Chorionic Gonadotropin, Chorionic Somatomammotropin, and Prolactin in the Uterine Vein and Peripheral Plasma of Pregnant Rhesus Monkeys1 SCOTT W. WALSH,2 RICHARD C. WOLF, ROLAND K. MEYER, MICHEL L. AUBERT, AND HENRY G. FRIESEN University ofWisconsin Regional Primate Research Center and Department of Physiology, Madison, Wisconsin, Department of Pediatrics, University of California, San Francisco, and Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada existed between peripheral and uterine vein concentrations of either mCG or prolactin at any of the stages of gestation examined. At day 22, mCS was not detectable; however, at day 42 of gestation mCS titers averaged 1.5 fig/m\ and 2.3 /xg/ml in the peripheral and uterine vein plasma, respectively. A statistically significant mCS increase occurred by day 157, levels in the periphery and uterine vein averaging 11.0 /JLgJml and 16.3 /ig/ml, respectively. Uterine vein titers of mCS were significantly higher than peripheral titers at both days 42 and 157. Thus, the highest levels of mCG were present during early pregnancy, whereas the highest levels of mCS and prolactin were present during late pregnancy. (Endocrinology 100: 851, 1977)
ABSTRACT. Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference
E HAVE previously reported that progesterone concentrations are elevated in the uteroovarian vein which drains the ovary containing the corpus luteum (CL) at days 22 and 157 of pregnancy in the rhesus monkey, and further, that these levels are significantly increased over progesterone concentrations in the uterine vein. At day 42 of pregnancy, however, there is no statistical difference between the low levels of progesterone present in these vessels (1). These data suggest that the CL is actively secreting progesterone at days 22 Received December 17, 1975. 1 Publication number 16-028 of the Wisconsin Regional Primate Research Center. This work was supported in part by Grant RR-00167 from the National Institutes of Health, United States Public Health Service, to the Wisconsin Regional Primate Research Center, Grants 5-T01-HD00104-10 and HD-0748-03, awarded by the National Institute of Child Health and Human Development, DHEW, and by Grant 6300505A from The Ford Foundation. 2 Present address: Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005.
and 157, but at day 42 it is either inactive or its secretory activity is very low. The data also suggest that the CL is capable of a period of relatively high activity followed by a period of low activity or dormancy followed, subsequently, by a period of renewed function. A similar variability in CL activity does not occur during the normal menstrual cycle. In view of this variation in function, we sought to determine what hormone(s) might be responsible for CL activity at these various stages of pregnancy. Monkey chorionic gonadotropin, prolactin, and monkey chorionic somatomammotropin (placental lactogen) were considered as possible luteotropins involved during pregnancy and their concentrations were determined in both uterine venous and peripheral plasma at approximately days 22, 42 and 157 of pregnancy. Materials and Methods Seven adult female rhesus monkeys (Macaca mulatta) were caged with males of proven fertility for approximately 18 h during midcycle
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852
WALSH ET AL.
TABLE 1. mCG in the uterine vein and peripheral plasma in pregnant rhesus monkeys (expressed as ng equivalents NIH-LH-S10 per ml) Day 22 Animal number
Pa
UVb
1460 1348 1450 1444 1203 1477 1476
130.2 466.5 90.2 250.3 163.7 306.6 395.2
135.9 423.5 88.5 203.4 171.2 314.0 391.6
257.5 53.0
246.9 49.2
Mean ± SE
Day 42
Day 157
uvb ND 0.7 ND 0.6 ND 0.5 ND
ND 0.6 ND 0.3 ND 0.4 ND
uvb — — ND — 0.4 0.3 ND
— — ND 0.5 ND 0.5 ND
a
Peripheral vessel. Uterine vein. ND < 0.25 ng/ml. — = Not determined. b
with the day of mating considered as day 1 of pregnancy. Their housing facilities, diet and general care have previously been described (2). Laparotomies were performed under sterile conditions between days 21-24,41-44, and 155159 of pregnancy. Animals were anesthetized with a 2.5% solution of thiamylal sodium (Surital, Parke, Davis and Co.), following which, peripheral blood samples (5 ml) were obtained by femoral puncture. At the time of laparotomy, blood (7 ml) was collected from one of the main uterine veins, and a uteroovarian vein sample (7 ml) was also taken for another study. Heparinized blood samples were kept cold until the plasma was separated and were subsequently stored at - 2 0 C until the time of assay. Specific radioimmunoassays were used for the determination of monkey chorionic gonadotropin (mCG) (3,4) and monkey prolactin (5) with the following modifications for the prolactin assay: human [125I]iodo-PRL was used as the trace and the sensitivity of the assay has been improved to 1-2 ng/ml. Monkey chorionic somatomammotropin (mCS) was measured according to a modification of a previously described radioimmunoassay method (6). The mCS for iodination and standard preparations have been described (7). Five /xg mCS were iodinated with 1 mCi 125I using a standard chloramine T method and purified on a long column (55 x 0.9 cm) of Sephadex G-100, as described previously (8). Purification resulted in 3 peaks, the second one representing purified monkey [125I]iodo-CS. If [125I]iodo-CS was used more than 1 week after iodination, it was re-
