Corpus Luteum and Fetoplacental Functions in Monkeys Hypophysectomized During Late Pregnancy1 SCOTT W. WALSH,2 ROLAND K. MEYER, RICHARD C. WOLF, AND HENRY G. FRIESEN University of Wisconsin Regional Primate Research Center and Department of Physiology, Madison, Wisconsin, and Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada ABSTRACT. The hormonal regulation of corpus luteum (CL) function during late pregnancy was studied in hypophysectomized monkeys. Between days 149-154 of gestation, 9 days after hypophysectomy, progesterone in the uteroovarian vein (UOV), uterine vein (UV) and peripheral circulation (P) averaged 179.7 ng/ml, 38.9 ng/ml and 5.5 ng/ml, respectively. Amniotic fluid prolactin ranged from 2150-6700 ng/ml and monkey chorionic somatomammotropin (mCS) in mothers carrying live fetuses ranged from 11.4-30.8 pig/ml in the UV and P. Prolactin and monkey chorionic gonadotropin in the UV and P were low or nondetectable as was mCS in 2 mothers carrying dead fetuses. CL function was further studied 7 and 39 days after removal of the fetus alone or both the fetus and placenta. Placental delivery was extremely variable, ranging from 2->63 days post-fetectomy. Although progesterone was not detectable in the P 7 days after cesarean section in those animals in which both fetus and placenta were absent, surprisingly,

B

OTH hormonal and morphological evidence have been accrued establishing the functional activity of the corpus luteum (CL) during late pregnancy in the rhesus monkey (1-5). This activity during late pregnancy is apparently a recrudescence of function since the ovary containing the CL is relatively inactive with respect to progesterone secretion during an earlier stage of gestation (4,6). Since the hormonal

Received December 17, 1975. Publication number 16-029 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. 1

progesterone was measurable in the UOV (range 1.648.2 ng/ml). At 39 days, progesterone was either nondetectable or very low. We have interpreted these data to mean: 1) neither the maternal pituitary gland nor a live fetus is necessary for placental or corpus luteum production of progesterone during late pregnancy, 2) the presence of high levels of circulating prolactin and mCS are apparently not necessary for continued secretion of progesterone from the CL during late pregnancy, 3) the fetoplacental unit may be the source of the luteotropic stimulus of late pregnancy since progesterone in the UOV decreases markedly in the absence of the fetoplacental unit or disruption of the unit brought about by fetectomy, and 4) regression of the CL following cesarean section in hypophysectomized monkeys is exceedingly slow when compared to the precipitous regression characteristic of the CL of the nonfertile menstrual cycle. {Endocrinology 100: 845, 1977)

regulation of the CL of late pregnancy has not as yet been explored, a study was conducted to study CL function following hypophysectomy and cesarean section during late pregnancy. Materials and Methods Six pregnant rhesus monkeys (Macaca mulatta) were used in this study. Housing, diet and daily care of the animals have previously been described (7). Animals were hypophysectomized between days 140-145 of pregnancy. Nine days later, between 149-154 days, they were laparotomized and blood was collected from the uteroovarian and uterine veins and from the peripheral circulation via femoral puncture. A cesarean section was performed and only the fetus removed in 3 animals while both fetus and placenta were removed in the others. Animals were again laparotomized 7 and 39 days following cesarean section and blood was collected from the uteroovarian vein and from one of the femoral vessels. All surgical procedures were

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846

Endo • 1977 Vol 100 • No 3

WALSH ET AL.

performed aseptically between 0830 and 1300 h. Heparinized blood samples were kept cold until the plasma could be separated and the samples were subsequently stored at - 2 0 C until assayed. Progesterone was analyzed by a competitive protein binding method as previously described (4). Specific radioimmunoassays were used to determine monkey chorionic gonadotropin (mCG) (6,8), prolactin (9,10), and mCS (9). The technique used for hypophysectomy was similar to that previously described (11-13) except that bleeding from the basisphenoid bone was controlled with cotton pellets and bone wax instead of electrocautery. The anesthetic regimen used for hypophysectomy was a modification of the procedures suggested by Hill (14) for pregnant monkeys. The anesthetic protocol was directed towards preventing hypotension, promoting adequate oxygenation of maternal and fetal blood and preventing uterine contractions which could lead to interruption of placental perfusion and increase the chances of placental separation. Animals were initially anesthetized with a combination of ketamine hydrochloride (Vetalar, Parke-Davis), 15 mg/kg, im, and halothane (Fluothane, Ayerst Laboratories, Inc.) vaporized with an excess amount of oxygen (1.3% halothane/ 2-4 liters O^min). Atropine sulfate (0.05 mg/kg) was administered to prevent salivation. After muscle relaxation was achieved, an endotracheal tube was passed into the trachea for the continued administration of halothane which was maintained at a reduced concentration (0.50.8%/2-4 liters 02/min) for the duration of the operation. Sixty minutes after the initial dose of ketamine and for every 30 min thereafter, additional doses of ketamine were administered (5 mg/kg, im). Animals also received a continuous intravenous infusion (30 ml/h) of lactated Ringer's solution with 5% dextrose throughout the operation. In one animal (#1077), a 2.5% solution of thiamylal sodium (Surital, Parke-Davis) administered iv was used instead of ketamine as the initial anesthetic and, in addition, a higher concentration of halothane vaporized with a lower amount of oxygen was used for maintenance of anesthesia (5% halothane/2 liters O2/ min for induction and 0.8%/l liter 0 2 /min during the operation). Immediately following surgery, animals received benzanthine penicillin G suspension (Bicillin L-A, Wyeth), 300,000 units, im, and cortisone acetate (Cortone Acetate, Merck, Sharp and

