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Gamete Biology |
a Infertility Center, Department of Obstetrics and Gynecology, Ghent University Hospital, B-9000 Ghent, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, follicular development, gamete biology, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The normal sequence of germinal vesicle progression from dispersed chromatin to a nucleolar rim has been demonstrated in mouse follicles cultured in vitro [9]. The acquisition of meiotic competence during culture has been shown in mice by the ability of oocytes to undergo germinal vesicle breakdown [10]. However, although mouse follicles grown from preantral or primordial to mature stages in vitro yield oocytes that can be fertilized in vitro, the embryonic development is poor [1, 5, 7, 8]. The critical role of respectively nuclear and cytoplasmic elements is poorly understood and may influence subsequent embryonic development [11].
The aim of this study was to investigate the role of both nuclear and cytoplasmic components using a number of experiments involving a reciprocal transfer of the nucleus (chromosome spindle apparatus) between in vitro-matured oocytes from preantral follicles and in vivo-matured oocytes with embryonic development as the endpoint of the experiment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early preantral mouse follicles were mechanically isolated from the ovaries of 14-day-old C57Bl/6J x CBA/Ca F1 female mice. The follicles with a diameter of 110–160 µm were selected and cultured in a culture system for 10 days as described previously [6]. Matured oocytes with the first polar body extruded were collected 15 h after stimulation with 2.5 IU/ml hCG (Pregnyl; Organon, Oss, The Netherlands). The cumulus cells were removed by exposure to 200 IU/ml hyaluronidase (Sigma, Bornem, Belgium) in Hepes-buffered potassium simplex optimized medium (KSOM) [8] for 6 min. Denudation was completed by aspirating oocytes in and out of a pulled glass pipette. All experimental protocols and the use of animals were approved by the Animal Research Ethical Committee, Ghent University Hospital.
In Vivo-Matured Metaphase II Mouse Oocytes
In vivo-matured oocytes were obtained from 10- to 12-wk-old C57Bl/6J x CBA/Ca F1 female mice. Gonadotropin priming was performed with 5 IU eCG (Folligon; Intervet, Turnhout, Belgium) followed by 5 IU hCG (Chorulon; Intervet) given 48 h later. Cumulus-enclosed oocytes were collected 14–16 h after hCG administration. Cumulus cells were removed from the metaphase II (MII)-arrested oocytes by treatment with hyaluronidase. After rinsing in Hepes-buffered KSOM, MII oocytes were used in the following experiments.
Nuclear Transfer Procedure
Micromanipulation was performed in a cover of a plastic Petri dish (model 3002; Falcon, Becton Dickinson Labware, Leuven, Belgium) in 3-µl droplets of KSOM supplemented with 5 mg/ml BSA (Sigma). All needles were made from borosilicate glass (Drummond Scientific, Broomall, PA) drawn on a micropuller (PB-7; Narishige, Tokyo, Japan) and calibrated, cut, and fire polished on a microforge (MF-9; Narishige). Micromanipulation procedures were carried out on a cooled stage (Microscope Thermal Stage MTS-1; Techne, Wilten Instruments, Antwerp, Belgium) placed on an inverted microscope (Olympus IX-70; Omnilabo, Aartselaar, Belgium).
A slit was made in the zona pellucida by passing a glass microneedle tangentially into the perivitelline space and rubbing the oocyte against the holding pipette. Prior to enucleation, oocytes were exposed for 15 min to KSOM containing 7.5 µg/ml cytochalasin B (CCB; Sigma) and 5 mg/ml BSA. Enucleation of oocytes was performed by removing the MII spindle with a small amount of ooplasm (karyoplast) using a micropipette with a 20-µm inner diameter through a slit made in the zona [12]. In the mouse, the spindle is identifiable as a translucent region. Thereafter, the isolated karyoplast was inserted with the same tool into the perivitelline space of another previously enucleated MII oocyte (MII cytoplast). A grafted oocyte constitutes an isolated karyoplast and an isolated MII cytoplast. After insertion of the karyoplast, grafted oocytes were washed in fresh medium to remove CCB and cultured in KSOM containing 5 mg/ml BSA for at least 15 min prior to electrofusion.
A Model 830 Electro Cell Manipulator (Merck-Eurolab, VWR International, Leuven, Belgium) was used for cell fusion. Each grafted oocyte was manually aligned between two electrodes (0.4 mm apart) with the contact surface between the karyoplast and the cytoplast parallel to the electrodes. To induce fusion, a double 1.0 kV/cm direct current (DC) was delivered for 80 µsec followed by 1.5 kV/cm DC for 40 µsec in KSOM. After washing and culture for 30 min, fused oocytes were examined to confirm cell survival and fusion.
