JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 163257-280 (1990)

Ultrastructural Correlates of Meiotic Maturation in Mammalian Oocytes ROBERT W. McGAUGHEY, CATHERINE RACOWSKY, VIRGINIA RIDER, KENDALL BALDWIN, ALYCE A. DEMARAIS, AND SCOTT D. WEBSTER Department of Zoology, Arizona State University, Tempe, Arizona 85287

KEY WORDS

Oocyte, Maturation, Ultrastructure, Gap junction, Cortex, Mammal, Granulosa,

Actin

ABSTRACT

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase 11. Oocytes remain a t metaphase I1 until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze-fracture analysis, and replicated oocyte cortical patches, we demonstrate stage-specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.

INTRODUCTION The developmental process during which primary oocytes complete meiosis and acquire the capacity to become fertilized and begin embryogenesis is known a s oocyte maturation (Masui and Clarke, 1979). In most mammals, this process includes several individually defined events that occur just before and during the first meiotic division. Because this cell division produces the haploid female gamete through the segregation of homologous chromosomes between the secondary oocyte and the extruded polar body, it contributes significantly to the acquisition of genetic diversity among gametes and consequently among embryos derived from the mature oocytes. Therefore, from genetic and evolutionary perspectives, the most important developmental change during oocyte maturation is the reductional first meiotic division. Several cytological and molecular events which are closely linked to nuclear division accompany meiotic maturation in mammalian oocytes. These events include reorientation of the meiotic spindle, apparent cessation of transcriptional activity, organelle redistribution, cell surface changes, stage-specific changes in protein synthesis, and changes in the structural and perhaps functional associations between the oocyte and its surrounding follicle cell layers. Elucidation of fundamental mechanisms which regulate oocyte maturation has been the primary goal of much recent research. Current hypotheses regarding these regulatory mechanisms are based on work by Pincus and Enzmann (1935), who observed that rabbit oocytes matured spontaneously in simple culture medium once they were removed from the follicular environment. These early investigators concluded that mammalian ovarian follicles constituted a n inhibitory

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environment in which oocytes would not mature until follicular exposure to luteinizing hormone. The several currently held hypotheses which explain spontaneous oocyte maturation in mammals all are founded on the concept of follicular inhibitors, the arresting actions of which are overcome directly or indirectly by the interaction of gonadotropin (i.e., luteinizing hormone) with the follicle, several hours before ovulation. Various small molecules have been suggested as contributors to follicular inhibition of oocyte maturation. These molecules include cyclic adenosine monophosphate, inhibitory peptides and steroids (see McGaughey, 1983, for review), unidentified “factors” from follicular granulosa cells (Sato and Ishibashi, 1977; Tsafriri and Channing, 1975), and interactions between the oocyte and the surrounding follicle cells (Anderson and Albertini, 1976; Racowsky et al., 1989). In designing experiments to test various putative follicular inhibitors, investigators have selected several criteria by which the meiotic status of oocytes can be assessed. The simplest and most commonly employed criterion, however, is the presence or absence of a n intact oocyte nucleus or germinal vesicle (GV). I n mammals, the GV nucleus characterizes the immature oocyte; when that nucleus begins to disintegrate or break down (GVBD) and its chromatin condenses to form first meiotic chromosomal elements or bivalents, the oocyte is characterized as maturing. At the molecular level, mammalian oocyte maturaReceived J u n e 15, 1989; accepted in revised form December 6, 1989. Dr. Virginia Rider is now at Peabody Pavilion, Tufts University, 200 Westboro Road, North Grafton, MA 01536. Address reprint requests to Dr. Robert W. McGaughey, Department of Zoology, Arizona State University, Tempe, AZ 85287.

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tion is less well defined. Although developmental pat- were obtained from a local abattoir within a few minterns of polypeptide synthesis during maturation have utes of slaughter and handled as described previously been determined for the oocytes of several mammalian (McGaughey, 1978). Oocytes from small to medium folspecies (McGaughey and Van Blerkom, 1977; Schultz licles (1.0 mm to 5.0 mm diameter) were pooled in modand Wassarman, 1977; Van Blerkom and McGaughey, ified BMOC-3 medium (2A medium; McGaughey, 1978), the developmental significance of these changes 1977) and class A oocytes were selected (McGaughey et has not been fully determined. Furthermore, while the al., 1979) and cultured in complex medium (TCM; Tsarelative contributions to changes in patterns of protein friri and Channing, 1975). Groups of oocytes (20-25 synthesis from qualitative changes in translation and per group) were cultured at 37°C in a humid atmopost-translational modification have been examined sphere of 5% C 0 2 in air for varying periods of time (Van Blerkom, 1985), most of the polypeptides which (6-24 hours), after which they were separated into subundergo stage-specific (qualitative or quantitative) groups for cytogenetic determination of meiotic stage changes in synthesis have not yet been identified. Like- or for thin sectioning. A total of 85 oocytes were airwise, although it has been demonstrated that synthesis of ribonucleic acid probably ceases in mammalian dried (McGaughey, 1978; McGaughey and Polge, 1971; oocytes a t approximately the time of GVBD (Wassar- Rice and McGaughey, 1981) for cytogenetic analysis. man and Letourneau, 1976), neither the nature of RNA Maturation had been initiated in 50% (5 out of 10) of sequences synthesized prior to the onset of maturation the oocytes by 6 hours of culture, and in 90% (9 out of nor the presence or function of stored messenger RNA 10) by 24 hours. Over the entire culture period (6-24 in mammalian oocytes during maturation is com- hours), 65% of the air-dried oocytes exhibited evidence pletely understood. Other biochemical studies have of maturation. elucidated the lipid composition of immature oocytes Fifty oocytes, from the same groups in which the (Homa et al., 1986) and their requirements for energy incidence of maturation had been determined cytogemetabolism (Biggers, 1972). In summary, the complex netically, were fixed, embedded, and sectioned for developmental programming which operates in mam- transmission electron microscopy (TEM). Cultured and malian oocytes to regulate macromolecular synthesis uncultured oocytes selected immediately after harvestand the cellular changes associated with maturation is ing were washed in 2A medium and fixed, using the incompletely understood. stepwise procedure of McGaughey (1978), in a solution The process of mammalian oocyte maturation is of paraformaldehyde (2%, wlv) and glutaraldehyde thought to be regulated by a series of integrated, se- (2.5% vlv) in 0.1 M sodium phosphate buffer, pH 7.4, a t quential events. It seems likely that no single follicular room temperature. Fixed oocytes were washed in the inhibitor regulates this process in all mammalian spe- same buffer and post-fixed for 60 minutes in 1%oscies, nor even within a n individual species. As one of mium tetroxide in buffer. After dehydration through a the most fundamental events in all of developmental graded series of ethanol and propylene oxide, oocytes biology, the sequence of changes in a n immature oocyte were embedded in Embed 812 (Electron Microscopy that culminates in a mature, fertilizable gamete may Sciences) and sectioned. When the oocyte nucleus or continue to confound investigators for many years be- chromosomes were first detected in thick sections unfore a complete and simplified explanation can be de- der light microscopy, thin sections were cut, mounted veloped. If this is true, then future research into the on 300-mesh copper grids, stained with uranyl acetate regulation of mammalian oocyte maturation necessar- (2%, aqueous, wlv) and lead citrate (Reynolds, 1963), ily must focus on the most promising areas suggested and examined at 60 kV by TEM. by our current knowledge. Of the sectioned oocytes, five were excluded from the In this article, we present data on ultrastructural ultrastructura1 study because they were abnormal due changes in maturing mammalian oocytes, which indi- to nondisjunction a t the first meiotic division which cate promising areas for further work. Initially, we pro- resulted in diploid metaphase I1 configurations (Mcvide a n overview of the ultrastructure of oocyte matu- Gaughey, 1985). One other sectioned oocyte was also ration, in which we emphasize the major cytological excluded because it exhibited characteristics of atresia changes which occur during this process. Second, we (McGaughey et al., 1979). The remaining 44 thin-secpresent new findings concerning the junctional inter- tioned oocytes provided a n ultrastructural sequence of actions among the membrana granulosa cells directly timed, stage-specific characteristics from which was esunderlying the cumulus cell stalk during the critical tablished the pattern of developmental changes occurperiod in which oocytes become irreversibly committed ring during maturation. to mature. Finally, we provide new information regardThe ultrastructure of uncultured immature porcine ing the structural integrity of the mammalian oocyte oocytes, fixed immediately after release from the follicortex. These observations are based, respectively, on cle, is shown in Figure 1A. Immature oocytes contained thin-sectioned oocytes, on replicas from freeze-frac- a large nucleus (about 30 p,m in diameter) and a contured membrana granulosa “sheets,” and on replicated spicuous, electron-dense, spherical nucleolus. The nuoocyte cortical preparations. clear envelope was smooth and nuclear pores were prominent and uniformly distributed. The individual OVERVIEW OF PORCINE OOCYTE width for each of the two envelope membranes was 7.6 ULTRASTRUCTURE DURING MATURATION nm (7.6 t SE 0.4), while the region separating the This study was designed to analyze the fundamental inner and outer membranes, the perinuclear space, ultrastructural changes which occur in porcine oocytes ranged from 17.0 to 30.0 nm with a mean width of 26.3 during their maturation in culture. Porcine ovaries nm (26.3 +- SE 1.0).

