Microtubule and microfilament organization in maturing human oocytes

Transcription

1 Human Reproduction vol.13 no.8 pp , 1998 Microtubule and microfilament organization in maturing human oocytes Nam-Hyung Kim 1, Hyung Min Chung 2, Kwang-Yul Cha 2 and Kil Saeng Chung 1,3 1 Animal Resource Research Center, Kon-Kuk University, Kwangjin-gu, Mojin-dong, Seoul and 2 Infertility Medical Center, CHA General Hospital, College of Medicine, Pochun CHA University, Korea 3 To whom correspondence should be addressed Various stages of immature human oocytes were imaged for microtubule, microfilament and chromatin organization. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. The asters appeared to elongate and encompass the condensed chromatin. At metaphase I stage, microtubules were detected in the meiotic spindle. The meiotic spindle in metaphase II was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. After germinal vesicle breakdown, treatment with taxol induced numerous cytoplasmic foci of microtubules, mainly in the cortex of the oocyte. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found near the germinal vesicle position. After germinal vesicle breakdown, the microfilaments were seen in both the cortex and around the female chromatin. In conclusion, this study suggests that both microtubules and microfilaments are closely associated with the reconstruction and proper positioning of chromatin after germinal vesicle breakdown and during meiotic maturation in human oocytes. Key words: meiotic maturation/microfilaments/microtubules/ oocytes Introduction The resumption of meiosis in mammalian oocytes, including in humans, is a unique and complex process that involves germinal vesicle breakdown (GVBD), chromosomal condensation, polar body extrusion and formation of metaphase structures. These structural changes are associated with changes in the organization of microtubules and microfilaments during specific phases of the cell cycle. In the mouse oocyte, two discrete populations of centrosomes can be observed; one in the spindle pole and the other in the cytoplasm (Maro et al., 1985; Rime et al., 1987; Messinger and Albertini, 1991). During meiotic maturation, these two discrete populations of centrosomes coordinately regulate microtubule assembly for both nuclear and cytoplasmic events. Although cytoplasmic microtubules are not well observed in most mammalian oocytes, treatment with taxol, a drug that promotes microtubule assembly, results in the formation of subcortical asters that nucleate microtubules in rabbit (Yllera-Ferandez et al., 1992) and sheep (Le Guen and Crozet, 1989). Similarly, in the pig, microtubule asters were observed in taxol-treated oocytes after GVBD, which seems to be involved in the organization of spindle formation for metaphase (Kim et al., 1996a). The distribution of microfilaments has also been studied in the mammalian oocyte. In matured mouse (Maro et al., 1984; Schatten et al., 1986), rat (Zernicka-Goetz et al., 1993) and pig oocytes (Kim et al., 1996a), microfilaments are located mainly in the cell cortex overlying the meiotic spindle. This domain, rich in microfilaments, seems to be responsible for the maintenance of the meiotic spindle and chromosomes in a peripheral position (Webb et al., 1986; Kim et al., 1996a,b). Unlike in-vivo derived oocytes, abnormal microfilament assembly and improper positioning of metaphase chromatin were frequently observed in in-vitro matured oocytes (Kim et al., 1996 a,b). More incidences of these abnormalities are observed in improperly matured (Funahashi et al., 1996) or aged oocytes (Kim et al., 1996b), which probably results in abnormal embryonic development following fertilization. Human immature oocytes from unstimulated ovaries have been matured, fertilized in vitro, and have succeeded in forming healthy offspring following transfer to recipients during assistant reproductive technique programmes in a clinic setting (Cha et al., 1991, 1992). However, immature oocytes collected from either stimulated or unstimulated ovaries lead to very low pregnancy rates after their in-vitro fertilization (IVF) and embryo transfer (Cha et al., 1991). Improper culture conditions during maturation may cause insufficient maturation of cytoplasmic organelles, such as microtubules and microfilaments, which possibly results in developmental arrest following IVF and embryo transfer. Successful oocyte cryopreservation of either mature or immature human oocytes can overcome many of the legal and ethical problems, in addition to extending options for patient treatment. Because microtubular spindles of mature oocytes are sensitive to temperature changes, chromatin non-disjunction may occur during cooling, possibly resulting in aneuploidy (Van der Elst et al., 1988; Pickering et al., 1990) and digyny (Carroll et al., 1989). It has been suggested that freezing of immature oocytes at the germinal vesicle (GV) stage is an alternative approach to the cryopreservation of mature oocytes. By using oocytes in prophase I, aneuploidy could be reduced following freezing and thawing (Toth et al., 1994; Son et al., 1996). Despite the obvious role of the reorganization of both microtubules and microfilaments during maturation, little European Society for Human Reproduction and Embryology 2217

