NOTES AND COMMENTS
Acknowledgments This work was supported by a Grant of Centro di Studio per la Biologia e la Fisiopatologia muscolare of C.N.R., Padova, Italy, to assess useful models to study insulin effects on muscle. Thanks are due to Mr. G. Papi for care for the animals.
References 1. Rakieten, N., M. L. Rakieten, and M. V. Nadkami, Cancer Chemother Rep 29: 91, 1963. 2. Bergamini, E., Endocrinology 90: 1582, 1972.
789
3. Masiello, P., M. D. Thesis, University of Siena, June 1973. 4. Rerup, C., and F. Tarding, EurJ Pharmacol 7: 89, 1969. 5. Junod, A., A. E. Lambert, L. Orci, R. Pictet, A. E. Gonet, and A. E. Renold, Proc Soc Exp Biol Med 126: 201, 1967. 6. , , W. Stauffacher, and A. E. Renold, / Clin Invest 48: 2129, 1969. 7. Losert, W., A. Rilke, O. Loge, and K. D. Richter, Arzneimittel Forsch 21: 1643, 1971. 8. Stauffacher, W., I. Burr, A. Gutzeit, D. Beaven, J. Veleminsky, and A. E. Renold, Proc Soc Exp Biol Med 133: 194, 1970.
Duration of Chorionic Gonadotropin Production by the Placenta of the Rhesus Monkey GARY D. HODGEN, WENDELL H. NIEMANN, AND WILLIAM W. TULLNER Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014, and Laboratory of Experimental Medicine and Surgery in Primates, (LEMSIP), New York University Medical Center, New York, New York 10016 ABSTRACT. Concentrations of macaque chorionic gonadotropin (mCG) in placenta, blood and urine of rhesus monkeys have been measured by both radioimmunoassay and bioassay throughout gestation. mCG was easily detected and quantified in these specimens for a brief period in early pregnancy, but was not detectable between the 40th day of pregnancy and term in placental extracts, serum, or 40-fold urine concentrates. The apparent absence of mCG after the 40th day of pregnancy
P
REVIOUS reports from various laboratories have been in disagreement about the period of chorionic gonadotropin production in pregnant rhesus monkeys. Hobson et al. (1-3) have used both bioassay and a cross-reacting radioimmunoassay for hCG to report an approximately 25-fold rise in chorionic gonadotropin levels in extracts of rhesus monkey placental tissues between the 6th week of gestation and term. Also, urinary chorionic gonadotropin was reported to be measurable by bioassay near term (1). In contrast, we have not been able to detect any macaque chorionic gonadotropin (mGG) in serum or urine by bioassay after the 40th day of pregnancy (4-6). Even 80-fold concentrates of daily urine pools collected dur-
Received August 8, 1974.
makes these macaques a valuable model for pregnancy research, where the absence of chorionic gonadotropin is experimentally desirable. Unlike women and some higher primates, the functional status of the fetal, placental and maternal endocrine compartments of macaques can be studied in the absence of circulating chorionic gonadotropin during mid and late gestation. (Endocrinology 96: 789, 1975
ing the week preceding delivery lacked measurable mCG activity (6). The importance of resolving this disagreement centers around the appropriate use of these macaques in pregnancy research. Since chorionic gonadotropin is present throughout gestation in women (7-9), chimpanzees (10,11) and gorillas (12), the absence of chorionic gonadotropin from placenta, blood and urine of these macaques after the 40th day of pregnancy makes them a valuable model for pregnancy research where the absence of chorionic gonadotropin is experimentally desirable. Accordingly, the functional status of the fetal, placental and maternal endocrine compartments can be studied in the absence of circulating chorionic gonadotropin. In the present study we have used extracts of placental tissue, serum and urine collected at
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Endo • 1975 Vo! 96 • No 3
NOTES AND COMMENTS
790
various times throughout gestation in rhesus monkeys to determine mCG levels by both a highly sensitive and specific radioimmunoassay and a bioassay.
