In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles

DEVELOPMENTAL

BIOLOGY

In Vitro

17, 627-643 ( 1968 )

Induction

of Physiological

Rana pipiens Their

Oocytes Ovarian

Maturation

Removed

in

from

Follicles

L. DENNIS SMITH, R. E. ECKER, AND S. SUBTELNY Dioision of Biological and Medical Research, Argome Argonne, Illinois 60439; and Department of Zoology, lowa City, Iowa 52240 Accepted

December

National Laboratory, Unioersity of Iowa,

~29, 1967

INTRODUCTION

In amphibians, full-grown oocytes at the end of oogenesis remain in prophase of the first meiotic division. The induction of ovulation stimulates the breakdown of the large prophase nucleus (germinal vesicle) and the release of eggs from the ovary. The ovulated eggs continue meiosis to the second meiotic metaphase, which is the first time they can be fertilized and can be considered physiologically mature (Subtelny and Bradt, 1961; Smith et al., 1966). Physiological maturation and ovulation are thus coordinated events that are ordinarily stimulated by the same procedure. The experimental induction of ovulation in anuran amphibians is routinely accomplished by homoplastic pituitary injections or implantations. Moreover, a number of studies have shown that mammalian steroids are also potent inducers of ovulation in vitro (Bergers and Li, 1960; Wright, 1961; Edgren and Carter, 1963) and as an adjunct to pituitary induction in vivo (Witschi and Chang. 1959; Wright and Flathers, 1961). In most studies of this type, however, the emphasis has been on the number of eggs ovulated, little attention being paid to the stage of maturation of either the unovulated oocytes or the ovulated eggs themselves. Thus, current views on the induction of ovulation have been concerned with the effects of the various hormones on rupture of the ovarian follicles, not with the effects of hormones on the eggs themselves. 627

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ECKER,

AND

SUBTELNY

Recently, DettIaff et al. (1964) have reported studies on pituitarystimulated maturation of toad oocytes in vitro. In these experiments, they established that the presence of pituitary gonadotropins was required only until just before dissolution of the germinal vesicle. After this time, maturation continued in the absence of hormones (DettIaff et al., 1964). This hormone-dependent period occurs prior to the time of ovulation (see Smith et al., 1966). Schuetz (1967a) has also reported that germinal vesicle breakdown in isolated follicles of Rana pipiens can be induced in htro. In these experiments, ovulation apparently was not a prerequisite for the induction of germinal vesicle breakdown. Recently, we have observed that physiological maturation can be obtained in ovarian fragments, with both pituitary and steroid hormones, in the complete absence of ovulation (Subtelny et al., 1968). Taken together, these experiments suggest that the effect of hormones in the induction of maturation may be completely separated from their effects on ovarian follicles and, in fact, may not even require the presence of ovarian tissue. In the present investigation, we have examined the possibility that physiological maturation can be induced in full-grown oocytes dissected from their ovarian follicles prior to exposure to any hormone. In these experiments we have tested the effect on individual oocytes of both homoplastic pituitary preparations and the steroid hormones progesterone and deoxycorticosterone. All these hormones have been shown to induce maturation in oocytes contained within ovarian tissue ( Dettlaff et al., 1964; Wright, 1961; Schuetz, 1967a,b). MATERIALS

AND METHODS

Sexually mature Runu pipiens from Vermont were used in these studies. The experiments were begun in December with frogs that had been captured when they were going into hibernation; they were maintaind in the laboratory at 4°C. The bulk of the experiments were carried out on these “fall” frogs during January and February, prior to the normal breeding season. Some additional experiments were performed with frogs captured in the spring as they were coming out of hibernation. These animals were placed at 4°C immediately after they were received from the dealer and were used for experiments from late April through August. Adult females were pithed. Portions of the ovarian lobes from either

