced with fresh medium. Tissues were fixed in Tellyesniczky's fluid on day 5. Culture medium. Chemically defined medium MB 725/1 (Hyland Laboratories, Los Angeles, Calif.) plus 0.002% phenol red, to which 50 IU per ml penicillin G and 350 #g per ml l-glutamine had been added, was the basic medium. Hormones (cortisol, corticosterone, aldosterone, mammotrophin, somatotrophin and insulin) were added to the basic medium as described by Ichinose and Nandi (8). Bovine thyroxine was dissolved in 0.001 N NaOH and sterilized by passing it through a 0.45-/~m porosity Millipore filter. Final hormone concentrations were d-aldosterone, 1/~g per ml; cortisol, 1 gg per ml; corticosterone, 1 #g per ml; and thyroxine, 5 #g per ml. Evaluation criteria. Mammary glands were fixed, then stained as wholemounts with hematoxylin, and examined and photographed in methyl salicylate (91. No differences in response to culture were found between various gland pairs; therefore data from Nos. 2, 3 and 4 gland pairs were combined. Results of experiments were evaluated on the basis of gross morphological differences between the tissues of the various groups examined. Lobule development was graded from 0 to 3, subjectively, on iron hematoxylin-stained wholemount preparations: 0 = no lobules; 1 = few small lobules in less than half the area of the explant; 2 = lobules in about half the explant; 3 = half or more of the explant shows lobules typical of an early lactating mammary gland. Almost all glands which showed extensive normal lobuloalveolar differentiation also had morphologically abnormal areas. Maintenance occurred when epithelial continuity was preserved, and all histological structures present in the experimental control were present in the cultured explant. Degenerated tissues were dense or showed interruptions in epi-
FIG. 1. Effects of exposure of newborn BALB/c Crgl female mice to 17{~-estradiolon mammary alveolar development after 9 days of hormone pretreatment of hosts followed by subsequent organ culture with various hormone-supplemented media. E mice were injected daily with 10 pg 17/3-estradiolfor each of the first 5 days of life. N mice were uninjected controls. Pretreatment was begun at 4 weeks of age: 1 ~g 17/%estradioland 1 mg of progesterone were injected subcutaneously in aqueous microcrystal suspension for 9 consecutive days. See Materials and Methods for the dose of various hormones added to the culture medium. The following abbreviations have been used: E = 17/3-estradiol;P ----progesterone; A = aldosterone; B ----corticosterone; F = cortisol; M = mammotropin; S = somatotropin; I ----insulin; T ---- thyroxine. Figures above the bar indicate number of explants per group. Open bar indicates no alveoli. Dotted areas represent explants less than one-half of which were alveolar; and lined spaces indicate that onehalf to three-fourths of the area of the explant had alveoli. thelial continuity and decreased stainability. Structures described as being degenerated or maintained were classified on the basis of wholemount examination, and judgements were confirmed by histologic section (sectioned at 6 gm and stained with Masson's trichrome). Mann Whitney or Wilcoxon nonparametic tests were used to determine statistical significance (10, 11). RESULTS See Figs. 1 and 2.
Morphology of nonpretreated control. Tissues of 4-week initial controls and of 5.5-week-old final controls of E or N mice lacked alveoli or lobules. Five-and-a-half-week E tissues were larger and branched more than their N controls (7). Effect of 9 days of pretreatment with 17[~-estradiol and progesterone (Figs. 1-3). By the 9th day, E tissues had more lobules and alveoli than N tissues, although relative group numbers were too small to show statistical significance.
Culture with mammotropin, somatotropin, insulin and thyroxine (MSIT) (Figs. 1, 2, 4; Tables 1, 2). Alveolar and lobular development were similar in E and N groups.
479
CORTICOID RESPONSE
Culture with corticosterone, mammotropin, somatotropin, insulin and thyroxine ( B M S I T ) (Figs. 1, 2, 5, 7, 9-11; T a b l e s 1, 2}. T h e r e was significantly m o r e b o t h alveolar a n d l o b u l a r developm e n t in E g r o u p c o m p a r e d with N group. T h i s r e p r e s e n t e d a significant i m p r o v e m e n t in b o t h alveolar a n d l o b u l a r d e v e l o p m e n t over M S I T cultured controls. A l t h o u g h alveoli a n d lobules were well developed morphologically; functionally, secretion was sparse. E group h a d more secretion t h a n N g r o u p (Figs. 9, 10L
Culture with aldosterone, mammotropin, somatotropin, insulin and thyroxine ( A M S I T ) FIG. 2. Effects of exposure of newborn BALB/c Crgl female mice to 173-estradiol on mammary lobuloMveolar development after 9 days of hormone pretreatment of hosts followed by subsequent organ culture with various hormone-supplemented media. E mice were injected daily with 10 t~g 173-estradiol for each of the first 5 days of life. N mice were uninjected controls. Pretreatment was begun at 4 weeks of age: 173-estradiol t l #gl and progesterone ~1 mg} were injected subcutaneously in aqueous microcrystal suspension for 9 consecutive days. See Materials and Methods for the dose of various hormones added to the culture medium. The following abbreviations have been used: E = 173-estradiol; P = progesterone; B = corticosterone; A = aldosterone; F = cortisol; M = mammotropin; S = somatotropin; I = insulin; T = thyroxine. Figures above the bar indicate number of explants per group. Open bar indicates no lobules. Dotted areas represent explants less than one-half of which were lobular; lined spaces indicate that one-half to three-fourths of the area of the explant had lobules; and solid bars represent three-fourths to total lobular development of the explant.
