in Vitro Incorporation of.Acetate-l-'4C into the Phospholipids of Rabbit and Human Endometria z ROBERT J. MORIN and MARIA CARRION2, Departments of Pathology, Los Angeles County Harbor General Hospital, Torrance, California 90509 and UCLA School of Medicine, Los Angeles, California 90024 ABSTRACT

Endometria from nonpregnant and 6day pregnant rabbits and from humans in the proliferative and secretory phase s were incubated with 1-14C-acetate. ~4CO2 was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of 14C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of ~4C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria. Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolami,e of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease. INTRODUCTION

ALTERATIONS in endometrial lipids C YCLIC have been demonstrated histochemically in guinea pigs (1), ferrets (2), monkeys (3), and humans (4). Increased amounts of endometrial osmophilic fat have been found during early pregnancy in the rat (5,6), cat (7), and human (4). Quantitative chemical studies by Okey et al. (8) of the pig endometrium indicated that the concentrations of cholesterol and lecithin were highest during the luteal phase of the cycle. In the rat uterus, however, the period of max-. imal phospholipid content was found to occur during the follicular phase (9). Goswami et al (10) found that during estrus and in ovariectomized animals given estrogens that the relative amounts of triglycerides were lower, XThis investigation was supported by a granl: from the t:'or d F o u n d a t i o n .

2Ford Foundation Postdoctoral

Fellow.

and phospholipids were higher than in the diestrus period. In a chemical study of the rabbit endometrium by Ray and Morin (11), the endometrium of the implantation site in the 8-day pregnant rabbit showed lower percentages of total lipids than either the nongravid or the gravid interimplantation endometrium. The glycerides of the interimplantation areas were higher than those of the nongravid endometrium, and also showed higher proportions of linoleic and arachidonic acids. The implantation sites contained less glyceride and essential fatty acids than the interimplantation areas. Hormonal alterations occurring during pregnancy may possibly influence endometrial phospholipid metabolism. In the uterus of castrated rats, Davis and Alden found that estrogen treatment increased the concentration of phospholipids, whereas progesterone had no effect (9). EstradioI has been found to increase ~2p incorporation into the total phospholipids of subcellular particles of whole rat uterus (12), and also in another study to increase the synthesis of choline, ethanolamine and inositol containing phospholipids from 32p_ phosphate and 14C-acetate precusors (13). The objectives of the present experiments were to determine the content of specific phospholipids and phospholipid fatty acids in the endometrium and to study the alterations in concentration and metabolism of these phospholipids occurring during pregnancy in the rabbit and during the menstrual cycle in humans. EXPERIMENTAL

Twenty-four female New Zealand white rabbits, 4 months old, and fed a stock commercial diet (Purina) since weaning were divided into one group of 6 and another group of 18. S i x rabbits were mated to induce pregnancy. After 6 days the rabbits were anesthetized by intravenous injection of Sodium Pentobarbital. Uteri of the mated rabbits were examined and all found to be gravid. Endometrial samples from one pregnant rabbit and endometrium of approximately equal weight from three nonpregnant rabbits were each placed in separate flasks

