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Ovine placental lactogen stimulation of ornithine decarboxylase activity in brain and liver of neonatal rats
Life Sciences, Vol . 22, pp . 2073-2078 Priated in the U .S .A.
Pergamon Preae
OVINE PLACENTAL LACTOGEN STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY IN BRAIN AND LIVER OF NEONATAL RATS 1 S . R. Butler, T. W. Hurley 2 , S . M. Schanberg3 and S . Handwerger 4 Departments of Pharmacology, Pediatrics, Physiology and Medicine Duke University Medical Center Durham, North Carolina
27710
(Received is final form April 24, 1978)
Summary Ovine placental lactogen (oPL), growth hormone (oGH), prolactin (oPRL) and human placental lactogen (hPL) were administered intracisternally (ic) or intraperitoneally (ip) to 17 day old rats end brain and liver ODC activities determined four hours later . When given ic, oPL, oGH and oPRL caused significant increases in brain ODC activity, while hPL had no significant effect . After ip administration, oPL and oGH also caused a significant increase in brain as well as liver ODC activity but oPRL and hPL were without significant effect . The stimulation of polyamine metabolism by oPL together with earlier reports of its potent somatotropic effects and its high concentration in the fetus supports the hypothesis that oPL may be important in the regulation of fetal growth . Ovine placental lactogen (oPL) is a polypeptide hormone with chemical and biological similarities to ovine growth hormone (oGH) . When administered to immature hypophysectomized rats, oPL stimu lates weight gain, tibial growth, and plasma somatomedin concentrations with potencies comparable to bovine and ovine growth hormones (1,2,3) . Since oPL is present in high concentration in the fetus
1Supported by grants from the National institutes of Health (MH-13688 and HD-07447) and the National Foundation-March of Dimes (1-297) . 2Supported by U.S .P .H .S . postdoctoral fellowship (1-F32-HD05508-O1) . 3U .S .P .H .S . Research Scientist Awardee (MH-06489), to whom reprint requests should be addressed. 4U.S .P .H .S . Research Career Development Awardee (HD00065) . 0300-9653/78/0619-2073$02 .00/0 Copyright © 1978 Pergamon Press
2074
Ovine Placental Lactogen aad ODC Activity
Vol . 22, No . 23, 1978
during the second half of pregnancy (4), it has been suggested (3) that oPL may be important in the regulation o£ fetal growth . However, an effect of oPL on a specific cellular enzyme involved in growth and/or differentiation has not been demonstrated . An extensive literature indicates that the polyamines are important in the control of cellular growth and differentiation (5-8) and that the rate-limiting reaction in polyamine biosyntheT sis is catalyzed by the enzyme ornithine decarboxylase (ODC ; 9,10), Previous experiments from this laboratory (11) have shown that bovine growth hormone (bGH) and, to a lesser extent, bovine prolactin (bPRL) stimulate ODC activity in neonatal rat brain and liver, suggesting that these hormones may be involved in the growth and development of those organs . To investigate further the somatotropic properties of oPL, we have studied its effects on brain and liver ODC activity in 17 day old rats and have compared these effects to those produced by the administration of ovine growth hormone (oGH) and ovine prolactin (oPRL) . Materials & Methods D, L-1- 14 C-ornithine monohydrochloride (specific activity 43 mCi/mmole) was purchased from New England Nuclear, N^iris-(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) from Calbiochem, dithiothreitol (DTT) from P-L Biochemicals, Milwaukee, Wisconsin, pyridoxal-5'-phosphate from Sigma Chemical Company, hyamine (p-diisobutyl-cresoxyethoxyethyl dimethyl-benzyl ammonium hydroxide, 1 .0 M in ethanol) from Packard Instrument Company . oPL was prepared as previously described (3) . oGH (NIH-GHS9),oPR (NIH-P-S-10) and hPL were provided by the Hormone Distribution Program, NIAMDD . oGH and oPRL were further purified by chromatography on Sephadex 6-150 (12) . The oPL and oGH preparations used in the investigation were previously demonstrated