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Cryobiology 59 (2009) 90 95 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes q Alberto M. Luciano a, *, Sara Chigioni b, Valentina Lodde a, Federica Franciosi a, Gaia C. Luvoni b, Silvia C. Modina a a Department of Animal Sciences, Division of Veterinary Anatomy and Histology, Faculty of Veterinary Medicine, University of Milan, Italy b Department of Veterinary Clinical Sciences, Obstetrics and Gynecology, Faculty of Veterinary Medicine, University of Milan, Italy article info abstract Article history: Received 1 October 2008 Accepted 7 May 2009 Available online 19 May 2009 Keywords: Cat Oocyte Banking Vitrification Slow freezing Gap junctions Actin Tubulin Cytoskeleton Meiosis Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability. In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes. From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes. Ó 2009 Elsevier Inc. All rights reserved. Introduction Oocyte cryopreservation is becoming an integral part of assisted reproductive technologies (ART) both for the conservation of endangered species and for preserving the female genome of valuable laboratory models. In the past few decades, several attempts have been made to improve the efficiency of cryopreservation of carnivore oocytes. Despite a number of potential advantages, the contemporary techniques of oocyte cryopreservation in mammals are often marred by both low survival and development rates [2]. q Work supported by MIUR PRIN 2005 2005073071-003 and FIRST 2006. * Corresponding author. Address: Sezione di Anatomia ed Istologia Veterinaria, Dipartimento di Scienze Animali, Università degli Studi di Milano, Via Celoria, 10, 20133 Milano, Italy. Fax: +39 02 50317980. E-mail address: alberto.luciano@unimi.it (A.M. Luciano). The standard method used for mammalian oocyte cryopreservation is slow freezing [33], which implies slow cooling rates and the use of a relatively low concentration of cryoprotectant agents (CPAs). Slow freezing has the advantage of using low concentrations of cryoprotectants, which are associated with chemical toxicity and osmotic shock, but their ability to prevent ice-crystal formation at low concentrations is limited. The vitrification technique avoids the occurrence of ice formation. This latter method determines a sharp increase in viscosity by ultra-rapid cooling rate and a very high concentration of CPAs, permitting the transition of aqueous solution to a glass state, avoiding the passage through the crystalline state [41]. However, the toxicity at these concentrations is such that the cell can only be exposed to the cryoprotectant solution for a very short period of time. Slow and ultra-rapid protocols may severely affect both the integrity and the survival of the oocytes after thawing and 0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.05.002

A.M. Luciano et al. / Cryobiology 59 (2009) 90 95 91 irreversibly compromise their subsequent developmental capability in several species, including the domestic cat [23,26,31,34]. Conflicting results have been obtained after cryopreservation of mammalian oocytes at different maturational stages, since the effects of cryoinjuries and CPA toxicity can vary considerably depending on the meiotic state of the gametes [2,4,7,19,20]. Mature oocytes are sensitive to cryoinjury as chromosomes are arranged on the delicate metaphase II (MII) spindle and are not protected by a nuclear membrane. Low temperatures affect the meiotic spindle, causing depolymerization of microtubules with successive chromosomal aberration and aneuploidy [4,38]. Although a few previous studies have reported a higher success rate when cat oocytes were cryopreserved at the MII stage [26,29,31], immature oocytes are more readily accessible in significant numbers in ovaries without the need for preventive hormonal treatment to cause oocyte maturation, increasing the availability of gamete for research applications or innovative