PROSTAGLANDINS

STIMULATION OF PROSTAGLANDIN SYNTHETASE ACTIVITY IN RAT GRANULOSA CELLS BY GONADOTROPINS IN VIVOl --

M.R. Clark2, G.B.N. Chainy3, J.M. Marsh, and W.J. LeHaire

The Endocrine Laboratory, Departments of Obstetrics and Gynecology, and Biochemistry, University of Miami

School of Medicine, P.O. Box 016960 Fliami, Florida 33101

ABSTRACT

The effect of in vivo exposure to gonadotropin on prostaglan- din synthetase activityin rat granulosa cells was examined in two experimental settings, The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also mar- kedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous Ltl surge (about 4 p.m: on proestrus), in comparison with the activity at mid- night of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin in vivo produced essentially the same effect as we had reported?axr for isolated granulosa cells incubated with LH in vitro. The -- stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.

Supported in part by NIH grants HD-08747, tlD-03142, HD-05444, and by a Biomedical Research Support Grant from the NIH to the University of Miami, RR-05363-17.

Recipient of a Postdoctoral National Research Service Award from the NIH.

Recipient of a Postdoctoral Fellowship from the Indian Minis- try of Education and Social Welfare.

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INTRODUCTION

The preovulatory follicles of several species exhibit a marked increase in PGE and PGF content as an essential element of ovulation (reviewed in (I)). The predominant source of these prostaglandins in the rabbit is the granulosa cell of the follicle (2,3). The increase in follicular prostaglandins occurs after the endogenous preovulatory peak of serum gonado- tropin concentrations or after the administration of luteinizing hormone (LH) or human chorionic gonadotropin (hCG) (1). Pros- taglandins also increase in large isolated follicles in response to exogenous LH in vitro (1). -- In addition, we have recently shown that isolated rat granulosa cells will respond to LH in vitro with increased'PGE synthesis (4).

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The biochemical mechanism by which LH stimulates granulosa cell prostaglandin synthesis is not fully understood (1). Earlier studies using whole ovaries from mature rats indicated that only small (50-80%) changes in prostaglandin synthetase activity accompanied the large (5-30 fold) preovulatory rise in prostaglandin content (5). It appeared from this work that the major action of LH might be at a site other than prostaglan- din synthetase (e.g. phospholipase activation). We recently reported, however, that in isolated ,rat granulosa cells the stimulation of prostaglandin synthetase activity by LH in vitro is marked (about 5 fold) and comparable to the stimulat=nof PGE accumulation (4). In order to determine if this difference from the earlier studies (5) might be due to our use of an in vitro model (and to confirm our findings in a more physioloFca1 setting), we pursued the present investigation into the in vivo -- effects of gonadotropins on follicular PGE levels and granulosa cell prostaglhndin synthetase activity.

MATERIAL AND METHODS

Materials: Pregnant mare's serum gonadotropin (PMSG, Equinex) and human chorionic gonadotropin (hCG) were donated by Dr. J. Jewel1 of Ayerst Laboratories, Inc. The (5,6-3H) PGE, (68 Ci/ mmole) was purchased from New England Nuclear. Arachidonic acid (approx. 99% pure), penicillin G, and streptomycin sulfate were obtairied from Sigma Chemical Co. Medium 199 (Ml99) with 25 mM 4-(hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, Earle's salts, and L-glutamine was purchased from Grand Island Biological Co. Bovine serum albumin (BSA), fraction V, was obtained from Nutrition Biochemicals Corp. and defatted as describe.d (6).

Animals and Treatments: : Immature (26-28 day) and mature (60- 90 day) Sprague-Dawley rat s were housed as described previously (7)s with the modification that the mature rats were housed in- dividually. For the exper 'iments summarized in Figure 1, im-

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mature rats were injected subcutaneously (s.c.) with 20 I.U. of PMSG'at 9:00 a.m. At noon of the second day after PMSG treatment (51 h), .the animals received an injection (s.c.) of 20 IU of hCG and were then killed either immediately (0 h), or 5 and 10 h later. Usually 3 rats were included at zero time and 2 each at 5 and 10 h after hCG for each experiment. Six such experiments were carried out (the sample tubes from one PGE determination and one enzyme assay, each at 5 hours were lost)

To obtain the results reported in Figure 2, mature rats were monitored for two or more estrous cycles, up to and including the day of the experiment, by daily vaginal smears (8). A total of 22 animals were used for these experiments: four rats at each time except midnight on proestrus, when six rats were included. Each rat was selected at the appropriate time of day on diestrus or proestrus. The tissues from a single animal at each time were used for the determination of follicular fluid PGE and prostaglandin synthetase activity.