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Fig. 1. Electron micrographs of thin-sectioned, embedded porcine oocytes at the immature stage. A Structural organization of cytoplasmic organelles in the immature oocyte. Major components of the cytoplasm were vesicles (V), lipid droplets (L), and protein bodies (PI. These organelles were uniformly distributed throughout maturation. Mitochondria (M) were usually clustered and maintained a close spatial relationship with endoplasmic reticulum throughout maturation (arrowhead). Mitochondria1 cristae were concentric or transverse; hooded forms were not observed. x 10,400. B A typical junctional

complex (J) between the oocyte and surrounding cumulus granulosa cell. The junctions were typically electron-dense and the bulbous ends of the cumulus cell process (CP) contained granules (GR) of unknown composition. Disruption of the junctions began with dissolution of the nuclear envelope; however, intact complexes were observed through the first meiotic division. Note the filaments (or tubules) which were often present in the cortical region of the oocyte (arrowheads). x 26,265. ZP, zona pellucida; M, mitochondrion; N, nucleus.

Short microvilli covered the surface of the immature oocyte and numerous processes from surrounding cumulus granulosa cells penetrated the zona pellucida to contact the oocyte surface. Some processes were closely juxtaposed to the oocyte surface without specialized terminations, while others terminated at junctional complexes (Fig. 1B). Mitochondria, with cristae organized concentrically and occasionally transversely, were present in clusters throughout the cytoplasm in close association with nonordered endoplasmic reticulum (ER) exhibiting single cisternae (Fig. lA, arrow). However, the most prominent cytoplasmic components were 1) membrane-bound vesicles containing flocculant material, 2) lipid droplets whose contents appeared streaked, and 3) vesicles termed “protein bodies” (Cran, 1985; Szollosi and Hunter, 1973) which were scattered throughout the cytoplasm (Fig. 1A). Ultrastructural evidence of maturation was observed in oocytes fixed after the shorter culture periods (6-14 hours). The earliest observed characteristics of maturation were minor nuclear envelope convolutions, followed at later time periods by extreme convolutions and breaks (Figs. 2). Alteration in the contour of the

nuclear envelope was initiated proximally to the cortical region of the oocyte and, as maturation progressed, the entire envelope became highly convoluted and deeply invaginated into the nuclear interior. At the earliest stages of this nuclear change, pores were evident on the envelope and, in obliquely sectioned regions, they appeared circular with a diameter of about 90 nm (89.5 2 SE 3.0).As early maturation progressed, breaks in the envelope became more numerous and nuclear pores disappeared (Fig. 2). Two types of membrane fragment resulted from nuclear envelope dissolution: 1)simple cisternal fragments of various lengths arose from periodic breaks along the envelope (Fig. 2); and 2) breaks in the envelope a t highly convoluted regions resulted in transient quadruple membrane fragments (Fig. 3A) that were not observed after the first meiotic division. Occasionally ribosomes were visible on the nuclear membrane as it fragmented (Fig. 3B). By late diakinesis (after 10 hours of culture) the nuclear envelope fragments were indistinguishable from granular ER near the condensed metaphase I chromosomes (Fig. 3C). In addition to the notable changes in nuclear enve-

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Fig. 2. Electron micrograph of a sectioned porcine oocyte during early maturation. This oocyte exhibits both undulation of the nuclear membrane and the beginning of membrane fragmentation which occurs during GVBD (arrows). As meiosis resumes, mitochondria (MI aggregate in the interior of the oocyte to surround the cytoplasmic

side of the nuclear envelope. Golgi stacks (G), while sparse in the immature oocyte and during later stages of maturation, were frequently observed in the cytoplasm a t GVBD. Ribosome clusters were consistently observed throughout the oocyte cytoplasm at all stages of maturation. N, nucleus. x 17,307.

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Fig. 3. Electron micrograph of a sectioned porcine oocyte during GVBD. A: The deeply invaginated nuclear envelope (NE) formed transient quadruple membrane complexes. x 45,522. B: Ribosomes were visible on both sides of the envelope leaflets as the membrane

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dispersed (arrowheads). (M) mitochondria. x 66,744. C: Residual envelope became indistinguishable from granular endoplasmic reticulum near the condensed metaphase I chromosomes (CH), and a continuous membrane was no longer visible. x 17,208.