349

350

ROBERT J. MORIN AND MARIA CARRION

with 10 ml of Tyrode's solution (14). ( A total of six incubations with gravid endometria and six with nongravid endometria were done.) After addition of 50 /~c sodium 1-a4C-acetate (Nuclear-Chicago; Specific Activity 29.0 m e / m M ) to each, the flasks were incubated for 4 hr in an air atmosphere at 37.5C in a Dubnoff metabolic shaking incubator. Carbon dioxide was collected in a center well containing 20% NaOH. The flasks were then placed on ice, and after cooling to 0C the contents were centrifuged, washed twice with 20 ml 0.9% NaC1 for 20 min each time and recentrifuged. The tissues were then refrigerated overnight in 50 ml of 0.155 M unlabeled sodium acetate. The next day, after one additional washing with 0.9% NaC1 and centrifugation, the total endometrial lipids were extracted in a VirTis 45 Homogenizer with two successive 50 ml portions of methylal/methanol ( 4 : 1 ) . The combined extracts were evaporated to dryness in a rotary vacuum evaporator and under N2, redissolved in chloroform, filtered, and evaporated down to 0.5 ml. Silica Gel H (Brinkmann) plates 0.5 m m thick were prepared by the method of Skipski (15). After drying and heating at l l 0 C for 1 hr, the plates were placed in the developing mixture and the solvents allowed to rise to the top of the plates, in order to remove impurities. The plates were then reactivated for 1 hr at l l 0 C just prior to use. The total endometrial Iipids were applied as a band across the plate and the phospholipids separated using an ascending developing mixture of chloroform-methanol-acetic acid-water ( 2 5 : 1 5 : 4 : 2 ) . The lysophosphatidyl choline, sphingomyelin, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine, and phosphatidyl ethanolamine bands were collected with a vacuum zone extractor. Elution of the phospholipids from the silica gel was accomplished as previously described (14). Aliquots of the eluates from each fraction were analyzed for phosphorous content (16). Other aliquots were assayed for radioactivity using a PPO-POPOP in toluene scintillator and a Packard Model 3314 liquid scintillation spectrometer. Quenching was monitored by the channels ratio technique, and all counts corrected to 100% efficiency. The remainder of the fractions were then hydrolyzed and methylated (17) and the fatty acid composition of each phosphatidyl choline and phosphatidyl ethanolamine fraction was determined by gas-liquid chromatography in a Barber-Colman Model 10 Gas Chromatograph using diethylene glycol succinate on GasChrom P, 70-80 mesh, at 187C with an argon LIPIDS, V O L . 3, N O . 4

TABLE I Amounts of Individual Phospholipids in Rabbit Endometrium a Rabbit group Lysophosphatidyl choline Sphingomyelin Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine

Nongravid

Gr avid

2.04-1.3 5.84-2.4 19.0-t-4.6 5.3-4-2.9 11.74-4.2 14.34-3.2

2.64-1.8 4.44-1.1 15.74-5.2 6.14-1.2 9.64-2.7 12.44-4.5

aMilligrams phospholipid/g dry weight endometrium (means of determinations f r o m 6 incubations per group + standard deviations).

pressure of 24 lb. The fatty acids were identiffed by comparison of retention times to those of standards (obtained from the National Institutes of Health and Calbiochem) and by graphic representation of retention times. The areas under the peaks were estimated by triangulation. Only the major fatty acids (16:0, 16:1, 18, 18:1, 18:2, and 20:4) have been included in the tabulations. Other fatty acids, identified in amounts either too small or having too long a retention time to measure accurately, were 14, 18:3, 20:1, 20:3 (8,11,14) 3, 22:4, 22:5, and 22:6. The radioactive CO 2 evolved was determined by precipitating an aliquot of N a O H Center well solution with 10% BaC1 v The light precipitate of BaCO 3 which formed was trapped by filtration as a very thin layer on a disc of W h a t m a n No. 42 filter paper and then the paper with the precipitate counted in the PPOPOPOP scintillator as described above. In a second series of experiments, samples of human endometria were obtained immediately after hysterectomy for fibroids in women 30-40 yr of age. Four samples were obtained in approximately the midproliferative phase and four in the midsecretory phase of the menstrual cycle as determined both clinically and histologically. The samples were incubated with 50~zC 1-14C-acetate (Specific Activity 29.0 m C / m M ) in Tyrode's solution for 4 hr under the same conditions described above for the rabbit endometrial samples. Carbon dioxide collection and counting, washing of aortas, extraction, thin-layer chromatography, phosphorus analysis and liquid scintillation counting were conducted the same way as in the rabbit experiments. The probabilities (p) that apparent differences in the data were due to chance was calculated by the student's t test, and the P values are given in the tables and text. SEicosatrienoic acid with double bonds in the 8-9, 11-12 and 14-15 positions as counted from the carboxyl end of the molecule.

