Surface alterations of the bovine oocyte and its investments during and after maturation and...

MOLECULAR REPRODUCTION AND DEVELOPMENT 38421430 (1994)

Surface Alterations of the Bovine Oocyte and Its Investments During and After Maturation and Fertilization In Vitro HIROYUKI SUZUKI, XIANGZHONG YANG, AND ROBERT H. FOOTE Department of Animal Science, Cornell University, Ithaca, New York

ABSTRACT Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 h r of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 pm during maturation and increased to 106 pm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. o 1994 Wiley-Liss, Inc.

Key Words: Cumulus, Oocyte, Zona pellucida, SEM, Ultrastructure

INTRODUCTION For in vitro fertilization in cattle, cumulus-oocyte

complexes (COCs) are usually recovered from small antral follicles of ovaries obtained from slaughtered cattle or aspirated in vivo and matured in vitro. The COCs, liberated from their follicular environment and incubated in suitable culture media, are able to complete nuclear maturation spontaneously in vitro, similar to events that occur in vivo in several species (Edwards, 1965; Thibault, 1977; Thibault et al., 1987), including 0 1994 WILEY-LISS, INC.

cattle (Fukui and Sakuma, 1980; Hunter et al., 1972; Yang et al., 1993). However, in vitro fertilization of these oocytes matured in vitro and their subsequent development have been relatively inefficient as compared to those matured in vivo (Greve et al., 1987; Leib- fried-Rutledge et al., 1987; Moor and Trounson, 1977; Thibault, 1977; Thibault et al., 1987; van Blerkom and McGaughey, 1978). These events suggest that oocytes need to undergo cytoplasmic maturation as well as nuclear maturation for normal fertilization and development.

The COCs are maintained by delicate cell-to-cell connections among the cumulus cells and with the oocyte (Eppig, 1982; Eppig and Downs, 1984; Moor et al., 1980). The cumulus cells have been shown to be linked among each other, and the innermost layers, the corona radiata cells, often form cytoplasmic processes penetrating through the zona pellucida (ZP) and maintain- ing contact with the vitelline membrane of the oocyte (Allworth and Albertini, 1993; Anderson and Albertini, 1976; Assey et al., 1993; Gilula et al., 1978; Hyttel, 1987; Moor et al., 1980; Zamboni, 1974). Thus, cumulus-corona cell mass may play a role in regulating oocyte activity (Eppig and Downs, 1984; Gilula et al., 1978; Laurincik et al., 1992b; Moor et al., 1980). Like- wise, oocytes produce cumulus cell expansion enabling factor(s) to stimulate expansion of cumulus cells during maturation in rodents (Vanderhyden et al., 1990). Cu- mulus cell-oocyte coupling has been studied in the mouse (Eppig, 1982). Recent studies in cattle have also emphasized the relationship between cumulus cell expansion and oocyte maturation (Laurincik et al., 1992a,b).

Furthermore, the cumulus cells, the ZP, and the vitelline membrane of the oocytes may undergo some changes, along with the nucleus and cytoplasm, in the maturation process to achieve normal fertilization. The ZP and vitelline membrane have been investigated morphologically in relation to penetration and fusion

Received September 30,1993; accepted February 1,1994. H. Suzuki's permanent address is Laboratory of Animal Science, Hiro- saki University, 3 Bunkyo-cho, Hirosaki, Aomori 36, Japan. Address reprint requests to Dr. Xiangzhong Yang, 205 Morrison Hall, Animal Science Department, Cornell University, Ithaca, NY 14853- 4801.

422 H. SUZUKI ET AL.

by a spermatozoon during fertilization (reviewed by Hyttel et al., 1989; Talbot, 1985; Yanagimachi, 1988). The surface morphology in relation to oocyte maturation of these structures has also been investigated in some laboratory animals (Calafell et al., 1992; Jack- owski and Dumont, 1979; Phillips and Shalgi, 1980)

classified as fertilized or unfertilized, depending on the presence or absence of two polar bodies, before processing for SEM.

Removal of Cumulus Cells and the Zona Pellucida

and in humans (Familiari et al.,-l988, 1989; Motta et al., 1988). In addition, some investigators, employing SEM, reported structural alterations in the ZP and vitelline membrane of the mouse oocyte after fertilization (Jackowski and Dumont, 1979). Other investigators, however, were unable to observe such morphological changes under similar conditions in the mouse and hamster (Phillips and Shalgi, 1980), or in humans (Fa- miliari et al., 1988). Related information on the ultrastructural changes of the oocyte surface and its investments has been scarce in farm animals, particularly in cattle.

