0 1992 MUNKSGAARD
Melatonin inhibits mammary gland development in female mice Mediavilla MD, San Martin M , Sgnchez-Barcelo EJ. Melatonin inhibits mammary gland development in female mice. J . Pineal Res. 1992: 1 3: 1 3- 19.
~
Abstract: The objective of this study was to determine whether melatonin (aMT) influences the postnatal development of the mammary gland parenchyma in female mice from the time of weaning to adulthood. Twenty-one-day-old female BALBc mice were treated with daily subcutaneous injections of aMT (200 pg) or diluent, 3 hr before the onset of darkness (photoperiod LD 12: 12). At 3, 5 , 7, 9, 1 1 , and 13 weeks of age, batches of 20 animals (ten controls and ten aMT-treated) were sacrificed and the second pair of mammary glands were dissected to evaluate their degree of development. Melatonin decreased body weight gain from 2 weeks before until 2 weeks after the onset of puberty. Treatment with aMT also resulted in a lower DNA content and smaller area of the mammary gland from the time of puberty until the end of the study. In aMT-treated mice the phase of highly positive allometric growth began 2 weeks later, but ended at the same time as in controls ( 1 lth week of life). Finally, aMT decreased the development of terminal, lateral, and alveolar buds while it increased the number of terminal ducts per gland. We conclude that pharmacological doses of aMT ( I ) reduce body weight gain at the peripuberal age; (2) partially inhibit postnatal mammary gland development by reducing the number of epithelial structures representing sites of growth and increasing that of structures representing the final state of ductal growth in virgin animals; (3) delay the onset of and shorten the phase of rapid mammary growth occurring in early postpuberal age.
Introduction
Mammary gland development is the result of a complex interplay of many hormones and local growth factors [see reviews from Topper and Freeman, 1980; Tucker, 1981; Imagawa et al., 19901. Melatonin (aMT) modulates circulating levels of some of the gonadal and pituitary hormones [Reiter, 19801 that control mammogenesis. Consequently, a possible role of this pineal hormone in the control of mammary gland development could be expected. Although the influence of the pineal gland and its hormones on the growth of mammary tumors has been extensively studied [Danforth et al., 1983; Shah et al., 1984; Kothari, 1988; Sanchez-Barcelo et al., 1988; Cos et al., 1989; Blask et al., 19901, the possible role of melatonin in the development of normal mammary tissue is still poorly understood. Mishkinsky et al. [1966] found no differences in the mammary glands of 100-day-old female rats pinealectomized at weaning or left intact. However, normal rats exposed to continuous lighting from
M.D. Mediavilla, M. San Martin, and E.J. Sanchez-Barcelo Department of Physiology and Pharmacology, School of Medicine, University of Cantabria, Spain
Key words: melatonin-mammary gland-body weight-female BALBc mice-terminal end bud-lateral bud-terminal duct-alveolar bud Address reprint requests to E.J. Sanchez-Barcelo, Departamento de Fisiologia y Farmacologia, Facultad de Medicina, Universidad de Cantabria, 3901 1 Santander, Spain Received January 16, 1992; accepted April 16, 1992
birth show increased mammary development, which is prevented by administering melatonin [Mhatre et al., 19841. We have recently demonstrated that aMT, in vivo, reduces mouse mammary gland growth in response to estradiol + progesterone injections [Sanchez-Barcelo et al., 19901. Orally administered aMT was also effective in reducing mammary growth in heifers [Sanchez Barcelo et al., 19911. The influence of aMT on mammary gland growth could be secondary to aMT-induced changes in the serum concentration of mammotrophic hormones such as estrogens and prolactin [Reiter, 1980; Sanchez-Barcel6 et al., 19911. However, direct actions of aMT on mammary tissue have also been demonstrated by in vitro studies in which aMT at micromolar concentrations reduced the growth of mammary tissue induced by mammotrophic hormones added to the culture medium, whereas nanomolar concentrations had opposite effects [Sanchez-Barcelo et al., 19901. The objective of the present study was to deter-
13
Mediavilla et al.
mine whether aMT influences the physiological postnatal development (not induced by exogenous mammotrophic hormones) of mammary gland parenchyma in female mice from weaning to adulthood.
