0013-7227/79/1051-0297$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 1 Printed in U.S.A.
Serum Somatomedin Activity after Hypophysectomy and during Parturition in Fetal Lambs* M. W. BRINSMEAD AND G. C. LIGGINS Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand
ABSTRACT. Somatomedins, detectable by sulfation factor activity (SFA) and/or somatomedin-like receptor activity (SmLRA), were measured in the serum of 72 normal fetal lambs of varying gestational ages and in 12 fetuses before and after hypophysectomy. Three hypophysectomized fetal lambs were infused with GH and/or insulin. Three intact fetal lambs were infused with glucose. Parturition was induced by infusion of synthetic ACTH into 5 fetal lambs. SmLRA and SFA were compared to a pool of adult sheep serum arbitrarily designated 1.00 U/ml. Serum SmLRA correlated significantly with gestational age (r = 0.65; P < 0.001), the linear regression line passing through 0.5 U/ml at 50 days and 2 U/ml at 150 days of gestation.
Serum SFA also correlated with gestational age (r = 0.40; n = 19; 0.1 > P > 0.05). Serum SmLRA and SFA were unchanged by hypophysectomy of the fetus and GH infusion but were reduced by insulin infusion. Plasma SmLRA correlated negatively with plasma insulin during glucose infusion (r = -0.67; P < 0.05). During induction of parturition with ACTH, serum SmLRA fell by a mean of 38% and concentrations correlated negatively with plasma cortisol (r = -0.62; P < 0.001). It is concluded that serum somatomedin activity in fetal sheep is not GH dependent. It is possible that somatomedin activity in cord blood after spontaneous delivery may not accurately reflect concentrations prevailing in fetal life. {Endocrinology 105: 297, 1979)
O
VER the past decade, a number of potent mitogens for cell multiplication have been described and characterized (1). Among these are the somatomedins, a family of related polypeptides which are insulin-like in structure and function and are thought by some investigators to mediate some of the actions of GH on growth (2, 3). Somatomedin activity in human cord blood increases over the last trimester of pregnancy (4) and correlates with birth size (4, 5). This suggests that these hormones may have a role in promoting fetal growth or that they may reflect the rate of growth processes in the fetus. However, nothing is known about the regulation of fetal somatomedins. In extrauterine life, the pituitary has a dominant influence over serum somatomedin activity (2, 3). Concentrations of serum somatomedins in hypopituitary rats and man are 30-50% of those in intact controls and can be restored by GH therapy (6, 7). This study was undertaken to assess the influence of the fetal pituitary on somatomedin activity in fetal lambs. Somatomedin concentrations were also studied in intact and hypophysectomized lambs during induced parturition, since it is possible that rising cortisol and estro-
gen concentrations which precede parturition (8) may affect serum somatomedin activity (9, 10). Materials and Methods Multiplication-stimulating activity (MSA) was purified from
the medium of the BRL-3A clone of rat liver cells (generously provided by Professor Temin, University of Wisconsin, Madison) by ion exchange and gel chromatography (11). The MSA used for iodination and standard in this study has been previously described (12). It had three major bands on polyacrylamide gel electrophoresis. Its potency was approximately twice that of a similarly prepared MSA produced by M. Rechler, NIH, and 1 mg was equivalent to 75 mU insulin in a bioassay using rat adipocytes. It contained no detectable immunoreactive insulin. Porcine insulin (615-D63-10; 25.4 U/mg) was donated by Dr. R. J. Hosley, Lilley Research Laboratories; ovine GH hormone (oGH; NIH-GH-S11; 0.56 IU/mg) was provided by the Hormone Distribution Officer, NIH; and insulin-like growth factors (IGFs I and II) were generous gifts from Professor R. Humbel, Biochemistry Institute, University of Zurich. Hormone assays Serum sulfation factor activity (SFA) was determined using SO4 uptake by porcine cartilage, as previously described (4). Sera at two dilutions (10% and 40%) were compared to four dilutions (5%, 10%, 20%, and 40%) of pooled serum from two normal rams arbitrarily given a potency of 1.00 U/ml SFA. Potency was calculated by the method of Finney (13) from four points of standard and test serum having the greatest parallel-
35
Received April 28,1978. Address all correspondence and requests for reprints to: Dr. Maxwell Brinsmead, Department of Obstetrics and Gynaecology, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, QLD. 4029, Australia. * This work was supported by the Medical Research Council of New Zealand.