Endo i 1977 Vol 100 < No 3
purified utilizing the same type of column. A double antibody method was used with the addition of the antisera and trace on the first day. Either rabbit-C3 or guinea pig-43 anti-mCS sera was used at a final dilution of 1:50,000 or 1:60,000, respectively. Incubation was carried out at room temperature for 4 days before addition of the second antibody. Further incubation for 1-2 days at 4 C resulted in complete precipitation. Maximum binding, in the absence of unlabeled mCS, ranged from 30 to 50%. The mean minimum detectable dose was 0.82 ± 0.03 ng mCS and half displacement of the maximum binding was achieved with 3.50 ± 0.21 ng mCS (n = 6). Complete parallelism was observed between serial dilutions of pregnant or fetal monkey plasma and the mCS standard curve. The prolactin and mCS data were statistically analyzed by Yates' method of weighted squares of means and independent orthogonal comparisons subsequent to log X + 1 transformation to achieve homogeneity of variances (9). The mCG data were analyzed by a paired t test.
Results
Plasma levels of mCG (Table 1) were elevated in both the uterine vein and periphery at day 22 of pregnancy. The mean concentrations were not different from each other (P > .05). Levels at days 42 and 157 were either nondetectable or less than 0.7 ng/ml. There was no difference among the conTABLE 2. Prolactin (ng/ml) in the uterine vein and peripheral plasma in pregnant rhesus monkeys Day 22
Day 42
Day 157
number
pa
UVb
pa
UVb
1460 1348 1450 1444 1203 1477 1476
185 — 1,100* 158 175 140 125
235 200 175 100 190 180 153
170 195 1,650* 128 190 210 425
140 362 180 128 185 215 155
632 >900 >900 5,832 2,990
495 2,106 1,620 3,320 7,290
314 158
176 16
424 208
195 30
>2,251 991
2,966 1,172
Mean ± SE
pa
UV
a
Peripheral vessel. Uterine vein. — = Not determined. * No explanation can be offered for these aberrant values; excluding these values the X ± SE at day 22 is 157 ± 11; at day 42, 220 ± 43. b
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LUTEOTROPINS IN THE PREGNANT MONKEY centrations of prolactin (Table 2) at days 22 and 42 (P > .05); however, by day 157 prolactin was significantly elevated (P < .01). Peripheral and uterine vein concentrations were not different at any of the stages of pregnancy (P > .05). With one exception mCS was nondetectable in plasma samples obtained at day 22 of pregnancy (Table 3). By day 42, however, mCS was present in all samples with uterine venous concentrations being significantly higher than peripheral plasma concentrations (P < .05). mCS then increased significantly by day 157 (P < .01) with uterine levels again higher than peripheral titers (P < .05). Discussion The high levels of mCG in the uterine vein and periphery at day 22 of pregnancy together with the relatively low levels of prolactin and nondetectable levels of mCS indicate that mCG is the luteotropic hormone of early pregnancy and is responsible for the elevated levels of progesterone present in the uteroovarian vein at this time (1). The concept that mCG is the initial luteotropic stimulus of pregnancy has been proposed previously by many individuals and there is compelling evidence to support this concept (10-15). We have also shown, however, that uterine vein progesterone as well as uterine and uteroovarian vein estradiol are elevated at day 22 of pregnancy (1,3). Consequently, mCG might induce the secretion of both progesterone and estrogens from the ovary and/or CL and, in addition, be responsible for the initiation of steroidogenic activity by the placenta. In regard to CL function, Knobil (13) and Atki nson etal.( 15) have suggested that mCG stimulates both estrogen and progesterone secretion during early pregnancy in the monkey. Data consonant with this theory have been reported by Bosu and Johansson (16) who demonstrated that the administration of human chorionic gonadotropin to monkeys during the late luteal phase results in a delay in the onset of menstruation and in a maintenance of elevated estrogen and
853
TABLE 3. mCS (/n,g/ml) in the uterine vein and peripheral plasma in pregnant rhesus monkeys Day 22 A n i1 i1 YI a1 11C&1
i l l l
Day 42
Day 157
b
number
pa
UV b
pa
uv
1460 1348 1450 1444 1203 1477 1476
ND ND ND ND ND ND ND
ND
0.84 1.10 3.00 0.94 1.40 1.90 1.54 1.53 0.28
ND
0.02 ND ND ND ND
Mean ± SE a b
Peripheral vessel. Uterine veini.
