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Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes John Zhang *, Hui Liu Reproductive Endocrinology and Infertility, New Hope Fertility Center, New York, NY, USA * Corresponding author.
E-mail address: [email protected] (J Zhang). Dr Zhang completed his medical degree in at the Zhejiang University School of Medicine, and subsequently received his Master’s Degree at Birmingham University in the UK. In 1991, Dr Zhang earned his PhD. in In-Vitro Fertilization (IVF), and, after studying and researching the biology of mammalian reproduction and human embryology for nearly ten years, became the first Fellow in the Division of Reproductive Endocrinology and Infertility of New York University’s School of Medicine in 2001. Dr Zhang continues his research in non-embryonic stem cell research, long-term cryopreservation of oocytes, and oocyte reconstruction by nuclear transfer.
Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMXtreated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF. Abstract
© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cytoplasm, germinal vesicle, meiotic maturation, meiotic spindle, nucleus, oocyte
http://dx.doi.org/10.1016/j.rbmo.2015.03.012 1472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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Introduction The mammalian ovary contains a large supply of inactive germ cells that reside in primordial follicles and two categories of oocyte. The first category constitutes oocytes that are unable to respond to maturation signals in vivo or in vitro and remain arrested in the diplotene stage (Fulka et al., 1998). The second category makes up a fully grown group of oocytes that respond to gonadotrophins and mature to metaphase II in pre-ovulatory follicles and in culture (Fulka et al., 1998). Oocyte maturation is a complex process involving both the progression of the meiotic cycle and the reprogramming of cytoplasmic events (Fulka et al., 1998; Karnikova et al., 1998; Liu et al., 1999; Moor et al., 1998). Some of the structural changes in the cytoplasm include an increase in the number of mitochondria, structural modification to the Golgi apparatus and accumulation of ribosomes (Fulka et al., 1998; Heacox and Schroeder, 1981). The end-point of oocyte maturation is the release of a mature metaphase II (MII) oocyte that is competent to support normal embryonic development. Both nuclear and cytoplasmic deficiencies have been shown to be responsible for poor oocyte quality by contributing to meiotic defects and subsequent impaired embryo development (Fulka et al., 2001; Liu et al., 2000, 2003; Liu and Keefe, 2004; Moor et al., 1998). Germinal vesicle (GV) transfer techniques have represented useful tools for studying the interaction between the nucleus and the cytoplasm in the oocyte maturation process in mammals (Chiang et al., 2012; Cohen et al., 1997; Dekel and Beers, 1978; Fulka et al., 1998, 2001; Heacox and Schroeder, 1981; Levron et al., 1996; Li et al., 2001; Liu et al., 1999, 2000; Zhang et al., 1999). Furthermore, GV transfer between different mammalian species has demonstrated that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at the MII stage (Li et al., 2001). It has previously been reported in humans (Zhang et al., 1999) and mice (Liu et al., 2000, 2003) that normal meiosis can occur after the transfer of GV into an enucleated host oocyte. It has also been shown in mice that oocytes reconstructed by GV transfer into a cytoplasm of the same developmental stage mature normally in vitro through MII (Liu et al., 1999). Studies to date have evaluated the in-vitro maturation capability of reconstructed oocytes by GV transfer (Heacox and Schroeder, 1981; Li et al., 2001; Zhang et al., 1999) but did not address data on meiotic maturation such as polar body (PB) extrusion, meiotic spindle assembly or chromatid separation. Abnormal oocyte spindle morphology is associated with human female infertility, and advanced maternal age has been attributed to spindle abnormalities such as abnormal chromosome alignment and a microtubule matrix that compromises the meiotic spindle (Battaglia et al., 1996). The regulatory mechanisms responsible for assembly of the meiotic spindle in the cytoplasm are significantly altered in older women, leading to high prevalence of aneuploidy (Battaglia et al., 1996). Whether the GV transfer technique could represent a therapeutic option for meiotic spindle abnormalities remains to be determined. It is well established that pharmacological activation of cAMP-dependent protein kinases by 3-isobutyl-1-methylxanthine (IBMX) suppresses meiotic
resumption (Dekel and Beers, 1978). The present authors (Liu et al., 2003) and others (Eppig and Schroeder, 1989) have demonstrated that mouse oocytes treated with IBMX at the GV stage in vitro have arrested during their meiotic maturation, and when released from IBMX were able to produce normal embryonic development. The purpose of this study was to explore the role of the cytoplasm on meiotic spindle assembly in oocytes arrested by IBMX in vitro and to assess whether cytoplasm replacement via GV transfer restores meiotic maturation, in particular spindle assembly in the IBMXarrested oocytes. We postulated that a healthy cytoplasm is required for GV breakdown and meiosis resumption.
Materials and methods Mouse oocytes The CB6F1 mice used in this experiment were purchased from Charles River Laboratory (Boston, MA). Mice were subjected to a 14L:10D cycle for at least 1 week before use. Animals were cared for according to the procedure approved by University Animal Welfare Committee (UAWC). GV-stage oocytes were collected from 6- to 8-week-old CB6F1 mice through puncturing the ovarian follicles 48 h following priming with 5 IU pregnant mare serum gonadotrophin (PMSG, Sigma, St Louis, MO). GV oocytes, stripped off cumulus granulosa cells with a diameter around 80 µm, were used in this experiment. GV oocytes were incubated in human tubal fluid (HTF) medium supplemented with 10% fetal calf serum (FCS) (HyClone, ThermoFisher Sceintific, Waltham, MA) and 50 µg/ml of IBMX (Sigma, St Louis, MO) constituting the treatment group, thus preventing GV from breakdown by increasing cytosolic cAMP following inhibition of phosphodiesterase. Mouse oocytes were arrested at GV stage in vitro following culture in IBMX for 6, 24 and 48 h, respectively. The arrested oocytes were then released from IBMX by changing the medium to IBMX-free medium. The dose of IBMX used was chosen from previously published data (Liu et al., 2003; Van Cauwenberge and Alexandre, 2000). These treatments were used for various periods of time and then GV were released from IBMX and cultured in IBMX-free HTF medium (F-HTF) for 15 h for meiosis resumption. Oocytes without IBMX treatment constituted the control group.
Human GV oocyte GV-stage oocytes were obtained from patients (25–42 years old) who were undergoing IVF with intracytoplasmic sperm injection (ICSI) after ovarian stimulation with gonadotrophins. All patients were treated with human chorionic gonadotrophin (HCG; 5000 or 10,000 IU) 36 h before transvaginal oocyte retrieval. Institutional Review Board (IRB) approval was obtained on July 23 1998 from New York University School of Medicine (NYUMC-IBRA Protocol H 6902). Following an IRB approval, participants consented and GV oocytes that were unsuitable for ICSI and routinely discarded were used in this study.