Experimental Group Design
Five study groups of mouse oocytes were used (Fig. 1): A) grafted oocytes containing an in vitro-matured MII cytoplast and a karyoplast from an in vivo-matured oocyte, B) grafted oocytes containing an in vivo-matured MII cytoplast and a karyoplast from an in vitro-matured oocyte, C) grafted oocytes containing an in vivo-matured MII cytoplast and a karyoplast from an in vivo-matured-oocyte, D) in vivo-matured oocytes, and E) in vitro-matured oocytes.
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Oocyte Activation and In Vitro Parthenogenetic Embryo Culture
Fused oocytes in groups A, B, and C and matured oocytes (MII stage) in groups D and E were subjected to activation for 4 h in Ca2+-free KSOM containing 10 mM SrCl2 (Sigma) and 1 µg/ml cytochalasin D (Sigma). Sr2+ ions were used to induce activation and cytochalasin D was used to inhibit second polar body emission and to generate diploid parthenogenetic embryos.
Following activation, oocytes showing pronuclei were cultured in KSOM containing 5 mg/ml BSA for parthenogenetic embryo development. Assessment of in vitro embryo development was performed at 24 h (two-cell stage), 72 h (morula/early blastocyst), and 96 h (blastocyst) after the onset of activation.
Counting the Number of Blastocyst Cells
Blastocyst-stage embryos obtained at 96 h postactivation were fixed for determination of cell number. Fixation and staining were performed according to the method described elsewhere [13]. Embryos were transferred to 75 mM KCl hypotonic solution for 8–10 min and then into precooled (-25°C) fixative (1–2 ml 3:1 methanol:acetic acid) in a watchglass for 5–20 sec. After fixation, the embryos were transferred to a glass slide with a small amount of fixative. Air-dried slides were stained in 20% Giemsa solution (Sigma) for 20 min at room temperature. Blastocyst cells were counted under a light microscope (Olympus CH-2; Omnilabo) with a 100x objective under immersion oil.
Data Analysis
Percentages of oocytes at various stages of development were calculated from three independent replicate experiments. Percentages were compared between groups by contingency table analysis followed by chi-square subtests. Blastocyst cell numbers in different groups (mean ± SEM) were compared using a one-way ANOVA followed by a Newman-Keuls t-test. The difference was considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The overall efficiency of the electrofusion of the spindle karyoplast and MII cytoplast to form the reconstituted oocytes was 62.1–77.8% (Table 1). Group A (in vitro cytoplast coupled with in vivo karyoplast) had a higher success rate of fusion, but the difference compared with the other groups was not significant.
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Development of Reconstituted Oocytes after Parthenogenetic Activation
In group D, 74 of 77 (97.4%) in vivo-matured oocytes were activated and cleaved to the two-cell stage. Subsequently, >90% of the parthenogenetic embryos developed to the morula (94.6%) and blastocyst (91.9%) stages (Table 1). For the in vitro-matured oocytes from preantral follicles (group E), a total of 57 MII oocytes were activated with Sr2+, but only 27 (47.4%) of the 57 oocytes were activated and cleaved to two-cell embryos. Of these two-cell embryos, 48.1% and 29.6% developed to the morula and blastocyst stages, respectively. In groups B and C, 36 of 45 (80%) and 28 of 36 (77.8%) of the reconstituted oocytes formed two-cell embryos after activation, respectively. The two-cell embryos from both groups developed to morulae (91.7% and 92.9%, respectively) and blastocysts (86.1% and 89.3%, respectively). The reconstituted oocytes in group C (technical control) had a significantly lower rate of activation compared with group D (77.8% vs. 97.4%, respectively; P = 0.007). However, once activation had taken place, further development was equally successful in the two groups. In group A, however, 24 of the 49 reconstituted oocytes cleaved (48.9%), a significantly lower percentage that that obtained in group B (P = 0.004). The subsequent parthenogenetic embryo development was quite poor; only 6 of 24 cleaved embryos developed to the morula stage (25%), and none of these morulae reached the blastocyst stage (Table 1).