to bind to growth hormone receptors from rabbit liver membranes and to increase plasma somatomedin concentrations in hypophysectomized rats . Also, hPL and oPRL preparations were previously demonstrated to bind to prolactin receptors on membranes from rabbit mammary tissue . Hormones were dissolved in normal saline, pH 8 .0, immediately prior to intracisternal (ic) or intraperitoneal (ip) injection . Gravid, Sprague-Dawley rats were obtained from Zivic Miller Company one week before delivery, housed individually, and given standard laboratory chow and water ad libitum . After delivery, all pups were allowed to remain with thébio goal mother until 17 days of age at which time the experiments were performed. Earlier studies in our laboratory (11) showed that basal ODC activity is relatively low but could be maximally stimulated 17 days postpartum . For intracisternal injections, peps were removed from the mother, lightly anesthetized with ether, and injected with hormone or vehicle in a total volume of 10 pl as described by Schanberg, et al (13) . The pups were returned to the mother and were sacri~ce~ by decapitation four hours later as it has been shown that peak ODC activity is reached four hours after hormone treatment (11) . The brain and liver were rapidly removed and homogenized in a 20-fold excess (V/W) of 0 .01 M TES, pH 7 .7 . The homogenates were
Vol . 22, No . 23, 1978
Ovine Placental Lactogen and ODC Activity
2075
centrifuged at 27 .000 x g for 20 minutes, and ODC activity in the supernatant was assayed by the liberation of radiolabelled CO from 1- 14 C-ornithine as previously described (14), except tha~ the reaction mixture was made 0.5 mM with DTT. For each tissue assayed, the counts of 14C02 evolved per 30 minutes per gram wet weight of tissue was calculated and the results expressed as a percent of the mean ODC activity in the brain or liver of rats receiving saline alone . The statistical difference between the ODC responses to saline and the various hormones was calculated by the analysis of variance . Results When oPL or oGH were administered intracisternally in doses of 1 .25 ug each, increases in brain ODC activity of 80 and 1208, respectively, were observed (Fig . 1) . The stimulation following oPRL administration, though modest, was statistically significant (p < 0 .05) but the small increase following hPL treatment was not significantly different from control values obtained in litter mates treated with normal saline alone . The larger increases following oPL and oGH treatment, while significantly (p < 0.001) different from control values, were not significantly different from each other . 160
FIG . 1 Effect of is administered oPR, hPL, oPL and oGH on rat brain ODC activity . 17 day old pups were given 1 .25 u9 of each hormone or an equal volume of saline and killed 4 hours later. Data are expressed as mean + SE of the  stimulation over control, with N's of 6-12 in each group . Control ODC activity was 0 .8 nCi/30 min ./g tissue .
2076
Ovine Placental Lactogen and ODC Activity
Vol . 22, No . 2 3, 1978
The intraperitoneal injection of 100 ug of oPL or oGH caused an increase in ODC activity in both brain and liver . In brain (Fiq . 2), both oPL and oGH evoked significant elevations of 5050$ while oPRL and hPL (100 ug each) were without significant effect . In liver (Fig . 3), increases in ODC activity of greater than 5008 were observed after intraperitoneal injection of oPL or oGH, while oPRL and hPL caused much smaller changes that were not significantly different from control values .
FIG . 2 Effect of ip administered oPR, hPL, oPL and oGH on rat brain ODC activity . 17 day old pups were given 100 ug of each hornione or an equal volume of saline and killed 4 hours later . Control ODC activity was 0 .8 nCi/30 min ./g tissue, with N's of 6-12 in each group .
FIG . 3 Effect of ip administered oPR, hPL, oPL and oGH on rat liver activity . 17 day old pups were given 100 ug of each hormone or an equal volume of saline and .killed 4 hours later . Control liver ODC activity was 24 nCi/30 min ./g tissue, with N's of 6-12 in each group .
