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Collagen of Rat Mammary Glands During Post-Lactational Involution
T E C H N I C A L NOTES
1185
Collagenof Rat Mammary GlandsDuring Post-tactational Involution Abstract
Nucleic acid and collagen content of parenchymal and fat pad segments of mammary glands were determined on rats killed at Days 0, 5, 15, 30, and 60 post-weaning. By 5 days post-weaning, involution of the mammary gland parenehyma was accompanied by large and rapid decreases in nucleic acid (DNA and RNA) of the tissue. Conversely, DNA and RNA content of the fat pad segment increased following weaning. By the 15th day of involution, DNA and RNA in both the parenchyma and fat pad segments were similar to amonnts in virgin animals. Parenchymal hydroxyproline (collagen), on the other hand, did not decrease until the 15th day at which time it leveled off to amounts comparable to virgin controls (4.0 rag/100 g body wt). Fat pad hydroxyproline increased for 15 days following weaning reaching amounts in virgin mammary glands (1.6 rag/100 g body vet). The collagenons framework of the mammary gland involuted slowly compared to cell numbers but, nevertheless, reached amounts in virgin glands at about the same time as the nucleic acid components of the g/and (15 days). Introduction
Post-lactational involution of the rat mammary gland is accompanied by large and rapid decreases in nucleic acid (2, 12) and represents atrophying of the duct and lobulealveolar systems (1). A return to the virgin state usually occurs between Days 10 to 20 of involution. On the other hand, the collagenous framework of the mammary gland parenchyma appears to be more resistant to the processes of involution. Removing rat pups from lactating mothers at various stages of lactation resulted, on the average, in only a 12~ decrease in hydroxyproline of the mammary gland (a measure of connective tissue collagen) 4 days later whereas deoxyribonucleic acid (cell numbers) decreased 57?o (9). It is not known whether the collagenous framework of the mammary gland, like the lobulealveolar systems, also involutes to the virgin state. Received for publication February 15, 1972.
The present experiment, therefore, was designed to determine changes in mammary collagen of both parenchymal and fat pad portions of the gland during post-]actational involution. Materials and Methods
Primiparous Sprague-Dawley rats in advanced gestation were housed in individual cages. On Days 1 and 3 postpartum, litter size was adjusted to 10 pups. Litter weights were taken on the third day. On the 16th day of lactation, litters were removed and weighed. Groups of mother rats were then killed immediately following weaning and on Days 5, 15, 30, and 60 post-weaning. All rats other than those killed immediately after weaning were ear-notched for identification and housed six to a cage. In addition, one group of virgin females was used to establish a base line for virgin females. At autopsy, the six abdominal inguinal mammary glands were removed and only that portion of the fat pad tissue surrounding the periphery of the glands was removed. Mammary lymph node.s were not discarded. The parenehymal portion did not consist solely of parenchymal cells but in all likelihood contained, to a lesser extent, adipose cells, leukocytes, fibroblasts and other connective tissue cells (5, 7). Parenchyanal and fat pad portions were then defatted by successive changes in 95~ ethyl alcohol, chloroform-methanol (1:2) and ether. Dried tissues were weighed, ground to a fine powder, and stored at --20 C until analyzed for nucleic acid (11) and hydroxyproline (OHP) (10). Results and Discussion
Total litter weight gain from the 3rd to the 16th day of lactation and final body weight of mother rats for all animals (Table 1) did not differ significantly (P > .05) among the various treatment groups. This indicated uniform distribution of rats among the various groups, thereby minimizing possible bias attributable to these two measures. l?arenchymal weight (mg/100 g body wt) decreased 16 and 71% by 5 and 15 days postweaning whereas fat pad weight increased 9 and 41%. By the 15th day of involution, both the parenehyrnal and fat pad weights approached amounts which were not significantly different (P > .05) from virgin controls. JOURNAL OF DAIRY SCIENCE VOL. 55, NO, 8
1186
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JOURNAL o r
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55,
No.
