Eur. J. Biochem. 61, 605-611 (1976)

Localization of a Prostaglandin F,, Receptor in Bovine Corpus luteurn Plasma Membranes William S. POWELL, Sven HAMMERSTROM, and Ben@ SAMUELSSON Department of Chemistry, Karolinska Institutet, Stockholm (Received April 26/ September 12, 1975)

The distribution of a prostaglandin F,, receptor in various subcellular fractions from bovine corpora lutea obtained by differential and gradient centrifugation paralleled very closely the distribution in these fractions of 5’-nucleotidase, a marker enzyme for plasma membranes. The fractions most enriched in the receptor and 5’-nucleotidase were relatively free of mitochondria and lysosomes but were contaminated to some extent by elements of the endoplasmic reticulum. From these results it can be concluded that the prostaglandin F,, receptor is localized on the plasma membranes of the corpus luteurn cells. A simple method is described for the purification of plasma membranes from bovine corpora lutea by differential centrifugation. Prostaglandin F,, has a luteolytic effect in many mammalian species [I] and has been shown to have a physiological role as a luteolytic hormone in the sheep [2]. Receptors specific for this prostaglandin have recently been demonstrated in ovine [3], bovine [4] and human [5] corpora lutea. The prostaglandin-receptor complex has been solubilized from a particulate fraction obtained from bovine corpora lutea [6]. For further studies on the mechanism of the luteolytic effect of prostaglandin F,, it is important to know the localization of its receptor within the cell. In the present report evidence is presented for the localization of this receptor on the bovine corpus luteum plasma membranes. A simple procedure for the preparation of partially purified plasma membranes from this tissue by differential centrifugation is also described. MATERIALS AND METHODS [9fi-3H]Prostaglandin F,, (1 Ci/mmol) was prepared as described previously [7]. Unlabeled prostaglandin F,, was obtained from Dr J. Pike of the Upjohn Co. (Kalamazoo, Michigan). A Packard Tri Carb, model 3385, liquid scintillation spectrometer, equipped with automatic external standardization was used for radioactivity measurements. Ultraviolet/visible absorbance measurements were made with a Zeiss PMQ I1 or a Cary 118 C spectrophotometer. Centrifugations were carried out using either a Sorvall RC2-B centrifuge or a Beckman L5-65

ultracentrifuge. All centrifugal forces were calculated for the middle of the centrifuge tube. Protein was determined by the method of Lowry et al. [8] with bovine serum albumin as standard. Enzyme Assays

The activities of succinate dehydrogenase [9], NADH cytochrome c reductase [lo], NADPH-cytochrome c reductase [lo], glucose 6-phosphatase [ll], glucose 6-phosphate dehydrogenase [12], 5’-nucleotidase [13], acid phosphatase [9], B-glucuronidase [14] and N-acetyl-/?-glucosaminidase [15] were determined as described in the literature. For the determination of the activities of the last three enzymes, tissue fractions (containing 0.3 M sucrose) were centrifuged at 100000 x g in a Beckman no. 40 rotor for one hour. The pellets were resuspended by homogenization in an amount of distilled water equal to the original volume and frozen and thawed 10 times. After recentrifugation as described above, the two supernatants were combined and the pellet was resuspended as before. The enzyme activities in the supernatant (soluble enzyme) and pellet (bound enzyme) were then determined. Determination of Concentration of Receptor Sites

The concentrations of the prostaglandin F,, receptor in the tissue fractions were determined as described previously [4] from Scatchard plots [I61 after incubation with [9B-3H]prostaglandinF,, in the

Prostdgkdndin F,, Receptor in Bovine Corpus luteum Plasma Membranes

606

presence (non-specific binding) or absence (total binding) of an excess of unlabeled prostaglandin FZa. Preparation of Partiully Purified Plasma Membranes by Differential Centrifugation

