0400-8166/90/0022-0827/$10.00

TISSUE AND CELL, 1990 22 (6) 827-851 @ 1990 Longman Group UK Ltd.

S. E. WERT* and W. J. LARSENt

PREENDOCYTOTIC ALTERATIONS IN CUMULUS CELL GAP JUNCTIONS PRECEDE MEIOTIC RESUMPTION IN THE RAT CUMULUS-OOCYTE COMPLEX Keywords:

Oocyte maturation, dihydrocytochalasin

intercellular communication. B, freeze-fracture electron

membrane microscopy

fusion, gonadotropins.

ABSTRACT. Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles; 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%. while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.

Introduction

Biggers, 1972). Results obtained by several laboratories suggest that this inhibitor is either secreted into the follicular fluid or is transmitted directly from the surrounding follicle cells to the oocyte cytoplasm through an extensive network of gap junctions that interconnects the entire follicle cell population with its enclosed oocyte (Tsafriri et al., 1982; Eppig and Downs, 1984; Tsafriri and Pomerantz, 1986). It has also been suggested that the disruption of this latter communication pathway between the source of the inhibitor, the membrana granulosa cell compartment, and its target, the oocyte, may be a pivotal event in the control of meiotic resumption during the preovulatory period (Gilula et al., 1978; Dekel and Beers, 1980; Dekel et al., 1981). Quantitative ultrastructural studies in our laboratory have provided support for this

Numerous reports over the past five decades

have implicated ovarian follicle cells in the regulation of meiotic arrest and maturation in the mammalian oocyte (Reviewed by Biggers, 1972; Tsafriri et al., 1982; Eppig and Downs, 1984; Schultz, 1985; Tsafriri and Pomerantz, 1986; Larsen and Wert, 1988). The observation that fully-grown oocytes undergo spontaneous meiotic resumption shortly after removal from the follicle supports the idea that these follicle cells produce a factor that is inhibitory to meiotic progress (Pincus and Enzmann, 1935; Edwards, 1965; * Department of Pediatrics, Division of Neonatology and t Department of Anatomy and Cell Biology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, USA. t To whom correspondence should be addressed. Received

23 August

1990 827

WERT AND LARSEN

latter hypothesis (Larsen et al., 1986, 1987). We have demonstrated that there is a temporal correlation in uiuo between the rapid down-regulation of gap junctions within the cumulus cell mass and meiotic resumption of the oocyte 2-3 hr after an ovulatory stimulus (Larsen et al., 1986). Gap junctions within the membrana granulosa are also down-regulated but less dramatically and over a longer period of time beginning just prior to meiotic resumption (Larsen et al., 1987). On the other hand, the nearly complete loss of gap junctions at the oocyte surface does not occur until later in the preovulatory period after meiotic resumption is complete (Larsen et al., 1987). These results have led us to suggest that the down-regulation of cumulus-tocumulus cell gap junctions plays a central role in signalling the oocyte to resume meiotic progress by interrupting the transfer of gapjunction-mediated inhibitory factors from the membrana granulosa cell compartment to the oocyte via its surrounding cumulus cells. This hypothesis is based primarily upon the very tight temporal correlation between cumulus cell gap junction down-regulation and germinal vesicle breakdown (GVBD) in the oocyte (Larsen et al., 1986). Results obtained by other laboratories, however, suggest that mammalian oocytes actually become committed to resume meiosis 3O60 min prior to GVBD (Dekel and Beers, 1978; Schultz et al.. 1983; Aberdam et al.. 1987; Racowsky et al., 1989), a morphological observation that is normally interpreted as the first sign of meiotic resumption. The observations reported here, which demonstrate significant morphological reorganization of cumulus cell gap junction plaques 1-2 hr prior to their loss and to the initiation of meiotic resumption, may reconcile this apparent contradiction. Earlier studies from our laboratory have provided evidence that both membrana granulosa and cumulus cell gap junctions in the preovulatory mammalian follicle are removed from the cell surface by an endocytotic process that is facilitated by actin and clathrin and results in the fusion of gap junction cytoplasmic vesicles with lysosomes prior to their degradation (Larsen and Tung, 1978; Larsen et al., 1979; Larsen et al.. 1981; Larsen et al., 1987; Murray et al., 1981; Risinger and Larsen, 1981, 1983; Wert and Larsen, 1989). The more recent studies of

gap junction behavior described in this report suggest that follicle cell gap junctions may undergo a variety of transformations at the cell surface prior to endocytosis. These include 1) the transformation of gap junction plaques from a planar to an irregular topography; 2) the lateral dispersion of junctional particles within the membrane; and 3) a phenomenon reported here for the first time, the clearing of non-junctional particles and the apparent fusion of apposed membranes around the periphery of large preendocytotic gap junction plaques. This report documents these preendocytotic alterations in cumulus cell gap junctions in intact follicles that have responded to an ovulatory stimulus and demonstrates that significant quantitative alterations in these parameters occur an hour or more before junctional endocytosis and GVBD. Similar changes in junctional behavior preceding GVBD are also observed in cumulus-oocyte complexes (COCs) removed from hormonally primed animals and incubated in culture medium follicle-stimulating supplemented with hormone (FSH). We suggest that these changes reflect initial steps in the functional down-regulation of cumulus cell gap junctions in these systems and conclude that junctional remodelling commits these junctions to their subsequent uptake through the actinfacilitated endocytotic process we have previously described (Larsen and Tung. 107X; Larsen et al., 1979; Wert and Larsen. 1Y8Y). If gap junction endocytosis in the cumulus cell mass disrupts the transmission of meiosis inhibitory factors to the oocyte, then the initiation of this process through the mechanisms described in this report may, in turn, commit the oocyte to meiotic resumption. Materials and Methods Animals Three-week-old Sprague Dawley rats (Harlan, Indiana) were Indianapolis, injected with 2Oi.u. of pregnant mare’s serum gonadotropin (PMSG) (Gestyl@. Diosynth. Chicago. Illinois) to induce follicular growth. Rats utilized for in uoo studies were then injected 48 hr later with 10 i.u of human chorionic gonadotropin (hCG) (Sigma Chemical Co.. St. Louis, MO) to induce ovulation. Stock solutions of PMSG and hCG were prepared in sterile physio-

PREENDOCYTOTIC BEHAVIOR OF GAP JUNCTIONS

logical saline and delivered intraperitoneally to each animal in 0.1 ml volumes. Groups of animals were housed 3-4 per cage with a 1ight:dark cycle of 14:lO hr and fed Purina rat chow and water ad libitum. Animals were killed by cervical dislocation or decapitation in accordance with established NIH guidelines for the use and care of experimental animals (Smith et al., 1986). Cell isolation and culture COCs were isolated from responsive follicles

