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Serum progesterone and corpus luteum function in pregnant pigtailed monkeys (Macaca nemestrina )
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SERUM PROGESTERONE AND CORPUS LUTEUM FUNCTION IN PREGNANT PIGTAILED MONKEYS (Macaca nemestrina) Varadaraj Chandrashekar, Richard C. Wolf, Donald J. Dierschke Samuel A. Sholl, William E. Bridson and James R. Clark Wisconsin Regional Primate Research Center University of Wisconsin Madison, Wisconsin 53706, U.S.A. Received 7-25-80
ABSTRACT
Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 ? 2.21 ng/ml and decreased progressively to 7.96 + 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 + 2.45 ng/ml on day 37, thereafter gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 f 1.48 ng/ml on the day of delivery to 2.16 + 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the uteroovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing 3H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy. Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation. These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear. INTRODUCTION In women and rhesus monkeys a functional corpus luteum (CL) is indispensable for the initial maintenance of pregnancy (1,2).
However,
in the rhesus monkey the activity of the CL decreases before midgestation but is subsequently "rejuvenated" before parturition (3,4).
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Similarly, in pregnant women the CL has been shown to be active near term (5).
Nevertheless, bilateral ovariectomy in the rhesus, as early
as day 21 of pregnancy, does not result in termination of gestation and a similar effect is observed in women (6,7).
Therefore, a functional
CL is not required for maintenance of gestation during the major portion of pregnancy.
However, the CL is present and maintained through-
out pregnancy but the significance of its functional changes during gestation in primates is not known.
Furthermore, no previous study has
assessed CL function during gestation in the pigtailed monkey.
The
present investigation was undertaken to evaluate the functional changes of the CL and its control during pregnancy and early lactation in this species in the hope of providing new insight to this problem. MATERIALS AND METHDDS Animals, Collection of Blood Samples and Ovarian Tissues -even adult pigtalledmonkeys (Maca?%%%&.trina7_were utilized in this study. Housing, diet and handlingof blood samples have been described previously (8). Female monkeys were placed with a male of proven fertility for three days during midcycle when sex skin coloration was at its peak (9). Two days before sex skin color breakdown was designated as the first day of pregnancy. Pregnancy was confirmed by a hemagglutination inhibition test for urinary monkey chorionic gonado-. tropin (mCG) and increasing serum levels of immunoreactive mCG during early gestation (10,ll). In five monkeys two subsequent pregnancies were studied after providing adequate rest following normal duration of In one phase of the study, peripheral blood samples (3-4 ml) lactation. were obtained by venipuncture of a femoral vein between 0300 and 1000 h from 5 monkeys with intact ovaries and 2 monkeys ovariectomized bilaterally on day 35 of pregnancy, Blood samples were taken daily on days 10 through 30 and again from day 150 to day 5 postpartum. Between days 31 and 149 of pregnancy, as well as from days 6 to 25 postpartum, blood was obtained 3 times a week. In another phase of study, five monkeys were laparotomized under aseptic conditions at an average of 25 (range 25-26), 80 (78-81) and 159 (157-160) days of pregnancy. Animals were sedated initially with ketamine hydrochloride (Vetalar, Parke-Davis; 15 mg/kg BW, im) and subsequently anesthetized with halothane (Fluorothane, Ayerst Laboratories, Inc.) vaporized with an excess amount of oxygen (1 to 1.5% halothane/2 liters of Oz/min). On day 25 of gestation, the CL was marked by injecting a drop of sterile india ink into the luteal tissue from a 1 ml syringe fitted with a 27-gauge needle. During each laparotomy, blood
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(8-10 ml) was obtained from a uterine vein and the uteroovarian vein ipsilateral to the CL-containing ovary. Five ml of peripheral blood were also taken at this time. From one of these monkeys and two additional animals one or both ovaries were removed at day 150 of pregnancy, fixed in Bouin's fluid for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraplast. Sections were taken at a thickness of 6 to 8 pm (two ovaries were serially sectioned), stained with hematoxylene and eosin, and evaluated histologically. Monkeys were also subjected to laparotomy on day 35 (N=Z) or day 159 (N=2) of gestation and ovariectomized to obtain ovarian tissues for radioisotopic incubation and histological studies, as well as to investigate pregnancy maintenance in the absence of ovaries. Hormone Assays Serum progesterone (P) concentrations were measured by RIA as described previously (8), with additional validation for utilizing this procedure for pregnant pigtailed monkeys. An antiserum (912) produced in a rabbit against a hemisuccinate of lla-hydroxyprogesterone linked to bovine serum albumin was used. The cross-reactivity of this antiserum has been reported elsewhere (12). In addition, 5a-pregnane-3,20-dione was found to cross-react (22.4%) with this antiserum. A further check was made to evaluate the specificity of this antiserum by comparing the assay results of varying amounts of serum obtained from pregnant pigtailed macaques which were tested as follows: (a) petroleum ether extraction only; (b) petroleum ether extraction and chromatography on Sephadex LH-20 columns using a heptane:benzene:methanol (85:5:4 by vol) solvent system. Chromatographic purification resulted in lower P levels (4.89 + 0.16 ng/ml vs 7.04 + 0.18 ng/ml, P < O.Ol), hence, all samples were extracted with petroleum ether and further purified by chromatography. Varying quantities of serum obtained from pregnant pigtailed monkeys produced a curve which was parallel with various amounts of P standards. The sensitivity of the assay was 5.4 pg. Serum concentrations of