The objective of this study was to evaluate by SEM the ultrastructure and surface morphology of the cumulus/corona cells, the ZP, and the vitelline membranes of immature, in vitro matured, and in vitro fertilized bovine oocytes. Changes in diameter of these oocytes were also recorded for comparison during SEM observations.

MATERIALS AND METHODS Collection of Oocytes

The COCs were collected by aspirating small antral follicles (2-5 mm in diameter) on ovaries obtained from a slaughterhouse. An immature group of COCs was processed for SEM immediately after collection. This consisted of 11 cumulus-intact oocytes, 15 ZP-intact oocytes with a small number of corona cells, and 33 oocytes from which the ZP had been removed as de- scribed later. The remaining COCs were incubated in tissue culture medium 199 (TCM 199; Earle’s salts) containing 25 mM Hepes and 7.5% fetal calf serum with added 0.5 Fg/ml oFSH, 5 p.g/ml oLH (NIDDH), and 1 p,g/ml estradiol a t 39°C in 5% COz and 95% air (Yang et al., 1993). COCs were harvested and processed after maturation incubation at 10 hr (10 cumulus-intact, 15 ZP-intact, and 30 ZP-free oocytes) and 22 hr (5 cumulus-intact, 10 ZP-intact, and 33 ZP-free oocytes). Oocytes of the 10 and 22 hr maturation groups are referred to as maturing and matured oocytes.

Inseminated oocytes were obtained following in vitro fertilization of oocytes a t 1 hr (5 cumulus-intact and 12 ZP-intact oocytes), 3 hr (5 cumulus-intact and 12 ZP- intact oocytes), 6 hr (5 cumulus-intact, 11 ZP-intact, and 23 ZP-free oocytes), or 20 hr (10 cumulus-intact, 24 ZP-intact, and 26 ZP-free oocytes) post insemination. After 6 hr of spermloocyte coincubation in Brackett’s defined medium (Brackett and Oliphant, 19751, sam- ples were taken and the remaining COCs were trans- ferred to fresh TCM 199 without hormones and cultured for a total of 20 hr or longer (not included) for further development, as done routinely in our laboratory (Yang et al., 1993). The inseminated oocytes were

The cumulus cells were removed by pipetting COCs in medium containing 0.2% hyaluronidase. Cumulus/ corona cells were removed completely when examining ZP-free oocytes. These were removed only partially when examining ZP-intact oocytes in order to obtain information about surface characteristics of the corona radiata cells.

The ZP was removed using a modified procedure from Yang et al. (1990). Oocytes were exposed to prewarmed, acidified (pH = 2.5) Dulbecco’s phosphate-buffered sa- line (DPBS, Gibco, Grand Island, NY) containing 0.1% polyvinyl alcohol (PVA, Sigma, St. Louis, MO) for 1 min and 0.5% pronase for 3-5 min. This allowed complete removal of ZP residuals on the oocytes. The ZP- free oocytes were incubated for 20 min in calcium and magnesium-free DPBS at 39°C.

SEM Observations Oocytes with or without their cumulus investments

or ZP in each group were fixed for 1 hr in 3% glutaral- dehyde and 0.5% paraformaldehyde in Hanks’ balanced salt solution (HBSS) with 0.1% PVA. They were washed in three changes of HBSS and placed on small glass coverslips (6 x 6 mm) coated with 0.1% po1y-L- lysine solution (Sigma). The oocytes on the coverslips were postfixed in 1% osmium tetroxide in HBSS for 1 hr. After rinsing, the coverslips were incubated in 2% tannic acid solution for 2 hr, rinsed, and then reosmi- cated for 1 hr in 1% osmium tetroxide in distilled water. The specimens were then dehydrated in a series of in- creasing concentrations of ethanol, critical point dried, and sputter coated with gold. Observations were made with a Zeiss DSM-960 scanning electron microscope at an accelerating voltage of 10 kV.

Diameter of Oocytes The diameters of ZP-free oocytes of various stages

were measured during observation with SEM. Diame- ters were measured systematically. The measurements were subjected to a one-way analysis of variance. Dun- can’s multiple range test was used to detect statistical differences among means.