48 hr after PMSG injection (0 hr) or l-3 hr after an ovulatory dose of hCG as previously described (Larsen et al., 1986, 1987; Wert and Larsen, 1989). COCs were collected in Leibovitz’s L1.5 complete tissue culture medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 10 pg/ml hydrocortisone (Sigma Chemical Co., St. Louis, MO), 0.01 u/ml of NPH human insulin isophane (Lilly, Indianapolis, IN), 60 ,ug/ml of penicillin-G (Sigma Chemical Co., St. Louis, MO), lOOpg/ml of streptomycin sulfate (GIBCO, Grand Island, NY), and 2.5pg/ ml fungisone (GIBCO, Grand Island, NY). COCs from preovulatory follicles were fixed for freeze fracture analysis within lo-15 min of isolation. Junctional parameters did not change significantly during a subsequent 15 min culture period at room temperature, suggesting that there were no detectable changes in gap junction morphology, area, or number during the dissection period (Wert and Larsen, 1989). For in vitro analyses, COCs were liberated into L15 complete medium with or without the addition of 10% FBS, 0.005 Armour units/ml of porcine FSH (Sigma Chemical Co., St. Louis, MO), or 10 PM dihydrocytochalasin B (Sigma Chemical Co., St. Louis, MO) as previously described (Wert and Larsen, 1989). In both model systems, at least 30-4OCOCs were pooled from the ovaries of 3-4 primed animals for each data point described below. Analysis of meiotic resumption

Isolated COCs were fixed for freeze fracture as described below and then scored for GVBD by examining them at magnifications of 200-320X with a Zeiss Photomicroscope II equipped with differential interference contrast optics (Wert and Larsen, 1989).

829

Thin-section electron microscopy COCs were fixed in a freshly prepared

solution of 0.2% tannic acid (Mallinckrodt, Paris, Kentucky, #KJEB)and2.5% glutaraldehyde in 0.05 M sodium cacodylate (pH7.2) at room temperature for 60 min. The cells were washed in 0.05 M sodium cacodylate buffer with 10% sucrose, post-fixed in 1% osmium tetroxide in 0.05 M sodium cacodylate (pH7.4) for 30 min, and washed again in sodium cacodylate buffer before being dehydrated in a graded series of ethanols (70100%). COCs were then rinsed in propylene oxide for 15 min, allowed to sit overnight in a 1: 1 solution of propylene oxide and Araldite (Cargille Laboratories, Cedar Grove, NJ), embedded the next day in 100% Araldite, and cured for 48 hr at 60°C under a slight vacuum. Ultrathin sections were cut with a diamond knife on a Sorvall MT2-B ultramicrotome and were mounted on 300 mesh copper grids prior to staining with 0.5% uranyl acetate and Reynold’s lead citrate. Quantitative freeze-fracture

analysis

COCs were fixed at room temperature

for 10-15 min in 2.5% glutaraldehyde in 0.05 M sodium cacodylate with 2% sucrose at pH 7.2. After washing in 0.05 M sodium cacodylate buffer, the cells were infiltrated with 30% glycerol for 1 hr and then sandwiched between 2, gelatin-filled (6%), Balzer’s mirror-image holders. The samples were frozen at -155°C in liquid Freon, inserted into a mirror-image device, fractured in a Balzer’s 301 freeze fracture apparatus (Balzer’s High Vacuum Corp., Nashua, NH) at -117°C and vacuums of 2 x 10m6-8 X lo-’ torr, and replicated with platinum and carbon. The replicas were then coated with 1% pyroxylin in amyl acetate, floated off the specimen holder onto double distilled water, cleaned in Chlorox, rinsed in double distilled water, mounted on 300 mesh copper grids, and examined in a JEOL 1OOCX electron microscope. For each timepoint, 100-400 micrographs of all protoplasmic (P) fracture face membrane from at least 2 different replicas were photographed at a magnification of x 10,000 (Larsen et al., 1986; Wert and Larsen, 1989). Gap junction areas were measured with a Zeiss Videoplan image analysis system from tracings of individual negatives enlarged to ~40,000. Approximately 10% (-25 ym2) of

WERT

830

Figs. 1-4. the course

Four

distinct

or non-rectdinear

Fig. 2. Crystalline

or rectilinear

separated

3. Loose

endocytotic hg.

at the cumulus

ccl1 surface

during

PPPs containing

tightly

packed

but randomly

crganzcd

x167,ooO.

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PPPs were observed

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Fig. 1. Disordered particles.

gap junction

AND

PPP,

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4. Mxed

PPPs containing

by particle-free containing

containing

PPPs containing

aisles. varying

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tightly

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LARSEN

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OF GAP JUNCTIONS

one cumulus cell’s total surface area was present as P fracture face in each negative. The total membrane area scrutinized was equivalent in area to at least six complete cell surfaces for each experimental timepoint. Measurements of all P face gap junctions, as well as both P and E (exoplasmic) fracture face components of junctional plaques when present in conjunction with one another in a micrograph, were analyzed to assess particle packing patterns (PPPs), junctional area distributions, average junctional areas, and junctional number per cell. Gap junctions were classified into four separate PPPs based upon the following criteria: (1) tightly packed junctional plaques containing randomly packed particles in contact with one another (disordered or nonrectilinear gap junctions) (Fig. 1); (2) organized junctional plaques containing rows or islands of tightly packed particles separated by particle-free aisles (crystalline or rectilinear gap junctions) (Fig. 2); (3) loosely packed junctional plaques containing varying degrees of dispersed particles arranged in plaque-like configurations (Fig. 3); and (4) mixed junctional plaques containing areas of both tightly and loosely packed particles (Fig. 4). The number of gap junction particles per pm* for each PPP was estimated from the measurements of cumulus cell gap junctions found on five randomly selected negatives for 3 of the 4 observable PPPs (i.e., tight, organized, and loose PPPs). Particle densities for mixed junctions were not quantitated since the proportion of tightly packed to loosely packed areas varied from one junction to the next for this PPP. Each negative was enlarged to X72,000 and a grid of squares measuring 10 mm* was superimposed on each print. Gap junction particles from 5 randomly selected grid squares overlying each gap junction plaque were counted, and the results were reported as the average number of particles per pm* of gap junction area. Statistical differences between particle packing densities were analyzed by ANOVA and Duncan’s Multiple Range Test using the SAS computer program (SAS Institute, Inc. 1982). Significant differences among particle packing densities were approximated at alpha = 0.05.