TABLE 2. Serum and urinary mCG iminunoreactivities throughout pregnancy often rhesus monkeys mCG-HRP equivalents Serum
Ten pregnant rhesus monkeys were caged, fed and maintained under standard laboratory conditions similar to those described earlier (13). Peripheral serum and 24-h urine specimens were collected at weekly intervals or more frequently from the time of mating until one week post-partum. Urine specimens were concentrated 40-fold by the kaolin-acetone procedure previously described (10). Placentas from 16 Macaco mulatta ranging from 22 days gestation to term were collected either by caesarean section or as soon as feasible following natural delivery. Placental homogenates were prepared according to the procedure of Hobson (1). Placental extracts, serum, and urine concentrates were assayed both in the mCG radioimmunoassay of Hodgen et al. (13), sensitive to 0.2 mlU of hCG (relative to biological activity of Second International Standard hCG) and a mouse uterine weight bioassay (4), sensitive to 0.1 IU of hCG (equivalent to biological activity of Second International Standard hCG). A kaolin-acetone extract of pooled rhesus pregnancy urine containing mCG was used as a provisional reference preparation (mCG-HRP) for dose interpolation in the radioimmunoassay since no standard reference preparation for immunoreactive rnCG was TABLE
1. Immunoreactivity (RIA)
and
biological
(MUW) activity of mCG in extracts of rhesus monkey placentas
Monkey C-139 672-VV C-144 9 744-T P-179 O-604 R-410 672-S 72-A 334-A 246-A 585-C R-162 R-172 R-156
MUW IU hCG
Days after fertilization
RIA fig mCG-HRP equivalents/ mg of wet tissue
equivalents/ mg wet tissue
22 23 24 26 72 116 119 140 146 166 162 166 171 166 168 162
12.21 17.25 9.69 2.66 ND* ND ND ND ND ND ND ND ND ND ND ND
1.3 2.2 1.1 1.2 ND* ND ND ND ND ND ND ND ND ND ND ND
* mCG activity not detectable.
Urine 0*8/24 1.)
(Aig/ml)
Materials and Methods
Days after fertilization 0-7 8 9 10 11 12 13 16 21 25 30
35 40 45-173**
Mean
Range
ND* 0.1 0.7 1.5 3.4 8.7 23.3 226 733 313 10.4 0.4 0.1 ND
ND-1.1 ND-1.8 ND-3.9 ND-11.0 1.3-53.1 3.9-112 32.6-757 103-1,547 19.1-516 6.2-23.5 ND-2.1 ND-1.0 —
Mean ND 12 91 123 186 566 744 1,789 10,033 3,455 488
37 9 ND
Range
ND-130 ND-274 ND-411 ND-603 120-1,131 155-3,086 241-13,073 2,159-34,111 2,900-10,107 101-3,642 ND-299 ND-107 —
* mCG not detectable. ** Samples collected at weekly intervals. available (13). Bioassay design was 3 x 3 using 5 mice per group. Doses of placental extracts were equivalent to either 5, 50 or 500 mg equivalents of wet tissue. Statistical evaluations of the assays were performed by the methods of Rod bard and Levvald (14).
Results The data summarized in Table 1 show that mCG was present in measurable quantities in extracts of placentas collected between 22 and 26 days of gestation using both the radioimmunoassay and the bioassay. However, we were unable to detect any ehorionie goitadotropin in extracts of placentas taken between the 72nd day of pregnancy and parturition. Table 2 illustrates the patterns of mCG concentrations in serum and urine from the time of fertilization until delivery. Uniformly, the mCG immunoreactivity rose from the 8th to the 21st day after fertilization; and then steadily declined to undetectable levels by the 45th day of pregnancy. Both serum and urine lacked measurable levels of mCG during the remainder of gestation.
Discussion The findings of this investigation, as well as those of others (4-6, 15-17), are in contrast with observations reported by Hobson et al. (1-3).