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BRINSMEAD AND LIGGINS
298
ism. Serum dilution (x axis) was converted to logio before calculation. Sulfate uptake (y axis) was transformed to loge when necessary to achieve parallelism. The slope of the test serum differs significantly from the standard when the F (mean slope) is significant at the 5% level. All sera from one animal were tested in the same assay. MSA labeled with 125I was used as ligand in a receptor assay for somatomedin peptides, as previously described (12). The receptor was a particulate suspension of cell membranes prepared from human placenta by the method of Cuatrecasas (14). This assay detects the somatomedins IGF I and II and another basic somatomedin-like peptide (15). The total activity is given the name somatomedin-like receptor activity (SmLRA). SmLRA is expressed in units per ml, and 1.00 U is defined as the receptor displacement activity of 1 ml serum from the same pool used as standard in the SFA assay. Serum from one hypophysectomized animal was assayed using [125I]iodo-MSA, and a receptor prepared from fetal sheep liver by the same method that was used to obtain placental receptor. SmLRA was assayed in unextracted sera. Parallelism of displacement of [125I]iodo-MSA by sheep serum and unlabeled MSA was assessed with sera from all adult sheep and from each hypophysectomized lamb before and after hypophysectomy. Other sera were assayed in duplicate using 5 jiil sheep serum/ incubation and compared to a standard curve using unlabeled MSA. The potency and parallelism of this MSA was assessed independently against the serum standard. Sera from each animal were tested in the same assay. Likewise, all sera from the normal fetuses were tested in one assay. The intraassay variation was 7.3% and the interassay variation (n = 13) was 21%. Duplicates of greater than 20% variation were rejected. Plasma insulin was determined by RIA (16) using porcine insulin as a standard. oGH was determined by RIA using oGH prepared by Dr. A. L. Wallace as a standard (17). The sensitivity of this assay was 5 ng/ml oGH. Plasma cortisol was determined by competitive protein-binding assay, as previously described (8) (18). Plasma glucose was measured by the glucose oxidase method using an autoanalyzer. Gel chromatography Fetal sheep serum was chromatographed on Sephadex G-100 in ascending fashion (19). Details of the column size and conditions are given in Fig. 3. Animal experiments Experiments were performed with the approval of the committee for animal experimentation. Three rats underwent hypophysectomy by the parapharyngeal route under halothane anesthesia. Blood was collected preoperatively from the jugular vein and 2-7 days postoperatively by cardiac puncture. Serum, separated by centrifugation, was stored frozen at -13 C until assayed. Completeness of hypophysectomy was judged by macroscopic dissection and by the absence of detectable PRL in postoperative serum samples. Blood was obtained by jugular venesection of 13 adult sheep (8 nonpregnant females, 2 rams, and 3 castrated males). Mixed arterial and venous blood was obtained from the umbilical cord of 72 fetuses of ewes of various breeds and gestational ages undergoing slaughter at a local abbattoir. Gestational age was
Endo Vol 105
1979 No 1
estimated from the fetal crown-anus length for fetuses of less than 30 cm length and by radiology of the hind limb in the remainder (20). Serum was obtained and stored as described above. Catheterization of one carotid artery and jugular vein was performed in 18 fetal lambs from the pregnancies of known duration between 104-130 days of gestation (8). Samples of carotid blood were taken. Fifteen fetuses then underwent cryosurgical hypophysectomy by the transfrontal approach (21). In three multiple pregnancies, 1 twin was left intact. After catheterization of the maternal jugular vein, the ewes were returned to individual cages and fed and watered ad libitum. Daily or every second day sampling from the fetal carotid artery (6 ml) was performed at approximately the same time each day until termination of the experiment by cesarean section, induced delivery (see below), or death of the fetus or ewe. Heparinized catheters were flushed with saline before sampling, and 3-ml aliquots of collected blood were allowed to clot at 30 C for 1 h. Serum, separated by centrifugation, was stored at —13 C until assayed