8
OF
DAIRY
SCIENCE
This atrophying of the mammary parenchyma and increase of the fat pad was reflected in changes in total DNA content (cell numbers) of these tissues. Deoxyribonucleic acid content (rag/100 g body wt) of the parenchymal portion decreased 30 and 72% by Days 5 and 15 post-weaning and approached amounts in virgin glands between Days 15 and and 60 post-weaning. This supports the results of others (1, 2, 12) who found a return of the mammary gland to the virgin state between Days 10 and 20 post-weaning. In contrast, fat pad DNA (mg/100 g body wt) increased twofold by 15 days post-weaning and reached maximum between Days 15 and 60. These values were higher than in lactating glands (P < .05) and were comparable to those in virgin mammary glands (P > .05). The increase in fat pad DNA and decrease in parenchymal DNA following weaning agrees with reported results (6) and reflects a greater proportion of cells in the fat pad as a result of rapidly regressing pareamhyma. During lactation the mammary parenchyrna appeared to proliferate at the expense of the fat pad. Parenchyrnal RNA (a measure of protein synthetic activity), however, did not approach virgin amounts until 30 days post-weaning, suggesting that the protein synthetic activity of the epithelial cells at 15 days, although greatly reduced from Iactational amounts, still retained a higher degree of activity when compared to virgins. Fat pad RNA following weaning rema/ned unchanged for the first 5 days, increased at Day 15 (P < .01) and then leveled off between Days 15 and 60. In general, changes in fat pad RNA paralleled changes in DNA for this tissue. Unlike DNA and tlNA which decreased substantially by Day 5 post-weaning, parenchymal OHP did not degrade to any significant extent (P < •01) until the 15th day of involution at which time it was 23% less than at the 16th day of lactation. This: suggested that although collagen was more resistant to the process of involution than parenehyma/ cells, it was not totally resistant to involution as suggested by earlier work (3). Fat pad OHP remained unchanged until 5 days post-weaning, then increased (P < .01) at 15 days. Values between Days 15 and 60 were greater (P < .01) than at Day 16 of lactation but were not different from virgin animals. Like fat pad DNA, increase in fat pad collagen probably reflected an expansion of the extraparenchymal segment of the mammary gland as a result of rapid regression of the parenchyma. However, this inca'ease in fat pad collagerL following weaning did not ac-
TECHNICAL NOTES
count for all of the collagen lost from the parenchymal segment. When parenchymal and fat pad segments were combined in total mammary gland O H P (Table 1), loss of collagen (P .~ .01) was evident at 15 days post-weaning. Since both soluble and insoluble forms of collagen exist within tissues (4), it is possible that this loss of collagen during involution represented a greater loss of the soluble fraction than the insoluble. It has been suggested that collagen, once deposited, is rather inert and probably lasts for the life of a tissue (13). Combined with previous results (8, 9), it appears that collagen in the mammary gland of the rat fluctuates in response to the physiological state of the animal. It increases rapidly following puberty, levels off at approximately 60 days of age, and remains essentially unchanged thereafter in the virgin rat (8). Following conception, there is another phase of inca'ease lasting until the 12th day of pregnancy after which time it again levels off (8). Following parturition there is another spurt of hyperplasia reaching peaks around the 16th day of lactation (9). Atrophy of the collagenous framework occurs following weaning until amounts approximating the virgin state are reached. M. J. PAAPE, A. J. KRAL, and R. H. MILLER Animal Science Research Division, USDA, Beltsville, Maryland 20705 References
