Acta physiol. scand. 1979. 105.239-247 From the Department of Physiology, University of Goteborg, Sweden

Lack of specific prostaglandin antagonistic effects of 7-oxa-13-prostynoic acid on ovarian metabolism in vitro

BY CLAES MAGNUSSON, GUNNAR SELSTAM and KURT AHRBN

Received 3 July 1978

Abstract

MAGNUSSON, C., G. SELSTAM and K. AHRBN. Lack of specific prostaglandin antagonistic effects of 7-oxa-I3 prostynoic acid on ovarian metabolism in vitro. Acta physiol. scand. 1979. 105. 239-247.

Prostaglandins (PGs) have been proposed to function as obligatory intermediates in the action of luteinizing hormone (LH) (Kuehl et al. 1970). This was based on experiments on isolated mouse ovaries where the substance 7-oxa-13-prostynoic acid (7-oxa-13-PA) was found to block the effect of both PGE, and LH on ovarian cyclic AMP formation and progesterone synthesis. We have studied whether 7-oxa-13-PA acts as a P G antagonist also on the isolated rat ovary, where PCs of the E type have many effects. Prepubertal rat ovaries were incubated up to 4 h in modified Krebs bicarbonate buffer containing glucose with PCE, and 7-oxa-13-PA, alone and in combination. PGE, stimulated the uptake of the non-utilizable amino acids a-aminoisobutyric acid (AIB) and cycloleucine in an equally wide dose-range as has earlier been found for the stimulation of lactic acid and cyclic AMP production as well as protein synthesis. 7-oxa-13-PA alone, in concentrations exceeding 25 pg/ml, stimulated lactic acid production, but inhibited the incorporation of labelled leucine and the uptake of AIB. The uptake of cycloleucine was slightly stimulated. 7-oxa-13-PA did not antagonize the PGE, effect on lactic acid production. 7-oxa-13-PA diminished the PGE, effect on the uptake of amino acids and the incorporation into protein, but only in concentrations where it in itself inhibited the protein synthesis. These results show that 7-oxa-13-PA does not act as a PG-antagonist in the prepubertal rat ovary, since it cannot block the effects of PGs without having marked inherent effects.

Extensive work is being done in order to elucidate the role of prostaglandins (PGs) in the mechanism of action of various hormones. For this purpose several types of inhibitors of the action of PGs have been developed (for ref. see Sanner 1974). In the late sixties Fried and co-workers synthesized a group of substances with close structural similarity to PGs (Fried et al. 1969). Several of these 7-oxa-prostaglandin analogues inhibited PG-induced contractions of isolated intestinal smooth muscle preparations in a competitive manner. The most specific PG inhibitors were the analogues with a triple bond in the 13, 14-position. Later workers, however, did not get the same clear-cut results. Flack (1970) considered 7- oxa-l3-prostynoic acid (7-oxa-13-PA) as the only specific PG inhibitor among these ana- logues, but in several other experimental systems even this substance had no inhibitory effect.

239

240 CLAES MAGNUSSON, GUNNAR SELSTAM AND KURT A H R ~ N

In 1971 Bennett and Posner reported that 7-oxa-13-PA did not specifically inhibit the PG- induced contractions of several different intestinal smooth muscle preparations. In spite of these contradictory findings, 7-oxa-13-PA has been widely used as a PG antagonist in several different experimental systems (see e.g. Sanner 1974).