for SmLRA and SFA. Duplicate aliquots of 3 ml fetal blood were placed into tubes containing EDTA. The plasma, separated by immediate centrifugation, was stored frozen until assayed for GH. After autopsy, the fetal head was fixed in formalin and decalcified with 15% formic acid. Serial sections were cut coronally through the pituitary fossa and examined histologically. Three hypophysectomized fetal lambs underwent infusion of oGH and/or insulin. Two were infused via the jugular catheter. The first received 1, 2, and 4 mg oGH during 8 h at each dose and then 4 mg oGH over 24 h. On another occasion, this animal received 4 mg oGH mixed with 3 U insulin over 24 h. Later still, this animal received 5 U insulin over 72 h. The second fetal lamb received 4 mg oGH over 24 h. Fetal carotid blood was collected at intervals from these two animals and assayed for SmLRA, SFA and GH or insulin and glucose. The third fetus was infused via the carotid catheter. It received 9 and 26 U insulin, each during 96 h. The blood sampled from the carotid catheter of the third fetus was contaminated with insulin and was unsuitable for SmLRA assay (12). Serum SFA was measured, since this assay is not influenced by insulin (22). Parturition was induced by infusion of synthetic ACTH-(124) (Synacthen, Ciba Pharmaceutical Co., Summit, NJ) at a rate of 125 /xg/day into the jugular vein in five fetal lambs (three hypophysectomized and two intact). Carotid blood (3 ml) was collected at 12-h intervals until delivery and assayed for serum SmLRA and plasma cortisol. Three intact fetal lambs, catheterized at 110-120 days of gestation, underwent glucose infusion (80 mg/min) for 3 h via their jugular catheters. Hourly blood samples were taken from the carotid artery and plasma was assayed for SmLRA, insulin, and glucose. Student's t test or the paired t test was used for statistical comparisons. Correlations were tested by Pearson's correlation coefficient (r).
Results Serum somatomedin activity in sheep In a preliminary study, 19 of 20 serum samples from normal fetal lambs of varying gestational ages displaced
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:40 For personal use only. No other uses without permission. . All rights reserved.
SOMATOMEDINS IN FETAL LAMBS
[125I]iodo-MSA in parallel with unlabeled MSA in the receptor assay. Eleven of 13 serum samples from nonpregnant adult sheep were similarly parallel. Overall, less than 5% of fetal sheep sera were rejected because of nonparallelism. However, 6 of 9 serum samples from pregnant ewes did not displace [125I]iodo-MSA in parallel with unlabeled MSA. Heparinized fetal plasma was also nonparallel in this assay. The mean (±SD) serum SmLRA concentration in 11 adult sheep was 1.27 ± 0.21 U/ml. This was significantly greater than that of pooled serum from 9 human adults (0.31 U/ml). The potency of human serum compared to sheep serum was similar when SmLRA was assayed with [125I]iodo-MSA and a receptor prepared from fetal sheep liver (data not shown). In contrast, sheep serum was significantly less potent than human serum in promoting 35 SO4 uptake by cartilage (Fig. 1). The 10% concentration of sheep serum sometimes resulted in 35SO4 uptake below that of buffer alone, and the slope of the response was significantly less than that of human serum. SmLRA concentrations in the serum of fetal sheep rose from approximately 0.5 U/ml at 50 days to approximately 2 U/ml at term (150 days; Fig. 2). SFA and gestational age were also positively correlated, but this did not reach statistical significance (r = 0.40; n = 19; 0.1 >P>0.05). SmLRA of fetal .sheep serum, when chromatographed
on Sephadex G-100 at neutral pH, eluted in two major peaks, both of which preceded the elution peak of [125I]iodo-MSA (mol wt, ^10,000; Fig. 3). In three experiments, the recovery was 101-110%, and no small molecular weight somatomedin-like activity was detected. Serum somatomedin activity after hypophysectomy Concentrations of SmLRA in the serum of the three hypophysectomized rats fell from a mean (±SD) of 1.99 ± 0.39 U/ml preoperatively to 0.55 ± 0.23 U/ml postoperatively. The change was significant (P < 0.05, by paired t test).
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GESTATIONAL AGE (DAYS)
FIG. 2. SmLRA in the serum of fetal lambs of varying gestational ages. The coefficient of correlation between SmLRA and gestational age is 0.65 (n = 72; P < 0.001).
