Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes

DEVELOPMENTAL

84,455-464

BIOLOGY

(1981)

Biochemical Studies of Mammalian Oogenesis: Metabolic Cooperativity between Granulosa Cells and Growing Mouse Oocytes T. HELLER,

DAVID Department

of Biology, Received

DANIEL University

December

M. CAHILL, of Pennsylvania,

1, 1980; accepted

AND RICHARD Philadelphia,

in revised

form

January

M. SCHULTZ

Pennsylvania

19104

15, 1981

Freeze fracture

and lanthanum tracer experiments have shown that gap junctions exist throughout folliculogenesis begranulosa cells and growing mouse oocytes (Anderson and Albertini, J. Cell Biol. 71,680-686, 1976). The following lines of experimentation in the present study suggest that metabolic cooperativity exists between granulosa cells and their enclosed oocytes, i.e., gap junctions are functional, and that in most cases examined, greater than 35% of the metabolites present in follicle-enclosed oocytes were originally taken up by the granulosa cells and transferred to the oocyte via gap junctions: (1) When incubated with various radiolabeled compounds, follicle-enclosed oocytes contained more intracellular radioactivity than did oocytes with no attached granulosa cells (denuded oocytes); (2) for two radiolabeled ribonucleosides examined, the distribution of phosphorylated metabolites in follicle-enclosed oocytes resembled that of granulosa cells and differed significantly from that in denuded oocytes; (3) pulse-chase experiments with radiolabeled ribonucleosides revealed that during the chase period more radioactivity became associated with the follicle-enclosed oocyte; (4) treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and oocytes; and (5) a series of control experiments using (a) medium conditioned by granulosa cells and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not permitted demonstrated that contact between follicle cells and oocytes was necessary for observing metabolic cooperativity. Metabolic cooperativity was also found between follicle cells and oocytes in the two culture systems which support growth of mouse oocytes in vitro. The fact that oocytes do not grow well, if at all, in the absence of follicle cells and the large contribution of nutrients apparently furnished to the oocyte by the granulosa cells is consistent with the concept that gap junction mediated metabolic cooperativity between follicle cells and their enclosed oocytes is vital for mammalian oocyte growth. tween

INTRODUCTION

During its growth phase, the mouse oocyte is arrested at the dictyate stage of the first meiotic prophase and increases in diameter from about 20 pm to about 80 pm. This large increase in volume is accompanied by the synthesis and accumulation of organelles and macromolecules (Jahn et al., 1976; Schultz and Wassarman, 1977; Wassarman and Josefowicz, 1978; Schultz et al., 19’79; Bachvarova and DeLeon, 1980; Sternlicht and Schultz, 1981). Presumably, these constituents are used to support early embryogenesis to some extent, as is the case in lower species (Davidson, 1976). In addition, it is during the growth phase that oocytes undergo a marked and progressive biochemical and morphological differentiation (Weakley, 1968; Szollosi, 1972; Anderson, 1974; Wassarman and Josefowicz, 1978; Schultz et al., 1979). Associated with these changes during oocyte growth is the acquisition of meiotic competence; when oocytes reach approximately 60 pm in diameter they acquire the ability to resume spontaneous meiotic maturation in vitro. The frequency at which oocytes resume meiosis in-

creases with increasing oocyte size (Sorensen and Wassarman, 1976). Only oocytes that have successfully completed growth and meiotic maturation are capable of being fertilized and giving rise to normal development (Thibault, 1972). While oocyte growth is crucial for normal fertilization and development, little is known about the factors that regulate growth of mammalian ooeytes. Due to their spherical shape and resulting minimal surface area to volume ratio, oocyte growth could potentially be limited by uptake of nutrients. It has long been proposed that follicle cells regulate oocyte growth by providing nutrients to the oocyte (Zamboni, 1970). An analogous nutritional role has been well documented for nurse cells in lower species (Anderson, 1974; Telfer, 1975). Throughout its growth phase, the mouse oocyte is invested by one or more layers of follicle cells. Freeze fracture and lanthanum tracer studies have shown that gap junctional contacts between granulosa cells and oocytes are present (Anderson and Albertini, 1976) before oocyte directed synthesis of the xona pellucida has occurred (Bleil and Wassarman, 1980). These junctions, which are

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456

DEVELOPMENTAL

BIOLOGY

maintained throughout folliculogenesis and zona deposition, are small and numerous (Anderson and Albertini, 1976). It is generally accepted that gap junctions provide channels by which small molecules pass from one cell directly into another (Lowenstein, 1979). This results in the phenomenon called metabolic cooperativity or metabolic coupling (Pitts, 1977). Through metabolic cooperativity mediated via gap junctions with adjacent granulosa cells, the oocyte could in effect recruit additional plasma membrane to meet its nutritive requirements. To date, there is no experimental evidence that gap junctions between granulosa cells and their enclosed growing oocytes are functional. However, evidence exists that gap junctions between the fully grown dictyate oocyte and its attached cumulus cells are functional. Cumulus cells and oocytes are ionically coupled and iontophoretically injected fluorescein dye is transferred between the oocyte and cumulus cells (Gilula et al., 1978). A coordinate decrease in the number of gap junctional contacts and extent of ionic coupling is observed as ovulation approaches. After ovulation, cumulus cells surround the oocyte but cumulus cell microvilli have retracted from the oocyte surface and ionic coupling is no longer observed (Gilula et al., 1978). Results from several previous studies suggested that naturally occurring metabolites pass from cumulus cells via gap junctions to the fully grown dictyate oocyte (Cross and Brinster, 1974; Wassarman and Letourneau, 1976; Moor et aZ., 1980; Heller and Schultz, 1980). This metabolic cooperativity is not found between ovulated oocytes and their surrounding cumulus cells (Cross and Brinster, 1973; Moor et al., 1980; Heller and Schultz, 1980). The naturally occurring union between granulosa cells and oocytes provides an ideal system to study the physiological role of intercellular communication between two heterologous cell types. The results from experiments described in this report suggest that gap junction mediated metabolic cooperativity exists between granulosa cells and their enclosed growing oocytes. These results are discussed in terms of the physiological role granulosa cells might exert upon oocyte growth. MATERIALS