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Fig. 4. Electron micrograph of sectioned maturing porcine oocytes after formation of the first meiotic spindle. A The presumed residual nuclear envelope (ER) appeared to be structurally associated with the spindle. CH, condensed metaphase I chromosome. X 11,538. B: Elec-

tron-dense granules (arrowheads) were observed a t the polar regions of the spindle. The function of these granular components is unknown; however, their time of appearance is consistent with being microtubule organizing centers. x 34,992.

lope ultrastructure during early maturation (6-14 hours of culture), redistribution of several cytoplasmic organelles was observed. Golgi stacks were sparse in the immature oocyte and a t later stages of maturation; however, during early maturation, Golgi bodies in stack-like configurations were evident in the oocyte cortex and adjacent to the convoluted nuclear envelope (Fig. 2). Mitochondria became localized in the internal region of the oocyte leaving the oocyte cortex relatively free of these organelles. Mitochondria1 morphology was not altered during maturation, and a close association between mitochondria and dilated ER was maintained. After 6 hours of culture, numerous cortical granules had accumulated beneath the oocyte plasma membrane; the beginning of this accumulation was associated chronologically with convolution and disruption of the nuclear envelope. After extended culture periods (16-24 hours), no evidence of a continuous nuclear envelope was seen and the residual envelope (resembling granular ER) adjacent to, but separated from, the first meiotic spindle apparatus was observed (Fig. 4A). The spindle was composed of microtubules with lateral projections spaced along their lengths. These lateral projections

were approximately 19 nm (18.8 r+ SE 1.1)in length and 7.0 nm (7.4 r+ SE 0.4) in width. Centrioles were absent although the polar regions of the spindle contained electron-dense granules (Fig. 4B). The polar spindle granules had a n average diameter of 33 nm ( & SE 2.0). Vesicles, ER, and mitochondria were closely associated with the first meiotic spindle but had dispersed from the spindle region in oocytes a t telophase I. Orientation of the first meiotic spindle was dependent upon chromosomal position in the oocyte. Spindle microtubules were oriented parallel to the oocyte surface when the chromosomes were directly beneath the oocyte plasma membrane; however, in oocytes with chromosomes located at some distance from the plasma membrane, the microtubules were oriented perpendicular to the oocyte surface. Polar body formation was initiated by invagination of the oocyte plasma membrane near the condensed chromosomes. The extruded polar body contained cytoplasm, chromosomes, cortical granules, mitochondria. and microtubules (Fig. 5A). The haploid group of chromosomes remaining in the oocyte after polar body abstriction was aligned on the second meiotic spindle and, in contrast to the recently formed first meiotic spindle,

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the surrounding cytoplasm was relatively free of organelles. Microtubules forming the second meiotic spindle exhibited lateral projections identical with those described above for the first meiotic spindle microtubules. The cumulus cells associated with oocytes a t progressively later stages of maturation were enriched with lipid droplets. These droplets were associated with mitochondria within the cumulus cells and in the processes abutting the oocyte plasma membrane (Fig. 5B). Also shown in Figure 5B is a n apparently intact junctional complex between a cumulus cell process and the surface of a n oocyte which had progressed to metaphase I. Although retraction and degeneration of these cumulus cell processes was seen to begin during the period when the nuclear envelope was undergoing dissolution, a few of these processes remained intact throughout the 24 hour culture period. At the later stages of maturation the cortical region of the oocyte was relatively free of organelles. Golgi stacks and mitochondria were notably absent. Lipid droplets and membrane-bound vesicles remained prominent throughout the oocyte cytoplasm a t each stage of maturation. Ultrastructural changes a t specific meiotic stages have been determined for porcine oocytes which represent a system that has been well defined a t the cytogenetic (McGaughey and Polge, 1971), physiologic (Racowsky, 19851, and molecular (McGaughey and Van Blerkom, 1977; McGaughey et al., 1979) levels. Since 90% of the cultured oocytes underwent maturation, we feel confident that these observations parallel the structural changes typical of oocyte maturation in vivo. Similarly, investigations with mouse oocytes reveal no significant differences in fine structure between oocytes allowed to mature in culture and those undergoing maturation in vivo (Calarco et al., 1972). In a n attempt to correlate cytoplasmic structural changes with specific stages of nuclear maturation, we noted three changes that occurred consistently, and which therefore may serve a s structural markers for cytoplasmic maturation. These include 1) the migration of cortical granules into the oocyte cortex at the time of nuclear disintegration, 2) the movement of mitochondria into the interior of the oocyte and association of these relocated mitochondria with the nuclear membrane as i t disintegrates, and 3) the dispersal of all organelles including mitochondria, ER, and residual nuclear envelope vesicles away from the first meiotic metaphase spindle before the meiotic division is completed. In several mammalian species, cortical granules have been reported to be synthesized by the Golgi (Szollosi, 1967; Zamboni, 1970). Our study of porcine oocytes clearly showed that stacked Golgi configurations were rare in the immature oocyte and after GVBD. Furthermore, cortical granules completed their localization beneath the plasma membrane only a t very late stages of maturation, as was also observed by Cran and Cheng (1985). Stacked configurations of Golgi are reported to be rare during maturation of bovine oocytes after disintegration of the nuclear envelope (Kruip e t al., 1983). The suggestion that porcine oocytes contain specialized

