In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

543

IN VITRO EVALUATION OF CORPUS LUTEUM -FUNCTION OF CYCLING AND PREGNANT RHESUS MONKEYS: PROGESTERONE PRODUCTION BY DISPERSED LUTEAL CELLS

Richard L. Stouffer, Wilbert E. Nixon, Bela J. Gulyas, David K. Johnsonl, and Gary D. Hodgen Section on Endocrinology, Reproduction Research Branch, National Institute of Child Health and Human Development, Auburn Building, Room 203, and Veterinary Resources Branchl, National Institutes of Health, Bethesda, Maryland 20014 Received:

2,/b/75 ABSTRACT

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term in vitro studies of progesterone production by suspensions of collagenase-ispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the fncubatlon medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level 163.7 + 13.1 versus 24.7 f 5.5 ng progesterone/ml/5x lo4 cells/ 3 Rr, X + S.E., n = 6 ; p < 0.05). However, luteal cells from early pregnancy (23-26 days after Pertflfzatfon) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 f 2.4 versus 24.7 + 5.5 ng/m1/5 x lo4 cells/3 hr, n = 3 ; p c 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108-118 days gestation ) luteal cells exhibited partially renewed function, and near the time of parturition (163-166 days gestation) basal and HCGstimulated progesterone secretion (30.2 f 5.6 and 63.0 f 13.0 ng/m1/5 x lo4 cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when sterofdogenfc activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitivesteroidogenesfs similar to that of the corpus luteum of the menstrual cycle. INTRODUCTION A principal physiologic function of the primate corpus luteum (CL) is the synthesis and secretion of progesterone. Expression of this function during the luteal phase of the menstrual cycle apparently requires tonic pituitary gonadotropin support in the form of luteinizing

VoZwne 27, Nwnber 4

s

TIIILOSDI

April,

1976

S

TDICOXD=

hormone (1). Moreover, during early pregnancy in the rhesus monkey, a gonadotropin of placental origin, macaque chorionic gonadotropin (PEG), provides the stimulus for continued luteal progesterone production for an additional 7-10 days (2). After the first three weeks of pregnancy, the macaque CL undergoes structurally regressive changes (3,4) and presumably relinquishes the steroidogenic functions, which are vital to the maintenance of normal pregnancy, to the developing placenta. In the rhesus monkey, the ovaries can be removed as early as the 21st day of gestation without altering the normal pattern of plasma progesterone concentrations (5) or terminating pregnancy (6). Nevertheless, recent reports have suggested that the CL of pregnancy in this species reestablishes its functional capability near the time of parturition, as evidenced in part by elevated ovarian venous progesterone concentration (7, 8), fine structuralanalysis of the CL (8,9), and increased luteal 3Bhydroxy-steroid dehydrogenase activity (10). The present study was initiated to provide a more direct evaluation of corpus luteum function in both cycling and pregnant rhesus monkeys through short term in vitro analysis of progesterone production by suspensions of dispersed luteal cells. Basal and gonadotropin (HCG)-stimulated progesterone production was compared between luteal cells obtained from monkeys in the luteal phase of the menstrual cycle and at early, middle, ana late pregnancy. MATERIALS AND METHODS LuteaZ CeZZ Preparations The corpus luteum was removed at laparotomy from cycling rhesus monkeys (Macaca rmdatta) 15-19 days after the onset of menses (n = 6) and from pregnant monkeys at 23-26 (n = 3), 108-118 (n = 4) ana 161-166 (n = 3) days gestation. These intervals correspond to mid-luteal phase of the menstrual cycle ana early, middle, ana term pregnancy, respectively. The corpus luteum was transported to the laboratory in Ham's FlO nutrient