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Vol. 105, No. 1 Printed in U.S.A.
Serum Somatomedin Activity after Hypophysectomy and during Parturition in Fetal Lambs* M. W. BRINSMEAD AND G. C. LIGGINS Postgraduate School of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand
ABSTRACT. Somatomedins, detectable by sulfation factor activity (SFA) and/or somatomedin-like receptor activity (SmLRA), were measured in the serum of 72 normal fetal lambs of varying gestational ages and in 12 fetuses before and after hypophysectomy. Three hypophysectomized fetal lambs were infused with GH and/or insulin. Three intact fetal lambs were infused with glucose. Parturition was induced by infusion of synthetic ACTH into 5 fetal lambs. SmLRA and SFA were compared to a pool of adult sheep serum arbitrarily designated 1.00 U/ml. Serum SmLRA correlated significantly with gestational age (r = 0.65; P < 0.001), the linear regression line passing through 0.5 U/ml at 50 days and 2 U/ml at 150 days of gestation.
Serum SFA also correlated with gestational age (r = 0.40; n = 19; 0.1 > P > 0.05). Serum SmLRA and SFA were unchanged by hypophysectomy of the fetus and GH infusion but were reduced by insulin infusion. Plasma SmLRA correlated negatively with plasma insulin during glucose infusion (r = -0.67; P < 0.05). During induction of parturition with ACTH, serum SmLRA fell by a mean of 38% and concentrations correlated negatively with plasma cortisol (r = -0.62; P < 0.001). It is concluded that serum somatomedin activity in fetal sheep is not GH dependent. It is possible that somatomedin activity in cord blood after spontaneous delivery may not accurately reflect concentrations prevailing in fetal life. {Endocrinology 105: 297, 1979)
O
VER the past decade, a number of potent mitogens for cell multiplication have been described and characterized (1). Among these are the somatomedins, a family of related polypeptides which are insulin-like in structure and function and are thought by some investigators to mediate some of the actions of GH on growth (2, 3). Somatomedin activity in human cord blood increases over the last trimester of pregnancy (4) and correlates with birth size (4, 5). This suggests that these hormones may have a role in promoting fetal growth or that they may reflect the rate of growth processes in the fetus. However, nothing is known about the regulation of fetal somatomedins. In extrauterine life, the pituitary has a dominant influence over serum somatomedin activity (2, 3). Concentrations of serum somatomedins in hypopituitary rats and man are 30-50% of those in intact controls and can be restored by GH therapy (6, 7). This study was undertaken to assess the influence of the fetal pituitary on somatomedin activity in fetal lambs. Somatomedin concentrations were also studied in intact and hypophysectomized lambs during induced parturition, since it is possible that rising cortisol and estro-
gen concentrations which precede parturition (8) may affect serum somatomedin activity (9, 10). Materials and Methods Multiplication-stimulating activity (MSA) was purified from
the medium of the BRL-3A clone of rat liver cells (generously provided by Professor Temin, University of Wisconsin, Madison) by ion exchange and gel chromatography (11). The MSA used for iodination and standard in this study has been previously described (12). It had three major bands on polyacrylamide gel electrophoresis. Its potency was approximately twice that of a similarly prepared MSA produced by M. Rechler, NIH, and 1 mg was equivalent to 75 mU insulin in a bioassay using rat adipocytes. It contained no detectable immunoreactive insulin. Porcine insulin (615-D63-10; 25.4 U/mg) was donated by Dr. R. J. Hosley, Lilley Research Laboratories; ovine GH hormone (oGH; NIH-GH-S11; 0.56 IU/mg) was provided by the Hormone Distribution Officer, NIH; and insulin-like growth factors (IGFs I and II) were generous gifts from Professor R. Humbel, Biochemistry Institute, University of Zurich. Hormone assays Serum sulfation factor activity (SFA) was determined using SO4 uptake by porcine cartilage, as previously described (4). Sera at two dilutions (10% and 40%) were compared to four dilutions (5%, 10%, 20%, and 40%) of pooled serum from two normal rams arbitrarily given a potency of 1.00 U/ml SFA. Potency was calculated by the method of Finney (13) from four points of standard and test serum having the greatest parallel-
35
Received April 28,1978. Address all correspondence and requests for reprints to: Dr. Maxwell Brinsmead, Department of Obstetrics and Gynaecology, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, QLD. 4029, Australia. * This work was supported by the Medical Research Council of New Zealand.