Material and methods Animals and housing conditions
In all experiments, female BALBc mice (Iffa Credo, France) were used. Animals were housed in our vivarium at 23 5 1°C room temperature and exposed to a LD12:12 photoperiod. Food and tap water were provided ad libitum. Experimental design
From 21 days of age, animals received a daily subcutaneous injection of aMT (Sigma) (200 pg) or the diluent (controls) 3 hr before the onset of the period of darkness. This dosage of melatonin was chosen after preliminary experiments had shown it to be the most effective [Sanchez-Barcelo et al., 19901. At 3, 5 , 7, 9, 1 1 , and 13 weeks of age, batches of 20 animals (ten controls and ten aMTtreated) were sacrificed. The second (thoracic) pair of mammary glands were dissected. Of each pair of glands, one was fixed to be used in morphometric evaluation of development and the contralateral mammary gland was weighed fresh and then frozen at -20°C until it was processed to determine the DNA content as an index of the number of cells [Lewin, 19571. From the beginning of the experiment, animals were weighed weekly in order to establish the rate of body weight gain. The onset of puberty was determined by the vaginal opening and first estrus. In postpubertal animals, vaginal smears were analyzed in order to verify that the estrous cycle stage distribution was not different in control and experimental animals, thus avoiding possible differences between groups due to cyclic changes in mammary DNA synthesis [Sinha and Tucker, 19661. Morphometric analysis
Glands were analyzed in wholemount preparations. After excision, glands were placed on dacron rafts, fixed in acetic:ethanol (25:75),stained with alumcarmine, and mounted with acrylic resin on slides. In each gland, the total number of terminal end buds (TEB), lateral buds (LB), terminal ducts (TD), and alveolar buds (AB) was counted under a light microscope with the aid of a grid. The mammary area, taken as the area enclosed by a line traced
14
around the periphery of the ducts, was measured with a planimeter. Measurement of DNA content
Each gland was homogenized with a Polytron for 20 sec in phosphate-buffered saline (0.05 M Na,PO,, 2.OM NaCl, 0.003 M EDTA) at a final concentration of 10 mg tissue/ml and centrifuged (2,500 rpm, 10 min). Duplicated aliquots (100 p1) were used to measure DNA following the procedure based on the enhancement of fluorescence of bisbenzimidazole (Sigma) on binding to DNA, as described by Labarca and Paigen [ 19801. Highly polymerized calf thymus DNA (Sigma) was used as standard. In order to analyze mammary gland growth relative to body growth, a plot of the log,, of mammary gland DNA content against the log,, of the surface areaof the body (estimated as BWz’3) was performed [Nagasawa et al., 19671. The quotient (Q) between the increase in the log,, of the DNA and the increase in the log,,, of the body surface area for a given time interval indicates whether growth of the gland is faster than (positively allometric, Q > l ) , similar to (isometric, Q = l), or slower than (negatively allometric, Q < l ) body growth as a whole. The quotient Q at each time interval (from week A to week B) was calculated as: QA.B = (log,, DNA, -log,, DNA,)/(log,, BW, - log,, BWJ. Statistical analysis
Data were processed by ANOVA and Student’s t-test for differences between means (control vs. melatonin) [Zar, 19841. Results
The age of vaginal opening (VO) and first estrus (FE) was not significantly different between control and melatonin treated mice (Controls: VO = 43.0 It 1.5 days, FE = 44.9 S 3.3 days; aMTtreated: VO = 42.6 k I . 1 days, FE = 45.07 5 3.4 days). Figure 1A shows the changes of body weight from the third week of age (start of the experiment). A significant reduction was found in the body weight of mice treated with aMT but only from the onset of puberty (7th week of age). When the rate of growth (expressed as body weight increase in grams per week) is considered, four different stages can be observed (Fig. 1B): (1) after the first week of treatment (weeks 3 to 4 of life), the body weight increase was higher in aMT-treated animals than in controls; (2) from week 4 to week 6 , the rate of body weight gain decreased in both groups of animals,
Melatonin and mammary gland growth 70
-
0-0 *--•
E E
Control Melotonin
50
d
10
I
3
4
5
6
7
8
9
1011
3
5
9
7
11
13
1 2 1 3
Age (weeks)
F i g. 2 . Area of the second thoracic mammary gland of female BALBc mice receiving melatonin (200 kgianimaliday) or vehicle from 3 weeks of age. Differences between means as in Figure 1 .
