Spontaneous cortical granule release and alteration of zona pellucida properties during and after meiotic maturation of mouse oocytes.код для вставкиСкачать
THE ANATOMICAL RECORD 237518426 (1993) Spontaneous Cortical Granule Release and Alteration of Zona Pellucida Properties During and After Meiotic Maturation of Mouse Oocytes AKIKO OKADA, KENICHIROU INOMATA, AND TAKESHI NAGAE Second Department of Anatomy, Toho University School of Medicine, Omori-Nishi, O h - k u , Tokyo 143 (A.O., K J . ) ; and Department of Obstetrics and Gynecology, Teikyo University School of Medicine, University Hospital Mizonokuchi, Kanagawa 213 (T.N.), Japan ABSTRACT Exocytosis of cortical granules (CGs) and the concomitant electron density changes of the zona pellucida (ZP)in the absence of sperm penetration were investigated in mouse oocytes processed with tannic acid containing fixation at various stages during and after maturation. After fusion of the CG membrane with the plasma membrane, the CG contents became very electron-dense, due to tannic acid. CG material is seen to be made up of coarse granular structures which gradually change to fine amorphous structures, which accumulate within the developing perivitelline space (PVS). When the coarse CG material attaches to the ZP, small domains exhibiting higher electron density appeared, and the number of these domains gradually increased. Release of CG was observed from metaphase I through metaphase 11. In metaphase I to immediately after ovulation, the higher electron density of ZP and CG release was restricted to the cortical area overlying the meiotic spindle. Finally, the CG-free domain formed itself overlying the meiotic spindle as a result of CG release. However, in oviductal ova, CG release additionally occurred in the hemisphere opposite the spindle. At this stage the entire PVS was well developed and contained numerous fine electron-dense materials. Moreover, the inner half of the ZP increased in electron density as well. This change in electron density of the ZP might be associated with released CG material. These results suggest that the “partial cortical reaction” may play an important role in conditioning the ZP prior to ZP reaction. Q 1993 Wiley-Liss, Inc. Key words: Oocyte, Meiotic maturation, Cortical granule, Exocytosis, Zona pellucida In ovulated mature ova, exocytosis of cortical granules (CGs) takes place immediately after penetration by sperm (Szollosi, 1967; Gulyas, 1980). The material released from the CGs changes the properties of the overlying zona pellucida (ZP).This change is called the zona reaction (Braden et al., 1954). Changes in the ZP andlor the ovum cell membrane are believed to play a major role in the prevention of polyspermic fertilization (reviewed by Austin, 1961; Yanagimachi, 1977). Recently, spontaneous exocytosis of the CGs has been reported in immature ovarian oocytes after resumption of meiosis and in mature ova after ovulation in several mammalian species, including the human (Rousseau et al., 1977), mouse (Nicosia et al., 1977; Okada et al., 1990; Ducibella et al., 1990), and hamster (Okada et al., 1986). These studies also have revealed that both maturing and mature ovulated ova possess a CG-free or CG-poor domain in their cell cortices overlying the meiotic spindle (Nicosia et al., 1977; Gulyas, 0 1993 WILEY-LISS, INC 1980). In the meta I1 of hamster ova, this domain is formed as the result of CG release prior to fertilization (Okada et al., 1986). Several lines of evidence based on physiological and biochemical studies indicate that the properties of ZP are modified during oocyte maturation and sperm penetration of ZP (Iwamatsu and Chang, 1972; Oehninger et al., 1991): the solubility of ZP by protease during in vitro culture of follicular oocyte in serum-free medium (Defelici and Shiracusa, 1982) and oviductal ova in vivo (Aonuma et al., 1978), and the transition of ZP2 to ZP2, in a serum-free medium (Ducibella et al., 1990). Additionally, the only morphological changes to the ZP are reported to be an increase in electron density of the inner half of the ZP (Sathannanthan and Trounson, Received December 19, 1991;accepted June 9,1993. CG R E L E A S E AND ZP ALTERATION DURING MATURATION 1982a; Familiari et al., 1992). However, it is still uncertain why andlor how the ZP is modified during maturation. Standard