the comparative behavior of mammalian eggs in vivo and in vitro. iii. factors controlling the growth...

THE C'ORIPARATIVE BEHAVIOR O F RlARIRIALIAK EGGS IN VIVO AND I N VITRO

111. FACTORS CONTROLLING THE G R O W T H O F THE RABBIT

BLASTOCYST

GREGORY PINCUS AND PIT. T. WERTHESSEN Biological Laboraiorirs, Haroard University, Cantbridge

SEVEN FIGURES

It has been shown that the growth of the rabbit blastocyst is peculiarly dependent upon factors external to the egg itself (Burdick and Pincus, '35 ; Pincus and Kirsch, '36 ; Pincus, '36). Neither the removal of the ovary, nor the injection of oestrogens will, for example, noticeably affect the cleavage process in vivo, but both procedures profoundly alter the course of blastocyst growth. Similarly, rabbit ova grown under the ordinary conditions of tissue culture will cleave regularly and at the normal rate, but collapse during the early growth stage of the blastocpst inevitably occurs (Lewis and Gregory, '29; Pincus, '36). I n this paper we present an account of certain investigations designed to elucidate the nature of the physiological control of the markedly sensitive blastocpst stage.

Our experiments have taken two courses: first, we hare endeavored to discover the simplest variation of ordinary tissue culture procedure that would lead to normal blastocyst expansion ; second, we have attempted to influence the course of ovum growth in utero chiefly by the injection of biologically active substances which might, on theoretical grounds, affect the growth process. M7e have devised a technique of explanta- tion which permits blastocyst growth in vitro to occur a t

This investigation was aided by a grant from the Josiali hfiep, Jr. Foundation.

1

T H E J O V R N A L OF EXPRRIMENTAL z o 6 m n , \ O L i 8 . NO. 1 FEBRUARY, 1938

2 GREGORY PINCUS AND N. T. WERTHESSEN

almost the rate observed in vivo. Ova placed in culture just hefore the onset of the growth stage over a period of 7 to 10 days show a regular development and espansion without the herniation and collapse observed by Lewis and Gregory ('29) and Pincus ( '36). I n a previous paper ( Pincus and Werthes- sen, '37) we have shown that the extent of blastocyst growth in ovariectomized rabbits is directly proportional to the dosage of injected progesterone. Our studies in vivo have largely centered about this finding, and involve a n aiialysis of the effect of progesterone upon uterus and ovum development. We have been able to dissociate ovum growth from uterine growth and to define to some extent the peculiar uterine metabolism associated with blastocys t development.

METHODS

The fertilized ova used for culturing were ordinarily taken from the fallopian tubes at 68 to 77 hours after copulation. They are then in late morula to early blastula stages and have scarcely formed the blastocyst cavity. Three types of cul- tures have been employed: l) watch glasses placed in a moist chamber (Pincus, '30) and containing a culture medium con- sisting of 20 to 25 drops of rabbit blood plasma or serum; 2) Carrel flasks containing 6 to 8 cc. of blood serum and stop- pered with rubber bulbs; 3) modified Carrel flasks through which blood serum is circulated constantly. The third type of culture is diagrammed in figure 1. It consists of a Carrel flask (A) with two openings (B and C), one (B) is an inlet for the circulating fluid, the other (C) is an outlet into which a sintered glass filter (D) has been fused. The filter is in- serted to prevent the egress of the ova. The serum passes into a capillary tube (E). Into this tube (E) a gas mixture consisting of N,, 7770, O,, 19% and C02, 3% filters through cotton (a t F) at a constant slow rate from a reserve tank. This gas mixture aerates the serum and at the same time lifts the fluid into the serum reservoir ( G ) . The building up of excessive gas pressure is avoided by an outlet (H) in the reservoir set well above the serum level. The fluid is inserted

COMPARATIVE BEHAVIOR O F MAMMALIAN EGGS 3

into the reservoir G through the cotton-stoppered tube I, which is then closed with a pinch cock. It then falls by gravity through the serum outlet into the Carrel flask. A pinch cock at (J) prevents the serum from leaving the circulatory tube E. Ordinarily we use about 30 cc. of rabbit serum in the

Fig. 1 Dingrani of the apparatus used for continuous circulation of a fluid culture medium. A, the culture flask; B, culture flask inlet; C, culture flask outlet; D, sintered glass filter; E, inlet capillary for gas mixture and outlet capillary for fluid; F, cotton plugged gas mixture inlet; G, fluid reservoir; H, cotton plugged gas mixture outlet; I, inlet to fluid reservoir. See text.

circulating cultures. A set of cultures can be placed in series using the same gas supply by connecting the gas outlet (H) to the gas inlet (F) of each succeeding circulation apparatus.