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NOTES AND COMMENTS We have been unable to detect any immunoreactive or biologically active chorionic gonadotropin in extracts of placental tissue, peripheral serum, or 40-fold urine concentrates during middle or late gestation in rhesus monkeys. This disparity is difficult to understand since we have used both a highly sensitive radioimmunoassay and a bioassay similar to the one described (1). In addition, special precautions were taken to duplicate the placental tissue extraction procedure outlined (1). Two possible explanations for these divergent findings are that discrepancies could arise on the basis of the well known differences in immunoreactive hormone concentrations when different antisera are used or that the antigenic properties of chorionic gonadotrophin synthesized in the rhesus monkey placenta are altered after the 40th day of gestation. However, neither of these reasons for differences can account for the discrepancies in biological activities of placental tissue extracts since both the endpoint and minimal effective doses were the same for bioassays utilized in the two laboratories. The present results are consistent with several studies reported earlier from our laboratory (4-6) and others using various bioassays (1517) and a radioimmunoassay (18) for gonadotropins in serum and urine. Together, these findings show that the placenta of the rhesus monkey synthesizes mCG for only a brief period in early pregnancy (from approximately the 8th to 40th day after fertilization) and that this synthetic process is not sustained or renewed at any later time during the same gestation. Although the temporal pattern of serum and urinary mCG levels is similar to that found in women and higher primates during early pregnancy, the absence of measurable mCG during middle and late gestation in rhesus monkeys markedly separates this macaque from women (7-9), chimpanzees (10,11) and gorillas (12) as regards the duration of chorionic gonadotropin production by the placenta. These reports have been reviewed recently (19). Accordingly, our findings suggest that rhesus monkeys, and prob-
791
ably other macaques and baboons, may be particularly appropriate research models for studying some functions of fetal, placental and maternal endocrine tissues where the presence of chorionic gonadotropin may be considered an undesirable, potentially interfering substance in the interval from midpregnancy to parturition.
Acknowledgments We thank Mr. Charles Turner, Mr. Donald Barber, Ms. Aline O'Connor and Mr. David Hildebrand for their technical contributions.
References 1. Hobson, B. M. In Bishop, M. W. H. (ed.), Advances in Reproductive Physiology, vol. 5, Academic Press, New York, 1970, p. 68. 2. , and L. Wide, J Endocrinol 55: 363, 1972. 3. , and L. Wide, 7 Endocrinol 60: 75, 1974. 4. Tullner, W. W., and R. Hertz, Endocrinology 78: 204, 1966. 5. Tullner, W. W. Endocrinology 82: 874, 1968. 6. Hodgen, G. D., M. L. Dufau, K. J. Catt, and W. W. Tullner, Endocrinology 91: 896, 1972. 7. Loraine, J. A., and J. H. Gaddum,/ Endocrinol 6: 319, 1950. 8. Taymor, M. L., Clin Obstet Gynecol 10: 303, 1967. 9. Varma, K. L., L. Larraga, and H. A. Selenkow, Obstet Gynecol 37: 10, 1971. 10. Nixon, W. E., G. D. Hodgen, W. H. Niemann, G. T. Ross, and W. W. Tullner, Endocrinology 90: 1105, 1972. 11. Clegg, M. T. and M. Weaver, Proc Soc Exp Biol Med 139: 1170, 1972. 12. Tullner, W. W. and C. W. Gray, Proc Soc Exp Biol Med 128: 954, 1968. 13. Hodgen, G. D., W. W. Tullner, J. L. Vaitukaitis, D. N. Ward, and G. T. Ross, J Clin Endocrinol Metab 39: 417, 1974. 14. Rodbard, D. A., and J. C. Lewald, Ada Endocrinol (Kbh) (Suppl) 64: 79, 1970. 15. Delfs, E., Anat Rec (Suppl) 79: 17, 1941. 16. van Wagenen, G., and M. E. Simpson, Proc Soc Exp Biol Med 90: 346, 1955. 17. Arslan, M., R. K. Meyer, and R. C. Wolf, Proc Soc Exp Biol Med 125: 349, 1967. 18. Knobil, E., Biol Reprod 8: 246, 1973. 19. Tullner, W. W., In Luckett, W. P. (ed.), Contributions to Primatology. Reproductive Biology of the Primates, vol. 3, S. Karger, Basel, 1974, p. 235.
