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Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture
Cryobiology 60 (2010) 229–234
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Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture q N. Cocchia a,*, F. Ciani b, M. Russo a, R. El Rass b, I. Rosapane a, L. Avallone b, G. Tortora a, R. Lorizio a a b
Department of Veterinary Clinic Sciences, University of Naples Federico II, Via F. Delpino, 1 – 80137 Naples, Italy Department of Biological Structures, Functions and Technologies, University of Naples Federico II, Via F. Delpino, 1 – 80137 Naples, Italy
a r t i c l e
i n f o
Article history: Received 30 July 2009 Accepted 12 January 2010 Available online 15 January 2010 Keywords: Oocyte Vitrification Germinal vesicle Cryoprotectant Dimethylsulfoxide Assisted reproductive techniques In vitro embryo production Blastocyst Domestic cat
a b s t r a c t Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P < 0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P < 0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P < 0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC. Ó 2010 Elsevier Inc. All rights reserved.
Introduction A great deal of progress has been made in recent years toward the development of assisted reproductive techniques (ART) for species conservation [32,31]. Most wild felid species are classified as rare, vulnerable or endangered due to poaching and habitat loss. It has been demonstrated that the domestic cat can serve as a successful recipient of embryos from closely related, small, non-domestic cats, as shown by the birth of Indian desert cat kittens and African wildcat kittens after IVF-derived embryo transfers in female domestic cats [12,31]. The domestic cat is often used as a model from which ART can be developed in felidae species [30,36]. Gamete cryopreservation represents an important tool for the development of efficient ART, and oocyte cryopreservation could facilitate the preservation of genetic resources in domestic and wild animals. Vitrification is an alternative procedure that has been investigated for the preser-
q Work supported by research funds of Veterinary Clinical department and research funds of Specialty School of Theriogenology, Naples University. * Corresponding author. Fax: +39 81 2536019. E-mail address: [email protected] (N. Cocchia).
0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.01.003
vation of mammalian embryos as well as oocytes. This procedure shortens the period of exposure to cryoprotectant solutions through immediate plunging of material into liquid nitrogen, achieving rapid cooling rates [37]. This process results in material solidification without ice crystal formation, minimising injury to cumulus-oocyte complexes (COC). Conflicting results have been obtained after oocyte cryopreservation at different maturation stages [7]. The freezing of immature oocytes could expand oocyte sources for ART programmes and experimental studies. Cryopreservation of oocytes at this stage could induce injury to oocyte membranes or cumulus cells [13,33], which play an important role in oocyte maturation via the gap junction [2,6,8,11,25]. Through gap junctions, cumulus cells facilitate the passage of nutrients, inhibitory substances and small molecules from follicle cells to the oocyte and are essential for oocyte growth and differentiation [4,21]. In addition, the presence of multiple layers of tightly compacted cumulus cells around immature oocytes may alter the rate and extent of dehydration, which, in turn, could worsen the degree of cytoplasmic damage [21]. Cryopreservation steps can be detrimental to the functionality of cumulus-oocyte communication in mouse [34], cow [22], buffalo [35] and porcine oocytes [40]. The immature cat oocyte
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shows an unusual resistance to osmotic stress can be used to investigate the fundamental role of cumulus-oocyte communications when tolerance limits are exceeded [5]. Cat oocytes have peculiar physical characteristics that increase the difficulty of developing successful cryopreservation methods compared to gametes of other species [20]. Domestic cat oocytes have high lipid droplet content in the ooplasm [14]; thus, oocyte permeability to cryoprotectant solutions may be lower than in oocytes of other species [19,23,27,38]. Only a few studies have investigated cat oocyte cryopreservation, and the few successes were only obtained for mature oocyte cryopreservation [20]. In the first study [21], mature and immature oocytes were cryopreserved by slow cooling, but no blastocysts were obtained after in vitro fertilisation (IVF). In the second study [26], matured cat oocytes were vitrified in straws and, after IVF with frozen–thawed epididymal spermatozoa, the first two blastocysts were obtained [26]. In a recent study, the first attached cat blastocysts were obtained from matured cat oocytes that were vitrified using a cryo-loop system [24]. Another very recent study reported blastocyst production from vitrified GV cat oocytes exposed to resveratrol (Res) in order to compact the decondensed chromatin contained in the large GV of cat oocytes [5]. Despite the importance of cryoprotectant penetration to avoid intracellular ice crystal formation, the greater cryoprotectant concentrations in vitrification solutions are toxic and may cause osmotic injury [3]. Suggestions to minimise the toxicity of vitrification solutions include the use of less toxic substances, association with different cryoprotectants, previous exposure to lesser concentrations of cryoprotectants and reduction of exposure time to vitrification solutions [16,37]. The major penetrating cryoprotectants for oocyte cryopreservation are ethylene glycol (EG), glycerol (GLY), dimethylsulfoxide (Me2SO), propylene glycol (PrOH) and acetamide [28]. Another common permeating CPA, 1,2-ethanediol (EG) [28], is also suitable for less permeable immature oocytes, as demonstrated in cattle [1]. A recent study investigating bovine oocyte vitrification demonstrated that a solution of EG + Me2SO is a favourable cryoprotectant combination, as the Me2SO (MW = 78.13) molecule is smaller and consequently more permeable than the glycerol molecule (MW = 92.1) [41]. The aim of this study was to assess the efficiency of immature domestic cat oocyte vitrification in OPSs (Open Pulled Straws) using a mixture of GE and Me2SO and evaluating the survival rate of COC cells and embryo development rates after vitrification/ thawing, IVM and IVF by fresh epididymal cat sperm. Materials and methods All chemicals were purchased from Sigma–Aldrich (Milan, Italy) unless otherwise stated.