1rl

\7i.tro

MATURATION

IN

FROG

OOCYTES

629

side of the animal were removed, rinsed in amphibian Ringer’s solution (see Hamburger, 1960), and stored in Ringer’s solution for subsequent use ( Fig, 1A). In anurans, the oocyte undergoes oogenesis within the ovarian follicle, which is composed of follicle cells, a thecal layer. and a surface epithelium (see Wischnitzer, 1966). Individual oocytes were dissected from their ovarian follicles with watchmaker’s forceps (Fig. 1B). To facilitate subsequent discussion, we have designated these free oocytes as afolliculate. This does not imply, however, that they are totally free of follicle cells, as will become apparent later. The afolliculate oocytes were exposed to the hormone preparations for varying periods of time, washed, and placed into Ringer’s solution for examination at various times after hormone exposure. Control oocytes were maintained in Ringer’s solution without hormone exposure. All manipulations were carried out at room temperature, which varied from 18 to 20°C. For electron microscopy, afolliculate oocytes or pieces of ovary were fixed in 2% glutaraldehyde (Sabatini et al., 1963) in 0.05 hl phosphate buffer, pH 7.4, at 23°C. After 1 hour, individual ovarian follicles or afolliculate oocytes were cut in half, and the animal hemispheres were placed in fresh fixative for a total time of 4-6 hours. They were then washed in the phosphate buffer, postfixed in 1% phosphate-buffered osmium tetroxide at 0°C for 1.5 hours, dehydrated in ethanol and propylene oxide and embedded in a mixture of Araldite 6005 and Epon 812 (Mollenhauer, 1964). Thin sections cut with a Huxley microtome were stained in sequence with 2% many1 acetate and 0.25% lead citrate (Venable and Coggeshall, 1965) and examined in a Siemens Elmiskop 1 electron microscope. Pituitary suspensions were prepared by macerating four male pitllitarics in 40 ml of Ringer’s solution. This preparation, containing the equivalent of l/10 of a pituitary per milliliter was then diluted with Ringer’s solution to obtain the lower concentrations reported in the experiments. The steroids hormones, progesterone and deoxycorticosteronc acetate (DOCA), are insoluble in aqueous media. Progesterone was obtained as an aqueous suspension of 25 mg/ml, and the DOC4 was obtained at a concentration of 5 mg/ml dissolved in cottonseed oil (Upjohn Company, Kalamazoo, Michigan). For the experiments, progesterone was prepared at a stock concentration of 2 pg/ml 1~ dilution with Ringer’s solution (the hormone was soluble at this concentration ) and further dilutions were made from this. DOC,4

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SMITH,

ECKER,

AND

SUBTELNY

FIG. 1. (A) Ovarian fragment from a mature female showing full-grown (B ) Individual oocytes which have been oocytes within their ovarian follicles. ( C ) Afolliculate oocytes which have removed from their ovarian follicles. matured, in uitro, to the first meiotic metaphase. Note the strings of cells on the egg surface. (D ) Individual body-cavity eggs at approximately the same stage of maturation as the oocytes in (C). Note the smooth egg surface and the lack of the strings of cells. Magnification: A and B, X approximately 7.5; C and D, X approximately 13.

In

Vitro

MATURATION

IN

FROG

OOCYTES

631.

was prepared by adding 0.5 mg (0.1 ml of the oil preparation) to 250 ml of Ringer’s solution and emulsifying with a Waring blender. Lesser concentrations were then prepared by diluting the emulsion with Ringer’s Several criteria were used to determine whether or not oocytes undergo maturation changes after exposure to hormone: First, following breakdown of the germinal vesicle, the stages of meiosis were observed by the presence of the first and second maturation metaphase spindles. Each is marked externally by the appearance of a black dot on the surface of the animal hemisphere (Subtelny and Bradt, 1961). Second, physiological maturity of oocytes that showed a second dot was demonstrated by their ability to respond to the activation stimulus produced by pricking the eggs with a clean glass needle. The external signs of activation include elevation of the vitelline membrane. rotation of the egg within the membrane, changes in the appearance and elasticity of the egg surface, and elimination of the second polar body. Finally, when oocytes did not exhibit any of the aforementioned signs of activation, they were simply dissected to determine the presence or absence of the germinal vesicle. Additional evidence for the complete physiological maturation of the hormone-treated eggs was provided by artificial insemination. Oocytes which have not passed through the oviducts do not possess any jelly envelopes, and it has not been possible to fertilize jelly-free eggs (see review by Shaver and Barth, 1960). Therefore jellyless oocytes which previously had been induced to mature in vitro were transplanted into ovulating “foster” females (see Arnold and Shaver, 1962; Lavin, 1964). Approximately 24 hours after the recipient female had been induced to ovulate (at lS”C), it was anesthetized and the body cavity opened. All body-cavity eggs were removed along with the ovary from one side of the animal. This reduced considerably the number of host eggs which would enter the oviduct and be stored in the uterus. Donor oocytes in the first meiotic division, approximately 24 hours after hormone treatment, were vitally stained for 1 minute in 0.1% neutral red in Ringer’s and placed into the open body cavity of the foster female. The female was then sutured, maintained at lS°C, and stripped the next day of as many eggs as possible. After fertihzation, the red donor eggs were separated from the unstained eggs of the foster female, and the stained eggs were observed for cleavage and later development.