pa
UV b
2.30 1.90 3.20 1.00 2.80 3.10 1.90
10.20 11.20 9.60 8.40 15.80
15.00 15.80 12.00 12.00 26.60
2.31 0.30
11.04 1.27
16.28 2.69
ND < 0.020 /ig/ml. — = Not determined.
progesterone concentrations in the peripheral blood which are remarkably similar to levels observed during early pregnancy. The functional activity of the CL during late pregnancy in the monkey has been well established within the past few years by both morphological (17,18) and hormonal data (1,17,19,20). Moreover, this activity appears to be a recrudescence of function with regard to progesterone secretion. We have observed relatively high levels of progesterone in the uteroovarian vein at day 157 of pregnancy in the monkey which are significantly higher than those in the uterine vein (1). At day 42 of gestation, however, progesterone is low in both vessels and the mean concentrations are not statistically different. We interpreted these data to mean that the CL is relatively inactive or dormant at day 42, but by day 157 the activity of the CL is again increased. Although mCG is reputed to be the luteotropic stimulus during early pregnancy, it would be tenuous to attribute this function to it during late pregnancy since mCG is either nondetectable or less than 0.7 ng/ml in both the uterine vein and periphery at day 157. Low levels reported as mCG may actually be LH since the antibody used for the assay of mCG also cross-reacts with LH. In the presence of mCG the cross-reaction is minimal, mCG being approximately 50 times as potent as monkey LH in competing for binding sites (G. N. Rao, personal com-
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854
WALSH ET AL.
munication); however, in the absence of mCG, LH can be detected. Hobson has reported the presence of mCG in urine and placental extracts during late gestation in the rhesus monkey (21,22). Numerous other investigators, however, have not been successful in detecting this hormone in plasma, urine or placental extracts during mid or late pregnancy (4,14,15,23-27). The relative increase in circulating prolactin levels we observed during late pregnancy confirms the recent report of Weiss et al. (28). These investigators reported a substantial increase in peripheral prolactin levels beginning approximately 2 weeks prior to parturition. The concentrations of prolactin we have reported, however, are considerably higher than those reported by Weiss et al. This can be explained in that the collection of uterine vein samples in our study necessitated surgical intervention. Friesen and colleagues (29) and Butler et al. (30) have shown that anesthesia is a potent stimulus for prolactin release and when this is compounded by surgery, prolactin levels increase even further (29). Consequently, the concentrations of prolactin we have reported are much higher than those present in pregnant monkeys. Since both prolactin and mCS are considerably elevated at day 157, it is tempting to speculate that either one or both of these hormones might be exerting a luteotropic effect during late gestation. Their physiological role in normal CL and placental function during late pregnancy, however, is obscure. Neither is apparently necessary for continued secretion of progesterone from these organs. We have observed that the ovary containing the CL and the placenta continue to secrete progesterone during late pregnancy even in animals in which the circulating levels of prolactin and mCS are nondetectable as the result of a combination of hypophysectomy and fetal death (3,31). This does not, however, preclude the possible involvement of these hormones in the normal regulation of progesterone secretion.
Endo • 1977 Vol 100 • No 3
Although prolactin has not been demonstrated to have luteotropic properties in the monkey, experiments designed to test this action have focused on the CL of the menstrual cycle (10,32,33). However, the possibility that the CL of late pregnancy may respond to different stimuli than the CL of the nonfertile cycle cannot be ignored. Knobil (13) and Weiss et al. (34) have proposed that prolactin might also exert a luteotropic effect in lactating monkeys. They have observed that peripheral prolactin and peripheral and ovarian venous progesterone concentrations are higher in lactating than in nonlactating monkeys. Recently, indirect evidence has been reported which suggests that prolactin may also be involved in the luteotropic process of cycling monkeys. Ergocryptine, a substance which blocks the release of prolactin, when administered during the luteal phase resulted in a decrease in peripheral levels of progesterone and estrogen and in a premature onset of menstruation (35). With regard to prolactin, there was no statistical difference between mean peripheral and uterine vein levels (Table 2). Examination of individual animals, however, reveals that in some animals uterine vein prolactin is higher than peripheral prolactin, whereas in other animals it is lower. This discrepancy may be due to differences in placental affinity for prolactin (36,37). A comparison of uterine vein and peripheral levels of mCG and mCS reveals that the concentration of mCS in the uterine vein is significantly higher (P < 0.05) than in the periphery, whereas there is no statistical difference for these sampling sites for mCG. Since both hormones are secreted by the placenta, one would expect the uterine vein levels to be higher than those in the peripheral blood. The absence of such an anticipated difference with respect to mCG cannot be explained, although it may be related to a considerably longer halflife of mCG as opposed to mCS.