mCG were assayed by RIA as described previously (ll), with minor modifications (13). Values are expressed in relation to the weight of the rhesus monkey chorionic gonadotropin reference preparation (mCG-VC; [biological potency of 1 IU hCG equivalents/mg lyophilized material]) (14). Varying quantities of sera collected from pregnant rhesus as well as from pregnant pigtailed macaques produced inhibition curves parallel to that of varying amounts of mCG-VC. The sensitivity of the assay was 1.03 pg of the reference standard. Serum concentrations of LH were measured by RIA (15), with minor modifications (16). Varying amounts of pooled sera obtained from an ovariectomized pigtailed monkey (representing endogenous LH) produced an inhibition curve which was not different from that of varying quantities of a purified rhesus monkey LH reference preparation (WDP-X-81-1720). The LH values are expressed in relation to the weight of this reference preparation (biological potency equivalent to 2.90 U/ml NIH-LH-Sl) (17). The sensitivity of the assay was 0.35 ng/tube. Ovarian Tissue Incubation and Purification of Metabolites The-and follicles were separated fr$i the ovary that had been removed during laparotomy. Weighed fragments of luteal tissue (l-2 mn thick) or secondary follicles were placed in small flasks (3-5/flask)
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containing 1 ml of 0.1 M Krebs-Ringer bicarbonate buffer (pH 7.4) with 3.5 mM glucose-6-phosphate, 0.3 mM NADP, 0.3 mM NAD, 0.5 IU glucose-6phosphate dehydrogenase, 2 mg glucose and 0.25 $i [7-3H]cholesterol (purified chromatographically, 21.6 Ci/mmole, New England Nuclear Corporation). Incubations were conducted in a Dubnoff metabolic incubator at 37°C for 2 h in the presence of 0,/C02 (95:5). At 30 min intervals each medium was aspirated, frozen in dry ice-acetone and fresh medium with the substrate was added to the flask containing the tissue. Incubation media without tissue fragments served as controls. Tritiated cholesterol, pregnenolone (3f+hydroxy-5-pregnen-20-one) and P were isolated from the incubation media and purified by methods described previously (18). Statistics Peripheral hormone concentrations were analyzed by using a two-way analysis of variance (19). Since the between vein variance of P concentrations at different stages of pregnancy differed considerably, a Kruskal-Wallis test (20) was utilized to assess the significance of differences in serum levels of this steroid in uteroovarian and uterine veins. A probability of less than 0.05 was considered significant. RESULTS Twelve live births occurred between days 162-180 of pregnancy regardless of whether ovaries were present or removed on either day 35 or 150 of gestation.
One animal with intact ovaries gave birth to a well-
developed, but dead infant on day 160 of pregnancy.
The remaining three
monkeys (one ovariectomized bilaterally and the other two ovariectomized unilaterally on day 150 of gestation) delivered dead infants 1-2 days after surgery.
Based on the weights and general condition of these in-
fants, it was inferred that the neonates died during delivery. The mean peripheral serum concentrations of mCG, LH and P during pregnancy and early lactation are illustrated in Fig. 1.
Circulating
levels of P on day 10 of pregnancy were 12.98 f. 2.21 ng/ml and decreased progressively to 7.96 ? 1.27 ng/ml on day 21 when high levels of mCG were present.
The concentrations of P increased again on day 27 of ges-
tation and reached peak levels on day 37 (18.48 * 2.45 ng/ml), thereafter decreasing to a nadir near midgestation.
About 10 days before par-
turition P levels increased again (P < 0.05) in relation to P concentra-
S tions at midgestation.
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487
Concentrations of P decreased precipitously
from 6.62 f 1.48 ng/ml on the day of delivery to 2.16 + 0.43 ng/ml on day 2 postpartum and remained at low levels throughout the remainder of this study.
Postpartum observations were limited to monkeys with suck-
ling infants.
Fig.
Mean chcuRa;tingcancentutianh06 chohionicgonadotiapin 1. (mCG),LH and pmgcuntemne (it SEMI duhg pegnancy (N=Sland eahey &a&a.tionIN=41in p-igigtaieed monheyn.
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Immunorea~tiy~ mCG in the peripheral circulation was detected in all animals on day 70 of pregnancy, rose to maximum amounts on days 18 to 24 of pregnancy, then declined to low concentrations on day 40.
This
hormone was not detectable throughout the remainder of the study.
Since
N39
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TDX?.OLD=
the antiserum used to measure LH (GDN-15) cross-reacted with mCG (Zl), LH was assayed from day 40 of pregnancy.
There was no significant dif-
ference in circulating levels of LH during gestation and lactation. The patterns of P secretion in animals without ovaries were similar to intact animals (Fig. 2).
In ovariectomized monkeys, although
infants were suckling, P was not detectable after parturition. Throughout pregnancy serum concentrations of P in uteroovarian and uterine veins were higher (P < 0,001) than in the femoral vein (Fig. 3). Progesterone levels in uteroovarian venous blood exceeded those of the uterine vein at days 80 and 159 of pregnancy, while the opposite was found at day 25 of gestation (Fig. 3, Table 1).
I
1
I
I
I
I
I
I
UTEROOVARIAN
40
-
-
XI20
VEIN
-. -
FEMORAL
10 -
* I 20
1 40
I 60
* T 1 00
I 100
.-. -+
VEIN I 120
I 140
t I60
DAY OF PREGNANCY
vein behum concctiaF&j. 3. Uehoovahian, LLtehinc and peniphm.aX tioio~nod phcrgc&e.&ane (+ SEhl) in phegnant p.igRaieed monhcgb wtih inZataotavanieh.
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TIIROIDSS TABLE 1
DIFFERENCE BETWEEN UTEROOVARIAN AND UTERINE VEIN SERUM CONCENTRATIONS 0~ PROGESTERONE (NG/ML) IN PREGNANT PIGTAILED MONKEYS
Animal Number
Day of Pregnancy 25
80
159 +ll.R
N38
-40.6
+25.Ja
N43
-13.7
+9.1
N44
-115.1
t199.4
N45
-142.9
t18.8
t3.5
N46
-7.3
t21.2
t34.3
a A pRti sign indica.ten that bAemid .EeveRs Wehc avtian utin Rhan in Lhe tietin@. vein.
kigheh
+Pl.5 t20.7
irz the
tiehc-
Incubation studies revealed that both the CL and follicles synthesized pregnenolone and P from cholesterol at days 35 and 159 of pregnancy (Table 2).