PGs exert many different actions in the reproductive sphere. The E type has been shown to mimic many effects of gonadotropins on ovarian metabolism. Thus, in the ovary they can stimulate steroid synthesis (Pharriss et a/. 1968, Marsh 1970, Speroff and Rarnwell 1970), the formation of cyclic 3’,5’-adenosine monophosphate (CAMP) (Kuehl eta/ . 1970, Marsh 1970, Ahren et a/. 1974, Selstam et a/. 1974), glycolysis (Perklev and Ahren 1971) and the amino acid transport and incorporation into protein (Ahren and Perklev 1973). It was therefore of great interest when Kuehl and co-workers (1970) reported that 7-oxa-13-PA (50-75 pglml) inhibited not only the PG-induced cAMP formation in the mouse ovary but also the stimula- tory effect of LH on the cAMP formation. In contrast to these findings no inhibition of the stimulatory effect of LH or PGs on cAMP formation was found in porcine granulosa cells (Kolena and Channing 1972) or rat ovaries (Lamprecht ef a/. 1973). Ellsworth and Armstrong (1974) could not find that 7-oxa-13-PA affected the luteinization of granulosa cells in trans- planted rat ovarian follicles in response to LH or PGE,. Channing (1972) could find such an inhibition on luteinization of monkey granulosa cells, but when added together with human chorionic gonadotrophin (HCG) or PGE,, 7-oxa-13-PA produced necrotic changes in the cells.

Tn the light of these studies it was considered to be valuable to study more in detail if 7-oxa-13-PA has PG-agonistic and/or PG-antagonistic action. The effects of 7-oxa-l3-PA, alone and in combination with a prostaglandin, on the carbohydrate and amino acid me- tabolism were studied in an experimental systemoften used in our laboratory, the prepubertal rat ovary. Some preliminary results have been reported earlier (AhrCn and Perklev 1973).

Materials and Methods Animals Rats, 23 days old, of the Sprague-Dawley strain, were used. A standard pellet diet was given and water ad libitum. The rats were obtained from Anticimex Ltd, Stockholm and deprived of food 18-24 h before the experiment.

Chemicals and hormones Generally tritiated or-aminoisobutyric acid (AIB-3H), AIB-1-14C, 1-aminocyclopentane-I-carboxylic acid- 14C (cycl~leucine-~~C), generally labelled I e ~ c i n e - ~ H and le~cine-’~C, and tritiated cyclic 3 ’,S’-adenosine monophosphate (3H-cAMP) were obtained from New England Nuclear Co., Boston, Mass., USA. The non-utilizable amino acids A1B and cycloleucine had a final concentration in the incubation medium of 0.1 mM. AIB-3H had an activity of 0.33-1.65 pCi/ml, AIB-14C 0.057-0.2 pCi/ml and cyc l~ leuc ine-~~C 0.025-0.1 pCi/ml. The labelled natural amino acids I e ~ c i n e - ~ H and leucine-14C were used in a final concen- tration of 0.01 mM and had an activity of 1.98 pCi/ml and 0.2 yCi/ml, respectively. cAMP dependent protein kinase and protein kinase inhibitor for the cAMP assay were obtained from Sigma Ltd. All other chemicals were of analytical grade, and purchased from Sigma Ltd and Merck Co.

PGE, and PGE, were supplied by Ono Pharmaceutical Co. Ltd, Osaka, Japan and PGF,, by Upjohn Co. The 7-oxa-13-PA was kindly given to us by Prof. J. Fried, Dept. of Chemistry and Biochemistry, Univ. of Chicago, Ill., USA. The PGs and the 7-oxa-13-PA were dissolved in ethanol (1 p l ethanol per p g PG and 0.2 pl per pg 7-oxa-13-PA). Aliquots of these solutions were added directly to the incubation media. Because ethanol in concentrations exceeding 10 ,ug/ml incubation medium in itself significantly inhibited

PROSTAGLANDIN EFFECT ON OVARIAN METABOLISM 241

lactic acid production and increased cycloleucine uptake, concentrations higher than 20 p g PGE,/ml or 75pg 7-oxa-l3-PA/ml were not tested. Bovine LH (NIH-LH-B8) was generously supplied by the Endocrino- logy Study Section of the National Institute of Health, Bethesda, Md, USA.

Experimental procedure The rats were killed by cervical fracture and the ovaries were rapidly removed and chilled in buffer. They were dissected free of extraneous tissues under microscope, rinsed, blotted on filter paper and each pair of ovaries was put into a 10 ml Erlenmeyer flask. The ovaries were incubated at 37°C for 30-240 min in 1 ml modified Krebs-Ringer bicarbonate buffer (1.25 mM CaCI,) pH 7.4, equilibrated with 95 % O,+ 5 % CO,, with addition of 1 mg glucose/ml. The labelled substances, the PGs and 7-oxa-13-PA were added in dif- ferent combinations as indicated in Results.