Bovine ovaries from pregnant animals, or estimated to be between days 6 and 16 of the estrus cycle from the macroscopic appearance of their corpora lutea, were collected at a slaughterhouse in Uppsala. Luteal tissue was immediately removed from the remainder of the ovary by scraping with a razor blade and was placed in ice-cold 0.3 M sucrose containing 1 mM NaHCO,. After transportation to the laboratories in Stockholm, which took 1 h, the tissue was homogenized in 10 vol. (ml/g of fresh tissue) of the same medium with 6 strokes of the loose pestle of a Dounce homogenizer (Kontes Glass Co., Vineland, N.J.). The mixture was filtered, first through 1 layer and then through 4 layers of cheesecloth and the filtrate was centrifuged at 6000 x g for 10 min in the SS-34 rotor of a Sorvall RC2-B centrifuge at 0 "C. The pellet was resuspended in 8 vol. of medium and recentrifuged under the same conditions. The two supernatants were combined and centrifuged at 35 000 x g for 30 min in the same rotor, giving a doublelayered pellet. The top layer was removed with a spatula, resuspended in 4 volumes of medium, and recentrifuged at 35 000 x g for 30 min. The supernatant was then removed by aspiration, care being taken not to remove any of the loose top layer of the pellet, which was separated from the bottom layer by swirling in 0.5 vol. of medium and then resuspended by homogenization to give a fraction containing partially purified p l a ~ t i i membranes. ;~ The bottom layers from the two 35000 x g centrifugations were combined and resuspended in 1 vol. of medium whilst the combined

supernatants were centrifuged at 80000 x g for 90 min in the no. 30 rotor of a Beckman L5-65 ultracentrifuge at 0 "C. The supernatant was then centrifuged in a Beckman no. 65 rotor at 270000 x g for 3 h at 0 "C. The enzyme activities and receptor concentrations were determined for each of the fractions (Fig. 1). The distributions of the constituents among the various fractions are given in Table 1. Purification o j Plasma Membranes by Sucrose-Gradient Centrifugation

Bovine corpora lutea were collected as described above in ice-cold 0.5 M sucrose containing 1 mM NaHCO,. The tissue was homogenized and filtered as before and the filtrate was adjusted to a concentration of 0.25 M in sucrose by the addition of 1 mM NaHCO,. The mixture was centrifuged at 10000 x g for 15 min in the SS-34 rotor of a Sorvall RC2-B centrifuge at 0 "C. The pellet was washed once, resuspended in 1Ovol. of 0.25 M sucrose, 1 mM NaHCO,, and centrifuged in the same rotor at 1000 x g for 10 min. This pellet was washed once and the two supernatants were combined and centrifuged at 1OOOOxg for 15 min giving a double-layered pellet. The supernatant was removed carefully so as not to remove any of the loose top layer of the pellet, which was separated from the bottom layer by swirling in several ml of the same medium and finally resuspended by homogenization in a total of 4vol. of medium. After recentrifugation as described above, the top layer was resuspended in 5 vol. of the same medium. 10 ml of this mixture (about 0.5 mg protein/ml) were then layered on top of 25 ml of a sucrose gradient (27-45% in distilled water) with a cushion (5 ml) of 50% sucrose in distilled water in a 1 by 3.5 inch cellulose nitrate centrifuge tube. After centri-

Table 1. Distribution of constituents in various fractions obtained upon differential centrifugation of bovine corpus luteum homogenates 6p, 6000 x g pellet; 35b, bottom layer of the 35000xg pellet; 35t, top layer of the 35000xg pellet; 80p, 80000xg pellet; 270p, 270000xg pellet; 270s, 270 x g supernatant Constituent