297
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BRINSMEAD AND LIGGINS
298
ism. Serum dilution (x axis) was converted to logio before calculation. Sulfate uptake (y axis) was transformed to loge when necessary to achieve parallelism. The slope of the test serum differs significantly from the standard when the F (mean slope) is significant at the 5% level. All sera from one animal were tested in the same assay. MSA labeled with 125I was used as ligand in a receptor assay for somatomedin peptides, as previously described (12). The receptor was a particulate suspension of cell membranes prepared from human placenta by the method of Cuatrecasas (14). This assay detects the somatomedins IGF I and II and another basic somatomedin-like peptide (15). The total activity is given the name somatomedin-like receptor activity (SmLRA). SmLRA is expressed in units per ml, and 1.00 U is defined as the receptor displacement activity of 1 ml serum from the same pool used as standard in the SFA assay. Serum from one hypophysectomized animal was assayed using [125I]iodo-MSA, and a receptor prepared from fetal sheep liver by the same method that was used to obtain placental receptor. SmLRA was assayed in unextracted sera. Parallelism of displacement of [125I]iodo-MSA by sheep serum and unlabeled MSA was assessed with sera from all adult sheep and from each hypophysectomized lamb before and after hypophysectomy. Other sera were assayed in duplicate using 5 jiil sheep serum/ incubation and compared to a standard curve using unlabeled MSA. The potency and parallelism of this MSA was assessed independently against the serum standard. Sera from each animal were tested in the same assay. Likewise, all sera from the normal fetuses were tested in one assay. The intraassay variation was 7.3% and the interassay variation (n = 13) was 21%. Duplicates of greater than 20% variation were rejected. Plasma insulin was determined by RIA (16) using porcine insulin as a standard. oGH was determined by RIA using oGH prepared by Dr. A. L. Wallace as a standard (17). The sensitivity of this assay was 5 ng/ml oGH. Plasma cortisol was determined by competitive protein-binding assay, as previously described (8) (18). Plasma glucose was measured by the glucose oxidase method using an autoanalyzer. Gel chromatography Fetal sheep serum was chromatographed on Sephadex G-100 in ascending fashion (19). Details of the column size and conditions are given in Fig. 3. Animal experiments Experiments were performed with the approval of the committee for animal experimentation. Three rats underwent hypophysectomy by the parapharyngeal route under halothane anesthesia. Blood was collected preoperatively from the jugular vein and 2-7 days postoperatively by cardiac puncture. Serum, separated by centrifugation, was stored frozen at -13 C until assayed. Completeness of hypophysectomy was judged by macroscopic dissection and by the absence of detectable PRL in postoperative serum samples. Blood was obtained by jugular venesection of 13 adult sheep (8 nonpregnant females, 2 rams, and 3 castrated males). Mixed arterial and venous blood was obtained from the umbilical cord of 72 fetuses of ewes of various breeds and gestational ages undergoing slaughter at a local abbattoir. Gestational age was
Endo Vol 105
1979 No 1
estimated from the fetal crown-anus length for fetuses of less than 30 cm length and by radiology of the hind limb in the remainder (20). Serum was obtained and stored as described above. Catheterization of one carotid artery and jugular vein was performed in 18 fetal lambs from the pregnancies of known duration between 104-130 days of gestation (8). Samples of carotid blood were taken. Fifteen fetuses then underwent cryosurgical hypophysectomy by the transfrontal approach (21). In three multiple pregnancies, 1 twin was left intact. After catheterization of the maternal jugular vein, the ewes were returned to individual cages and fed and watered ad libitum. Daily or every second day sampling from the fetal carotid artery (6 ml) was performed at approximately the same time each day until termination of the experiment by cesarean section, induced delivery (see below), or death of the fetus or ewe. Heparinized catheters were flushed with saline before sampling, and 3-ml aliquots of collected blood were allowed to clot at 30 C for 1 h. Serum, separated by centrifugation, was stored at —13 C until assayed for SmLRA and SFA. Duplicate