Corpus luteum function during the early postpartum interval in lactating rhesus monkeys: In vivo and in vitro response to exogenous gonadotropin

73 CORPUS LUTEUM FUNCTION DURING THE EARLY POSTPARTUM INTERVAL IN LACTATING RHESUS MONKEYS: IN ~NO

AND IN ~Z'?CJRESPONSE TO EXOGENOUS GONADOTROPIN Richard L. Stouffer, Laura A. Bennett*, Wilbert E. Nixon, and Gary D. Hodgen

Section on Endocrinology, Reproduction Research Branch, National Institute of Child Health and Human Development, Auburn Building, Room 203, National Institutes of Health, Bethesda, Md. 20014, and *Office of Pesticide Programs, Environmental Protection Agency, Arlington, Va. 22202. Received:

10/18/76 ABSTRACT

The response of the postpartum corpus luteum to exogenous gonadotropin was studied in 12 lactating rhesus monkeys iven daily injections of either human chorionic gonadotropin (HCG, n = 6 3 or saline (control, n = 6) for 4 days imnediately following parturition. Peripheral blood samples were collected daily. On the 5th day postpartum, luteectomy was performed and progesterone production by dispersed luteal cells was examined. Whereas progesterone in the peripheral circulation of control monkeys progressively declined between days 1 and 5 postpartum, progesterone levels increased significantly (~~0.025) with the onset of HCG treatment and remained significantly (~~0.025) elevated above the controls throughout the period of HCG treatment. However, despite the daily administration of HCG, circulating progesterone levels declined (~~0.05) between days 3 and 5 postpartum. The weight of the corpus luteum excised from HCG-treated macaques was significantly (p
rhesus monkey undergoes structurally regressive changes (1,2) and relinquishes its essential steroidogenic functions to the developing placenta (3). However, it is reported that the corpus luteum of this

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macaque reestablishes progesterone synthetic capability near the end of gestation (4-7) and remains somewhat functional well into the postpartum interval in lactating monkeys, as indicated by circulating progesterone levels (S,9). Using an in vitro system to evaluate corpus luteum function, we observed that luteal cells isolated from rhesus monkeys at term pregnancy exhibited basal and gonadotropin-stimulatedprogesterone production similar to that of cells isolated during the mid-luteal phase of the menstrual cycle

(7).

These findings supported the in vivo evidence of re-

juvenated luteal function in late pregnancy (h-6) and suggested that luteal cell activity during late pregnancy or following parturition may be responsive to gonadotropic hormones. The present study examines corpus luteum function during the early postpartum interval in rhesus monkeys and evaluates postpartum luteal response to exogenous gonadotropin both ii?viva and in vi&o.

The two

parameters utilized to assess corpus luteum function were peripheral circulating progesterone concentrations and progesterone production by isolated luteal cells during short term incubation. F?fWJXRIALS AND MEZ'HODS Postpurtwn Rhesus Monkeys Twelve postpartum rhesus monkeys (Macaca rmlatta) were studied following natural delivery between days ~53-1.67 of gestation. The monkeys were housed, fed, and maintained as described earlier (10) and allowed to nurse their offspring during the study. Counting the day after delivery as the first day postpartum, six animals received daily intramuscular injections of 1000 IU of human ehorionic gonadotropin (HCG; Pregnyl, Organon) for the first four days postpartum. The remaining six animals served as controls and received saline injections during the same four day period. On the fifth day postpartum, the corpus luteum was excised from the monkeys at laparotomy under ketamine hydrochloride ('Vetalar,Parke-Davis, 15-20 mg/kg im) anesthesia. Blood samples (3 ml) were obtained by femoral puncture during the week preceding delivery, the postpartum interval immediately prior to

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the daily injection or luteectomy, and 48 hours after surgery. The serum was stored at -15C until analyzed for progesterone content. Dispersion and Incubation of LuteaZ Cells The procedure employed for isolation of rhesus monkey luteal cells has been reported previously (7). This technique involves dispersion of luteal tissue fragments by a combination of 0.2% collagenase (CLS grade, Worthington Biochemical Company) digestion and gentle mechanical disruption. Suspensions of dispersed luteal cells were incubated as previously described (7). Cells were incubated for 3 hours at a final concentration of 1.25~10~ viable cells/O.25 ml of Ham's FlO medium (GIBCO) in silicone-treated 12x75 mm glass tubes (Corning). Highly purified HCG (CKLl9, 11,600 IU/mg, 2nd Int. Std. HCG) was added to the cell suspensions in Ham's FlO medium. Control and HCGtreated suspensions were routinely performed in replicates of 3 or 4. Cells from one corpus luteum were utilized in each incubation study. Neasurement of Progesterone Progesterone production by luteal cells in vitro was determined by measuring the progesterone content of the incubation medium according to the radioimmunoassay technique developed by De'Villaet al. (11) as modified in this laboratory (7). Peripheral serum progesterone concentration was determined using the same technique (7) after extraction of 0.2 ml aliquots of serum with petroleum ether (12). Statistical Analysis Significant differences between saline and HCGtreatment groups were determined by the Student's t test. Analyses of within animal data utilized the paired t test. RESULTS