831

Results Qualitative responses of cumulus cell gap junctions to an ovulatory stimulus or to isolation in culture medium

In the present study we have examined the morphology of cumulus cell gap junctions in COCs harvested from PMSG-primed animals stimulated with hCG or isolated into culture medium prior to junctional loss. Careful analysis of electron micrographs from these early time points (&2 hr) reveals that the larger gap junctions undergo at least three major structural transformations in response to an ovulatory stimulus in vivo or to incubation in culture medium containing FSH. These transformations include 1) the change from an originally planar topography to irregular topographical profiles, 2) the lateral dispersal of gap junction particles within the membrane, and 3) the irregular fusion of particle-free, non-junctional membranes from apposing cells at the periphery of junctional aggregates. The first two changes have been described previously in rabbit membrana granulosa cell gap junctions in preovulatory follicles (Larsen et al., 1981), but the fusion and ruffling of membrane at the edges of preendocytotic gap junctions is reported here for the first time. Topographical changes. Junctional topography undergoes a radical change in response to an ovulatory stimulus in vivo or to isolation of COCs into FSH-containing culture medium. Virtually all control cumulus cell gap junctions in PMSG-primed animals are rigidly planar in character or demonstrate a minimal regular curvature (Figs 5, 6). This topographical regulatory is lost, particularly in large junctions, in specimens stimulated l-2 hr previously with hCG or in complexes isolated into FSH-containing medium 1530 min earlier. A large proportion of these junctions develop bumps, blebs, endocytotic pits and corrugated irregularities and exhibit more loosely packed or dispersed gap junction particles (Figs 7, 8, 11). Particle packing patterns. A radical shift in particle packing patterns and densities is observed prior to the loss of cumulus cell gap junctions in hCG- or FSH-treated COCs. In control complexes from PMSG-primed animals, the majority of gap junction plaques

PREENDOCYTOTIC

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OF GAP JUNCTIONS

are composed of tightly clustered gap junction particles. These can be classified further into either tightly-packed, randomly organized gap junctions (Fig. 5) or into tightlypacked, highly organized or crystalline gap junctions composed of closely packed islands or rows of 10-80 particles separated by particle-free aisles (Fig. 6). This latter morphological type of gap junction is prominent on the cumulus cell membrane of control complexes but disappears entirely from the cumulus cell surface of hCG-stimulated complexes and isolated COCs incubated with FSH. Loosely packed gap junctions are prominent in uivo 2 hr after hCG stimulation and exhibit varying degrees of dispersed particles arranged in plaque-like configurations (Fig. 8). Large mixed junctions containing areas of both tightly and loosely packed particles are prominent 1 hr after hCG stimulation (Fig. 7) and may actually represent early remodeling of tightly packed junctions into the loosely organized junctional plaques found 1 hr later. Both mixed and loosely organized junctions are also observed in isolated COCs incubated with serum and FSH. The particle packing density of tightly packed cumulus cell gap junctions in control complexes is significantly higher than that observed in the loosely organized gap junctions of COCs stimulated l-2 hr earlier with hCG (Table 1). Measurements of randomly selected micrographs of loosely organized gap junctions found in cultured COCs also indicate a significant difference in particle packing density in response to incubation with serum and FSH (3220 per ,um* t SE 74, p < 0.05). In both systems, small residual gap junctions are found at the cumulus cell surface after the majority of gap junction membrane has been lost and now represent only 6%-U% of the total gap junction area

833

originally observed in the control complexes. Although most of these small junctional fragments exhibit tightly packed PPPs, many examples of loose PPPs and dispersing particles can be found (Figs 9, 10). These are often arranged into linear or circular configurations of 20-30 small junctions. RufJEing and fusion of non-junctional membrane at the periphery of cumu1u.r gap junctions. A dramatic effect of hCG stimulation of COCs in viva and of the incubation of

mature COCs in culture is the apparent fusion and ruffling of particle-free non-junctional membrane at the periphery of many cumulus cell gap junctions. This response is an obvious and common feature in these specimens and has been observed repeatedly in all cumulus cell membranes from intact follicles stimulated l-2.5 hr earlier with hCG (Figs 11, 12) and in COCs isolated into FSHcontaining medium 15-30 min earlier (Figs 14, 15). These ruffled membranes represent regions of membrane fusion between the surfaces of apposing cells, as indicated by the complete ablation of the intercellular space in these regions. Quantitation of particle packing patterns and membrane fusion in response to hCG in vivo

We have carried out a quantitative analysis of changes in PPPs and membrane fusion and ruffling of cumulus cell gap junctions responding to an ovulatory stimulus in vivo. Examination of cumulus cell membrane in Otime controls, at 1 hr, 2 hr, and 2.5 hr reveals significant changes in these parameters prior to cumulus cell gap junction down-regulation and GVBD in the oocyte.

Fig. 5. Large, tightly packed gap junction found at the cumulus cell surface prior to an ovulatory stimulus. Typically, there is about 1 such large junction per cell which contributes to -50% of the total gap junction membrane area per cell (Larsen er al., 1987). Junctional area = 2.65 pm*. ~48,000. Fig. 6. Tightly-packed, crystalline gap junction separated by particle-free aisles. Such organized membranes and contribute to 25% of the total gap to 0% following an ovulatory stimulus. Junctional

with particles organized into rows that are junctions are found predominantly on 0 hr junction area. This value subsequently drops area = 3.65 pm2. x48,lJW.

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835

Table 1. Particle densities as a function of particle packing patterns in cumulus cell gap junctions PPP

No. Particles/ym* Gap Junction Area

N

6431.6 2 155* 5933.7 2 154* 3514.0 + 150*

25 25 25

Tight: Random Tight: Ordered Loose

Particle densities were determined as described in Materials and Methods. Values are the mean r SEM from counts performed on a total of 25 randomly selected 10 mm’ grid squares spread over 5 randomly selected negatives of gap junctions from each PPP category. All 3 PPPs are statistically different from one another based on Duncan’s Multiple Range Test with alpha = 0.05.