assisted reproduction techniques. Immature oocytes are arrested in the diplotene stage of prophase I (germinal vesicle stage GV), and the chromatin is protected by a nuclear envelope. At this stage, no spindle apparatus is present; therefore, they are considered more resistant to cold-induced damage. Even though the majority of studies have been conducted with MII oocytes, the interest in GV stage oocytes has an additional justification. However, immature oocytes are directly connected to the surrounding granulosa cells by the transzonal projections and their structural and functional integrity is very sensitive to cryopreservation [3,8,18,36]. Recently Comizzoli et al. investigated the osmotic tolerance of immature cat oocytes, and the impact of anisosmotic environment on cumulus oocyte communications when tolerance limits are exceeded [8]. Given the above observations, the aim of this study was to assess a suitable cryopreservation protocol for immature cat oocytes, capable of preserving the functional coupling with cumulus cells as well as both nuclear and cytoplasmic competence after thawing. Materials and methods Chemicals and reagents All chemicals and reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA), unless otherwise stated. Collection of feline oocytes Ovaries were harvested from pubertal queens during routine ovariectomy or ovario-hysterectomy at local veterinary clinics. Ovaries were stored at +38 C in phosphate-buffered saline (PBS) with a mixture of antibiotics and antimycotic (100 IU/ml of penicillin G sodium, 0.1 mg/ml of streptomycin sulfate and 0.25 lg/ml of amphotericin B) for 1 2 h before processing. Cumulus oocyte complexes (COCs) were released by repeatedly puncturing of the ovarian cortex and only COCs with darkly pigmented ooplasm, surrounded by one or more layers of cumulus cells (grade 1) were selected for the experiments. Cryopreservation of immature feline oocytes Slow freezing and thawing Slow freezing was carried out as previously reported [26]. Selected COCs were exposed for 20 min at room temperature, to the cryoprotectant solution, which consisted of Dulbecco s PBS/ DPBS supplemented with 5 mm glucose, 0.3 mm sodium pyruvate, 0.2 M sucrose, 1.5 M ethylene glycol (EG) and 20% fetal bovine serum. After equilibration in the freezing solution, oocytes were loaded in 0.25 ml straws (five per straw) and placed in a programmable freezer (DB1 Embryo Freeze Biotronics Ltd., Leominster, UK). The straws were cooled at a rate of 2 C/min from +20 Cto 7 C. After 5 min, each straw was seeded manually with chilled forceps, held at 7 C for an additional 5 min and cooled at a rate of 0.3 C/ min to 30 C, before being plunged into liquid nitrogen. Oocytes were thawed by exposing to air for 10 s and then in a +38 C water bath for 30 s. The COCs were recovered from the straw and equilibrated for 5 min within the freezing medium, then transferred into fresh maturation medium. Vitrification and warming Oocyte vitrification was carried out following the open pulled straw method (OPS, Minitube, Tiefenbach, Germany) [41]. All procedures were performed on a heated stage (40 C) in a warm room (25 28 C). Groups of 3 5 immature COCs were initially equilibrated for 1 min in M199, supplemented with HEPES 20 mm and 20% fetal bovine serum (HM). After equilibration, COCs were incubated for 30 s in HM supplemented with 10% EG and 10% dimethyl sulfoxide (DMSO) then successively transferred into HM supplemented with 20% EG and 20% DMSO and 1 M sucrose for 25 s, during which the COCs were loaded into OPS and successively submerged into liquid nitrogen. Oocyte warming was performed by placing the end of the straws directly into HM supplemented with 0.3 M sucrose. The vitrified medium liquified within 1 2 s, where upon the medium entered the straw. The COCs flowed out of the straw into the 0.3 M sucrose supplemented HM where they remained for 1 min. Subsequently, oocytes were transferred and washed for 5 min into 0.2 M sucrose