Collection

AND

METHODS

of Mouse Follicles

Follicles were obtained from juvenile, randomly bred, Swiss albino mice (Swiss Webster, Ace) by puncturing isolated ovaries bathed in standard egg culture medium (SECM) (Biggers, 1971) with fine steel needles under a dissecting microscope. Intact follicles were harvested with a mouth-operated micropipet and washed a minimal essential medium (Earle’s salts) supplemented with pyruvate (100 pg/ml), bovine serum albumin (BSA) (3

VOLUME

84,198l

mg/ml), 10 mil4 HEPES, pglml (MEIWBSA). Collection

pH 7.3, and Gentamicin

(10

of Mouse Oocytes

Follicles were obtained as described above. Oocytes were liberated from their follicles by a modification of the method of Eppig (1977a). Follicles were incubated in calcium and magnesium-free SECM containing 0.05% collagenase (Type III, Worthington) and 0.3% BSA for 30 min at 37°C. After this incubation the oocytes were liberated from their follicles by repeated pipetting with a mouth-operated micropipet whose inner diameter was slightly larger than that of the oocyte. Oocytes free of adhering granulosa cells (denuded or naked oocytes) were washed in MEM/BSA. All manipulations involved in collection of follicles and oocytes were performed with siliconized glassware. Oocyte Culture

in Vitro

The oocyte culture systems of Eppig (197713) and Bachvarova et al. (1980) were used. In the Eppig system, ovaries obtained for g-day-old mice were shredded into small pieces with fine steel needles and incubated in MEM containing 0.1% collagenase (Type III, Worthington) for 15 to 30 min at 37°C. At the end of the incubation, an equal volume of fetal calf serum (FCS) was added and follicles were liberated from the ovarian tissue by repeated pipetting with a Pasteur pipet. The follicles were harvested with a mouth-operated micropipet and seeded in plastic culture dishes (35 mm, Corning or Falcon) in clusters each composed of about 25 to 50 follicles. Culturing was performed in Waymouth’s 752/l medium containing 5% FCS, pyruvate (100 pg/ml), and Gentamitin (10 pg/ml) at 37°C in a humidified atmosphere of 5% CO, in air. After 1 day of culture, the follicle aggregates attached to the culture dish. Oocytes that were released with time were discarded. The medium was changed every third day. In the system of Bachvarova et al. (1980), ovaries (lo20) obtained from g-day-old juvenile mice were incubated in SECM lacking BSA but containing 0.2% Pronase(Ca1 Biochem) for 20 min at 37°C. The ovaries, washed once with MEM/lO% FCS and then placed in MEM./lO% FCS in a plastic culture dish (35 mm, Corning or Falcon), were dissociated into ovarian cells, follicles, and free oocytes by repeated pipetting with a Pasteur pipet. The nondissociated ovarian fragments were removed and the cells cultured at 37°C in a humidified atmosphere of 5% CO, in air. After 1 day of culture, the ovarian cells formed a monolayer on which rested naked oocytes. The medium, MEM/lO% FCS containing pyruvate (100 pglml) and Gentamicin (10 pg/ml) was changed every third day.

HELLER, CAHILL, AND SCHULTZ

Assay for Metabolic Cooperativity Cells and Oocytes

between Follicle

Metabolic cooperativity between follicle cells and their enclosed oocytes was determined by culturing for a given period of time in the same culture dish denuded and follicle-enclosed oocytes in 200 ~1 of MEIWBSA containing the radiolabeled compound under investigation. Cell culture was performed under paraffin oil in a humidified atmosphere of 5% CO, in air at 37°C. Following culture, denuded oocytes and oocytes liberated from their follicles were washed as separate groups in SECM and transferred to separate polypropylene tubes. Oocytes were solubilized in 25 ~1 of 1 N NaoH for 10 min at room temperature, the solution acidified with 30 ~1 of 1 N HCl and subjected to liquid scintillation counting in 3 ml of Liquistint (National Diagnostics). Samples of about 10 oocytes were routinely counted. The exact conditions of these experiments are described in the table legends. Fractionation of Metabolites and Choline

of Uridine,

Guanosine,

Uridine and guanosine. Denuded and follicle-enclosed oocytes were cultured overnight in the same culture dish in MEWBSA containing 50 &X/ml of either [3H]uridine or [3H]guanosine. Denuded oocytes, oocytes liberated from their follicles and the resulting follicle cells were washed as separate groups with SECM to remove extracellular radioactivity and transferred to separate polypropylene tubes. The appropriate ribonucleoside and its mono-, di-, and triphosphate derivative (10 nmole of each) were added and cellular structure disrupted by two cycles of freezing and thawing. The entire sample was spotted on polyethyleneimine cellulose (PEI-cellulose). Uridine and guanosine and their phosphorylated metabolites were separated by first developing the chromatogram with water. The chromatogram was then dried and developed in the same direction with 0.75 M LiCl for uridine or 1.0 M LiCl for guanosine. In each case, the ribonucleoside and its phosphorylated metabolites were located under ultraviolet light, excised from the chromatogram, and transferred to a 6 ml scintillation vial. The samples, eluted from the PEI-cellulose with 0.4 ml of 0.7 M MgCl, in 10 mM Tris-HCl, pH 7.4, with shaking for 30 min at room temperature, were subjected to liquid scintillation counting in 3 ml of Liquiscint. In each case, about 20 oocytes were used (or the follicle cells from about 20 follicles). Choline. Denuded and follicle-enclosed oocytes were cultured overnight in the same culture dish in MElWBSA containing 50 &i/ml of [3H]choline. Denuded oocytes, oocytes liberated from their follicles and the resulting follicle cells were washed as separate groups with SECM

457

Gmnulosa Cell -0ocyte Interactions

and transferred to separate polypropylene tubes. Radiolabeled phospholipids were separated from their radiolabeled aqueous precursors (e.g., choline, phosphoryl choline, CDP-choline) by adding 25 ~1 of 154 mM NaCl to each sample and extracting with 75 ~1 of chloroformmethanol (2: 1, v:v). The phases, separated by centrifugation, were removed from the tube with a mouthoperated micropipet and transferred to scintillation vials. The organic phase was taken to dryness under a stream of air. Samples were counted in 3 ml of Liquistint. The metabolites in the aqueous phase following chloroform-methanol extraction were fractionated as follows: choline (300 pg) and phosphorylcholine (100 pg> were added to the sample which was spotted on Whatman chromatography paper. Choline and phosphorylcholine were resolved from one another by paper electrophoresis of 500 V for 15 min using 50 mM NH4HC03, pH 7.5, as buffer. The standards were located by spraying the chromatogram with 0.05% bromcresol in 95% ethanol. The dried chromatogram was cut into l-cm portions. Each fraction was placed in a scintillation vial, eluted with 0.5 ml of water for 30 min at room temperature with shaking, and subjected to liquid scintillation counting after adding 5 ml of Liquiscint. RESULTS