However, increased capability of synthesis of these
steroids was observed in CL tissue at day 159 of pregnancy. Gross examination of ovaries on day 25 of gestation revealed welldeveloped and vascularized CL with distinct luteal tissue.
However,
the same CL were not grossly apparent in the majority of monkeys when examined during midgestation
(day 80) except for the india ink marking
which appeared as a small spot on the surface of the ovary.
Although
the CL were not grossly visible in the ovaries from 3 of 4 monkeys examined on day 159 of gestation, histclogically small CL were located inside the tunica albuginea of the ovary.
A well-developed CL was
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TDROXDTABLE 2
TOTAL AMOUNT OF STEROIDS SYNTHESIZED DURING A TWO HOUR INCUBATION WITH [7-3H]CHOLESTEROL 3 H-Pregnenolone
Day of Pregnancy
(DPM
Tissue
159 (2)
10~~)
2.93b
Controlsa 35 (2)c
x
3
H-Progesterone (DPM x 10-2) 0.21
Corpus Luteum
12.00
5.35
Follicles
12.20
5.25
Corpus Luteum
27.10
9.18
Follicles
31.85
2.21
2 'Incubtioti uLthoti tibue
dhagmeti; N=3
,"Mean vaihen Nwnbeh 05 monkey& examined present in the fourth animal.
These CL contained india ink marking and
the majority of the granulosa luteal cells were well-delineated, with spherical nuclei, which were centrally located in the granulated cytoplasm. DISCUSSION It has been known for many years that a functional CL is essential for initiation and maintenance of early pregnancy in the rhesus monkey since ovariectomy soon after nidation was followed by termination of pregnancy, but gestation continued in these monkeys if treated with P (1).
Furthermore, both morphological and hormonal studies revealed
that the CL is active during early stages of pregnancy (22,23,24).
In
the pigtailed monkey circulating concentrations of P were elevated during the initial period of gestation indicating that the CL is functional
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at this stage of pregnancy.
TIIROXDD In this species, as in the rhesus (23),
P concentrations increased again near day 27, reaching maximum amounts on day 37 of pregnancy.
That this increase in P arises from the feto-
placental unit is suggested by our observation that serum concentrations of P did not decline rapidly following ovariectomy on day 35 of gestation.
It is also possible that the fetoplacental unit is secreting P
as early as day 25 of pregnancy as evidenced by higher serum concentrations of this steroid,in the uterine vein than in the uteroovarian vein. Similarly, ovariectomy on day 35 of pregnancy did not alter the subsequent normal course of pregnancy or the patterns of P secretion, indicating that the ovaries have minimal influence on gestation after this time and that the fetoplacental unit is the primary source of P thereafter.
The elevated concentrations of this steroid during late
pregnancy also seem to arise from the fetoplacental unit.
This view is
substantiated by elevated levels of P at late gestation in animals with or without ovaries.
In the rhesus monkey it has been shown that removal
of the placenta but not the fetus results in reduction of serum concentrations of P (251, revealing the importance of the placenta in the production of this steroid.
However, in the present study at days 80 and
159 of pregnancy higher serum levels of P in the uteroovarian vein than in the uterine vein support the concept that the CL secrete P during both mid and late gestation.
Perhaps P produced by the CL during these
stages of pregnancy is not reflected in the peripheral circulation due to the dilution of this steroid in circulation. thesis of 'H-progesterone from
3
Increased in vitro syn-
H-cholesterol by luteal fragments as
well as the presence of active luteal cells, regardless of CL size, on day 159 of gestation, suggests that the CL is capable of synthesizing P
S during late pregnancy.
493
T~ROXDrn
Similarly, the presence of detectable amounts
of P follow ing parturit ion in intact, but not in ovariectomized animals indicates that the CL function continues during lactation, as in rhesus monkeys
(26,27).
The possibility that a new CL is formed in late preg-
nancy is not compatible with the present observation that the only CL present on day 159 of gestation still contained the india ink, injected on day 25 of pregnancy. It is not known which hormones are responsible for the maintenance of CL function during pregnancy in the pigtailed monkey.
However, in the
rhesus, evidence exists to suggest that mCG is luteotropic during the initial period of gestation (14,23,28).
In the pigtailed macaque, on day 10
of pregnancy high concentrations of P and detectable amounts of mCG were present in peripheral circulation, indicating that mCG may be luteotropic in function during early pregnancy.
Nevertheless, the circulating concen-
trations of P gradually decreased during early pregnancy while mCG titers increased.
This relationship was also observed in the rhesus and may be
due to refractoriness of the CL to continued stimulation by mCG after the dramatic initial response (23,29).
Chorionic gonadotropin does not ap-
pear to be responsible for the CL function after day 40 of pregnancy since following this period mCG was undetectable.
The presence of constant
levels of LH in circulation suggests that this gonadotropin may play a role in maintenance of CL function during mid and late gestation. In humans, the general patterns of P secretion during pregnancy and following parturition (30,31), is similar to that described herein for the pigtailed monkey and as described previously for the rhesus monkey (3,4,22,23,24,29). in women.
However, the absolute amounts of P are much higher
This species variation may be explained on the basis of dif-
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ferences in the levels of steroid-binding globulins present in these primates (32,33,34).