electron microscopic fixation methods are inadequate for staining difficult to observe glycoprotein structures (Sturgess et al., 1978) such as CG materials. On the other hand, ruthenium red (polycationic inorganic dye) intensely stains CG released materials (Gordon et al., 1975). However, ruthenium red does not easily pass through the plasma membrane (Luft, 1971); consequently, it is used primarily for staining of extracellular materials. According to Maupin and Pollard (1983), tannic acid staining in combination with fixation enhances electron density, both of intracellular structures (Anderson et al., 1975; Mizuhira and Futaesaku, 1973) and extracellular structures of surface coats, etc. (Roubos and van der Wal-Divendal, 1980).Tannic acid is considered to serve as a mordanting agent of osmium-treated structures. This preferential staining is particularly evident in “exocytosing CG after fertilization” (Takeuchi and Takeuchi, 1985). Therefore, this staining technique facilitates the visualization of the premature CG release process, both from qualitative and quantitative aspects. In this study, we undertook to observe the behavior of the CG in the ooplasm, as well as the releasing process of the CGs. We also investigated the interactions of released CG materials and ZP, employing tannic acid methods, in maturing oocytes and mature ova of the mouse. MATERIALS AND METHODS Collection of Oocytes and Ova Mature female ICR strain mice, 8-15 weeks old, were given an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Teikoku Hormone Mfg., Tokyo, Japan) followed 42-48 hr later by 5 IU human chorionic gonadotropin (hCG; Teikoku Hormone Mfg., Tokyo, Japan). Four to 12 hr following hCG injection, the ovaries were removed and placed in dishes containing a modified Krebs-Ringer’s solution, BWW medium (Biggers et al., 1971), containing 0.1% bovine serum albumin (Fraction V, Sigma Chem., St. Louis, MO). Maturing oocytes were recovered from the ovaries by puncturing the antral follicles with a stainless steel needle under a dissecting microscope. Mature, ovulated ova were collected from 1) the ovarian surface or bursae 13-14 hr after hCG injection when ovulation was in progress and 2) the oviducts by cutting the wall of the ampulla with a pair of needles between 14 and 17 hr after hCG injection. Electron Microscopy For thin sectioning, the oocytes and ova with cumulus cells were fixed for 1 hr a t 22°C with Karnovsky’s paraformaldehyde-glutaraldehyde fixative containing 2% tannic acid (Takeuchi and Takeuchi, 1985). After the specimens were washed with 0.1 M cacodylate buffer (pH 7.2), they were left overnight a t 4°C in the same buffer and then postfixed for 1 hr a t 4°C with Dalton’s chrome-osmium fixative (pH 7.2). Fixed specimens were dehydrated in an ethanol series and embedded in Spurr’s resin. Thin sections were stained with uranyl acetate and lead citrate. 519 RESULTS Ovarian Oocytes at Metaphase I All seven oocytes collected from the ovary 8-9 hr after hCG injection exhibited a CG-free domain in their cortices overlying the meiotic spindle and the beginning of a small PVS. Simultaneously, the CG area could also be seen. A CG-free domain, just above the chromosome (Ch) area, and the initial PVS formation were observed (Fig. l.A,B). The PVS formation area revealed a discontinuously narrow or absent PVS (8 hr after hCG injection) (Fig. 1.B). The coarse granular materials attached to the ZP exhibited higher electron density (Fig. lA, arrowheads). At the more developed stage (9 hr after hCG injection), the cortical surface of the oocyte seemed to form a valley-like structure with caverna (Fig. 2). These areas of PVS contain high-electron-dense coarse granular structures (arrows) and fine granular structures (Fig. 2). Additionally, the CG cavernous structure has high electron-dense remnants. These remnants were observed on the surface of the periphery of the CG-free domain. Release of dense material was not observed from the processes of the corona cells and granulosa cells. The process of CG release is shown in the following stages: 1. Approaching stage: Some CGs were observed migrating to the area immediately under the ooplasm prior to membrane fusion (Fig. 3A, arrow, and B at high magnification). 