Blood serum is obtained in large quantity by bleeding from the carotid artery after ether anesthesia.

4 GREGORY PINCUS AND N. T. WERTHESSEN

The progesterone control of ovum growth in vivo has been investigated using chiefly the technique of ovariectomy and hormone injection previously reported (Pincus and Werthes- sen, ’37), e.g., ovariectomy at 18 to 20 hours after copulation, hormone administration twice daily for 3 days beginning the clay after ovariectomy and examination of eggs and uterus on the fourth day after ovariectomy (fifth day after copulation).

TABLE 1 The droeIopment in watch glass cultures o f rabbit ova explanted f rom t he oviducts

at 68 t o 77 hours a f t e r fertile copulation

SERIES NO.

l a

l b

l c

I d

Oa

Ob

2c Od

9e Of

“g

211

MEDIUM

Plasma plus lecithin

Plasma plus progesterone in lecithin

Plasma plus lecithin

Plnsnia plus progesterone in lecithin

Plasma Plasma plus

vitnniin C Plasma Plasma plus

Plasma Plasma plus

vitainin C Plasma plus

vitamin C Plasma

splenic extract

UXRER O F

EQGS

DAYS !ULTURED

2

0

3

3

1.5 1.5

9 I

2

3

3

3

5

MEAN OVUM DIAMETER AT CONCLUSION OF CULTURE PERIOD (/L)

363

392

3 74

277

...

. . .

236 173

300 358

. . .

. . .

R E X i K K S

Herniation in both ova

Herniation in both ova

Herniation in six ova, collapse by the fourth day of culturing

Herniation with collapse on the fourth day of culturing

Slight expansion Slight expansion

Herniated Herniated

Herniation in all ova Herniation in all ova

Slight expansion ; her- niation in ten ova

Slight expansion till third day of eultur- ing followcd by col- lapse of ova

* The ova are 170 to 220 p in diameter a t the time of explantation. Progesterone, 0.5 mg.

COMPARATIVE BEHAVIOR OF MAMMALIAN EGGS 3

RESULTS

Experiineizts in vitro Ova grown in watch glasses in late morula and early blasto-

cyst stages show only a moderate increase in size over a period of several days (table 1). Furthermore normal blastocyst expailsion does not occur; the eggs become herniated (fig. 2) and finally collapse. 1

Allen and Corner (’29) showed that progestin injection into rabbits ovariectomized shortly after fertilization of their 0173

Fig. 2 Photomicrograph of a typical herniated blastocyst from a watch glass culture; 3 days’ growth.

carried the ova through successfully to implantation. Through the courtesy of Dr. George W. Corner we obtained a supply of crystalline progesterone which we added to our culture medium in order to determine whether the hormone would stimulate blastocyst growth directly. I n order to maintain the hormone in solution i t was taken up in lecithin and added to the plasma cultures in this form. Control cultures con- tained lecithin with no dissolved progesterone. The data of these experiments presented in table 1 (series la to Id), dem- onstrate no significant growth stimulation due to the additiou

6 GREGORY PINCUS A N D N. T. WERTHESSEN

of progesterone, since the ova of control cultures grow to about the same size as those containing progesterone. Fur - thermore, typical herniation and eventual collapse occur in all the ova of this series. Lack of growth stimulation can scarcely be attributed to insufficient hormone since the amount added to the cultures is sufficient to cause marked ovum growth in viro (Pincus and Werthessen, '37).

Pincus and Bcrkman ( '37) found an increase in the ascorbic acid content of the rabbit uterus occurring during the period of blastocyst growth. On the assumption that the ascorbic acid is the secondary product of progesterone action upon the uterus this substance was added to certain watch glass cul- tures to see if growth stimulation occurred. Since ascorbic acid is rapidly oxidized in ordinary blood serum (Barron, Barron and Klemperer, '37) cystein was also dissolved in the blood plasma to inhibit the oxidation. N o very obvious dif- ference between the control cultures and those containing vitamin C was noted (table 1, series 2) . There appeared to be a slight advantage over a 3-day period in the ova of the vitamin C cultures, but this was not marked. It is still possi- ble that the ascorbic acid was not sufficiently protected by the cystein to have enough time to exert its presumable stimu- lating effect. Herniation certainly was not avoided to any extent (table 1).