Distribution in fraction ~

~~~

6P

35b

34 21 24 63 9 53 49 21 50 40

11

270p

~

35t --

Protein Prostaglandin F,, receptor 5'-Nucleotidase Succinate dehydrogenase NADPH-cytochrome c reductase NADH-cytochrome c reductase P-Glucuroniddse (soluble) ~-Glucuronidase(bound) N-Acetyl-p-glucosaminidase(soluble) N-Acetyl-p-glucosaminidase (bound)

.

3 19 34 1 2 1 6

14

1

2

24

2

1

I

-.

~~

6 23 34 2 3 13 2 8

20 26 22 4 34 40 20

~-

6

-

1 1 8 5

Recovery

270s

41 95 14

10 34

~-

101 83 118 88 114 122 102 69 101 72

607

W. S. Powell, S. Hammerstrom, and B. Samuelsson

p- Gucuronidase P-Glucuronidase (bound)

(soluble)

NADPH -cytochrorne c reductase c) .r

NADH -cytochrome c reductase

35 t

6

Succinate dehydrogenase

3

0

0 0

100 0 Protein in fraction (Yototal)

50

100

Fig. 1. Distribution patterns of constituents after fractionation of bovine corpus luteum homogenates by differential centrifugatiop. On the ordinate is plotted the relative specific activity (the activity per mg of protein in the fraction/the activity per mg of protein in the homogenate) and on the abscissa is given the percentage of the total protein of each of the fractions (cumulatively from left to right). The fractions were prepared as described in the Materials and Methods section under “Preparation of Partially Purified Plasma Membranes by Differential Centrifugation”. The shaded bars represent the fraction most enriched in plasma membranes. 6p, 6000 x g pellet; 35b, bottom layer of the 35000xgpellet; 3 3 , top layer ofthe 35000xgpellet; XOp, 80000xgpellet; 270p, 270000xgpellet; 270s, 270000xg supernatant; n.d., not determined

fugation at 96000 x g for 10 h in an SW-27 rotor in a Beckman L5-65 ultracentrifuge, l-ml fractions were collected and their absorbances at 650 nm and refractive indices were measured. Fractions were then pooled as indicated in Fig. 3 and the activities of several enzymes and the concentrations of the prostaglandin FZo!receptor sites were determined for the pooled fractions (Fig. 2). The 10000x g supernatants obtained from the differential centrifugation step were combined and centrifuged in a Beckman no. 30 rotor at 80000 x g for 90 min. The activities of several enzymes and the concentrations of prostaglandin F2. receptor sites for the fractions obtained by differential centrifugation are also given in Fig. 2.

RESULTS Fig. 1 shows the distributions of a prostaglandin F,, receptor and several marker enzymes in fractions obtained from the differential centrifugation of homogenates from bovine corpora lutea. On the ordinate is given the purification of the constituent with respect to its activity in the homogenate and on the abscissa, the percentage of the total. protein present each of the fractions. Table 1 givens the distributions by percentage of the receptor and marker enzymes in the different fractions along with their recoveries. The absolute activity of each of these constituents in bovine corpus luteum homogenates is given in Table 2.

Prostaglandin F,, Receptor in Bovine Corpus luteurn Plasma Membranes

608

Table 1. Absolute activities of constituents in bovine corpus luteum homogenates Enzyme activities are expressed in nkat (nmol/s)/rng of protein and the receptor concentration in prnol/mg protein. One gram of corpus luteum yielded 73 mg of protein after homogenization and filtration Constituent

EC

Activity or concentration

A

B NADPH -cytochrome c reductase

1 l

0

nkat (or prnol)/ mg protein Prostaglandin F,, receptor 5‘-Nucleotidase Succinate dehydrogenase NADPH-cytochrome c reductase NADH-cytochrome c reductase /3-Glucuronidase (soluble) /3-Glucuronidase (bound) N-Acetyl-j-glucosaminidase (soluble) N -Acetyl-j-glucosaminidase (bound)

3.2.1.31 3.2.1.31

(0.82) 2.7 0.32 0.75 8.3 0.017 0.017

3.2.1.30

0.47

3.2.1.30

0.72

3.1.3.5 1.3.99.1

1.6.2.3 1.6.2.1

5’-Nucleotidase is localized mainly on the plasma membranes of many types of cells [17] and it is assumed here that this is also the case with bovine corpora lutea. It has been shown that the distribution of this enzyme in subcellular fractions from bovine COYpora lutea corresponds to the distribution of a gonadotropin receptor in these fractions and it was concluded that the receptor is localized on the corpus luteum plasma membranes [18]. This was supported by electron microscopic evidence [18]. There is also immunological [19], autoradiographic [20] and biochemical [adenyl cyclase assumed to be a plasma membrane marker (cf. [17])] [21] evidence suggesting the localization of the gonadotropin receptor on corpus luteum plasma membranes. Since it has the same subcellular localization as the gonadotropin receptor [18] it would appear that 5’-nucleotidase is also localized on the