Dohme), 25 mg, im. Continued replacement therapy of glucocorticoids was not necessary. All animals received: Purina monkey chow, fresh fruit, a sandwich made with a mixture of unsulfurized molasses, Similac (Ross Laboratories) and water (2:5:2) daily. Tap and sugar water (50 g dextrose [Cerelose Dextrose, CPE International]/liter) were available. Hypophysectomy was judged to be complete by several factors including: lack of detectable amounts of LH-like material in the maternal plasma, extremely low or nondetectable levels of plasma prolactin, and examination of the sella turcica of 3 monkeys which died or were sacrificed following completion of the study. Roentgenography of fetal bone development was used to estimate fetal age for those fetuses found dead in utero (15-17).

Results

At days 149-154 of gestation, 9 days after hypophysectomy, progesterone was substantially elevated over peripheral concentrations in both the uteroovarian and uterine veins with uteroovarian vein levels markedly higher than uterine vein levels in all but 1 animal (Table 1). Mean concentrations for the 3 sampling sites were significantly different from each other (P < .05, one way analysis of variance). The CL was a large pinkish, well vascularized structure on the surface of the ovary. Its histological appearance was very similar to the CL of the luteal phase, although there were scattered areas in which the cells were not well rounded and contained flattened nuclei and scanty cytoplasm. Monkey chorionic somatomammotropin was considerably higher in mothers with live fetuses than in mothers with dead fetuses (Table 2). Uterine and peripheral prolactin concentrations were low or nondetectable, whereas amniotic fluid prolactin averaged 3370 ng/ml (Table 3). Uterine and peripheral levels of monkey chorionic gonadotropin were not measurable. Four apparently normal healthy fetuses and 2 dead macerated fetuses weighing between 340-470 g were recovered at cesarean section (Table 4).

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847

HORMONES IN HYPOX PREGNANT MONKEYS

T A B L E 1. Progesterone levels (ng/ml) following hypophysectomy during late pregnancy in the rhesus monkey Cesarean section— Days 149-154 of gestation!

Day 7 Postcesarean section

Day 39 Postcesarean section

number

pa

UV b

UOVC

pa

UV b

UOVC

pa

1077* D-81 d N-96 1450* 1434" G~46e

4.5 5.3 6.4 1.4 6.7 8.9

16.8 35.1 7.7 136.3 26.8 10.7

198.2 60.7 210.2 227.6 24.8 356.4

ND ND ND ND 4.1

— — — — 6.3

41.9 48.2 1.6 4.6 30.5

ND ND ND ND 3.9

Mean ±SE

5.5 1.0

38.9 19.9

179.7 49.3

UV b

UOVC

ND 1.0 ND

ND 1.8

4.2

a

Peripheral vessel. Uterine vein. Uteroovarian vein. d Fetectomized at cesarean section, placenta delivered within 4 days. e Fetectomized at cesarean section, placenta surgically removed 63 days later. — = Not determined. N D < 1.0 ng/ml. * Fetus was dead. t Mean progesterone levels (ng/ml) in intact monkeys at approximately days 149-154 of gestation are: b

c

2.9 4.6 5.1 4.1

UV b

UOVC

21.4

43.2 69.8 1,061.1 159.1

9.0 13.5 23.0

Placental delivery following fetectomy was extremely variable ranging from 2— >63 days post-fetectomy (Table 5). The placenta of 1 of the animals (#G-46) was removed surgically 63 days after fetectomy. Upon removal, the placenta was found to be firmly attached to the uterus and was normal in appearance except for some areas of necrosis present on the fetal surface. Seven days after cesarean section, progesterone was measurable in the uteroovarian vein but not in the peripheral circulation (Table 1). The CL was still a prominent structure on the ovary with good vascularization; it appeared pinkish with some scattered yellowish areas. Prolactin and mCS were either low or nondetectable (Tables 2 and 3) and mCG was again not measurable. In the monkey still carrying her placenta (#G-46), both progesterone and mCS were present.