After incubation the ovaries were rinsed, weighed, and homogenized in 10 % trichloroacetic acid (TCA) and centrifuged. Aliquots from the supernatant and incubation media were taken for radioactivity deter- mination according to Nilsson and Selstam (1974). The uptake of the labelled amino acids is given as the distribution ratio at the end of the incubation period, calculated according to Ahrkn and Rubinstein (1965).

The TCA precipitates were used for determination of the incorporation of radioactivity into protein as described earlier (Nilsson and Selstam 1974) and expressed as DPM/pg protein. The protein content was determined according to Lowry et al. (1951).

Lactic acid accumulation in the medium was determined at the end of the incubation period according to Lundholm et al. (1963 a, b). The tissue content of cAMP was determined according to Gilman (1970). The content of cAMP in the medium was determined by a modification of this procedure a s has been described earlier (Selstam et al. 1974).

Statistical procedure Mean values are given & standard error of the means. All groups presented in figures or mentioned in the text represent the mean of 5-15 pairs of ovaries. The values were compared by analysis of variance, followed by Student-Newman-Keul's multiple range test for comparison between individual groups (Woolf 1968). A p-value of less than 0.05 was considered significant.

TABLE 1. Effects of different prostaglandins and fatty acids on lactic acid production and amino acid uptake by prepubertal rat ovaries. The ovaries were incubated for 2 h in modified Krebs-Ringer bicar- bonate buffer containing 5.5 mM glucose. The lactic acid was measured at the end of the incuba- tion period. The amino acid uptake is given as distribution ratio a t the end of the incubation period.

Test substance Oldml)

Lactic acid production (pg/ml tissue) CPM/ml intracellular water

AIB-3H distribution ratio

( CPM/ml medium

Expt. I Control PGE,

PGE,

Expt. 2 Control PGE, Arachidonic acid

Palmitic acid

0.1

0. I

0.1

10

10

10

0.1 0.1

0.1 50

50

1.58k0.17 2.37f0.1 I** 2.64+0.17** 2.49+0.17** 2.73+0.17** 1.48 f 0.08 N.S. 2.92+ 0.13**

I.lS+ 0.07 1.95f0.09** 1.07f0.05 N.S. 1.16+0.07 N.S. 1.30k0.06 N.S. 0.63 kO.O9**

14.8 f 0.4 18.8k0.6** 22.9 k 0.5** 20.4+ 1 2 * * 21.8+1.2** 16.1k0.4 N.S. 19.6+ 1.2..

11.2k0.4 14.9+ 1.0** 10.5k0.4 N.S. 11.3k0.3 N.S. 10.7f0.3 N.S. 11.3f0.7 N.S.

~

Levels of significance us. control: ** = p < 0.01; N.S.= not significant.

21 - 795812

242

15-

0 e .- 0

c

10 - c

P -c ti 0 ._

CLAES MAGNUSSON. GUNNAR SELSTAM AND KURT A H R ~ N

51 O J 1 4 1 I I 1 ,

0 ,001 .01 .I I 10 20

PGE, (vglrnl)

Fig. 1. Effect of PGE, on the uptake of AIB and cyclo- leucine in prepubertal rat ovaries. The ovaries were in- cubated for 2 h in modified Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose, 0.1 mM AIB-3H and 0.1 m M cycl~leucine-~~C. The uptake of amino acid is given as distribution ratio at the end of the incubation period. Levels of significance of the PGE, effect: *= p i 0 . 0 5 , **=p<O.OI, N.S.=not significant.

Results Effects of PGs

The effects of PGEl, PGE, and PGF,, on lactic acid production and uptake of AIB were compared (Table I). The potency of PGE, and PGE, were equal, while PGF,, was less potent. The uptake of cycloleucine was also measured after 2 h of incubation. PGEl, PGEz and PGF2, showed effects similar to those on the AIB-uptake (not shown in the Table). The PG precursor arachidonic acid and the fatty acid palmitic acid had no stimulatory effect on any of the parameters (Table I).