Vol. 22, No . 23, 1978
Ovine Placental Lactogen and ODC Activity
2077
Discussion At the doses tested, oPL was comparable to oGH in its ability to stimulate ODC activity in neonatal rat brain and liver . These results are the first demonstration of an effect of oPL on a cellu lar enzyme involved in growth . oPRL stimulated ODC activity in brain and liver to a much lesser extent than oPL. The failure of hPL to stimulate ODC activity in brain or liver after ip or is injection was not surprising since our earlier studies (11) showed that the is injection of 200 times more hPL than the 1 .5 ug dose used in this study caused only a small increase in ODC activity . The fact that oPL and oGH cause much greater stimulation of ODC activity in liver and brain than does an equivalent dose of hPL is consistent with other reports (15) that, in the rat, hPL is a much less potent growth promoting stimulus than growth hormone in several bioassays . The potency of oPL, on the other hand, is comparable to that of growth hormone in the weight gain and tibia width assays as well as in its ability to elevate circulating somatomedin concentrations (2,16,17) . The stimulation of ODC activity by oPL is consistent with its other growth hormonelike activities and emphasizes again the differences in somatotropic potency between oPL and the primate placental lactogens . Whether these differences indicate a fundamental divergence in the physiology of the primate and subprimate placental lactogens is unclear . Marked stimulation of brain ODC activity followed ip and is injection of oPL and oGH . Stimulation of brain ODC activity by both hormones after is injection is consistent with the hypothesis that oPL and oGH can cross the blood-cerebrospinal fluid barrier in 17 day old rats . The possibility that both hormones may give rise in the brain and/or peripherally to an intermediate which directly stimulates ODC activity cannot be ruled out . Nevertheless, the large increase in brain ODC activity after is injection of 1 .5 ug of oPL or oGH (Fig . 1) and the fact that the induction of a similar increase in brain ODC activity occurred after ip injection of 100 ug of either hormone (Fig . 2) support the interpretation that both hormones probably act directly on brain tissue . These findings demonstrate that polypeptide hormones including oPL can increase ODC activity in neonatal rat brain and liver . Since ODC activity is thought to be critically involved in cellu lar growth and development, its stimulation by oPL supports the hypothesis that oPL may have a significant role in the regulation of fetal growth . References l. 2. 3.
S . HANDWERGER, W . MAURER, J . BARBETT, T . W. HURLEY and R. E . FELLOWS, Endo . Res . Commun . 1 403-413 (1974) . T . W . HURLEY, L. E . UNDERWOOD, S . HANDWERGER, A . J. D'ERCOLE, R. FURLANETTO and R . E . FELLOWS, Endocrinol . 101 1635-1638 (1977) . T . W. HURLEY, W . MAURER, W. HANDWERGER, and R. E . FELLOWS, In Walter, R. and J. Meienhoffer (eds .), Pe tides : Chemist , Structure and Biology , p. 583-588, Ann Arbor Sc ence Pu s ers, Ann Arbor (1975) .
2078
4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
16 . 17 .
Ovine Placental Lactogen and ODC Activity
Vol . 22, No . 23, 1978
S . HANDWERGER, C . CRENSHAFl r W, ~ " MP~URER r J " BP~RRETT r T, ~. HURLEY, A. COLANDER and R . E . FELLOWS, T. Endocrinol , _72 27-34 (1977) . S . S . COHEN, Introduction to the Pql an[ines r Frentice Hall, Englewood Cli s D. H . RUSSELL, Pol amines in Normal and Neo lactic Growth, Raven Press, New York 9 ). U. BACHRACH, Function of Naturall Occurrin Pol amines, Academic Press, New York (1 73 . D. H . RUSSELL and S . H . SNYDER, Endocrinol . 84 223-228 (1969) . A. E . PEGG and H . G . WILLIAMS -ASHMAN,BoheYn . J . _108 533539 (1968) . D. H . RUSSELL and S . H . SNYDER, Proc . Nat . Acad . Sci . U,S .A . 60 1420-1427 (1968) . L. J . ROGER, S . M. SCHANBERG and R . E . FELLOWS, Endocrinol . 95 904-911 (1974) . R. E. FELLOWS and A. D . ROGOL, J. Biol . Chem . _244 1567-1575 (1969) . S . M . SCHANBERG, J . J . SCHILDBRAUT and I . J . ROPIN, J . Pharmacol . Exp . Ther . 15 7 311-318 (1967) . T . R. ANDERSON anc~S . M . SCHANBERG, J . Neurochem _19 1471-1481 (1972) . S . L . RAPLAN and M . M . GRUMBACH , In J . B . Josimovich, M. Reynolds and E . Lobo (eds .), Lactoénic Hormones, Fetal Nutrition and Lactation , p . 183-192, Jo Whey & ons, New Yor (1974) . J . S . D . CHAN, H . A . ROBERTSON and H . G. FRIESEN, Endocrinol . 98 65-76 (1976) . T. W . HURLEY, Doctoral dissertation, Duke University (1976) .