Thus, the pigtailed macaque may be added to the
list of primate species in which the CL retains functional capacity throughout gestation, but from the present study no answer can be found to the questions regarding the mechanism nor physiological role for this extended luteal function. ACKNOWLEDGMENTS This work was supported by Grant RR-00167 from the National Institutes of Health, United States Public Health Service, and Grant 6300505B,C from The Ford Foundation. Publication number 20-005 of the Wisconsin Regional Primate Research Center. The gifts of oLHB antiserum (H-26) utilized for mCG RIA from Dr. G.D. Hodgen, an ovine LH antiserum (GDN-15) from Dr. G.D. Niswender used for LH determinations, progesterone antiserum (912) from Dr. A.H. Surve, and rhesus monkey LH reference preparation (WDP-X-81-1720) from Dr. W.D. Peckham are most gratefully acknowledged. Reagents for hemagglutination inhibition tests for urinary mCG were kindly supplied by the Hormone Distribution Branch, NIAMDD, NIH. Part of the technical assistance was provided by Ms. P.A. Meller and Ms. K. Rygasewicz. We also appreciate Mr. D.E. Cowley and Mr. R.A. Vertein for their excellent animal care. REFERENCES Meyer, R.K., Wolf, R.C. and Arslan, M. Proc. 2nd Int. Congr. Primat., Atlanta, Georgia, RECENT ADVANCES IN PRIMATOLOGY, Vol. 2, Karger, Basel/New York p. 30 (1969). 2. Tulsky, A.S. and Koff, A.K. FERTIL. STERIL. g: 118 (1957). 3. Treloar, O.L., Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY -91: 665 (1972). Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY -93: 686 4. Koering, M.J., (1973). 5. LeMaire, W.W., Conly, P.W., Moffett, A. and Cleveland, W.W. AM. J. OBSTET. GYNECOL. 108: 132 (1970). Melinkoff, E. AM. J. OBSTET. GYNECOL. 60: 437 (1950). 76: Tullner, W.W. and Hertz, R. ENDOCRINOLOGY 78: 1076 (1966). REPROD. -18: 779 8. Clark, J.R., Dierschke, D.J. and Wolf, R.C.BIOL. (1978). Czaja, J.A., Eisele, S.G. and Goy, R.W. FED. PROC. 34: 1680 (1975). 1;: Hodgen, G.D. and Ross, G.T. J. CLIN. ENDOCRINOL. MEi?iB. 38: 927 (1974). 11. Hodgen, G.D., Tullner, W.W., Vaitukaitis, J.L., Ward, D.N. and Ross, G.T. J. CLIN. ENDOCRINOL. METAB. 39: 457 (1974). 12. Surve, A.H., Bacso, I., Brinckerhov, J.H. and Kirsch, S.J. BIM. REPROD. 15: 34.3 (1976). 13. WehrenbeG, W.B., Dierschke, D.J. and Wolf, R.C. BIOL. REPROD. 20: 601 (1979). 1.
S 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33. 34.
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Chandrashekar, V., Meyer, R.K., Bridson, W.E. and Wolf, R.C. BIOL. REPROD. E: 889 (1979). Niswender, G.D., Monroe, S.E., Peckham, W.D., Midgley, Jr., A.R., Knobil, E. and Reichert, Jr., L.E. ENDOCRINOLOGY 38: 1327 (1971). Toivola, P.T.K., Bridson, W.E. and Robinson, J.A. ENDOCRINOLOGY 102: 1815 (1978). Krman, G.A., Niswender, G.D., Gay, V.L., Reichert, Jr., L.E. and Midgley, Jr., A.R. ENDOCRINOLOGY 92: 618 (1973). Sholl, S.A. STEROIDS 24: 703 (197q. Winer, B.J. STATISTICS PRINCIPLES IN EXPERrMENTAL DESIGN. 2nd Ed., McGraw-Hill Book Co., New York (1971). Kruskal, W.H. and Wallis, W.A. 3. AM. STAT. ASSOC. 47: 583 (1952). Hobson, W., Faiman, C., Dougherty, W.J., Reyes, F.I.and Winter, J.S.D. FERTIL. STERIL. 26: 93 (1975). Koering, M.J., Wolf, R.CFand Meyer, R.K. BIOL. REPROD. 2: 254 (1973). Atkinson, L.E., Hotchkiss, J., Fritz, G.R., Surve, A.H., Fleill, J.D. and Knobil, E. BIOL. REPROD. 12: 335 (1975). Walsh, S-W., Wolf, R.C. and Meyer, EK. E~~DOCRINOLOGY -95: 1704 (1974). Tullner, W.W. and Hodgen, G.D. STEROIDS 3: 887 (1974). Weiss, G., Dierschke, D.J., Karsch, F.J., Hotchkiss, J., Butler, W.R., and Knobil, E. ENDOCRINOLOGY 93: 954 (1973). Weiss, G., Butler, W.R., Hotchkiss, x, Dierschke, D.J. and Knobil, E. PROC. SOC. EXP. Blot. MED. 151: 113 (1976). Sundaram, K., Chang, C.C., Laurence, K.A., Brinson, A.O., Atkinson, L.E., Segal, S.J. and Ward, D.N. CONTRACEPTION 14: 639 (1976). Knobil, E. BIOL. REPROD. 8: 246 (1973). Weiss, G., O'Byrne, E.M., Fiochman, J.A., Goldsmith, L.T., Rifkin, L. and Steinetz, B.G. OBSTET. GYNECOL. 50: 679 (1977). Johansson, E.D.B. ACTA ~NDOCRINOL. (KBHJ-61: 607 (1969). Seal, U.S. and Doe, R.P. In METABOLIC EFFETS OF GONADAL HORMONES AND CONTRACEPTIVE STEROIDS, Eds. H.A. Salhanick, D.M. Kipnis and Ee;mean;e Wiele, Plenum Press,_New York p. 277 (1969). Hagemenas, F. and Kittinger, G.W. ENDOCRINOLOGY -90: 325 (1472j: Schiller, H.S., Holm, R.A. and Sackett, G.P. AM. J. PHYSIOL. 234: E489 (1978).