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forms of Golgi as sites of cortical granule synthesis (Cran, 1985) remains to be confirmed. In oocytes of rabbits, mice and cows, disruption of junctional complexes between the oocyte and cumulus cells begins before migration of cortical granules (Hytte et al., 1986; Szollosi et al., 1978). In our study, we found that although disruption of junctions between the oocyte and cumulus cells began when the nuclear envelope began to disintegrate, some morphologically intact junctional complexes remained at least through metaphase I. Although with our analysis we were unable to determine the functional state of these junctions, the possibility exists t h a t total uncoupling is not required for cortical granule migration in the porcine oocyte. Junctional association among follicle cells and with the oocyte is addressed in more detail in the following section. Similar to our observation with porcine oocytes (see also Cran, 19851, perinuclear mitochondrial aggregation occurs in the mouse oocyte during maturation in vivo (Calarco et al., 1972; Van Blerkom and Runner, 1984). Cran (1985) suggested that mitochondrial movement to the interior of the oocyte may signal metabolic independence of the oocyte from the cumulus granulosa cells. If this interpretation is correct, mitochondrial movement into the interior of the pig oocyte begins near the time of nuclear disintegration and probably precedes uncoupling of the oocyte from the cumulus cells (Cran, 1985; Moor e t al., 1981; Motlik e t al., 1986; Racowsky and Satterlie, 1985). Our observations are consistent with the suggestion of Van Blerkom and Runner (1984) that mitochondrial aggregation in mammalian oocytes may provide energy for formation andlor function of the first meiotic spindle. We observed mitochondria, ER, and the residual nuclear envelope associated with the newly formed first meiotic spindle. These organelles sequester calcium (reviewed by Inoue, 1981) for regulation of microtubule assembly (Welsh et al., 1979), and may provide the calcium necessary for oocytes to complete the first meiotic division (Paleos and Powers, 1981; Racowsky, 1986). At the beginning of the first meiotic division, we observed that the cytoplasm surrounding the spindle apparatus contained a decreased number of cytoplasmic organelles and that, by telophase, it was free of not only mitochondria but of all cytoplasmic organelles, Failure of cytoplasmic organelle dispersion from the cytoplasm surrounding the first meiotic spindle has been correlated with abnormal spindle orientation and function (Szollosi and Gerard, 1983; Van Blerkom and Runner, 1984). Together, these observations suggest that dispersion of cytoplasmic organelles following formation of the first meiotic spindle is a crucial step toward completion of mammalian oocyte maturation. Ultrastructural studies, using thin-sectioned oocytes, allow for the definition and analysis of stage-specific fine structural changes that occur during oocyte maturation. Some of these developmental changes, such as early disintegration of the nuclear envelope and reorganization of organelles, can be observed only at the level of resolution allowed by TEM. Thus, in experiments designed to examine regulatory mechanisms op-

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I. Although disruption of most junctional complexes occurred with nuclear envelope dissolution, some intact junctions (J)were observed at later stages. Osmiophilic lipid droplets (0)were seen in cumulus cell processes only after dissolution of the nuclear envelope; however, vesicles and extracted lipid droplets (L) were prominent in the oocyte a t all stages of maturation. Cortical granules were localized directly beneath the oocyte plasma membrane at these later stages of maturation. x 27,810.

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Fig. 6 . Freeze-fracture replica of planar surface of a hamster oocyte revealing gap junction (GJ) and sheared off microvilli (MV) on the oocyte surface and microvilli extending from the oolemma (00)into the zona pellucida (ZP). x 33,440.

erating very early in maturation, it is essential to distinguish between oocytes beginning maturation and those remaining in the immature state. Although the precise mechanisms by which oocyte organelles and nuclear elements migrate during maturation are unclear, evidence has been presented that in the mouse, these organelle migrations may be under the influence of microtubules and actin microfilaments (Longo and Chen, 1985; Mar0 et al., 1986; Van Blerkom and Bell, 1986). It should be noted also that not all mammalian oocytes exhibit the same patterns of organelle migration during meiotic maturation (reviewed by Van Blerkom, 1989). Several of the observations made in this study reveal cellular changes which are developmentally significant, suggesting an underlying mechanism that regulates each change. These mechanisms, however, cannot be directly determined by TEM. For example, the reorganization of mitochondria and cortical granules is likely to be controlled by cytoskeletal elements in the oocyte; however, these cytoskeletal elements are not clearly resolved by standard TEM procedures (Capco and McGaughey, 1986). Similarly, the dynamics of the

junctional complexes between oocytes and surrounding cumulus granulosa cells, although observable in thin sections, can be studied more definitively by other ultrastructural methods including freeze-fracture analysis. In the following two sections we present the results of investigations carried out with specialized ultrastructural methods designed to examine the intercellular junctional associations among the cellular compartments of the ovarian follicle, and the cortical cytoskeleton of mammalian oocytes.

FOLLICULAR GAP JUNCTION INTEGRITY AND OOCYTE MEIOTIC STATUS Since the early suggestion that the cells forming the cumulus oophorus provide metabolic support for the growth and development of the enclosed oocyte (Paladino, 1890), numerous studies have confirmed both nutritional and regulatory roles for these somatic cells. Furthermore, electron microscope (Bjorkman, 1962; Gilula et al., 1979; Sotelo and Porter, 1959) and freezefracture (Anderson and Albertini, 1976; Gilula et al., 1978; Larsen et al., 1986; Wert and Larsen, 1989) studies have revealed that gap junctions, the structures

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Fig. 7. Representative oocyte-cumulus gap junctions in freeze-fracture replicas of hamster oocytecumulus complexes immediately after follicular release prior to the ovulatory gonadotropic stimulus. A Gap junctions organized as particle aggregates in more or less circular arrays. x 68,040. B: Gap junctions organized as particle aggregates in more or less linear arrays. x 80,190.

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Fig. 8. Representative cumulus cell gap junctions observed in freeze-fracture replicas of hamster oocyte-cumulus complexes immediately after follicular release prior to the ovulatory gonadotropic stimulus. A Particle-packing shown to be non-rectilinear as seen on the P-fracture face and pitted E-face. x 53,460. B: Particle-packing

shown to be ordered into rectilinear aggregates that are separated by particle-free aisles. x 65,063. C: Particle packing in rectilinear aggregates as in B, but with bordering regions of non-rectilinear particlepacking. x 65,063.

implicated in cell-to-cell communication and metabolic cooperativity, are indeed present at the points of contact both of the cumulus cells and oocyte, and of the

cumulus cells themselves. In addition, several studies have revealed an elaborate gap junctional network throughout the follicular membrana granulosa prior to

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Fig. 9. Membrana granulosa cell gap junction observed in a freeze-fracture replica of membrana cell “sheet” dissected from a hamster follicle prior to the ovulatory gonadotropic stimulus. Note the irregular configuration quite typical of these gap junctions. E- and P-fracture faces are indicated. x 26.364.

the gonadotropic surge (rabbit: Albertini and Anderson, 1974; Larsen and Tung, 1978; rat: Larsen et al., 1987). Thus, within the mammalian follicle there is a n extensive syncytium involving the membrana granulosa cells, the cumulus granulosa cells, and the oocyte that provides the structural basis for transfer of nutritional and regulatory molecules from the somatic cells into the oocyte. Ultrastructural analyses have revealed that oocytecumulus gap junctions usually occur a t the ends of those cumulus cell processes that interact with the planar surface of the oocyte membrane among the microvilli (Fig. 6). These junctions typically comprise either one or more small clusters of particles arranged around a particle-free region (Fig. 7A), or linear arrays of one or more rows of particles (Fig. 7B). The individual areas of oocyte-cumulus junctions are small in comparison with those of other junctional populations within the follicle; in the rat, these areas range from 0.0001 to 0.091 pm2 (Larsen et al., 1987). In contrast to these heterologous junctions, cumulus-cumulus junctions are frequently circular or slightly irregularly shaped plaques in which the particles are organized into either polygonal domains (non-rectilinear packing, Fig. 8A) or parallel rows separated by particle-free aisles (rectilinear packing, Fig. 88) that may be bordered by non-ordered regions a t the edges of the junctional plaque (Fig. 8C). These homologous junctions vary greatly in size; in the unstimulated rat follicle, the vast majority are less than 0.1 pm“ although typically one junction per cumulus cell is larger than 2.5 km2 (Larsen et al., 1986). Membrana granulosa junctions tend to exhibit even greater pleiomorphism than cumulus junctions (Fig. 9) and may be very extensive