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LUTEOTROPINS IN THE PREGNANT MONKEY References 1. Walsh, S. W., R. C. Wolf, and R. K. Meyer, Endocrinology 95: 1704, 1974. 2. Blomquist, A. J., and H. F. Harlow, Proc Anim Care Panel 11: 57, 1961. 3. Walsh, S. W., Ph.D. Thesis, University of Wisconsin-Madison, 1975. 4. Rao, G. N., R. E. Larson, S. W. Walsh, and R. K. Meyer, Fed Proc 34: 324, 1975 (Abstract). 5. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci USA 68: 1902, 1971. 6. Vinik, A. I., S. L. Kaplan, and M. M. Grumbach, Endocrinology 92: 1051, 1973. 7. Shome, B., and H. G. Friesen, Endocrinology 89: 631, 1971. 8. Aubert, M. L., M. M. Grumbach, and S. L. Kaplan, Acta Endocrinol (Kbh) 77: 460, 1974. 9. Steel, R. G. D., and J. H. Torrie, Principles and Procedures of Statistics, ed. 1, McGraw-Hill, New York, 1960. 10. Hisaw, F. L., Yale J Biol Med 17: 119, 1944. 11. Meyer, R. K., In Diczfalusy, E., and C. C. Standley (eds.), The Use of Non-Human Primates in Research on Human Reproduction, WHO Research and Training Centre on Human Reproduction, Stockholm, 1972, p. 214. 12. Neill, J. D., and E. Knobil, Endocrinology 90: 34, 1972. 13. Knobil, E., Biol Reprod 8: 246, 1973. 14. Hodgen, G. D., W. W. Tullner, J. L. Vaitukaitis, D. N. Ward, and G. T. Ross, / Clin Endocrinol Metab 39: 457, 1974. 15. Atkinson, L. E., J. Hotchkiss, G. R. Fritz, A. H. Surve, J. D. Neill, and E. Knobil, Biol Reprod 12: 335, 1975. 16. Bosu, W. T. K., and E. D. B. Johansson, IntJ Fertil 19: 28, 1974. 17. Koering, M. J., R. C. Wolf, and R. K. Meyer, Endocrinology 93: 686, 1973. 18. Gulyas, B. ].AmJ Anat 139: 95, 1974.
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19. Treloar, O. L., R. C. Wolf, and R. K. Meyer, Endocrinology 91: 665, 1972. 20. Macdonald, G. J., K. Yoshinaga, and R. O. Greep, AmJPhys Anthropol 38: 201, 1973. 21. Hobson, B. M., Adv Reprod Physiol 5: 67, 1971. 22. Hobson, B. M.,Folia Primatol 18: 463, 1972. 23. Tullner, W. W., and R. Hertz, Endocrinology 78: 204, 1966. 24. Arslan, M., R. K. Meyer, and R. C. Wolf, Proc Soc Exp Biol Med 125: 349, 1967. 25. Hodgen, G. D., M. L. Dufau, K. J. Catt, and W. W. Tullner, Endocrinology 91: 896, 1972. 26. Hobson, W., C. Faiman, W. J. Dougherty, F. I. Reyes, and J. S. D. Winter, Fert Steril 26: 93,1975. 27. Hodgen, G. D., W. H. Niemann, and W. W. Tullner, Endocrinology 96: 789, 1975. 28. Weiss, G., W. R. Butler, J. Hotchkiss, D. J. Dierschke, and E. Knobil, Proc Soc Exp Biol Med 151: 113, 1976. 29. Friesen, H., B. Shome, C. Belanger, P. Hwang, H. Guyda, and R. Myers, Excerpta Medica Int Cong Series 244: 224, 1971. 30. Butler, W. R., L. C. Krey, K.-H. Lu, W. D. Peckham, and E. Knobil, Endocrinology 96: 1099, 1975. 31. Walsh, S. W., R. K. Meyer, R. C. Wolf, and H. G. Friesen, Endocrinology 100: 845, 1977. 32. Rothchild, I.,7 Reprod Fertil (Suppl) 1: 49, 1966. 33. Macdonald, G. J., and R. O. Greep, Fertil Steril 23: 466, 1972. 34. Weiss, G., D. J. Dierschke, F. J. Karsch, J. Hotchkiss, W. R. Butler, and E. Knobil, Endocrinology 93: 954, 1973. 35. Espinosa-Campos, J., W. R. Butler, and E. Knobil, Proc 57th Mtg Endocrine Soc, New York, 1975, p. 82 (Abstract). 36. Josimovich, J. B., G. Weiss, and D. L. Hutchinson, Endocrinology 94: 1364, 1974. 37. Josimovich, J. B., and H. Tobon, Proc 56th Mtg Endocrine Soc, Atlanta, 1974, p. A-253 (Abstract).