2692
SERUM PROGESTERONE AND CORPUS LUTEUM FUNCTION IN PREGNANT PIGTAILED MONKEYS (Macaca nemestrina) Varadaraj Chandrashekar, Richard C. Wolf, Donald J. Dierschke Samuel A. Sholl, William E. Bridson and James R. Clark Wisconsin Regional Primate Research Center University of Wisconsin Madison, Wisconsin 53706, U.S.A. Received 7-25-80
ABSTRACT
Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 ? 2.21 ng/ml and decreased progressively to 7.96 + 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 + 2.45 ng/ml on day 37, thereafter gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 f 1.48 ng/ml on the day of delivery to 2.16 + 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the uteroovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing 3H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy. Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation. These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear. INTRODUCTION In women and rhesus monkeys a functional corpus luteum (CL) is indispensable for the initial maintenance of pregnancy (1,2).
However,
in the rhesus monkey the activity of the CL decreases before midgestation but is subsequently "rejuvenated" before parturition (3,4).
Volume 36, Number 4
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Similarly, in pregnant women the CL has been shown to be active near term (5).
Nevertheless, bilateral ovariectomy in the rhesus, as early
as day 21 of pregnancy, does not result in termination of gestation and a similar effect is observed in women (6,7).
Therefore, a functional
CL is not required for maintenance of gestation during the major portion of pregnancy.
However, the CL is present and maintained through-
out pregnancy but the significance of its functional changes during gestation in primates is not known.
Furthermore, no previous study has
assessed CL function during gestation in the pigtailed monkey.
The
present investigation was undertaken to evaluate the functional changes of the CL and its control during pregnancy and early lactation in this species in the hope of providing new insight to this problem. MATERIALS AND METHDDS Animals, Collection of Blood Samples and Ovarian Tissues -even adult pigtalledmonkeys (Maca?%%%&.trina7_were utilized in this study. Housing, diet and handlingof blood samples have been described previously (8). Female monkeys were placed with a male of proven fertility for three days during midcycle when sex skin coloration was at its peak (9). Two days before sex skin color breakdown was designated as the first day of pregnancy. Pregnancy was confirmed by a hemagglutination inhibition test for urinary monkey chorionic gonado-. tropin (mCG) and increasing serum levels of immunoreactive mCG during early gestation (10,ll). In five monkeys two subsequent pregnancies were studied after providing adequate rest following normal duration of In one phase of the study, peripheral blood samples (3-4 ml) lactation. were obtained by venipuncture of a femoral vein between 0300 and 1000 h from 5 monkeys with intact ovaries and 2 monkeys ovariectomized bilaterally on day 35 of pregnancy, Blood samples were taken daily on days 10 through 30 and again from day 150 to day 5 postpartum. Between days 31 and 149 of pregnancy, as well as from days 6 to 25 postpartum, blood was obtained 3 times a week. In another phase of study, five monkeys were laparotomized under aseptic conditions at an average of 25 (range 25-26), 80 (78-81) and 159 (157-160) days of pregnancy. Animals were sedated initially with ketamine hydrochloride (Vetalar, Parke-Davis; 15 mg/kg BW, im) and subsequently anesthetized with halothane (Fluorothane, Ayerst Laboratories, Inc.) vaporized with an excess amount of oxygen (1 to 1.5% halothane/2 liters of Oz/min). On day 25 of gestation, the CL was marked by injecting a drop of sterile india ink into the luteal tissue from a 1 ml syringe fitted with a 27-gauge needle. During each laparotomy, blood
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485
(8-10 ml) was obtained from a uterine vein and the uteroovarian vein ipsilateral to the CL-containing ovary. Five ml of peripheral blood were also taken at this time. From one of these monkeys and two additional animals one or both ovaries were removed at day 150 of pregnancy, fixed in Bouin's fluid for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraplast. Sections were taken at a thickness of 6 to 8 pm (two ovaries were serially sectioned), stained with hematoxylene and eosin, and evaluated histologically. Monkeys were also subjected to laparotomy on day 35 (N=Z) or day 159 (N=2) of gestation and ovariectomized to obtain ovarian tissues for radioisotopic incubation and histological studies, as well as to investigate pregnancy maintenance in the absence of ovaries. Hormone Assays Serum progesterone (P) concentrations were measured by RIA as described previously (8), with additional validation for utilizing this procedure for pregnant pigtailed monkeys. An antiserum (912) produced in a rabbit against a hemisuccinate of lla-hydroxyprogesterone linked to bovine serum albumin was used. The cross-reactivity of this antiserum has been reported elsewhere (12). In addition, 5a-pregnane-3,20-dione was found to cross-react (22.4%) with this antiserum. A further check was made to evaluate the specificity of this antiserum by comparing the assay results of varying amounts of serum obtained from pregnant pigtailed macaques which were tested as follows: (a) petroleum ether extraction only; (b) petroleum ether extraction and chromatography on Sephadex LH-20 columns using a heptane:benzene:methanol (85:5:4 by vol) solvent system. Chromatographic purification resulted in lower P levels (4.89 + 0.16 ng/ml vs 7.04 + 0.18 ng/ml, P < O.Ol), hence, all samples were extracted with petroleum ether and further purified by chromatography. Varying quantities of serum obtained from pregnant pigtailed monkeys produced a curve which was parallel with various amounts of P standards. The sensitivity of the assay was 5.4 pg. Serum