GV transfer and electromembrane fusion Oocytes were exposed to modified HTF medium (mHTF) with 10% FCS supplemented with 7.5 µg/ml cytochalasin B (CB;
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
ARTICLE IN PRESS Cytoplasm replacement restores meiosis Sigma, C6762) for 15 min at room temperature in order to disrupt microfilament and increase plasma membrane flexibility before manipulation. The dish was placed onto the stage of Olympus X71 inverted microscope equipped with Narishige micromanipulators. A slot was made on the zona pellucida of each oocyte by applying a sharp-tipped pipette to penetrate the zona and grind the zona against the wall of the holding pipette. This allowed the enucleation pipette to pass through the zona slot and approach the GV before gently applying negative pressure to aspirate the GV. Once a GV was separated from the cytoplasm, it was transferred into the perivitelline space of an enucleated recipient GV oocyte. The membrane fusion between GV and cytoplasm was initiated by placing it into fusion medium (0.3 mol/l mannitol, 0.1 mmol/l CaCl2 and 0.05 mmol/l MgSO4) between platinum electrodes alignment in response to AC (6–8 V) for 5–10 s before electrical pulse (1.8–2.5 kV/cm DC for 50 µs) delivered by a Model 2001 Electro Cell Manipulator (BTX, Holliston, MA). The formed complexes were then rinsed three times in mHTF, and then incubated in F-HTF medium at 5% CO2 and 37°C. Membrane fusion usually occurred 30 min after electric pulse.
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Immunofluorescent staining for meiotic spindle Dissolution of zona pellucida and permeation of oocyte membrane were required for indirect immunofluorescent staining for microtubule. Oocytes were briefly treated with Tyrode’s acid solution to dissolve the zona pellucida and then fixed with phosphate-buffered saline (PBS) containing 3.7% paraformaldehyde for 30 min. Oocytes were then treated with 0.25% Triton X-100 for 20 min at room temperature to permeate the membrane. Oocytes were finally rinsed two times with PBS containing 0.25% bovine serum albumin (BSA: Sigma, St Louis, MO) to remove the free aldehydes and get prepared for double staining. Microtubules were localized with 1:2000 mouse monoclonal antibody to β-tubulin (Sigma, St Louis, MO). The primary antibody was detected using 1:400 fluorescein isothiocyanatelabelled secondary goat anti-mouse IgG (Sigma, St Louis, MO). Each antibody was applied overnight at 4°C and rinsed with PBS between antibody applications. Oocytes were rinsed with 5 µg/ml Hoechst 33342, a cell-permeable DNA stain that is excited by UV light and emits blue fluorescence at 460 to 490 nm allowing simultaneous detection of the chromosomes. Each oocyte was mounted on a glass slide covered with a coverlid and examined using Olympus BX61 fluorescence microscope.
Treatment groups Three types of oocyte were reconstructed by GV transfer: (i) GV of non-IBMX-arrested oocyte was transferred to a cytoplasm (C) of non-IBMX-arrested oocyte (GV/C) serving as control to ensure that the GV transfer technique per se did not affect meiosis; (ii) GV of non-IBMX-arrested oocyte with cytoplasm of IBMX-arrested oocyte (GV/CIBMX) (Figure 1); and (iii) GV of IBMX-arrested oocyte with cytoplasm of non-IBMXarrested oocyte (GVIBMX/C) (Figure 1). In other experiments, GV transfer between mouse and human oocytes was performed in order to construct xenooocytes. Thus, human GV/mouse cytoplasm (H/M) and mouse GV/human cytoplasm (M/H) oocytes were reconstructed. Mouse GV with mouse cytoplasm was reconstructed and served as controls because mouse oocytes are known to fully mature while human oocytes are known to poorly mature in vitro. The oocytes were then cultured for 15–36 h in vitro and examined for meiotic maturation.
Figure 1
Birefringent spindle The meiotic spindle of living oocytes was imaged using an Olympus IX71 equipped with a polarizer and an analyser. The living oocytes were placed into an mHTF droplet in a glass bottom dish and analysed by bright field observation. During analysis, the holding pipette was applied to aspirate the oocyte lightly with the assistance of an injection pipette in order to rotate the oocyte and ultimately locate the birefringent spindle signal. Because the meiotic spindle is a temperature-sensitive structure, all procedures were performed at a temperature of 37°C.
Statistics Data were analysed using chi-squared or Fisher exact probability tests as appropriate. Statistical significance was
Schematic diagram of GV transfer between IBMX-treated oocyte and a control non-treated oocyte.
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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determined at P < 0.05. Sample size calculation was performed using a power of 80% and alpha error of 0.05. In order to detect 50% change in PB extrusion between the reconstructed oocytes by GV transfer, a sample size of 15 oocytes were needed in each group.
Results The effect of IBMX arrest on PB extrusion in mouse oocytes The meiotic maturation, as shown by the first PB extrusion, was evaluated 14–16 h after being released from IBMX. As seen in Table 1, oocytes treated by IBMX for 6 h displayed meiotic ability similar to control oocytes with 98% completing meiotic maturation as shown by PB at both 0 and 6 h. However, when IBMX treatment time was extended to 24 and 48 h, oocytes exhibited reduced ability for meiotic maturation of the oocytes extruding the first PB compared with controls (P < 0.01) where only 68% of oocytes had PB extrusion at 24 h and only 44% of oocytes had PB extrusion at 48 h of IBMX treatment (P < 0.01) (Table 1).
The effect of IBMX arrest on meiotic spindle of mouse oocyte After being released from IBMX and matured in vitro, oocytes were visualized for meiotic spindle by indirect immunofluorescent staining. Following IBMX treatment for 6 h, 67 out of 69 (97%) oocytes meiotically matured by releasing a PB, 99% of which displayed normal meiotic spindle (Table 2 and Figure 2A) with a barrel shape and a slightly narrow poles, and had chromosomes aligned in the equatorial plate of the spindle (Figure 2a). In contrast, following IBMX treatment for
Table 1
48 h, only 50 out of 90 (56%) of oocytes meiotically matured, 32% of which displayed normal spindle and 68% of which exhibited abnormal spindle (P < 0.001 compared with IBMX treatment for 6 h) (Table 2 and Figure 2B, 2C, 2D, 2E). The abnormalities observed following IBMX arrest for 48 h included multiple poles of spindle (Figure 2B), disrupted spindle (Figure 2C, 2D) or no spindle (Figure 2E). Other abnormalities included chromosome misalignment, such as aggregated and scattered chromosomes (Figure 2b, 2c, 2d), or decondensed chromosomes (Figure 2e). Additionally, the majority of oocytes that had IBMX arrest for 48 h showed various abnormal spindle structures (Table 2).