Shifts in particle packing patterns prior to gap junction loss and GVBD. Our analysis of

non-hCG-stimulated cumulus cells reveals that the percentage of junctional area devoted to tightly packed particles is relatively high compared to junctions with loosely packed particles (Fig. 16). In these cells, 89% of the total gap junction area consists of tightly packed junctional particles, 26% of which (by area) are highly organized into rows or islands of particles separated by particle-free aisles. Four percent of the total gap junction area in these cells exhibit loose PPPs of randomly dispersed particles, while 7% contain a mixture of both tight and loose PPPs. Within 1 hr after an ovulatory stimulus the area of junctions with tightly packed particles decreases by 50%, while the total area of junctions exhibiting a mixed pattern increases seven fold to 47% of the total gap junction area. Highly organized junctions, so plentiful in control animals, virtually disappear and the incidence of junctions containing loosely packed particles remains low at 9% of the total gap junction area. By 2 hr, however, gap junctions containing loosely packed particles increase to 42% of the total

gap junction area at the expense of both tightly packed and mixed junctions. 2.5-3 hr after stimulation, when 63%-94% of the gap junction membrane has been removed from the cell surface (Larsen et al., 1986), the distribution of these four PPPs once again approaches that observed in the non-hCGstimulated COC. Significant differences in gap junction mean areas and numbers per cell are also found between these four PPPs (Fig. 17). Mixed junctions are consistently the largest gap junctions at the cell surface with an overall mean of 2.05 pm*. Organized junctions, when present, are the second largest with a mean size of 0.75 pm*, followed by loosely packed junctions that average 052pm* in area. Although tightly packed junctions are the most numerous, they are also the smallest, averaging only 0.18 pm* in size. These small junctions increase proportionally at 2.5 hr just after the largest gap junctions disappear from the cell surface (Fig. 17). Since the total gap junction area is decreased by 63% at this time point, this increase in small, tightly packed gap junctions may reflect the incomplete endocytosis of the

Fig. 7. Large, mixed gap junction exhibiting both tight and loose particle packing patterns. Note evidence of endocytotic pits (arrows) in areas of low particle packing density. Mixed junctions are found at the cumulus cell surface 1 hr after hCG injection and represent 47% of the total gap junction area. These are the largest junctions found at the cell surface with a mean size of 2.05 pm* and a median of 1.50 pm’. Junctional area = 5.38 pm2. Corresponding gap junction pits can also be seen on the E-fracture face. ~48,000. Fig. 8. Large, loosely packed gap junction typically found 2 hr after an ovulatory stimulus. Junctions with this particle packing pattern represent 42% of the total gap junction area surveyed at this time point. Note evidence of endocytotic pits and forming vesicles (arrows) and unusual, particle-free, ruffled membrane at the junctional periphery (arrowheads). Junctional area = 4.46pm*. Corresponding gap junction pits can also be seen on the E-fracture face. x48$00.

836

WERT AND LARSEN

Figs. 9-10. Clusters of small cumulus cell gap Junctions found in COCs isolated 2.5 hr after hCG injection. Although most of the junctional remnants at this timepoint exhibit tight PPPs (arrows). particle-free areas of ruffled membrane (arrowhead) can be found in association with loose PPPs in 20% of these small junctions x46.250.

Fig. 11 This large, mixed gap junction exhibits unusual. particle-free ruffled or puckered membrane around its periphery (arrows). Note the thin line at the edges of these ruffles (arrowheads) where the E- and P-fracture faces are tightly opposed. Junctions exhibiting ruffled borders represent 44% of the total gap junction area 2 hr after hCG injection. Junctional area = 5.38pm*. Corresponding gap junction pits can also be seen on the E-fracture face. x48.000. Fig. 12. Cross section of a large cumulus cell gap junction showing areas of fusion (arrows) and ruffling and splitting of the adjacent cell membranes at one end of &hegap junction profile. x97,000. Fig. 13. Cumulus cell gap junctions are internalized as they are lost from the cell surface and appear as gap junction vesicles within the cell’s cytoplasm. x97,OW.

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839

I, i!

a ;; $ E 8 8

n n

80

0

TIGHT: RANDOM TIGHT: ORDERED DISPERSED MIXED

60 40 20 0

% GJ LOSS: %GVSD:

0

1

215

S

0

0

0

63

94

0

0

10

47

M

HR IN VW0

Fig. 16. Shifts in gap junction PPPs occur l-2 hr prior to cumulus cell gap junction loss. Note the shift from highly organized and tight PPPs to mixed and loosely packed patterns between hours 0 and 2. Over 60% of cumulus cell gap junction membrane is lost between hours 2 and 2.5 and by 3 hr this value has risen to 94% (Larsen er nl., 1986). By 3 hr, gap junction PPPs are again approaching that seen in the unstimulated mature follicle (0 hr). Note that reorganization of cumulus cell gap junction PPPs is initiated -1 hr prior to the onset of GVBD in the oocyte.

larger mixed and loosely packed junctions previously seen at 2 hr. Membrane ruffling and fusion. These shifts in particle packing patterns are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the periphery of many junctions as described above. One hour after hCG stimulation, 29% of the total gap junction area is surrounded by these unusual membrane perturbations (Fig. Da). By 2 hr, this value reaches a maximum of 44% and then decreases to 22%, 2.5 hr after hCG stimulation when more than half of the cell’s total gap junction membrane has been lost from the cell surface. No regions of membrane fusion are observed in cumulus cell

Figs 14-15. Morphological

changes

in cumulus

membranes 3 hr after hCG stimulation, when gap junction loss is virtually complete. In general, membrane fusion profiles are associated with the larger mixed and loosely organized gap junctions found at all 3 timepoints. However, both the mean and median areas of gap junctions surrounded by these fusion profiles decrease dramatically between 1 and 2.5 hr after an ovulatory stimulus (Fig. 18b), once again reflecting the loss of large gap junctions from the cell surface (Larsen et al., 1987). Quantitation qf particle packing patterns and membrane fusion in COCs incubated in vitro

Cumulus cell gap junctions are lost rapidly from the cell surface when isolated COCs are

cell gap junctions

in response

to FSH.

Fig 14. Example of a down-regulating cumulus cell gap junction observed in COC’s incubated for 30 min with FSH. Note the more loosely packed, randomly dispersed particles with unusual puckering of the membrane around the periphery of the junction. The intercellular space narrows significantly at the edges of the junction (arrows) indicating the close apposition and fusion of neighboring cell membranes at this point. ~50,000. Fig. 15. Higher magnification of FSH-treated junctions. Note again the randomly dispersed gap junction particles surrounded by puckered membranes and the extremely close apposition of membrane in these regions with virtually no intercellular space between (arrows). Corresponding gap junction pits can also be seen on the E-fracture face. ~60,750.