We soon found that prolonged blastocyst growth might occur in certain types of culture containing only blood serum and with no special addition of hormone or vitamin. Good growth with typical expansion and little or no herniation was observed in Carrel flasks containing 6 to 8 cc. of serum or in our fluid circulating cultures. The data on ovum growth of typical series in both types of culture a re given in table 2," and presented graphically in figures 3 and 4.

We are indebted to Dr. A. Szent-Gyorgyi for the ascorbic acid used in these

The data experiments.

are adequately illustrated in the figures. 'Tables 2, 3 and 4 of this paper are on file at The Wistsr Institute.

COMPARATIVE BEHAVIOR O F MAMMALIAN EGGS 7

I n figure 3 the curve to the left is the mean of all the data of Gregory and Castle (’31) on the normal growth of rabbit blastocysts during the 4 days preceding implantation ; the curve on the right represents the mean diameters of eight ova of rabbit no. P57 as grown in our circulating culture. It will be noted that the cultured ova eventually attained’ the diam- eters of normal pre-implantation blastocysts, but that this required S+ days of culturing compared with 4 days of growt!i in utero. Furthermore these ova during the last 2 to 3 days

Fig. 3 Comparative growth of rabbit blastocysts in vivo and in vitro. The curve at the left shows normal growth in vivo (data of Gregory and Castle, ’31), that at the right the growth of the ova of rabbit no. Pi57 (table 2) in the circu- lating apparatus. Abscissa, time in hours after ovulation ; ordinate, mean ovum diameter in micra.

of culture developed peculiar ‘blebs’ of trophoblast tissue on the surface and exhibited also inwardly migrating cords of trophoblast cells. The germinal disk appeared to develop more rapidly than the expansion, as if the process of embry- onic differentiation occurred at a rate independent of ovum growth. Thus the primitive streak appeared at the fifth day of culturing when the blastocysts had not yet attained full size. We have not, however, studied the development of the emliryonic area in detail. Our attention has been chiefly con-

8 GREGORY PINCUS AND N. T. WERTHESSEN

fined to an examination of the conditions governing ovum growth.

We find, for example, that the circulating medium leads to better ovum growth than simple Carrel flask explantatioii (fig. 4, lower curves, and animal no. P53, table 2). The source of the seruni seems to make little difference provided it is fairly fresh. Thus we have cmployed the serum of females in

nourn F ig .4 Tile growth of rabbit blastocysts in various types of cultures. Lower

r,urves: 0 , ova of rabbit no. P57 grown in the circulating apparatus; 0, ova of rabbit no. P57 grown in a Carrel flask, serum from oestrous female. Upper curves: 0, ova of rabbit no. P74 in a Carrel flask, serum from pseudopregnant doe; 0 , ova of rabbit no. P74 grown in the circulating apparatus, serum from pseudopregnant doe refrigerated 11 days (table 2 ) . Abscissa, time in hours after ovulation ; ordinate, mean oruin diameter in micra.

heat (animal no. P57) and 1 to 3 days pseudopregnant (ani- mals nos. P74, P53 and P50) as ~ c l l as male serum (animal no. P50), with good growth in all cases. In one instance (animal no. P74 and fig. 4, upper curve) the use of serum kept on ice for 11 days resulted in an eventual inhibition of ovum growth. Although the serum was circulated, the control cul- ture with fresh serum in a Carrel flask showed better growth in this instance.

COMPARATIVE BEHAVIOR O F MAMMALIAN EGGS 9

These data tell us merely that the sort of growth obtained in the uterus of a normally pregnant female may be approxi- mated by certain types of serum cultures. The serum medium is roughly equivalent to the progesterone controlled medium of the uterus. We have taken fertilized eggs from a uterus free of progesterone control and find that they will also grow markedly in serum culture. The eggs were obtained on the fifth day after mating from the uteri of females ovariecto- mized a t 18 to 20 hours after copulation. These ova remain

Fig. 5 Pliotomierograph of a rabbit blastocgst removed from the uterus of :in ovariectomized female 4 days after ovariectomy, e.g., 5 days after copulation. Note absence of typical expansion cavit,y and presence of large crystalline deposits.

in the uterus for 2 days in a slightly expanded condition. They have a mean diameter of about 280 p (table 3) compared with a diameter of over 1OOOp in normally developing ova (fig. 1). Furthermore they a re often partially covered with a peculiar crystalline deposit which is evidently laid down in the tubes shortly after ovariectomy, since the crystals are usually located nest the egg in the first layers of the albumen coating (fig. 5 ) . The albumen is gradually accumulated in the course of the downward passage of the ova ( Pincus, '36).