concentrations of mCG were assayed by RIA as described previously (ll), with minor modifications (13). Values are expressed in relation to the weight of the rhesus monkey chorionic gonadotropin reference preparation (mCG-VC; [biological potency of 1 IU hCG equivalents/mg lyophilized material]) (14). Varying quantities of sera collected from pregnant rhesus as well as from pregnant pigtailed macaques produced inhibition curves parallel to that of varying amounts of mCG-VC. The sensitivity of the assay was 1.03 pg of the reference standard. Serum concentrations of LH were measured by RIA (15), with minor modifications (16). Varying amounts of pooled sera obtained from an ovariectomized pigtailed monkey (representing endogenous LH) produced an inhibition curve which was not different from that of varying quantities of a purified rhesus monkey LH reference preparation (WDP-X-81-1720). The LH values are expressed in relation to the weight of this reference preparation (biological potency equivalent to 2.90 U/ml NIH-LH-Sl) (17). The sensitivity of the assay was 0.35 ng/tube. Ovarian Tissue Incubation and Purification of Metabolites The-and follicles were separated fr$i the ovary that had been removed during laparotomy. Weighed fragments of luteal tissue (l-2 mn thick) or secondary follicles were placed in small flasks (3-5/flask)
S
486
TIJROXDrn
containing 1 ml of 0.1 M Krebs-Ringer bicarbonate buffer (pH 7.4) with 3.5 mM glucose-6-phosphate, 0.3 mM NADP, 0.3 mM NAD, 0.5 IU glucose-6phosphate dehydrogenase, 2 mg glucose and 0.25 $i [7-3H]cholesterol (purified chromatographically, 21.6 Ci/mmole, New England Nuclear Corporation). Incubations were conducted in a Dubnoff metabolic incubator at 37°C for 2 h in the presence of 0,/C02 (95:5). At 30 min intervals each medium was aspirated, frozen in dry ice-acetone and fresh medium with the substrate was added to the flask containing the tissue. Incubation media without tissue fragments served as controls. Tritiated cholesterol, pregnenolone (3f+hydroxy-5-pregnen-20-one) and P were isolated from the incubation media and purified by methods described previously (18). Statistics Peripheral hormone concentrations were analyzed by using a two-way analysis of variance (19). Since the between vein variance of P concentrations at different stages of pregnancy differed considerably, a Kruskal-Wallis test (20) was utilized to assess the significance of differences in serum levels of this steroid in uteroovarian and uterine veins. A probability of less than 0.05 was considered significant. RESULTS Twelve live births occurred between days 162-180 of pregnancy regardless of whether ovaries were present or removed on either day 35 or 150 of gestation.
One animal with intact ovaries gave birth to a well-
developed, but dead infant on day 160 of pregnancy.
The remaining three
monkeys (one ovariectomized bilaterally and the other two ovariectomized unilaterally on day 150 of gestation) delivered dead infants 1-2 days after surgery.
Based on the weights and general condition of these in-
fants, it was inferred that the neonates died during delivery. The mean peripheral serum concentrations of mCG, LH and P during pregnancy and early lactation are illustrated in Fig. 1.
Circulating
levels of P on day 10 of pregnancy were 12.98 f. 2.21 ng/ml and decreased progressively to 7.96 ? 1.27 ng/ml on day 21 when high levels of mCG were present.
The concentrations of P increased again on day 27 of ges-
tation and reached peak levels on day 37 (18.48 * 2.45 ng/ml), thereafter decreasing to a nadir near midgestation.
About 10 days before par-
turition P levels increased again (P < 0.05) in relation to P concentra-
S tions at midgestation.
TDROXDS
487
Concentrations of P decreased precipitously
from 6.62 f 1.48 ng/ml on the day of delivery to 2.16 + 0.43 ng/ml on day 2 postpartum and remained at low levels throughout the remainder of this study.
Postpartum observations were limited to monkeys with suck-
ling infants.
Fig.
Mean chcuRa;tingcancentutianh06 chohionicgonadotiapin 1. (mCG),LH and pmgcuntemne (it SEMI duhg pegnancy (N=Sland eahey &a&a.tionIN=41in p-igigtaieed monheyn.
488
S
TIIROSDRB
Immunorea~tiy~ mCG in the peripheral circulation was detected in all animals on day 70 of pregnancy, rose to maximum amounts on days 18 to 24 of pregnancy, then declined to low concentrations on day 40.
This
hormone was not detectable throughout the remainder of the study.
Since
N39
S
489
TDX?.OLD=
the antiserum used to measure LH (GDN-15) cross-reacted with mCG (Zl), LH was assayed from day 40 of pregnancy.
There was no significant dif-
ference in circulating levels of LH during gestation and lactation. The patterns of P secretion in animals without ovaries were similar to intact animals (Fig. 2).
In ovariectomized monkeys, although
infants were suckling, P was not detectable after parturition. Throughout pregnancy serum concentrations of P in uteroovarian and uterine veins were higher (P < 0,001) than in the femoral vein (Fig. 3). Progesterone levels in uteroovarian venous blood exceeded those of the uterine vein at days 80 and 159 of pregnancy, while the opposite was found at day 25 of gestation (Fig. 3, Table 1).
I
1
I
I
I
I
I
I
UTEROOVARIAN
40
-
-
XI20
VEIN
-. -
FEMORAL
10 -
* I 20
1 40
I 60
* T 1 00
I 100
.-. -+
VEIN I 120
I 140
t I60
DAY OF PREGNANCY
vein behum concctiaF&j. 3. Uehoovahian, LLtehinc and peniphm.aX tioio~nod phcrgc&e.&ane (+ SEhl) in phegnant p.igRaieed monhcgb wtih inZataotavanieh.
S
490
TIIROIDSS TABLE 1
DIFFERENCE BETWEEN UTEROOVARIAN AND UTERINE VEIN SERUM CONCENTRATIONS 0~ PROGESTERONE (NG/ML) IN PREGNANT PIGTAILED MONKEYS
Animal Number
Day of Pregnancy 25
80
159 +ll.R
N38
-40.6
+25.Ja
N43
-13.7
+9.1
N44
-115.1
t199.4
N45
-142.9
t18.8
t3.5
N46
-7.3
t21.2
t34.3
a A pRti sign indica.ten that bAemid .EeveRs Wehc avtian utin Rhan in Lhe tietin@. vein.
kigheh
+Pl.5 t20.7
irz the
tiehc-
Incubation studies revealed that both the CL and follicles synthesized pregnenolone and P from cholesterol at days 35 and 159 of pregnancy (Table 2).