632

SMITH,

ECKER,

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SUBTELNY

RESULTS

Full-grown ovarian oocytes removed from their follicles prior to exposure to either progesterone or DOCA were induced to mature in the continuous presence of the hormones (Table 1). Doses of either of the steroids of 0.1-2 pg/ml induced maturation in 100% of the cases, and concentrations much lower than this were effective in inducing maturation in a significant percentage of the cases. Pituitary hormones also induced maturation in oocytes removed from their follicles but, by contrast, not as efficiently as the steroids. An optimum dose of l/320 of a pituitary per milliliter induced maturation in only 45% of the exposed oocytes. With doses either greater or less than this, the TABLE 1 CONTINJOUS EXPOSIJRE OF AFOLLICULATE OOCYTES TO PROGESTERONE, DOCA, OR PITUITARY

Hormone

Progesterone

DOCA

Pituitary

Ringer’s

Dose

w/ml 2 1 0.2 0.1 0.02 0.01 0.002 2 1 0.2 0.1 0.02 0.01 0.002 per ml l/10 l/20 l/40 l/80 l/160 l/320 l/640 l/2560 None

Number of eggs

Number of eggs with intact germinal vesicle

Number of eggs with dot and activated

52 150 55 40 15 15 20 25 20 60 50 15 15 15

0 0 0 0 4 2 20 0 0 0 0 0 0 11

52 150 55 40 11 13 0 25 20 60 50 15 15 4

40 20 110 20 113 20 95 44 125

36 20 106 19 78 11 76 44 125

3 0 4 1 35 9 19 0 0

In

vitro

MATURATION

IK

FROG

OOCTTES

633

percentage of exposed oocytes that were induced to mature decreased markedly. It should be pointed out that the results summarized in Table 1 were obtained from experiments carried out in January. Similar experiments carried out later in the spring, during the breeding season, showed that the effect of the steroids was the same. Howevcar. at this time, the optimum dose of pituitary induced maturation in as much as 90% of the exposed oocytes. Apparently, the induction of maturation, in r;itro, with pituitarv i suspensions is subject to seasonal variations of the type reported for the induction of ovulation in C;:I-O ( see Wright and Flathers, 1961) . The temporal sequence of maturational events induced by in z;itrc) exposure to any of the hormones is essentially the same as that ob served after the stimulation of maturation in. ho (see Smith cf (11.. 1966). At 18”C, the germinal vesicle breaks down at about 16-20 hours after the initial hormone exposure. At about 24 hours, the first meiotic division occurs, and by about a day and a half after exposure the eggs have reached the second meiotic metaphase and can bc activated. Full-grown oocytes, after dissection from their ovarian follicles, have, a rough, pebbled appearance when viewed through the dissecting microscope (Fig. 1B). Control oocytes which are maintained in Ringer’s solution retain this appearance indefinitely. Oocytes that ha\~ been induced to mature, however, undergo a distinct change during the course of maturation. At about the time of the first meiotic division these oocytes have an appearance similar to that of body cavity eggs obtained from an ovulating female; both ha1.e a smooth, somewhat shinv egg surface, but the oocytes induced to mature in oitro contain striigs of cellular material attached to their surface (Fig. 1C.D). Figure 2 shows an electron micrograph of a full-grown oocvte withill its ovarian follicle. Three cell or tissue layers can be obseried en\~loping the oocyte; the surface epithelium, the thecal or connective tissura layer, and the follicle cell layer (see \Vischnitzer. 1966). In the fllllgrown oocyte, the region between the follicle cells and the surfaces 0: the oocytc is occupied bv the vitelline mr>mbrane. The follicle c~l]]s contain processes (macrovilli) which penetratcl into the substance of the vietellinc membrane, and the oocyte contains numc’rous micro\+lli which ASO extend into the region of the \,itelline membrane. ~hc follicle cells and the oocyte are thus bound into an intimate relationship throllgh the mediation of the vitelline membrane.