0-0
o- -
BALBc mice receiving melatonin (200 kg/animal/day) or vehicle (controls). Differences between means are expressed: a, P
Melatonin inhibits mammary gland development in female mice Mediavilla MD, San Martin M , Sgnchez-Barcelo EJ. Melatonin inhibits mammary gland development in female mice. J . Pineal Res. 1992: 1 3: 1 3- 19.
~
Abstract: The objective of this study was to determine whether melatonin (aMT) influences the postnatal development of the mammary gland parenchyma in female mice from the time of weaning to adulthood. Twenty-one-day-old female BALBc mice were treated with daily subcutaneous injections of aMT (200 pg) or diluent, 3 hr before the onset of darkness (photoperiod LD 12: 12). At 3, 5 , 7, 9, 1 1 , and 13 weeks of age, batches of 20 animals (ten controls and ten aMT-treated) were sacrificed and the second pair of mammary glands were dissected to evaluate their degree of development. Melatonin decreased body weight gain from 2 weeks before until 2 weeks after the onset of puberty. Treatment with aMT also resulted in a lower DNA content and smaller area of the mammary gland from the time of puberty until the end of the study. In aMT-treated mice the phase of highly positive allometric growth began 2 weeks later, but ended at the same time as in controls ( 1 lth week of life). Finally, aMT decreased the development of terminal, lateral, and alveolar buds while it increased the number of terminal ducts per gland. We conclude that pharmacological doses of aMT ( I ) reduce body weight gain at the peripuberal age; (2) partially inhibit postnatal mammary gland development by reducing the number of epithelial structures representing sites of growth and increasing that of structures representing the final state of ductal growth in virgin animals; (3) delay the onset of and shorten the phase of rapid mammary growth occurring in early postpuberal age.
Introduction
Mammary gland development is the result of a complex interplay of many hormones and local growth factors [see reviews from Topper and Freeman, 1980; Tucker, 1981; Imagawa et al., 19901. Melatonin (aMT) modulates circulating levels of some of the gonadal and pituitary hormones [Reiter, 19801 that control mammogenesis. Consequently, a possible role of this pineal hormone in the control of mammary gland development could be expected. Although the influence of the pineal gland and its hormones on the growth of mammary tumors has been extensively studied [Danforth et al., 1983; Shah et al., 1984; Kothari, 1988; Sanchez-Barcelo et al., 1988; Cos et al., 1989; Blask et al., 19901, the possible role of melatonin in the development of normal mammary tissue is still poorly understood. Mishkinsky et al. [1966] found no differences in the mammary glands of 100-day-old female rats pinealectomized at weaning or left intact. However, normal rats exposed to continuous lighting from
M.D. Mediavilla, M. San Martin, and E.J. Sanchez-Barcelo Department of Physiology and Pharmacology, School of Medicine, University of Cantabria, Spain
Key words: melatonin-mammary gland-body weight-female BALBc mice-terminal end bud-lateral bud-terminal duct-alveolar bud Address reprint requests to E.J. Sanchez-Barcelo, Departamento de Fisiologia y Farmacologia, Facultad de Medicina, Universidad de Cantabria, 3901 1 Santander, Spain Received January 16, 1992; accepted April 16, 1992
birth show increased mammary development, which is prevented by administering melatonin [Mhatre et al., 19841. We have recently demonstrated that aMT, in vivo, reduces mouse mammary gland growth in response to estradiol + progesterone injections [Sanchez-Barcelo et al., 19901. Orally administered aMT was also effective in reducing mammary growth in heifers [Sanchez Barcelo et al., 19911. The influence of aMT on mammary gland growth could be secondary to aMT-induced changes in the serum concentration of mammotrophic hormones such as estrogens and prolactin [Reiter, 1980; Sanchez-Barcel6 et al., 19911. However, direct actions of aMT on mammary tissue have also been demonstrated by in vitro studies in which aMT at micromolar concentrations reduced the growth of mammary tissue induced by mammotrophic hormones added to the culture medium, whereas nanomolar concentrations had opposite effects [Sanchez-Barcelo et al., 19901. The objective of the present study was to deter-
13
Mediavilla et al.
mine whether aMT influences the physiological postnatal development (not induced by exogenous mammotrophic hormones) of mammary gland parenchyma in female mice from weaning to adulthood.