Pergamon Preae
OVINE PLACENTAL LACTOGEN STIMULATION OF ORNITHINE DECARBOXYLASE ACTIVITY IN BRAIN AND LIVER OF NEONATAL RATS 1 S . R. Butler, T. W. Hurley 2 , S . M. Schanberg3 and S . Handwerger 4 Departments of Pharmacology, Pediatrics, Physiology and Medicine Duke University Medical Center Durham, North Carolina
27710
(Received is final form April 24, 1978)
Summary Ovine placental lactogen (oPL), growth hormone (oGH), prolactin (oPRL) and human placental lactogen (hPL) were administered intracisternally (ic) or intraperitoneally (ip) to 17 day old rats end brain and liver ODC activities determined four hours later . When given ic, oPL, oGH and oPRL caused significant increases in brain ODC activity, while hPL had no significant effect . After ip administration, oPL and oGH also caused a significant increase in brain as well as liver ODC activity but oPRL and hPL were without significant effect . The stimulation of polyamine metabolism by oPL together with earlier reports of its potent somatotropic effects and its high concentration in the fetus supports the hypothesis that oPL may be important in the regulation of fetal growth . Ovine placental lactogen (oPL) is a polypeptide hormone with chemical and biological similarities to ovine growth hormone (oGH) . When administered to immature hypophysectomized rats, oPL stimu lates weight gain, tibial growth, and plasma somatomedin concentrations with potencies comparable to bovine and ovine growth hormones (1,2,3) . Since oPL is present in high concentration in the fetus
1Supported by grants from the National institutes of Health (MH-13688 and HD-07447) and the National Foundation-March of Dimes (1-297) . 2Supported by U.S .P .H .S . postdoctoral fellowship (1-F32-HD05508-O1) . 3U .S .P .H .S . Research Scientist Awardee (MH-06489), to whom reprint requests should be addressed. 4U.S .P .H .S . Research Career Development Awardee (HD00065) . 0300-9653/78/0619-2073$02 .00/0 Copyright © 1978 Pergamon Press
2074
Ovine Placental Lactogen aad ODC Activity
Vol . 22, No . 23, 1978
during the second half of pregnancy (4), it has been suggested (3) that oPL may be important in the regulation o£ fetal growth . However, an effect of oPL on a specific cellular enzyme involved in growth and/or differentiation has not been demonstrated . An extensive literature indicates that the polyamines are important in the control of cellular growth and differentiation (5-8) and that the rate-limiting reaction in polyamine biosyntheT sis is catalyzed by the enzyme ornithine decarboxylase (ODC ; 9,10), Previous experiments from this laboratory (11) have shown that bovine growth hormone (bGH) and, to a lesser extent, bovine prolactin (bPRL) stimulate ODC activity in neonatal rat brain and liver, suggesting that these hormones may be involved in the growth and development of those organs . To investigate further the somatotropic properties of oPL, we have studied its effects on brain and liver ODC activity in 17 day old rats and have compared these effects to those produced by the administration of ovine growth hormone (oGH) and ovine prolactin (oPRL) . Materials & Methods D, L-1- 14 C-ornithine monohydrochloride (specific activity 43 mCi/mmole) was purchased from New England Nuclear, N^iris-(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) from Calbiochem, dithiothreitol (DTT) from P-L Biochemicals, Milwaukee, Wisconsin, pyridoxal-5'-phosphate from Sigma Chemical Company, hyamine (p-diisobutyl-cresoxyethoxyethyl dimethyl-benzyl ammonium hydroxide, 1 .0 M in ethanol) from Packard Instrument Company . oPL was prepared as previously described (3) . oGH (NIH-GHS9),oPR (NIH-P-S-10) and hPL were provided by the Hormone Distribution Program, NIAMDD . oGH and oPRL were further purified by chromatography on Sephadex 6-150 (12) . The oPL and oGH preparations used in the investigation were previously demonstrated to bind to growth hormone receptors from rabbit liver membranes and to increase plasma somatomedin concentrations