Effects of fetectomy on plasma estrogens and progesterone in monkeys (Macaca mulatta)

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EFFECTS OF FETECTOMY ON PLASMA ESTROGENS AND PROGESTERONE IN MONKEYS (Macaca mulatta)

William W. Tullner and Gary D. Hodgen Section on Endocrinology, Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Ma. 20014 ABSTRACT

Received: 8/9/74

Effects of fetectomy on peripheral plasma levels of estrogens and progesterone were studied at 10 weeks (3 monkeys) and 16 weeks gestation (4 monkeys). Fetectomy was followed by a decrease in maternal peripheral plasma estradiol 17-B (E2) at a time when E2 levels remained elevated in Estrone (El) levels, initially low at fetectomy intact pregnant monkeys. (10 weeks), were maintained at similar low levels in contrast to elevated concentrations observed in normal animals during the final 30 days of In the absence of the fetus, progesterone (P) levels were pregnancy. similar to those of the normal pregnancies. After removal or delivery of the placenta, P levels decreased rapidly. Maternal hypophysectomy in one animal (10 weeks) produced a transient decrease in EP followed by recovery to control levels by 16 weeks. Subsequent fetectomy (16 weeks) was followed by a precipitous decline in maternal E2 levels. In conclusion, results indicate: a fetal origin, possibly from adrenal precursors, for the major contritution of E2 during the last 3 months and E1 during the last month of gestation; and placental production of most of the P found in peripheral plasma of pregnant rhesus monkeys.

INTRODUCTION The lack of effect of early ovariectomy on the normal course of pregnancy in the rhesus monkey has been described (1). Further observation during normal pregnancy in this species demonstrated that delivery of fetus and placenta was followed by a sharp decline in plasma levels of progesterone (2,3), estradiol -178 and urinary estrone (3). In order to evaluate the contribution of the fetus and the placenta to maternal plasma levels of estrogens aa

progesterone, we have fetectcxnizedrhesus monkeys

at ten weeks and at sixteen weeks gestation, leaving the placentas intact. One monkey was hypophysectomized at 10 weeks and fetectomized at 16 weeks. MATRRIALS AND METHODS Adult female rhesus monkeys were maintained under conditions previously described (3). A commercial primate diet was fed once daily and

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supplemented 3 times weekly with fruit and vegetables. Water was available --* ad lib Mating and determination of pregnancy have been described (3). Four monkeys were fetectomized at 107-119 days gestation. Another group of 3 monkeys was fetectomized at 69-73 days. Sodium pentobarbital was given intravenously at 30 mg/kg. body wt. to maintain anesthesia. Fetectomy was carried out under aseptic conditions as described by van Wagenen and Newton (4) except that the uterine wall was trans-illuminated before incision in order to avoid damage to the primary and secondary placentas as well as the major uterine blood vessels. Hypophysectomy was carried out using the technique of Hnobil and Greep (5). Heparinized blood samples were drawn from the femoral vessels at regular intervals throughout the experimental period. Plasma was frozen at -lOC until assayed. Plasma progesterone (P) concentrations were determined by the radioimmunoassay method of Speiler, Webb, Saldarini, and Coppola (6). Antiserum was provided through the courtesy of Dr. Saldarini. Plasma estradiol-178 (Ez) and estrone (El) were immunoassayed employing the procedure described in (3) with the following modifications. EI and Ez were separated on Sephadex LH-20 using a benzene-methanol solvent system (S13) described by Carr et al. (7) The estrone antibody used for radioimmunoassaywas raised by immunization of rabbits with estrone coupled at position 17 with bovine serum albumin. RESULTS A series of 4 rhesus monkeys was fetectomized at 107-119 days gestation.

After fetectomy, a marked decrease in plasma Ez concentration oc-

curred. An example of this effect is shown in Fig. la.

In contrast,

plasma Ez levels in normal pregnant animals followed the pattern observed in earlier studies and remained elevated throughout this period of pregnancy. Results from a normal animal in this series is presented in Fig. lb.

Plasma P concentrations showed minimal alterations in the period

following fetectomy (Fig. 2a) and did not differ significantly from levels observed during the same interval in normal pregnancy (Fig. 2b).

Placen-

tas were removed surgically just prior to the expected time of delivery. Progesterone consistently declined to basal levels after removal of the placenta.

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1000

889

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90149

r

Estradiokl7P

Fete&my (II6 days1

600

1

e 400 0”

200

i

0

I

I

I

20

40

60

1

60 GESTATION

I

I

I

I

100 (DAYS)

120

140

160

0 I60

Fig. la. Plasma estradiol levels in a rhesus monkey fetectomized at 116 clays gestation.