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ABSTRACT. Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference
E HAVE previously reported that progesterone concentrations are elevated in the uteroovarian vein which drains the ovary containing the corpus luteum (CL) at days 22 and 157 of pregnancy in the rhesus monkey, and further, that these levels are significantly increased over progesterone concentrations in the uterine vein. At day 42 of pregnancy, however, there is no statistical difference between the low levels of progesterone present in these vessels (1). These data suggest that the CL is actively secreting progesterone at days 22 Received December 17, 1975. 1 Publication number 16-028 of the Wisconsin Regional Primate Research Center. This work was supported in part by Grant RR-00167 from the National Institutes of Health, United States Public Health Service, to the Wisconsin Regional Primate Research Center, Grants 5-T01-HD00104-10 and HD-0748-03, awarded by the National Institute of Child Health and Human Development, DHEW, and by Grant 6300505A from The Ford Foundation. 2 Present address: Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005.
and 157, but at day 42 it is either inactive or its secretory activity is very low. The data also suggest that the CL is capable of a period of relatively high activity followed by a period of low activity or dormancy followed, subsequently, by a period of renewed function. A similar variability in CL activity does not occur during the normal menstrual cycle. In view of this variation in function, we sought to determine what hormone(s) might be responsible for CL activity at these various stages of pregnancy. Monkey chorionic gonadotropin, prolactin, and monkey chorionic somatomammotropin (placental lactogen) were considered as possible luteotropins involved during pregnancy and their concentrations were determined in both uterine venous and peripheral plasma at approximately days 22, 42 and 157 of pregnancy. Materials and Methods Seven adult female rhesus monkeys (Macaca mulatta) were caged with males of proven fertility for approximately 18 h during midcycle
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852
WALSH ET AL.
TABLE 1. mCG in the uterine vein and peripheral plasma in pregnant rhesus monkeys (expressed as ng equivalents NIH-LH-S10 per ml) Day 22 Animal number
Pa
UVb
1460 1348 1450 1444 1203 1477 1476
130.2 466.5 90.2 250.3 163.7 306.6 395.2
135.9 423.5 88.5 203.4 171.2 314.0 391.6
257.5 53.0
246.9 49.2
Mean ± SE
Day 42
Day 157
uvb ND 0.7 ND 0.6 ND 0.5 ND
ND 0.6 ND 0.3 ND 0.4 ND
uvb — — ND — 0.4 0.3 ND
— — ND 0.5 ND 0.5 ND
a
Peripheral vessel. Uterine vein. ND < 0.25 ng/ml. — = Not determined. b
with the day of mating considered as day 1 of pregnancy. Their housing facilities, diet and general care have previously been described (2). Laparotomies were performed under sterile conditions between days 21-24,41-44, and 155159 of pregnancy. Animals were anesthetized with a 2.5% solution of thiamylal sodium (Surital, Parke, Davis and Co.), following which, peripheral blood samples (5 ml) were obtained by femoral puncture. At the time of laparotomy, blood (7 ml) was collected from one of the main uterine veins, and a uteroovarian vein sample (7 ml) was also taken for another study. Heparinized blood samples were kept cold until the plasma was separated and were subsequently stored at - 2 0 C until the time of assay. Specific radioimmunoassays were used for the determination of monkey chorionic gonadotropin (mCG) (3,4) and monkey prolactin (5) with the following modifications for the prolactin assay: human [125I]iodo-PRL was used as the trace and the sensitivity of the assay has been improved to 1-2 ng/ml. Monkey chorionic somatomammotropin (mCS) was measured according to a modification of a previously described radioimmunoassay method (6). The mCS for iodination and standard preparations have been described (7). Five /xg mCS were iodinated with 1 mCi 125I using a standard chloramine T method and purified on a long column (55 x 0.9 cm) of Sephadex G-100, as described previously (8). Purification resulted in 3 peaks, the second one representing purified monkey [125I]iodo-CS. If [125I]iodo-CS was used more than 1 week after iodination, it was re-
Endo i 1977 Vol 100 < No 3
purified utilizing the same type of column. A double antibody method was used with the addition of the antisera and trace on the first day. Either rabbit-C3 or guinea pig-43 anti-mCS sera was used at a final dilution of 1:50,000 or 1:60,000, respectively. Incubation was carried out at room temperature for 4 days before addition of the second antibody. Further incubation for 1-2 days at 4 C resulted in complete precipitation. Maximum binding, in the absence of unlabeled mCS, ranged from 30 to 50%. The mean minimum detectable dose was 0.82 ± 0.03 ng mCS and half displacement of the maximum binding was achieved with 3.50 ± 0.21 ng mCS (n = 6). Complete parallelism was observed between serial dilutions of pregnant or fetal monkey plasma and the mCS standard curve. The prolactin and mCS data were statistically analyzed by Yates' method of weighted squares of means and independent orthogonal comparisons subsequent to log X + 1 transformation to achieve homogeneity of variances (9). The mCG data were analyzed by a paired t test.