in the unstimulated mature antral follicle. The largest membrana gap junction in such follicles has been reported to be 10.2 km2 in rat (Larsen e t al., 1987), 18.2 pm2 in hamster (Racowsky et al., 19891, and of the order of 31 pm2 in rabbit (Larsen e t al., 1981). During the last decade, considerable attention has been focused upon the possible role of these follicular gap junction specializations in the regulation of oocyte meiotic maturation. Indeed, one of the currently popular hypotheses for the regulation of oocyte meiotic status involves a central role for such gap junctions. This hypothesis proposes that 1) maintenance of follicular gap junction integrity sustains meiotic arrest by enabling transfer of a “meiotic arrester” from granulosa cells into the oocyte, and 2 ) physical disruption of the follicular gap junction network by the preovulatory surge of gonadotropin interrupts this transfer of “arrester” into the oocyte, thereby permitting meiotic resumption (Dekel and Beers, 1978, 1980). Given the anatomy of the follicle, there are several potential locations where transfer of a granulosa cell “arrester” into the oocyte may be regulated. These locations include the junctional connections between 1) the oolemma and the innermost layer of cumulus cells, 2) the cells of the cumulus oophorus, 3) the region of membrana granulosa cells directly underlying the cumulus cell stalk, and 4)the remainder of the membrana granulosa cell population. The above hypothesis has been tested predominantly with cultured oocyte-cumulus complexes after removal from their follicles (i.e., liberated oocyte-cumulus complexes) and with application of one of three assay systems designed to determine the extent of functional heterologous coupling within the complex. These as-

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says depend upon assessment of transfer from the cumulus mass into the oocyte of radiolabeled metabolites (usually of choline or uridine; metabolic coupling assays), fluorescent dye (usually lucifer yellow; fluorescent dye coupling assays; Fig. 101, or ionic current

Fig. 10. Fluorescent dye coupling revealed between a hamster oocyte and surrounding cumulus mass after lucifer yellow was injected iontophoretically into the oocyte following follicular release prior to the ovulatory gonadotropic stimulus. A Phase-contrast microscopy. B: Fluorescence microscopy. x 350.

Fig. 11. Examples of membrana gap junctional plaques comprising particle densities at the extremes and in the intermediate range observed during the period of irreversible commitment in hamster oocytes following the gonadotropin stimulus of hCG. x 51,594. A: Particles arranged in tightly packed organization (376 particles/ym2 gap junction membrane) from the control group with 0% oocytes irreversibly committed to mature and sacrificed a t 0 hour post-hCG. E- and P-faces are indicated. B Particles packed a t intermediate density (137 particlesipm’ gap junction membrane) from the group in which 1-19% oocytes were irreversibly committed to mature and which were sacrificed at a mean time of 0.79 hour post-hCG. E-face indicated by arrows. C : Particles arranged in loosely packed organization (67 particledpm” of gap junction membrane) from the group of animals in which 68233% of oocytes were irreversibly committed to mature and which were sacrificed at a mean time of 2.35 hour post-hCG.

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(ionic coupling assays). The majority of findings obtained from the metabolic coupling assays have contradicted the above hypothesis (mouse: Eppig, 1982; Heller et al., 1981; rat: Racowsky, 1984; pig: Racowsky, 1985; sheep: Moor and Cran, 1980), by showing that GVBD occurs before the onset of heterologous gap junction closure, as determined by a reduction in transfer of

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Fig. 12. Apparent cytoplasmic membrana granulosa cell gap junctional vesicle in replica obtained from membrana sheets dissected from hamsters at a mean time of 1.88 hour post-hCG. Both E- and P-faces are exposed. x 50,119.

metabolite from the cumulus compartment to the oocyte. Nevertheless, the results of two metabolic and fluorescent dye studies with hamster oocyte-cumulus complexes indicated a temporal correlation between heterologous uncoupling and GVBD (Racowsky and Satterlie, 1985,19871, suggesting a possible species difference in this regard. However, as previously discussed by Larsen et al. (1987), the only coupling which is directly assessed by application of any of the abovementioned assays is that between the oocyte and the innermost layer of cumulus cells. It is possible, therefore, that dissociation between these heterologous cell membranes simply occurs more rapidly in the cultured hamster oocyte-cumulus complex than in complexes of the other species under the conditions examined. Given the inherent shortcomings in the application of metabolic, fluorescent dye, or ionic coupling assays to our understanding of follicular gap junctional control of oocyte meiotic status, several recent studies have employed the more definitive approach of freeze-fracture electron microscopy. In combination with quantitative morphometric analyses, this approach has enabled tracking of follicular gap junction dynamics following either follicular release and culture of the oocyte-cumulus complex (Wert and Larsen, 1989) or exposure of the preovulatory follicle to human chorionic gonadotropin (Larsen et al., 1986, 1987). Taken together, the results of these studies with the rat have shown a temporal correlation between gap junctional down-regulation in both the cumulus and membrana granulosa membrane and the period of

Fig. 13. Apparent particle dispersion (A) and particle removal (B,C) in membrana granulosa cell gap junctions. Note particles distant from the junctional area (arrows in A), and apparently undergoing endocytosis in a non-rectilinear (B) and rectilinear (C) region (indicated by arrows). x 209,322. Fig. 14. Electron micrographs of platinum-replicated porcine oocyte cortical patches. A: A relatively thin cortical patch showing the bare polylysine-coated surface of the coverslip (PL), regions of inner plasma membrane surface (PM), and the diverse array of cortical filaments and spherical vesicles. x 17,025. B A thicker patch of cortex with a n extensive, interconnected network of branched filaments and bundles of filaments intersecting vesicular structures. x 40,063.