{Figs. 1, 6, 8, 13; T a b l e s 1, 2L B o t h alveoli a n d lobules were b e t t e r developed in E g r o u p mice t h a n in N controls. Alveolar a n d l o b u l a r developm e n t were i m p r o v e d significantly in the N a n d E groups c u l t u r e d w i t h A M S I T over d e v e l o p m e n t in groups c u l t u r e d with M S I T or B M S I T .
Culture with cortisol, mammotropin, somatotropin, insulin and thyroxine ( F M S I T ) (Figs. 1, 2, a n d 12; T a b l e s 1, 2 }. Alveolar d e v e l o p m e n t was similar in N a n d E g r o u p s after culture with F M S I T a n d showed n o significant i m p r o v e m e n t over t h e g r o u p cultured w i t h A M S I T . L o b u l a r dev e l o p m e n t was e n h a n c e d in tissues of E g r o u p mice cultured with F M S I T over their N controls. B o t h E a n d N g r o u p tissues cultured with F M S I T h a d more lobules t h a n tissues cultured with A M S I T , B M S I T or M S I T controls. Secretion was m o s t a b u n d a n t in t h e F M S I T group. Secretion was e n h a n c e d in E g r o u p as opposed to controls.
TABLE 1 COMPARISON OF SIGNIFICANTDIFFERENCES IN ALVEOLARDEVELOPMENTOF MAMMARYEXPLANTS FROM
NEONATALLYESTROGEN-INJECTEDAND CONTROLMICE AFTER CULTURE WITH VARIOUSCORTICO1DSa P value for normal i N } controls Hormone supplement to basic medium P values for neonatally estrogenized ~E } mice P value for N versus E
0.001
MSIT
EFFECTS OF PERINATAL ESTROGEN ON MOUSE MAMMARY RESPONSE TO CORTICOIDS IN VITRO M. R. WARNER 1ANDR. L. WARNER 2
SUMMARY
Effects of three adrenal corticoids on in vitro mammary differentiation were compared in neonatally estrogenized (E) and uninjected control (N) B A L B / c Crgl female mice. E mice were injected with 10/~g of 17/3-estradiol on each of the first 5 days of life. At 4 weeks of age, all mice were pretreated with 1/~g 17/3-estradiol and 1 mg progesterone for 9 consecutive days. Groups of 10 or more entire mammary glands then were organ-cultured for 5 days at 37~ on chemically defined medium in 95% 02/5% CO2 with various combinations of hormones: mammotropin (M), somatotropin (S), insulin (I), thyroxine (T); and corticosterone (B), aldosterone (A), or cortisol (F). Differentiation followed a quantitative pattern of M S I T < B M S I T < A M S I T < F M S I T for both E and N tissues. E tissues formed more alveoli and more lobules in vitro than N tissues with each of the corticoids tested. These findings may have pathologic significance.