in hypophysectomized rats . Also, hPL and oPRL preparations were previously demonstrated to bind to prolactin receptors on membranes from rabbit mammary tissue . Hormones were dissolved in normal saline, pH 8 .0, immediately prior to intracisternal (ic) or intraperitoneal (ip) injection . Gravid, Sprague-Dawley rats were obtained from Zivic Miller Company one week before delivery, housed individually, and given standard laboratory chow and water ad libitum . After delivery, all pups were allowed to remain with thébio goal mother until 17 days of age at which time the experiments were performed. Earlier studies in our laboratory (11) showed that basal ODC activity is relatively low but could be maximally stimulated 17 days postpartum . For intracisternal injections, peps were removed from the mother, lightly anesthetized with ether, and injected with hormone or vehicle in a total volume of 10 pl as described by Schanberg, et al (13) . The pups were returned to the mother and were sacri~ce~ by decapitation four hours later as it has been shown that peak ODC activity is reached four hours after hormone treatment (11) . The brain and liver were rapidly removed and homogenized in a 20-fold excess (V/W) of 0 .01 M TES, pH 7 .7 . The homogenates were
Vol . 22, No . 23, 1978
Ovine Placental Lactogen and ODC Activity
2075
centrifuged at 27 .000 x g for 20 minutes, and ODC activity in the supernatant was assayed by the liberation of radiolabelled CO from 1- 14 C-ornithine as previously described (14), except tha~ the reaction mixture was made 0.5 mM with DTT. For each tissue assayed, the counts of 14C02 evolved per 30 minutes per gram wet weight of tissue was calculated and the results expressed as a percent of the mean ODC activity in the brain or liver of rats receiving saline alone . The statistical difference between the ODC responses to saline and the various hormones was calculated by the analysis of variance . Results When oPL or oGH were administered intracisternally in doses of 1 .25 ug each, increases in brain ODC activity of 80 and 1208, respectively, were observed (Fig . 1) . The stimulation following oPRL administration, though modest, was statistically significant (p < 0 .05) but the small increase following hPL treatment was not significantly different from control values obtained in litter mates treated with normal saline alone . The larger increases following oPL and oGH treatment, while significantly (p < 0.001) different from control values, were not significantly different from each other . 160
FIG . 1 Effect of is administered oPR, hPL, oPL and oGH on rat brain ODC activity . 17 day old pups were given 1 .25 u9 of each hormone or an equal volume of saline and killed 4 hours later. Data are expressed as mean + SE of the  stimulation over control, with N's of 6-12 in each group . Control ODC activity was 0 .8 nCi/30 min ./g tissue .
2076
Ovine Placental Lactogen and ODC Activity
Vol . 22, No . 2 3, 1978
The intraperitoneal injection of 100 ug of oPL or oGH caused an increase in ODC activity in both brain and liver . In brain (Fiq . 2), both oPL and oGH evoked significant elevations of 5050$ while oPRL and hPL (100 ug each) were without significant effect . In liver (Fig . 3), increases in ODC activity of greater than 5008 were observed after intraperitoneal injection of oPL or oGH, while oPRL and hPL caused much smaller changes that were not significantly different from control values .
FIG . 2 Effect of ip administered oPR, hPL, oPL and oGH on rat brain ODC activity . 17 day old pups were given 100 ug of each hornione or an equal volume of saline and killed 4 hours later . Control ODC activity was 0 .8 nCi/30 min ./g tissue, with N's of 6-12 in each group .
FIG . 3 Effect of ip administered oPR, hPL, oPL and oGH on rat liver activity . 17 day old pups were given 100 ug of each hormone or an equal volume of saline and .killed 4 hours later . Control liver ODC activity was 24 nCi/30 min ./g tissue, with N's of 6-12 in each group .