634

SMITH,

ECKER, AND SUBTELNY

FIG. 2. Full grown oocyte within its ovarian follicle. The follicle consists of the outer surface epithelium (SE ), the middle theta ( T), and the inner follicular epithelium, which, in this particular case, is represented almost entirely by the follicle cell nucleus (FCN). Macrovilli (MAV) from the follicIe cell and microvihi (MN) from the oocyte extend into the substance of the vitelline membrane (VM). Also shown are cortical granules ( CG ), yolk platelets (YP), pigment granules (PG), and lipid droplets ( L ).

In

vi.ttrO

MATURATION

IN

FHOG

OOCYTES

635

Figure 3 shows an electron micrograph of a similar full-grown ()ocytc’ which has been dissected manually from its ovarian envelopes. The surface epithelial and thecal layers have been removed. It is evident, however, that follicle cells are still associated with the oocyte. The follicle cell processes appear to still be at least partially connectd with the vitelline membrane. The membrane itself has been stretched out as a result of the dissection but has retained its integrity sufficientI>to be connected still to the oocyte through the microvilli. Apparently. the intimate relationship between the microvilli, thr vitelline membrane, and the macrovilli has prevented a complete removal of the follicle cell layer. At later times, the villi withdraw from the vitelline membrane. Thus, ovulated eggs contain only a vitelline membrane with no adherent follicle cells ( Wischnitzer, 1966). Possibly the strings of cells observed on oocytes induced to mature in vitro (Fig. 1C ) arc’ the result of the same mechanisms; villi withdraw from the vitellinc membrane, and the follicle cells roll up on the egg surface. Additional experiments were carried out to determine whether short exposures to hormones could induce maturation in afolliculate oocytes. In these experiments, no attempt was made to compare the different hormones; progesterone alone was tested. The data from these experiments are summarized in Table 2 and represent a summar:, of experiments performed in February and May. Exposure of individual afolliculate oocytes to progesterone, at a dose of 1 pg/ml, for as brief a time as 5 minutes induced physiological maturation in an average of 28% of the oocytes. Increasing the exposure time to progesterone increased the percentage of oocytes undergoing maturation. and an exposure time of 1 hour was sufficient to induce all the oocytes to undergo maturation. We have been able to induce maturation in afolliculate oocvtes, as a routine procedure. bv a l-hour exposure to progesterone at’virtually any time of the year. In the results shown in Table 2, the criteria of physiological maturation included a positive response to an activation stimulus, Additional evidence that a I-hour exposure to progesterone induces physiological maturation was obtained by artificial insemination of such oocytes. Approximately 24 hours after a l-hour exposure of afolliculate oocytes to progesterone, the stimulated eggs were transplanted into “foster” females which previously had been induced to ovulate, This enabled the afolliculate oocytes to acquire jelly layers and thus become capable of being fertilized. All the eggs had undergone germinal vesicle break-

636

SMITH,

ECKER,

AND

SUBTELNY

FIG. 3. Afolliculate oocyte of the type shown in Fig. 1C. The surface epithelium and theta layers have been removed. Follicle cells (FC) are still present. Two macrovilli ( MAV) from the follicle cell and numerous microvilli ( MIV) from the oocyte are still protruding into the vitelline membrane (VM ), which has been extended by the dissection. CG, cortical granule; FCN, follicle cell nucleus; PG, pigment granule.

In

Vi.ttrO MATURATIOhTABLE

637

IN FROG OOCYTES

2 Numhr,r of cans :wti\xt,.d 1; “h i.i !I4 20

.5 minutes

10 rnitrutes 1-5millrltes 30 millrItes 46 millrItes lx) milllltes 90 minutes 120 milrute:: Continlloris

II1 .il 1 (i.i IOI

(4X hours) so expos11re.

IO0 (5)

100 (looy~i

0

0

I:inyrr’s (‘oIIlroIs ‘1 I)ow trf progesterone, 1 pg/ml. .) /’ For rsample, 60 (3) means 60 eggs from n total 111’ ” sfq):ir:itt~ experimellts.

down prior to transplantation and exhibited a distinct black dot. Had such eggs been left in Ringer’s solution for an additional 16-24 hours, they would have reached the second meiotic metaphase and would have responded to an activation stimulus. The data in Table 3 show that such eggs can also be inseminated and will undergo cleavage and subsequent development. A total of 681 oocytes in first meiotic mctaphast were transplanted into foster females. Of 307 eggs reco\.ercd approximately a day later, 229 (75%) underwent cleavage after artificial insemination and 201 (88X of the cleaved eggs) developed into normal tadpoles. Host eggs from the foster females which \vere inseminated along with the donor oocytes showed greater than 95% cleava;e and subsequent development. Thus, the percentages of the oocytes which cleaved and developed after transfer into foster females is lower than eggs induced to mature in ciao. This difl‘erencc probabl\~ hXEI,OI’MENT