bovine corpus luteum plasma membranes. It can be seen from Fig. 1 that the purifications of the receptor (6-fold) and of 5’-nucleotidase (1I-fold) were maximal in the fraction designated 35t (the top layer of the pellet obtained by centrifuging the 6000 x g supernatant at 35000 x g). None of the other marker enzymes tested showed maximal purification in this fraction with the exception of membrane bound figlucuronidase, which, however was rather evenly distributed throughout all the fractions. Succinate dehydrogenase, a marker enzyme for mitochondria [9], was concentrated mainly in the 6000 xg pellet and in the bottom layer of the pellet obtained upon centrifugation of the 6000 x g supernatant at 35000 x g. The membrane-bound activity of NADPH-cytochrome c reductase, which, in liver, is associated mainly with the endoplasmic reticulum [22] was purified to the greatest extent in the 8OOOO x g pellet. Most

Prostaglandin F2= receptor

0

50 Protein in fraction

100

0 50 100 Protein recovered from gradient

(‘lo total) (Ole total) Fig. 2. Distribution patterns o ] constituents after fructionation of’ bovine corpus luteum homogenates by differential centrifugation ( A ) and gradient centrifugation ( B ) . (A) See description in the Materials and Methods section under “Preparation of Plasma Membranes by Sucrose Gradient Centrifugation”. The data are plotted as in Fig. 1. The shaded bars represent the fraction most enriched in plasma membranes. l p , 1OOOxg pellet; lob, bottom layer of 10000 x g pellet; lot, top layer of 10000 x g pellet; 80p, 80000 x g pellet; 80s, 80000xg supernatant. (B) Gradient centrifugation as described in the Materials and Methods section. On the ordinate is plotted the relative specific activity (the activity per mg of protein in the gradient fraction/the activity per mg of protein in the homogenate) and on the abscissa is given the percentage of the total protein recovered from the gradient ofeach of the fractions (cumulatively from left to right). The fractions are arranged in decreasing order of density from left to right. The shaded bars represent the fraction most enriched in plasma membranes. The numbers assigned to each of the fractions refer to Fig. 3

of the activity of this enzyme in homogenates of bovine corpora lutea,.however, was associated with the soluble fraction, which is not case with liver [22]. It is possible that this could be a non-specific activity involving some other enzyme. In agreement with a previous report [I 81 the activity of glucose 6-phosphatase in fractions from bovine corpora lutea was

609

W. S. Powell, S. Hammerstrom, and B. Saniuelsson

I

5

rl

0

10

20

-.-

30

Volune ( m i )

Fig. 3. Density gradient centrifugation of bovine corpus luteum hornogenates (as described in the Materials and Methods section). ( x ) Sucrose density; (0)absorbance at 650 nm. Fraction volumes were 1 ml except for the first, which was 4 ml and was composed mainly of the cushion of 50% sucrose. The various 1-ml fractions were pooled as indicated to give 8 larger fractions

found to be too low to enable its use as a reliable marker enzyme for the endoplasmic reticulum. NADH-cytochrome c reductase activity was distributed throughout both the heavy and light fractions indicating that it is present in both the mitochondria and the endoplasmic reticulum as is the case for liver [22]. The activity of b-glucuronidase was determined after freeze-thawing the fractions followed by centrifugation. The activity remaining in the supernatant (soluble B-glucuronidase) was highest in the fraction designated 35b while the activity in the pellet (bound 8-glucuronidase) showed a wider distribution. Similar results were obtained with N-acetyl-p-glucosaminidase although in this case the distributions of both the soluble and bound activities were shifted towards the heavier fractions. The acid phosphatase activities in these fractions were rather low and this enzyme was not considered to be a convenient marker for lysosomes in this tissue. Since the activities of b-glucuronidase and N-acetyl-b-glucosaminidase which can be released by freeze-thawing are indicative of the presence of lysosomes (cfi [22]) it can be seen that fraction 35t contains a very small proportion of the lysosomes present in the homogenate. Attempts to further purify the plasma membrane vesicles present in fraction 35t by sucrose gradient centrifugation were not successful since these components behaved quite similarly to vesicles derived