( I ) Anderson, R. R., and C. W. Turner. 1963. Involution of the mammary gland duct and lobule-alveolar systems in the mouse. Proc. Soe. Exp. Biol. Med., 113:333. (2) Grifllth, D. R., and C. W. Turner. 1961. Normal and experimental involution of rat mammary gland. Proc. Soc. Exp. BioL Med., 107:668. (3) Harkness, M. L. R., and R. D. Harkness. 1956. The effect of pregnancy and lacta-
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
1187
tion on the collagen content of the mainmary gland of the rat. J. Physiol., 132:476. Kowalewski, K., and S. Yong. 1968. Effect of growth hormone, an anabolic steroid, and cortisone npon various fractions of skin and bone hydroxyproline in mice. Canadian J. Physiol. and Pharmacol., 46:591. Nicoll, C. S., and H. A. Tucker. 1965. Estimates of parenehymal, stromal, and lymph node deoxyribonucleic acid in mammary glands in C3H/Crgl/2 mice. Life Sci., 4:993. Paape, M. J. 1967. Influence of pregnancy and dry period on lactation in the rat; Ph.D. Dissertation. Michigan State University, East Lansing. Paape, M. J., and C. Desjardins. 1971. Nursing duration and suckling intensity: effects on plasma corticosterone, circulating leukocytes, and mannnary nucleic acids. Proe. Soc. Exp. Biol. Med., 138:12. Paape, M. J., and Y. N. Sinha. 1971. Nucleic acid and collagen content of mammary glands between 30 and 80 days of age in normal and ovariectomized rats and during pregnancy. J. Dairy Sci., 54:1068. Paape, M. J., and H. A. Tucker. 1969. Mammal T nucleic acid, hydroxyproline, and hexosamine of pregnant rats during lactation and post-lactational involution. J. Dairy Sci., 52:380. Prockop, D. J., and S. Udenfriend. 1960. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem., 1:228. Tucker, H. A. 1964. Influence of number of suckling young on nucleic acid content of lactating rat mammary gland. Proe. Soc. Exp. Biol. Med., 116:218. Tucker, H. A., and R. P. Reeee. 1963. Nucleic acid content of rat mammary glands during post-laetational involution. Proe. Soe. Exp. Biol. Med., 112:1002. White, A., P. Handler, and E. L. Smith, 1964. Principles of Biochemistry. McGrawHill, New York City, pp. 770, 3rd edition.
JOURNAL OF DAIRY SCIENCE VOL,
55, NO.
gl
1185
Collagenof Rat Mammary GlandsDuring Post-tactational Involution Abstract
Nucleic acid and collagen content of parenchymal and fat pad segments of mammary glands were determined on rats killed at Days 0, 5, 15, 30, and 60 post-weaning. By 5 days post-weaning, involution of the mammary gland parenehyma was accompanied by large and rapid decreases in nucleic acid (DNA and RNA) of the tissue. Conversely, DNA and RNA content of the fat pad segment increased following weaning. By the 15th day of involution, DNA and RNA in both the parenchyma and fat pad segments were similar to amonnts in virgin animals. Parenchymal hydroxyproline (collagen), on the other hand, did not decrease until the 15th day at which time it leveled off to amounts comparable to virgin controls (4.0 rag/100 g body wt). Fat pad hydroxyproline increased for 15 days following weaning reaching amounts in virgin mammary glands (1.6 rag/100 g body vet). The collagenons framework of the mammary gland involuted slowly compared to cell numbers but, nevertheless, reached amounts in virgin glands at about the same time as the nucleic acid components of the g/and (15 days). Introduction
Post-lactational involution of the rat mammary gland is accompanied by large and rapid decreases in nucleic acid (2, 12) and represents atrophying of the duct and lobulealveolar systems (1). A return to the virgin state usually occurs between Days 10 to 20 of involution. On the other hand, the collagenous framework of the mammary gland parenchyma appears to be more resistant to the processes of involution. Removing rat pups from lactating mothers at various stages of lactation resulted, on the average, in only a 12~ decrease in hydroxyproline of the mammary gland (a measure of connective tissue collagen) 4 days later whereas deoxyribonucleic acid (cell numbers) decreased 57?o (9). It is not known whether the collagenous framework of the mammary gland, like the lobulealveolar systems, also involutes to the virgin state. Received for publication February 15, 1972.