In

‘V+VO

Studies

Circulating serum progesterone concentrations in saline (control) and HCGtreated rhesus monkeys during the final week of gestation and the early postpartum interval are illustrated in Fig. 1.

Progesterone

levels were similar in both groups prior to the initiation of HCG treatment.

In the controls, progesterone levels declined significantly

(~~0.025) between day 1 and day 5 postpartum. In contrast, the first day of HCG administration significantly (pcO.025) increased circulating progesterone levels (3.3kO.7 ng/ml, %SE;

day 2 postpartum) above those

76

DAYS ~STPARTUM

Fig. 1. Mean i&SE, n=61 progestez+oneconcentration in peripheral serwn of Zactating rhesus monkeys administered saline (closed circles) or HCG (open eircZes1 durZng the postpartum intierval. Arrows indicate the days Of treatment. Luteectoq (LX) was perfomed on day 5 postpartum. G reJ%XXS to the fina week of gestation. of the previous day (1.810.4 n&ml).

The progesterone concentrations in

the lEGtreated group were significantly greater than those of the controls from day 2 (3.3kO.7 vs. 1.750.3 ng/ml, pc0.05) through day 5 (2.ko.6

vs.

1.0+0,2 ng/ml, p
tinued daily HCG administration, progesterone levels declined significant3_yfp
Cells dispersed

from corpora lutea of HCGtreated macaques reached 30~ in diameter and appeared under Nomaxski differential interference contrast optics to contain more large lipid droplets than did comparable postpartum luteal

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TabZe 1.

~rzvitro progesterone production by ZuteaZ ceZZs isolated from -Zactatkg, ~o~~ar~ rhesus monkeys fo~~~~ng 4 dqs of saline or HCG treatment.- reZation&ip to corpus Zutem weight and peripheraZ sewni progesterone concentrat
Parameter

N

Treatment -in viva HCG Saline

Luteal Cell Progesterone Production (ng/ml)* Control

5

16.3+ 4.5

28.4+7.03

1000 ng HCG/ml

5

36.Ozt14.8

45.728.8

Luteal Wet Weight (mg)

6

34.7rt

5.9

59.0*3.g4

Serum Progesterone (ng/ml)

6

1.02

0.2

2.4k0.6~

IPerformed on day 5 postpartum 21ncubation of dispersed luteal cells described in text 3Not significantly different from saline treatment, ~~0.10 4p
Cells from saline-treatedmonkeys produced

under control conditions (nutrient media only;

11.6.3k4.5 ng/niL/5x104cells/3 hr, %SE)

and responded to the addition of

1000 ng HCG/ml with a significant (pcO.025) increase in progesterone production (36.094.8 rig/m..).Under control in tiitroconditions, mean progesterone production (28.4k7.0

ng/ml.)

by luteal cells from HCG-treated

macaques was greater than that of cells from saline-treated animals, although this difference was not significant fp
ng/ml).

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.SALINE POSTPARTUM .-----

-

/

aHCG POSTPARTUM / /

5

t’

10

I

IO

HCG CONCENTRATION

l@

l(r

nglml

Effect of increasing HCG concentrations on in progestep Fig. 2. -- vitro one production by ZuteaZ ceZZs isolated from eontrot lcilosed circlesl and XCG-treated (open eireZesl rhesus monkeys on day 5 postpartum. Vu.& ues are t&e mean of three expetiments. The broken tine tzypifges the response of luteal ceZZs isolated during the mid-luteaZ phase of the mensMiuZ cycle or term pregnancy. Production is expressed as a percent of progesterone produced in the absence of HCG -in vitro. The effect of increasing concentrations of HCG on in vitro terone

proges-

production by luteal cells from saline and HCGtreated postpartum

rhesus monkeys is shown in Fig. 2.