Fig. 14

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GVBD, but no such correlation with respect to oocyte-cumulus gap junctions. Interestingly, the extent of junctional down-regulation was greater within the cumulus oophorus than among the membrana granulosa cells, leading to the postulate that the cumulus oophorus provides a bridge that regulates the conduction of meiotic “arrester” from the more extensive population of granulosa cells to the oocyte (Larsen et al., 1987; Wert and Larsen, 1989). The above freeze-fracture studies with the rat, in conjunction with earlier oocyte-cumulus complex follicular hemisection co-cultures with the pig (Sato and Ishibashi, 1977; Tsafriri and Channing, 1975), point to the potential importance of intercellular communication a t the interface of the cumulus mass and the underlying membrana granulosa cells. In a recent in vivo study with the golden Syrian hamster, we extended these previous studies with rat to investigation of the gap junctional response to the ovulatory stimulus within the membrana granulosa cells directly underlying the cumulus cell stalk (referred to here as the membrana “sheet”) (Racowsky et al., 1989). The experiments were designed specifically to determine whether junctional down-regulation occurs in this discrete population of granulosa cells during the period in which the oocyte becomes irreversibly committed to undergo meiotic resumption. This study, therefore, directly addressed the hypothesis of Dekel and Beers, since implicit in this hypothesis is the proposal that junctional disruption occurs prior to any overt signs of GVBD and thus concomitantly with the period of irreversible commitment. Thirty mature golden Syrian hamsters were used for this study, in which ovarian stimulation had been induced by 30 I.U. pregnant mare’s serum gonadotropin at metestrus. Animals were sacrificed on the morning of proestrus a t various times from 0 to 2.5 hours after a n ovulatory injection of 25 I.U. human chorionic gonadotropin (hCG). The oocytes from one ovary of each hamster were cultured in 0.2 mM 3-iso-butyl-1-methylxanthine (IBMX) in TCM medium (Racowksy, 1984) to determine the percentage of oocytes irreversibly committed to mature. The second ovary provided the samples for freeze-fracture; ovarian segments were excised and fixed overnight in 0.05 M Na Cacodylate, pH 7.3, containing 2.5% glutaraldehyde. The fixed, large preovulatory follicles were subsequently microdissected to obtain membrana “sheets.” Samples from individual animals were pooled for freeze-fracture analysis with respect to the percent of oocytes irreversibly committed to mature. In addition to the two extremes of 0 and loo%, five ranges of percent commitment were established. The isolated membrana granulosa “sheets” were placed in 1.0 mlO.05 M Na Cacodylate buffer in a small Falcon dish (No. 1008), 1.0 ml of 30% glycerol solution was added to the dish, and the cell segments were incubated for 30 minutes. After a n additional 1hour incubation in fresh 30% glycerol solution, approximately 10 membrana “sheets” were sandwiched between two goldhickel flat-top specimen holders, frozen in melting Freon 22, and placed in a Balzers double replica device. Specimens were fractured in a Balzers 400 Freeze Etch Unit (Balzers Corp., Nashua, NH) at

-llo“C, etched for 10 seconds, and replicated with platinum and carbon. After cleaning in sodium hypochlorite, sulfuric acid and deionized water, replicas were surveyed by TEM at 80 kV. Quantitative morphometric analyses were applied to tracings of electron microscope negatives using a Zeiss Videoplan Image Analysis System. Total gap junction membrane areas and total surveyed membrane areas were measured, and from these measurements the percentage of total membrane occupied by gap junctional area (termed the fractional gap junction area) was derived. In addition, the average gap junctional particle density for each commitment period was determined using the following procedure. A tem late with square holes of area 2.25,4.00, and 6.25 mm was laid over a n EM negative such that the largest possible area of the gap junction could be viewed. Three areas were analyzed where possible, although five areas were included where there was great variability in particle density in any one junctional area. An effort was made to take measurements from scattered areas which were representative of the overall particle distribution. When the template was in position, the number of particles within the area and touching two adjacent sides of the square border was counted using a lupe and all counts were standardized to a n area (6.25 mm2) and a magnification (29,485 x ) from which the number of particles per p,m2 of gap junction was determined. These analyses revealed that the fractional gap junction area decreased progressively with increasing percent of oocytes irreversibly committed to mature with more than a 50% decrease in this parameter between 0 and 75% oocytes committed. In addition, the density of particle-packing was significantly and negatively correlated with the percent of committed oocytes. Figure 11shows examples ofjunctional areas that exhibited 1) tight particle-packing (376 particles/p,m2 of gap junctional area from the control group with 0% oocytes irreversibly committed); 2) intermediate particlepacking (137 particles/p,m2 of gap junctional area from the group of animals in which 1-19% oocytes were irreversibly committed); and 3) loose particle-packing (67 particles/p,m2 of gap junctional area from the group of animals in which 68-838 of oocytes were irreversibly committed). The cellular mechanisms responsible for this dramatic down-regulation in membrana gap junction integrity were not defined in this study with the hamster. However, our observations implicate both of the fundamental mechanisms for gap junction dis-

P

Fig. 15. Electron micrographs of platinum-replicated Syrian hamster oocyte cortical patches. ~ 4 0 , 0 6 3 .A A cortical view showing branched filaments of several different diameters, bifurcated filaments (BF), and cytoskeletal sheets (S) enmeshed loosely within the filamentous network. B: A localized, extensively filamentous network which characterizes hamster oocyte cortical patches. Note the variety of different thicknesses of filaments and bundles of filaments in this moderately thick patch. C: A thin cortical patch which contains a sheet (S). Note the high degree of branching of the filaments, the intimate association between filament arrays and vesicular structures and the dense pattern of fine filaments covering the inner surface of the plasma membrane in this preparation.

Fig. 15.

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Fig. 16. A high-magnification electron micrograph of a portion of one of the cytoskeletal sheets observed in a cortical patch from a hamster oocyte. The sheet appears to have torn horizontally near the bottom, probably as a result of the preparatory method. This view of

a sheet dramatically illustrates the pattern of rows formed by aligned particles which appear to constitute the substructure of this unique cytoskeletal element. x 117,000.

MEIOTIC MATURATION IN MAMMALIAN OOCYTES

posal that have been described for other systems (see Larsen et al., 1987, for review). First, we have observed more or less spherical structures in several of the replicas of the cytoplasm of these cells. These structures (Fig. 121, which are characterized by the P-face particles and E-face pits typical of gap junctional membrane a t the cell surface, appear identical with the cytoplasmic gap junctional vesicles described by Larsen and his colleagues (Larsen and Tung, 1978; Larsen et al., 1986, 1987). Such cytoplasmic vesicles are believed to arise from junctional interiorization which provides a natural endocytotic process for gap junctional disposal. The alternate mechanism of gap junction disruption which may occur in the above-described hamster system is that of particle dispersion within the plane of the membrane as proposed by Yancey et al. (1979) and Lane and Swales (1980) in regenerating rat liver and insect mesoglial cells, respectively. In support of this possibility, we have observed apparent particle dispersion at the edges of (Fig. 13A) and within (Fig. 13B,C) junctional plaques, and significant decreases in overall gap junction particle density with time post-hCG (Fig. 11). While a quantitative analysis of particle density in the immediate vicinity of the junctional areas was not undertaken in this study, we did not observe any parallel increase in density of surface membrane particles contemporaneous with the decrease in gap junctional particle density. Therefore, it seems probable that the particles are removed rapidly from the cell surface after dispersion. This quantitative freeze-fracture study with the hamster provides direct support for the Dekel and Beers hypothesis for the regulation of meiotic resumption in mammalian oocytes. This support is derived from our observations that concomitant with a n increase in the proportion of oocytes irreversibly committed to resume meiosis, there is a progressive downregulation of gap junctions among the membrana granulosa cells which underlie the cumulus cell stalk. Whether this down-regulation reflects that which occurs within the other cellular subpopulations in the hamster follicle remains to be determined. However, the present observations may have considerable physiological relevance since the membrana “sheet” occupies a critical location in the follicle by providing a bridge between the oocyte-cumulus complex and the remaining membrana granulosa.