Detection of Metabolic Cooperativity Cells and Growing Oocytes

between Follicle

Compared to ovaries of cycling mice, ovaries of juvenile mice yield more follicles at a given stage of follicle growth. This is due to a large and fairly synchronous wave of follicle growth in juvenile mice (Pederson, 1969). In most cases, the follicles used in this study were derived from ovaries of ll- to 12-day-old mice. After classifying follicles into three groups, i.e., small, medium, and large, follicle diameters, as measured with an ocular micrometer attached to an inverted microscope, were 71.4 f 7.4, 96.7 + 8.4, and 121.4 _+ 8.5pm, respectively. The diameters of the enclosed oocytes of these small, medium and large follicles were 43.9 t 2.4, 49.5 f 3.3, and 54.2 f 4.2 pm, respectively. The medium size class of follicles was used in most of the experiments described below. Metabolic cooperativity (used interchangeably with metabolic coupling) has been measured by others by nucleotide exchange (Pitts, 1977). Cells are labeled with radiolabeled uridine and residual intracellular radiolabeled uridine flushed out of the cells by repeated washing. RNA and uridine nucleotides are retained since they cannot permeate plasma membranes. These prelabeled cells are placed in contact with unlabeled cells which are later examined for intracellular radioactivity. Uridine

DEVELOPMENTALBIOLOGY VOLUME&I,1981

458 TABLE

1

METABOLICCOUPLINGBETWEENFOLLICLE CELLSAND GROWING MOUSEO~CYTES Cf Compound Uridine” Guanosine* Choline’ 2-Deoxyglucosed Methionine-sulfonee

(ave k SD) 13.6 6.7 5.9 10.5 1.2

+ 2 k k c

10.9 3.5 4.0 4.9 0.4

(1 Concentration of [5,6-3H]uridine (40-50 Wmmole, Amersham) was 50 j&i/ml. b Concentration of [8-3H]guanosine (7.7 Wmmole, Amersham) was 50 $.Xml. c Concentration of [methyPC]choline (50 mCi/mmole, Amersham) was 20 wCi/ml. d Concentration of 2-deoxy-n-[l-3H]glucose (19.5 Ciimmole, Amersham) was 50 $.X/ml. e Concentration of [Wlmethionine sulfone was 125 $IX!ml. [YS]Methionine (1325 Wmmole, Amersham) was converted to the sulfone derivative by per-formic acid oxidation (Hirs, 1967). [YS]Methionine sulfone was not incorporated into protein. After the culture period, greater than 95% of the intracellular radioactivity present in either denuded or follicle-enclosed oocytes was acid-soluble. ’ Denuded oocytes and follicles were cultured overnight in MEM/BSA containing the radiolabeled compound under investigation. Values of C were calculated as described in Results.

nucleotides, but not RNA, move from one cell to another presumably through gap junctions (Pitts and Simms, 1977). Passage of uridine nucleotides into the unlabeled cells is termed metabolic cooperativity. It is generally accepted that if metabolic cooperativity is observed, then gap junctions are present. Exceptions to this statement have never been found. In this laboratory, metabolic cooperativity between granulosa cells and their enclosed oocytes is assayed by incubating follicle-enclosed oocytes and denuded oocytes with a radiolabeled compound and comparing the amount of intracellular radioactivity in these oocytes (Heller and Schultz, 1980). Results from this type of experiment are expressed as a metabolic cooperativity ratio C, defined as (A - B)/B, where A is the number of cpm per follicle-enclosed oocyte and B is the number of cpm per denuded oocyte. While values of C greater than 0 indicate the existence of metabolic cooperativity, values of C greater than 0.5 are taken as being indicative of metabolic cooperativity, due to experimental error. Metabolic cooperativity between granulosa cells and growing mouse oocytes was examined with a number of different radiolabeled compounds and the resulting metabolic cooperativity ratios are given in Table 1. Compounds used included precursors for RNA (uridine and guanosine) and phospholipid (choline) biosynthesis, an amino acid analog (methionine sulfone), and an analog for

an energy source (2-deoxyglucose). The experimental error in the value of C for these different compounds usually was due to the difference in uptake of the radiolabeled precursor by the denuded oocyte. The lower the level of uptake by denuded oocytes, the greater the value of c. Oocytes, due to their large size and highly characteristic morphology, are easily separated from granulosa cells and individual oocytes can be biochemically analyzed. This permitted an analysis of metabolic cooperativity between granulosa cells and oocytes in single follicles. Though different degrees of metabolic cooperativity with radiolabeled uridine and choline were observed, every follicle-enclosed oocyte examined exhibited some degree of metabolic cooperativity (Table 2, columns under Coupled). Pulse-chase experiments were also consistent with metabolic cooperativity between granulosa cells and oocytes. Follicles were briefly labeled with a radiolabeled ribonucleoside and then washed free of extracellular radioactivity. The amount of radioactivity associated with the follicle-enclosed oocyte was analyzed immediately after the washings and after a 2 hr incubation in nonradioactive medium (Table 3). During the chase, intracellular levels of radioactivity increased in follicle-enclosed oocytes by about 74 and 79% when pulsed with uridine and guanosine, respectively. A previous study demonstrated that when incubated with various radiolabeled ribonucleosides, cumulus-enclosed dictyate oocytes had higher levels of intracellular radioactivity and greater proportions of the ribonucleoside di- and triphosphates when compared to denuded TABLE

2

ANALYSISOFMETABOLIC COOPERATIVITY IN SINGLECOUPLED AND UNCOUPLED FOLLICLE-ENCLOSED~OCYTES Value of c” Choline

Ave + SD

Uridine

Coupled

Uncoupled”

Coupled

Uncoupledb

11.9 19.5 16.8 13.5 16.7 13.3 15.0 19.1 13.1 15.4 2 2.7

3.2 3.9 0.9 2.5 2.7 5.9 0.4 1.8 2.1 2.6 * 1.6

7.3 7.1 7.1 6.5 7.7 6.4 8.1 9.4 7.9 7.5 i 0.9

1.7 0.3 0.0 1.7 0.9 0.5 2.1 0.8 1.9

1.1 f 0.8

a Values of C were calculated as described in Results. The culture period was for 2 hr and [methyl-3H]choline (30 Wmmole; Amersham) was used at a final concentration of 50 pCi/ml. *Uncoupling was performed as described in footnote g of Table 6.