RESULTS Fine Structure of Cumulus Investment

Immature oocytes. The SEM features of COCs of the immature group displayed a compacted arrangement of flattened smooth cells of cumulus oophorus (Fig. la). The cumulus cells possessed multiple, long thread-like cell processes, making contact with neigh- boring cells (Fig. la). The corona radiata cells were spherical and most had a smooth membrane surface

INVESTMENTS AND ULTRASTRUCTURE OF THE BOVINE OOCYTE 423

Fig. 1. Characteristics of cumulus investment of the cumulus- oocyte complexes (COCs) during oocyte maturation and fertilization in vitro. a: Surface features of COCs in the immature group. The cumulus cells had a smooth surface on which few microvilli (MV) and cytoplasmic blebs were observed. Note an extensive network of the thread- like cell processes among cumulus cells. Bar represents 5 pm. b Surface features of COCs after 10 hr of incubation. The thread-like cell processes were thicker, and the intercellular spaces among cumulus cells became wider when compared with the immature group. Some cumulus cells were covered with well-developed MV (arrow). Bar rep-

(Fig. 2a). Some ruptured cell membranes were occasionally found, probably due to processing. No obvious cell processes among the corona radiata cells were observed. However, residuals of cytoplasmic processes of

resents 5 pm. c: Surface features of COCs after 22 hr of incubation. The cumulus cells were characterized by a solid geometric architecture of thick thread-like cell processes. Note the much wider intercellular spaces and the thick cell projections among cumulus cells. Bar represents 5 wm. d Surface features of COCs 1 h r after insemination. Many spermatozoa are attached to the COCs. Note the tight arrangement of cumulus cells with much smaller intercehlar spaces among them compared to the maturing and matured oocytes. The thread-like cell processes became thinner when compared with those of the matured oocytes (Fig. la). Bar represents 5 pm.

these cells projected to the ZP were numerous (Fig. 2a and 2b).

Maturing and matured oocytes. After 10 hr of incubation, cumulus cells of the maturing oocytes became


less compacted and the intercellular spaces were more obvious (Fig. lb). Sometimes the cumulus cells were covered with well-developed cumulus microvilli (MV). However, cell contacts among cumulus cells were less tenuous and fewer in number. The thread-like cell processes among them appeared to become slightly thick- ened compared with those of the immature oocytes (compare Fig. la and lb). The corona radiata cells seemed to be slightly swollen with more prominent processes projected to the ZP. No membrane ruptures were found on corona radiata cells, which may suggest disas- sociation of cell contact with the cumulus cells.

At 22 hr of incubation, almost all oocytes were considered to be matured, based on cytological examina- tion in our maturation culture system (Yang et al., 1993). The SEM examinations revealed dramatic changes in the morphology of COCs. Cell contact among the cumulus cells became even looser with much wider intercellular spaces among them (Fig. lc). The thread-like cell processes, however, became much thicker than those in earlier stages (compare Fig. la+). Full expansion of the corona radiata cells was discern- ible. Individual cells became elongated bulb-shaped structures (Fig. 2e). No membrane ruptures of the corona radiata cells were observed, again suggesting the absence of their direct connections with the cumulus cells. Cytoplasmic processes of the corona radiata cells to the ZP decreased dramatically with practically no