Results
Plasma levels of mCG (Table 1) were elevated in both the uterine vein and periphery at day 22 of pregnancy. The mean concentrations were not different from each other (P > .05). Levels at days 42 and 157 were either nondetectable or less than 0.7 ng/ml. There was no difference among the conTABLE 2. Prolactin (ng/ml) in the uterine vein and peripheral plasma in pregnant rhesus monkeys Day 22
Day 42
Day 157
number
pa
UVb
pa
UVb
1460 1348 1450 1444 1203 1477 1476
185 — 1,100* 158 175 140 125
235 200 175 100 190 180 153
170 195 1,650* 128 190 210 425
140 362 180 128 185 215 155
632 >900 >900 5,832 2,990
495 2,106 1,620 3,320 7,290
314 158
176 16
424 208
195 30
>2,251 991
2,966 1,172
Mean ± SE
pa
UV
a
Peripheral vessel. Uterine vein. — = Not determined. * No explanation can be offered for these aberrant values; excluding these values the X ± SE at day 22 is 157 ± 11; at day 42, 220 ± 43. b
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LUTEOTROPINS IN THE PREGNANT MONKEY centrations of prolactin (Table 2) at days 22 and 42 (P > .05); however, by day 157 prolactin was significantly elevated (P < .01). Peripheral and uterine vein concentrations were not different at any of the stages of pregnancy (P > .05). With one exception mCS was nondetectable in plasma samples obtained at day 22 of pregnancy (Table 3). By day 42, however, mCS was present in all samples with uterine venous concentrations being significantly higher than peripheral plasma concentrations (P < .05). mCS then increased significantly by day 157 (P < .01) with uterine levels again higher than peripheral titers (P < .05). Discussion The high levels of mCG in the uterine vein and periphery at day 22 of pregnancy together with the relatively low levels of prolactin and nondetectable levels of mCS indicate that mCG is the luteotropic hormone of early pregnancy and is responsible for the elevated levels of progesterone present in the uteroovarian vein at this time (1). The concept that mCG is the initial luteotropic stimulus of pregnancy has been proposed previously by many individuals and there is compelling evidence to support this concept (10-15). We have also shown, however, that uterine vein progesterone as well as uterine and uteroovarian vein estradiol are elevated at day 22 of pregnancy (1,3). Consequently, mCG might induce the secretion of both progesterone and estrogens from the ovary and/or CL and, in addition, be responsible for the initiation of steroidogenic activity by the placenta. In regard to CL function, Knobil (13) and Atki nson etal.( 15) have suggested that mCG stimulates both estrogen and progesterone secretion during early pregnancy in the monkey. Data consonant with this theory have been reported by Bosu and Johansson (16) who demonstrated that the administration of human chorionic gonadotropin to monkeys during the late luteal phase results in a delay in the onset of menstruation and in a maintenance of elevated estrogen and
853
TABLE 3. mCS (/n,g/ml) in the uterine vein and peripheral plasma in pregnant rhesus monkeys Day 22 A n i1 i1 YI a1 11C&1
i l l l
Day 42
Day 157
b
number
pa
UV b
pa
uv
1460 1348 1450 1444 1203 1477 1476
ND ND ND ND ND ND ND
ND
0.84 1.10 3.00 0.94 1.40 1.90 1.54 1.53 0.28
ND
0.02 ND ND ND ND
Mean ± SE a b
Peripheral vessel. Uterine veini.
pa
UV b
2.30 1.90 3.20 1.00 2.80 3.10 1.90
10.20 11.20 9.60 8.40 15.80
15.00 15.80 12.00 12.00 26.60
2.31 0.30
11.04 1.27
16.28 2.69
ND < 0.020 /ig/ml. — = Not determined.