HELLER, CAHILL, AND SCHULTZ

oocytes (Heller and Schultz, 1980). Furthermore, cumulus cells readily phosphorylated all ribonucleosides tested; greater than 80% of the acid-soluble intracellular radioactivity was present as the ribonucleoside di- and triphosphates. It was postulated that cumulus-enclosed oocytes derived most of the ribonucleosides and their phosphorylated metabolites from their attached cumulus cells. According to this reasoning, the distribution of metabolites in follicle-enclosed oocytes should be similar to that of their attached granulosa cells and different from that of denuded oocytes. Accordingly, the distribution of uridine and guanosine metabolites in denuded and follicle-enclosed oocytes and granulosa cells was analyzed. As anticipated, the distribution of metabolites present in follicle-enclosed oocytes was similar to that of their attached follicle cells but differed from that in denuded oocytes (Table 4). This result is consistent with the hypothesis that radiolabeled precursor taken up and metabolized by granulosa cells can enter growing oocytes, presumably via gap junctions.

Granulosa

Cell -0ocyte

459

Znteractions

TABLE

4

DISTRIBUTION OF URIDINE AND GUANOSINE METABOLITES DENUDED AND FOLLICLE-ENCLOSED OOCYTES AND FOLLICLE

IN CELLS”

Percentage total cpm Compound Uridine UMP UDP UTP Guanosine GMP GDP GTP

Denuded oocyte 18.0 22.0 30.0 32.0 10.0 43.0 22.0 25.0

2 f + + f k k ”

5.0 3.0 8.0 6.0 12.0 25.0 11.0 17.0

Follicle-enclosed oocyte 14.0 7.0 16.0 63.0 3.0 3.0 8.0 86.0

f + + f k 2 2 +

10.0 0.0 6.0 2.0 3.0 2.0 5.0 11.0

Follicle cells 15.0 6.0 4.0 75.0 2.0 5.0 8.0 85.0

f f ” + f f * f

13.0 0.0 1.0 13.0 1.0 1.0 2.0 2.0

n The experiment was performed as described in Materials and Methods. The results for uridine are the average (-t range) of two experiments and those for guanosine are the average 2 SD of three experiments.

The metabolic fate of radiolabeled choline in denuded and follicle-enclosed oocytes was also determined. FolliTABLE 3 cles and denuded oocytes incubated with radiolabeled TRANSFER OF RADIOLABELED RIBONUCLEOSIDE FROM FOLLICLE choline were washed free of extracellular radioactivity CELLS TO GROWING OOCYTES DURING A 2 hr CHASE and the denuded oocytes and oocytes liberated from their follicles were subjected to chloroform-methanol extracIncubation Percentage Compound time (hr) cpm/oocyte increase tion. Compared to denuded oocytes, follicle-enclosed oocytes had about 21.5 + 9.0 (average + SD of three exUridine Intact 0 15.8 2 1.2 74 periments) times more cpm present in the organic phase. follicle” 2 27.5 ” 0.5 While this radiolabeled material was not characterized, it Guanosine Intact 0 8.4 2 15.0 79 follicle” was most likely phospholipid. Compared to denuded Uridine Uncoupled 0 3.3 oocytes, follicle-enclosed oocytes had about 2.7 + 1.1 follicle” 2 3.9 18 (average + SD of three experiments) times the number Uridine In vitro 0 12.5 f 0.3 2 21.0 + 0.3 68 of cpm present in the aqueous phase following chlorocultureC form-methanol extraction. Electrophoretic analysis of oocytes u Follicles obtained from 12-day-old mice were labeled for 15 min in this material for both free and follicle-enclosed MEIWBSA containing 50 @X/ml of either [3H]uridine or [3H]guanosine. revealed that greater than 95% of the radioactivity comiOocytes were liberated from one-half of the follicles and intracellular grated with phosphorylcholine (data not shown). radioactivity measured while the remaining follicles were washed with A series of control experiments demonstrated that the MEM/BSA for 2 hr. Follicle-enclosed oocytes were liberated and subincreased uptake of radioactivity by follicle-enclosed jected to liquid scintillation counting. The amount of intracellular radioactivity in denuded oocytes fell slightly during the chase (data not oocytes, compared to denuded oocytes, was due to physishown). The results for uridine are the average of two experiments cal contact between the two cell types and was not due to (+- range) while those for guanosine represent a single experiment. a diffusible factor secreted by the follicle cells that prob Follicles obtained from 12-day-old mice and uncoupled by the compounds tested. In procedure described in footnote g of Table 6 were labeled for 15 min in moted uptake of the radiolabeled MEIWBSA containing 50 $X/ml of [$H]uridine. The experiment was each instance in which denuded oocytes were cultured in then conducted as described in footnote a of this table. The results are medium conditioned by follicle cells or in which denuded from a single experiment. oocytes were cocultured with, but not allowed to come c Oocytes were cultured for 5 days using the Eppig system (see into contact with, follicle cells, no enhanced uptake of raMaterials and Methods). The cells were labeled for 15 min in MEIWBSA containing 50 &i/ml of [3H]uridine. The radioactive medium was diolabeled compound was observed (Table 5). removed from the culture dish and the cells repeatedly washed with MEIWBSA until the number of cpm in the medium fell to background levels. One-half of the oocytes were liberated from their attached follicle cells for analysis of intracellular radioactivity. The remainder of the experiment was conducted as described in footnote a of this table. The results are the average 2 SD of three separate measurements.