10 GREGORY PINCUS A N D N. T. WERTHESSEN

Despite their inhibited condition these ova increase in diam- eter some four to five times in the course of 6 days. This is illustrated in the data of table 3 and figure 6. Thus the five ova of rabbit no. B20 grew at about the sanie rate as the seven eggs of no. B21 despite the fact that the ova of the latter doe had received a mild growth stimulation as the result of the injection of a total of 0.21 mg. of progesterone during the 3 days preceding the removal of the ova from the uterus. I t

I

50 I00 I 50 HOURS

Fig. 6 The growth in culture of 5-day blastocysts taken from the uteri of ovariectomized does. 0 , ova of rabbit no. B20, serum from no. B20; 0 , ova of rabbit no. BP1, serum from no. BSO; 0, ova of rabbit no. B19, serum from a pseudopregnant female (table 3 ) . Abscissa, time of culturing in hours; ordinate, mean ovum diameter in micra.

is notable that the ova of rabbit no. B20 were grown in the serum of rabbit no. B20. In other words, this rabbit doe’s serum effectively stimulated the growth of her own ova whereas her uterine medium was unable to do so. In fact these ova, placed in the serum of their ovariectomized mother, showed a somewhat better growth than those of no. B19 (also ovariectomized) in the serum of a pseudopregnant female (fig. 6). The ova of all these cultures showed marked sigm of degeneration and collapse on the seventh day of culturing.

COMPARATIVE BEHAVIOR O F MAMMALIAN EGGS 11

Experiments in vivo The foregoing experiments imply that there is something

lacking in the uterus of an ovariectomized female which is supplied to the eggs in our serum cultures. We know that this deficiency in the ovariectomized animal can be overcome by the injection of progesterone (Allen and Corner, '29; Pincus and Werthessen, '37). Two effects of progesterone administration have been measured; these are 1) the increase in ovum size and 2) the increase in the glandular portion of the mucosa. We can show that these two phenomena are positively correlated. The data presented in table 4 are taken

Fig. 7 The relation between ovum size and pseudopregnant proliferation in ovarieetomized females receiving various dosages of progesterone (table 4 ) . Abscissa, the ratio of glandular area ( G ) to total mueosa area ( h l ) ; ordinate, mean ovum diameter in micra.

in part from Pincus and Werthessen ('37) and include a num- ber of additional observations not previously published. The ratio (G/M) of the area of the glandular portion ( G ) of the mucosa to the total mucosa area ( M ) may be taken as a direct measure of the growth of the glandular portion in uteri of different sizes on the assumption that the effect of progester- one stimulation is primarily a development of glandular tissue at the expense of the stroma, in any event the ratio G,/M is directly proportional to progesterone dosage in test ovari- ectomized females (Pincus and Werthessen, '37). I n figure 7 we plot the data of table 4. The correlation between mucosa and ovum growth is obvious.

13 GREGORY PINCUS AND N. T. U’ERTHESSEN

We may then consider the progesterone effect as follows : 1. Frogesteione + mucosa + glandular growth + O V U U ~ growth

or ovum growth

/ \ -0. Progesterone + mucow

I glandular gronth or

3. Progesterone + niucosa + glandular growth + ovuni growth

Tlie first alternative requires an effect of the hormone on the uterine metabolism of such a nature that endometrial pro- liferation ensues, and that this proliferation stimulates ovum growth ; the second alternative implies that a progesterone- induced metabolism results by two inclepenclent routes, 011 the one hand, in ovum growth and on the other in mucosa growth ; the third alternative implies that a common progesterone- induced metabolism results in both ovum and mucosa growth, and that these a re governed by exactly the same chain of events. W e put the ‘mucosa’ in each scheme because our experiments in r i t ro indicate 110 direct stimulation of the ova by the hormone and secondly because, under ordinary condi- tions, a prepared uterine endometrium is necessary for pro- gesterone action, c.g., priming by oestrin offers a mucosa most readily responsive to progesterone stimulation.