S

=EDEOfDrn

medium with 25 m&lHEPES buffer (GIBCO) at 4C. Penicillin (50 U/ml) and streptomycin (50 pg/ml) were added to this and all dissociation and incubation media. The luteal tissue was dissected free of any adventitial tissue, weighed, and minced in Ham's FlO-H'EPEZT medium utilizing two SUTgical scalpels fitted with No. 11 blades. The luteal tissue fragments were dispersed in Ham's FlO-HEPES medium containing 2% botine serum albumin (BSA) and 0.2% collagenase (Worthington CLS) at an initial concentration of 75 mg tissue/ml. Dispersion was performed at 37C in capped polyethylene tubes containing an atmosphere of 95% 02 - 5% CO2, with constant shaking at 150 cycles/min. Every five minutes an equal volume of Ham's FlO-HEPES containing 2% BSA was added, the tissue fragments dissociated by gentle aspiration in a 10 ml plastic pipette (Falcon Plastics), and the medium removed and replaced with fresh dissociation medium. After four medium renewals the dispersion process was discontinued with but a few small tissue fragments remaining. Dispersed cells released into the dissociation media were collected by low speed centriwation (100 g for 5 min). The collected cells were washed and resuspended in Ham's FlO-HEPES medium containing 2% BSA at 4C. Cell viability was assessed from the ability to exclude try-panblue: An aliquot of the cell suspension was added to a Levy hemocytometer (Hausser Scientific) to determine cell concentration. Short-tern Incubations and Progesterone Determination Dispersed luteal cells were incubated at a final concentration of 5 x lo4 viable cells/ml Ham's FlO-HEPES medium in 20 ml glass vials (Kimble) or 12 x 75 mm tubes (Corning). All glassware was silicone-treated. The final incubation volume in the glass vials and tubes was 1.0 and 0.25 ml, respectively. The incubations were performed for 3-6 hours at 37C under 95% 02 - 5% CO2 with constant shaking at 60 cycles/min. HCG (CRllg; 12,000 IU of 2nd IRP HCG/mg) was added to the cell incubations in 10 1.11 of Ham's FlO-HEPES medium. Control and HCGtreated incubations were routinely performed in at least triplicate; 50 ~1 of medium was removed from each sample at specified time intervals and stored at -15C prior to analysis for progesterone content. Progesterone content in the incubation media was determined by radioimmunoassay (RIA) of unextracted samples (11) according to a procedure modified from that of DeVilla et al. (12). The equivalent of 2-10 1.11 of medium was assayed in duplicate. Incubation in a total volume of 1.0 ml was carried out at 4C overnight in the presence of 1,2 - 3H-progesterone (NEN) aa anti-progesteroneantiserum. The antiserum was highly specific for progesterone, with little cross-reactivitywith 17-hydroxyprogesterone (0.35%) or other steroids, i.e., cortisol and testosterone (both < 0.1%). The sensitivity of the assay was 0.025 ng; the range of the st=a8,ra curve extended to 1.0 ng. Analysis of the RIA and potency estimates of sample unknowns was performed utilizing the computer program developed by Roabsrd and Lewald (13). Intra- and inter-assay variability were 7.7 aa 8.0 per cent, respectively. Accuracy was confirmed with samples for which progesterone values were known previously (5,lg).

RESULTS Cell suspensions prepared from rhesus monkey CL by treatment with collagenase contained predominately single isolated cells and some small tissue clumps of up to lo-20 cells. The yield of luteal cells ranged from 0.5 to 3.0 x lo6 cells/100 mg of tissue. Evidence of cell lysis was not observed in the dispersed cells and their integrity was maintained over the incubation period, as confirmed by either Nomarski differential interference contrast or phase contrast optics. Initial cell viability was greater than 90% and remained at this level during 3-6 hours in suspension. The kinetics of progesterone secretion by suspensions of rhesus monkey luteal cells are illustrated in Fig. 1.

Results obtained with luteal

cells from CL of cycling monkeys (not shown) and of term pregnant animals were similar. Under control conditions progesterone secretion was linear for up to 3 hours while incubated in Ham's FlO nutrient enriched medium. However, in the presence of HCG (100 ng/ml) progesterone secretion increased markedly as early as 30 minutes after the onset of incubation. Progesterone secretion in the presence of HCG was maximal during the first 3 hours of incubation but, thereafter it declined to the control rate despite the continued presence of chorionic gonadotropin. A lo-fold increase in HCG concentration (1000 ng/ml) failed to produce a greater stimulation of progesterone secretion. Progesterone secretion by suspensions of luteal cells from both cycling and pregnant monkeys under control conditions, and in the presence of 100 ng HCG/ml, is summarized in Fig. 2.