Uncoupling of Metabolic Cooperativity between Follicle Cells and Their Enclosed Oocytes

Different types of ionically coupled cells have been uncoupled by a variety of treatments which include replac-

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TABLE 5 EFFECT OF CONDITIONED MEDIUM AND C&CULTURING OF OOCYTES AND FOLLICLE CELLS UPON URIDINE UPTAKE BY DENUDED OOCYTES Sample

C (ave 2 SD)

Conditioned medium” Cocultures of follicle cells and free oocytes*

-0.40 2 0.20 0.03 * 0.30

* Follicle cells were isolated by treating follicles isolated from ovaries of 13.day-old mice with calcium and magnesium-free SECM containing 0.1% collagenase and 0.02% DNase I (Worthington) for 30 min at 37°C with agitation. After the incubation, the liberated oocytes were removed with a mouth-operated micropipet. The remaining follicle cells were harvested by centrifugation and resuspended in 1 ml of MEM/lO% FCS and cultured overnight. The next day follicle cells were removed by centrifugation and [3H]uridine was added in a volume to l/20 that of the conditioned medium (fmal concentration 50 pCi/ml). Naked oocytes, isolated from follicles obtained from ovaries of mice 12 days old, were added in a volume less than 10 ~1 to this conditioned medium, as well as, to fresh MEwlO% FCS containing [3H]uridine (final concentration, 50 $.Xml). Samples were cultured overnight. Following culture, oocytes were removed and washed as separate groups and analyzed for intracellular radioactivity as usual so that values of C could be computed. * Denuded oocytes were collected from follicles obtained from ovaries of mice 10 days old and placed in two culture dishes containing [3H]uridine (final concentration, 50 &i/ml). To one of the culture dishes were added about 300-400 follicles obtained from ovaries of mice 10 days old. The free oocytes and follicles were placed close to one another but they were not in direct physical contact. After culturing overnight, free oocytes from both samples were washed separately, and analyzed for intracellular radioactivity as usual so that values of C could be computed.

ing chloride with propionate (Asada and Bennett, 1971), lowering or elevating calcium concentration in the medium (Lowenstein et al., 1967) and increasing the osmolarity of the medium with various sugars (Lowenstein et al., 1967; Peracchia and Dulhunty, 1976). In some instances, loss of ionic coupling has been correlated with ultrastructural changes in gap junction morphology (Pappas et al., 1971; Peracchia, 1977). To determine whether uncoupling of metabolic cooperativity had been achieved, the metabolic cooperativity ratios were compared for treated and untreated (control) follicles. This was done by analyzing the value of C+/C where C+ is defined as (A’ - B)/B in which A’ is the number of cpm per follicle-enclosed oocyte isolated from treated follicles and B is the number of cpm per denuded oocyte and C is as previously defined. While values of C+/C less than 1 indicated uncoupling of metabolic cooperativity between folicle cells and their enclosed oocytes, only values of C+IC less than 0.85 were considered significant due to experimental error. Replacing chloride with propionate did not result in uncoupling (data not shown). Likewise, follicles cultured for 16 hr in MEM/lO% FCS containing either luteinizing

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hormone (5 pg/ml, NIH grade), follicle-stimulating hormone (5 pg/ml, NIH grade), or dibutyryl CAMP (1 mM> prior to performing the coupling assay revealed no sign of reducing metabolic cooperativity between follicle cells and oocytes whether the assay was performed in the presence or absence of these compounds (data not shown). Follicles treated with collagenase or hypertonic medium (sucrose treatment) remained metabolically coupled (Table 6). No uncoupling was found when follicles were treated sequentially with collagenase and sucrose. TABLE 6 EFFECT OF VARIOUS TREATMENTS USED TO INHIBIT METABOLIC COOPERATIVITY BETWEEN FOLLICLE CELLS AND OOCYTES Treatment Collagenase” Sucrose’ Collagenase + sucrose + MEM” Collagenase + EGTA’ Collagenase + EGTA + MEM” Sucrose + EGTAf Sucrose + EGTA + MEM’ Collagenase + sucrose/EDTA + EGTAQ Collagenase + sucrose/EGTA + MEMO

c+/c” 1.00 1.00 1.11 0.65 0.98 0.56 1.03 0.18 1.11

u Treated and untreated follicles and denuded oocytes were cultured for 2 hr in MEM/BSA containing [methyPH]choline (50 Ciimmole, Amersham) and values of metabolic cooperativity were computed as previously described. * Follicles were incubated for 20 min at 37°C in MEM/BSA containing 0.05% collagenase, washed in MEM/BSA, and then subjected to labeling with radiolabeled choline. t Follicles were incubated for 10 min at 37°C in MEM/BSA containing 0.5 M sucrose washed in MEM/BSA and then subjected to labeling with radiolabeled choline. d Follicles were incubated for 20 min at 3’7°C in MEM/BSA containing 0.05% collagenase, washed in MEM/BSA, and transferred to MEMYBSA containing 0.5 M sucrose. After incubating for 10 min, the follicles were returned to MEM/BSA and then subjected to labeling with radiolabeled choline. e Follicles were incubated for 20 min at 37°C in MEM/BSA containing 0.05% collagenase, washed in MEM/BSA, and transferred to calciumfree MEM/BSA containing 3 m&f EGTA. The follicles were incubated for 30 min at 37°C and then transferred to either MEM/BSA with radiolabeled choline or to calcium free MEM/BSA containing 3 n&f EGTA and radiolabeled choline. f Follicles were incubated for 10 min at 37” in MEM/BSA containing 0.5 M sucrose, washed in MEM/BSA, and then transferred to calciumfree MEM/BSA containing 3 mM EGTA. After incubating for 30 min at 37”C, the follicles were transferred to either MEM/BSA with radiolabeled choline or to calcium-free MEM/BSA containing 3 mJ4 EGTA and radiolabeled choline. y Follicles were incubated for 20 min at 37°C in MEM/BSA containing 0.05% collagenase, washed in MEM/BSA and transferred to calciumfree MEM/BSA containing 3 mM EGTA and 0.5 M sucrose. After incubating for 10 min at 37”C, the follicles were transferred to either MEM/BSA or calcium-free MEM/BSA containing 3 mM EGTA, maintained in these media for 30 min at 37”C, and then labeled with radiolabeled choline in these respective media.