I I , 0 60 120 180 240

Incubation time (min)

Fig. 2. Time-relationship of the uptake of AIB in prepubertal rat ovaries incubated with or without PGE, (0.1 ,ug/ml). The ovaries were incubated in modified Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose and 0.1 mM AIB-'*C or AIB-3H. The uptake of amino acid is given as distribution ratio a t the end of the incubation period. Levels of significance of the PGE, effect at the different times: **=p<O.OI, N.S. - not significant.

PROSTAGLANDIN EFFECT ON OVARIAN METABOLISM 243

Fig. 3. Effect of 7-oxa-13-PA alone and in com- bination with PGE, (0.1 pg/ml) on the lactic acid production by prepubertal rat ovaries. The ovaries were incubated for 2 h in modified Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose. The lactic acid was measured at the end of the incubation period. Levels of significance: * = p < 0.05, **=p<O.OI, N.S.=not significant. Levels of significance of the effect of the different con-

? 3.0 P

c. 7-oxa-13-PA o o 7-0x0-13-PA

+ PGE, (0 I pg/ml)

I I

0 25 50 75 7- oxa- 13- PA Cg/ml)

centrations of 7-oxa-13-PA alone are indicated at the lower curve. Levels of significance of the effect of added PGE, at the various concentrations of 7-oxa-13-PA are indicated at the upper curve.

2 7

PGE, stimulates lactic acid production in a dose-dependent way in the concentration range 0.025-0.8 ,ug/ml (Perklev and Ahrtn 1971). In the present study we found that PGE,- concentrations exceeding 0.8 ,ug/ml did not give any further stimulation of the lactic acid production (data not shown). PGE, also stimulated the uptake of the non-utilizable amino acids AIB and cycloleucine in the ovary in the same range of concentrations that stimulated lactic acid production (Fig. 1). The time-relationship of the effect of PGE, (0.1 ,ug/ml) on the AIB-uptake is shown in Fig. 2. The uptake is linear up to 4 h with time both with and without PGEI.

In control ovaries, the incorporation of I e ~ c i n e - ~ ~ C into ovarian protein after 2 h of incubation was 879 k22 DPM/pg protein and increased to 1 078 +42 DPM/,ug protein when stimulated by 0.1 pg PGEJml medium. No further increase in incorporation rate was seen with higher PGE, concentrations.

Effects of 7-oxa-13-PA 7-oxa-13-PA in concentrations ranging from 25 to 75 ,ug/ml stimulated the lactic acid produc- tion in a dose-related manner (Fig. 3), i.e. had an effect similar to that of PGE,. The effect of added PGE, was not abolished by 25 pg 7-oxa-13-PAlml. With higher concentrations of 7-oxa-l3-PA, in itself strongly stimulating the lactic acid production, PGE, could not give any further increase. In contrast to the effect on lactic acid production, 7-oxa-13-PA de- creased the uptake of AIB, i.e. an effect opposite to that of PGE, (Fig. 4 a). The effect of added PGEl was not abolished by 7-oxa-13-PA. On cycloleucine-uptake, 7-oxa-13-PA had a stimulatory effect (Fig. 4 b), but, as in the case of AIB-uptake, PGEl could stimulate the cycloleucine-uptake when 7-oxa-13-PA was present.

The protein synthesis was inhibited by 7-oxa-13-PA in a dose-dependent way. Again PGE, had a stimulatory effect (Fig. 5) , but only with 25 ,ug 7-oxa-13-PA/ml where it in itself did not decrease the protein synthesis. Since 7-oxa-13-PA inhibited the protein synthesis it was compared to a specific inhibitor of the protein synthesis in the ovary. Puromycin in a dose (500 pg/ml incubation medium) which inhibits the incorporation of labelled natural amino acids approx. 95 % (AhrCn and Rubinstein 1965), decreased the lactic acid production