\TTe can demonstrate that the third alternative is incorrect and that the second is more probable than the first. The pertinent data are presented in table 5. They represent es- periments in which progesterone has been injected by our standard technique in combination with oestrone, ascorbic acid, and glutathione. From the quantity of progesterone injected, we expect to find ova of a certain diameter (column 6) in the uterus and a glandular proliferation of a given de- gree (column lo) , and when these two measurements are combined an expected value of the derived index, 2, can be calculatecl (column 14). These expected values and the value of their standard errors are derived from our stanclard curve (Pincus and Wertliessen, ’37). Now if we can significantly affect either ovum diameter or G/M without altering the other,

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14 QREGORY PINCUS AND N. T. WERTHESSEN

then the third alternative is untenable since it assumes that both of these are invariably completely correlated, within the limits of error of the measurements. Using p = 0.023 as the significant level any difference between observed and calcu- lated values of 2 or greater indicates a significant alteration.

Actually we find a clear-cut demonstration of the dissocia- tion between ovum growth and glandular proliferation in the ascorbic acid series. Here the injection of ascorbic acid simul- taneously with progesterone results in an inhibition of egg growth with no inhibition of glandular development (there is in fact a small but statistically insignificant increase of G/M with the result that the index Z does not vary significantly from expectation, since the increased G/M value compensates for the decreased egg diameter value). The 7 value of 2.35 for the combined data on ovum size for animals PC-1 and PC-5 indicates a probability of about 1 in 100 that such an inhibition would occur by chance alone. It is somewhat sur- prising that ascorbic acid which increases in concentration in the uterus during the period of blastocyst growth acts as an inhibitor of blastocyst growth. Actually the effect of ascorbic acid is an indirect one as we shall see, but this is immediately suggested by the data on animals VC-1 and VC-2 in which the injection of ascorbic acid without the simultaneous injection of progesterone results in no significant alteration of either egg size or glandular growth.

The dissociation between egg growth and glandular pro- liferation is also made obvious in the data on the effects of the simultaneous injection of oestrone and progesterone. Oestrone inhibits both egg growth (compare Burdick and Pincus, '35) and pseudopregnant proliferation (Hisaw, '32), but the data on animals nos. PO-2 and PO-5 show that ovum growth is first affected significantly and that higher doses arc' required to affect endometrial proliferation (PO-22). A dosage of 1 O y of oestrone in combination with 0.42 mg. pro-

difference

'We have excluded the egg size measurements of animal PC-3 because these eggs were found in the tubes instead of the uterus and their mere presence in the tuba1 environment might be responsible for the growth inhibition.

COMPARATIVE BEUAVIOR OF MAMMALlAN EGOS 15

gesterone seems actually to effect a significant increase in glandular proliferation without affecting ovum growth (PO-4). This is presumably the well-known synergistio effect upon the endometrium of low oestrin doses with progesterone.

The data on the oestrone injected animals cast some doubt on our first alternative, e.g.,

progesterone + mucoaa + glandular growth + ovum growth.

We have shown (Pincus and Werthessen, '37) that the'ratio G/M is a much more sensitive indicator of progesterone activ- ity than is the egg diameter. But in the oestrone series egg diameter is first affected, not the G/M ratio. I n other words, it would seem as though the oestrone affects a progesterone induced reaction which supplies stimulation to both ova and endometrium, and that the stimulating products of this re- action show a higher partition coefficient for the endometrial processes. None the less, until we can obtain ovum growth in a uterus lacking a glandular endometrium we cannot be certain of this situation which is in effect our second alterna- tive.

We have attempted to induce ovum growth in an unstimu- lateduterus by the injection of glutathione alone (animal no. GL-l), but no significant increase in egg diameter occurred. None the less when glutathione is injected in combination with progesterone, the ovum diameter exceeds expectation (nos. GLP-1, GLP-2, GLP-3) and when all the data on these ani- mals is combined a markedly significant increase is had. The glandular area of the endometrium is also significantly in- creased.

If glutathione increases ovum size in a progesterone in- jected animal and ascorbic acid leads to a decrease, then the simultaneous injection of both compounds should restore normal ovum size. The data on animal PCG-1 (table 5) dem- onstrate this to be the case. The mean ovum diameter is much greater than in the animals receiving vitamin C plus progesterone.