HELLER, CAHILL, AND SCHULTZ

Follicles treated with collagenase and low calcium (EGTA treatment) exhibited a modest degree of uncoupling of metabolic cooperativity. This treatment was reversible; follicles treated with collagenase and EGTA and then returned to medium containing normal levels of calcium exhibited full metabolic cooperativity. A modest (and reversible) degree of uncoupling of metabolic cooperativity was also found when follicles were sequentially treated with sucrose and EGTA and then maintained in medium containing EGTA. Oocytes could be markedly uncoupled from their surrounding granulosa cells if these follicles were treated first with collagenase and then sucrose/EGTA and then kept in medium containing EGTA (Table 6). This treatment was reversible since follicles treated with collagenase and then sucrose/EGTA but returned to medium containing normal concentrations of calcium revealed no indication of uncoupling of metabolic cooperativity between follicle cells and their enclosed oocytes. Control experiments showed that this uncoupling treatment did not affect uptake of radiolabeled uridine or choline into denuded oocytes or intact follicles (after correcting for the extent of uncoupling) (data not shown). However, low concentrations of calcium in the medium drastically inhibited uptake of radiolabeled 2-deoxyglucose by denuded oocytes or by intact follicles. Therefore, this compound could not be studied further. Uncoupling of uridine and choline metabolic cooperativity was also analyzed in single oocytes isolated from follicles subjected to the collagenase/sucrose/EGTA regimen. Although a range of values for the extent of uncoupling was obtained, every treated follicle-enclosed oocyte exhibited a decrease in the value of C compared to the control (Table 2, columns under Uncoupled). Results from pulse-chase experiments were also consistent with the hypothesis that the uncoupling procedure disrupted gap junctions between follicle cells and oocytes. Uncoupled follicles were pulsed with radiolabeled uridine, washed free of extracellular radioactivity, and then incubated for 2 hr in nonradioactive medium. After a 2 hr chase, only an 18% increase in the number of cpm associated with the follicle-enclosed oocytes was observed (Table 3). Oocytes from intact follicles in which metabolic coupling was present revealed a 74% increase in the number of cpm associated with the oocyte during such a chase. The 18% increase observed in the oocytes derived from uncoupled follicles was consistent with the extent of uncoupling usually obtained @O-90%). Thus, on the average, one would anticipate an 11% increase in the number of cpm associated with the oocyte which is close to the observed 18% increase. If metabolic cooperativity for both uridine and choline (and their metabolites) is mediated by a common pathway, i.e., gap junctions, and if the uncoupling procedure

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TABLE 7 EXTENT OF URIDINE AND CHOLINE METABOLIC COOPERATIVITY IN THE SAME UNCOUPLED FOLLICLE-ENCLOSED OOCYTES~ Compound [3H]Uridine [W]Choline

C*

C+’

c+tc

5.9 4.5

1.2 0.8

0.20 0.18

a In both the uncoupled and control experiments, uridine and choline uptake by treated and untreated follicle-enclosed oocytes was compared to that of denuded oocytes cultured for 2 hr in MEIWBSA containing [3H]uridine (50 &i/ml) and [Wlcholine (20 $X/ml). Values for C+ and C were calculated as previously described. b Untreated follicles were cultured for 2 hr in MEM/BSA containing [3H]uridine (50 &i/ml) and [Wlcholine (20 pCi/ml). c Uncoupling was performed by the sequential collagenase-sucrose/ EGTA-EGTA treatment described in footnote g to Table 6. The treated follicles were cultured for 2 hr in calcium free MEM/BSA containing [3H]uridine (50 pCi/ml) and [Wlcholine (20 &i/ml).

does indeed disrupt these gap junctions, then the value of C+/C for both uridine and choline in the same set of follicles should be the same. This prediction was found to be the case. Treated and untreated follicles were cultured separately in medium containing both [14C]choline and [3H]uridine and values of C-t/C were calculated for each compound (Table 7). It is apparent that the value of C+/C for both uridine and choline was quite similar. Metabolic Cooperativity between Follicle Oocytes Growing in Vitro

Cells and

At present there exist two culture systems which support mouse oocyte growth in vitro. In the culture system reported by Eppig (1977b), gap junctions have been observed between granulosa cells and the growing oocytes. After allowing oocytes to grow in vitro for 2 to 7 days using this culture system, metabolic cooperativity between granulosa cells and oocytes was examined using several radiolabeled compounds (Table 8). In each case, metabolic cooperativity was found. Pulse -chase experiments were also consistent with the existence of metabolic cooperativity between the two cell types (Table 3). During the chase period the intracellular levels of radioactivity associated with the oocytes increased about 68%. A series of control experiments similar to those described in Table 5 demonstrated that the integrity of the connections between follicle cells and the oocyte was necessary for observation of metabolic cooperativity. Thus gap junctions between granulosa cells and growing mouse oocytes in this culture system are presumably functional. In the culture system described by Bachvarova et al. (1980), metabolic cooperativity was found between the ovarian cells in a monolayer and oocytes resting upon that monolayer (Table 8). Control experiments using me-

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TABLE 8 METABOLIC COOPERATIVITYBETWEEN FOLLICLE CELLSAND OOCYTES GROWNIN VITRO