The effect of GV transfer on meiotic spindle assembly and meiotic maturation in mouse oocytes After reconstruction and in vitro culture for 14–16 h without IBMX, the control oocytes exhibited meiotic maturation, with 98% of oocytes extruding the first PB (Table 3), indicating that the procedure of GV transfer did not influence the meiotic maturation. The GV/CIBMX reconstructed oocytes displayed a significantly reduced ability for meiotic maturation, with only 43% extruding a PB compared with controls and GVIBMX/C reconstructed oocytes (P < 0.001; Table 3). The majority (63%) of the mature GV/CIBMX reconstructed oocytes showed abnormal meiotic spindle (Table 4). These oocytes had scattered chromosomes across the spindle (Figure 3A, 3a), scattered chromosomes along the disrupted spindle (Figure 3B, 3b, 3C, 3c) and decondensed chromosomes as the spindle disappeared (Figure 3D, 3d). By contrast, the GVIBMX/C reconstructed oocytes showed similar ability of meiotic maturation to that observed in control oocytes (Figure 3E, 3e). These reconstructed oocytes had 100% (n = 35) maturing oocytes with extrusion of first PB (Table 4),
Maturation of mouse oocytes following release from IBMX arrest.
IBMX arrest (time) 0 h (control) 6h 24 h 48 h
Total no. of oocytes
GV n (%)
No PB n (%)
PB n (%)
110 104 105 149
0 0 5 8
2 2 29 75
108 102 71 66
(0) (0) (5) (5)
(2) (2) (28) (50)
(98)a (98)a (68)b (44)c
GV = germinal vesicle; PB = polar body indicating oocyte maturity. Data are presented as n or n (%). Chi-squared test: a versus b versus c; P < 0.01.
Table 2
Meiotic spindle analysis of IBMX-arrested mouse oocytes.
IBMX arrest time 48 h (90 total oocytes) 6 h (69 total oocytes)
14–16 h after release No PB PB No PB PB
(n = 40) (n = 50) (n = 2) (n = 67)
Normal spindle n (%)
Abnormal spindle n (%)
12 16 1 66
28 34 1 1
(30) (32)a (50) (99)b
(70) (68)c (50) (1)d
PB = polar body. Chi-squared test: a versus b and c versus d; P < 0.001.
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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20µm -------Figure 2 The effect of IBMX arrest on meiotic spindle in mouse oocytes. Indirect immunofluorescent staining for β-tubulin (A, B, C, D, E); Hoechst 33342 staining for chromosomes (a, b, c, d, e). Normal meiotic spindle observed in control metaphase II oocytes (A and a). Oocytes arrested with IBMX for 6 h were similar to controls (not shown). Abnormal meiotic spindle observed in metaphase II oocytes arrested with IBMX for 48 h (B, b, C, c, D, d, and E, e).
Table 3 The effect of GV transfer on restoration of meiotic maturation in reconstructed mouse oocytes. Types of oocyte (GV/C) GVIBMX/C GV/CIBMX GV/C (control)
Total no. of oocytes
No PB n (%)
PB n (%)
35 37 52
0 (0) 21 (57) 1 (2)
35 (100)a 16 (43)b 51 (98)a
C = cytoplasm; GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b; P < 0.001.
Table 4
The effect of GV transfer on assembly of meiotic spindle in mouse oocytes.
Type of oocyte (GV/C) GV/CIBMX (37 total oocytes) GVIBMX/C (35 total oocytes)
Oocyte No PB PB No PB PB
(n = 21) (n = 16) (n = 0) (n = 35)
Normal spindle n (%)
Abnormal spindle n (%)
5 6 0 32
16 10 0 3
(24) (38)a (0) (91)b
(76) (63)c (0) (9)d
C = cytoplasm; GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b and c versus d; P < 0.05.
91% (n = 32) of which displayed normal spindle and chromosome alignment (Table 4). Only 9% (n = 3) of these oocytes displayed abnormal meiotic spindles with two showing loose alignment of chromosomes at equatorial plate and one showing multiple microtubule stars, disappearance of meiotic spindle, and decondensed chromosomes in a pronucleus-like structure.
The meiotic behaviour of human GV karyoplast in cytoplasm of mice oocytes (xeno-oocyte) M/H oocytes displayed significantly reduced meiotic maturation ability with only 44% of these xeno-oocytes extruding
the first PB (P < 0.001; Table 5). Interestingly, all the H/M xeno-oocytes matured to MII and extruded the first PB (Table 5 and Figure 4D, 4d). Non-invasion live observation of oocytes under inverted microscope equipped with polarizer and analyser revealed that the birefringent spindle detected in mature H/M oocytes (Figure 4d) was similar to that of mature mouse control oocytes in size and shape (Figure 4b), whereas the birefringent spindle of mature M/H oocytes (Figure 4c) was similar to that of mature human oocytes (Figure 4a). Nine pairs of oocytes reconstructed by reciprocal exchange of GV between human and mouse were successfully prepared for cytogenetic analysis. Five out of nine H/M oocytes had 23 normal sets of chromosomes (Figure 5A). In
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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20µm -------Figure 3 Effect of cytoplasm replacement by GV transfer on meiotic spindle of mouse oocytes arrested by IBMX. Indirect immunofluorescent staining for β-tubulin (A, B, C, D, E); Hoechst 33342 staining for chromosomes (a, b, c, d, e). Abnormal meiotic spindle observed in oocytes reconstructed with GV/CIBMX (A, a, B, b, C, c, D, d). Normal meiotic spindle observed in oocytes constructed with GVIBMX/C (E, e). C = cytoplasm; GV = germinal vesicle.
Table 5 Maturation of xeno-oocytes reconstructed by GV transfer between human and mouse oocytes. Type of oocyte (GV/Cytoplasm) Human GV/mouse cytoplasm Mouse GV/human cytoplasm Mouse GV/mouse cytoplasm (control)
Number n
No PB n (%)
PB n (%)
25
0 (0)
25 (100)a
18
10 (56)
8 (44)b
52
1 (2)
51 (98)a
GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b; P < 0.001.
contrast, none of nine M/H oocytes had a normal number of chromosomes (Figure 5B). Additionally, precocious chromatid division was found in six M/H oocytes (Figure 5B), while none of the H/M oocytes displayed similar findings (Figure 5A). Other abnormalities observed included chromosome fragment (two H/M oocytes), chromosome decondensation (one M/H oocyte) and bivalent chromosomes (one M/H oocyte).