ENDOMETRIAL PHOSPHOLIPIDS

TABLE III Amounts of 1-14C-Acetate Incorporated into Individual Phospholipids of Rabbit Endometria a

TABLE II Specific Radioactivities of Individual Phospholipids of Rabbit Endometrium after Inct~bation with 1-a4C-Acetatea Rabbit group

Nongravid Gravid

Lysophosphatidyl choline Sphingomyelin Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Phosphatidylethanolamine

479• 350-+ 88 1,360-+221 427+ 76 794-+332 1,120-+245

260-+ 45 178-+ 64 742-+147 315-+- 70 6?2-+283 656-+207

P <0.01 <0.01 <0.01 <0.05 <0.6 <0.01

aDPM/mg, phospholipid (means of 6 incubations per group --t- standard deviations). RESULTS

T h e amounts o f the individual phospholipid fractions in the non-gravid and gravid end o m e t r i a are indicated in Table I. T h e order of concentration of phospholipids was phosphatidyl choline > phosphatidyl ethanolamine phosphatidyl serine ~ sphingomyelin phosphatidyl inositol ~ lysophosphatidyl choline. T h e r e were no significant differences in amounts of phospholipids in endometria of n o n p r e g n a n t as c o m p a r e d to pregnant rabbits. D u r i n g the incubation with 1-~4C-acetate the z4COz evolved was 120,400 _ 13,500 D P M / m g dry tissue f r o m the nongravid endometria and 125,000 _ 10,650 D P M / m g f r o m the gravid endometria. T h e specific activities of the individual rabbit endometrial phospholipids after incubation with ~4C-acetate are indicated in Table II. Phosphatidyl choline showed the highest specific activity and sphingomyelin and lysophosphatidyl choline, the lowest. P r e g n a n c y seemed to significantly decrease the specific activity of phosphatidyl choline and phosphatidyl ethan o l a m i n e and also the other phospholipids to a lesser extent (all differences were significant to P values of < 0.05 except for phosphatidyl sef i n e ) . T h e a m o u n t of 1-14C-acetate in /~/~ moles incorporated into each phospholipid are s h o w n in Table III. T h e results in general paralleled those for specific activity.

351

Rabbit group

Nongravid

Lysophosphatidyl choline Sphingomyelin Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine

14-+ 3 32-+ 8 403+65 35• 6 144+60 2500-54

Gr avid

P

11-+ 2 <0.1 12-4- 4 <0.01 181 -+36 <0.01 30-+ 7 <0.3 100-+42 <0.2 126-+40 <0.01

a#Iz moles 1-~4C-acetate incorporated/g dry weight of (means of 6 incubations per group +.~ standard deviations).

endometrium

T h e fatty acid compositions of the two highest concentration phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, are indicated in Table IV. T h e percentages of linoleic acid in both phosphatidyl choline and phosphatidyl ethanolamine were significantly higher in the pregnant than in the nonpregn a n t state (P < 0.01). In both phospholipids, palmitoleic acid was lower in gravid e n d o m e t r i a ( P < 0.01). T h e amounts of individual phospholipids in h u m a n endometria in the proliferative and secretory phases are indicated in Table V. The order of concentration of the phospholipids was phosphatidyl choline ~ phosphatidyl ethanolamine ~ phosphatidyl serine ~ sphingomyelin lysophosphatidyl choline > phosphatidyl inositol. T h e endometria of the proliferative phase showed a higher concentration of phosphatidyl choline than did the secretory phase ( e < 0.05). I n Table V I are indicated the specific activities of the individual h u m a n endometrial phospholipids after incubation with ~4C-acetate and in Table V I I the n u m b e r o f / ~ moles of acetate i n c o r p o r a t e d / g dry weight of tissue. O f the phospholipids, phosphatidyl choline incorporated the a4C-acetate most actively, in b o t h the proliferative and the secretory endometria. T h e specific activity and a m o u n t of acetate incorporated into phosphatidyl choline was lower in

TABLE IV Percentages of Fatty Acid Methyl Esters in Phosphatidyl Choline and Phosphatidyl Ethanolamine of Rabbit Endometriuma Rabbit group Fatty acid 16:0 16:1 18:0 18:1 18:2 20:4

Phosphatidyl choline Nongravid Gravid 16.0+__2,1 10.6-+1.7 21.2-+5.0 24.2-+2.4 12.5-+0.8 15.4-+3.5