244

15

.o IC e c C

= 'E

Q

.- t

n - .- m a 5

C

800 - - c (Y - ? 600-

0 ._ g cn \=

400- I

c 0

i a g 200- E

CLAES MAGNUSSON, GUNNAR SELSTAM AND KURT AHREN

a T b'

I 1

0 2 5 5 0 75 7- oxa -13-PA ( pg/mll

b

C. 7-OXO-13-PA

7 - 0x0-13- PA + PGE, (0 I pg/mll

0 ........... 0

0 2 5 5 0 75 7-0x0-13-PA (pg/ml)

Fig. 4 a and b. Effect of 7-oxa-13-PA alone and in combination with PGE, (0.1 pg/ml) on the uptake of AIB (a) and cycloleucine (b) in prepubertal rat ovaries. The ovaries were incubated for 2 h in modified Krebs-Ringer bicarbonate buffer containing 5.5 m M glucose and 0.1 mM AIB-SH or 0.1 rnM cycloleucine- l4C. The uptake of amino acid is given as distribution ratio a t the end of the incubation period. Levels of significance are indicated as in Fig. 3.

(control 1.16 k0.14; puromycin 0.34 1-0.06 pgfml w.w.), somewhat decreased the AIB uptake (control 8.5 k0.7; puromycin 6.4 k0.3) and increased cycloleucine uptake (control 4.8 k0.5; puromycin 6.7 ?0.3). Thus, on amino acid uptake and protein synthesis, 7-oxa-13-PA acted very similar to puromycin.

Due to the abovementioned marked inherent effects of 7-oxa-13-PA at concentrations above 25 pglml, it was considered meaningful to test the effects of 7-oxa-13-PA on the cAMP system only at lower concentrations. 7-oxa-13-PA (1 and 20 pglml) did in itself not change the cAMP content in the tissue nor the release of cAMP into the incubation medium (Table 11). When 1 pg 7-oxa-13-PA/ml was added in combination with a gonadotropin (LH,

.................. '1

+... .............

.....+ ..,..,

u 7-oxa-13-PA o...... o 7-oaa-13-PA

+ PGE, (0.1 pg/rnl)

o i I-- r-- 0 25 50 75

7 - oka - 13- PA ($g/ml)

Fig. 5 . Effect of 7-oxa-13-PA alone and in combina- tion with PGE, (0.1 pg/ml) on the incorporation of radioactivity from leucine-%H into ovarian protein. Prepubertal rat ovaries were incubated for 2 h in Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose and 0.01 mM leucine-SH. Levels of signifi- cance are indicated as in Fig. 3.

