0013-7227/78/1024-1317$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 4 Printed in U.S.A.
MAINTENANCE OF SERUM SOMATOMEDIN ACTIVITY IN HYPOPHYSECTOMIZED PREGNANT RATSl William H. Daughaday and Milan Kapadia Department of Medicine, Metabolism Division, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT. Pregnant female rats were hypophysectomized on the 14th day of pregnancy without fetal loss or premature delivery. Completeness of hypophysectomy was confirmed by measurements of serum growth hormone and prolactin. Serum somatomedin activities were measured by the hypophysectomized rat costal cartilage in vitro assay using a human serum standard. In this assay somatomedin activity of normal rat serum is about four times that of normal human serum. Forty-eight hours after hypophysectomy serum somatomedin was maintained at 3.9 + 1.0 units/ml. At this time
after operation somatomedin activity of nonpregnant hypophysectomized rat serum would be virtually undetectable. Somatomedin concentrations were still maintained on the 18th and 20th day of pregnancy. However, within 24 hours after delivery somatomedin had fallen to less than 0.6 units/ml and by three days post delivery were less than 0.2 units/ml. These observations indicate that the fetal-placental unit, probably by secreting a chorionic somatomammotropin, can maintain the concentration of growth hormone-dependent somatomedin in the absence of the pituitary.
The placentas of many mammalian species synthesize and secrete peptides with lactogenic and growth hormone-like properties. It is unresolved whether this chorionic somatomammotropin (CS) exerts a significant growth hormone-like effect in the mother. There is reason to believe that the human somatomammotropin (hCS) may contribute to the insulin resistance of pregnancy (1). In order to obtain more direct evidence of the ability of the fetalplacental unit to replace maternal pituitary secretion we have hypophysectomized (hypox) rats on the 14th day of pregnancy and measured serum somatomedin by hypophysectomized rat cartilage bioas-say. We have previously shown that hypophysectomy of nonpregnant rats leads to a rapid disappearance of somatomedin activity with a half-life of about 4 to 5 hours (2 ) .
nant female Sprague-Dawley rats were obtained from Charles River Breeding Laboratory. The first day of pregnancy was determined after timed matings by finding a vaginal plug. Hypophysectomy was carried out on the 14th day of pregnancy. The rats were shipped on the following day. The rats studied on the 16th day of pregnancy were decapitated and blood was collected in Boston, courtesy of Dr. Seymour Reichlin. All other studies were conducted in St. Louis. The hypophysectomized and control pregnant rats tolerated transport to St. Louis without fetal or maternal loss. Pregnancy continued to term without any replacement hormones. All animals delivered on either the 21st or 22nd day of gestation. The pups were removed from the mother after birth. Pups from hypophysectomized mothers were noted to contain a small amount of gastric milk. Blood was obtained by decapitation. After clotting, the serum was collected and stored at -17 C until assay. Before assay all rat sera, diluted 1:10 with assay medium, were heated to 65 C for 30 minutes. This heat
METHODS Normal and hypophysectomized pregISubmitted October 28, 1977
1317
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1318
RAPID COMMUNICATIONS
treatment reduced inhibitors occasionally found in rat serum. Somatomedin activity is unaffected (3). Completeness of hypophysectomy was determined by double antibody radioimmunoassay measurement of growth hormone and prolactin using standards and antisera distributed by the Rat Pituitary Distribution Program, NIAMDD. Somatomedin activity of serum was determined by the hypophysectomized rat costal cartilage method (4). In these assays 80 segments of cartilage were prepared from the rib cage of a single rat. Ten segments of cartilage were used for each concentration of standard normal human serum or test rat serum. In most assays a normal human serum pool (potency 1.17 U/ml) was tested in 1%, 4% and 9% dilutions. Because normal rat serum has about four times the somatomedin activity of human serum most rat sera were tested at 1% and 4% dilutions. Post-partum hypox rat serum was tested at 4% and 9%. The regression line generated by plotting response vs logarithm of the dose was calculated. Potency was determined by parallel line analysis by a previously reported method using a Hewlett Packard desk calculator (5). Lambda for the standard line varied from 0.17 to 0.50. RESULTS The completeness of hypophysectomy was determined by radioimmunoassay (Table 1). One rat, after hypophysectomy had a serum prolactin concentration of 250 ng/ml and a GH concentration of 125 ng/ml. This rat was excluded from the group. The remaining rats had GH levels which were very low or undetectable. Low levels of prolactin were detected in the sera of several of the hypophysectomized rats. In the control pregnant rats immunoassayable GH was easily detected. Prolactin levels in pregnancy were suppressed at 16 days and were elevated 1 day post-partum. The somatomedin activities of
Endo
Vol. 102, No. 4 Printed in U.S.A.