T 9890

IO00

800

I

4:

Ii+; M

GESTATION

(DAYS)

Fig. lb. Plasma estradiol levels during pregnancy in a normal rhesus monkey. M is day female caged with male.

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gr

Fig. 2a.

TDEOIDI

?Dl49

I

1

to M

20

40

60

60

100

GESTATION

120

140

0 160

I60

0AY3)

Plasma progesterone levels before and after fetectomy at 1.16

days. 9

9690

Parturlhon 1

1

M

Fig. 2b.

.

I

to

I

20

40

60

80 GESTATION

100 (DAYS)

120

140

160

160

Plasma progesterone levels during normal pregnancy.

In a second series of 3 monkeys fetectomized earlier in pregnancy (G-73

days),

maternal plasma concentrations of El, E2 and P were measured.

E2 levels declined 2- to lo- fold after fetectomy and were reduced to lower levels after spontaneous delivery of the placentas.

~958 illustrate this pattern (Fig. 3a). time of fetectomy

Results from monkey

El levels which were low at the

(less than 100 pg/ml) showed only small changes through-

out the remainder of pregnancy

(Fig. 3a).

In the normal monkey however,

similar low El concentrations were maintained until late pregnancy when the pattern was characterized by a rise and marked peak equal ing the elevated levels of E2 (3b).

to

or exceed-

Plasma P levels were in the normal

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891

600-

0 P-958 500

-

400-

-

Estmdid - 178

o---9

Ewcm

1 LOe &? 200

-

IOO-

‘0

20t

40I

80

60

100 I20

140

i69

J 160

GESTATION (Days)

Fig. 3a. Alterations in peripheral plasma levels of estradiol and estrone after early fetectorny.

800

-

Eslradiil-

o--q

Estm

OL4l38

176

140

160

la0

GESTATION IOAYSYS)

Fig. 3b. Peripheral plasma course of normal.pregnancy.

levels

of estradiol and

estrone during the

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892

PREGNANT

MONKEY

'IF-EOIDI

P-958

Fetectomy

GESTATION

Fig. ka.

(days)

Plasma progesterone levels before and after fetectomy at 73 days.

9 L466

I

20

I

40

ii

I

I

60

80 GESTATION

Fig. 4b. Plasma progesterone sus monkey.

1

I

100

120

140

1

J

160

180

(DAYS)

levels throughout pregnancy in a normal rhe-

range until placental delivery.

After this, P concentrations were low

(Fig. 4a and b). Of seven fetuses, 3 were female and four male. ferences were apparent.

No sex dependent dif-

In both series, placentas were grossly normal at

the time of delivery or placentectomy.

Placental weights of fetectomized

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animals at delivery were comparable to those of normal animals (Table 1).

Table 1 Growth of the placentas in rhesus monkeys after fetectomy

Placental Weight(g.)

Day

Monkey

Fetectomy

Delivery

Primaq

Secondary

P-317

Intact

---__

163

79

66

~-488

Intact

_____

164

90

69

D-149

116

171

82

69

9

110

166

59

49

D-172

119

167

99

80

G-555

107

167

69

58

P-958*

73

163

--_

m-e

p-563

69

116**

73

61

P-168"

72

159

---

___

*Portions of placentas had been eaten prior to their recovery. **Aborted

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One monkey (P954) was h~ophysecto~zed

at 73 days, fetectomized at

107 days and placentas removed by hysterotomy at 158 days gestation. AIthough plasma Ez levels decreased after hypophysectomy, they were elevated again by 105 days but declined sharply following fetectomy at 107 days. El levels remained low and relatively unchanged throughout this period (Fig. 5a). Plasma P rose 3-fold after hypophysectomy and these elevated levels persisted until removal of the placenta. This was followed by an abrupt decline in P concentration (Fig. 5b). DISCUSSION Earlier reports from this and other laboratories have described alterations in peripheral blood levels of estrogens and progesterone throughout gestation in the normal monkey (2,3,9). On the basis of results obtained after ovariectomy, it was demonstrated that the ovaries had little effect on maternal plasma levels of estrogens (El and E2) or P after 3 weeks gestation (2,8,9). Pregnancy continues normally in the absence of the ovaries with estrogen and progesterone levels similar to those of normal gestation. Moreover these ovariectomized mothers were able to maintain lactation and nurse young until weaning (5 l/2 months). Following parturition, an abrupt decline in P concentrations in maternal peripheral blood has been reported (2,3,9). The present finding, that placentectomy resulted in a similar fall in plasma P level, supports the observations that the placenta is the major source of P throughout the latter 4 l/2 months of pregnancy. Within 6 to 14 days after fectectomy, decreased maternal plasma concentrations of E2 were found although plasma El levels were relatively unchanged from the low preoperative levels even during the final month of