Discussion Human oocytes are arrested at the prophase/GV stage before birth and do not resume meiotic maturation until ovulatory events at puberty. Additionally, the aberrant meiotic spindle, especially the aberrant metaphase I spindle, may cause meiotic errors and oocyte aneuploidy, leading to spontaneous abortion and subfertility (Chiang et al., 2012). Abnormal oocyte spindle morphology is associated with human female subfertility and advanced maternal age has been attributed to spindle abnormality (Battaglia et al., 1996; Sakurada et al., 1996; Volarcik
et al., 1998). Because the interaction between the nucleus and the cytoplasm is important in the oocyte maturation process in mammals (Chiang et al., 2012; Cohen et al., 1998; Dekel and Beers, 1978; Fulka et al., 1998; Heacox and Schroeder, 1981; Li et al., 2001; Liu et al., 1999, 2000; Willadsen et al., 1999; Zhang et al., 1999), this study aimed at further exploring the role of the cytoplasm in GV breakdown, PB extrusion, meiotic spindle assembly and chromosome alignment by using GV transfer technique in oocytes meiotically arrested by IBMX. The results indicated that the structural and functional integrity of the GV and the recipient cytoplasts (enucleated GV oocytes) are not compromised by the micromanipulation and electrofusion procedures, because the meiotic resumption of reconstructed GV oocytes (not treated with IBMX) did not differ from that of non-manipulated controls. Specifically, the metaphase components of the mature reconstructed GV oocytes showed normal PB extrusion, chromosomal structures and meiotic spindles. This observation provides further support that GV transfer constitutes an appropriate cell model to investigate the contributions of cytoplasmic and nuclear factors in normal and abnormal meiosis. In this study, mouse oocytes arrested by IBMX for 24 and 48 h displayed a decline in their ability for meiotic maturation, reflected by an increase in aberrant meiotic spindles. The results also indicated that GVIBMX/C, but not GV/CIBMX, reconstructed oocytes displayed meiotic ability similar to that of control oocytes, indicating the importance of the cytoplasm in meiosis resumption in arrested oocytes. These data indicate that cytoplasm replacement by GV transfer may rescue the nucleus from oocytes whose cytoplasm has been affected. A safety assessment is needed before the clinical implementation of GV transfer. To date, limited data from human oocytes support the hypothesis that cytoplasm is affected in the ageing process (Zhang et al., 1999). Reconstructed human oocytes have been reported to be capable of completing normal meiotic maturation (Takeuchi et al., 2001; Zhang et al., 1999). For instance, the present authors have demonstrated that in four
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20µm -------Figure 4 Birefringent spindle observed in H/M and M/H types of xeno-oocytes at metaphase II. Metaphase II oocytes of human (A, a) and mouse (B, b) as well as metaphase II reconstituted xeno-oocytes of M/H (C, c) and H/M (D, d). Birefringent spindle in metaphase II oocytes of human (a), mouse (b), and in metaphase II reconstituted xeno-oocytes of M/H (c) and H/M (d). GV = germinal vesicle; H/M = oocyte reconstituted by human GV and mouse cytoplasm; M/H = oocyte reconstituted by mouse GV and human cytoplasm.
Figure 5 Cytogenetic analysis of mature H/M and M/H xeno-oocytes. Twentry-three sets of univalent chromosomes found in metaphase II H/M xeno-oocytes (A). Premature division of chromatids detected in metaphase II M/H xeno-oocyte (B). GV = germinal vesicle; H/M = oocyte reconstituted by human GV and mouse cytoplasm; M/H = oocyte reconstituted by mouse GV and human cytoplasm.
out of seven human oocytes, reconstructed from GV of old oocytes (from women aged >38 years) with cytoplasm of young oocytes (from women aged A mutation causes MELAS/ Leigh overlap syndrome presenting with acute auditory agnosia. Mitochondr. DNA 26, 208–212. Levron, J., Willadsen, S., Bertoli, M., Cohen, J., 1996. The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm. Hum. Reprod. 11, 1287–1292. Li, G.P., Chen, D.Y., Lian, L., Sun, Q.Y., Wang, M.K., Song, X.F., Meng, L., Schatten, H., 2001. Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals. J. Exp. Zool. 289, 322–329. Liu, H., Wang, C.W., Grifo, J.A., Krey, L.C., Zhang, J., 1999. Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis. Hum. Reprod. 14, 2357–2361. Liu, H., Zhang, J., Krey, L.C., Grifo, J.A., 2000. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Hum. Reprod. 15, 1997–2002. Liu, H., Chang, H.C., Zhang, J., Grifo, J., Krey, L.C., 2003. Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term. Hum. Reprod. 18, 1903–1907. Liu, L., Keefe, D.L., 2004. Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice. Biol. Reprod. 71, 1724– 1729. Moor, R.M., Dai, Y., Lee, C., Fulka, J., Jr., 1998. Oocyte maturation and embryonic failure. Hum. Reprod. Update 4, 223–236. Sakurada, K., Ishikawa, H., Endo, A., 1996. Cytogenetic effects of advanced maternal age and delayed fertilization on firstcleavage mouse embryos. Cytogenet. Cell Genet. 72, 46–49. Takeuchi, T., Gong, J., Veeck, L.L., Rosenwaks, Z., Palermo, G.D., 2001. Preliminary findings in germinal vesicle transplantation of immature human oocytes. Hum. Reprod. 16, 730–736. Van Cauwenberge, A., Alexandre, H., 2000. Effect of genistein alone and in combination with okadaic acid on the cell cycle resumption of mouse oocytes. Int. J. Dev. Biol. 44, 409–420. Volarcik, K., Sheean, L., Goldfarb, J., Woods, L., Abdul-Karim, F.W., Hunt, P., 1998. The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum. Reprod. 13, 154–160. Willadsen, S., Levron, J., Munne, S., Schimmel, T., Marquez, C., Scott, R., Cohen, J., 1999. Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum. Reprod. 14, 470–475. Zhang, J., Wang, C.W., Krey, L., Liu, H., Meng, L., Blaszczyk, A., Adler, A., Grifo, J., 1999. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil. Steril. 71, 726–731. Declaration: The author reports no financial or commercial conflicts of interest. Received 2 January 2015; refereed 19 March 2015; accepted 27 March 2015.