from the endoplasmic reticulum. By using a different procedure for the differential centrifugation step, however, it was possible to achieve a greater separation of the 5’-nucleotidase and prostaglandin F,, receptor activities from the NADPH-cytochrome c reductase activity at the expense of greater contamination with succinate dehydrogenase (Fig. 2). Thus the receptor was purified 3.5-fold and 5‘-nucleotidase 6-fold in the top layer of the pellet obtained by centrifuging the lo00 x g supernatant at loo00 x g. Fig. 3 shows the results of centrifugation of this fraction on a sucrose gradient. The various fractions were pooled as indicated according to their absorbances at 650 nm. The mitochondria (i.e. succinate dehydrogenase) were purified to the greatest extent in fraction 5 (mean density 1.160). Maximal purification of the prostaglandin F,, receptor (8-fold compared to the original homogenate) and 5’-nucleotidase (20-fold) occurred in fraction 7 (mean density 1.135 g/ml). NADPHcytochrome c reductase activity, although distributed throughout the gradient, was also purified to some extent in this fraction.

DISCUSSION

A simple method for the partial purification by differential centrifugation of plasma membranes from bovine corpora luteu has been developed. Using this procedure 5’-nucleotidase was purified 11-fold with respect to its activity in the homogenate with a yield of 29%. Relatively small amounts of mitochondria and lysosomes but significant amounts of endoplasmic reticulum vesicles were present in this fraction as judged by the activities of marker enzymes for these organelles. Several other methods for the purificacion of plasma membranes from bovine corporu luteu utilizing sucrose gradient centrifugation have recently appeared [18, 21, 23, 241 but in only two of these have values for the purification of marker enzymes been given. Gospodarowicz [18] similarly reported an 11-fold purification of 5’-nucleotidase activity with a yield of 8% in a fraction of purified plasma membranes. Purification of a luteinizing hormone receptor in this fraction was about 20-fold, about 3 times the purification of the prostaglandin F,, receptor reported here. Mitochondria were virtually absent from this fraction but the amount of contamination by elements of the endoplasmic reticulum is not clear since the marker enzyme employed was glucose 6-phosphate dehydronase. In our hands this enzyme appeared almost exclusively in the 80000 x g supernatant as previously reported for bovine and human corporu futea [25]. Plasma membranes have also been purified from bovine corporu lutea by centrifugation on sucrose gradients of a low speed (2000 x g ) pellet giving purifi-

610

Prostaglandin F2. Receptor in Bovine Corpus futeum Plasma Membranes

cations for a human chorionic gonadotrophin receptor, adenylate cyclase and Na, K-dependent ATPase of 8, 6 and 3-fold respectively [21]. We could detect only low concentrations of 5’-nucleotidase and the prostaglandin F,, receptor in such fractions, however, and found it necessary to use higher centrifugal forces to sediment significant-amounts of the plasma membranes present in the homogenate. It can be seen from Fig. 1 that the distribution of the prostaglandin F,, receptor in the various fractions obtained by differential centrifugation resembles very closely that of 5’-nucleotidase, strongly suggesting that the receptor is localized on the plasma membranes of the corpus luteum cells. Furthermore, the marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (soluble /3-glucuronidase and soluble N acetyl-P-glucosaminidase) and the endoplasmic reticulum (membrane-bound-NADPH-cytochromec reductase) clearly show different distributions with the former two organelles being concentrated in heavier fractions and the latter present to the greatest extent in the 80000 x g pellet. The significance of the membrane-bound activities of /3-glucuronidase and N acetyl-P-glucosaminidase is not quite clear. P-Glucuronidase is present in the endoplasmic reticulum of rat [l 1,221and mouse [26] liver and, from the experiments reported here it appears that this is also the case in the bovine corpus luteum. The activity of this enzyme does not quite parallel that of NADPH-cytochrome c reductase, however, so it is possible that there could also be an additional explanation such as the adsorption of the soluble enzyme to subcellular organelles. The membrane-bound activity of N-acetyl-/3-glucosaminidase is clearly concentrated in the heavier fractions and therefore does not reflect the presence of this enzyme to any great extent in the endoplasmic reticulum. It is possible that this may be at least partially explained by adsorption of the soluble enzyme to subcellular organelles as was found to be the case when rat spleen was homogenized in 0.25 M sucrose (but not when homogenized in 0.2 M KCl) [27]. It was not possible to purify the plasma membranes in fraction 35t to a much greater extent using sucrose