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895

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600-

500-

-

Estradid

*+

Estrolw

0 P-954

- 17,9

4OOFetactomy (107 daya) d $ r

300-

200

-

loo-

‘0

L

I

,

I

20

40

60

I

100 Days)

60 GESTATION

120

140

160

160

Fig. 5a. Peripheral plasma concentrations of estradiol and estrone in a rhesus monkey hypophyse&omizeZ. in early pregnancy (73 days) followed by fetectomy at 107 days.

12

I

I

I

Maternal Hypophysactomy

IO -

Oo

I

I

I

20

40

I al

I 80

I

I

0 P954

Fetectomy 107 days)

(

I loo

GESTATION

I

1

Placentas Removed (I 56 days)

I

I

120

140

& 160

I I60

,

(days)

Fig. 5b. Progesterone levels in peripheral plasma of monkey shown in 5b (above).

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gestation. This is in marked contrast to the rapid rise in El occurring in normal pregnancies 2 to 4 weeks prior to parturition (Fig. 3b). During this peak production, El levels occasionally exceed those of EP. The ab. sence of this increase in plasma El concentration following surgical removal of the fetus indicates a causal relationship between fetus and maternal plasma El levels that may depend on fetal adrenocortical hormones. Fetal gonads may not be involved since sex of the fetus had no apparent effect on El levels in the pre-partum period. It has been reported that both plasma estrogen and P levels decrease beginning about 1 day after fetal cord ligation (9).

In the data pre-

sented here, El levels were not markedly changed after fetectomy and plasma P concentrations did not decline but followed the pattern seen during this period in normal gestation. The decreased P levels after cord ligation, with the fetus remaining in utero (9),

suggest abnormal

placental function that is not seen after surgical removal of the fetus. It has been reported that after removal of the fetal rhesus monkey, the mother maintains characteristic features of pregnancy including intense sex skin color, generalized edema and weight gain (4). The present study confirms these observations. One cannot draw conclusions from the single pregnancy in which hypophysectomy was followed by fetectomy except to note that fetectomy in this monkey produced a similar decline in E2 as that found in the series described above. Also

low El levels observed prior to hypophysectomy were

apparently not altered by this procedure or fetectomy. The hormonal contribution of the several compartments will require more detailed study particularly in view of the intimate relationship

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between mother, fetus and placenta with regard to metabolism of steroid intermediates demonstrated in women (10). Results presented here indicate a causal relationship between fetal ablation and markedly reduced maternal.levels of E2 during the latter two-thirds of gestation and the lack of elevated El in late pregnancy plasma. However, experimental evidence is necessary to determine the role of the fetal and maternal adrenals in providing precursors for placental conversion to estrogens.

ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Mr. Don Barber, Ms. Mary Collins and Mr. Charles Turner. REXFZENCES 1. 2.

3. 4. 2: 7.

Tullner, W.W. and Hertz, R., ENDOCRINOLOGY 78, 1076 (1966). Neill, J.D., Johansson, E.D.B. and Knobil, E ., ENDOCRINOLOGY 84, 45 (1969). Hodgen, G.D., Dufau, M.L., Catt, K.J., and Tullner, W.W., ENDOCRINOLOGY 91, 896 (1972). vanwsgenen, G. and Newton, W.H., SURG. GYN. OBST. 77, 539 (1943). Knobil, E. and Greep, R.O., RECENT PROGR. HORMO. P&S. 15, 1, (1959). Speiler, J.M., Webb, R.L., Saldarini, R.J. and Coppola, J.A., STEROIDS 19, 751 (1972). Carr, B.R., Mikhail, G. and Flickinger, G.L., J.CLIN. ENDOCR. 33, 358

(1971). 8. Hodgen, G.D. and Tullner, W.W., Abstract, 56th ANNUAL MEETING of the ENDOCRINE SOCIETY, p. A-248 (June 1974). 9. BOSU, W.T.K., Johansson, E.D.B. and Gemzell, C., ACTA ENDOCR. (KBH) 75, 601 (1974). 10.

Diczfalusy, E., AMER. J. OBSTET. GYNECOL. 119, 419 (1974).