13.5+3.7 5.8-+4-1.5 15.6__+4.4 29.1-+-4.9 19.8-+2.7 16.2-+2.6

Phosphatidyl ethanolamine Nongravid Gravid 10.9• 11.5-+t-2.3 23.6-+3.0 25.1-+4.7 10.0-4-1.9 18.6-+2.1

10.3+__2.0 6.8• 17.7_+5.8 28.2-+2.5 17.6-+2.3 19.5-+3.0

apercentage of each fatty acid methyl ester (means of 6 determinations per group • standard deviations). LIPIDS, VOL. 3, NO. 4

352

ROBERT J. MORIN AND MARIA CARRION TABLE V Am6unts of Individual Phospholipids in Human Endometria a

Phase Lysophosphatidyl choline Sphingomyelin Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine

TABLE VII Amounts of 1-14C-Acetate Incorporated into Individual Phospholipids of Human Endometriuma

Proliferative

Secretory

2.6-+0.9 3.1-+0.9 20.5-+2.3 2.0-+0.7 7.2 -+2.1 8.6-+ 1.9

1.9+0.5 2.5-+ 1.1 15.0-+2.7 2.5-+0.9 5.6 -+1.8 9.1 -+2.5

aMilligram phospholipid/g dry weight endometrium (means of determinations from 4 incubations for each phase 4- standard deviations). the s e c r e t o r y t h a n in the p r o l i f e r a t i v e phase. T h e activity in p h o s p h a t i d y l e t h a n o l a m i n e in the s e c r e t o r y p h a s e s h o w e d a lesser b u t also significant decrease. T h e proliferative e n d o m e t r i a evolved m o r e a4CO2 ( 2 , 2 3 0 + 460 D P M / m g d r y tissue) t h a n did the secretory e n d o m e t r i a ( 1 , 2 7 0 + 355). DISCUSSION

I n the r a b b i t a n d h u m a n e n d o m e t r i u m , as has b e e n f o u n d in m o s t o t h e r m a m m a l i a n organs ( 1 8 ) , p h o s p h a t i d y l choline was the pred o m i n a n t class of phospholipid. I n the p r e s e n t e x p e r i m e n t s o n the f e m a l e r a b b i t e n d o m e t r i u m in vitro, i n c o r p o r a t i o n of l~C-acetate was highest into p h o s p h a t i d y l choline, similar to the h i g h in vivo i n c o r p o r a t i o n of 14C-acetate into p h o s p h a t i d y l choline of r a t liver ( 1 9 ) a n d in vitro into r a b b i t testes ( 2 0 ) a n d a o r t a ( 2 1 ) . T h e d e c r e a s e d i n c o r p o r a t i o n of acetate into the p h o s p h o l i p i d classes of the gravid r a b b i t e n d o m e t r i a was n o t due to i n c r e a s e d o x i d a t i o n a n d r e s u l t i n g decreased availability of substrate, as i n d i c a t e d b y the similar a m o u n t of 14CO2 p r o d u c e d b y e n d o m e t r i a f r o m the n o n p r e g n a n t a n d p r e g n a n t groups. A n i n c r e a s e d o x i d a t i o n of f o r m e d f a t t y acids in the gravid e n d o m e t r i a also c a n n o t e x p l a i n the o b s e r v e d differences, since this w o u l d h a v e p r o d u c e d m o r e ~4CO2. TABLE VI Specific Radioactivities of Individual Phospholipids of Human Endometrium after Incubation with 1-a4C-Acetatea Phase Lysophosphatidyl choline Sphingomyelin Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine

Proliferative Secretory 770+162 564+133 608+ 109 793+ 190 4,020+623 2,850+448 568--+153 689• 2,280-+3492,080+323 2,180___496 1,270-+290

P <0.1 <0.2 <0.05 <0.3 <0.5 <0.02

aDPM/mg phospholipid (means of 4 incubations of each phase -+ standard deviations). LIPIDS, VOL. 3, No. 4