2 N.-H.Kim et al. information is available on this subject for human oocytes. A more complete understanding of the fundamental events which occur during maturation would help to provide insight into strategies for improving cryopreservation and clinical IVF procedures using immature human oocytes. In this study we imaged microtubule and microfilament assembly in various stages of human immature oocytes. In addition, the origin and spatial distribution of cytoplasmic centrosomal material 2218

3 Microtubules and microfilaments in human eggs (centrosomal material or probably γ-tubulin) in human immature oocytes were also examined after taxol treatment. Materials and methods Source of human immature and mature oocytes Before beginning the study, approval was obtained from the Institutional Review Board at the CHA General Hospital. Oocytes (n 36) at various stages were donated from unstimulated or stimulated ovaries of consenting patients undergoing gynaecological surgery and IVF procedures. Twenty-seven GV stage oocytes were obtained from unstimulated ovaries. Twenty oocytes among them were cultured for h in Ham s F-10 (Gibco, Grand Island, NY, USA) with 20% fetal cord serum and 2 mm hypoxanthine. Ovarian stimulation was achieved by desensitization in the previous luteal phase using gonadotrophin-releasing hormone agonist (buserelin; Hoechst AG, Seoul, Korea) followed by ovarian stimulation with human menopausal gonadotrophin (HMG, Pergonal; Serono, Seoul, Korea). When at least two follicles measuring 18 mm in diameter were detected, ovulation was induced with 10ˆ000 IU human chorionic gonadotrophin (Profasi; Serono). Immature oocytes were aspirated by puncturing the ovarian follicles (2 5 mm in diameter) with a 21 gauge needle attached to a syringe filled with Ham s F-10 medium. Only oocytes with a compact cumulus cell mass were included in the study. All oocyte preparation steps were performed at 37 C in an atmosphere of 5% CO 2 in air. Immunofluorescence microscopy Microtubules and DNA were detected by indirect immunocytochemical techniques described by Kim et al. (1996a). Briefly, the eggs were permeabilized in a modified buffer M (Simerly and Schatten, 1993) for 20 min at 37 C, fixed in methanol at 20 C for 10 min and stored in phosphate-buffered saline (PBS) containing 0.02% sodium azide and 0.1% bovine serum albumin (BSA) for 2 7 days at 4 C. Microtubule localization was performed using α-tubulin monoclonal antibody (Sigma). Fixed oocytes were incubated for 90 min at 37 C with antibody diluted 1:300 in PBS. After several washes with PBS containing 0.5% Triton X-100 and 0.5% BSA, oocytes were incubated in a block solution (Simerly and Schatten, 1993) at 37 C for 1 h. The blocking was followed by incubation in fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody (Sigma). DNA was observed by exposure to 10 µg/ml propidium iodide (Sigma). To detect distribution of microfilaments, the oocytes were cultured in FITC-labelled phalloidin (1 µg/ml) for 1 h. Stained oocytes were then mounted under a coverslip with antifade mounting medium (Universal Mount; Fisher Scientic Co., Huntsville, AL, USA) to retard photobleaching. Slides were examined using a laser-scanning confocal microscope (BIO-RAD MRC 1024). All images were recorded and archived on an erasable magnetic optical diskette and downloaded to a dye sublimation printer (Sony, Tokyo, Japan) using Adobe Photoshop Software (Adobe, Mountain View, CA, USA). Taxol treatment Stock solution of 1 mm taxol (Sigma) in dimethylsulphoxide was used. The stock solution was stored at 20 C and diluted to 1 µm in medium containing 0.4% BSA immediately prior to treatment of oocytes. Results Twenty-seven GV oocytes from unstimulated ovaries were obtained. Of these, 20 oocytes progressed to the GVBD between 24 and 36 h of culture. They were found to be at stages between early GVBD and metaphase II (six at GVBD, eight at metaphase I, three at anaphase telophase I and three at metaphase II). Additionally, four intermediate (three at GVBD and one metaphase I) and five metaphase II stage oocytes were donated from the stimulated ovaries. Microtubule organization during meiotic maturation Well-organized microtubules were not detected in oocytes at the GV stage (n 2, Figure 