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ARTICLE
Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes John Zhang *, Hui Liu Reproductive Endocrinology and Infertility, New Hope Fertility Center, New York, NY, USA * Corresponding author.
E-mail address: [email protected] (J Zhang). Dr Zhang completed his medical degree in at the Zhejiang University School of Medicine, and subsequently received his Master’s Degree at Birmingham University in the UK. In 1991, Dr Zhang earned his PhD. in In-Vitro Fertilization (IVF), and, after studying and researching the biology of mammalian reproduction and human embryology for nearly ten years, became the first Fellow in the Division of Reproductive Endocrinology and Infertility of New York University’s School of Medicine in 2001. Dr Zhang continues his research in non-embryonic stem cell research, long-term cryopreservation of oocytes, and oocyte reconstruction by nuclear transfer.
Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMXtreated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF. Abstract
© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cytoplasm, germinal vesicle, meiotic maturation, meiotic spindle, nucleus, oocyte
http://dx.doi.org/10.1016/j.rbmo.2015.03.012 1472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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Introduction The mammalian ovary contains a large supply of inactive germ cells that reside in primordial follicles and two categories of oocyte. The first category constitutes oocytes that are unable to respond to maturation signals in vivo or in vitro and remain arrested in the diplotene stage (Fulka et al., 1998). The second category makes up a fully grown group of oocytes that respond to gonadotrophins and mature to metaphase II in pre-ovulatory follicles and in culture (Fulka et al., 1998). Oocyte maturation is a complex process involving both the progression of the meiotic cycle and the reprogramming of cytoplasmic events (Fulka et al., 1998; Karnikova et al., 1998; Liu et al., 1999; Moor et al., 1998). Some of the structural changes in the cytoplasm include an increase in the number of mitochondria, structural modification to the Golgi apparatus and accumulation of ribosomes (Fulka et al., 1998; Heacox and Schroeder, 1981). The end-point of oocyte maturation is the release of a mature metaphase II (MII) oocyte that is competent to support normal embryonic development. Both nuclear and cytoplasmic deficiencies have been shown to be responsible for poor oocyte quality by contributing to meiotic defects and subsequent impaired embryo development (Fulka et al., 2001; Liu et al., 2000, 2003; Liu and Keefe, 2004; Moor et al., 1998). Germinal vesicle (GV) transfer techniques have represented useful tools for studying the interaction between the nucleus and the cytoplasm in the oocyte maturation process in mammals (Chiang et al., 2012; Cohen et al., 1997; Dekel and Beers, 1978; Fulka et al., 1998, 2001; Heacox and Schroeder, 1981; Levron et al., 1996; Li et al., 2001; Liu et al., 1999, 2000; Zhang et al., 1999). Furthermore, GV transfer between different mammalian species has demonstrated that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at the MII stage (Li et al., 2001). It has previously been reported in humans (Zhang et al., 1999) and mice (Liu et al., 2000, 2003) that normal meiosis can occur after the transfer of GV into an enucleated host oocyte. It has also been shown in mice that oocytes reconstructed by GV transfer into a cytoplasm of the same developmental stage mature normally in vitro through MII (Liu et al., 1999). Studies to date have evaluated the in-vitro maturation capability of reconstructed oocytes by GV transfer (Heacox and Schroeder, 1981; Li et al., 2001; Zhang et al., 1999) but did not address data on meiotic maturation such as polar body (PB) extrusion, meiotic spindle assembly or chromatid separation. Abnormal oocyte spindle morphology is associated with human female infertility, and advanced maternal age has been attributed to spindle abnormalities such as abnormal chromosome alignment and a microtubule matrix that compromises the meiotic spindle (Battaglia et al., 1996). The regulatory mechanisms responsible for assembly of the meiotic spindle in the cytoplasm are significantly altered in older women, leading to high prevalence of aneuploidy (Battaglia et al., 1996). Whether the GV transfer technique could represent a therapeutic option for meiotic spindle abnormalities remains to be determined. It is well established that pharmacological activation of cAMP-dependent protein kinases by 3-isobutyl-1-methylxanthine (IBMX) suppresses meiotic
resumption (Dekel and Beers, 1978). The present authors (Liu et al., 2003) and others (Eppig and Schroeder, 1989) have demonstrated that mouse oocytes treated with IBMX at the GV stage in vitro have arrested during their meiotic maturation, and when released from IBMX were able to produce normal embryonic development. The purpose of this study was to explore the role of the cytoplasm on meiotic spindle assembly in oocytes arrested by IBMX in vitro and to assess whether cytoplasm replacement via GV transfer restores meiotic maturation, in particular spindle assembly in the IBMXarrested oocytes. We postulated that a healthy cytoplasm is required for GV breakdown and meiosis resumption.
Materials and methods Mouse oocytes The CB6F1 mice used in this experiment were purchased from Charles River Laboratory (Boston, MA). Mice were subjected to a 14L:10D cycle for at least 1 week before use. Animals were cared for according to the procedure approved by University Animal Welfare Committee (UAWC). GV-stage oocytes were collected from 6- to 8-week-old CB6F1 mice through puncturing the ovarian follicles 48 h following priming with 5 IU pregnant mare serum gonadotrophin (PMSG, Sigma, St Louis, MO). GV oocytes, stripped off cumulus granulosa cells with a diameter around 80 µm, were used in this experiment. GV oocytes were incubated in human tubal fluid (HTF) medium supplemented with 10% fetal calf serum (FCS) (HyClone, ThermoFisher Sceintific, Waltham, MA) and 50 µg/ml of IBMX (Sigma, St Louis, MO) constituting the treatment group, thus preventing GV from breakdown by increasing cytosolic cAMP following inhibition of phosphodiesterase. Mouse oocytes were arrested at GV stage in vitro following culture in IBMX for 6, 24 and 48 h, respectively. The arrested oocytes were then released from IBMX by changing the medium to IBMX-free medium. The dose of IBMX used was chosen from previously published data (Liu et al., 2003; Van Cauwenberge and Alexandre, 2000). These treatments were used for various periods of time and then GV were released from IBMX and cultured in IBMX-free HTF medium (F-HTF) for 15 h for meiosis resumption. Oocytes without IBMX treatment constituted the control group.
Human GV oocyte GV-stage oocytes were obtained from patients (25–42 years old) who were undergoing IVF with intracytoplasmic sperm injection (ICSI) after ovarian stimulation with gonadotrophins. All patients were treated with human chorionic gonadotrophin (HCG; 5000 or 10,000 IU) 36 h before transvaginal oocyte retrieval. Institutional Review Board (IRB) approval was obtained on July 23 1998 from New York University School of Medicine (NYUMC-IBRA Protocol H 6902). Following an IRB approval, participants consented and GV oocytes that were unsuitable for ICSI and routinely discarded were used in this study.