MAINTENANCE OF SERUM SOMATOMEDIN ACTIVITY IN HYPOPHYSECTOMIZED PREGNANT RATSl William H. Daughaday and Milan Kapadia Department of Medicine, Metabolism Division, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT. Pregnant female rats were hypophysectomized on the 14th day of pregnancy without fetal loss or premature delivery. Completeness of hypophysectomy was confirmed by measurements of serum growth hormone and prolactin. Serum somatomedin activities were measured by the hypophysectomized rat costal cartilage in vitro assay using a human serum standard. In this assay somatomedin activity of normal rat serum is about four times that of normal human serum. Forty-eight hours after hypophysectomy serum somatomedin was maintained at 3.9 + 1.0 units/ml. At this time
after operation somatomedin activity of nonpregnant hypophysectomized rat serum would be virtually undetectable. Somatomedin concentrations were still maintained on the 18th and 20th day of pregnancy. However, within 24 hours after delivery somatomedin had fallen to less than 0.6 units/ml and by three days post delivery were less than 0.2 units/ml. These observations indicate that the fetal-placental unit, probably by secreting a chorionic somatomammotropin, can maintain the concentration of growth hormone-dependent somatomedin in the absence of the pituitary.
The placentas of many mammalian species synthesize and secrete peptides with lactogenic and growth hormone-like properties. It is unresolved whether this chorionic somatomammotropin (CS) exerts a significant growth hormone-like effect in the mother. There is reason to believe that the human somatomammotropin (hCS) may contribute to the insulin resistance of pregnancy (1). In order to obtain more direct evidence of the ability of the fetalplacental unit to replace maternal pituitary secretion we have hypophysectomized (hypox) rats on the 14th day of pregnancy and measured serum somatomedin by hypophysectomized rat cartilage bioas-say. We have previously shown that hypophysectomy of nonpregnant rats leads to a rapid disappearance of somatomedin activity with a half-life of about 4 to 5 hours (2 ) .
nant female Sprague-Dawley rats were obtained from Charles River Breeding Laboratory. The first day of pregnancy was determined after timed matings by finding a vaginal plug. Hypophysectomy was carried out on the 14th day of pregnancy. The rats were shipped on the following day. The rats studied on the 16th day of pregnancy were decapitated and blood was collected in Boston, courtesy of Dr. Seymour Reichlin. All other studies were conducted in St. Louis. The hypophysectomized and control pregnant rats tolerated transport to St. Louis without fetal or maternal loss. Pregnancy continued to term without any replacement hormones. All animals delivered on either the 21st or 22nd day of gestation. The pups were removed from the mother after birth. Pups from hypophysectomized mothers were noted to contain a small amount of gastric milk. Blood was obtained by decapitation. After clotting, the serum was collected and stored at -17 C until assay. Before assay all rat sera, diluted 1:10 with assay medium, were heated to 65 C for 30 minutes. This heat
METHODS Normal and hypophysectomized pregISubmitted October 28, 1977
1317
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 13:31 For personal use only. No other uses without permission. . All rights reserved.
1318
RAPID COMMUNICATIONS
treatment reduced inhibitors occasionally found in rat serum. Somatomedin activity is unaffected (3). Completeness of hypophysectomy was determined by double antibody radioimmunoassay measurement of growth hormone and prolactin using standards and antisera distributed by the Rat Pituitary Distribution Program, NIAMDD. Somatomedin activity of serum was determined by the hypophysectomized rat costal cartilage method (4). In these assays 80 segments of cartilage were prepared from the rib cage of a single rat. Ten segments of cartilage were used for each concentration of standard normal human serum or test rat serum. In most assays a normal human serum pool (potency 1.17 U/ml) was tested in 1%, 4% and 9% dilutions. Because normal rat serum has about four times the somatomedin activity of human serum most rat sera were tested at 1% and 4% dilutions. Post-partum hypox rat serum was tested at 4% and 9%. The regression line generated by plotting response vs logarithm of the dose was calculated. Potency was determined by parallel line analysis by a previously reported method using a Hewlett Packard desk calculator (5). Lambda for the standard line varied from 0.17 to 0.50. RESULTS The completeness of hypophysectomy was determined by radioimmunoassay (Table 1). One rat, after hypophysectomy had a serum prolactin concentration of 250 ng/ml and a GH concentration of 125 ng/ml. This rat was excluded from the group. The remaining rats had GH levels which were very low or undetectable. Low levels of prolactin were detected in the sera of several of the hypophysectomized rats. In the control pregnant rats immunoassayable GH was easily detected. Prolactin levels in pregnancy were suppressed at 16 days and were elevated 1 day post-partum. The somatomedin activities of
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