Luteal cells isolated on day 5 post-

partum, from either saline or HCGtreated monkeys, did not respond to low levels (1 ng/ml) of HCG.

Moreover, in vivo

exposure to HCG during

the postpartum interval resulted in approximately a 50-fold reduction in luteal cell sensitivity to HCG in vitro. Accordingly, the addition of 100 ng HCG/ml significantly {p
The current investigation confirms this observa-

tion and provides additional evidence that the major source of

progesterone in early puerperium is the corpus luteum. The in vitro demonstration of the progesterone synthetic activity of luteal cells isolated on day 5 postpartum provides direct evidence of the steroidogenic capability of the corpus luteum of the postpartum rhesus monkey. Furthermore, luteectomy on day 5 postpartum was followed by a marked decline in serum progesterone to follicular phase levels (eO.3 n&ml). These findings support the conclusion of Weiss and coworkers (8) that, on the basis of histological appearance and ovarian venous progesterone concentrations, the corpus luteum is mo~hologic~ly

and ~ction~ly

intact in lactating, postpartum rhesus monkeys. Nevertheless, there are several indications that luteal function declines with time after parturition in the rhesus monkey.

The level of circulating progesterone in

saline-treated monkeys decreased steadily during the 5 day interval following delivery. !Phisgradual decline is reportedly sustained through the first postpartum month (9,13). In addition, luteal cells isolated on day 5 postpartum typically exhibited less than half the basal and gonadotropin-stimulatedprogesterone synthetic capacity of luteal cells isolated at term pregnancy (7). Corpus luteum function in lactating, postpartum rhesus monkeys is responsive to exogenous gonadotropin. Administration of HCG to these macaques significantly increased circulating levels of progesterone. Moreover, luteal cells isolated on day 5 postpartum responded to the addition of HCG in ?uitrowith enhanced progesterone production. Gonadotropins of pituitary and placental origin have been shown to play an important role in the expression of luteal function in the rhesus monkey during the menstrual cycle and early pregnancy, respectively (9,lO). The present and earlier (7) findings suggest that luteal function during

late gestation and the early postpartum interval in this macaque may also be dependent on gonadotropic support.

Accordingly, the observation

that luteal cells isolated in early puerperium exhibited reduced sensitivity to HCG in vitro, compared to luteal cells isolated at term pregnancy or mid-luteal phase of the menstrual cycle (171, suggests that declining luteal function during the postpartum interval may be due to the diminished ability of the corpus luteum to bind or respond to endogenous gonadotropin. In a previous study on corpus luteum function in human postpartum subjects (181, LeMaire and coworkers suggested that the stimulatory effect of exogenous HCG on postpartum luteal steroidogenesis

supports an

essential role for chorionic gonadotropin in the maintenance of the corpus luteum of pregnancy.

Although the current studies on luteal func-

tion in a nonhuman primate do not relate directly to this hypothesis in

mm, our data indicate that the corpus luteum of the rhesus monkey exhibits active steroidogenic function in late pregnancy

(7) and during

the early postpartum interval despite nondetectable levels of endogenous macaque chorionic gonadotropin

(MCG).

MCG was not detected in the pe-

ripheral blood, urine concentrates, or placental extracts from these monkeys after day 45 of gestation

(191, as compared to the continued

presence of HCG throughout gestation in man (20). ministration

Furthermore, the ad-

of HCG to postpartum monkeys appeared to result in only a

transient stimulation, rather than maintenance of luteal function.

Cir-

culating progesterone levels declined between day 3 and day 5 postpartum despite continued HCG treatment. A transient stimulatory effect on corpus luteum function has also been observed during the administration of HCG to rhesus monkeys in the

luteal phase of the menstrual cycle (21-23) and during the production of endogenous MCG in early pregnancy (10).

Together, these studies sug-

gested that the gradual fall in luteal function despite the continued presence of chorionic gonadotropin was due to the development of a state of refractoriness (22) to further gonadotropic stimuli by the corpus luteurn. The current observation that isolated postpartum luteal cells exhibited markedly reduced sensitivity to HCG in vitro following 4 days of exposure to HCG in

viva

may support this proposed gonadotropin-re-

fractory status of the corpus luteum following prolonged exposure to high levels of gonadotropin. The biological importance of active, gonadotropin-sensitiveluteal function during late gestation and the postpartum interval in these macaques may not relate to either pregnancy or lactation, since the ovaries are expendable as early as day 21 of gestation without discernable effects on the survival and growth of nursing infants (3,24). Rather, the presence of a functional corpus luteum may serve to delay the re-initiation of ovulatory menstrual cyoles. Anovulation and lactational amenorrhea are known to occur frequently in postpartum women, especially when nursing is extensive (25). These findings correlate with evidence that circulating progesterone levels remain elevated considerably longer in postpartum monkeys which are lactating, than in those which do not nurse (8).