Material and methods Animals and housing conditions
In all experiments, female BALBc mice (Iffa Credo, France) were used. Animals were housed in our vivarium at 23 5 1°C room temperature and exposed to a LD12:12 photoperiod. Food and tap water were provided ad libitum. Experimental design
From 21 days of age, animals received a daily subcutaneous injection of aMT (Sigma) (200 pg) or the diluent (controls) 3 hr before the onset of the period of darkness. This dosage of melatonin was chosen after preliminary experiments had shown it to be the most effective [Sanchez-Barcelo et al., 19901. At 3, 5 , 7, 9, 1 1 , and 13 weeks of age, batches of 20 animals (ten controls and ten aMTtreated) were sacrificed. The second (thoracic) pair of mammary glands were dissected. Of each pair of glands, one was fixed to be used in morphometric evaluation of development and the contralateral mammary gland was weighed fresh and then frozen at -20°C until it was processed to determine the DNA content as an index of the number of cells [Lewin, 19571. From the beginning of the experiment, animals were weighed weekly in order to establish the rate of body weight gain. The onset of puberty was determined by the vaginal opening and first estrus. In postpubertal animals, vaginal smears were analyzed in order to verify that the estrous cycle stage distribution was not different in control and experimental animals, thus avoiding possible differences between groups due to cyclic changes in mammary DNA synthesis [Sinha and Tucker, 19661. Morphometric analysis
Glands were analyzed in wholemount preparations. After excision, glands were placed on dacron rafts, fixed in acetic:ethanol (25:75),stained with alumcarmine, and mounted with acrylic resin on slides. In each gland, the total number of terminal end buds (TEB), lateral buds (LB), terminal ducts (TD), and alveolar buds (AB) was counted under a light microscope with the aid of a grid. The mammary area, taken as the area enclosed by a line traced
14
around the periphery of the ducts, was measured with a planimeter. Measurement of DNA content
Each gland was homogenized with a Polytron for 20 sec in phosphate-buffered saline (0.05 M Na,PO,, 2.OM NaCl, 0.003 M EDTA) at a final concentration of 10 mg tissue/ml and centrifuged (2,500 rpm, 10 min). Duplicated aliquots (100 p1) were used to measure DNA following the procedure based on the enhancement of fluorescence of bisbenzimidazole (Sigma) on binding to DNA, as described by Labarca and Paigen [ 19801. Highly polymerized calf thymus DNA (Sigma) was used as standard. In order to analyze mammary gland growth relative to body growth, a plot of the log,, of mammary gland DNA content against the log,, of the surface areaof the body (estimated as BWz’3) was performed [Nagasawa et al., 19671. The quotient (Q) between the increase in the log,, of the DNA and the increase in the log,,, of the body surface area for a given time interval indicates whether growth of the gland is faster than (positively allometric, Q > l ) , similar to (isometric, Q = l), or slower than (negatively allometric, Q < l ) body growth as a whole. The quotient Q at each time interval (from week A to week B) was calculated as: QA.B = (log,, DNA, -log,, DNA,)/(log,, BW, - log,, BWJ. Statistical analysis
Data were processed by ANOVA and Student’s t-test for differences between means (control vs. melatonin) [Zar, 19841. Results
The age of vaginal opening (VO) and first estrus (FE) was not significantly different between control and melatonin treated mice (Controls: VO = 43.0 It 1.5 days, FE = 44.9 S 3.3 days; aMTtreated: VO = 42.6 k I . 1 days, FE = 45.07 5 3.4 days). Figure 1A shows the changes of body weight from the third week of age (start of the experiment). A significant reduction was found in the body weight of mice treated with aMT but only from the onset of puberty (7th week of age). When the rate of growth (expressed as body weight increase in grams per week) is considered, four different stages can be observed (Fig. 1B): (1) after the first week of treatment (weeks 3 to 4 of life), the body weight increase was higher in aMT-treated animals than in controls; (2) from week 4 to week 6 , the rate of body weight gain decreased in both groups of animals,
Melatonin and mammary gland growth 70
-
0-0 *--•
E E
Control Melotonin
50
d
10
I
3
4
5
6
7
8
9
1011
3
5
9
7
11
13
1 2 1 3
Age (weeks)
F i g. 2 . Area of the second thoracic mammary gland of female BALBc mice receiving melatonin (200 kgianimaliday) or vehicle from 3 weeks of age. Differences between means as in Figure 1 .
0-0
o- -
BALBc mice receiving melatonin (200 kg/animal/day) or vehicle (controls). Differences between means are expressed: a, P