progesterone concentrations in the peripheral blood which are remarkably similar to levels observed during early pregnancy. The functional activity of the CL during late pregnancy in the monkey has been well established within the past few years by both morphological (17,18) and hormonal data (1,17,19,20). Moreover, this activity appears to be a recrudescence of function with regard to progesterone secretion. We have observed relatively high levels of progesterone in the uteroovarian vein at day 157 of pregnancy in the monkey which are significantly higher than those in the uterine vein (1). At day 42 of gestation, however, progesterone is low in both vessels and the mean concentrations are not statistically different. We interpreted these data to mean that the CL is relatively inactive or dormant at day 42, but by day 157 the activity of the CL is again increased. Although mCG is reputed to be the luteotropic stimulus during early pregnancy, it would be tenuous to attribute this function to it during late pregnancy since mCG is either nondetectable or less than 0.7 ng/ml in both the uterine vein and periphery at day 157. Low levels reported as mCG may actually be LH since the antibody used for the assay of mCG also cross-reacts with LH. In the presence of mCG the cross-reaction is minimal, mCG being approximately 50 times as potent as monkey LH in competing for binding sites (G. N. Rao, personal com-
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WALSH ET AL.
munication); however, in the absence of mCG, LH can be detected. Hobson has reported the presence of mCG in urine and placental extracts during late gestation in the rhesus monkey (21,22). Numerous other investigators, however, have not been successful in detecting this hormone in plasma, urine or placental extracts during mid or late pregnancy (4,14,15,23-27). The relative increase in circulating prolactin levels we observed during late pregnancy confirms the recent report of Weiss et al. (28). These investigators reported a substantial increase in peripheral prolactin levels beginning approximately 2 weeks prior to parturition. The concentrations of prolactin we have reported, however, are considerably higher than those reported by Weiss et al. This can be explained in that the collection of uterine vein samples in our study necessitated surgical intervention. Friesen and colleagues (29) and Butler et al. (30) have shown that anesthesia is a potent stimulus for prolactin release and when this is compounded by surgery, prolactin levels increase even further (29). Consequently, the concentrations of prolactin we have reported are much higher than those present in pregnant monkeys. Since both prolactin and mCS are considerably elevated at day 157, it is tempting to speculate that either one or both of these hormones might be exerting a luteotropic effect during late gestation. Their physiological role in normal CL and placental function during late pregnancy, however, is obscure. Neither is apparently necessary for continued secretion of progesterone from these organs. We have observed that the ovary containing the CL and the placenta continue to secrete progesterone during late pregnancy even in animals in which the circulating levels of prolactin and mCS are nondetectable as the result of a combination of hypophysectomy and fetal death (3,31). This does not, however, preclude the possible involvement of these hormones in the normal regulation of progesterone secretion.
Endo • 1977 Vol 100 • No 3
Although prolactin has not been demonstrated to have luteotropic properties in the monkey, experiments designed to test this action have focused on the CL of the menstrual cycle (10,32,33). However, the possibility that the CL of late pregnancy may respond to different stimuli than the CL of the nonfertile cycle cannot be ignored. Knobil (13) and Weiss et al. (34) have proposed that prolactin might also exert a luteotropic effect in lactating monkeys. They have observed that peripheral prolactin and peripheral and ovarian venous progesterone concentrations are higher in lactating than in nonlactating monkeys. Recently, indirect evidence has been reported which suggests that prolactin may also be involved in the luteotropic process of cycling monkeys. Ergocryptine, a substance which blocks the release of prolactin, when administered during the luteal phase resulted in a decrease in peripheral levels of progesterone and estrogen and in a premature onset of menstruation (35). With regard to prolactin, there was no statistical difference between mean peripheral and uterine vein levels (Table 2). Examination of individual animals, however, reveals that in some animals uterine vein prolactin is higher than peripheral prolactin, whereas in other animals it is lower. This discrepancy may be due to differences in placental affinity for prolactin (36,37). A comparison of uterine vein and peripheral levels of mCG and mCS reveals that the concentration of mCS in the uterine vein is significantly higher (P < 0.05) than in the periphery, whereas there is no statistical difference for these sampling sites for mCG. Since both hormones are secreted by the placenta, one would expect the uterine vein levels to be higher than those in the peripheral blood. The absence of such an anticipated difference with respect to mCG cannot be explained, although it may be related to a considerably longer halflife of mCG as opposed to mCS.