No. observations per mean

Reference (4,6) (1) (2) (3)

Thirty-nine days following cesarean section, the CL was still apparent on the surface of the ovary, although its size appeared smaller than previously. It was slightly pinkish with more yellowish areas present than at 7 days post-cesarean section; vascularization was minimal. Progesterone was nondetectable, except in 1 animal (#N-96) in which it was barely measurable in the uteroovarian vein (Table 1). In the monkey which retained her placenta, progesterone was measurable in all 3 vessels and the CL appeared much as it did at 7 days post-cesarean section. Discussion Both the ovary containing the CL and the placenta were secreting progesterone between days 149-154 of pregnancy in the hypophysectomized monkeys as evidenced by the elevated levels present in the utero-

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TABLE 2. Monkey chorionic somatomammotropin levels (/xg/ml) following hypophysectomy during late pregnancy in the rhesus monkey Cesarean section — Days 149-154 of gestation!

Day 7 Postcesarean section

Animal number

pa

|jyb

pa

UVb

1077* D-81 N-96 1450* 1434 G-46

2.0 11.4 19.8 2,251

2,966

5

(6,9)

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HORMONES IN HYPOX PREGNANT MONKEYS

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TABLE 4. Hypophysectomy during late pregnancy in the rhesus monkey Day of gestation

Fetus

Animal number

Hypophysectomy

Cesarean section

Gestational age

Condition

Sex

(g)

1077* D-81 N-96 1450 1434 G-46

145 145 145 141 145 140

154 154 154 150 154 149

140-147

dead live live dead live live

male male male female female female

463 370 470

154 154

133-140 154 149

Weight

360 440 340

* Anesthetic regimen differed from that followed for the other animals. See text.

structure with scattered areas of vascularization, and in 1 animal (#N-96) progesterone was detected in the uteroovarian vein. Apparently the involution of the CL following pregnancy in the hypophysectomized monkey is very slow compared to the rapid decline of the CL of the non-fertile menstrual cycle (21-23). Slow regression of the CL following pregnancy seems to occur in intact animals as well, since progesterone is elevated in the ovarian vein 26 days postpartum (24,25). Despite the absence of the maternal pituitary gland, amniotic fluid prolactin concentrations were high (Table 3) and essentially the same as those present in normal monkeys (26). Although Josimovich et al. (26) have concluded that the maternal circulation may be a major source of amniotic fluid prolactin in the monkey, they have also demonstrated that approximately 30% of labeled prolactin injected into the amniotic fluid disappears within 3.5 h suggesting that the prolactin in the amniotic fluid is turning over at a moderate rate. Therefore, if the maternal pituitary gland is the major source of amniotic fluid prolactin, one would expect prolactin in the amniotic fluid to be extremely low 9 days after hypophysectomy. It is possible, however, that removal of the pituitary gland results in an inhibition or marked reduction in the transport of prolactin out of the amniotic fluid. The fetal pituitary is another possible source of prolactin, but in one of the mothers carrying a dead fetus (#1450) amniotic fluid prolactin was

actually higher than in mothers with live fetuses. Consideration should also be given to the possibility that amniotic fluid prolactin originates from a source other than the maternal or fetal pituitary glands. Monkey chorionic somatomammotropin levels in mothers with live fetuses were high between days 149-154 (Table 2) and were essentially the same as in intact monkeys during late pregnancy (6,9,27,28, Table 2). On the other hand, mCS was low or not detectable in mothers with dead fetuses (#1077, #1450) and was low 7 days after removal of the fetus in 1 of the animals (#G-46) even though the placenta remained in utero. Friesen (27) and Belangeret al. (28) have also observed a sharp decline in mCS following fetectomy in normal monkeys. Although the placenta is considered to be the primary source of mCS (29), the continued elevated secretion of mCS is dependent on the presence of a live fetus. TABLE 5. Placental delivery following fetectomy in rhesus monkeys hypophysectomized during late pregnancy Day of placental delivery Animal number

Postcesarean section

Gestation

Placental weight (g)

D-81 1434 G-46*

4 2 >63

158 156 >212

102 163 184

* Placenta surgically removed.

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WALSH ETAL.