However, increased capability of synthesis of these
steroids was observed in CL tissue at day 159 of pregnancy. Gross examination of ovaries on day 25 of gestation revealed welldeveloped and vascularized CL with distinct luteal tissue.
However,
the same CL were not grossly apparent in the majority of monkeys when examined during midgestation
(day 80) except for the india ink marking
which appeared as a small spot on the surface of the ovary.
Although
the CL were not grossly visible in the ovaries from 3 of 4 monkeys examined on day 159 of gestation, histclogically small CL were located inside the tunica albuginea of the ovary.
A well-developed CL was
S
491
TDROXDTABLE 2
TOTAL AMOUNT OF STEROIDS SYNTHESIZED DURING A TWO HOUR INCUBATION WITH [7-3H]CHOLESTEROL 3 H-Pregnenolone
Day of Pregnancy
(DPM
Tissue
159 (2)
10~~)
2.93b
Controlsa 35 (2)c
x
3
H-Progesterone (DPM x 10-2) 0.21
Corpus Luteum
12.00
5.35
Follicles
12.20
5.25
Corpus Luteum
27.10
9.18
Follicles
31.85
2.21
2 'Incubtioti uLthoti tibue
dhagmeti; N=3
,"Mean vaihen Nwnbeh 05 monkey& examined present in the fourth animal.
These CL contained india ink marking and
the majority of the granulosa luteal cells were well-delineated, with spherical nuclei, which were centrally located in the granulated cytoplasm. DISCUSSION It has been known for many years that a functional CL is essential for initiation and maintenance of early pregnancy in the rhesus monkey since ovariectomy soon after nidation was followed by termination of pregnancy, but gestation continued in these monkeys if treated with P (1).
Furthermore, both morphological and hormonal studies revealed
that the CL is active during early stages of pregnancy (22,23,24).
In
the pigtailed monkey circulating concentrations of P were elevated during the initial period of gestation indicating that the CL is functional
S
492
at this stage of pregnancy.
TIIROXDD In this species, as in the rhesus (23),
P concentrations increased again near day 27, reaching maximum amounts on day 37 of pregnancy.
That this increase in P arises from the feto-
placental unit is suggested by our observation that serum concentrations of P did not decline rapidly following ovariectomy on day 35 of gestation.
It is also possible that the fetoplacental unit is secreting P
as early as day 25 of pregnancy as evidenced by higher serum concentrations of this steroid,in the uterine vein than in the uteroovarian vein. Similarly, ovariectomy on day 35 of pregnancy did not alter the subsequent normal course of pregnancy or the patterns of P secretion, indicating that the ovaries have minimal influence on gestation after this time and that the fetoplacental unit is the primary source of P thereafter.
The elevated concentrations of this steroid during late
pregnancy also seem to arise from the fetoplacental unit.
This view is
substantiated by elevated levels of P at late gestation in animals with or without ovaries.
In the rhesus monkey it has been shown that removal
of the placenta but not the fetus results in reduction of serum concentrations of P (251, revealing the importance of the placenta in the production of this steroid.
However, in the present study at days 80 and
159 of pregnancy higher serum levels of P in the uteroovarian vein than in the uterine vein support the concept that the CL secrete P during both mid and late gestation.
Perhaps P produced by the CL during these
stages of pregnancy is not reflected in the peripheral circulation due to the dilution of this steroid in circulation. thesis of 'H-progesterone from
3
Increased in vitro syn-
H-cholesterol by luteal fragments as
well as the presence of active luteal cells, regardless of CL size, on day 159 of gestation, suggests that the CL is capable of synthesizing P
S during late pregnancy.
493
T~ROXDrn
Similarly, the presence of detectable amounts
of P follow ing parturit ion in intact, but not in ovariectomized animals indicates that the CL function continues during lactation, as in rhesus monkeys
(26,27).
The possibility that a new CL is formed in late preg-
nancy is not compatible with the present observation that the only CL present on day 159 of gestation still contained the india ink, injected on day 25 of pregnancy. It is not known which hormones are responsible for the maintenance of CL function during pregnancy in the pigtailed monkey.
However, in the
rhesus, evidence exists to suggest that mCG is luteotropic during the initial period of gestation (14,23,28).
In the pigtailed macaque, on day 10
of pregnancy high concentrations of P and detectable amounts of mCG were present in peripheral circulation, indicating that mCG may be luteotropic in function during early pregnancy.
Nevertheless, the circulating concen-
trations of P gradually decreased during early pregnancy while mCG titers increased.
This relationship was also observed in the rhesus and may be
due to refractoriness of the CL to continued stimulation by mCG after the dramatic initial response (23,29).
Chorionic gonadotropin does not ap-
pear to be responsible for the CL function after day 40 of pregnancy since following this period mCG was undetectable.
The presence of constant
levels of LH in circulation suggests that this gonadotropin may play a role in maintenance of CL function during mid and late gestation. In humans, the general patterns of P secretion during pregnancy and following parturition (30,31), is similar to that described herein for the pigtailed monkey and as described previously for the rhesus monkey (3,4,22,23,24,29). in women.
However, the absolute amounts of P are much higher
This species variation may be explained on the basis of dif-
S
494
TDROXDB
ferences in the levels of steroid-binding globulins present in these primates (32,33,34).