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Acknowledgments This work was supported by a Grant of Centro di Studio per la Biologia e la Fisiopatologia muscolare of C.N.R., Padova, Italy, to assess useful models to study insulin effects on muscle. Thanks are due to Mr. G. Papi for care for the animals.
References 1. Rakieten, N., M. L. Rakieten, and M. V. Nadkami, Cancer Chemother Rep 29: 91, 1963. 2. Bergamini, E., Endocrinology 90: 1582, 1972.
789
3. Masiello, P., M. D. Thesis, University of Siena, June 1973. 4. Rerup, C., and F. Tarding, EurJ Pharmacol 7: 89, 1969. 5. Junod, A., A. E. Lambert, L. Orci, R. Pictet, A. E. Gonet, and A. E. Renold, Proc Soc Exp Biol Med 126: 201, 1967. 6. , , W. Stauffacher, and A. E. Renold, / Clin Invest 48: 2129, 1969. 7. Losert, W., A. Rilke, O. Loge, and K. D. Richter, Arzneimittel Forsch 21: 1643, 1971. 8. Stauffacher, W., I. Burr, A. Gutzeit, D. Beaven, J. Veleminsky, and A. E. Renold, Proc Soc Exp Biol Med 133: 194, 1970.
Duration of Chorionic Gonadotropin Production by the Placenta of the Rhesus Monkey GARY D. HODGEN, WENDELL H. NIEMANN, AND WILLIAM W. TULLNER Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014, and Laboratory of Experimental Medicine and Surgery in Primates, (LEMSIP), New York University Medical Center, New York, New York 10016 ABSTRACT. Concentrations of macaque chorionic gonadotropin (mCG) in placenta, blood and urine of rhesus monkeys have been measured by both radioimmunoassay and bioassay throughout gestation. mCG was easily detected and quantified in these specimens for a brief period in early pregnancy, but was not detectable between the 40th day of pregnancy and term in placental extracts, serum, or 40-fold urine concentrates. The apparent absence of mCG after the 40th day of pregnancy
P
REVIOUS reports from various laboratories have been in disagreement about the period of chorionic gonadotropin production in pregnant rhesus monkeys. Hobson et al. (1-3) have used both bioassay and a cross-reacting radioimmunoassay for hCG to report an approximately 25-fold rise in chorionic gonadotropin levels in extracts of rhesus monkey placental tissues between the 6th week of gestation and term. Also, urinary chorionic gonadotropin was reported to be measurable by bioassay near term (1). In contrast, we have not been able to detect any macaque chorionic gonadotropin (mGG) in serum or urine by bioassay after the 40th day of pregnancy (4-6). Even 80-fold concentrates of daily urine pools collected dur-
Received August 8, 1974.
makes these macaques a valuable model for pregnancy research, where the absence of chorionic gonadotropin is experimentally desirable. Unlike women and some higher primates, the functional status of the fetal, placental and maternal endocrine compartments of macaques can be studied in the absence of circulating chorionic gonadotropin during mid and late gestation. (Endocrinology 96: 789, 1975
ing the week preceding delivery lacked measurable mCG activity (6). The importance of resolving this disagreement centers around the appropriate use of these macaques in pregnancy research. Since chorionic gonadotropin is present throughout gestation in women (7-9), chimpanzees (10,11) and gorillas (12), the absence of chorionic gonadotropin from placenta, blood and urine of these macaques after the 40th day of pregnancy makes them a valuable model for pregnancy research where the absence of chorionic gonadotropin is experimentally desirable. Accordingly, the functional status of the fetal, placental and maternal endocrine compartments can be studied in the absence of circulating chorionic gonadotropin. In the present study we have used extracts of placental tissue, serum and urine collected at
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Endo • 1975 Vo! 96 • No 3
NOTES AND COMMENTS
790
various times throughout gestation in rhesus monkeys to determine mCG levels by both a highly sensitive and specific radioimmunoassay and a bioassay.