A recent report by Goodman and coworkers

(12) provided physiological evidence that the presence of a functional corpus luteum during the menstrual cycle of the rhesus monkey suppressed follicular growth. Accordingly, the secretory activity of the postpartum corpus luteum of lactating monkeys may constitute an important natural mechanism for deferral of the next ovulation and the risk of an

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initiation of another pregnancy. ACKNOWLEDDEMENT

The expert technical assistance of Mr. Donald Barber is greatfully acknowledged. ~~R~&ES E.W.) Bartelmez, C.W., and Hartma, C.G., Am, J. Anat. 59, 433 (1936). Koering, M.J., Wolf, R.C., and Meyer, R.K., BIOL. REPROD. 9, 254 (1973). Hodgen, G.D., and Tullner, W.W., STEROIDS 25, 275 (1975). Treloar, O.L., Wolf, R.C., and Meyer, R.K., END#CR~NO~~GY 91, 665 f1912‘1, Koerlng, X.5., Wolf, R.C., and Meyes, R-K., ~~~C~~N~L~G~ 93, 686 (1-973). Gtiyas, B.S., AX. J. ANAT. 139, 95 f1914f. Stouffer, R.L., Nixon, W.E., Gtiyas, B.J., Johnson, D.K., and Hodgen, G.D., STEROIDS 27, 543 (1976). Weiss, G., Dierschke, D.J., Karsch, J., Hotchklsa, J., Butler, W.R., and Knobil.,E., ENDOCRINOLOGY 9& 954 (1973). Knobii, E., BIOL. REPROD. 8, 246 (1973). Hodgen, G-D., Tullner, W.W., Vaitukaitis, J.L., Wara, D.N., and Ross, G.T., J. CLIN. ~~C~~N~L* z%ETAB.39, 457 (X974.)* &%illa, G.O., Jr., Roberts, K., Wiest, W.C., &%khail, G., and Plickenger, G., 5. CLIN. EN~C~~N~L. METAB. 35, b58 jf_9'j'2f. Goodman, A.L., Nixon, W.E., Sahnson, D.K., and Hndgen, G.D. ENDOCRINOLOGYY in press. Weiss, G., Butler, W.R., Hot&kiss, J., Dierschke, D.J., and Knobil, E., PRQC. SOC. EXP. BIG&. MED. 151, I.13(1976). Hodgen, G.D., Wilks, J.W., Vaitukaitls, J.L., Chen, BC., Papkoff, H .) and ROSS, G.T., ~~CR~N~LOG~ 99, 1x7 {19763. Phoenix, C.K., J. CLAN. Besko, J.&L, Koezing, M.J.% Gay, R.W., 8-r~& ~~RIN~~. ITS. 41, 120 (1915). Monroe, S.E., Atkinson, L.E., and Knob%l, E., EN~~CRINOL~~Y 87, 453 (19701. Stouffer, R.L., Nixon, W.E., Gulyrzs,B.J., and Hndgen, G.D., ENDOCRINOLOGY, in press. LeMaire, W.J., Conly, P.W., Moffctt, A., Spellacy, W.N., Cleveland, W.W., and, Savard, K., Am. J. Ob&4?%. Gynec. 110, 612 (19-Q). Hoodgen,G.D., Neimann, W.H., and Tullner, W.W., END~~HIN~~O~Y 96, 1% Ii%?). Loraine, J.A., and Gari&Xlzi, J.H., J. ~ND~C~~N~L. 6, 319 (1950). Hisaw, F.L., YALE J. BIOL. MZD. 21, Xl9 j1944f. Neili, J.D., and Knobil, E., ENDOCRINOLOGY 90, 34 (1972). Surve, A.B., Hayrington, F.E., and Elton, R.L., PROC. SOC. EXP. BIOL. MED. 144, 963 (1973). Tullner, W.W., and Hertz, R., ENDOCRINOLOGY 78, lO'T6(1.966). Perez, A., Bela, P., Masnick, G.S., and Potter, R.G., AM. J. OBSTET. GYNECOL. 1118,1041 f19121. Corner,