Cells from mid-luteal phase

of the menstrual cycle secreted progesterone under control conditions (24.7 f 5.5 ng/ml/5 x lo4 cells/3 hr; X f S.E., n = 6) and responded to

S

547

TDEOXDI

123456 lncubstion Period Murs) Nms coume of progesterone secretion (?&ml) by a suepension F%g. 1. of Zuteat 0eZZe obtcrtned from the COTpus lUteUrn Of the rheeus nwnkeu T&e concentrat%on wa8 5 2 lo4 uetl8/mt. P=gnayY. _l%e ceZZ _ _ ceZZ8 were incubated in the abeence (czosed cfrcZe8l and presence of

a+_p=m

or 1000

ng/mZ CcZosed equree) 2nd TRP RCG).

100 ng/mZ (open drcti8) pum’fied hCG (12,000 IV&;

HCG

with a significant (p < 0.05)

(63.7 + 13.1

ng/ml/5

x lo4

cells/3

of highZy

increase in progesterone production hr).

In contrast, lutesl cells of

early pregnancy produced significantly less (p < 0.05) progesterone under control conditions than cells from cycling monkeys (3.6 sus

24.7

+ 5.5

n&l./5

x lo4

+ 2.4

ver-

cells/3 hr; n = 3), and did not respond to

HCG with enhanced steroidogenesis. Although the data from luteal cells obtained during mid-pregnancy were more variable, the results suggest limited gonadotropin sensitivity with progesterone secretion intermediate between that of luteal cells from early and late gestation. Lute&l cells of late pregnancy exhibited both basal and HE-stimulated progesterone secretion (30.2 f 5.6 and 63.0 + 13.0 &ml/5

x lo4 cells/3 hr,

respectively, n = 3) similar to those of cells from cycling monkeys.

S

548

TDROXD6

Fig. 2. Progesteronesecret;ion by suspensions of luteaZ csZZs obtained from rhesus monkeys dting the ZuteaZ phase of the menstruaZ cyete and Luteat 0eZZs were ineubatsdat a eady, ntZddZe, and term pregnancy.

concentrat&m of 5 x 104 ceZzS/mtfor tfireehours in the ahence (ctossd erbars1 and presence (openbars) of 100 ng h'CG/mt.Mean and St&d GOT of 3-6 separdte experiments per category.

The procedure described for dispersal of rhesus monkey corpus luteum is simple, reproducible, and adequate for dissociating this small tissue (< 150 mg wet weight) into a population consisting primarily of isolated, viable luteal cells. Moreover, the ability of gonadotropin to stimulate progesterone secretion by luteal cell suspensions supports the conclusion that these cells retain their functional as well as structural integrity. The secretion of progesterone in vStr0 by luteal cells from nonpregnant monkeys was expected in view of their origination from CL during the mid-secretory phase of the menstrual cycle. The enhancement of progesterone secretion from these cells by HCG is in accord with reports that a~nistrat~on

of this gonadotropin to rhesus monkeys during the

lute&t phase of the menstrual.cycle is followed by a dramatic rise in

plasma progesterone concentration and extended luted function (14,15). Therefore, HCG was utilized routinely to evaluate gonadotropin-sensitive steroidogenesisby rhesus monkey luteal cells in vitro. Subsequent studies have also shown these cells to be sensitive to macaque chorionic gonadotropin (unpublished). Basal and HCGstimulated progesterone aecretion by suspensions of luteal cells from both cycling and term pregnancy monkeys are similar to that reported by Gospodarowicz and Goapodarowicz (16) for bovine luteal cells in vitro. The ability of luteal cells from pregnant rhesus monkeys to secrete progesterone in vitro was a function of the period of gestation. In early pregnancy (days 23-26),

when the post-implantationprogesterone

surge was declining despite continued high lwels of MCG (2), dispersed luteal cells secreted little progesterone and were unresponsive to HCG. This lack of functional activity probably was not related to any structural damage incurred by the cells during the dissociation procedure. Rather, it seems reasonable to conclude that this is the first in vitro observation of the refractory state described for the primate corpus luteum following prolonged exposure to chorionic gonadotropin in viva. Neil1 and Knobil (15) have reported previously that administration of HCG to rhesus monkeys is only temporarily luteotropic despite continued hormone injections. More recently, Hodgen et al. (2) observed that the increase in plasma progesterone concentration, induced by endogenous MCG, around the time of implantation is only transitory despite the continued presence of chorionic gonadotropin. Thus, the lack of in tdtrr,response to HCG by luteal cells obtained between days 23-26 of pregnancy may be related to a refractory condition developed following prolonged exposure to MCG in viva.