OF ,h;S

TRISSFERREI)

TO I“OSTER 12E\1,41.Eh

638

SMITH,

ECKEB,

AND

SUBTELNY

results from the manipulations to which the afolliculate oocytes were exposed prior to transfer. Dissection of ovarian oocytes from their follicles and prolonged exposure of such oocytes to Ringer’s solution does not cause breakdown of the germinal vesicle or any outward manifestations of maturation. Likewise, prolonged residence of such oocytes in Ringer’s solution (48 hours) does not prohibit a subsequent positive response to hormonal stimulation. In several cases, oocytes were dissected from their ovarian follicles and stored in Ringer’s solution for 2 days. These oocytes were then exposed to progesterone (60 minutes) and maintained an additional 2 days in Ringer’s solution, All the stimulated oocytes underwent physiological maturation. At least one other culture medium, however, appeared to have a deleterious effect on oocytes removed from their follicles. In two separate experiments, for example, more than 100 oocytes were exposed to progesterone and then incubated in Steinberg’s solution (see Hamburger, 1960). All the oocytes underwent germinal vesicle breakdown and all possessed black dots 2 days after the hormone stimulation. None of the eggs, however, gave any sign of being activated after being pricked with a clean glass needle. An additional experiment suggested that eggs exposed to Steinberg’s solution for as little as 24 hours after hormone stimulation cannot respond to insemination. A total of 140 oocytes were dissected from their follicles, exposed to progesterone for 1 hour, and then maintained in Steinberg’s solution for approximately a day. All the oocytes, in first meiotic metaphase, were then transplanted into foster females, stripped from the frog the next day, and inseminated with a fresh sperm suspension. Of 70 eggs recovered from the females, not one showed any sign of activation or underwent cleavage. Morrill (1965) h as shown that the hormones which induce ovulation in amphibians also bring about changes in ion permeability of the oocyte membrane. He has suggested the possibility that certain maturational events result from changes in the intracellular ionic environmentchanges which result from the action of hormones on the egg membrane. Possibly Steinberg’s solution is an unfavorable medium for the maintenance of proper intracellular environments during maturation. DISCUSSION

The induction of maturation in vitro can be brought about by both pituitary hormones and steroid hormones. Long ago it was reported

In.

Vitro

MATURATION

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6398

that eggs, dissected from the ovaries of Rana pipiens, were induced to undergo maturation by exposure to pituitary hormones in vitro (Heilbrunn et al., 1939). Recently, in vitro studies on toad oocytes have shown that pituitary-induced maturation requires the continuous presence of hormones only until just before dissolution of the germinal vesicle; after this, maturation continues in the absence of hormones (Dettlaff et al., 1964). Removal of follicular tissue during the hormonedependent period did not prevent breakdown of the germinal vesicle as long as the oocytes were maintained in the presence of pituitaq gonadotropins. Additional experiments by Dettlaff (1966) showed that Actinomycin D suppressed maturation throughout the hormonrdependent period, an observation suggesting that the synthesis of RNA’s specific for the maturation process occurred during this period. These experiments indicated that pituitary gonadotropins induced maturation by acting through the oocyte nucleus, the germinal vesicle (Dettlaff, 1966; Dettlaff et al., 1964). Wright ( 1961) exposed ovarian fragments to both pituitary suspensions and progesterone for short periods of time (6 hours) and compared the amount of ovulation obtained. He found progesterone to be much more effective. This led him to the suggestion that pituitnr! hormones induce ovulation by stimulating secretion of a steroid which, on its own, causes ovulation. While the nature of such a steroid was not specified, it is interesting to note that progesterone has been identified as a constituent of amphibian ovarian tissue (Chiefi and Lnpo, 1963 ) . Recently, we have observed that maturation can be induced in the complete absence of ovulation, both in vitro and in Vito, by exposing ovarian tissue to pituitary suspensions and to steroid hormones. In this case, the steroids were more effective than the pituitary suspensions (Subtelny et al., 1968). Using isolated Rana pipiens follicles, Schuetz (1967a,b) showed that breakdown of the germinal vesicle was accomplished by continuous exposure to progesterone and pituitary suspensions. In these experiments, neither removal of follicular tissue before hormone treatment nor treatment with actinomycin D prevented steroid-induced breakdown of the germinal vesicle. Removal of follicular tissue inhibited pituitary-induced germinal vesicle breakdown in about 50% of the cases, however, and treatment witi actinomycin D at the time of pituitary stimulation prevented maturation ( Schuetz, 1967b). These experiments show that both pituitam