supplemented HM then equilibrated for 5 min in HM. For both protocols, after warming, oocytes were selected on the basis of the integrity of the oolemma and the zona pellucida and used for the following experiments. Assessment of cytoskeleton organization In order to assess the effect of cryopreservation protocols on cytoskeleton organization after thawing or warming, the distribution of microtubules and microfilaments in oocytes cryopreserved with both protocols was analyzed by three-color fluorescence staining, as previously described [30]. Briefly, cumulus cells were removed and denuded oocytes were fixed in 0.3% Triton X-100 and 2% paraformaldehyde in DPBS at 37 for 1 h. After incubation for 30 min at room temperature in DPBS containing 1% BSA, 1% of Normal Donkey Serum and 0.3% Triton X-100, the oocytes were incubated overnight at 4 C with an anti-a-tubulin monoclonal antibody diluted 1:200 in DPBS, and then with a fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG antibody raised in donkey (Jackson Immunoresearch lab., Inc., West Grove, PA, USA) for 1 h at room temperature. To detect the distribution of microfilaments, the oocytes were then exposed to tetramethylrhodamine B isothiocyanate (TRITC) labeled phalloidin (1 lg/ml for 30 min at 37 C). DNA was stained with DAPI (1 lg/ml in DPBS; Molecular Probes Invitrogen, Milan, Italy). As a control, some oocytes were treated with unlabeled phalloidin before incubation with labeled filaments marker then incubated directly with the secondary antibody, without pre-incubation with the primary anti-tubulin antibody. All samples were then mounted with an anti-fade medium (Vectashield Vector lab., Burlingame, CA, USA) and observed using a conventional epifluorescence microscope (Eclipse E 600, Nikon, Tokyo, Japan). Oocytes were classified according to morphological evidence, as previously described, both in epifluorescence and confocal LS microscopy [30,38]. A group of freshly isolated COCs was processed as described above and used as a control.

92 A.M. Luciano et al. / Cryobiology 59 (2009) 90 95 Evaluation of functional coupling between cumulus cells and oocyte Immediately after warming, the intracellular communications between the oocyte and cumulus cells were assessed by lucifer yellow dye (LY) microinjection, as previously indicated [22]. A 3% solution of LY in 5 mm of lithium chloride was pressure injected into the oocyte and the spread of dye into surrounding cumulus cells was monitored using an inverted fluorescence microscope (Nikon Diaphot Nikon Corp., Tokyo, Japan). A microinjection apparatus (Narishige, CO LTD, Tokyo, Japan) was used to guide the holding and injecting of micropipettes into a 50 ll drop of TALP Hepes covered by mineral oil [25]. Analysis of gap junction functionality was performed within 10 min after injection by observing the spread of LY from oocyte to cumulus cells. Gap junction mediated oocyte cumulus cell communications were classified as open when complete LY diffusion in corona radiata cells was detected; partially open, when less than 50% of the circumference of the oocyte showed signs of dye diffusion between ooplasm and corona radiata cells and closed, when LY remained confined within the ooplasm [22]. A group of freshly isolated COCs was LY-injected as described above and used as a control. In vitro maturation and assessment of meiotic progression After warming, COCs from both cryopreservation protocols were matured in Petri dishes in 100 ll drops under mineral oil (from 5 to 10 oocytes per drop) and incubated at 38.5 C under 5% CO 2 in air for 24 h. The maturation medium was Krebs s Ringer Bicarbonate (mkrb) with antibiotics (100 IU/ml of penicillin G potassium and 100 lg/ml of streptomycin sulfate), 3 mg BSA/ml and gonadotropins (0.5 IU FSH/ml and 0.5 IU LH/ml, Pluset, Bio98, Bologna, Italy). After 24 h of culture, cumulus cells of matured oocytes were removed by gentle mechanical pipetting with a small bore glass pipette. Denuded oocytes