Fig. 2. Characteristics of corona radiata cells and the ZOM pellucida (ZP) during oocyte maturation and fertilization in vitro. a: ZP- intact immature oocyte with some corona cells. The corona cells were spherical and some had ruptured membranes. The ZP showed a mesh- like appearance. Much cellular debris was present. Bar represents 5 pm. b Higher magnification of the immature ZP surface. A fibrous mesh-like network structure of the ZP was apparent. Note club- shaped invaginations (arrows) located in the mesh of the ZP. Bar represents 2 pm. c: ZP-intact oocyte after 10 hr of incubation. Corona radiata cells were swollen. Note many terminals of the cell cytoplasmic processes penetrating into the ZP. Bar represents 5 pm. d Higher magnification of ZP surface after 10 hr of incubation. The mesh-like structure of the ZP was similar to the immature oocyte, but with more prominent cytoplasmic processes invaginations. Note well-developed invaginations penetrating into the ZP. Bar represents 2 pm. e: ZP- intact oocyte with some corona cells after 22 hr of incubation. The corona cells became fully expanded and elongated to a bulb-shaped structure. Note fewer invaginations of cytoplasmic processes at this stage. Bar represents 5 pm. E Higher magnification of the ZP surface of an oocyte after 22 hr of incubation. Few invaginations of cytoplasmic processes were found on the ZP at this stage. Note the wider mesh arrangement of the fine fibrous structure. Bar represents 2 pm. g: ZP-intact oocyte with some corona cells 1 hr after insemination. The corona cells became spherical but with a shrunken, wrinkled surface. ZP surface changes were also discernable. Bar represents 5 pm. h ZP surface of an oocyte 6 hr after insemination. The surface of the ZP was more smooth and less porous. Note a spermatozoon penetrating into the ZP. The sperm tail was visible. The sperm head may be stopped in the inner part of the ZP. Bar represents 2 pm. i: ZP surface of an oocyte 20 hr after insemination. The ZP surface became a solid, mesh-less structure. Cumulus cells displayed MV and blebs. Note phagocytosis of a spermatozoon by cumulus cells (arrow) and numerous depressions behind the arrow, probably caused by sperm enzymatic digestion. Bar represents 5 pm.

residuals of cytoplasmic processes found on the ZP a t this stage (compare Fig. 2b,d,fl.

Inseminated oocytes. One hour after insemination the COCs showed a compact arrangement of cumulus cells similar to the immature oocytes (compare Fig. l a and Id). The intercellular spaces among the cumulus cells became much less prominent than those of the maturing or matured oocytes (compare Fig. lb-d). However, in contrast to the immature oocytes, the cumulus cells of the inseminated oocytes remained spherical (compare Fig. l a and Id). The corona radiata cells became smaller with a shrunken wrinkled surface (Fig. 2g). No residuals of cytoplasmic processes were found on the ZP.

Fine Structure of the Zona Pellucida Immature oocytes. The ZP of immature oocytes

was characterized by a fibrous network, with numerous wide meshes and deep holes. Often large numbers of residuals of the cytoplasmic processes were found to be embedded in the meshes and holes of the ZP (Fig. 2b). Under higher magnification, these processes were usually noticed as club-shaped invaginations that pene- trated into the meshes of the ZP (Fig. 2b). They were probably the remaining parts of the cumulus and corona radiata cell processes, which normally extend through the ZP into the oocyte.

Maturing and matured oocytes. Maturing oocytes at 10 hr of incubation manifested an increased number of cytoplasmic processes to the ZP. Predomi- nant and rope-like processes were observed in 4 of the 15 oocytes examined (Fig. 2c). In general, these processes were more developed than those in the immature oocyte group (compare Fig. 2c,d vs. Fig. 2a,b). No re- markable changes were found in the mesh structure.

At 22 hr of maturation, the corona radiata cells possessed much fewer cytoplasmic processes and even fewer invaginated into the ZP (Fig. 2c,e). The fibrous network of the ZP appeared to become finer, and the meshes and holes seemed less deep (Fig. 20.

Inseminated oocytes. At 1 hr after insemination, changes in the mesh structure in the ZP were discern- ible. Practically no invaginations of the cytoplasmic processes were observed. The mesh of the ZP decreased in size, probably due to fusion of several layers of the fibrous network. Similar features of the COCs and the ZP were observed for oocytes 3 and 6 hr after insemination. The mesh arrangement of the ZP of the oocytes at 6 hr without a second polar body resembled that observed in the group matured for 22 hr. At 20 hr after insemination, the cumuluskorona cells displayed long MV and many cytoplasmic blebs on the ZP surface (Fig. 2i). No meshes were found at this stage, and sometimes cup-shaped depressions were observed on the surface of the ZP (Fig. 2i). Occasionally phagocytosis of the sperm cells could be found on the ZP (Fig. 29.