denuded oocytes. Moor et al. (1980) found that cumulusenclosed sheep oocytes took up more radiolabeled uridine, inositol, and choline than did denuded oocytes. Heller and Schultz (1980) reported that cumulus-enCulture system Compound C closed dictyate oocytes took up more radiolabeled riboUridine 7.4 * 0.9 EmiP nucleosides than did denuded dictyate oocytes and that Choline 3.0 L 1.6 the distribution of phosphorylated metabolites of these 2-Deoxyglucose 9.3 -r- 0.8 ribonucleosides differed in cumulus-enclosed dictyate Bachvarova” Uridine 5.2 k 1.3 ZDeoxyglucose oocytes compared to denuded dictyate oocytes. More11.5 * 1.7 over, several of these studies revealed that the extent of c Oocytes were allowed to grow in vitro for 3 days according to the this metabolic cooperativity decreased during meiotic method of Eppig (1977b). A portion of the oocytes was transferred to maturation (Cross and Brinster, 1973; Moor et al., 1980; another dish and freed of adhering follicle cells (see Materials and Heller and Schultz, 1980), a period during which gap Methods-collection of mouse oocytes). These free oocytes and the junctions between the two heterologous cell types are follicle-enclosed oocytes were then cultured in separate dishes overnight in MEM/BSA containing 50 &i/ml of the radiolabeled compound disrupted due to retraction of cumulus cell microvilli under investigation. Values of C were calculated as described in from the oocyte surface (Gilula et al., 1978). Results. Results are average t SD for four separate measurements. In the present study, several lines of experimentation b Oocytes were allowed to grow in vitro for 3 days according to the method of Bachvarova et al. (1980). A portion of the free oocytes was suggest that gap junctions between granulosa cells and growing oocytes are also functional. (1) When incubated transferred to another dish. These free oocytes and oocytes resting upon the ovarian monolayer were cultured in separate dishes overnight with various radiolabeled compounds, follicle-enclosed in MEM/lO% FCS containing 50 ~Ci/ml of the radiolabeled compound oocytes contained more intracellular radioactivity than under investigation. Values of C were calculated as described in did denuded oocytes. (2) For the two radiolabeled riboResults. The results are the average ? SD for 15 and 5 separate nucleosides examined, the distribution of phosphorylated measurements for uridine and 2-deoxyglucose, respectively. metabolites in follicle-enclosed oocytes resembled that of the follicle cells and differed significantly from that in dium conditioned by the ovarian monolayer demondenuded oocytes. (3) Pulse-chase experiments with rastrated that the enhanced uptake of radioactivity found diolabeled ribonucleosides revealed that more radioactivfor oocytes resting on the monolayer, compared to that of ity became associated with the follicle-enclosed oocyte free oocytes, was not due to a diffusable and stable factor secreted by the follicle cells (data not shown; results from similar experiments are described in Table 5). TABLE 9 TRANSFEROFRADIOACTIVITYFROMTHEPRELABELEDOVARIAN Results from experiments in which the ovarian monoMONOLAYERTO~OCYTES RESTINGONTHATMONOLAYER layer was prelabeled with radiolabeled uridine prior to adding naked oocytes on top of that monolayer were conConditions of culture Time (hr) cpmloocyte sistent with metabolic cooperativity between these ovarian cells and oocytes (Table 9). Oocytes initially containOvarian monolayer prelabeled 0 0.1 with radiolabeled uridine” 18 1.2 ing no radioactivity were found to contain significant Medium derived after 18 hr amounts of radioactivity after the incubation with prelaexposure to prelabeled beled ovarian cells. Control experiments in which free 18 ovarian monolayeld 0.1 oocytes were added to the medium conditioned by the ovarian monolayer and denuded oocytes revealed no sig” Oocyte and ovarian cell cultures were initiated using Sday-old mice nificant increase in radioactivity associated with these as described by Bachvarova et al. (1980). Free oocytes were removed from the sample prior to the attachment of the ovarian cells which free oocytes (Table 9). DISCUSSION

Results from previous studies suggested that gap junction mediated metabolic cooperativity exists between cumulus cells and the fully grown dictyate oocyte. Cross and Brinster (1974) observed that dictyate oocytes with attached cumulus cells took up significantly more radiolabeled leucine than did dictyate oocytes without attached cumulus cells. Wassarman and Letourneau (1976) found that uptake of radiolabeled uridine by cumulus-enclosed dictyate oocytes was much greater than that by

were then maintained for 2 days. The monolayer was incubated for 3 hr in MEMBSA containing 50 @Z/ml of [3H]uridine. Extracellular radioactivity was reduced to essentially background levels by repeated washing with MEMBSA. The monolayer was then bathed in MEM/lO% FCS containing 1 mgiml of uridine and free oocytes obtained from g-day-old mice were added to the monolayer. Oocytes were removed immediately or after 18 hr of culture, washed, and processed for determination of intracellular radioactivity. About 50 oocytes were used in each instance. * The medium from the above experiment was removed from the culture dish. Naked oocytes obtained from Sday-old mice were added and cultured in this medium for 18 hr. Oocytes were washed and processed for determination of intracellular radioactivity. About 50 oocytes were used in each instance,

HELLER, CAHILL, AND SCHULTZ

during the chase period. (4) Treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and their enclosed oocytes. (5) A series of control experiments using (a) follicle cell conditioned medium and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not allowed, demonstrated that physical contact between the two cell types was necessary for observing metabolic cooperativity. The equation C/(C + l), where C is the value for the extent of metabolic cooperativity can be used to determine the minimum mole percentage of the radiolabeled compound and its metabolites in the oocytes which originated in the follicle cells. This assumes that the increased amounts of intracellular radioactivity present in follicleenclosed oocytes, compared to that in denuded oocytes, is due to uptake by follicle cells of a particular radiolabeled compound and transfer of that compound and its metabolites via gap junctions to the oocyte. (An explanation as to why this is a minimum value can be found in Heller and Schultz, 1980). In most cases examined, greater than 85% of the metabolites derived from the medium and present in follicle-enclosed oocytes apparently was derived from the granulosa cells. This large somatic cell contribution of nutrients used in gamete growth is consistent with the hypothesis that ooeyte growth may, to some extent, be dependent upon this type of heterologous cell interaction. At present two culture systems have been developed which support mouse oocyte growth in vitro (Eppig, 1977b; Bachvarova et al., 1980). In both of these systems growth appears to be normal in the oocytes display an increased level of CO, evolution from exogenous pyruvate, manifest most of the ultrastructural changes associated with oocyte growth, and acquire the ability to resume spontaneous meiotic maturation in vitro. In the system reported by Eppig (1977b), follicles derived from collagenase digested ovaries attach to the culture dish within a day. Though some granulosa cells migrate away from the follicle, oocytes remained invested by at least one layer of granulosa cells. Gap junctions have been found between these granulosa cells and their enclosed oocytes (Eppig, 197713). Results from the present study suggest that these gap junctions are functional; metabolic cooperativity was found using several radiolabeled compounds. Furthermore, this coupling was found at all stages of oocyte growth in culture (data not shown). In the culture system reported by Bachvarova et al. (1980), mouse ovaries are treated with Pronase and then mechanically disrupted. Ovarian cells attach to the culture dish within a day and denuded oocytes rest upon this monolayer. While gap junctions between the ovarian cells and oocytes have not been looked for morphologi-