gradient centrifugation because of the difficulty of removing endoplasmic reticulum vesicles. Although a slight purification of 5’-nucleotidase activity was achieved, there was no significant purification of the prostaglandin FZareceptor, possibly due to degradation occurring during the centrifugation, since the recovery of the receptor from sucrose gradients was usually about 25% lower than that of 5’-nucleotidase. When a lower speed pellet (the top layer of the pellet obtained by centrifugation of the 1OOOxg supernatant at 10000 x g) was used, however, the major contaminants were mitochondria and these were readily separated from the plasma membranes by sucrose gradient centrifugation. In this way it was possible to

purify 5’-nucleotidase 20-fold (3% yield) and the prostaglandin FZ, receptor 8-fold (2% yield) in frdction (Fig.2). Almost all the mitochondria (i.e. succinate dehydrogenase) were removed from this fraction, although there was still a significant amount of contamination by endoplasmic reticulum vesicles (i.e. NADPH-cytochrome c reductase). From Fig. 3 it can be seen that the equilibrium density of the fraction most enriched in mitochondria (fraction 5) is 1.160 g/ml while that of the plasma membrane fraction (fraction 7) is 1.135 g/ml. The value obtained for plasma membranes is similar to values (1.12 - 1.14 g/ml) reported for the equilibrium density of 5’-nucleotidase associated with the light subfraction of liver plasma membranes [28-301. No evidence was found for a second heavier subfraction of plasma membranes from the corpus luteum corresponding to the fraction with a density of about 1.18 g/ml which was isolated from liver homogenates and consisted of long membrane strips with junctional complexes [29]. The higher values previously reported [18] for the densities (1.14 1.16 and 2.16-1.18 g/ml) in sucrose gradients of plasma membranes from bovine corpora lutea may have been due to the inclusion of 1 mM CaCl, and 25 mM Tris in the medium employed in these experiments. We found that the presence of these salts had marked effects in both differential and gradient centrifugation experiments, causing the particles to behave as though they were larger and denser, presumably due to aggregation. Prostaglandin El receptors were also reported to be localized on the plasma membranes of liver [31] and corpus luteum [32] cells. In the former case [31] high affinity receptors for this prostaglandin were not present in subcellular fractions containing nuclei, rough microsomes, Golgi complexes or mitochondria. In conclusion, our results clearly demonstrate that the prostaglandin F,, receptor in bovine corpus luteurn is located on the plasma membranes of the luteal cells. The next step in the luteolytic action of prostaglandin F,, is not known, although several hypotheses, including effects on intracellular enzymes [33, 341, receptors [35, 361 and lysosomes [37] have been suggested. W. S. P. is the holder of a Postdoctoral Fellowship from the Medical Research Council of Canada. This work was supported by Grants from the World Health Organization.

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61 1 20. Rajaniemi, H. & Vanha-Perttula, T. (1972) Endocrinology, 90, 1-9. 21. Menon, K. M. J. & Kiburz, J. (1974) Biochem. Biophys. Res. Commun. 56,363- 371. 22. Amar-Costesec, A., Beaufay, H., Wibo, M., Thines-Sempoux, D., Feytmans, E., Robbi, M. & Berthet, J. (1974) J. Cell Biol. 61,201 -212. 23. Haour, F. & Saxena, B. B. (1974) J. Eiol. C h t m 249, 21952205. 24. Rao, C. V. (1974) J. B i d . Chem. 249,2864-2872. 25. Nielson, M. H. & Warren, J. C. (1965) Acta Endocrinol. 4, 58 - 64. 26. Paigen, K. (1961) Exp. Cell Res. 25, 286-301. 27. Bowers, W. E. & de Duve, C. (1967) J. Cell Biol. 32, 339- 348. 28. Coleman, R., Michell, R. H., Finean, J. B. & Hawthorne, J. N. (1967) Biochim. Biophys. Acta, 135, 573- 579. 29. Evans, W. H. (1970) Biochem. J . 116, 833-842. 30. Beaufay, H., Amar-Costesec, A., Thines-Sempoux, D., Wibo, M., Robbi, M. & Berthet, J. (1974) J. Cell Biol. 61,213-231. 31. Smigel, M. & Fleischer, S. (1974) Biochim. Biophys. Acta, 332, 358 - 373. 32. Rao, C. V. (1973) Prostaglandins, 4, 567- 576. 33. Behrman, H. R., Macdonald, G . J . & Greep, R. 0. (1971) Lipids, 6,791 - 796. 34. Strauss, J. F. & Stambaugh, R. L. (1974) Prostaglandins, 5, 73-85. 35. Hichens, M., Grinwich, D. L. & Behrman, H. R. (1974) Prostaglandins, 7,449 -458. 36. Jacobson, H., Obrams, G. I. & Swartz, D. P. (1974) Ferril. Steril. 25, 293. 37. Weiner, R. & Kaley, G. (1972) Nat. New B i d . 236, 46-47.

W. S . Powell, Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1Al S. Hammerstrom and B. Samuelsson, Kemiska Institutionen 11, Karolinska Institutet, Solnavagen 1, S-10401 Stockholm, Sweden