2. CG membrane fusion and opening stage: Just after membrane fusion occurred, the CG opens into the PVS (arrowhead, Fig. 3 0 . Enhanced electron-dense materials were observed from opened CGs by tannic acid stain (Fig. 3C). Occasionally, the content of second exocytosing granules (enhanced electron dense granule) was observed (Fig. 3C, arrow). Intraooplasmic intact CGs were only moderately stained with tannic acid stain (Fig. 3D). 3. CG dispersing stage: The CGs were dispersed within the PVS, and the cavernous structure was often observed at the releasing area (Fig. 3D-GI. The high electron density spot of the ZP was shown to be very close to the released CG material (Fig. 3H, circled area, and I). The material was not only found in the PVS but also seemed to be produced by penetration of the CG material into the overlying ZP (Fig. 31). In the cortical area adjacent to the CG-free domain, two types of CGs, differing in the electron density of their contents, were observed (Fig. 35, arrow and arrowhead). In the hemisphere opposite the CG-free domain, on the other hand, there was no evidence of CG release nor of CG material in the narrow PVS. Moreover, the narrow PVS was found to be clear (Fig. 3K). CG Ch FPB PVS SP ZP Abbreviations cortical granule chromosome first polar body perivitelline space meiotic spindle zona pellucida Fig. 1 (top). A, B Sections of ovarian oocytes at metaphase I, collected 8 hr after hCG injection. A: CG-free cortex near the chromosomes (Ch) and an adjacent cortical area with CGs (arrows), surrounded by the discontinuous perivitelline space (PVS) and overlying ZP (ZP). Small electron dense regions (arrowheads) are observed in the inner zone of the ZP. Scale bar: 1 pm. B: Part of a CG-free domain over the chromosomes (Ch) under high magnification. Discontinuous narrow perivitelline space or the complete absence of the perivitelline space was observed. Scale bar: 0.5 pm. Figs. 2.3.A-L Sections of ovarian oocytes at Meta I, collected 9 hr after hCG injection. Fig. 2 (bottom). The valley-like cortical surface is part of the CG-free cortex. The released high-electron-dense CG material ( t ) contained within the small PVS and ZP can be seen. Scale bar: 0.5 pm. Fig. 3. A The CG domain and adjacent CG-free domain. CGs just below the ooplasm are seen. Scale bar: 1 pm. B: One CG approaching the ooplasm is seen under high magnification B. Scale bar: 1 pm. C: Part of the CG-releasing domain and continuous PVS are seen. Soon after CG membrane and ooplasm fusion, this membrane opened mouth (V)and enhanced high-electron-dense material are observed by tannic acid stain. Moreover, just below the released CG, the second exocytosing granule is also observed ( t ). Scale bar: 0.5 pm. D Within the caverna structure of releasing cortex. Granule-like high-electrondense materials (V)and another intact CG of moderately low electron density (G+) are seen in the cortical surface of the oocyte. Scale bar: 0.5 pm. E-G: Released CG within PVS dispersed (AA),and the electron high density of fine materials is seen. Scale bar: 0.5 pm. 522 A. OKADA ET AL. Fig. 3. H, I: The recently released CG domain, the released CG materials within PVS, and small high electron density of ZP and CG free domain are observed. H: CG-free surface domain on left side and just released CG materials within right side are visible. On the right side of the ZP a small part showing increased electron density in the circled area and a fine granule like structure are observed within the PVS. Scale bar: 1 pm. I: Released CG material which is close to the inner part of the ZP is seen. Under high magnification (in the circled area H) only the small area of the internal ZP appears to have become high in electron density (-1. Scale bar: 0.5 pm. J The part of CG domain adjacent to the CG releasing domain is rich in CG. Here two types of CG are seen, one of high electron density (A)and another one of light electron density ( t ). Scale bar: 1 pm. CG RELEASE AND ZP ALTERATION DURING MATURATION 523 Fig. 3.K In the CG (arrow) domain and the overlying ZP in the hemisphere opposite the spindle no high-electron-densematerial and no change of the inner part of the ZP are seen. Scale bar: 1 pm. L The narrow PVS is clear; compare to the spindle pole of the CG-releasing domain (H,I). Under high magnification (L), no CG granule released materials or cortical caverna could be seen. Scale bar: 0.5 pm. Even on higher magnification of the inner surface