GV transfer and electromembrane fusion Oocytes were exposed to modified HTF medium (mHTF) with 10% FCS supplemented with 7.5 µg/ml cytochalasin B (CB;
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
ARTICLE IN PRESS Cytoplasm replacement restores meiosis Sigma, C6762) for 15 min at room temperature in order to disrupt microfilament and increase plasma membrane flexibility before manipulation. The dish was placed onto the stage of Olympus X71 inverted microscope equipped with Narishige micromanipulators. A slot was made on the zona pellucida of each oocyte by applying a sharp-tipped pipette to penetrate the zona and grind the zona against the wall of the holding pipette. This allowed the enucleation pipette to pass through the zona slot and approach the GV before gently applying negative pressure to aspirate the GV. Once a GV was separated from the cytoplasm, it was transferred into the perivitelline space of an enucleated recipient GV oocyte. The membrane fusion between GV and cytoplasm was initiated by placing it into fusion medium (0.3 mol/l mannitol, 0.1 mmol/l CaCl2 and 0.05 mmol/l MgSO4) between platinum electrodes alignment in response to AC (6–8 V) for 5–10 s before electrical pulse (1.8–2.5 kV/cm DC for 50 µs) delivered by a Model 2001 Electro Cell Manipulator (BTX, Holliston, MA). The formed complexes were then rinsed three times in mHTF, and then incubated in F-HTF medium at 5% CO2 and 37°C. Membrane fusion usually occurred 30 min after electric pulse.
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Immunofluorescent staining for meiotic spindle Dissolution of zona pellucida and permeation of oocyte membrane were required for indirect immunofluorescent staining for microtubule. Oocytes were briefly treated with Tyrode’s acid solution to dissolve the zona pellucida and then fixed with phosphate-buffered saline (PBS) containing 3.7% paraformaldehyde for 30 min. Oocytes were then treated with 0.25% Triton X-100 for 20 min at room temperature to permeate the membrane. Oocytes were finally rinsed two times with PBS containing 0.25% bovine serum albumin (BSA: Sigma, St Louis, MO) to remove the free aldehydes and get prepared for double staining. Microtubules were localized with 1:2000 mouse monoclonal antibody to β-tubulin (Sigma, St Louis, MO). The primary antibody was detected using 1:400 fluorescein isothiocyanatelabelled secondary goat anti-mouse IgG (Sigma, St Louis, MO). Each antibody was applied overnight at 4°C and rinsed with PBS between antibody applications. Oocytes were rinsed with 5 µg/ml Hoechst 33342, a cell-permeable DNA stain that is excited by UV light and emits blue fluorescence at 460 to 490 nm allowing simultaneous detection of the chromosomes. Each oocyte was mounted on a glass slide covered with a coverlid and examined using Olympus BX61 fluorescence microscope.
Treatment groups Three types of oocyte were reconstructed by GV transfer: (i) GV of non-IBMX-arrested oocyte was transferred to a cytoplasm (C) of non-IBMX-arrested oocyte (GV/C) serving as control to ensure that the GV transfer technique per se did not affect meiosis; (ii) GV of non-IBMX-arrested oocyte with cytoplasm of IBMX-arrested oocyte (GV/CIBMX) (Figure 1); and (iii) GV of IBMX-arrested oocyte with cytoplasm of non-IBMXarrested oocyte (GVIBMX/C) (Figure 1). In other experiments, GV transfer between mouse and human oocytes was performed in order to construct xenooocytes. Thus, human GV/mouse cytoplasm (H/M) and mouse GV/human cytoplasm (M/H) oocytes were reconstructed. Mouse GV with mouse cytoplasm was reconstructed and served as controls because mouse oocytes are known to fully mature while human oocytes are known to poorly mature in vitro. The oocytes were then cultured for 15–36 h in vitro and examined for meiotic maturation.
Figure 1
Birefringent spindle The meiotic spindle of living oocytes was imaged using an Olympus IX71 equipped with a polarizer and an analyser. The living oocytes were placed into an mHTF droplet in a glass bottom dish and analysed by bright field observation. During analysis, the holding pipette was applied to aspirate the oocyte lightly with the assistance of an injection pipette in order to rotate the oocyte and ultimately locate the birefringent spindle signal. Because the meiotic spindle is a temperature-sensitive structure, all procedures were performed at a temperature of 37°C.
Statistics Data were analysed using chi-squared or Fisher exact probability tests as appropriate. Statistical significance was
Schematic diagram of GV transfer between IBMX-treated oocyte and a control non-treated oocyte.
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determined at P < 0.05. Sample size calculation was performed using a power of 80% and alpha error of 0.05. In order to detect 50% change in PB extrusion between the reconstructed oocytes by GV transfer, a sample size of 15 oocytes were needed in each group.
Results The effect of IBMX arrest on PB extrusion in mouse oocytes The meiotic maturation, as shown by the first PB extrusion, was evaluated 14–16 h after being released from IBMX. As seen in Table 1, oocytes treated by IBMX for 6 h displayed meiotic ability similar to control oocytes with 98% completing meiotic maturation as shown by PB at both 0 and 6 h. However, when IBMX treatment time was extended to 24 and 48 h, oocytes exhibited reduced ability for meiotic maturation of the oocytes extruding the first PB compared with controls (P < 0.01) where only 68% of oocytes had PB extrusion at 24 h and only 44% of oocytes had PB extrusion at 48 h of IBMX treatment (P < 0.01) (Table 1).
The effect of IBMX arrest on meiotic spindle of mouse oocyte After being released from IBMX and matured in vitro, oocytes were visualized for meiotic spindle by indirect immunofluorescent staining. Following IBMX treatment for 6 h, 67 out of 69 (97%) oocytes meiotically matured by releasing a PB, 99% of which displayed normal meiotic spindle (Table 2 and Figure 2A) with a barrel shape and a slightly narrow poles, and had chromosomes aligned in the equatorial plate of the spindle (Figure 2a). In contrast, following IBMX treatment for
Table 1
48 h, only 50 out of 90 (56%) of oocytes meiotically matured, 32% of which displayed normal spindle and 68% of which exhibited abnormal spindle (P < 0.001 compared with IBMX treatment for 6 h) (Table 2 and Figure 2B, 2C, 2D, 2E). The abnormalities observed following IBMX arrest for 48 h included multiple poles of spindle (Figure 2B), disrupted spindle (Figure 2C, 2D) or no spindle (Figure 2E). Other abnormalities included chromosome misalignment, such as aggregated and scattered chromosomes (Figure 2b, 2c, 2d), or decondensed chromosomes (Figure 2e). Additionally, the majority of oocytes that had IBMX arrest for 48 h showed various abnormal spindle structures (Table 2).