840

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DISPERSED MIXED

Li

2:s %GJ LOSS:

0

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94

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60 0

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HR IN VIVO

Fig. 17. Fluctuations in cumulus cell gap junction mean area (A) and number a function of PPPs and in response to an ovulatory dose of hCG. Mixed junctions larger than junctions exhibiting other PPPs, but are only found at a frequency Tightly packed junctions, on the other hand, are not only much smaller but numerous than those in any other category.

incubated at 37°C in tissue culture medium (Wert and Larsen, 1989). This loss occurs before GVBD and is enhanced by the addition of serum or serum and FSH. On the other hand, the addition of dihydrocytochalasin B to the medium completely inhibits gap junction loss, suggesting that an actinbased contractile system is involved in the endocytotic removal of gap junction membrane from the cell surface (Wert and Larsen, 1989). Shifts in gap junction PPPs and the appearance of membrane fusion profiles at the gap junction periphery have now been quantitated in these isolated COCs. While the appearance of ruffled membranes is correlated with the disappearance of gap junctions from the cell surface, only those cells

per cell (B) as are consistently of l-3 per cell. are also more

exposed to FSH exhibit loosely packed or dispersing gap junction particles. Particle packing patterns vary in response to tissue culture additives. A quantitative analy-

sis of gap junction PPPs found on the cumulus cell surface was performed for COCs incubated under three different culture conditions (Fig. 19). Each experimental condition yielded distributions of gap junction PPPs that differed from one another. In serum free medium (Fig. 19a), the percentage of tightly packed junctions (both organized and randomly-packed) remains at about 90% of the total gap junction area throughout the initial period of gap junction loss (&30min) and subsequent periods of

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0

0 % GJ LOSS: 0 %GVBD:

0

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50

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Fig. 18. Quantitative analysis of cumulus cell gap junction areas associated with ruffled membrane profiles. (A) Total gap junction surface area associated with ruffled membrane increases prior to loss from the cell surface in hCG-stimulated follicles. (B) Analysis of the means reveals that ruffled membranes tend to be associated with medium-sized gap junctions prior to gap junction loss

gap junction reestablishment (30-60 min) and stabilization (60-120min). In fact, the percentage of organized gap junctions increases from 29% to 60% of the total gap junction area coincident with the reappearance of gap junctions at the cell surface. As previously reported, the addition of serum to the medium enhances cumulus cell gap junction loss which results in a slower but more sustained course of junctional loss than that observed in serum free medium (Wert and Larsen, 1989). Quantitative analysis of gap junction PPPs in this group (Fig. 19b) reveals an increase in the amount of gap junction membrane composed of mixed junctions containing areas of both tightly packed and dispersed particles. The percentage of tightly packed junctions (both organized and randomly-packed) decreases

initially from 90% to 60% of the total gap junction area, while that composed of mixed PPPs increases from 7% to 34%. Once again the organized, or rectilinear, PPP contributes more to the total amount of tightly packed junctional membrane than was observed at any time in uivo. (Compare in uivo levels of O-26% with those in this group of 40-60%.) The addition of FSH to serum-containing medium accelerates and augments cumulus cell gap junction loss in comparison to that observed in the control groups described above (Wert and Larsen, 1989). In this case, the shifts in PPPs are very similar to that observed in uiuo in the hCG-stimulated COC (Fig. 19c and 16 respectively). Tightly packed junctions drop to 27% of the total gap junction area within 15 min of exposure to FSH, while loose and mixed junctions increase

WERT

R42

0 %GJ

LOSS:

%GVBD:

6.

0

0.5

1

2

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71

43

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with different tissue culture in serum-free medium exhibit the culture medium increases PPPs. (C) Finally, in FSHafter hCG stimulation with a

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Fig. 20. The incidence of gap junction membranes associated with membrane fusion profiles and ruffling in isolated COCs incubated with different tissue culture additives. Gap junction loss. shown here as a decrease in gap junction fractional area (Wert and Larsen, 1989), is plotted as well. (A] Serum-free medium (B) Serum-containing medium (C) FSH-containing medium. Fusion profiles composed of smooth, particle-free, ruffled membrane at the periphery of cumulus cell gap junctions are observed in all of the above treatment groups where gap junction loss is also observed.

WERT AND LARSEN

dramatically to a combined value of 71% of the total junctional area. Highly organized junctions almost are nonexistent, representing only 2% of the total gap junction area. The percentages of gap junction membrane exhibiting mixed and loose PPPs remain high until 2 hr into the incubation period when over 80% of the cells’ gap junction membrane has disappeared from the surface. As in vivo, the distribution of PPPs subsequent to this loss reapproaches that seen inthe control complexes at 0 hr. COCs incubated for 16 hr with dihydrocytochalasin B to prevent gap junction loss, however, exhibit only tightly packed and organized gap junction. Membrane rufffing and fusion. Fusion profiles composed of smooth, particle-free, ruffled membrane at the periphery of cumulus cell gap junctions are observed in all of the above treatment groups where gap junction loss is also observed (Fig. 20). In FSH-treated COCs, ruffled and fusing membranes are seen primarily in association with loose and mixed PPPs. These fusion profiles are also seen, however, in association with the highly organized gap junctions that are found predominantly in COCs incubated in medium without added FSH. Finally, no evidence of membrane fusion is seen in those COCs incubated for 16 hr with dihydrocytochalasin B to inhibit gap junction loss.

Discussion Gap junction remodelling is correlated with commitment to resume meiosis in vivo

In 193.5, Pincus and Enzmann demonstrated that meiotic arrest in mammalian oocytes is maintained by an inhibitory factor or factors within the follicle. These investigators found that fully-grown, meiotically competent oocytes resumed meiotic maturation when they were surgically removed from the follicular environment and cultured in a nutrient medium. Normally, meiosis resumes in the follicle-enclosed rat oocyte within 2-3.5 hr following an ovulatory stimulus (Tsafriri and Kraicer, 1972; Dekel et al., 1979; Larsen and Wert, 1986). It has been suggested that the stimulus to resume meiotic progress in mice and rats results from a drop in the con-