Surface Characteristics of Vitelline Membranes Immature oocytes. Two types of ultrastructures

were observed on the surface of the immature oocyte,


the tongue-shaped protrusions (TSPs), and the microvilli (MV). TSPs were observed as predominant or semipredominant structures on the vitelline surface in 21 out of 33 immature oocytes examined. As Figure 3a-c shows, the distribution of TSPs varied from a uni- form dense pattern (Fig. 3a) to being loosely distributed and mixed with MV over the entire surface of the oocyte (Fig. 3c). The oocytes were densely populated with TSPs (11/21, 52%) and had few microvilli (MV) on their sur- faces (Fig. 3a). Some MV appeared to be sprouted from the top of the TSPs (Fig. 3b,c). In oocytes with relatively fewer TSPs (10/21, 48%) two types of MV were observed, the typical, cylinder-like MV and the “sprouted” MV (Fig. 3c). Some of the TSPs had notched tips, which looked like the teeth of a comb (Fig. 3b,c). This phenomenon was particularly observed on oocytes with a mixture of TSPs and MV.

Maturing and matured oocytes. The vitelline surface of the oocytes incubated for 10 or 22 hr were characterized by a very dense distribution of MV with scattered TSPs (Fig. 3d). The TSPs with notched tips were more frequently observed on the maturing oocytes (10 hr incubation), indicating possible transformation of TSPs to MV during maturation. The MV were evenly spaced and distributed on the surface of the matured oocytes. No MV-free area overlying the metaphase spindle, the so-called nipple region reported in the mature mouse oocyte (Ziomek, 1987), was observed on mature bovine oocytes. There were no other indications of surface polarization found at this stage oocyte.

Matured oocytes had expelled the first polar body in a majority of the oocytes examined a t 22 hr of incubation (26/33), similar to our previous report (Yang et al., 1993). The polar body was covered with no or very few MV. Small amounts of cell debris were usually found at the base of the polar body. In the oocytes without a first polar body after 22 hr of incubation, more cytoplasmic blebs were often noted on the vitelline surface. Further- more, the MV of these oocytes were fewer and smaller as compared with those of the oocyte with a first polar body (not shown).

Inseminated oocytes. The vitelline surface morphology of the oocytes after in vitro fertilization (6 and 20 hr) resembled that of matured oocytes, characterized by highly densely populated MV and occasionally with scattered TSPs (Fig. 30. MV of some inseminated oocytes (20 hr post insemination) were less populated than those in the matured oocytes. A convex protrusion was found on two of the oocytes examined at 6 hr after insemination. The protrusion areas contained no MV, as shown in Figure 3e. These probably were fertilized oocytes in the telophase stage of the second meiosis that were ready to release the second polar body. The distribution pattern of MV of the unfertilized oocytes was less dense than that of the fertilized oocytes, and resembled that observed in oocytes that failed to mature after incubation.

At 6 hr post insemination, depressions on the ZP surface of some oocytes, occasionally with spermatozoa1 penetration but blocked at the inner part of the ZP,

were observed (Fig. 2h, i). A tail from a penetrating spermatozoon was also found on the vitelline surface of an oocyte at 6 hr after insemination (Fig. 30.

Size of Oocytes The analysis of variance showed significant differ-

ences among the immature, matured, and inseminated oocyte groups (Table 1). Diameters of the immature oocytes were 98.8 -t 1.3 pm (mean k SE), which decreased to 79.9 k 1.8 pm after 10 hr of maturation in vitro, remained small upon completion of the first meiosis at 22 hr (81.6 k 0.3 pm), and then “expanded” to 105.8 ? 0.9 p m 20 hr after insemination. However, light microscopic measurements of the fresh oocytes throughout these stages remained similar in diameter (data not shown). The size difference between mature and inseminated oocytes could not be due to fertilization because fertilized and unfertilized oocytes (fertilization judged by the presence of a second polar body) exhibited no difference in size. Thus, data in the 20 hr group represent pooled observations of inseminated oocytes regardless of the fertilization status.

DISCUSSION Fine Structure of the Cumulus Investment and

the Zona Pellucida Our results demonstrated that cumulus morphology,

intercellular spaces, and cell to cell communications (processes) changed dramatically during oocyte maturation and fertilization. Changes in surface morphology of individual cumulus cells during maturation in vitro observed here were generally in agreement with those reported by Laurincik et al. (1992a). These findings were also similar to previous SEM reports on oocytes in the mouse (von Weymarn et al., 1980; Nogues et al., 1988), hamsters (Phillips and Shalgi, 19801, and humans (Familiari et al., 1988; Motta et al., 1988). How- ever, in the present study full expansion of the corona cells was observed and elongation of these cells occurred after maturation of oocytes. This observation contrasted with that by Laurincik et al. (1992a1, in which these authors reported absence of full expansion of corona radiata cells, even after 24 hr of maturation culture. The reason for this discrepancy may be due to differences in the culture system used and the use of a culture medium without addition of gonadotropins (All- worth and Albertini, 1993; Laurincik et al., 1992a; Yang et al., 1993). It is known that the gonadotropic hormones induce expansion of cumulus cells in cattle (Allworth and Albertini, 1993; Yang et al., 1993) as well as other species (Flbchon et al., 1986; Vanderhy- den et al., 1990).