were fixed and stained with bis-benzimide (Hoechst 33342). Briefly, denuded oocytes were stained with a solution of sodium citrate: ethanol (3:1) containing 10 lg Hoechst/ml. Oocytes were placed on a slide with a minimum amount of medium and then covered by 10 ll of Hoechst solution. After 5 min of incubation in the dark, the Hoechst solution was removed and the oocytes were covered with an anti-fade reagent (Gel Mount TM, biømeda corp, Foster City, CA, USA). The oocytes were overlaid with a coverslip supported by four droplets of vaseline paraffin and then observed under a fluorescent microscope (Axiovert 100, Zeiss, Arese, Italy) at 400 magnification for the evaluation of meiotic progression. Oocytes were classified as follows: GV: all oocytes that did not progress beyond the germinal vesicle stage; intermediate: all oocytes from GVBD to anaphase I; mature: all oocytes at telophase I and metaphase II (MII) stages. Oocytes which could not be identified as being at any of these stages, due to faint or invisible staining, were considered degenerate. A group of freshly isolated COCs was cultured for 24 h as described above and used as a control. Statistical analysis All experiments were replicated at least three times. Data were compared with chi-square test. Probabilities of less than 0.05 were considered statistically significant. Results Cytoskeleton organization after slow freezing and vitrification procedures The overall oocyte morphological integrity after warming was not different between the two groups and ranged between 80% and 90%. Analysis of cytoskeleton elements of cryopreserved oocytes was conducted to address the effectiveness of cryopreservation procedures on cytoplasmic organization after warming. In Fig. 1, the representative patterns of cytoskeletal element distribution, are Fig. 1. Representative images of cytoskeleton patterns of cryopreserved immature oocytes after warming, showing microfilaments in red, microtubules in green, and DNA in blue. Pattern A: microfilaments diffused in the ooplasm with a slightly greater concentration beneath the oolemma (A 0 ), microtubules uniformly distributed throughout the ooplasm (A 00 ) and germinal vesicle stage chromatin (A 000 ). Pattern B: microfilaments and microtubules moderately aggregated in the ooplasm (B 0,B 00 ) and germinal vesicle stage chromatin (B 000 ). Pattern C: microfilaments and microtubules abnormally distributed in the ooplasm (C 0,C 00 ) and DNA condensed into a single clump (C 000 ). Bar represents 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

A.M. Luciano et al. / Cryobiology 59 (2009) 90 95 93 Table 1 The effect of cryopreservation method on the pattern of cytoskeleton organization after warming. Cryopreservation method No. of oocytes examined No. of oocytes (%) No. of oocytes (%) No. of oocytes (%) Pattern A Pattern B Pattern C Control 24 22 (91.7 c ) 2 (8.3 a ) Slow freezing 30 24 (80 b ) 6 (20 b ) Vitrification 24 4 (16.6 a ) 16 (66.8 c ) 4 (16.6) (A, normal; B, irregular; C, abnormal). a, b, c; values with different superscripts within columns are significantly different (p < 0.05). shown. Pattern A represents the correct distribution and organization of cytoskeletal elements in immature GV stage oocytes, characterized by microfilaments distributed homogeneously throughout the ooplasm, with a slightly greater concentration just beneath the oolemma (Fig. 1A 0 ), and by finely labeled microtubules distributed uniformly into the cytoplasm (Fig. 1A 00 ). Examples of oocytes showing irregular distribution of microfilaments and microtubules after thawing or warming is represented in pattern B, where cytoskeletal elements are irregularly diffused in the cytoplasm (Fig. 1B 0 and B 00 ). Abnormal distribution, (pattern C) was characterized by mostly aggregate microfilaments and microtubules, with clusters scattered in the ooplasm (Fig. 1C 0 and C 00 ), and DNA occasionally condensed into a single clump (Fig. 1C 000 ). As shown in Table 