Key words: perinatal; differentiation; hormones; mammary; in vitro. INTRODUCTION
with a daily dose of 10/~g of 17/~-estradiol in 0.02 ml aqueous microsuspension subcutaneously into the intrascapular region for 5 consecutive days, starting within 24 hr after birth. Warner (7) has shown that this dose of estradiol to newborn mice results in altered growth of mammary glands. Pretreatment. Beginning at 4 weeks of age, both the perinatally estradiol-injected group and control mice were injected daily for 9 days with 1 /~g of 17/3-estradiol and 1 mg of progesterone in 0.2 ml aqueous microsuspension subcutaneously in the intrascapular region (8k Experimental groups. M a m m a r y glands were compared in order to determine the effect of neonatal administration of estradiol on in vivo and in vitro hormone responsiveness of mammary tissues. Pretreated mice were killed 24 hr after the last hormone injection. Several mammary glands from each mouse were fixed immediately in 15% formalin to determine the effect of pretreatment MATERIALS AND METHODS on morphology. The remaining glands were Animals. Newborn and immature B A L B / c grown in organ culture with various hormone supCrgl female mice were studied. Mice were housed, plements. Each experiment was repeated at least five to a cage, with their mothers. With the excep- once. tion of uninjected controls, all mice were injected Hormone source. Hormones used as pretreatment and in culture media were as follows: 17/3'To whom reprint requests should be sent at the De- estradiol (Schering Corp., Bloomfield, N.J.); partment of Cell Biology, Baylor College of Medicine, cortisol and corticosterone (Sigma Chemical Co., Houston, Texas 77030. 2Department of Neurobiology and Anatomy, Univer- St. Louis, Mo.); progesterone and thyroxine sosity of Texas Medical School, Houston, Texas 77025. dium (Nutritional Biochemical Corp., Cleveland, 477 Exposure of mice to 17/3-estradiol early in life (E mice) induces an increased susceptibility of their reproductive tissues (ovary, uterus, vagina, cervix and mammary glands) to tumors in later life (1-3L Increased tumor incidence in one of these organ systems, the mammary gland, could result from alterations in tissue level sensitivity to hormones. Indeed, we have described a decrease in estrogen and progesterone pretreatment requirement in E mice, together with an enhanced response to several combinations of steroid and protein hormones (4k Nandi and Bern (5) have outlined in vivo mammary responses to corticoids, and Rivera (6) has provided similar data for in vitro studies. The following experiments were performed to see how tissues of neonatally estradiolinjected mice respond in vitro to major corticoids in terms of these established norms.
478
WARNER AND WARNER
Ohio,; d-aldosterone (Ciba Pharmaceutical Co., Summit, N.J.); mammotrophin {ovine) and somatotrophin {bovine) (Hormone Research Laboratory, University of California, Berkeley}; and insulin (Calbiochem, Los Angeles, Calif.). Culture method. Whole mammary glands were explanted according to the method described by Ichinose and Nandi (8). Culture dishes were incubated in polystyrene chambers in a water-jacketed incubator at 37~ An atmosphere of 95% 02/5% CO2, saturated with water vapor, was provided. Gas flow was adjusted to maintain a pH of about 7.4 in the medium. At day 2.5 to 3 of incubation, medium was removed and replaced with fresh medium. Tissues were fixed in Tellyesniczky's fluid on day 5. Culture medium. Chemically defined medium MB 725/1 (Hyland Laboratories, Los Angeles, Calif.) plus 0.002% phenol red, to which 50 IU per ml penicillin G and 350 #g per ml l-glutamine had been added, was the basic medium. Hormones (cortisol, corticosterone, aldosterone, mammotrophin, somatotrophin and insulin) were added to the basic medium as described by Ichinose and Nandi (8). Bovine thyroxine was dissolved in 0.001 N NaOH and sterilized by passing it through a 0.45-/~m porosity Millipore filter. Final hormone concentrations were d-aldosterone, 1/~g per ml; cortisol, 1 gg per ml; corticosterone, 1 #g per ml; and thyroxine, 5 #g per ml. Evaluation criteria. Mammary glands were fixed, then stained as wholemounts with hematoxylin, and examined and photographed in methyl salicylate (91. No differences in response to culture were found between various gland pairs; therefore data from Nos. 2, 3 and 4 gland pairs were combined. Results of experiments were evaluated on the basis of gross morphological differences between the tissues of the various groups examined. Lobule development was graded from 0 to 3, subjectively, on iron hematoxylin-stained wholemount preparations: 0 = no lobules; 1 = few small lobules in less than half the area of the explant; 2 = lobules in about half the explant; 3 = half or more of the explant shows lobules typical of an early lactating mammary gland. Almost all glands which showed extensive normal lobuloalveolar differentiation also had morphologically abnormal areas. Maintenance occurred when epithelial continuity was preserved, and all histological structures present in the experimental control were present in the cultured explant. Degenerated tissues were dense or showed interruptions in epi-
FIG. 1. Effects of exposure of newborn BALB/c Crgl female mice to 17{~-estradiolon mammary alveolar development after 9 days of hormone pretreatment of hosts followed by subsequent organ culture with various hormone-supplemented media. E mice were injected daily with 10 pg 17/3-estradiolfor each of the first 5 days of life. N mice were uninjected controls. Pretreatment was begun at 4 weeks of age: 1 ~g 17/%estradioland 1 mg of progesterone were injected subcutaneously in aqueous microcrystal suspension for 9 consecutive days. See Materials and Methods for the dose of various hormones added to the culture medium. The following abbreviations have been used: E = 17/3-estradiol;P ----progesterone; A = aldosterone; B ----corticosterone; F = cortisol; M = mammotropin; S = somatotropin; I ----insulin; T ---- thyroxine. Figures above the bar indicate number of explants per group. Open bar indicates no alveoli. Dotted areas represent explants less than one-half of which were alveolar; and lined spaces indicate that onehalf to three-fourths of the area of the explant had alveoli. thelial continuity and decreased stainability. Structures described as being degenerated or maintained were classified on the basis of wholemount examination, and judgements were confirmed by histologic section (sectioned at 6 gm and stained with Masson's trichrome). Mann Whitney or Wilcoxon nonparametic tests were used to determine statistical significance (10, 11). RESULTS See Figs. 1 and 2.