Thus, the pigtailed macaque may be added to the
list of primate species in which the CL retains functional capacity throughout gestation, but from the present study no answer can be found to the questions regarding the mechanism nor physiological role for this extended luteal function. ACKNOWLEDGMENTS This work was supported by Grant RR-00167 from the National Institutes of Health, United States Public Health Service, and Grant 6300505B,C from The Ford Foundation. Publication number 20-005 of the Wisconsin Regional Primate Research Center. The gifts of oLHB antiserum (H-26) utilized for mCG RIA from Dr. G.D. Hodgen, an ovine LH antiserum (GDN-15) from Dr. G.D. Niswender used for LH determinations, progesterone antiserum (912) from Dr. A.H. Surve, and rhesus monkey LH reference preparation (WDP-X-81-1720) from Dr. W.D. Peckham are most gratefully acknowledged. Reagents for hemagglutination inhibition tests for urinary mCG were kindly supplied by the Hormone Distribution Branch, NIAMDD, NIH. Part of the technical assistance was provided by Ms. P.A. Meller and Ms. K. Rygasewicz. We also appreciate Mr. D.E. Cowley and Mr. R.A. Vertein for their excellent animal care. REFERENCES Meyer, R.K., Wolf, R.C. and Arslan, M. Proc. 2nd Int. Congr. Primat., Atlanta, Georgia, RECENT ADVANCES IN PRIMATOLOGY, Vol. 2, Karger, Basel/New York p. 30 (1969). 2. Tulsky, A.S. and Koff, A.K. FERTIL. STERIL. g: 118 (1957). 3. Treloar, O.L., Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY -91: 665 (1972). Wolf, R.C. and Meyer, R.K. ENDOCRINOLOGY -93: 686 4. Koering, M.J., (1973). 5. LeMaire, W.W., Conly, P.W., Moffett, A. and Cleveland, W.W. AM. J. OBSTET. GYNECOL. 108: 132 (1970). Melinkoff, E. AM. J. OBSTET. GYNECOL. 60: 437 (1950). 76: Tullner, W.W. and Hertz, R. ENDOCRINOLOGY 78: 1076 (1966). REPROD. -18: 779 8. Clark, J.R., Dierschke, D.J. and Wolf, R.C.BIOL. (1978). Czaja, J.A., Eisele, S.G. and Goy, R.W. FED. PROC. 34: 1680 (1975). 1;: Hodgen, G.D. and Ross, G.T. J. CLIN. ENDOCRINOL. MEi?iB. 38: 927 (1974). 11. Hodgen, G.D., Tullner, W.W., Vaitukaitis, J.L., Ward, D.N. and Ross, G.T. J. CLIN. ENDOCRINOL. METAB. 39: 457 (1974). 12. Surve, A.H., Bacso, I., Brinckerhov, J.H. and Kirsch, S.J. BIM. REPROD. 15: 34.3 (1976). 13. WehrenbeG, W.B., Dierschke, D.J. and Wolf, R.C. BIOL. REPROD. 20: 601 (1979). 1.
S 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33. 34.
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495
Chandrashekar, V., Meyer, R.K., Bridson, W.E. and Wolf, R.C. BIOL. REPROD. E: 889 (1979). Niswender, G.D., Monroe, S.E., Peckham, W.D., Midgley, Jr., A.R., Knobil, E. and Reichert, Jr., L.E. ENDOCRINOLOGY 38: 1327 (1971). Toivola, P.T.K., Bridson, W.E. and Robinson, J.A. ENDOCRINOLOGY 102: 1815 (1978). Krman, G.A., Niswender, G.D., Gay, V.L., Reichert, Jr., L.E. and Midgley, Jr., A.R. ENDOCRINOLOGY 92: 618 (1973). Sholl, S.A. STEROIDS 24: 703 (197q. Winer, B.J. STATISTICS PRINCIPLES IN EXPERrMENTAL DESIGN. 2nd Ed., McGraw-Hill Book Co., New York (1971). Kruskal, W.H. and Wallis, W.A. 3. AM. STAT. ASSOC. 47: 583 (1952). Hobson, W., Faiman, C., Dougherty, W.J., Reyes, F.I.and Winter, J.S.D. FERTIL. STERIL. 26: 93 (1975). Koering, M.J., Wolf, R.CFand Meyer, R.K. BIOL. REPROD. 2: 254 (1973). Atkinson, L.E., Hotchkiss, J., Fritz, G.R., Surve, A.H., Fleill, J.D. and Knobil, E. BIOL. REPROD. 12: 335 (1975). Walsh, S-W., Wolf, R.C. and Meyer, EK. E~~DOCRINOLOGY -95: 1704 (1974). Tullner, W.W. and Hodgen, G.D. STEROIDS 3: 887 (1974). Weiss, G., Dierschke, D.J., Karsch, F.J., Hotchkiss, J., Butler, W.R., and Knobil, E. ENDOCRINOLOGY 93: 954 (1973). Weiss, G., Butler, W.R., Hotchkiss, x, Dierschke, D.J. and Knobil, E. PROC. SOC. EXP. Blot. MED. 151: 113 (1976). Sundaram, K., Chang, C.C., Laurence, K.A., Brinson, A.O., Atkinson, L.E., Segal, S.J. and Ward, D.N. CONTRACEPTION 14: 639 (1976). Knobil, E. BIOL. REPROD. 8: 246 (1973). Weiss, G., O'Byrne, E.M., Fiochman, J.A., Goldsmith, L.T., Rifkin, L. and Steinetz, B.G. OBSTET. GYNECOL. 50: 679 (1977). Johansson, E.D.B. ACTA ~NDOCRINOL. (KBHJ-61: 607 (1969). Seal, U.S. and Doe, R.P. In METABOLIC EFFETS OF GONADAL HORMONES AND CONTRACEPTIVE STEROIDS, Eds. H.A. Salhanick, D.M. Kipnis and Ee;mean;e Wiele, Plenum Press,_New York p. 277 (1969). Hagemenas, F. and Kittinger, G.W. ENDOCRINOLOGY -90: 325 (1472j: Schiller, H.S., Holm, R.A. and Sackett, G.P. AM. J. PHYSIOL. 234: E489 (1978).