The effect of GV transfer on meiotic spindle assembly and meiotic maturation in mouse oocytes After reconstruction and in vitro culture for 14–16 h without IBMX, the control oocytes exhibited meiotic maturation, with 98% of oocytes extruding the first PB (Table 3), indicating that the procedure of GV transfer did not influence the meiotic maturation. The GV/CIBMX reconstructed oocytes displayed a significantly reduced ability for meiotic maturation, with only 43% extruding a PB compared with controls and GVIBMX/C reconstructed oocytes (P < 0.001; Table 3). The majority (63%) of the mature GV/CIBMX reconstructed oocytes showed abnormal meiotic spindle (Table 4). These oocytes had scattered chromosomes across the spindle (Figure 3A, 3a), scattered chromosomes along the disrupted spindle (Figure 3B, 3b, 3C, 3c) and decondensed chromosomes as the spindle disappeared (Figure 3D, 3d). By contrast, the GVIBMX/C reconstructed oocytes showed similar ability of meiotic maturation to that observed in control oocytes (Figure 3E, 3e). These reconstructed oocytes had 100% (n = 35) maturing oocytes with extrusion of first PB (Table 4),
Maturation of mouse oocytes following release from IBMX arrest.
IBMX arrest (time) 0 h (control) 6h 24 h 48 h
Total no. of oocytes
GV n (%)
No PB n (%)
PB n (%)
110 104 105 149
0 0 5 8
2 2 29 75
108 102 71 66
(0) (0) (5) (5)
(2) (2) (28) (50)
(98)a (98)a (68)b (44)c
GV = germinal vesicle; PB = polar body indicating oocyte maturity. Data are presented as n or n (%). Chi-squared test: a versus b versus c; P < 0.01.
Table 2
Meiotic spindle analysis of IBMX-arrested mouse oocytes.
IBMX arrest time 48 h (90 total oocytes) 6 h (69 total oocytes)
14–16 h after release No PB PB No PB PB
(n = 40) (n = 50) (n = 2) (n = 67)
Normal spindle n (%)
Abnormal spindle n (%)
12 16 1 66
28 34 1 1
(30) (32)a (50) (99)b
(70) (68)c (50) (1)d
PB = polar body. Chi-squared test: a versus b and c versus d; P < 0.001.
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20µm -------Figure 2 The effect of IBMX arrest on meiotic spindle in mouse oocytes. Indirect immunofluorescent staining for β-tubulin (A, B, C, D, E); Hoechst 33342 staining for chromosomes (a, b, c, d, e). Normal meiotic spindle observed in control metaphase II oocytes (A and a). Oocytes arrested with IBMX for 6 h were similar to controls (not shown). Abnormal meiotic spindle observed in metaphase II oocytes arrested with IBMX for 48 h (B, b, C, c, D, d, and E, e).
Table 3 The effect of GV transfer on restoration of meiotic maturation in reconstructed mouse oocytes. Types of oocyte (GV/C) GVIBMX/C GV/CIBMX GV/C (control)
Total no. of oocytes
No PB n (%)
PB n (%)
35 37 52
0 (0) 21 (57) 1 (2)
35 (100)a 16 (43)b 51 (98)a
C = cytoplasm; GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b; P < 0.001.
Table 4
The effect of GV transfer on assembly of meiotic spindle in mouse oocytes.
Type of oocyte (GV/C) GV/CIBMX (37 total oocytes) GVIBMX/C (35 total oocytes)
Oocyte No PB PB No PB PB
(n = 21) (n = 16) (n = 0) (n = 35)
Normal spindle n (%)
Abnormal spindle n (%)
5 6 0 32
16 10 0 3
(24) (38)a (0) (91)b
(76) (63)c (0) (9)d
C = cytoplasm; GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b and c versus d; P < 0.05.
91% (n = 32) of which displayed normal spindle and chromosome alignment (Table 4). Only 9% (n = 3) of these oocytes displayed abnormal meiotic spindles with two showing loose alignment of chromosomes at equatorial plate and one showing multiple microtubule stars, disappearance of meiotic spindle, and decondensed chromosomes in a pronucleus-like structure.
The meiotic behaviour of human GV karyoplast in cytoplasm of mice oocytes (xeno-oocyte) M/H oocytes displayed significantly reduced meiotic maturation ability with only 44% of these xeno-oocytes extruding
the first PB (P < 0.001; Table 5). Interestingly, all the H/M xeno-oocytes matured to MII and extruded the first PB (Table 5 and Figure 4D, 4d). Non-invasion live observation of oocytes under inverted microscope equipped with polarizer and analyser revealed that the birefringent spindle detected in mature H/M oocytes (Figure 4d) was similar to that of mature mouse control oocytes in size and shape (Figure 4b), whereas the birefringent spindle of mature M/H oocytes (Figure 4c) was similar to that of mature human oocytes (Figure 4a). Nine pairs of oocytes reconstructed by reciprocal exchange of GV between human and mouse were successfully prepared for cytogenetic analysis. Five out of nine H/M oocytes had 23 normal sets of chromosomes (Figure 5A). In
Please cite this article in press as: John Zhang, Hui Liu, Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.03.012
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20µm -------Figure 3 Effect of cytoplasm replacement by GV transfer on meiotic spindle of mouse oocytes arrested by IBMX. Indirect immunofluorescent staining for β-tubulin (A, B, C, D, E); Hoechst 33342 staining for chromosomes (a, b, c, d, e). Abnormal meiotic spindle observed in oocytes reconstructed with GV/CIBMX (A, a, B, b, C, c, D, d). Normal meiotic spindle observed in oocytes constructed with GVIBMX/C (E, e). C = cytoplasm; GV = germinal vesicle.
Table 5 Maturation of xeno-oocytes reconstructed by GV transfer between human and mouse oocytes. Type of oocyte (GV/Cytoplasm) Human GV/mouse cytoplasm Mouse GV/human cytoplasm Mouse GV/mouse cytoplasm (control)
Number n
No PB n (%)
PB n (%)
25
0 (0)
25 (100)a
18
10 (56)
8 (44)b
52
1 (2)
51 (98)a
GV = germinal vesicle; PB = polar body. Chi-squared test: a versus b; P < 0.001.
contrast, none of nine M/H oocytes had a normal number of chromosomes (Figure 5B). Additionally, precocious chromatid division was found in six M/H oocytes (Figure 5B), while none of the H/M oocytes displayed similar findings (Figure 5A). Other abnormalities observed included chromosome fragment (two H/M oocytes), chromosome decondensation (one M/H oocyte) and bivalent chromosomes (one M/H oocyte).