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cally, metabolic cooperativity was found. Since it is generally accepted that if metabolic cooperativity is observed between two cells, then gap junctions are present and functional, weak and/or transient gap junctions probably exist between the ovarian cells and oocytes in this culture system. That such junctions could form is likely; preliminary results have shown that metabolic cooperativity can be reestablished in vitro between follicle cells and oocytes (Eppig and Schultz, unpublished observations). While metabolic cooperativity between granulosa cells and growing mouse oocytes has been found in intact follicles and in both in vitro culture systems, the role of metabolic cooperativity in oocyte growth remains to be firmly established. In the Eppig system, oocyte growth occurs only when granulosa cell-oocyte gap junctional contacts are present; in cocultures of free oocytes and follicle cells in which heterologous cell contact did not occur, no or only minimal oocyte growth was observed (Eppig, 1979). In the system described by Bachvarova et al. (1980) metabolic cooperativity occurs between the ovarian monolayer and oocytes and oocyte growth occurs oniy in the presence of follicle cells. However, oocyte growth, in terms of change in volume per unit time (albeit at a rate l/5 that of the control culture), was reportedly found when oocytes were separated from the ovarian cell monolayer by a 0.7-mm layer of agarose (Bachvarova et al., 1980). It is not known if metabolic cooperativity can exist under such circumstances. Current studies correlating the rate of oocyte growth with the extent of metabolic cooperativity should help to clarify the role of metabolic cooperativity between granulosa cells and oocytes in oocyte growth. This research was supported by grants from The Rockefeller Foundation and National Science Foundation (PCM-79-03973) to R.M.S. The authors would like to thank Nicola Neff for helpful discussions and constructive criticisms. R.M.S. would like to thank John Eppig for introducing him to the techniques of oocyte growth in vitro, helpful discussions, and the opportunity to view the beautiful Maine coast at Bar Harbor. REFERENCES ANDERSON, E. (1974). Comparative aspects of the ultrastructure of the female gamete. Znt. Rev. Cytol. (Suppl.) 4, l-70. ANDERSON, E., and ALBERTINI, D. F. (1976). Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. Cell

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ASADO, Y., and BENNETT, M. V. (1971). Experimental alteration of coupling resistance at an electrotonic synapse. J. Cell Biol. 49, 159172. BACHVAROVA, R., BARAN, M. M., and TEJBLUM, A. (1980). Development of naked growing mouse oocyte in vitro. J. Exp. Zool. 211, 159-169. BACHVAROVA, R., and DELEON, V. (1980). Polyadenylated RNA of mouse ova and loss of maternal RNA in early deveiopment. Develop. Biol. 74, 1-8.

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BIGGER& J. D. (1971). New observations on the nutrition of the mammalian oocyte and the preimplantation embryo. In “The Biology of the Blastocyst” (R. J. Blandeon, ed.), pp. 319-382. University of Chicago Press, Chicago. BLEIL, J. D., and WASSERMAN, P. M. (1980). Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Proc. Nut. Acud. Sci. USA 77, 1029-1033. CROSS, P. C., and BRINSTER, R. L. (1974). Leucine uptake and incorporation at three stages of mouse oocyte maturation. Exp. Cell Res. 86, 43-46. DAVIDSON, E. H. (1976). “Gene Activity in Early Development,” 2nd ed. Academic Press, New York. EPPIG, J. J. (1977a). Analysis of mouse oogenesis in vitro: oocyte isolation and utilization of exogenous energy sources by growing oocytes. J. Exp.

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PERACCHIA, C. (1977). Gap junctions. Structural changes after uncoupling procedures. J. Cell Biol. 72, 628-641. PERACCHIA, C., and DULHUNTY, A. F. (1976). Low resistance junctions in crayfish: Structural changes with functional uncoupling. J. Cell Biol. 70, 419-439. PITTS, J. D. (1977). Direct communication in animal cells. In “International Cell Biology” (B. R. Brinkley and K. R. Porter, eds.), pp. 4249. The Rockefeller University Press, New York. PITTS, J. D., and SIMM& J. W. (1977). Permeability of junctions between animal cells: Intercellular transfer of nucleotides but not of macromolecules. Exp. Cell Res. 104, 153-163. SCHULTZ, R. M., LETOURNEAU, G. E., and WASSARMAN, P. M. (1979). Program of early development in the mammal: Changes in the patterns and absolute rates of tubulin and total protein synthesis during oocyte growth in the mouse. Develop. Biol. 73, 120-133. SCHULTZ, R. M., and WASSARMAN, P. M. (1977). Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse. J. Cell Sci. 24, 167-194. SORENSEN, R. A., and WASSARMAN, P. M. (1976). Relationship between growth and meiotic maturation of the mouse oocyte. Develop. Biol.

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SZOLLOSI, D. (1972). Changes of cell organelles during oogenesis in mammals. In “Oogenesis” (J. D. Biggers and A. W. Schultz, eds.), pp. 47-64. University Park Press, Baltimore. Enzymol. 11,59-61. TELFER, W. H. (1975). Development and physiology of the oocytenurse cell syncytium. In “Advances in Insect Physiology” (J. E. TreJAHN, C. L., BARAN, M. M., and BACHVAROVA, R. (1976). Stability of hens, M. J. Berridge, and V. B. Wigglesworth, eds.), Vol. 11, pp. RNA synthesized by the mouse oocyte during its major growth 223-319. Academic Press, New York. phase. J. Exp. Zool. 197, 161-172. THIBAULT, C. G. (1972). Final stages of mammalian oocyte maturation. LOWENSTEIN, W. R. (1979). Junctional intercellular communication In “Oogenesis” (J. D. Biggers and A. W. Schultz, eds.), pp. 397-411. and the control of growth. Biochim. Biophys. Acta 560, l-65. University Park Press, Baltimore. LOWENSTEIN, W. R., NAKAS, M., and SOCOLAR, S. J. (1967). JuncWASSARMAN, P. M., and JOSEFOWICZ, W. J. (1978). Oocyte developtional membrane uncoupling. Permeability transformations at a cell ment in the mouse: A ultrastructural comparison of oocytes isolated membrane junction. J. Gen. Physiol. 50, 1865-1891. at various stages of growth and meiotic competence. J. Morphol. MOOR, R. M., SMITH, M. W., and DAWSON, R. M. C. (1980). Measure156, 209-236. ment of intercellular coupling between oocytes and cumulus cells WASSARMAN, P. M., and LETOURNEAU, G. E. (1976). RNA synthesis using intercellular markers. Exp. Cell Res. 126, 15-29. in fully-grown mouse oocytes. Nature (London) 261, 73-74. PAPPAS, G. D., ASADA, Y., and BENNETT, M. V. L. (1971). MorphologWEAKLEY, B. S. (1968). Comparison of cytoplasmic lamellae and memical correlates of increased coupling resistance at an electrotonic synbrane elements in oocytes of five mammalian species. Z. Zellforsch. apse. J. Cell Biol. 49, 173-188. Mikrosckop. Anat. 85, 109-123. PEDERSEN, T. (1969). Follicle growth in the immature mouse ovary. ZAMBONI, L. (1970). Ultrastructure of mammalian oocytes and ova. Acta Endocrin. 62, 117-132. Biol. Reprod. Suppl. 2, 44-63.