Later in gestation (108-118 days) dispersed luteal cells exhibited renewed steroidogenic function. Eventually, near the time of parturition

(l.63-166 days gestation) both basal and HCGstimulated progesterone production in Vitro were equivalent to that of cells from the mid-luteal phase of the menstrual cycle. Thus, following a period of diminished activity, around the fourth week of gestation, luteal cells of the pregnant rhesus monkey progressively reacquire their steroidogenic capability and at term exhibit gonadotropin-sensitivesteroidogenesis similar to that of cells from the mid-luted phase of the menstrual cycle. The current in vitro demonstration of the reacquisition of steroid secreting function by rhesus monkey luteal cells at term pregnancy strongly supports the hypothesis of Treloar et al. (7) that the morphological regrowth of the macaque CL in late gestation is accompanied by a rejuvenation of the steroidogenic capacity of the tissue. The observations with the macaque are in accord with in

viva

(17) and in vitro (18)

evidence indicating that the human corpus luteum of pregnancy is capable of progesterone production well after the time when the ovary can be extirpated without terminating gestation and at term pregnancy. The mechanisms involved in the rejuvenation of luteal function in the macaque during late gestation are unknown (7). Moreover, the physiological role of this tissue, in view of the dominant steroidogenic capabilities of the fetal-placental unit (5,19),remains speculative. The preparation of isolated luteal cells described here provides a model system for the in vitro study of primate luteal function and allows direct assessment of the effects of potential luteotropic and luteolytic agents on the corpus luteum at various periods during its lifespan.

ACKNOWLEDGEMENTS The expert technical assistance of Mr. Donald Barber is greatfully acknowledged.

REZZRENCES 1. 2. 3.

4. 2: 7. 8.

Knobil, E., BIOL. REl%OD. 8, 246 (1973). Hodgen, G.D., Tullner, W.W., Yaitukaitus, J.L., Ward, D.N., and Ross, G.T., J. CLIN. ENDOCRINOL. METAB. 39, 45'7(1974). Corner, G.W., Bartelmez, G.W., and Hartman, C.G., AMER. 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). l'ullner,W.W., and Hertz, R., ENDOCRINOLOGY 78, 1076 (1966). Treloar, O.L., Wolf, R.C., and Meyer, R.K., ENDOCRINOLOGY 91, 665 (1972). Koering, M.J., Wolf, R.C., and Meyer, R.K., ENDOCRINOLOGY 93, 686

(1973). 9. Gulyas, B.J., AM. J. ANAT, 139, 95 (1974). 10. 11. 12. 13.

14. 15. 16. 17. 18.

Shall, S.A., Wolf, R.C., and Colas, A.E., ENDOCRINOLOGY 94, 908 (1974). Goldenberg, R.L., Bridson, W.E., and Kohler, P.O., BIOCHEM. BIOPHYS. RES. corn..48, 101 (1972). Delrilla,G.O., Jr., Roberts, K., Wiest, W.G., Mikhail, G., and Flickinger, G., J. CLIN. ENWCRINOL. MEIXB. 35, 458, 1972. Rodbard, D., and Lewald, J.E., ACTA ENDOCRINOL. 64, Suppl. 147, 79 (1970). Hisaw, F.L., YALE J. BIOL. MED. 17, 119 (1944). Nelll, J.D., and Knobil, E., ENDOCRINOLOGY 90, 34 (1972). Gospodarowicz, D., and Gospodarowicz, F., ENDOCRINOLOGY 90, 1427 (1972). LeMaire, W.J., Conly, P.W., Moffett, A., and Cleveland, W.W., AMEX?. J. OBSTET. GYNEC. 108, 132 (1970). LeMaire, W.J., Rice, B.F., and Savard, K., J. CLIN. ENDOCR. 28,

1249 (1968). 19. Tullner, W.W., and Hodgen, G.D., STEROIDS 24, 887 (1974).