Phase

Proliferative Secretory

Lysophosphatidyl choline 31 -+ 7 17-+ 4 Sphingomyelin 29-+ 5 31-+ 7 Phosphatidyl choline 1,280-+198 665_+104 Phosphatidylinositol 20+" 4 27+ 5 Phosphatidyl serine 255-+ 39 181+" 28 Phosphatidyl ethanolamine 292-+ 66 180+ 41

P <0.02 <0.7 <0.01 <0.1 <0.05 <0.05

a Micromicromoles 1-14C-acetate incorporated/g dry weight of endometrium (meaas of 4 incubations from each phase -+ standard deviations). E s t r o g e n s h a v e b e e n d e m o n s t r a t e d to increase total p h o s p h o l i p i d synthesis in the liver a n d uterus ( 1 2 , 2 2 ) . I n the w h o l e uterus, estrogens were f o u n d to i n c r e a s e the synthesis of choline, e t h a n o l a m i n e a n d inositol containing p h o s p h o l i p i d s (11 ). S o m e studies h a v e i n d i c a t e d t h a t estrogens m a y increase the hepatic synthesis of p a r t i c u l a r species of lecithins ( 2 3 ) . T h e p r e s e n t in v i t r o e x p e r i m e n t s w i t h the r a b b i t e n d o m e t r i u m , h o w e v e r , h a v e s h o w n t h a t d u r i n g p r e g n a n c y , w h e n t h e r e is a n increase in c i r c u l a t i n g estrogens, the p r e d o m i n a n t effect is a decrease in the i n c o r p o r a t i o n of 14C-acetate into m o s t p h o s p h o l i p i d classes. T h i s p r o b a b l y r e p r e s e n t s a d e c r e a s e d n e t synthesis, a l t h o u g h f a c t o r s s u c h as p e r m e a b i l i t y c h a n g e s a n d v a r i a t i o n in i n t e r m e d i a t e pool size of substrate h a v e n o t b e e n r u l e d o u t b y the p r e s e n t e x p e r i m e n t s ( t h e s e factors were also n o t r u l e d o u t in the a b o v e cited studies b y o t h e r investigators). T h e difference b e t w e e n the p r e s e n t results a n d t h o s e of the f o r m e r studies m a y b e due to the h i g h levels of p r o g e s t e r o n e or to the i n c r e a s e d g o n a d o t r o p i c h o r m o n e s d u r i n g p r e g n a n c y . C o n c u r r e n t a d m i n i s t r a t i o n of estradiol a n d p r o g e s t e r o n e to o v a r i e c t o m i z e d female rats seems to p r o d u c e a lesser effect o n uterine s u b c e l l u l a r p h o s p h o l i p i d d i s t r i b u t i o n t h a n c a n b e p r o d u c e d b y the e s t r o g e n a l o n e ( 2 4 ) . I n the m o u s e uterus, p r o g e s t e r o n e appears to a n t a g o n i z e the i n c r e a s e d c o n c e n t r a t i o n of p h o s p h o l i p i d s p r o d u c e d b y estrogens ( 1 2 ) . T h e p r e d o m i n a n t effect of p r e g n a n c y o n the p h o s p h o l i p i d f a t t y acids in the r a b b i t was a n i n c r e a s e d p r o p o r t i o n of linoleic acid in p h o s phatidyl choline and phosphatidyl ethanolamine. A s i m i l a r increase was also f o u n d in a p r e v i o u s s t u d y in the total glycerides of the i n t e r - i m p l a n t a t i o n areas of the 4 a n d 8 day gravid r a b b i t e n d o m e t r i u m . Since linoleic acid is a n essential f a t t y acid, a n d c a n n o t b e synthesized b y t h e animal, it is possible t h a t the i n c r e a s e d p r o p o r t i o n of linoleic acid in the glycerides a n d p b o s p h o l i p i d s of the e n d o m e -