The present experiment, therefore, was designed to determine changes in mammary collagen of both parenchymal and fat pad portions of the gland during post-]actational involution. Materials and Methods
Primiparous Sprague-Dawley rats in advanced gestation were housed in individual cages. On Days 1 and 3 postpartum, litter size was adjusted to 10 pups. Litter weights were taken on the third day. On the 16th day of lactation, litters were removed and weighed. Groups of mother rats were then killed immediately following weaning and on Days 5, 15, 30, and 60 post-weaning. All rats other than those killed immediately after weaning were ear-notched for identification and housed six to a cage. In addition, one group of virgin females was used to establish a base line for virgin females. At autopsy, the six abdominal inguinal mammary glands were removed and only that portion of the fat pad tissue surrounding the periphery of the glands was removed. Mammary lymph node.s were not discarded. The parenehymal portion did not consist solely of parenchymal cells but in all likelihood contained, to a lesser extent, adipose cells, leukocytes, fibroblasts and other connective tissue cells (5, 7). Parenchyanal and fat pad portions were then defatted by successive changes in 95~ ethyl alcohol, chloroform-methanol (1:2) and ether. Dried tissues were weighed, ground to a fine powder, and stored at --20 C until analyzed for nucleic acid (11) and hydroxyproline (OHP) (10). Results and Discussion
Total litter weight gain from the 3rd to the 16th day of lactation and final body weight of mother rats for all animals (Table 1) did not differ significantly (P > .05) among the various treatment groups. This indicated uniform distribution of rats among the various groups, thereby minimizing possible bias attributable to these two measures. l?arenchymal weight (mg/100 g body wt) decreased 16 and 71% by 5 and 15 days postweaning whereas fat pad weight increased 9 and 41%. By the 15th day of involution, both the parenehyrnal and fat pad weights approached amounts which were not significantly different (P > .05) from virgin controls. JOURNAL OF DAIRY SCIENCE VOL. 55, NO, 8
1186
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JOURNAL o r
DAIRY
SCIENCE V O L .
55,
No.
8
OF
DAIRY
SCIENCE
This atrophying of the mammary parenchyma and increase of the fat pad was reflected in changes in total DNA content (cell numbers) of these tissues. Deoxyribonucleic acid content (rag/100 g body wt) of the parenchymal portion decreased 30 and 72% by Days 5 and 15 post-weaning and approached amounts in virgin glands between Days 15 and and 60 post-weaning. This supports the results of others (1, 2, 12) who found a return of the mammary gland to the virgin state between Days 10 and 20 post-weaning. In contrast, fat pad DNA (mg/100 g body wt) increased twofold by 15 days post-weaning and reached maximum between Days 15 and 60. These values were higher than in lactating glands (P < .05) and were comparable to those in virgin mammary glands (P > .05). The increase in fat pad DNA and decrease in parenchymal DNA following weaning agrees with reported results (6) and reflects a greater proportion of cells in the fat pad as a result of rapidly regressing pareamhyma. During lactation the mammary parenchyrna appeared to proliferate at the expense of the fat pad. Parenchyrnal RNA (a measure of protein synthetic activity), however, did not approach virgin amounts until 30 days post-weaning, suggesting that the protein synthetic activity of the epithelial cells at 15 days, although greatly reduced from Iactational amounts, still retained a higher degree of activity when compared to virgins. Fat pad RNA following weaning rema/ned unchanged for the first 5 days, increased at Day 15 (P < .01) and then leveled off between Days 15 and 60. In general, changes in fat pad RNA paralleled changes in DNA for this tissue. Unlike DNA and tlNA which decreased substantially by Day 5 post-weaning, parenchymal OHP did not degrade to any significant extent (P < •01) until the 15th day of involution at which time it was 23% less than at the 16th day of lactation. This: suggested that although collagen was more resistant to the process of involution than parenehyma/ cells, it was not totally resistant to involution as suggested by earlier work (3). Fat pad OHP remained unchanged until 5 days post-weaning, then increased (P < .01) at 15 days. Values between Days 15 and 60 were greater (P < .01) than at Day 16 of lactation but were not different from virgin animals. Like fat pad DNA, increase in fat pad collagen probably reflected an expansion of the extraparenchymal segment of the mammary gland as a result of rapid regression of the parenchyma. However, this inca'ease in fat pad collagerL following weaning did not ac-