The anesthetic protocol used in this study appeared to favor survival of the fetus. Of the 5 animals receiving low levels of halothane vaporized with excess amounts of oxygen and periodic injections of ketamine, only one fetus was found dead at the time of cesarean section and its death, based on bone development, was estimated to have occurred prior to hypophysectomy of the mother (Table 4). The sixth animal (#1077), whose anesthetic regimen differed significantly from the other 5 and included a thiobarbiturate, delivered a dead fetus whose death was estimated to have occurred following hypophysectomy. Barbiturates, either alone or in combination with halothane, were used in the anesthetic protocols for hypophysectomy in 3 other animals not included in this study. In each case pregnancy was terminated either by abortion or maternal death shortly after surgery. Delivery of the placenta following fetectomy was extremely variable (Table 5). Two monkeys delivered the placenta close to the average gestational length of 165 days; however, the third monkey retained her placenta until 212 days of gestation at which time it was surgically removed. Some investigators have noted this same sort of variability in intact monkeys (30, Slob and Wolf, unpublished); however, other investigators have observed that the placenta is delivered near to the expected time of parturition following fetectomy in normal monkeys (31, 32). References 1. Treloar, O. L., R. C. Wolf, and R. K. Meyer, Endocrinology 91: 665, 1972. 2. Koering, M. J., R. C. Wolf, and R. K. Meyer, Endocrinology 93: 686, 1973. 3. Macdonald, G. J., K. Yoshinaga, and R. O. Greep, Am J Phys Anthropol 38: 201, 1973. 4. Walsh, S. W., R. C. Wolf, and R. K. Meyer, Endocrinology 95: 1704, 1974. 5. Gulyas, B. J., Am J Anat 139: 95, 1974. 6. Walsh, S. W., Ph.D. Thesis, University of Wisconsin-Madison, 1975.

Endo • 1977 Vol 100 • No 3

7. Blomquist, A. J., and H. F. Harlow, Proc Anim Care Panel 11: 57, 1961. 8. Rao, G. N., R. E. Larson, S. W. Walsh, and R. K. Meyer, Fed Proc 34: 324, 1975 (Abstract). 9. Walsh, S. W., R. C. Wolf, R. K. Meyer, M. L. Aubert, and H. G. Friesen, Endocrinology 100: 851, 1977. 10. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci USA 68: 1902, 1971. 11. Smith, P. E., Endocrinology 55: 655, 1954. 12. Knobil, E., and R. O. Greep, Recent Prog Horm Res 15: 1, 1959. 13. Kraemer, D. C., G. T. Moore, and N. C. Vera Cruz, In Hafez, E. S. E. (ed.), Comparative Reproduction of Nonhuman Primates, Charles C Thomas, Springfield, 1971, p. 473. 14. Hill, J. D., In Proceedings of the Workshop in the Clinical Care of Nonhuman Primates, March 7-8, National Institutes of Health, Bethesda, 1973. 15. Van Wagenen, G., and C. W. Asling, Am J Anat 103: 163, 1958. 16. Van Wagenen, G., and C. W. Asling, Am J Anat 114: 107, 1964. 17. Kerr, G. R., J. H. Wallace, C. F. Chesney, and H. A. Waisman, Growth 36: 59, 1972. 18. Tullner, W. W., B. J. Gulyas, and G. D. Hodgen, Steroids 26: 625, 1975. 19. Tullner, W. W., and G. D. Hodgen, Steroids 24: 887, 1974. 20. Gulyas, B. J., L. Yuan, W. W. Tullner, and G. D. Hodgen, Biol Reprod 14: 613, 1976. 21. Corner, G. W., G. W. Bartelmez, and C. G. Hartman, Am J Anat 59: 433, 1936. 22. Comer, G. W., C. G. Hartman, and G. W. Bartelmez, Contrib Embryol Carneg Inst 31: 117, 1945. 23. Koering, M. J., Am J Anat 126: 73, 1969. 24. Weiss, G., D. J. Dierschke, F. J. Karsch, J. Hotchkiss, W. R. Butler, and E. Knobil, Endocrinology 93: 954, 1973. 25. Knobil, E., Biol Reprod 8: 246, 1973. 26. Josimovich, J. B., G. Weiss, and D. L. Hutchinson, Endocrinology 94: 1364, 1974. 27. Friesen, H., B. Shome, C. Belanger, P. Hwang, H. Guyda, and R. Myers, Excerpta Medica bit Cong Series 244: 224, 1971. 28. Belanger, C , B. Shome, H. Friesen, and R. E. MyersJ Clin Invest 50: 2660, 1971. 29. Friesen, H. G., S. Suwa, and P. Pare, Recent Prog Horm Res 25: 161, 1969. 30. Lanman, J. T., R. Thau, K. Sundaram, A. Brinson, and R. Bonk, Endocrinology 96: 591, 1975. 31. Dorfman, R. I., and G. Van Wagenen, Surg Gynecol Obstet 73: 545, 1941. 32. Van Wagenen, G., and W. H. Newton, Surg Gynecol Obstet 77: 539, 1943.

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Corpus luteum and fetoplacental functions in monkeys hypophysectomized during late pregnancy.

Corpus Luteum and Fetoplacental Functions in Monkeys Hypophysectomized During Late Pregnancy1 SCOTT W. WALSH,2 ROLAND K. MEYER, RICHARD C. WOLF, AND H...
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