THE CORTEX OF MAMMALIAN OOCYTES The cortical region of oocytes is considered instrumental in developmental events which follow oocyte maturation. In this region, cortical granules accumulate during late stages of maturation (our thin-section study discussed above; Cran and Cheng, 1985; Szollosi, 1967). This is the cytoplasmic area of the oocyte first encountered by the penetrating sperm nucleus, and the region in which the meiotic spindle operates during the first and second meiotic divisions. Following fertilization, cleavage furrow formation is apparently regulated by the cortical region of the zygote. Later in development, the cortical regions of embryonic blastomeres play important roles in compaction of the morula

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through intercellular contact (Van Blerkom and Motta, 1979). During meiotic maturation, the cortical cytoplasm is relatively devoid of major organelles (see thinsection study discussed above) but contains accumulations of actin (Ducibella et al., 1977; Lehtonen and Badley, 1980). Work with nonmammalian oocytes suggests that the cortical region contains developmental information which is localized in specific areas. This information controls the differentiation of these specialized regions following compartmentalization during cleavage (see Capco and Larabell, 1989, for review). We have begun to characterize the ultrastructure of the mammalian oocyte cortex by examining platinumshadowed replicas of cortical patches prepared from porcine and Syrian hamster oocytes. We adopted the procedure of Aggeler e t al. (19831, in which cells are allowed to attach to polylysine-coated glass coverslips and are subsequently blown away with a stream of buffer to leave adherent patches of cortical material. These cortical preparations provide information a s to the inner layer of the oocyte plasma membrane, the filamentous network underlying the plasma membrane, vesicular organelles intermingled with the cortical filaments, and other organelles which are more loosely associated with the cortical filaments. Porcine oocytes were collected and matured in vitro during 24 hours of culture in 2A medium. Immature hamster oocytes were harvested after ovarian stimulation as described above but without hCG treatment, and placed in 2A-AA medium (2A containing four amino acids; Gwatkin and Haidri, 1973) for 21 to 23 hours to allow for maturation in vitro. Oocytes from both species were subjected to the same basic methods of cortical patch preparation. Oocytes were removed from culture, and the zonae pellucidae removed by brief incubation in pronase-CB (0.1%, w/v; pig oocytes) or trypsin (0.1%, w/v; hamster oocytes). Oocytes were then washed in 2A medium, transferred to polylysinecoated coverslips, and incubated in a n intracellular buffer (100 mM KC1,5 mM MgCl,, 3 mM EGTA and 20 mM HEPES, pH 6.8; Aggeler et al., 1983). When the zona-free oocytes had attached to the surface of the coated coverslip, buffer was taken up into a mouthoperated glass pipette and a stream of buffer was blown onto the top surface of each oocyte. This resulted in disruption of the oocytes, and removal of all but the adherent plasma membrane patches with associated cortex. The cortical patches were fixed in 2% glutaraldehyde in intracellular buffer, washed in buffer, dehydrated through a n ethanol series and dried through the critical point of freon or CO,. After fixation and critical point drying, the cortical patches were shadowed with platinum and carbon and examined in the TEM. A two-step procedure was used to label actin microfilaments in the cortical patches from hamster oocytes before fixation. The adherent cortical patches first were extracted for 5 minutes in a buffer containing detergent (0.5’70,v/v, Triton X-100, 100 mM NaC1, 300 mM sucrose, 3 mM MgCl,, 10 mM PIPES, 5 mM EGTA, 1.2 mM phenylmethylsulfonylfluoride,pH 6.8) to remove soluble proteins (Capco and McGaughey, 1986). After washing the extracted patches three times in intracellular buffer, the patches were placed in a solution

Fig. 17.