TECHNICAL NOTES
count for all of the collagen lost from the parenchymal segment. When parenchymal and fat pad segments were combined in total mammary gland O H P (Table 1), loss of collagen (P .~ .01) was evident at 15 days post-weaning. Since both soluble and insoluble forms of collagen exist within tissues (4), it is possible that this loss of collagen during involution represented a greater loss of the soluble fraction than the insoluble. It has been suggested that collagen, once deposited, is rather inert and probably lasts for the life of a tissue (13). Combined with previous results (8, 9), it appears that collagen in the mammary gland of the rat fluctuates in response to the physiological state of the animal. It increases rapidly following puberty, levels off at approximately 60 days of age, and remains essentially unchanged thereafter in the virgin rat (8). Following conception, there is another phase of inca'ease lasting until the 12th day of pregnancy after which time it again levels off (8). Following parturition there is another spurt of hyperplasia reaching peaks around the 16th day of lactation (9). Atrophy of the collagenous framework occurs following weaning until amounts approximating the virgin state are reached. M. J. PAAPE, A. J. KRAL, and R. H. MILLER Animal Science Research Division, USDA, Beltsville, Maryland 20705 References
( I ) Anderson, R. R., and C. W. Turner. 1963. Involution of the mammary gland duct and lobule-alveolar systems in the mouse. Proc. Soe. Exp. Biol. Med., 113:333. (2) Grifllth, D. R., and C. W. Turner. 1961. Normal and experimental involution of rat mammary gland. Proc. Soc. Exp. BioL Med., 107:668. (3) Harkness, M. L. R., and R. D. Harkness. 1956. The effect of pregnancy and lacta-
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
1187
tion on the collagen content of the mainmary gland of the rat. J. Physiol., 132:476. Kowalewski, K., and S. Yong. 1968. Effect of growth hormone, an anabolic steroid, and cortisone npon various fractions of skin and bone hydroxyproline in mice. Canadian J. Physiol. and Pharmacol., 46:591. Nicoll, C. S., and H. A. Tucker. 1965. Estimates of parenehymal, stromal, and lymph node deoxyribonucleic acid in mammary glands in C3H/Crgl/2 mice. Life Sci., 4:993. Paape, M. J. 1967. Influence of pregnancy and dry period on lactation in the rat; Ph.D. Dissertation. Michigan State University, East Lansing. Paape, M. J., and C. Desjardins. 1971. Nursing duration and suckling intensity: effects on plasma corticosterone, circulating leukocytes, and mannnary nucleic acids. Proe. Soc. Exp. Biol. Med., 138:12. Paape, M. J., and Y. N. Sinha. 1971. Nucleic acid and collagen content of mammary glands between 30 and 80 days of age in normal and ovariectomized rats and during pregnancy. J. Dairy Sci., 54:1068. Paape, M. J., and H. A. Tucker. 1969. Mammal T nucleic acid, hydroxyproline, and hexosamine of pregnant rats during lactation and post-lactational involution. J. Dairy Sci., 52:380. Prockop, D. J., and S. Udenfriend. 1960. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem., 1:228. Tucker, H. A. 1964. Influence of number of suckling young on nucleic acid content of lactating rat mammary gland. Proe. Soc. Exp. Biol. Med., 116:218. Tucker, H. A., and R. P. Reeee. 1963. Nucleic acid content of rat mammary glands during post-laetational involution. Proe. Soe. Exp. Biol. Med., 112:1002. White, A., P. Handler, and E. L. Smith, 1964. Principles of Biochemistry. McGrawHill, New York City, pp. 770, 3rd edition.
JOURNAL OF DAIRY SCIENCE VOL,
55, NO.
gl