Preparation of Samples and Assays: At the appropriate times the animals were sacrificed by cervical dislocation. The ovaries were removed and the granulosa cells with the follicular fluid were collected at 0" C by follicular puncture, as previously described (4). The cell suspension was centrifuged at 250 x g for 3 minutes and the supernatant used for assessment of fol- licular fluid PGE. The term "follicular fluid" was defined here as the fluid expressed from the ovary after puncture of the fol- licles for collection of granulosa cells. For comparison, the follicular fluid PGE values were expressed as nglovary, although they thus are not equivalent to the total PGE in the ovary. The cells were homogenized and assayed for prostaglandin syn- thetase activity in whole homogenates as recently reported (4). PGE was determined by radioimmunoassay (4,7,9). The data were examined by analysis of variance and Duncan's Yew Multiple Range Test (10) and are represented as mean f S.E. Comparisons with p < 0.05 were considered significant.

RESULTS AF!D DISCUSSION

The administration of hCG to PMSG-treated immature rats produced a significant increase in ovarian follicular fluid PGE 5 h after the hCG injection (Fig. 1). A parallel increase in the prostaglandin synthetase activity of granulosa cells isolated from the same ovaries was also observed. Similarly, 10 h after the hCG injection both follicular fluid PGE and prostaglandin synthetase activity were significantly elevated. The values of both‘parameters at 10 h were slightly but not significantly lower than those at 5 h.

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f “0 -7 sx --__--__-__ gY 2: 5 _-----__--_

z s -----------

(4 cl o_m-_

0

--- _ --- 6 2 5

--- A --s 9

--- 2m

TIME AFTER hCG (hours)

Figure 1. Follicular Fluid PGE and Prostaglandin Synthetase Activity in Granuloss Cells from Immature Rats Treated with PMSG and hCG.

The follicular fluid PGE and the granulosa cell prostaglandin synthetase activity were also determined at various times on the day of proestrus for rats exhibiting four-day estrus cycles (Fig. 2). A peak in plasma LH occurs at about 4 p.m. on proes- trus, and ovulation takes place between 1 and 4 a.m. on the following day in such animals (8). The data in Figure 2 show that follicular fluid PGE and granulosa cell prostaglandin syn- thetase activity are low prior to (noon) or at the time of the LH surge (4 p.m.). Both parameters then increased significantly at the times sampled between the LH surge and ovulation (i.e. at 8 p.m. and midnight proestrus). The follicular fluid PGE levels followed a pattern similar to that previously reported by our laboratory using whole follicles (8). To verify that the observed increases were restricted to the day of proestrus and not due to a diurnal variation, determinations were also made at midnight of the day preceding proestrus (diestrus). No eleva- tions of fluid PGE or prostaglandin synthetase activity were seen at midnight on diestrus (Fig. 2).

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12:oo .. 12:oo 4:oo MIDNIGHT NOON P.M.

8:00

_ _--___ 4

(6)

12:oo P.M. MIDNIGHT

DIESTRUS 4 PROESTRUS 6

TIME OF DAY (hours)

Figure 2. Follicular Fluid PGE and Prostaglandin Synthetase Activity in Granulosa Cells from Mature Rats.

It is known that LH produces ovarian hyperenia (ll), and that blood platelets can produce prostaglandins (12). The possibility was considered, therefore, that differential contamination by platelets of samples before and after exposure to gonadotropin might contribute to the changes in enzyme activity observed. To test this possiblity, blood from cut ovarian vessels was ex- pressed into modified medium 199 (4) by applying gentle pressure with a blunt spatula to four ovaries excised from PMSGltreated inunature rats. By this procedure blood was collected into medium in the same manner as would occur when granulosa cells were collected (but without puncturing follicles, so no granulosa cells were expressed). The resultant red blood cells were collected and prepared for enzyme assay as had been done with granulosa cells. (The platelets sediment with erythrocytes under these conditions, since no anticoagulant is present - unpublished observation.) MO prostaglandin synthetase activity was detected in such preparations, even though the amount of blood (as deter-

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mined by counting erythrocytes) was approximately twice that found in the usual granulosa cell preparations. The platelets which. contaminate such cell preparations, therefore, did not contribute to the enzyme activity measured.

In two physiological settings, i.e. the PMSG-hCG treated im- mature rat and the mature proestrus rat, therefore, we have found that a major effect of LH/hCG on the production of prostaglandins is a stimulation of prostaglandin synthetase activity. The present findings support our previous similar observations after in vitro exposure of rat granulosa cells to LH (4). The reason -__ why we have observed a much larger stimulation of prostaglandin synthetase activity than that reported by others (5), therefore, does not appear to be due to our earlier use of an in vitro model (4).