Morphology of nonpretreated control. Tissues of 4-week initial controls and of 5.5-week-old final controls of E or N mice lacked alveoli or lobules. Five-and-a-half-week E tissues were larger and branched more than their N controls (7). Effect of 9 days of pretreatment with 17[~-estradiol and progesterone (Figs. 1-3). By the 9th day, E tissues had more lobules and alveoli than N tissues, although relative group numbers were too small to show statistical significance.
Culture with mammotropin, somatotropin, insulin and thyroxine (MSIT) (Figs. 1, 2, 4; Tables 1, 2). Alveolar and lobular development were similar in E and N groups.
479
CORTICOID RESPONSE
Culture with corticosterone, mammotropin, somatotropin, insulin and thyroxine ( B M S I T ) (Figs. 1, 2, 5, 7, 9-11; T a b l e s 1, 2}. T h e r e was significantly m o r e b o t h alveolar a n d l o b u l a r developm e n t in E g r o u p c o m p a r e d with N group. T h i s r e p r e s e n t e d a significant i m p r o v e m e n t in b o t h alveolar a n d l o b u l a r d e v e l o p m e n t over M S I T cultured controls. A l t h o u g h alveoli a n d lobules were well developed morphologically; functionally, secretion was sparse. E group h a d more secretion t h a n N g r o u p (Figs. 9, 10L
Culture with aldosterone, mammotropin, somatotropin, insulin and thyroxine ( A M S I T ) FIG. 2. Effects of exposure of newborn BALB/c Crgl female mice to 173-estradiol on mammary lobuloMveolar development after 9 days of hormone pretreatment of hosts followed by subsequent organ culture with various hormone-supplemented media. E mice were injected daily with 10 t~g 173-estradiol for each of the first 5 days of life. N mice were uninjected controls. Pretreatment was begun at 4 weeks of age: 173-estradiol t l #gl and progesterone ~1 mg} were injected subcutaneously in aqueous microcrystal suspension for 9 consecutive days. See Materials and Methods for the dose of various hormones added to the culture medium. The following abbreviations have been used: E = 173-estradiol; P = progesterone; B = corticosterone; A = aldosterone; F = cortisol; M = mammotropin; S = somatotropin; I = insulin; T = thyroxine. Figures above the bar indicate number of explants per group. Open bar indicates no lobules. Dotted areas represent explants less than one-half of which were lobular; lined spaces indicate that one-half to three-fourths of the area of the explant had lobules; and solid bars represent three-fourths to total lobular development of the explant.
{Figs. 1, 6, 8, 13; T a b l e s 1, 2L B o t h alveoli a n d lobules were b e t t e r developed in E g r o u p mice t h a n in N controls. Alveolar a n d l o b u l a r developm e n t were i m p r o v e d significantly in the N a n d E groups c u l t u r e d w i t h A M S I T over d e v e l o p m e n t in groups c u l t u r e d with M S I T or B M S I T .
Culture with cortisol, mammotropin, somatotropin, insulin and thyroxine ( F M S I T ) (Figs. 1, 2, a n d 12; T a b l e s 1, 2 }. Alveolar d e v e l o p m e n t was similar in N a n d E g r o u p s after culture with F M S I T a n d showed n o significant i m p r o v e m e n t over t h e g r o u p cultured w i t h A M S I T . L o b u l a r dev e l o p m e n t was e n h a n c e d in tissues of E g r o u p mice cultured with F M S I T over their N controls. B o t h E a n d N g r o u p tissues cultured with F M S I T h a d more lobules t h a n tissues cultured with A M S I T , B M S I T or M S I T controls. Secretion was m o s t a b u n d a n t in t h e F M S I T group. Secretion was e n h a n c e d in E g r o u p as opposed to controls.
TABLE 1 COMPARISON OF SIGNIFICANTDIFFERENCES IN ALVEOLARDEVELOPMENTOF MAMMARYEXPLANTS FROM
NEONATALLYESTROGEN-INJECTEDAND CONTROLMICE AFTER CULTURE WITH VARIOUSCORTICO1DSa P value for normal i N } controls Hormone supplement to basic medium P values for neonatally estrogenized ~E } mice P value for N versus E
0.001
MSIT