16 GREGORY PINCUS AND N. T. WERTHESSEN

DISCUSSION

We may, upon the basis of these experiments, present the following hypothesis : Glutathione stimulates both blastocyst expansion and glandular proliferation, and it is made availa- ble for these processes in vivo by the action of progesterone. Since glutathione is presumably the primary organic agent for maintaining ascorbic acid in the reduced state (Hopkins and Morgan, '36 ; Rarron, Barron and Klemperer, '37 ; Bor- sook, Davenport, Jeffreys and Warner, '37), we can esplain the ascorbic acid inhibition of ovum growth in vivo by assum- ing that the glutathione that would normally be used for ovum stimulation is largely used in maintaining the ascorbic acid in the reduced ~ t a t e . ~ It is, as it were, shunted away from the eggs; it is apparently less easily withdrawn from the reactions concerned with glandular proliferation, since ascorbic acid injection in the doses we have employed scarcely affects glandular proliferation. Oestrone may act like ascorbic acid by shunting glutathione into those reactions which are oestrone-initiated or by inhibiting directly the progesterone- induced reactions.

We cannot explain the improved growth of ova in vitro on the basis of the observations reported in this paper. Since ascorbic acid is readily oxidized in blood serum (Barron, Barron and Klemperer, '37) and normally not very concen- trated therein, we might deduce that its does not cause the loss of appreciable amounts of glutathione and that the serum glutathione presumably present in excess is the primary stimulant of blastocyst expansion. But we do not know the actual concentrations of glutathione in our serum cultures, and Hopkins and Morgan ('36) have shown that under cer- tain conditions at least glutathione is rapidly oxidized in vitro. We place these facts on record, therefore, in anticipa- tion of further exploitation of the suggestions they promote.

SInjected ascorbic acid is accumulated to some extent in the uterus (Pineus and Berkman, unpublished data).

COMPARATIVE BEHAVIOR OF MAMMALIAN EGGS 17

SUMMARY

Rabbit ova taken from the fallopian tubes in late morula and early blastocyst stages exhibit a limited :mount of growth in watch glass cultures eventuating in trophoblast herniation and collapse. Growth is not improved nor herniation pre- vented by the addition of either progesterone or ascorbic acid to the medium in such cultures, although the injection of pro- gesterone into ovariectomized females leads to blastocyst ex- pansion in vivo, and ascorbic acid normally increases in concentration in utero during the period of blastocyst growth. Regular blastocyst expansion without herniation is obtainable in vitro in Carrel flasks containing 6 to 8 cc. of rabbit blood serum and growth to normal preimplantation size occurs (but at less than the normal rate) in culture flasks through which a properly aerated serum medium is continually circu- lated. Ova from ovariectomized females which have remained in an unexpanded state in the uterus for 2 days will exhibit typical expansion when explanted to flask cultures and this growth will occur in male serum or in the serum of normal or ovariectomized females. This is taken to demonstrate that the growth inhibition resulting from ovariectomy is not due to a loss of inherent growth potency in the ova, but to the removal of some necessary uterine condition. Experiments designed to study this necessary uterine condition involved the ovariectomy of rabbit does carrying fertilized tuba1 ova and the comparison of ovum growth and glandular prolifera- tion in the endometrium when certain biologically active substances are injected. When progesterone is injected in varying doses into such females ovum growth and glandular proliferation are closely correlated. Oestrone injected in com- bination with progesterone inhibits ovum growth in low dosages and glandular proliferation in higher dosages. Ascor- bic acid injected alone (in the dosages employed) has no de- tectable effect upon either ovum growth or glandular pro- liferation, but in combination with progesterone it inhibits the former and does not affect the latter. Glutathione alone is also without effect, but in combination with progesterone both

18 GREQORY PINCUS AND N. T. WERTHESSEN

ovum size and glandular growth are increased. When ascor- bic acid, glutathione and progesterone are injected together the ascorbic acia effect is overcome. It is concluded that glutathione can be made available for both ovum stimulation and pseudopregnant proliferation as the result of progester- one action and that ascorbic acid inhibits egg growth in vivo by shunting glutathione from the reactions leading to egg stimulation into reactions involving the reduction of ascorbic acid ; oestrone presumably inhibits glutathione action by a similar shunting or by inhibition of the progesterone effect directly.

LITERATURE CITED

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