TABLE 2. Serum and urinary mCG iminunoreactivities throughout pregnancy often rhesus monkeys mCG-HRP equivalents Serum
Ten pregnant rhesus monkeys were caged, fed and maintained under standard laboratory conditions similar to those described earlier (13). Peripheral serum and 24-h urine specimens were collected at weekly intervals or more frequently from the time of mating until one week post-partum. Urine specimens were concentrated 40-fold by the kaolin-acetone procedure previously described (10). Placentas from 16 Macaco mulatta ranging from 22 days gestation to term were collected either by caesarean section or as soon as feasible following natural delivery. Placental homogenates were prepared according to the procedure of Hobson (1). Placental extracts, serum, and urine concentrates were assayed both in the mCG radioimmunoassay of Hodgen et al. (13), sensitive to 0.2 mlU of hCG (relative to biological activity of Second International Standard hCG) and a mouse uterine weight bioassay (4), sensitive to 0.1 IU of hCG (equivalent to biological activity of Second International Standard hCG). A kaolin-acetone extract of pooled rhesus pregnancy urine containing mCG was used as a provisional reference preparation (mCG-HRP) for dose interpolation in the radioimmunoassay since no standard reference preparation for immunoreactive rnCG was TABLE
1. Immunoreactivity (RIA)
and
biological
(MUW) activity of mCG in extracts of rhesus monkey placentas
Monkey C-139 672-VV C-144 9 744-T P-179 O-604 R-410 672-S 72-A 334-A 246-A 585-C R-162 R-172 R-156
MUW IU hCG
Days after fertilization
RIA fig mCG-HRP equivalents/ mg of wet tissue
equivalents/ mg wet tissue
22 23 24 26 72 116 119 140 146 166 162 166 171 166 168 162
12.21 17.25 9.69 2.66 ND* ND ND ND ND ND ND ND ND ND ND ND
1.3 2.2 1.1 1.2 ND* ND ND ND ND ND ND ND ND ND ND ND
* mCG activity not detectable.
Urine 0*8/24 1.)
(Aig/ml)
Materials and Methods
Days after fertilization 0-7 8 9 10 11 12 13 16 21 25 30
35 40 45-173**
Mean
Range
ND* 0.1 0.7 1.5 3.4 8.7 23.3 226 733 313 10.4 0.4 0.1 ND
ND-1.1 ND-1.8 ND-3.9 ND-11.0 1.3-53.1 3.9-112 32.6-757 103-1,547 19.1-516 6.2-23.5 ND-2.1 ND-1.0 —
Mean ND 12 91 123 186 566 744 1,789 10,033 3,455 488
37 9 ND
Range
ND-130 ND-274 ND-411 ND-603 120-1,131 155-3,086 241-13,073 2,159-34,111 2,900-10,107 101-3,642 ND-299 ND-107 —
* mCG not detectable. ** Samples collected at weekly intervals. available (13). Bioassay design was 3 x 3 using 5 mice per group. Doses of placental extracts were equivalent to either 5, 50 or 500 mg equivalents of wet tissue. Statistical evaluations of the assays were performed by the methods of Rod bard and Levvald (14).
Results The data summarized in Table 1 show that mCG was present in measurable quantities in extracts of placentas collected between 22 and 26 days of gestation using both the radioimmunoassay and the bioassay. However, we were unable to detect any ehorionie goitadotropin in extracts of placentas taken between the 72nd day of pregnancy and parturition. Table 2 illustrates the patterns of mCG concentrations in serum and urine from the time of fertilization until delivery. Uniformly, the mCG immunoreactivity rose from the 8th to the 21st day after fertilization; and then steadily declined to undetectable levels by the 45th day of pregnancy. Both serum and urine lacked measurable levels of mCG during the remainder of gestation.
Discussion The findings of this investigation, as well as those of others (4-6, 15-17), are in contrast with observations reported by Hobson et al. (1-3).