1, significantly fewer oocytes presented an irregular distribution of cytoskeleton (20% with pattern B) when cryopreserved by slow freezing, compared to vitrified oocytes (66.8%), while most of the slow cooled oocytes showed a normal pattern after thawing (80%). In contrast, the majority of oocytes preserved by vitrification showed pattern B (66.8%) or C (16.6%) after warming. However, both cryopreservation procedures induced a significant decrease in the percentage of oocytes showing a normal pattern, compared to the control. Functional status of communication between oocytes and cumulus cells after slow freezing and vitrification The percentages of cumulus oocyte complexes with a pattern of open, partially open and closed communications, evaluated in each group after thawing or warming, are shown in Table 2. Overall, both cryopreservation procedures were accompanied by a decrease of gap junction mediated communications, compared to the control. The interruption of communications was more pronounced in the vitrification group than in COCs cryopreserved by slow freezing (81% and 62.8%, respectively, p > 0.05). However, the percentage of oocytes with partial gap junction mediated communications in cumulus oocyte complexes cryopreserved by slow freezing, was significantly higher than for vitrified gametes. The total percentage of oocytes with open or partially open gap junctions was significantly higher in COCs cryopreserved by slow freezing than vitrified oocytes (37.2% and 19%, p < 0.05). Meiotic competence after slow freezing and vitrification procedures Table 3 presents the rates of nuclear maturation of oocytes cryopreserved by slow freezing and vitrification after warming, respectively, after 24 h of culture. Oocytes cryopreserved by the slow freezing protocol showed a significantly higher overall capability of resuming meiosis (intermediate and mature), than vitrified oocytes (39.2 vs. 26.8, p < 0.05). Moreover, ability to complete the first meiotic division was significantly higher in oocytes cryopreserved by slow freezing than in oocytes stored by vitrification (32.5% vs. 14.1%, respectively, p < 0.05). However, the meiotic competence of oocytes cryopreserved by either slow freezing or vitrification, was significantly lower than that of the control group. Discussion The present study demonstrates that immature cat oocytes were better cryopreserved by the slow freezing protocol. Compared to vitrification technique, slow freezing appears to be the less damaging method for GV stage COCs cryopreservation, since it efficiently protected the gap junction functionality between oocyte and cumulus cells and preserved both the cytoskeleton organization and meiotic competence of the cryopreserved gametes. To the best of our knowledge, this present study evaluated the effects of different cryopreservation protocols on the cytoskeleton organization and functional coupling of immature cat oocytes, for the first time. Microfilaments and microtubules, which play an Table 2 The effect of different methods of cryopreservation on oocyte cumulus cells coupling after warming. Cryopreservation method No. of oocytes examined No. of oocytes (%) No. of oocytes (%) No. of oocytes (%) Open Partial Close Control 55 25 (45.5 b ) 11 (20 b ) 19 (34.5 a ) Slow 43 6 (14 a ) 10 (23.2 b ) 27 (62.8 a,b ) Vitrified 42 5 (11.9 a ) 3 (7.1 a ) 34 (81 b ) a,b; values with different superscripts within columns are significantly different (p < 0.05). Table 3 The effect of different methods of cryopreservation on meiotic competence. Cryopreservation method No. of oocytes examined No. of oocytes (%) No. of oocytes (%) No. of oocytes (%) No. of oocytes (%) GV Intermediate Mature Degenerated Control 59 12 (20.3 a,b ) 1 (1.7 a ) 42 (71.2 c ) 4 (6.8 a ) Slow 74 10 (13.5 a ) 5 (6.7 a,b ) 24 (32.5 b ) 35 (47.3 b ) Vitrification 71 21 (29.6 b ) 9 (12.7 b ) 10 (14.1 a ) 31 (43.6 b ) a, b, c; values with different superscripts within columns are significantly different (p < 0.05).