640

SMITH,

ECKER,

AND

SUBTELNY

hormones and steroids can induce maturation in vitro, although not equally well, and possibly not even by the same mechanism. A cytological examination of oocytes dissected from their ovarian follicles shows that not all the follicle cells have been removed (Figs. 2 and 3). In fact, we cannot say with certainty that any of the follicle cells have been removed. We have observed regions of the oocyte in which follicle cells are not present, but this could be the result of the procedure by which the oocytes were prepared for electron microscopy. Apparently, the intimate relationship between the follicle cell processes, the vitelline membrane, and the microvilli of the oocyte (Fig. 2) makes the manual removal of follicle cells virtually impossible without causing extensive damage to the oocyte itself. This observation may provide an explanation for the differences observed in pituitaryinduced and steroid-induced maturation in vitro. For example, earlier data on pituitary-induced maturation, in vitro, using oocytes removed from their follicular envelopes (Dettlaff et al., 1964; Schuetz, 1967b) are subject to an alternative interpretation. It is possible that the pituitary hormones were acting through the mediation of follicle cells not removed. This would help explain several other observations; the differential effect of actinomycin D in the inhibition of pituitary(Dettlaff, 1966) versus progesterone-induced maturation (Schuetz 1967b), the decrease in the percentage of maturation obtained after removal of follicular tissue (Schuetz, 1967b), and our observations on the seasonal variation and limited response after continuous pituitary exposure (Table 1). On the other hand, the wide dose range over which progesterone and DOCA are effective (Table 1) , the seasonal independence of their efficiency, and the effectiveness of short exposures (Table 2), all are consistent with the hypothesis that steroids act directly on the oocyte to induce maturation. A 5minute exposure of oocytes to progesterone induced maturation in almost one-third of the cases; a 1 hour exposure was always successful, These oocytes were washed thoroughly in Ringer’s solution after the hormone exposure. Assuming that no hormone remained adsorbed to the egg surface, we must conclude that the effect of progesterone on the oocyte is very rapid and is brought about long before the breakdown of the germinal vesicle. In this connection, it should be emphasized that breakdown of the germinal vesicle occurred at the same time after a S-minute exposure to progesterone, in vitro, as after the induction of maturation

In

VittrO

MATURATIOS

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641

with pituitary, in oiuo. The hormone-dependent time with steroids is thus in the order of minutes. Steroid-induced maturation has became a routine procedure in our laboratory and has been used successfully on thousands of oocytes. Using this procedure, we have been able to perform a variety of manipulations on ooevtes prior to hormone stimulation with the assurance that the operated eggs can subsequently be induced to mature. Similar manipulations can be performed at almost anv time after the induction of maturation without the necessity of maintaining eggs in the conA variety of experimental studies on timmus presence of hormones. ooc\te maturation have thus become practical.

Full-grown oocytes dissected from their ovarian follicles can 1,~ in duced to undergo physiological maturation by exposure, in vitro, to the steroid hormones progesterone and cleoxyeorticosterone or to homoplastic pituitary suspensions. Evidence for the occurrence of physiological maturation includes (1) a positive response to an activation stimulus and (2) the ability to undergo cleavage and subseqtrent development. \I’hile all the hormones tested are capable of inducing maturation, the steroids are most effective. Exposure of afolliculatc f or 1 hour is routinely oocytcs to progesterone (1 pg/ml) successful in inducing maturation in 100% of the cases at virtually any time of the vear. The effectiveness of pituitary preparations did not reach 100%: varied considerably, and exhibited seasonal dependence. These results support the hypothesis that steroids act directly on the oocytr to induce maturation whereas pituitary hormones act through the mediation of follicular tissue, ~otr, addcc~ iti proof: Since the acceptance of this manuscript for pnldication. two papers have appearecl concerned with the in uitro induction of maturation in Ram pipiem oocytes (Schuetz, A. W., 1967, J. Exptl. Zool. 166, 347-354; Masui. I’., 1967, J. Exptl. Zool. 166, 365-376). Th e results of these papers support the hvpothcsis that steroid hormones act directly on the oocyte to induce maturatior, while pitllit;rr\- hormones act through the mediation of follicle cells. The authors wish to thank Mrs. Marilyn A. Williams and Miss Anita Konrcn) for invalnalde technical assistance. We particularl!~ wish to thank Mrs. Willi;~m~ who performed the electron microscopy.