ENDO3tETRIAL PHOSPHOLIP'IDS trium facilitates the acquisition of this fatty acid by the embryo. In one previous study (25), human endomet r i u m w a s f o u n d to i n c o r p o r a t e less ~4C-acet a t e i n t o t h e t o t a l p h o s p h o l i p i d s d u r i n g t h e secretory than during the proliferative phase of t h e m e n s t r u a l cycle. T h e p r e s e n t e x p e r i m e n t s h a v e c o n f i r m e d t h i s f i n d i n g a n d i n d i c a t e in addition that the lesser incorporation into phosp h a t i d y l c h o l i n e , a n d a l s o to s o m e e x t e n t i n t o p h o s p h a t i d y l e t h a n o l a m i n e is w h e r e t h e d e crease occurs. The secretory endometrium was a l s o f o u n d to o x i d i z e a ~ C - a c e t a t e at a s l o w e r rate than the proliferative endometrium. I n t h e h u m a n , as in t h e p r e g n a n t r a b b i t , t h e decreased incorporation of ~*C-acetate into the endometrial phospholipids that was observed in t h i s s t u d y d u r i n g t h e s e c r e t o r y p h a s e o f t h e cycle may represent an antagonism by progesterone of the prior estrogenic stimulation of phospholipid synthesis during the proliferative phase. T h e p o s s i b i l i t i e s a l s o e x i s t as in t h e r a b b i t that a decreased permeability of the endom e t r i a l cell m e m b r a n e s to a c e t a t e o r a n inc r e a s e d i n t e r m e d i a t e p o o l size o f a c e t a t e in t h e e n d o m e t r i u m d u r i n g t h e s e c r e t o r y p h a s e in humans may have been responsible for the observed decreased incorporation of 14C-acetate into the phospholipids. Further experimentation will b e r e q u i r e d to e x c l u d e t h e s e p o s s i b i l i t i e s . REFERENCES 1. Nicol, T., and R. S. Snell, J. Obstet. Gynec. Brit. Empire. 61, 216 (1954). 2. Nicol, T., and B. Vernon-Roberts, Ibid. 70, 851 (1963).

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3. Van Dyke, H. B., and G. Chen, Am. J. Anat. 66, 411 (1940). 4. Gillman, J., S. Afr. J. Med. Sci. 6, 59 (1941). 5. Alden, R. H., Anat. Res. 97, 1 (1947). 6. Lobel, B. L., and H. W. Dearie, Endocrinology 70, 567 (1962). 7. Dawson, A. B., and B. A. Kosters, Am. J. Anat. 75, I (1944). 8. Okey, R., W. R. Bloor and G. W. Corner, J. Biol. Chem. 86. 307 (1930). 9. Davis, J. S., and R. H. Alden, Anat. Res. 134, 725 (1959). 10. Goswami, A., A. B. Kar and S. R. Chowdbury, J. Reprod. Fertility 6, 287 (1963). 11. Ray, S. C., and R. J. Morin, Proc. Soc. Exptl. Biol. Med. 120, 849 (1965). 12. Gorski, J.. and J. A. Nicolette. Arch. Biochem. Biophys. 103, 418 (1963). 13. Aizawa, Y., and G. C. Mueller, J. Biol. Chem. 236, 381 (1961). 14. Layton, L. L., Cancer 3, 725 (1950). 15. Skipski, V. P., R. F. Peterson and M. Barclay, Biochem. J. 90, 378 (1964). 16. Zilversmit, D. B., and A. K. Davis, J. Lab. Clin. Med. 35, 155 (1950). 17. Morin, R. J., S. Bernick, J. F. Mead and R. B. Alfin-Slater, J. Lipid Res. 3 432 (1962). 18. Youngs, J. N.. and W. E. Cornatzer, Comp. Biochem. Physiol. 9, 257 (1963). 19. Morin, R. J., Biochem. Biophys. Acta 144, 594 (1967). 20. Morin, R. ft., Proc. Soc. Exptl. Biol. Med. 126, 229 (1967). 21. Morin, R. J., J. Atheroscler. Res., In Press, 1968. 22. Ranney, R. E., and S. E. Weiss, Endocrinology 62, 828 0958). 23. Lyman. R. L., J. Tinoco P. Bouchard, G. Sheehan and R. Ostwald, Biochim. Biol~hys. Acta 137, 107 (1967). 24. Elftman, H., Endocrinology 62. 410 (1958). 25. Merrill, J. A. and N. T. Werthessen, Am. J. Obstet. Gynecol. 96, 619 (1966). [Received Jan. 15, 1968]

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