1A). However, dense staining of tubulins was observed in the cortex and around the GV. After GVBD, a small microtubule aster was observed near the condensed chromatin (n 3, Figure 1B). During the prometaphase stage, microtubule asters were found in association with each chromatin particle (Figure 1C). The asters were elongated and formed spindle chromatin at metaphase I (n 3, Figure 1D). During the anaphase I and telophase I stage (n 1), microtubules were found as a midbody in the well-organized spindle (Figure 1E). In oocytes at the metaphase II stage (n 3), microtubules were observed in the second meiotic spindle and around the polar body (Figure 1F). The meiotic spindle was symmetrical and barrel-shaped, containing anastral broad poles and was located peripherally and radially (Figure 1F). Overall pole-to-pole distance of the microtubule spindle in metaphase I and metaphase II was and µm respectively. Effect of taxol The addition of taxol induced numerous microtubule foci, mainly around the GV in oocytes at the GV stage (n 2, Figure 1G). Following GVBD, cytoplasmic foci of microtubules formed an interlacing matrix in the cortex (n 3, Figure 1. Immunofluorescence localization of microtubules in human oocytes. Green: microtubules; red: chromatin; yellow: area of microtubules and DNA overlapping. Bar 25 µm. (A) At germinal vesicle (GV) stage, well-developed microtubules were not seen in the cytoplasm. Dense staining of tubulins was observed in the cortex of oocytes. (B) After germinal vesicle breakdown (GVBD), microtubules were produced near the condensed chromatin. (C) The asters elongated and encompassed the condensed chromatin. (D) At metaphase I, microtubules were seen in the meiotic spindle. (E) In telophase, microtubules were found in the midbody. (F) Metaphase plate and polar body at metaphase II stage. Microtubules were detected in the spindle and some in the cortex and around the polar body. (G) Treatment with taxol induced numerous microtubule foci around the germinal vesicle (GV) and some scattered in the cortex. (H, I) Same oocytes with different focal points; with focus at centre of oocyte (H, ~20 µm from surface) and with focus at cortex (I, ~5 µm from surface). Following germinal vesicle breakdown, an interlacing microtubule network was formed in the cortex of taxol-treated oocytes. (J, K) Interlacing microtubule assembly was organized in the cytoplasm (mainly in the cortex) in taxol-treated metaphase I (J) and metaphase II (K) stage oocytes. (L) In a taxol-treated metaphase II stage oocyte, the spindle of microtubules was broken and numerous microtubule asters lay in the cytoplasm. M metaphase; PB polar body. 2219

4 N.-H.Kim et al. Figure 2. Immunofluorescence localization of microfilaments in maturing human oocytes. Blue: microfilaments; red: DNA. Bar 25 µm. (A) At germinal vesicle stage, microfilaments were observed around the oocyte cortex and around the germinal vesicle. (B) After germinal vesicle breakdown, the microfilaments were concentrated to the chromatin. (C, D) Same oocyte at different focal points. When focused on the centre of oocytes (~30 µm from the surface), microfilaments were seen around the cortex (C). In the cortex of oocytes (~5 µm from cortex), a microfilament-rich area was seen (D). (E, F) Metaphase I stage oocytes. In one oocyte, thick microfilaments were seen around the metaphase plate (E). In another metaphase I oocyte, the metaphase plate was seen in the oocyte centre, and a thick microfilament domain was seen in the metaphase plate (F). (G) During anaphase to telophase, thick microfilaments were observed between chromatids. (H, I) In metaphase II stage oocytes, microfilament furrows (arrows) were observed between chromatin and polar body. Figure 1H,I). In metaphase oocytes (one at metaphase I and two at metaphase II), an interlacing microtubule network was observed in the whole cytoplasm (Figure 1J,K). In a metaphase II oocyte, the microtubules were dispersed in the spindle, and many microtubular foci were observed in the cytoplasm (Figure 1L). Microfilament organization during meiotic maturation In GV stage oocytes, the microfilaments were observed in both cortex and around vesicles (n 3, Figure 2A). Following 2220 GVBD, the microfilaments were concentrated closer to the chromatin and seemed to move toward the cortex of the oocytes (n 4, Figure 2B,C,D). In metaphase I stage oocytes (n 2), two domains existed in the egg cortex, a thick and a thin microfilament domain (Figure 2E,F). Chromosomes were located in the thick microfilament domain. Microfilament-rich cleavage furrows were observed between daughter chromatids at anaphase I to telophase I (n 2, Figure 2G). After polar body extrusion, microfilaments were seen around the metaphase chromatin and polar body (n 3, Figure 2H,I).