of ZP, electron-dense structures were not observed (Fig. 3L). Ovulated Ova at Metaphase II Ovulated ova with the first polar body, collected from the ovarian bursae 12-13 hr after hCG injection, exhibited a well developed CG-free cortex in the vicinity of the meiotic spindle (Fig. 4A). In the cortex adjacent to the CG-free domain, CG release was observed as a burst of electron-dense CG material (Fig. 4A, arrowhead). The PVS was enlarged near the polar body and fine and high electron-dense material accumulated in it (Fig. 4A). A number of electron-dense spots appeared within the ZP overlying the hemisphere containing the spindle (Fig. 4A, B). The coarse granular materials released from the CGs were still observed frequently in the PVS. In the hemisphere opposite the spindle, neither CG release nor electron-dense structural changes in the PVS or ZP could be found a t this stage, and the PVS formation was still small (not shown). Unfertilized, Oviductal Ova Arrested at Metaphase II In ova collected from the oviducts 17-19 hr after hCG injection, the PVS was seen to be fully developed. At this stage, CG release was finally observed from the CG area in the hemisphere opposite the spindle. Fine, amorphous materials were seen to accumulate within the entire PVS. The inner half of the ZP, not only over the CG-free domain (Fig. 5A) but also over other areas of the ovum cortex, had a somewhat higher electron density than the outer half (Fig. 5B, delimited by arrows). These observations are summarized in the diagram in Figure 6. DISCUSSION The fully mature (arrested a t metaphase 11) mouse and hamster ova have a CG-free domain (Szollosi, 1967; Nicosia et al., 1977, Gulyas, 1980). In the hamster oocyte, the formation of this CG-free domain was observed to be result of CG release (Okada et al., 1986). In the mouse oocyte, use of a fluorescent microscopic observation showed a decrease in the number of granules stained with Lens culinaris agglutinin during maturation (Ducibella et al., 1990). However, CG release during meiotic development of the mouse oocyte is still not clear. In the present study we found that during maturation in the follicular and the post-ovulatory stage, the CG release is performed by a unique mechanism, which occurs over a long period (6 hr or more) in the absence of sperm stimulus. After fertilization, it is well known that the ZP prevents polyspermic fertilization by releasing CG material. However, it is reported that artificial premature CG release offers no prevention of sperm penetration, 524 A. OKADA ET AL. Fig. 4. A, B Sections of just-ovulated ova at metaphase I1 with the first polar body (FPB), collected 12-13 hr after hCG injection from ovarian bursae or oviducts. Scale bar: 1 pm. A CG-free cortical domain and adjacent CG domain (upper right) near the meiotic spindle (Sp). Electron-dense material (arrowhead) representing a burst of CG material, is found on the surface of the CG domain. The PVS has enlarged near the first polar body (FPB). The material in the PVS is finer and denser than at metaphase I (cf. Fig. 1C). No CGs are found within the first polar body. Note that a number of electron-dense spots are distributed within the ZP. B: Part of the ZP in the same section as that presented in Fig. 3A under higher magnification. The electrondense macula-like structures (spots) apparently represent densely stained ZP components. either ZP perletration or oocyte membrane fusion of sperm (Wolf et al., 1979). At the Meta I stage, human (Marrs et al., 1984;Trounson et al., 1982),mouse (Iwamatsu and Chang, 19721, and pig oocytes (Hunter, 1976) studies report that the ZP reveals polyspermic penetration. Therefore, the effect of CG material on ZP seems different between premature CG release and ZP reaction. Why such a difference between polyspermic and monospermic penetration of the ZP occurs is still uncertain. The present study reveals two kinds of CGs, in terms of the electron density of their contents (Fig. 35). Nicosia et al. (1977)and Ducibella et al. (1988) have also demonstrated two kinds of CGs in the mouse oocyte with different electron densities. The human, monkey, hamster, and rabbit oocytes also have two kinds of CGs (Gulyas, 1980).We speculate that allowing polyspermy during maturation of ova and the prevention of polyspermy after fertilization are due to differences