Blastocyst Cells
The total number of cells in the blastocyst embryos from groups B, C, D, and E is displayed in Table 1. Parthenogenetic blastocyst embryos from in vivo MII oocytes (group D) contained 57.5 ± 1.7 cells 96 h after activation. A significantly lower number of cells was found in the parthenogenetic blastocysts from the in vitro-matured oocytes of group E (21.2 ± 2.2; P < 0.0001). Micromanipulation associated with nuclear transfer did not significantly compromise the capacity of cells to divide, as shown by the blastocyst cell number in group C (60.2 ± 2.9) compared with group D (P = 0.43). Reconstituted oocytes in group B developed to blastocyst embryos with 47.9 ± 3.5 cells, which is significantly lower than the number of cells in group C blastocysts (P = 0.019). However, group B blastocysts showed a higher developmental capacity than did the in vitro-matured oocytes in group E after parthenogenetic activation (P = 0.0002).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data support previously published data showing that in vitro-matured oocytes from preantral follicles lack cytoplasmic competence to support preimplantation embryo development [15, 16]. The difference from previous studies, however, is that we went beyond the pure microscopic observation of poor embryo development. By incorporating nuclear transfer, we have provided concrete evidence for the role of cytoplasmic factors. The next step would be to identify the cytoplasmic factors involved, which was beyond the scope of the present study. We tried to dissect the cytoplasm in its nuclear and nonnuclear components by investigating the contribution of the chromosome spindle complex and the cytoplasm sensu stricto.
Nuclear maturation is characterized by extrusion of a polar body. However, other events such as correct chromosome segregation or genomic imprinting, which are achieved in oocyte development in vivo, must be replicated in vitro. The vulnerability of imprinted genes to epigenetic errors can be demonstrated by nuclear manipulation, in vitro growth/maturation, and culture conditions [17, 18]. Blastocysts developing from the group of oocytes with in vivo cytoplasm and a spindle from an in vitro-matured oocyte had fewer cells in the blastocyst. Thus, nuclear maturation also plays a role in achieving developmental competence of the oocyte.
Electrofusion efficiency of the reconstituted oocytes largely depends on the developmental stage of the oocytes. The efficiency of fusion of a karyoplast containing an MII chromosome spindle apparatus is low [19]. This poor fusion rate may be due to the fact that the membrane of an MII karyoplast lacks microvilli [20], which play a critical role in the fusion of many cells [21]. The high level of maturation promoting factor in the MII oocytes also may block cell fusion [19]. In the current study, a double fusion protocol was applied by which 61.2%–77.8% of the grafted oocytes could be fused. This improvement is substantial compared with the results of previous studies (17.6%) [19]. However, double fusion may also have a detrimental effect on embryo development; about 20% of the reconstituted oocytes in group C were fragmented after parthenogenetic activation. A higher fusion rate was observed in group A than in groups B and C. A likely explanation for this difference is the fact that the perivitelline space of in vitro-matured oocytes (group A) was smaller than that of in vivo-matured oocytes. The couples (karyoplast and cytoplast) in group A were kept in very close contact, which might have facilitated the cell fusion process.
Activation of mammalian oocytes is normally triggered by sperm penetration. Oocyte activation consists of a sequence of morphological and molecular events that include release of Ca2+ and that culminate in the second meiotic division, extrusion of a second polar body, formation of pronuclei, DNA synthesis, and mitotic cleavage [22]. MII oocytes can also be activated parthenogenetically by electrical pulses [23], calcium ionophore, Sr2+, ethanol, cycloheximide (a protein synthesis inhibitor), or combinations of these treatments [24–28]. In this study, SrCl2 and cytochalasin D were used for production of diploid parthenogenetic embryos. Approximately 97% of the in vivo-matured oocytes were activated (group D) using this protocol compared with 47% of the in vitro-matured oocytes (group E). The MII spindle (translucent region) was still observed in some inactivated oocytes 4 h after Sr2+ treatment. The reconstituted oocytes in group B showed a similar activation rate and further parthenogenetic development compared with group C. The number of blastocyst cells in group B was lower than that in group C, which may be the result of the poor nuclear maturation of in vitro-matured oocytes.
The low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component. The oocytes reconstructed with the in vitro-matured MII chromosome spindle apparatus and an in vivo-matured MII cytoplast can be activated and can develop to the blastocyst stage, whereas in the reverse situation blastocyst formation is severely compromised. Further research will focus on media supplements that influence the functional competence of the cytoplasm of oocytes matured in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jun Liu, Infertility Center, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. FAX: 32 9 240 4972;jun.liu{at}rug.ac.be
Received: 9 June 2002.
First decision: 21 June 2002.
Accepted: 24 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  J.-Z. Wang, H.-S. Sui, D.-Q. Miao, N. Liu, P. Zhou, L. Ge, and J.-H. Tan Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes Reproduction, February 1, 2009; 137(2): 181 - 189. [Abstract] [Full Text] [PDF]
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S J Martins da Silva, J O Gardner, J E Taylor, A Springbett, P A De Sousa, and R A Anderson Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development Reproduction, April 1, 2005; 129(4): 423 - 434. [Abstract] [Full Text] [PDF]
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