MEIOTIC MATURATION IN MAMMALIAN OOCYTES

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of heavy meromyosin (Sigma) for 15-20 minutes. La- cles alternating with particle-free areas (Fig. 16). The beled patches were washed with intracellular buffer replica shown in Figure 16 is from a n unextracted corand fixed in 1% glutaraldehyde containing 0.2% tannic tical patch and therefore exhibits a sheet with associacid before dehydration and critical point drying. ated soluble proteins. Recent biochemical studies of deThe unextracted cortex of pig oocytes contains re- tergent-extracted hamster oocytes have shown that the gions of inner plasma membrane, a n extensive network sheets may constitute stores for the intermediate filaof filaments of varying diameter and a large number of ment protein cytokeratin (McGaughey and Capco, prominent vesicular structures of various sizes (Fig. 1989). Similar cytoskeletal elements have been ob14). In thinner cortical patches (Fig. 14A), the plasma served in detergent-extracted mouse oocytes (Mutchler membrane is more readily observed, and many small et al., 1988). Cytoskeletal sheets, which appear univesicular structures, probably cortical granules, are formly distributed in whorls in unfertilized eggs, beclosely associated with regions of the plasma mem- come highly organized in the peripheral cytoplasmic brane. In many regions of the cortex, filaments are regions of blastomeres of compacting morula-stage emfound to be connected to vesicular organelles and ap- bryos, and disappear from the outer, epithelial cells of pear to hold groups of them together in clusters. the trophectoderm at the early blastocyst stage (Capco Extensive associations among cortical filaments of and McGaughey, 1986). different sizes are more evident in thicker cortical Investigations are now underway to identify the filpatches (Fig. 14B). These thicker preparations demon- aments which constitute the networks we have obstrate the branched nature of many of the filaments, served in cortical preparations of mammalian oocytes. and show that filaments form bundles in regions where One such investigation employs a labeling method for they intersect vesicular structures. Cortical patches identification of actin. By utilizing heavy meromyosin from pig oocytes exhibit a more uniformly distributed, labeling, we have been able to demonstrate that the extensive filamentous network and larger vesicular cortex exhibits a n extensive network of actin microfilstructures than do hamster oocyte cortical patches. aments (Fig. 171, a n observation recently confirmed by Hamster oocytes exhibit unique cellular elements ultrastructural immunocytochemistry (Webster and called “sheets,” which are generally located in the sub- McGaughey, 1988). In addition, two types of filaments cortical region of the unfertilized hamster oocyte are present in the cortex which are smaller in diameter (Capco and McGaughey, 1986), as evidenced by their than microfilaments and which do not comprise actin. relative abundance in thicker cortical patches a s com- One type includes relatively long, branched filaments pared with their relative sparseness in thinner prepa- with a diameter approximately one-half that of a mirations. In platinum replicas, these sheets appear as crofilament (Fig. 17A). The second type is usually very planar elements with highly ordered substructure (Fig. short, has a diameter between one-eight and one-fourth 15A,C). As seen in Figure 15A, the sheets appear to be that of a microfilament (Fig. 17B), and generally forms loosely enmeshed among filaments; some of these fila- interconnections between microfilaments. ments actually lie directly across the surface of the CONCLUSIONS sheets. In this review, we have considered three different The diversity of cortical filaments in hamster oocytes is shown in Figure 15B. Areas of highly complex fila- approaches used to analyze the ultrastructure of mammentous networks are regionally localized in the ham- malian oocytes and follicular granulosa cells. By examster oocyte cortex as compared with a more uniform ining thin sections of embedded porcine oocytes at predistribution of these networks in the pig oocyte. The cisely determined stages of meiotic maturation, we degree to which the cortical filaments are branched have identified major nuclear and cytoplasmic changes and interconnected, however, is very similar in ham- correlated with this important developmental process. ster and pig oocytes. Thin cortical patches from ham- By utilizing freeze-fracture electron microscopy, we ster oocytes (Fig. 15C) reveal associations between have investigated the relationship between gap juncbundles of cortical filaments and spherical organelles, tion integrity among the membrana granulosa cells in addition to the dense network of filaments juxta- which underlie the cumulus cell stalk and oocyte commitment status. By employing isolated cortical patch posed to the inner surface of the plasma membrane. Cytoskeletal sheets are observed in cortical replicas preparations, we have begun to understand the ultraas planar elements composed of rows of uniform parti- structure, and to identify the constituent filaments, of the oocyte cortex. The standard method of TEM revealed that the following major developmental patterns occur in oocytes concomitant with the progression of meiotic maturation: 1) early dissolution of the nuclear envelope and Fig. 17. Electron micrographs of detergent-extracted hamster oocyte cortical patches after treatment with heavy meromyosin to formation of ER-like elements from disrupted nuclear identify actin microfilaments. A high proportion of the filaments in envelope remnants; 2) perinuclear migration of mitothese thin patches of extracted cortex are decorated by heavy meromyosin. Two different populations of unlabeled filaments can be iden- chondria during early stages of maturation and the tified in these preparations. Longer, branched filaments with a diam- subsequent migration of mitochondria and other oreter approximately one-half that of the actin microfilaments are seen ganelles away from the first meiotic spindle once it has at the top right and top center of A. The second type of unlabeled formed; 3) migration of cortical granules to positions filament is very short, often is observed to form interconnections bebeneath the plasma membrane; and 4) a n abundance of tween actin filaments, and has a very narrow diameter, as shown in junctional complexes between the cumulus cell proB (arrows). x 84,225.

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cesses and the oocyte plasma membrane in the immature oocyte, with a n apparent overall decrease in number of complexes with meiotic progression, but with some complexes maintained even a t the late stages of maturation. These prominent developmental changes, observed in thin sections, provide ultrastructural markers for specific stages of oocyte maturation in carefully timed experiments. Such markers are essential correlates to future studies of the molecular and cellular mechanisms by which mammalian oocyte maturation is regulated. Cytological changes, examined in this and other studies, can be employed to distinguish between immature oocytes and oocytes that are at very early stages of the maturation process (i.e., by observing whether the nuclear envelope has begun to convolute and invaginate). In addition, cytological characteristics can be used to assess the normalcy of oocytes maturing under varied experimental conditions (e.g., by observing mitochondria1 and other organellar migration patterns). One criticism of investigations using cultured mammalian oocytes to assess meiotic maturation is that certain culture conditions and agents thought to be physiological regulators of maturation may cause damage to oocytes, thereby causing degenerative maturation events instead of those which occur naturally in meiosis (Biggers and Powers, 1979). By incorporating ultrastructural analyses into such investigations, this criticism could be partially waived with the observation of patterns of developmental events which mimic those of healthy maturing oocytes. Elucidation of the role of intercellular coupling in the regulation of mammalian oocyte maturation will significantly enhance our understanding of this fundamental developmental process. Using freeze-fracture analysis, we have demonstrated that the commitment period, in which cellular and molecular programs for maturation are initiated, is highly correlated with a decrease in the degree of coupling among the membrana granulosa cells underlying the cumulus cell stalk. This correlation adds credence to the possibility that reduced efficiency of the transfer of small molecules between follicular cells plays a role in initiation of maturation. Such small molecules may be derived from granulosa cells and act as follicular meiotic “arresting” agents. Reduction in the transfer of these agents between granulosa cells and the oocyte may occur, bearing a causal relationship to commitment to maturation. Identification of the physiologically active “arresting” agents coupled with direct evidence that such agents are indeed transported through gap junctions will greatly enhance our understanding of meiotic regulation in mammalian oocytes. Although thin section analyses demonstrate a consistent pattern of organellar reorganization during meiotic maturation, the cellular mechanisms by which this reorganization is accomplished remain obscure. However, it is probable that mechanisms are involved that are similar to those which accompany such changes in somatic cells in which major restructuring of the cytoskeleton occurs (Ben-Ze’ev et al., 1980; Folkman and Greenspan, 1975; Reiter et al., 1985). If true,

then detailed knowledge of the oocyte cytoskeleton will be essential for a n understanding of the mechanisms of nuclear migration to the peripheral cytoplasm prior to polar body abstriction. Likewise, cytoskeletal elements may play a role in the movement of mitochondria and other organelles to their perinuclear position and later, away from the meiotic spindle. Cortical patch analysis, coupled with the study of embedment-free sections of detergent-extracted oocytes (Capco and McGaughey, 1986), hold great promise for uncovering the structural relationships between specific cytoskeletal elements and other cellular components.

ACKNOWLEDGMENTS We are grateful to Dr. Carolyn A. Larabell for preparation of freeze-fracture replicas, to Charles J. Kazilek for preparation of the photographic prints and to Larry Nienaber for breeding and maintaining the hamsters. This work was supported by grants HD15984 (to C.R.) and HD23686 (to R.W.M.) from the NIH, and by Biomedical Research Support Grant SO7 RR07112 (to C.R.), awarded to Arizona State University by the Biomedical Support Grant Program, Division of Research Resources, NIH.

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Ultrastructural correlates of meiotic maturation in mammalian oocytes.

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arr...
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