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LUTEOTROPINS IN THE PREGNANT MONKEY References 1. Walsh, S. W., R. C. Wolf, and R. K. Meyer, Endocrinology 95: 1704, 1974. 2. Blomquist, A. J., and H. F. Harlow, Proc Anim Care Panel 11: 57, 1961. 3. Walsh, S. W., Ph.D. Thesis, University of Wisconsin-Madison, 1975. 4. Rao, G. N., R. E. Larson, S. W. Walsh, and R. K. Meyer, Fed Proc 34: 324, 1975 (Abstract). 5. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci USA 68: 1902, 1971. 6. Vinik, A. I., S. L. Kaplan, and M. M. Grumbach, Endocrinology 92: 1051, 1973. 7. Shome, B., and H. G. Friesen, Endocrinology 89: 631, 1971. 8. Aubert, M. L., M. M. Grumbach, and S. L. Kaplan, Acta Endocrinol (Kbh) 77: 460, 1974. 9. Steel, R. G. D., and J. H. Torrie, Principles and Procedures of Statistics, ed. 1, McGraw-Hill, New York, 1960. 10. Hisaw, F. L., Yale J Biol Med 17: 119, 1944. 11. Meyer, R. K., In Diczfalusy, E., and C. C. Standley (eds.), The Use of Non-Human Primates in Research on Human Reproduction, WHO Research and Training Centre on Human Reproduction, Stockholm, 1972, p. 214. 12. Neill, J. D., and E. Knobil, Endocrinology 90: 34, 1972. 13. Knobil, E., Biol Reprod 8: 246, 1973. 14. Hodgen, G. D., W. W. Tullner, J. L. Vaitukaitis, D. N. Ward, and G. T. Ross, / Clin Endocrinol Metab 39: 457, 1974. 15. Atkinson, L. E., J. Hotchkiss, G. R. Fritz, A. H. Surve, J. D. Neill, and E. Knobil, Biol Reprod 12: 335, 1975. 16. Bosu, W. T. K., and E. D. B. Johansson, IntJ Fertil 19: 28, 1974. 17. Koering, M. J., R. C. Wolf, and R. K. Meyer, Endocrinology 93: 686, 1973. 18. Gulyas, B. ].AmJ Anat 139: 95, 1974.
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19. Treloar, O. L., R. C. Wolf, and R. K. Meyer, Endocrinology 91: 665, 1972. 20. Macdonald, G. J., K. Yoshinaga, and R. O. Greep, AmJPhys Anthropol 38: 201, 1973. 21. Hobson, B. M., Adv Reprod Physiol 5: 67, 1971. 22. Hobson, B. M.,Folia Primatol 18: 463, 1972. 23. Tullner, W. W., and R. Hertz, Endocrinology 78: 204, 1966. 24. Arslan, M., R. K. Meyer, and R. C. Wolf, Proc Soc Exp Biol Med 125: 349, 1967. 25. Hodgen, G. D., M. L. Dufau, K. J. Catt, and W. W. Tullner, Endocrinology 91: 896, 1972. 26. Hobson, W., C. Faiman, W. J. Dougherty, F. I. Reyes, and J. S. D. Winter, Fert Steril 26: 93,1975. 27. Hodgen, G. D., W. H. Niemann, and W. W. Tullner, Endocrinology 96: 789, 1975. 28. Weiss, G., W. R. Butler, J. Hotchkiss, D. J. Dierschke, and E. Knobil, Proc Soc Exp Biol Med 151: 113, 1976. 29. Friesen, H., B. Shome, C. Belanger, P. Hwang, H. Guyda, and R. Myers, Excerpta Medica Int Cong Series 244: 224, 1971. 30. Butler, W. R., L. C. Krey, K.-H. Lu, W. D. Peckham, and E. Knobil, Endocrinology 96: 1099, 1975. 31. Walsh, S. W., R. K. Meyer, R. C. Wolf, and H. G. Friesen, Endocrinology 100: 845, 1977. 32. Rothchild, I.,7 Reprod Fertil (Suppl) 1: 49, 1966. 33. Macdonald, G. J., and R. O. Greep, Fertil Steril 23: 466, 1972. 34. Weiss, G., D. J. Dierschke, F. J. Karsch, J. Hotchkiss, W. R. Butler, and E. Knobil, Endocrinology 93: 954, 1973. 35. Espinosa-Campos, J., W. R. Butler, and E. Knobil, Proc 57th Mtg Endocrine Soc, New York, 1975, p. 82 (Abstract). 36. Josimovich, J. B., G. Weiss, and D. L. Hutchinson, Endocrinology 94: 1364, 1974. 37. Josimovich, J. B., and H. Tobon, Proc 56th Mtg Endocrine Soc, Atlanta, 1974, p. A-253 (Abstract).
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