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NOTES AND COMMENTS We have been unable to detect any immunoreactive or biologically active chorionic gonadotropin in extracts of placental tissue, peripheral serum, or 40-fold urine concentrates during middle or late gestation in rhesus monkeys. This disparity is difficult to understand since we have used both a highly sensitive radioimmunoassay and a bioassay similar to the one described (1). In addition, special precautions were taken to duplicate the placental tissue extraction procedure outlined (1). Two possible explanations for these divergent findings are that discrepancies could arise on the basis of the well known differences in immunoreactive hormone concentrations when different antisera are used or that the antigenic properties of chorionic gonadotrophin synthesized in the rhesus monkey placenta are altered after the 40th day of gestation. However, neither of these reasons for differences can account for the discrepancies in biological activities of placental tissue extracts since both the endpoint and minimal effective doses were the same for bioassays utilized in the two laboratories. The present results are consistent with several studies reported earlier from our laboratory (4-6) and others using various bioassays (1517) and a radioimmunoassay (18) for gonadotropins in serum and urine. Together, these findings show that the placenta of the rhesus monkey synthesizes mCG for only a brief period in early pregnancy (from approximately the 8th to 40th day after fertilization) and that this synthetic process is not sustained or renewed at any later time during the same gestation. Although the temporal pattern of serum and urinary mCG levels is similar to that found in women and higher primates during early pregnancy, the absence of measurable mCG during middle and late gestation in rhesus monkeys markedly separates this macaque from women (7-9), chimpanzees (10,11) and gorillas (12) as regards the duration of chorionic gonadotropin production by the placenta. These reports have been reviewed recently (19). Accordingly, our findings suggest that rhesus monkeys, and prob-
791
ably other macaques and baboons, may be particularly appropriate research models for studying some functions of fetal, placental and maternal endocrine tissues where the presence of chorionic gonadotropin may be considered an undesirable, potentially interfering substance in the interval from midpregnancy to parturition.
Acknowledgments We thank Mr. Charles Turner, Mr. Donald Barber, Ms. Aline O'Connor and Mr. David Hildebrand for their technical contributions.
References 1. Hobson, B. M. In Bishop, M. W. H. (ed.), Advances in Reproductive Physiology, vol. 5, Academic Press, New York, 1970, p. 68. 2. , and L. Wide, J Endocrinol 55: 363, 1972. 3. , and L. Wide, 7 Endocrinol 60: 75, 1974. 4. Tullner, W. W., and R. Hertz, Endocrinology 78: 204, 1966. 5. Tullner, W. W. Endocrinology 82: 874, 1968. 6. Hodgen, G. D., M. L. Dufau, K. J. Catt, and W. W. Tullner, Endocrinology 91: 896, 1972. 7. Loraine, J. A., and J. H. Gaddum,/ Endocrinol 6: 319, 1950. 8. Taymor, M. L., Clin Obstet Gynecol 10: 303, 1967. 9. Varma, K. L., L. Larraga, and H. A. Selenkow, Obstet Gynecol 37: 10, 1971. 10. Nixon, W. E., G. D. Hodgen, W. H. Niemann, G. T. Ross, and W. W. Tullner, Endocrinology 90: 1105, 1972. 11. Clegg, M. T. and M. Weaver, Proc Soc Exp Biol Med 139: 1170, 1972. 12. Tullner, W. W. and C. W. Gray, Proc Soc Exp Biol Med 128: 954, 1968. 13. Hodgen, G. D., W. W. Tullner, J. L. Vaitukaitis, D. N. Ward, and G. T. Ross, J Clin Endocrinol Metab 39: 417, 1974. 14. Rodbard, D. A., and J. C. Lewald, Ada Endocrinol (Kbh) (Suppl) 64: 79, 1970. 15. Delfs, E., Anat Rec (Suppl) 79: 17, 1941. 16. van Wagenen, G., and M. E. Simpson, Proc Soc Exp Biol Med 90: 346, 1955. 17. Arslan, M., R. K. Meyer, and R. C. Wolf, Proc Soc Exp Biol Med 125: 349, 1967. 18. Knobil, E., Biol Reprod 8: 246, 1973. 19. Tullner, W. W., In Luckett, W. P. (ed.), Contributions to Primatology. Reproductive Biology of the Primates, vol. 3, S. Karger, Basel, 1974, p. 235.
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