centration of a meiosis inhibitory factor within the oocyte itself just prior to meiotic resumption (Schultz et al., 1983; Freter and Schultz, 1984; Racowsky, 1984; Aberdam et al., 1987). Schultz et al. (1983) have demonstrated that the concentration of cyclic adenosine monophosphate (CAMP), a well characterized inhibitor of GVBD (Cho et al., 1974), drops rapidly about 30-60 min prior to meiotic resumption in mouse oocytes of intact follicles. Other experiments have correlated this drop in oocyte CAMP levels with the regulation of phosphodiesterase activity within the oocyte (Bornslaeger et al., 1986) and with levels of CAMP dependent protein kinase and the metabolism of oocyte phosphoproteins (Bornslaeger ef al., 1986). Studies of isolated mouse COCs demonstrate that the concentration of CAMP within the oocyte also drops significantly prior to GVBD (Schultz et al., 1983; Vivarelli et al., 1983; Aberdam er al., 1987). Indeed, CAMP concentrations in the oocyte appear to drop concurrently with the commitment of the oocyte to resume meiotic maturation under the conditions employed in these studies (Schultz et al., 1983). Briefly. COCs are isolated into medium containing a concentration of isobutylmethylxanthine (IBMX) sufficient to arrest meiotic maturation for several hours. When these complexes are removed from IBMX-containing medium and cultured for 30 min before being returned to IBMXcontaining medium, meiotic arrest is maintained. If, however, the period of incubation in IBMX-free medium is extended to 60 min before the return of the COCs to IBMXcontaining medium, the oocytes will resume meiotic maturation. Significant decreases in oocyte CAMP levels after 45-60 min in IBMX-free medium suggest that commitment to meiotic maturation parallels decreasing CAMP levels. Similar observations have also been reported for rat oocytes (Dekel and Beers, 1978; Dekel and Beers, 1980; Aberdam et al., 1987). These experiments suggest that oocytes in isolated COCs become committed to meiotic resumption in response to changes induced by a depression in CAMP concentration. Based upon several related studies, Bornsiaeger et al. (1986) have suggested that this event involves the dephosphorylation of proteins that regulate meiotic progress in some as yet unknown manner. Taken together, these results suggest that

845

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preovulatory oocytes become committed to meiotic resumption between 30 and 60 min prior to GVBD and that CAMP, or a regulator of CAMP, in the oocyte may control meiotic maturation through the regulation of CAMP-dependent protein kinases and protein dephosphorylation (Schultz et al., 1983; Bornslaeger et al., 1986). Conversely, mechanisms which maintain CAMP concentrations at preovulatory levels prevent meiotic maturation and may be dependent upon the transfer of CAMP from the surrounding follicle cells to the oocyte via their gap junctions (Bornslaeger and Schultz, 1985; Salustri et al., 1985; Aberdam et al., 1987). Other inhibitory factors have also been proposed including a low-molecularweight peptide, oocyte maturation inhibitor (OMI), of less than 2000 Daltons isolated from follicular fluid (Tsafriri and Pomerantz, 1986); changes in steroid metabolism in the follicle (Racowsky, 1983, 1985; Kaji et al., 1987); and hypoxanthine, also a component of follicular fluid (Downs et al., 1985; 1986; Eppig et al., 1983; Eppig and Downs, 1987). Since it has been suggested that the effect of OMI and CAMP on the oocyte are mediated by its surrounding cumulus cells (Hillensjo et al., 1979; Tsafriri et al., 1982; Eppig et al., 1983; Eppig and Downs, 1984), the presence of an extensive network of gap junctions between these cells (Albertini and Anderson, 1974; Gilula et al., 1978, Anderson and Albertini, 1976; Amsterdam et al., 1976) has stimulated speculation that these intercellular junctions serve as a pathway for the transfer of follicle-cell-generated inhibitory factors to the oocyte (Gilula et al., 1978; Anderson, 1979). Consequently, it has also been postulated that a disruption of this pathway could (1) interfere with the transfer of these inhibitors to the oocyte, thus stimulating meiotic resumption (Gilula et al., 1978; Dekel and Beers, 1980; Moor et al., 1980), or (2) reinforce a previous signal to resume meiosis, thus allowing meiotic maturation to go to completion (Schultz, 1985; Larsen and Wert, 1988). We have proposed that meiotic resumption is dependent upon the disruption of this communication pathway and interruption of the transfer of potential inhibitor from the membrana granulosa cell compartment to its target within the oocyte. Although we have shown that the rapid down-regulation of gap junctions within the

cumulus oophorus could provide this functional signal (Larsen et al., 1986), gap junction loss does not occur until GVBC is initiated, 30-60 min after decreases in CAMP and other related metabolic changes in the oocyte are observed (Schultz et al., 1983; Bornslaeger et al., 1986). In the present study, however, we have demonstrated that morphological changes in gap junctions at the cumulus cell surface occur at least 90 min prior to GVBD in the preovulatory rat COC. Furthermore, we believe that these morphological modifications represent structural changes that prepare and commit these gap junctions to endocytotic removal. Commitment of cumulus cell gap junctions to endocytotic removal may therefore be relevant to the commitment to resume meiosis that is hypothesized to occur in oocytes 3060 min prior to GVBD. Gap junction remodeling and membrane fusion precede endocytosis in vivo

The changes in cumulus cell gap junctions that are initiated 90min before their disappearance and the onset of GVBD include the development of an irregular topography with lateral dispersion of junctional particles within the membrane and the clearing of nonjunctional particles with fusion of apposing cell membranes at the periphery of large gap junction plaques. The rigid crystalline topography of large cumulus cell gap junctions disappears in vivo within 1 hr following an ovulatory stimulus. Junctions develop corrugations and blebs that may reflect a change in the membrane fluidity of gap junction plaques that is required to form invaginations and endocytotic vesicles. Topographical changes in large preendocytotic gap junction caps have been described previously in rabbit granulosa cells (Larsen and Tung, 1978; Larsen et al., 1979; Larsen et al., 1981), and clathrin bristle coats have been observed enclosing some of these irregular junctional blebs (Larsen et al., 1979). These observations support the suggestion that deformation of the gap junction membrane is required for internalization. In addition, internalized gap junction vesicles become enveloped by a single layer of 5 nm filaments that are decorated with subfragment-l of heavy meromyosin, diagnostic for actin (Larsen et al., 1979). The role of actin in gap