Discussion Human oocytes are arrested at the prophase/GV stage before birth and do not resume meiotic maturation until ovulatory events at puberty. Additionally, the aberrant meiotic spindle, especially the aberrant metaphase I spindle, may cause meiotic errors and oocyte aneuploidy, leading to spontaneous abortion and subfertility (Chiang et al., 2012). Abnormal oocyte spindle morphology is associated with human female subfertility and advanced maternal age has been attributed to spindle abnormality (Battaglia et al., 1996; Sakurada et al., 1996; Volarcik
et al., 1998). Because the interaction between the nucleus and the cytoplasm is important in the oocyte maturation process in mammals (Chiang et al., 2012; Cohen et al., 1998; Dekel and Beers, 1978; Fulka et al., 1998; Heacox and Schroeder, 1981; Li et al., 2001; Liu et al., 1999, 2000; Willadsen et al., 1999; Zhang et al., 1999), this study aimed at further exploring the role of the cytoplasm in GV breakdown, PB extrusion, meiotic spindle assembly and chromosome alignment by using GV transfer technique in oocytes meiotically arrested by IBMX. The results indicated that the structural and functional integrity of the GV and the recipient cytoplasts (enucleated GV oocytes) are not compromised by the micromanipulation and electrofusion procedures, because the meiotic resumption of reconstructed GV oocytes (not treated with IBMX) did not differ from that of non-manipulated controls. Specifically, the metaphase components of the mature reconstructed GV oocytes showed normal PB extrusion, chromosomal structures and meiotic spindles. This observation provides further support that GV transfer constitutes an appropriate cell model to investigate the contributions of cytoplasmic and nuclear factors in normal and abnormal meiosis. In this study, mouse oocytes arrested by IBMX for 24 and 48 h displayed a decline in their ability for meiotic maturation, reflected by an increase in aberrant meiotic spindles. The results also indicated that GVIBMX/C, but not GV/CIBMX, reconstructed oocytes displayed meiotic ability similar to that of control oocytes, indicating the importance of the cytoplasm in meiosis resumption in arrested oocytes. These data indicate that cytoplasm replacement by GV transfer may rescue the nucleus from oocytes whose cytoplasm has been affected. A safety assessment is needed before the clinical implementation of GV transfer. To date, limited data from human oocytes support the hypothesis that cytoplasm is affected in the ageing process (Zhang et al., 1999). Reconstructed human oocytes have been reported to be capable of completing normal meiotic maturation (Takeuchi et al., 2001; Zhang et al., 1999). For instance, the present authors have demonstrated that in four
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20µm -------Figure 4 Birefringent spindle observed in H/M and M/H types of xeno-oocytes at metaphase II. Metaphase II oocytes of human (A, a) and mouse (B, b) as well as metaphase II reconstituted xeno-oocytes of M/H (C, c) and H/M (D, d). Birefringent spindle in metaphase II oocytes of human (a), mouse (b), and in metaphase II reconstituted xeno-oocytes of M/H (c) and H/M (d). GV = germinal vesicle; H/M = oocyte reconstituted by human GV and mouse cytoplasm; M/H = oocyte reconstituted by mouse GV and human cytoplasm.
Figure 5 Cytogenetic analysis of mature H/M and M/H xeno-oocytes. Twentry-three sets of univalent chromosomes found in metaphase II H/M xeno-oocytes (A). Premature division of chromatids detected in metaphase II M/H xeno-oocyte (B). GV = germinal vesicle; H/M = oocyte reconstituted by human GV and mouse cytoplasm; M/H = oocyte reconstituted by mouse GV and human cytoplasm.
out of seven human oocytes, reconstructed from GV of old oocytes (from women aged >38 years) with cytoplasm of young oocytes (from women aged A mutation causes MELAS/ Leigh overlap syndrome presenting with acute auditory agnosia. Mitochondr. DNA 26, 208–212. Levron, J., Willadsen, S., Bertoli, M., Cohen, J., 1996. The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm. Hum. Reprod. 11, 1287–1292. Li, G.P., Chen, D.Y., Lian, L., Sun, Q.Y., Wang, M.K., Song, X.F., Meng, L., Schatten, H., 2001. Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals. J. Exp. Zool. 289, 322–329. Liu, H., Wang, C.W., Grifo, J.A., Krey, L.C., Zhang, J., 1999. Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis. Hum. Reprod. 14, 2357–2361. Liu, H., Zhang, J., Krey, L.C., Grifo, J.A., 2000. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Hum. Reprod. 15, 1997–2002. Liu, H., Chang, H.C., Zhang, J., Grifo, J., Krey, L.C., 2003. Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term. Hum. Reprod. 18, 1903–1907. Liu, L., Keefe, D.L., 2004. Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice. Biol. Reprod. 71, 1724– 1729. Moor, R.M., Dai, Y., Lee, C., Fulka, J., Jr., 1998. Oocyte maturation and embryonic failure. Hum. Reprod. Update 4, 223–236. Sakurada, K., Ishikawa, H., Endo, A., 1996. Cytogenetic effects of advanced maternal age and delayed fertilization on firstcleavage mouse embryos. Cytogenet. Cell Genet. 72, 46–49. Takeuchi, T., Gong, J., Veeck, L.L., Rosenwaks, Z., Palermo, G.D., 2001. Preliminary findings in germinal vesicle transplantation of immature human oocytes. Hum. Reprod. 16, 730–736. Van Cauwenberge, A., Alexandre, H., 2000. Effect of genistein alone and in combination with okadaic acid on the cell cycle resumption of mouse oocytes. Int. J. Dev. Biol. 44, 409–420. Volarcik, K., Sheean, L., Goldfarb, J., Woods, L., Abdul-Karim, F.W., Hunt, P., 1998. The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum. Reprod. 13, 154–160. Willadsen, S., Levron, J., Munne, S., Schimmel, T., Marquez, C., Scott, R., Cohen, J., 1999. Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum. Reprod. 14, 470–475. Zhang, J., Wang, C.W., Krey, L., Liu, H., Meng, L., Blaszczyk, A., Adler, A., Grifo, J., 1999. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil. Steril. 71, 726–731. Declaration: The author reports no financial or commercial conflicts of interest. Received 2 January 2015; refereed 19 March 2015; accepted 27 March 2015.
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