642

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REFERENCES ARNOLD, J. F., and SHAVER, J. R. (1962). Interfemale transfer of eggs and ovaries in the frog. Exptl. Cell Res. 27, 150-153. BERGERS, A. C. J., and LI, C. H. (1960). Amphibian ovulation in vitro induced by mammalian pituitary hormones and progesterone. Endocrinology 66, 255259. CHIEFFI, G., and LUPO, C. ( 1963). Identification of sex hormones in the ovarian extracts of Torpedo murmorata and Bufo vulgaris. Gen. Comp. Endocrinol. 3, 149-152. DETTLAFF, T. A. ( 1966). Action of actinomycin and puromycin upon frog oocyte maturation. .I. Embryol. Exptl. Morph&. 16, 183-195. DETTLAFF, T. A., NIKITINA, L. A., and STROEVA, 0. G. (1964). The role of the germinal vesicle in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei. J. Embryol. Exptl. Morpbl. 12, 851-873. EDGREN, R., and CARTER, D. L. ( 1963). Studies on progesterone induced in vitro ovulation of Rana pipiens. Gen. Comp. Endocrinol. 3,526-528. HAMBURGER, V. ( 1960). “A Manual of Experimental Embryology,” rev. ed., p. 35. Univ. Chicago Press, Chicago, Illinois. HEILBRUNN, L. V., DAUGHERTY, K., and WILBUR, K. M. (1939). Initiation of maturation in the frog egg. Physiol. Zool. 12, 97-100. LAVIN, L. H. (1964). The transfer of coelomic eggs between frogs. 1. Embryol. Exptl. Morphol. 12, 457463. MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114. MORRILL, G. A. ( 1965). Water and electrolyte changes in amphibian eggs at ovulation. Exptl. Cell Res. 40, 664-667. SABATINI, D. D., BENSCH, K., and BARRNETT. R. J., (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58. SCHUETZ, A. W. ( 1967a). Effect of steroids on germinal vesicle of oocytes of the frog (Rana pipiens) in vitro. Proc. Sot. Exptl. Biol. Med. 124, 1307-1310. SCHLJETZ, A. W. ( 196713). Mechanism of progesteroneand pituitary-induced germinal vesicle breakdown in oocytes of Rana pip-iens. J. Cell Biol. 35, 123A. SHAVER, J. R., and BARCH, S. H. (1960). Experimental studies on the role of jelly coat material in fertilization in the frog. Acta Embryol. MorphoE. Exptl. 3, 180-189. SMITH, L. D., ECKER, R. E., and SUBTELNY, S. (1966). The initiation of protein synthesis in eggs of Ranu pipiens. Proc. Natl. Acad. Sci. U.S. 56, 1724-1728. SUBTELNY, S., and BRADT, C. (1961). Transplantation of blastula nuclei into activated eggs from the body cavity and from the uterus of Rana pipiens. II. Development of the recipient body cavity eggs. Develop. Biol. 3, 96-114. SUBTELNY, S., SMITH, L. D., and ECKER, R. E. (1968). Maturation of ovarian frog eggs without ovulation. J. Exptl. Zool., in press. VENABLE, J. H., and COGGESHALL, R, (1965). A simplified lead citrate stain for use in electron microscopy. .I. Cell Biol. 25, 407408.

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S. (1966). The ultrastructure of the cytoplasm of the developing amphibian egg. Advan. Morphogenesis 5, 131-179. WITSCHI, E., and CHANG, C. Y. (1959). Amphibian ovulation and spermiation. In “Comparative Endocrinology” (A. Gorbman, ed. ), pp. 149-161. Wiley, New York. WRIGHT, P., and FLATHERS, A. R. (1961). Facilitation of pituitary induced frog ovulation bv progesterone in early fall. Proc. Sot. Exptl. Biol. Med. 106, 246247. . WRIGHT, P. (1961). Induction of ovulation in vitro in Rana pipierts with steroids. WISCHNITZER,

Gen. Comp. Endocrinol.

1, 20-23.