WERT AND LARSEN

junction endocytosis has been supported recently by our finding that dihydrocytochalasin B, a microfilament disruptor, completely inhibits the removal of cumulus cell gap junctions in cultured COCs (Wert and Larsen 1989). One force which may contribute to the maintenance of the crystalline rigidity of gap junctions at the cell surface is lateral connexon-to-connexon binding. This cohesion may be reflected in the tight particle-to-particle packing pattern typically observed in unstimulated cumulus cell gap junctions. Subsequent to an ovulatory stimulus in vivo or to isolation of COCs into FSH-containing culture medium, however, gap junction connexons disperse. Consequently, it is not unreasonable to suggest that disaggregation of the gap junction plaque may contribute to the loss of rigid crystallinity normally apparent in unstimulated cumulus cell junctions. Consistent with this suggestion is the observation that rigidity and tight particle packing are coincident features of the small population of gap junctions left after the dramatic down-regulation of junctions from the cumulus cell surface that occurs between 2 and 3 hr after hCG stimulation. Secondly, the dispersal of gap junction particles that results in profiles of loosely packed gap junction plaques is consistent with previously published studies of topographical changes in liver cell gap junctions during their disappearance from the cell surface following partial hepatectomy in the rat (Yancey et al., 1979). Furthermore, studies correlating the loss of intercellular communication (induced by carbon dioxide treatment) with the lateral dispersal of gap junction particles within the granulosa cell plasma membrane (Lee et al., 1982) suggests that changes in PPPs reflect the functional status of gap junctions in this system. If this relationship were to hold true for the large, loosely packed, preendocytotic junctional aggregates seen in the current study, then almost 50% of the cumulus cell gap junction population could become nonfunctional 60-90 min prior to its loss from the cell surface and to the initiation of GVBD in vivo. Although this would not result in the complete loss of intercellular communication within the cumulus oophorus, as is demonstrated by the continued presence of metabolic coupling among the cells in isolated preovulatory COC’s (Moor et al., 1981; Salu-

stri and Siracusa, 1983; Racowsky and Satterlie, 1985; Heller and Schultz, 1980; Eppig. 1982), it might dilute the inhibitory effect of the surrounding membrana granulosa cells on meiotic maturation by influencing the net rate of molecular diffusion from the compartment to another or the net transfer of regulatory substances to the oocyte. Over the years, an attempt has been made in many different model systems to correlate gap junction PPPs with metabolic or electrical coupling of adjacent cells. Results of such comparisons, however, have been inconsistent and vary with (1) the tissue or cell type under investigation, (2) the uncoupling agent employed in these studies, and (3) the fixation technique used to preserve these tissues for ultrastructural analysis. For example, in rapidly frozen tissues, coupled cells tend to be characterized by loosely packed gap junction particles, while tightly packed, crystalline gap junctions are observed under physiological conditions that uncouple cells, such as the elevation of intracellular calcium levels (Perrachia, 1980: Raviola et al., 1980). Other investigators. however, have reported particle dispersal in rapidly frozen cardiac purkinje fibers in response to elevated intracellular calcium levels (Shibata and Page, 1981) or no change at all in quick frozen chick embryo lens epithelial cells treated with a variety of metabolic uncouplers (Miller and Goodenough. 1985). Chemical fixation with glutaraldehyde. itself an uncoupling agent, usually results in more condensed, or tightly packed gap junction particles and thus may complicate interpretation of correlations between gap junction morphology and physiology (Bennett and Goodenough, 1978: Sikerwar and Malhotra, 1981; Miller and Goodenough, 1985). Gap junctions from different tissues can vary. however, in their response to glutaraldehyde such that hepatocyte and stomach epithelial gap junctions fixed in glutaraldehyde cannot be distinguished from those visualized after rapid freezing. while gap junctions from cornea1 endothelium and ciliary epithelium tend to crystallize in response to chemical fixation (Raviola er ul.. 1980). In addition, gap junctions from ventricular myocytes fixed with either glutaraldehyde or by quick freezing remain compact and closely packed despite their physiological state (Page er al., 1981). On the

PREENDOCYTOTIC

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OF GAP JUNCTIONS

other hand, gap junction loss and particle dispersal has been correlated in a variety of model systems with tissue differentiation and remodeling (Fujisawa et al., 1976; Lane and Swales, 1978, 1980; Larsen et al., 1981; Larsen and Risinger, 1985; Larsen et al., 1987; Larsen and Wert, 1988), or with cell disaggregation and proliferation (Yancey et al., 1979; Campbell and Albertini, 1981; Murray et al., 1981; Preuss et al., 1981; Risinger and Larsen, 1981). The dispersal of gap junction particles in these systems, as well as in the present study, is observed in glutaraldehyde fixed tissues and is probably not an artifact related to tissue fixation, since this treatment should have the opposite effect, i.e. that of aggregating gap junction particles. In the present study gap junction dispersal is correlated with exposure of the COC to hormonal stimuli and preceeds a massive loss of gap junction membrane from the cell surface via an endocytotic mechanism that can be blocked by dihydrocytochalasin B (Wert and Larsen, 1989). The shift from tightly packed to loosely packed gap junction plaques, which peaks 2 hr after hCG stimulation in ho, may also explain the transient increase in gap junction fractional area observed at this timepoint m our initial studies of gap junction loss in the preovulatory COC (Larsen et al., 1986, 1987). The ruffling and fusion of apposed cell-cell membranes around the periphery of cumulus cell gap junctions is a new finding that has not been described in previous reports on endocytotic mechanisms. For this reason, we were initially concerned that this configuration represented an artifact of tissue preparation. We have since considered the possibility that this dramatic alteration in junctional morphology represents a structural modification that is significant to the endocytotic process that is so dramatically synchronized in these preparations. As previously described, endocytotic removal of gap junctions from the cell surface appears to involve the uptake of junctional membrane into one cell of the original cell pair forming a gap junction connection (Larsen and Risinger, 1985). Topologically, then, one cell essentially takes a bite out of the other one at the gap junction, and the cytoplasm enclosed within the resulting vesicle represents cytoplasm from the other cell. If this process gives rise to these endocytotic

847

vesicles, then it is logical to assume that membrane at the peripheral margins of the gap junction must first fuse to prevent leakage of cytoplasmic contents from either of the two cells as the invaginated junctional membrane is removed from the surface. The molecular conformation of the membrane in this region of the junction may then be more prone to fission as the rim of the junctional invagination draws together prior to the release of the junctional vesicle into the cytoplasm. This initial fusion event may facilitate the instantaneous sealing of nonjunctional membrane in both cells at the completion of gap junction vesicle formation, maintaining the individual integrity of the newly uncoupled and disaggregated cells. In support of this interpretation is the observation that cumulus cell gap junctions whose endocytotic removal is prevented by dihydrocytochalasin B (Wert and Larsen, 1989) never exhibit these particle-poor regions of membrane fusion. In addition, similar regions of deformed, particle-free membrane have been observed with freeze-fracture techniques during the fusion and exocytosis of secretory granules at the cell surface (reviewed by Neutra and Schaeffer, 1977), during zoospore encystment in Phytophthora palmvora (Pinto da Silva and Nogueira, 1977), during myoblast fusion (Kalderon and Gilula, 1979), and during the acrosome reaction in ram (Flechon et al., 1986) and guinea pig (Friend et al., 1977) spermatozoa. Gap junction remodelling in cultured COCs