PROSTAGLANDIN EFFECT ON OVARIAN METABOLISM 245

TABLE 11. Influence of 7-oxa-13-PA on the stimulatory effect of LH on the cAMP content in the ovary and the incubation medium. The ovaries were incubated for 2 h in modified Krebs Ringer bicarbonate buffer containing 5.5 mM glucose.

~~~~ _____

7-oxa-13-PA cAMP (pmole/pg protein) (pdml)

control NIH-LH-B8 ( 1 ,ug/ml)

tissue medium tissue medium

0 15+ 1 N.D.= 72+ 8 3502 120 1 1451 N.S. N.D. 14k 10 N.S. 166k32 N.S.

20 20+2 N.S. N.D. 166?27** 127?36*

a N.D.=not detectable. Levels of significance of the effect of 7-oxa-13-PA: * = p < 0.05, ** = p < 0.01, N.S. = not significant.

1 ,ug/ml), no effect was seen. At 20 ,ug/ml, however, an increase in the tissue content with a contemporaneous decrease in the release of cAMP was found.

Discussion One of the important questions in the reproductive field is what roles the different PGs may play, When studying the function of PGs, several different experimental approaches have been used. Among these, we have further studied several gonadotropic effects that can be mimicked by the PGs, and we have also tested a proposed inhibitor, 7-oxa-13-PA on these parameters.

Gonadotropins stimulate amino acid uptake and lactic acid production (Hamberger and A h r h 1967, Nilsson and Selstam 1974). In the present paper it was shown that a clear effect and a wide dose-response was achieved by PGE, on the uptake of the amino acids AIB and cycloleucine, as well as production of lactic acid. Thus, on these parameters the ovary is as sensitive to PGs of the E type as it is on others, such as steroidogenesis and contractility. The effects of PGs of the F type observed in our study were much smaller.

Another way of testing the effects of PGs is by the addition of antagonists. One obvious criterion for an inhibitory substance is that the antagonist in inhibitory concentrations should have no or at least a less pronounced effect than the agonist, when competing for the receptor site. In this study, the PG analogue 7-oxa-13-PA could not fulfil this criterion; it could only inhibit the PGE-effects in concentrations where it in itself had a marked stimulatory or inhibitory effect.

In this study we found no significant effect of 7-oxa-13-PA on amino acid transport and incorporation, or lactic acid production in concentrations up to 25 pg/ml. This result on AIB transport is consistent with findings by Sato et al. (1972) in the thyroid.

In combination with LH, 7-oxa-13-PA exerted an effect on the cAMP system. The release of cAMP from the ovary was decreased and the tissue content increased. This redistribution may be due to a non-specific effect on the membrane, since in these concentrations 7-oxa- 13-PA does not change adenylate cyclase activity as has been shown for many tissues by Hynie et al. (1975).

The question arises whether the different effects of 7-oxa-13-PA are dependent upon each

246 CLAES MAGNUSSON, GUNNAR SELSTAM AND KURT A H R ~ N

other, e.g. are the effects secondary to a general impairment of membrane function or to the inhibition seen in protein synthesis? As for the latter suggestion, inhibitors of protein synthesis can inhibit AIB transport (Ahren and Rubinstein 1965) and stimulate cycloleucine transport (Ahrkn, Hillensjo and Selstam, unpublished), both findings confirmed in this study. It is therefore quite possible that the similar effects of 7-oxa-13-PA on these amino acids might be secondary to the inhibited protein synthesis. On the other hand an impaired membrane function may explain the change in amino acid transport as well as the decrease in cAMP release and could perhaps also lead to an increased demand for energy substrates as indicated by the increased lactic acid production.

In order to study the role of PGs in the ovary, however, it is also necessary to measure whether PG-content and synthesis are changed after gonadotropic stimulation. In the follicle and the ovary an increase of PGs has been shown after LH stimulation, but this increase is registered after an increase in cAMP content (LeMaire et al. 1973, Marsh et al. 1973, Moon et al. 1974). Inhibitors of PG synthetase, such as indomethacin and aspirin, are ineffective in inhibiting the LH effect on adenylate cyclase as discussed by Marsh (1975).

Another mode of procedure when testing a possible role for PGs in the gonadotropin action is to study whether there is an additivity or not between the PG and the gonado- tropin. Additivity would speak against a role of PG as an intermediate step in the gonado- tropin action, while a lack of additivity is an indication, but not evidence of a role of PG. Moreover, in the ovary, additivity studies are complicated by the many different cell types, which at least in part can be responsible for the different time-courses in cAMP response after LH and PGE, stimulation (Selstam et al. 1974).

In view of the findings that PGs mimic many of the actions of gonadotropins and that PG effects are related to changes in the cAMP system, it has been tempting to postulate a functional role for PGs in the ovarian metabolism. Inhibitors of PG action are one important tool for studying this role. Our conclusion from the present study, however, is that 7-0Xa- 13-PA is not appropriate in this respect, since it does not act as a PG inhibitor in the ovary.

The authors wish to thank Prof. J. Fried, Department of Chemistry and Biochemistry, University of Chicago, Ill., USA, for the gift of 7-oxa-13-PA and valuable suggestions. Thanks are also due to Leo Ltd, Helsingborg, Sweden, for the supply of PGs and to NIH for the supply of LH. The assistance given by Stina Oberg, B.A., is gratefully acknowledged. This work was supported by grants from the Swedish Medical Research Council (B77-14)<-27), Magnus Bergvall’s Foundation, Wilhelm and Martina Lund- gren’s Foundation and from the Faculty of Medicine, Goteborg.

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