aliquots of 3 ml fetal blood were placed into tubes containing EDTA. The plasma, separated by immediate centrifugation, was stored frozen until assayed for GH. After autopsy, the fetal head was fixed in formalin and decalcified with 15% formic acid. Serial sections were cut coronally through the pituitary fossa and examined histologically. Three hypophysectomized fetal lambs underwent infusion of oGH and/or insulin. Two were infused via the jugular catheter. The first received 1, 2, and 4 mg oGH during 8 h at each dose and then 4 mg oGH over 24 h. On another occasion, this animal received 4 mg oGH mixed with 3 U insulin over 24 h. Later still, this animal received 5 U insulin over 72 h. The second fetal lamb received 4 mg oGH over 24 h. Fetal carotid blood was collected at intervals from these two animals and assayed for SmLRA, SFA and GH or insulin and glucose. The third fetus was infused via the carotid catheter. It received 9 and 26 U insulin, each during 96 h. The blood sampled from the carotid catheter of the third fetus was contaminated with insulin and was unsuitable for SmLRA assay (12). Serum SFA was measured, since this assay is not influenced by insulin (22). Parturition was induced by infusion of synthetic ACTH-(124) (Synacthen, Ciba Pharmaceutical Co., Summit, NJ) at a rate of 125 /xg/day into the jugular vein in five fetal lambs (three hypophysectomized and two intact). Carotid blood (3 ml) was collected at 12-h intervals until delivery and assayed for serum SmLRA and plasma cortisol. Three intact fetal lambs, catheterized at 110-120 days of gestation, underwent glucose infusion (80 mg/min) for 3 h via their jugular catheters. Hourly blood samples were taken from the carotid artery and plasma was assayed for SmLRA, insulin, and glucose. Student's t test or the paired t test was used for statistical comparisons. Correlations were tested by Pearson's correlation coefficient (r).
Results Serum somatomedin activity in sheep In a preliminary study, 19 of 20 serum samples from normal fetal lambs of varying gestational ages displaced
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:40 For personal use only. No other uses without permission. . All rights reserved.
SOMATOMEDINS IN FETAL LAMBS
[125I]iodo-MSA in parallel with unlabeled MSA in the receptor assay. Eleven of 13 serum samples from nonpregnant adult sheep were similarly parallel. Overall, less than 5% of fetal sheep sera were rejected because of nonparallelism. However, 6 of 9 serum samples from pregnant ewes did not displace [125I]iodo-MSA in parallel with unlabeled MSA. Heparinized fetal plasma was also nonparallel in this assay. The mean (±SD) serum SmLRA concentration in 11 adult sheep was 1.27 ± 0.21 U/ml. This was significantly greater than that of pooled serum from 9 human adults (0.31 U/ml). The potency of human serum compared to sheep serum was similar when SmLRA was assayed with [125I]iodo-MSA and a receptor prepared from fetal sheep liver (data not shown). In contrast, sheep serum was significantly less potent than human serum in promoting 35 SO4 uptake by cartilage (Fig. 1). The 10% concentration of sheep serum sometimes resulted in 35SO4 uptake below that of buffer alone, and the slope of the response was significantly less than that of human serum. SmLRA concentrations in the serum of fetal sheep rose from approximately 0.5 U/ml at 50 days to approximately 2 U/ml at term (150 days; Fig. 2). SFA and gestational age were also positively correlated, but this did not reach statistical significance (r = 0.40; n = 19; 0.1 >P>0.05). SmLRA of fetal .sheep serum, when chromatographed
on Sephadex G-100 at neutral pH, eluted in two major peaks, both of which preceded the elution peak of [125I]iodo-MSA (mol wt, ^10,000; Fig. 3). In three experiments, the recovery was 101-110%, and no small molecular weight somatomedin-like activity was detected. Serum somatomedin activity after hypophysectomy Concentrations of SmLRA in the serum of the three hypophysectomized rats fell from a mean (±SD) of 1.99 ± 0.39 U/ml preoperatively to 0.55 ± 0.23 U/ml postoperatively. The change was significant (P < 0.05, by paired t test).
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GESTATIONAL AGE (DAYS)
FIG. 2. SmLRA in the serum of fetal lambs of varying gestational ages. The coefficient of correlation between SmLRA and gestational age is 0.65 (n = 72; P < 0.001).
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