We have shown previously that cumulus cell gap junctions are lost rapidly in uitro in response to a variety of culture conditions (Wert and Larsen, 1989). The kinetics of gap junction loss vary, however, with the incubation conditions. In serum free medium, 71% (by area) of the gap junction membrane present in freshly isolated COCs is lost within the first 30 min of incubation; 33% of the remaining junctional membrane is associated with membrane fusion profiles indicating that an endocytotic mechanism is involved in gap junction clearance from the cell surface. As mentioned previously, this hypothesis is supported by the complete inhibition of gap junction loss in response to incubation with dihydrocytochalasin B (Wert and Larsen, 1989). There is an increase, how-

WERT AND LARSEN

ever, in the amount of tightly organized gap junction membrane which suggests that particle dispersal is not necessarily required for endocytosis in vitro. The incidence of mixed or dispersing gap junction PPPs remain low at about 10% of the total gap junction membrane area. During the next 1.5 hr, the amount of junctional membrane at the cell surface increases and remains at half of its original level for as long as 16 hr in culture (Wert and Larsen, 1989). Gap junction loss in this experimental group may result initially from the shock of isolation, since gap junction membrane levels stabilize thereafter. Membrane fusion profiles, however, continue to be seen surrounding about 20% of the remaining junctional membrane. The clearance of cumulus cell gap junctions in serum-containing medium is slower but more extensive than that seen in serum free medium (Wert and Larsen, 1989). The incidence of mixed or partially dispersed gap junction PPPs in this second experimental group is increased by 30%-40% of the total cumulus cell gap junction membrane area, and 33%-56% of the junctional membrane present is associated with membrane fusion profiles. FSH accelerates and augments gap junction loss in comparison to these two previous experimental conditions. Over 80% of the cumulus cell gap junction membrane is lost within the first 30 min of incubation with FSH and remains low for up to 16 hr in culture (Wert and Larsen, 1989). The incidence of membrane fusion profiles remains high until 2 hr into the incubation period when 85% of the cumulus cell gap junction membrane has disappeared. A dramatic shift in the distribution of the PPPs from tight to mixed and loose PPPs occurs within 15 min of incubation, a shift that is almost identical to that seen in uiuo after hCG stimulation. This suggests that hCG and FSH have a significant effect on the lateral dispersion of gap junction particles in the membrane, a result which supports the possibility that gonadotropin-induced changes in membrane fluidity enhance endocytosis and gap junction clearance from the cell surface. Membrane fusion is a complex biological process that involves a number of different biochemical and biophysical events leading to membrane destabilization and changes in membrane fluidity (reviewed by Blumenthal, 1987). Observations concerning the action of

gonadotropic hormones on membrane fluidity, however, are few and somewhat contradictory. Although Kolena ef af. (1986) reported that PMSG increased membrane fluidity and the number of detectable LH/ hCG receptors simultaneously in rat testicular membranes, injection of hCG downregulated the LH-hCG receptor with no effects on membrane fluidity. These results differ from those of Strulovici et al. (1981) who reported that continued exposure of cultured granulosa cells to FSH (a condition that also up-regulates LH/hCG receptors) increased membrane viscosity. In the model system described here, endocytotic removal of remodeled gap junction membrane requires the internalization of a relatively large bimembranous structure without subsequent lysis of either cell contributing to the gap junctional contact. This is a requirement which would necessitate the efficient resealing or fusion/fission of the plasma membrane at the completion of each endocytotic event and could involve changes in membrane fluidity or destabilization of the plasma membrane prior to these events. Finally, cumulus cell gap junction levels in both hCG- and FSH-treated COCs remain low over the next 12-16 hrs (Larsen et al., 1986; Wert and Larsen, 1989), suggesting that gonadotropins have an inhibitory influence on gap junction gene expression in these terminally differentiating cells. This ultimately results in the complete disaggregation of the cumulus cell mass, and. in uivo, in the release of the mature oocyte from its association with the membrana granulosa cells of the follicle wall.

Conclusions

This report documents both qualitatively and quantitatively three major morphological transformations that occur in cumulus cell gap junctions prior to their disappearance from the cell surface. All three of these transformations are consistent with preparations for the endocytotic removal of gap junctions in this system. If such structural transformations influence the function of gap junctions at the cumulus cell surface, it is possible that they may also influence the propagation of membrana-granulosa-generated inhibitory signals to the oocyte well before the actual

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OF GAP JUNCTIONS

removal of gap junctions from the cell surface and the initiation of GVBD. Alternatively, it is conceivable that these early morphological transformations merely commit these junctions to an endocytotic sequence, thereby more indirectly committing the enclosed oocyte to the resumption of meiotic progress l-2 hr later.

Acknowledgements The authors are particularly grateful to Susan Eder and Barbara Burch for the typing of this manuscript and to George Brunner for his technical assistance. This study was supported by NSF grant PCM-8117927, NIH BRSG 9217, and U.S.D.A. 87-CRCR-l2574.

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Eppig. J. J., Freter, R. R., Ward-Bailey, P. F. and Schultz. R. M. 1983. Inhibition of oocytc maturation in the mouse: participation of CAMP. steroid hormones. and a putative maturation-inhibitory factor. Dco. &o/. 100, 39-49. FICchon. J. E.. Harrison, R. A. P.. FICchon. B. and Escaig. J. 1986. Mcmbranc fusmn cvcntb m the Ca’ +:wnophorcinduced acrosome reaction of ram spermatozoa. J. Ce//. Ski.. 81, 4.1-63. Freter, R. R. and Schultz. R. M. 1984. Regulation of murine oocytc meiosis: Evidcncc for a gonadotropin-induced. CAMP-dependent reduction in a maturation inhibitor. J. Cc// Ciol., 98, I I I%)_]128. Friend, D. S.. Orci. L.. Perrelet, A. and Yanagimachi. R. IY77. Membrane particle changes attcndmg the acro~omc reaction in guinea pig spermatozoa. J. Cell Biol.. 74, 561-577. Fujisawa, H., Morioka. H.. Watanabe, K. and Nakamura. H. 1976. A decay of gap junctwns in asaociatmn with ccl1 differentiation of neural retina in chick embryonic development. J. Cell Sci.. 22, 5X5-596. Gilula, N. 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