5 Microtubules and microfilaments in human eggs Discussion In the present study we imaged cytoskeletal organization and chromatin configuration in human oocytes maturing in vitro. Both microtubule and microfilaments are integrated and interact with chromosomal changes during oocyte maturation. Because of our limitation to use only human oocytes, we have used both stimulated and non-stimulated oocytes. In the present study we did not find any distinct difference in the cytoskeletal organization in oocytes from the stimulated and nonstimulated ovaries. Numerous taxol-induced microtubule foci were seen mainly around the GV. Following GVBD, the microtubule foci appeared to move to the cortex and formed the interlacing microtubule matrix. This result is similar to the observation by the Battaglia et al. (1996), who reported the increase in number of astral arrays of microtubules following GVBD in the human oocyte. These observations seem to support the previous hypothesis that the centrosomal material is probably associated with the GV membrane, which becomes dispersed as many foci within the cytoplasm at the time of GVBD (Schatten et al., 1986; Kim et al., 1996a). Although the mechanism is not known, the association of centrosomal material on the nuclear envelope seems to be the general strategy in mammalian eggs. Previously, Schatten et al. (1986) observed that after pronuclear apposition, microtubule foci migrated and aggregated near the pronuclear surface at the end of the first interphase as the cytoplasmic microtubules disassembled, leaving pronuclear sheaths of microtubules. More recently, Kim et al. (1996c) also observed that the microtubules concentrated around the female pronucleus following pronuclear apposition, and that the microtubules then disappeared from the cytoplasm. This probably was due to the association of centrosomal material on the nuclear envelope and its dissociation during nuclear envelope breakdown. Collectively, previous results and our observations suggest that the nuclear envelope at prophase may have the ability to retain centrosomal material in mammals, which is probably related with spindle formation of either the meiotic or mitotic metaphase. The meiotic spindles of mammalian oocytes, which lack centrioles, contain at their poles a band of electron-dense material from which the spindle microtubules emanate. The chromosomes appear to be important in regulating patterns of microtubule assembly in mammalian oocytes during maturation. Under the influence of the chromosomes, maternal centrosomal material determines the organization and shape of the spindle by nucleation of microtubules (Howlett et al., 1985; Maro et al., 1986). In the present study, microtubules are concentrated in the meiotic spindle at the metaphase I stage. This spindle is involved in the process of polar body extrusion. As in the mouse (Maro et al., 1985; Schatten et al., 1985), cow (Navara et al., 1994), and pig (Kim et al., 1996a), human meiotic spindles are symmetrical, barrel-shaped and contain anastral broad poles. In contrast, the rat meiotic spindle is elongated and cone-shaped with distinct poles (Albertini, 1987). The size (18 21 µm) of the meiotic metaphase spindle of human oocytes appeared to be larger than those (~10 12 µm) in the pig and cow. In a metaphase II stage oocyte, the microtubules were dispersed in the spindle, and many microtubular foci were observed in the cytoplasm (Figure 1 L). This is probably due to a disturbance of the tubulin equilibrium shifting to breakdown instead of steady-state equilibrium. We observed dense microfilaments in the cortex and around GV in human oocytes. As suggested previously in the pig and mouse (Longo and Chen, 1985; Kim et al., 1996a), the microfilaments around the GV seem to move the chromatin to the proper position after GVBD. At metaphase, the thick microfilaments were organized near the metaphase chromatin, which possibly induced polar body extrusion. This is similar to that reported for other species (Merry et al., 1995; Kim et al., 1996b). In mature rat (Zernicka-Goetz et al., 1993), mouse (Webb et al., 1986) and pig (Kim et al., 1996a,b; Funahashi et al., 1996) oocytes, microfilament-rich areas overlay