in the materials contained in the two kinds of CGs, as well as in the timing of their discharges. Further research is required to provide evidence for this speculation. CG release after fertilization is known to alter the ZP, which is called “ZP reaction” and “ZP hardening” (reviewed by Austin, 1961;Yanngimachi, 1977).Prcvious morphological reports did not observe a direct correlation between CG materials and the ZP. Our present study, using the tannic acid method, clearly showed that the initial change of the high electron density of ZP was observed on the Meta I spindle pole, very closely located just above the CG-release material (Fig. 3A,B). Additionally, in more advanced stages, the high electron density area of the ZP was always observed, depending on the particular stage of meiosis, a t the site where CG exocytosis was taking place simultaneously (see diagram, Fig. 6).Finally, in oviductal ova, CG release occurred over almost the entire cell surface, and the entire inner layer of the ZP had uniformly higher electron density (Fig. 5A,B). A similar change in only the inner part of the Z P has also been reported in unfertilized human ova (Sathananthan and Trounson, 1982a) and in mouse ova incubated in Con A (Longo, 1981). After fertilization, it is known that the alteration of ZP substance ZP2 to ZP2, (Bleil and Wassarman, 1981) and an increased resistance to enzymatic digestion is caused by the release of CG materials. The ZP hardening may result from cross-linking of tyrosine residues 525 CG RELEASE AND ZP ALTERATION DURING MATURATION Fig. 5. A, B: Sections of ovulated, unfertilized ova arrested a t metaphase 11, collected 19 hr after hCG injection (about 7 hr after ovulation). The further developed PVS contains fine amorphous materials. Note that the inner half of the ZP (delimited by arrows) has higher electron density than the outer half. Scale bar: 1 pm. A: CG-free A B cortical domain above the meiotic spindle. The cell surface is comparatively smooth. B: Cortex of the hemisphere opposite from that of the meiotic spindle. Numerous microvilli are densely distributed on the cell surface. C D Fig. 6. Correlation between cortical granule release (+) and conditioning of zona pellucida. in the Z P substance induced by ovoperoxidase of CG 1982) and oviductal oocyte in vivo (Aonuma et al., origin (Schnell and Gulyas, 1980). On the other hand, 1978) but the reason for Z P hardening during maturaduring maturation a concomitant decrease in the num- tion is still not clear. ber of CGs and conversion of ZP2 to ZP2, within serumThis study clearly shows a CG-free domain at Meta I free medium have been observed in mouse oocytes us- owing to the CG release in mouse oocyte. The CG-free ing electrophoretic methods (Ducibella et al., 1990). domain is formed again at Meta I1 by CG release. It Also during maturation, a gradual increase in sponta- was reported that in the mature mouse oocyte, 40% of neous Z P hardening was reported in the mouse oocyte the cortex is occupied by the CG-free domain (Ducibella within serum-free medium (DeFelici and Siracusa, et al., 1988).Additionally, after the second CG-free do- 526 A. OKADA ET AL. main formation at Meta 11, another CG release was observed from the CG domain in the mature mouse oocyte. Therefore, a number of CGs are released during maturation. Such released CG material plays a positive role or roles in the modification of the overlying structure, the ZP. The present observation shows CG release and gradual enhanced electron density of the inner layer of ZP which may have caused spontaneous ZP hardening. It was reported that, in monospermic fertilization of human oocytes, the effective block of polyspermy seems to occur in the inner half of the Z P (Sathananthan and Trounson, 1982b), since supplementary-reacted sperm were rarely found to penetrate this region (Soupart and Morgenstern, 1973). Therefore, the alteration of the inner layer of the Z P may play an important role in maturation of ZP. Only strong, active sperm may pass through the inner layer of the ZP. After fertilization, ZP reaction immediately occurs, with the ZP being previously conditioned by partially premature CG release. Gulyas, B.J. 1980 Cortical granules of mammalian eggs. Int. Rev. Cvtol.. -, .. .., 6,3357-392. - - . - - . -. -. 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