Vol. 22, No . 23, 1978
Ovine Placental Lactogen and ODC Activity
2077
Discussion At the doses tested, oPL was comparable to oGH in its ability to stimulate ODC activity in neonatal rat brain and liver . These results are the first demonstration of an effect of oPL on a cellu lar enzyme involved in growth . oPRL stimulated ODC activity in brain and liver to a much lesser extent than oPL. The failure of hPL to stimulate ODC activity in brain or liver after ip or is injection was not surprising since our earlier studies (11) showed that the is injection of 200 times more hPL than the 1 .5 ug dose used in this study caused only a small increase in ODC activity . The fact that oPL and oGH cause much greater stimulation of ODC activity in liver and brain than does an equivalent dose of hPL is consistent with other reports (15) that, in the rat, hPL is a much less potent growth promoting stimulus than growth hormone in several bioassays . The potency of oPL, on the other hand, is comparable to that of growth hormone in the weight gain and tibia width assays as well as in its ability to elevate circulating somatomedin concentrations (2,16,17) . The stimulation of ODC activity by oPL is consistent with its other growth hormonelike activities and emphasizes again the differences in somatotropic potency between oPL and the primate placental lactogens . Whether these differences indicate a fundamental divergence in the physiology of the primate and subprimate placental lactogens is unclear . Marked stimulation of brain ODC activity followed ip and is injection of oPL and oGH . Stimulation of brain ODC activity by both hormones after is injection is consistent with the hypothesis that oPL and oGH can cross the blood-cerebrospinal fluid barrier in 17 day old rats . The possibility that both hormones may give rise in the brain and/or peripherally to an intermediate which directly stimulates ODC activity cannot be ruled out . Nevertheless, the large increase in brain ODC activity after is injection of 1 .5 ug of oPL or oGH (Fig . 1) and the fact that the induction of a similar increase in brain ODC activity occurred after ip injection of 100 ug of either hormone (Fig . 2) support the interpretation that both hormones probably act directly on brain tissue . These findings demonstrate that polypeptide hormones including oPL can increase ODC activity in neonatal rat brain and liver . Since ODC activity is thought to be critically involved in cellu lar growth and development, its stimulation by oPL supports the hypothesis that oPL may have a significant role in the regulation of fetal growth . References l. 2. 3.
S . HANDWERGER, W . MAURER, J . BARBETT, T . W. HURLEY and R. E . FELLOWS, Endo . Res . Commun . 1 403-413 (1974) . T . W . HURLEY, L. E . UNDERWOOD, S . HANDWERGER, A . J. D'ERCOLE, R. FURLANETTO and R . E . FELLOWS, Endocrinol . 101 1635-1638 (1977) . T . W. HURLEY, W . MAURER, W. HANDWERGER, and R. E . FELLOWS, In Walter, R. and J. Meienhoffer (eds .), Pe tides : Chemist , Structure and Biology , p. 583-588, Ann Arbor Sc ence Pu s ers, Ann Arbor (1975) .
2078
4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
16 . 17 .
Ovine Placental Lactogen and ODC Activity
Vol . 22, No . 23, 1978
S . HANDWERGER, C . CRENSHAFl r W, ~ " MP~URER r J " BP~RRETT r T, ~. HURLEY, A. COLANDER and R . E . FELLOWS, T. Endocrinol , _72 27-34 (1977) . S . S . COHEN, Introduction to the Pql an[ines r Frentice Hall, Englewood Cli s D. H . RUSSELL, Pol amines in Normal and Neo lactic Growth, Raven Press, New York 9 ). U. BACHRACH, Function of Naturall Occurrin Pol amines, Academic Press, New York (1 73 . D. H . RUSSELL and S . H . SNYDER, Endocrinol . 84 223-228 (1969) . A. E . PEGG and H . G . WILLIAMS -ASHMAN,BoheYn . J . _108 533539 (1968) . D. H . RUSSELL and S . H . SNYDER, Proc . Nat . Acad . Sci . U,S .A . 60 1420-1427 (1968) . L. J . ROGER, S . M. SCHANBERG and R . E . FELLOWS, Endocrinol . 95 904-911 (1974) . R. E. FELLOWS and A. D . ROGOL, J. Biol . Chem . _244 1567-1575 (1969) . S . M . SCHANBERG, J . J . SCHILDBRAUT and I . J . ROPIN, J . Pharmacol . Exp . Ther . 15 7 311-318 (1967) . T . R. ANDERSON anc~S . M . SCHANBERG, J . Neurochem _19 1471-1481 (1972) . S . L . RAPLAN and M . M . GRUMBACH , In J . B . Josimovich, M. Reynolds and E . Lobo (eds .), Lactoénic Hormones, Fetal Nutrition and Lactation , p . 183-192, Jo Whey & ons, New Yor (1974) . J . S . D . CHAN, H . A . ROBERTSON and H . G. FRIESEN, Endocrinol . 98 65-76 (1976) . T. W . HURLEY, Doctoral dissertation, Duke University (1976) .