-- Additional possible explanations remain, however, in-

eluding our use of isolated granulosa cells instead of whole ovaries, or technical differences in the enzyme assay (4).

In this regard, Liedtke and Seifert have also examined a heterogenous human ovarian preparation for prostaglandins syn- thesis in response to LH and hCG (13). These authors were unable to demonstrate a stimulation of prostaglandin synthesis by gonadotropin. In this instance, however, the use of a hetero- genous preparation may not have been as important as the major methodological differences between the procedures of Liedtke and Seifert (13) and our own. Liedtke and Seifert incubated whole homogenates from frozen tissue in the presence of hCG. This use of whole homogenates instead of intact cells may have contributed to their negative results, since nearly all effects of gonadotropins have been observed when the hormones were added before homogenization (14). Thus it is not possible to compare our observations with those recently reported by Liedtke and Seifert (13).

An important role in LH regulation of granulosa cell pros- taglandin production must now be given to the stimulation of prostaglandin synthetase activity. In addition, it can be con- cluded that the previously described (4) convenient in vitro -- model utilizing isolated granulosa cells accurately reflects the in vivo action of LH on prostaglandin synthesis, and can, -- therefore, be used in further studies into the mechanism by which LH stimulates prostaglandin synthetase activity.

ACKNOWLEDGEMENTS

We gratefully recognize the contribution of prostaglandins by Dr. J. Pike of the tipjohn Co., and of PMSG (Equinex) and hCG (APL) by Dr. J. Jewel1 of Ayerst Laboratories. We appreciate the excellent technical assistance of Adela Gomez and the careful preparation of this manuscript by Lourdes Rodriguez and Maria A. Rodriguez.

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REFERENCES

LeMaire, W.J., M.R. Clark and J.Fl. Marsh. In: tiuman Ovula- tion: Mechanism, Detection, and Regulation (tlafez, E.S.E., ed.), Elsevier/North Holland, Amsterdam, 1978, p. 163.

Erickson, G.F., J.R.G. Challis and K.J. Ryan. Production of Prostaglandin F by Rabbit Granulosa Cells and Thecal Tissue. J. Reprod. Fertil. 49:133, 1977. -

Triebwasser, W.F., M.R. Clark, W.J. LeMaire, and J.M. Plarsh. Localization and In Vitro Synthesis of Prostaglandins in ?-- Components of Rabbit Preovulatory Graafian Follicles. Pros- taglandins 16:621, 1978. -

Clark, M.R., J.M. Marsh, and W.J. Lerlaire. Mechanism of Luteinizing Hormone Regulation of Prostaglandin Synthesis in Rat Granulosa Cells. J. Biol. Chem. 253:7757, 1978.

Bauminger, S. and H.R. Lindner. Periovulatory Changes in Ovarian Prostaglandin Formation and their Hormonal Control in the Rat. Prostaglandins 9_:737, 1975.

Goodman, D.S. Preparation of tiuman Serum Albumin Free of Long-Chain Fatty Acids. Science 125:1296, 1957.

Clark, ?l.R., J.Y. Marsh, and W.J. LeMaire. Stimulation of Prostaglandin Accumulation in Preovulatory Rat Follicles by Adenosine 3',5'-Monophosphate. Endocrinology 102:39, 1978.

LeMaire, W.J., R. Leidner ?nd J.M. Marsh. Pre and Post-Ovu- latory Changes in the Concentration of Prostaglandins in Rat Graafian Follicles. Prostaglandins 9_:221, 1975.

Jaffe, B.M., I-1.R. Behrman and C.U. Parker. Radioimmunoassay Measurement of Prostaglandins E, A and F in Human Plasma. J. Clin. Invest. 52:398, 1973. -

10. Duncan, D.B. Multiple Range and Multiple F Tests. Biometrics ll:l, 1955. -

11. Ellis, S. Bioassay of Luteinizing Hormone. Endocrinology 63:334, 1961. -

12. Smith, J.B. and A.L. Willis. Formation and Release of Pros- taglandins by Platelets in Response to Thrombin. Brit. J. Phamacol. 40:545P, 1970. -

13. Liedtke, M.P. and B. Seifert. Biosynthesis of Prostaglandins in Human Ovarian Tissues. Prostaglandins 16:825, 1978. -

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14. Channing, C.P. and A. Tsafriri. Mechanism of Action of Luteinizino Hormone and Follicle Stimulating Hormone on the Ovary in Vitro. Metabolism 26:413, 1977. -

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