Our observations on changes of the ZP during and after maturation and fertilization in vitro suggest a maturation change of the ZP fine structure and a dramatic change of the ZP mesh network after fertilization. These observations were in agreement with a recent study in the mouse in which a correlation between the morphology of the ZP surface and degree of oocyte maturity was reported (Calafell et al., 1992). Also, ZP

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TABLE 1. Average Diameter (pm) of Immature, In Vitro Matured, and In Vitro Fertilized Bovine Oocytes

Groups of oocvtes oocvtes Mean 2 SE Range

No, of Oocyte diameter (pm)

Immature, 0 hr 33 98.8" t 1.3 83-119 Matured, 10 hr 30 79.gb t 1.8 6%97 Matured, 22 hr 23 81.6b t 0.3 75-92 Inseminated, 6 hr 15 81.3b t 2.5 70-107 Inseminated, 20 hr 26 105.8" t 0.9 9S112

a,bscMeans with different superscripts are different (",'P < .05; a,b;b,cp < .01).

alterations were found in the mouse oocyte before and after fertilization (Jackowski and Dumont, 1979). On the other hand, some investigators found no changes in ZP surface morphology after fertilization in the mouse and hamster oocytes, and considered any ZP alterations as artifacts (Phillips and Shalgi, 1980). However, the fact that all the various groups of oocytes were subjected to the same procedure in the present study is interpreted to indicate that these ZP changes were related to physiological changes of the oocyte rather than artifacts. In addition, we have observed ZP alterations in cleaved embryos, including blastocysts, processed similarly to this study for SEM (Suzuki, Yang, and Foote, unpublished observation).

We hypothesize the fact that wide intercellular spaces among the cumulus cells and mesh-like arrangement of the ZP appear only after maturation is of evolu- tionary significance and should assist spermatozoa in becoming appropriately oriented to fertilize the oocyte. At 1 hr after insemination the Z P surface changed substantially, which probably corresponds to molecular changes during the zona reaction (Wassarman, 1988). It is well known that release of the contents of the cortical granules occurs rapidly in response to sperm penetration, preventing polyspermy. Prevention of penetration of accessory sperm is associated with modi- fication of ZP glycoproteins (reviewed by Y anagimachi, 1988; Wassarman, 1988).

Surface Characteristics of the Vitelline Membrane

The present observations on the surface of the vitelline membrane clearly showed that the bovine oocytes aspirated from small antral follicles varied in distribution of TSPs and MV. Prominent TSPs were more frequently observed in immature oocytes. After incubation for 10 or 22 hr, the oocyte surface was often densely covered with MV with scattered TSPs, indicating that surface characteristics of the oocyte changed from a TSP-predominant pattern to a MV-predominant pattern during maturation. Also transitional stages from TSPs to MV predominant patterns were observed. This and the inverse relationship between the TSPs and MV suggest that the typical densely populated MV on the oocytes reported here and elsewhere were probably derived from the TSP structures. This finding indi-

cates that the vitelline membrane undergoes certain surface changes while the oocyte is in the follicle. To what extent the status of the follicle (developing or atretic) affects this morphology is unknown. Also, the surface microvilli on the matured bovine oocyte were found as evenly distributed with no indications of polarity. This finding was different to that in the mouse (Ziomek, 1987) but similar to that of humans (Santella et al., 1992).