the meiotic spindle. Webb et al. (1986) showed that, after activation, the normal formation of a polar body is related to the existence of a microfilament-rich domain overlying the spindle. Longo and Chen (1985) studied the role of microfilaments during meiosis in mouse oocytes by treatment with the microfilament disrupting agent, cytochalasin B. This study showed that GVBD occurred at a normal rate and a meiotic spindle was found at the chromatin after GVBD. However, the meiotic spindle with chromatin failed to move to the oocyte cortex and extrusion of the polar body was inhibited. This result (Longo and Chen, 1985), together with our findings, suggests that microfilaments may be involved in chromosome movement to a peripheral position after GVBD, which seems to be important for further meiotic maturation. Previous results showed that normal assembly of microtubules and microfilaments in unfertilized oocytes is required for the successful fertilization process (Edwards, 1958; Maro et al., 1986; Kim et al., 1997). As shown in mouse (Webb et al., 1986), rat (Zernicka-Goetz et al., 1993) and pig (Kim et al., 1996a,b) oocytes, microfilament areas overlie the meiotic spindle of the human oocytes. The existence of a microfilament domain overlying the spindle is possibly related with normal polar body extrusion as suggested previously in the mouse and pig (Webb et al., 1986; Kim et al., 1996b, 1997). In aged or improperly matured oocytes, chromatin is located outside of microfilament-rich areas which seem to be related to abnormal embryonic development following fertilization (Webb et al. 1986; Funahashi et al., 1996; Kim et al., 1996b). George et al. (1996) observed changes of microtubule organization in aged oocytes making them unsuitable for use therapeutically after reinsemination or intracytoplasmic sperm injection. It was also known that defects of the microtubular system probably induce loss or gain of a single or a few chromosomes, leading to aneuploidy (Edwards, 1958) during conventional fertilization and intracytoplasmic sperm injection (Meng and Wolf, 1997). Changes in temperature, or use of dimethylsulphoxide as a cryoprotectant during freezing can disturb microtubule assembly in mature oocytes, perhaps decreasing the viability of human oocytes and early embryos following fertilization and cryopreservation (Pickering and Johnson, 1987; Pickering et al., 1988; George et al., 1996). 2221

6 N.-H.Kim et al. During normal gamete intra-fallopian transfer or IVF procedure, transient cooling of human oocytes leads to abnormal development following fertilization. High concentrations of cryoprotectant and/or improper cooling procedures possibly impair the function of cytoplasmic organelles controlling polar body extrusion and pronuclear formation in the oocytes which then results in the abnormal developmental patterns and lower incidence of embryonic development. Studies are in progress to determine the effect of various conditions imposed during cryopreservation procedures on the microtubule and microfilament organization in both animal and human oocytes. In summary, two sources of microtubules were observed in human maturing oocytes, and these are certainly involved in chromatin reconstruction during meiotic maturation. Microfilaments are involved in chromosomal movement to a peripheral position after GVBD which may be important for continued embryonic development after fertilization. Further understanding of factors regulating microtubule and microfilament organization during meiotic maturation could bring insight into strategies for improving cryopreservation and clinical IVF procedures using immature human oocytes. Acknowledgements We gratefully thank the anonymous patients who donated their excess and discarded oocytes for this research. We extend our gratitude to Dr Jerry Schatten for his encouragement and Mr Rick Roe for critical reading of the manuscript. References Albertini, D.F. (1987) Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes. Dev. Biol., 120, Battaglia, D.E., Klein, N.A. and Soules, M.R. (1996) Changes in centrosomal domains during meiotic maturation in the human oocyte. Mol. Hum. 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