94 A.M. Luciano et al. / Cryobiology 59 (2009) 90 95 important role during the oocyte maturation process, are very sensitive to the cooling process [7,27,32] and their integrity is essential for proper progression through meiosis [35,39]. In fact, tubulin is responsible for the constitution of the meiotic spindle and drives the proper alignment of chromosomes on the metaphase plate, while microfilaments are necessary for cortical granule migration and polar body expulsion, both crucial mechanisms for appropriate fertilization. Our morphological study demonstrated that the slow freezing method preserves the cytoskeleton organization better than vitrification. These findings are in agreement with the results of experiments on meiotic competence and are supported by the observation that the normal distribution of microfilaments and microtubules was associated with the progression through meiotic division, while an irregular or abnormal organization was related to a significantly lower meiotic capability after in vitro maturation. However, the integrity and cytoplasmic organization of oocytes are severely affected by both slow and ultra-rapid protocols. Our data confirm that the cooling process dramatically compromises the developmental capabilities of mammalian oocytes [2], notably in cats [26,31,43,44]. While immature oocyte cryopreservation is gaining attention as an option for preservation of fertility [2,28], one limiting factor is the presence of somatic cells. Several layers of cumulus cells surround the immature oocyte and these communicate with oocyte and with each other through gap junctions [6,22,24,25]. This functional coupling is essential to the transfer of nutrients and messenger molecules during oocyte growth and maturation [10,11]. Previous studies have demonstrated that there is a direct correlation between the persistence of functional coupling and the oocyte developmental potential after fertilization [8,22]. However, cryopreservation procedures have been reported to cause cumulus and corona cells loss and failure, in membrane integrity in immature mammalian oocytes [9,15,18,36,37,42]. In cats, Comizzoli et al. recently demonstrated that anisosmotic conditions exceeding the osmotic tolerance limit of immature oocytes during cryopreservation procedures, affect the structural and functional integrity of cumulus oocyte complexes [8]. Hyperosmotic or hyposmotic levels were detrimental to oocyte competence. In particular, hyperosmotic conditions caused a dramatic loss of cumulus oocyte communications. Our data confirmed previous findings, since both slow freezing and vitrification protocols caused a loss of functional coupling between somatic and germinal compartments. The percentage of open and partially open communications of frozen thawed or vitrified warmed immature cat oocytes, is lower than in freshly isolated oocytes. However, the percentage of partially open communications is higher in the slow freezing protocol compared to the vitrification procedure. Finally, results in terms of the meiotic competence of cryopreserved cat oocytes are still unsatisfactory, whichever method is adopted. Several hypotheses could contribute towards understanding the low efficiency of feline oocyte banking. Although cat oocytes are characterized by peculiar features, such as the high content of lipid droplets in the ooplasm, which complicates its cryopreservation [16], a possible explanation for our results could be the different osmotic challenge induced by the two protocols and the specific physiology of oocyte and cumulus cells investment. Oocytes and cumulus cells are different sized and require diverse freezing conditions in order to be preserved. Furthermore, cumulus cells naturally slow the penetration of CPAs and the output of water, and determine an uneven intracellular distribution of CPAs [14,17]. The slow freezing procedure brings a decrease in temperature at a controlled cooling rate in the presence of CPAs, which causes a gradual efflux of water from the cells. This procedure minimizes the osmotic shock and may prevent injuries to the oocyte cumulus cell complex. By contrast, vitrification leads to an extreme increase in viscosity in the presence of high concentration of CPAs, permitting the transition of aqueous solutions into a glassy state, bypassing the crystalline state [40]. However, the concentration of CPAs required for vitrification has been calculated as being 4 7 times higher than that required in slow freezing and may result in either osmotic or chemical toxicity [1,12]. For all of the above reasons, cryopreservation of immature oocytes after cumulus cells removal could represent an alternative, to overcome the critical structure of the immature female gamete, as recently demonstrated in cows. In this case, only one type of cell should be considered for the setting up of a cryopreservation protocol which accounts for a unique cellular surface-to-volume ratio. After warming, the subsequent embryonic development may be sustained by an appropriate culture system, which can support the absence of cumulus cells during oocyte maturation and fertilization, as demonstrated in cows [21,30]. In fact, a recent study demonstrated that the presence of exogenous cumulus cells positively affects oocyte maturation and fertilization in cats [13]. This further confirms our previous studies, demonstrating that companion exogenous cumulus cells (such as intact COCs or cell clumps) have a beneficial effect on resumption of in vitro nuclear maturation of denuded oocytes in cats [5] and cows [21]. In conclusion, our results demonstrate that cryopreservation by slow freezing is a suitable protocol for the banking of cat cumulus oocyte complexes, since it preserved the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence. 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