Ultrastructural and immunocytochemical studies have shown that the cumulus cell processes pass through the ZP with gap junctions contacting the surface of the ooplasm (Allworth and Albertini, 1993; Anderson and Albertini, 1976; Assey et al., 1993; Gi- lula et al., 1978; Hyttel, 1987; Moor et al., 1980; Zam- boni, 1974). These projections maintain the biochemi- cal communications between the oocyte and cumulus cells (Eppig and Downs, 1984; Gilula et al., 1978; Moor et al., 1980; Laurincik et al., 1992b; Vanderhyden et al., 1990). TSPs observed in the present study are interpreted to represent the oocyte's terminals of the cytoplasmic processes that were disconnected from the cumulus cells when the ZP were removed.

It has been demonstrated that uncoupling between the bovine oocyte and cumulus cell cytoplasmic processes occurred after 12 or 18 hr of culture when all junctional contact was disrupted (Hyttel et al., 1986b; Hyttel, 1987). In addition, changes in locations of cytoplasmic organelles, such as mitochondria and some ves- icles, occur a few hours after the final loss of gap junctions in vivo (Kruip et al., 1983; Hyttel et al., 1986a) and in vitro (Hyttel et al., 198613; Hyttel, 1987). Such organelle migrations may be linked with the loss of intercellular communications of cumulus cells and the oocyte, as reported here, and the cytoskeletal changes in the cytoplasmic processes as reported by others (All- worth and Albertini, 1993; Hyttel, 1987).

Volumetric Changes of Oocytes In the present study, the size of oocytes changed sig-

nificantly during maturation, and also after fertilization, as determined by measurements on fixed oocytes during SEM observations. Formation and enlargement of the perivitelline space after oocyte maturation have previously been reported (Kruip et al., 1983; Okada et al., 1986; Hyttel et al., 1988a; Xu et al., 1987). The direct measurements here of the oocyte volume changes and the indirect observations of the early studies showed a decrease in size of oocytes after maturation. This size decrease of the maturing and matured oocytes was probably due to physiological contractions of the oocyte cytoplasm rather than the release of the polar body, as no size differences were found between oocytes at 10 and 20 hr of maturation. The increase in size of the perivitelline space (decrease in the size of the ooplasm) may aid in disconnecting the junctions between the oocyte and cumulus cell cytoplasmic projections under physiological conditions. The volumetric decrease occurring as the oocyte matures may also be related to


the very dense distribution of MV on the vitelline surface as oocytes mature. The volumetric increase at 20 hr after insemination may be related to activation pre- ceding the first mitotic division. However, the biologi- cal significance of such volumetric changes in ooplasm during maturation and after fertilization remains to be determined, particularly as these measurements were made on fixed oocytes.

GENERAL DISCUSSION AND CONCLUSIONS The surface characteristics of the bovine vitelline

membrane changed from a TSP-predominant pattern to a MV-predominant pattern during in vitro maturation, suggesting that these “follicular” TSPs were retracted during maturation. In addition, TSPs seemed to be transformed morphologically into MV, resulting in the MV-predominant surface pattern seen in the in vitro matured and fertilized oocytes. The dense distribution pattern of MV with scattered TSPs after maturation was not observed in the cleaved bovine embryos reported previously (Koyama et al., 1993,1994). This typical MV-predominant surface pattern in matured oocytes with an associated increase in perivitelline space may be a prerequisite for sperm penetration and fertilization because the actual contribution of the MV to gamete membrane fusion has been observed in this species (Hyttel et al., 1988a,b), as well as in laboratory animals (reviewed by Yanagimachi, 1988). Large intercellular spaces among cumulus cells and the wide open mesh structure of the ZP after maturation of the oocyte may be of significance in facilitating sperm orientation and penetration to the oocyte. After fertilization oocyte surface features changed drastically. Those ultrastructural changes were probably associated with the block to polyspermy.

ACKNOWLEDGMENTS The authors thank all those who collected ovaries at

the slaughterhouse and the cooperation of Taylor Pack- ing Company. Dr. Suzuki was supported by funds from Hirosaki University. The pituitary hormones used were supplied by the National Hormone and Pituitary Program, NIDDKD, NICHHD, and USDA. This study was a part of the NICHHD National Cooperative Pro- gram on nonhuman in vitro fertilization and preimplantation development, and was funded by the Na- tional Institute of Child Health and Human Development, NIH, through Cooperative Agreement HD21939. This research was also supported in part by USDA NRI Competitive Grant 91-37203-6551. The authors are grateful to Dr. Barry Bavister and Dr. Don Wolf for their critical comments and to Deloris Bevins for assistance with preparation of the manuscript.

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