Oocyte vitrification After collection, COC were washed twice in HSOF and vitrified after a three-step exposure to vitrification solution. The vitrification solution used in this study was Holding medium (HM: HSOF with 6 mg/ml BSA) and Sucrose medium (SM: 1 M sucrose dissolved in HM) with the following cryoprotectant mixture: dimethylsulfoxide, Me2SO (Sigma D2650) (16.5% – 2.32 M final concentration) and ethylene glycol, EG (Sigma E9129) (16.5% – 2.94 M final concentration). Cryoprotectants were added at room temperature. The holding and vitrification solutions were placed into a 4-well dish as follows: 800 ll HM in wells 1 and 2, 850 ll HM with 75 ll Me2SO and 75 ll EG (Vitrification Solution 1; VS1) in well 3 and 670 ll SM with 165 ll EG and 165 ll Me2SO (Vitrification Solution 2; VS2) in well 4. The 4-well dish (4WD) was warmed for an additional 5–10 min. OPSs were marked (date, code, and other pertinent information) at their thick ends. The oocytes were placed (up to 30) into well 1 and were transferred after approximately 1 min to well 2. This well was used for oocyte storage during stepwise vitrification. A 20-ll droplet from well 4 was transferred close to the centre right side of the bottom of the 4WD. A set of two oocytes was transferred to well 3 with a minimal volume of medium [37]. Subsequently, the oocytes were transferred in the smallest possible volume into the 20-ll droplet of medium prepared in the centre of the 4WD. Rapidly, within 30 s, the oocytes were recovered into a glass pipette and transferred into an OPS [3]. The straws were immersed into LN2 with a continuous rapid movement, passing through the vapour very quickly. The straws were placed into 15-ml blue-cap Falcon centrifuge tubes that were punctured by an 18G needle to ensure LN2 penetration into the small holder, allowing them to float vertically. Before each new vitrification cycle, the medium in the 20-ll droplet was changed [37]. Oocytes were preserved at least 4 weeks before warming. Oocytes were warmed by immersing OPSs in well 1 containing 800 ll HM and 400 ll SM for 30 s and then washing three times in well 2 containing 800 ll HM and 400 ll SM, in well 3 containing 800 ll HM and 200 ll SM and in well 4 containing 800 ll HM. OPSs were taken out of LN2 after approximately 30 s and rapidly immersed into well 1 at an angle of 30–45° (measured from the horizontal position), remaining completely submerged in the vitrified liquid. Immediately after immersion, oocytes were transferred into wells 2, 3 and 4, with 5-min incubations in each well. All oocytes were placed into the appropriate dish, randomly divided and immediately stained with cFDA/Trypan blue (Experimental 1) or incubated in IVM medium in the same conditions for 24 h (Experimental 2).
Oocyte collection Viability staining of COC cells (CFDA/Trypan blue staining) Cat oocytes were recovered from ovaries obtained in the ASL hospital of Naples after ovariectomies of 45 mixed-breed domestic queens (Felis catus), between 1 and 8 years of age, from September 2008 to April 2009. Ovaries were kept in Dulbecco’s PBS supplemented with 75 lg/ml kanamycin at room temperature until oocyte collection. Within 3 h of excision, ovaries were minced with a scalpel blade in a 35 0.7-mm Petri dish, flushed by HEPES synthetic oviductal fluid (HSOF), and cumulus-oocyte complexes (COC) were collected. Grade I and II oocytes [20] were washed three times in HSOF and then randomly divided into 3 groups: (1) immediately stained with CFDA + Trypan blue, (2) subjected to IVM and (3) vitrified immediately (see below method).
The compound 5-carboxyfluorescein diacetate (cFDA) (Sigma Chemical Co.; C4916) is an esterase substrate that can be converted by non-specific esterases of living cells from a non-polar, non-fluorescent substance to a polar, fluorescent dye (CF). The cFDA molecule passes through cell membranes passively. The presence of CF in cells indicates plasma membrane integrity and can be used as general indicator of cell viability. In contrast, the Trypan blue molecule exclusively stains cells with broken plasma membranes. Trypan blue has been used in vital staining of various tissues. The reactivity of this dye is based on the fact that the chromophore is negatively charged and does not react with cells that have any damaged membrane. Staining facilitates the visualisation of cell
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morphology. Live (viable) cells do not take up the Trypan blue dye, while dead (non-viable) cells do take up this dye [29]. The assay was performed as previously described [10]. Fresh (Control 1) and vitrified/thawing oocytes (Experimental 1) were stained with cFDA by immersion into SOFaaBSA (Synthetic Oviductal Fluid with 10 ll/ml essential and 10 ll/ml non-essential amino acids and 6 mg/ml bovine serum albumin) with 11 mg/ml of cFDA for 15 min at 38.5 °C. After being washed with H–SOF, the oocytes were placed on a slide under a cover slip mounted with paraffin at a concentration of 5–10 oocytes per slide. Since the Trypan blue staining solution (Sigma Chemical Co.; T8154) was perfused onto the slide, excess dye was removed using a sterile paper. The slides were evaluated using an optical microscope. Blue staining of cells following CFDA/Trypan blue staining indicates that the cells were damaged. COC were classified as viable COC (viable oocytes and cumulus cells, CFDA stained) (Fig 1a and b) and non-viable COC (non-viable oocytes and cumulus cells, Trypan blue stained (Fig. 1c) or non-viable oocytes or cumulus cells, Trypan blue stained with viable, CFDA stained counterpart (Fig. 1d and e). In vitro maturation Fresh (Control 2) and vitrified/thawing oocytes (Experimental 2) were cultured (25–50 oocytes/ml) in synthetic oviductal fluid
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with amino acids and 6 mg/ml BSA (SOFaaBSA) containing 0.1 IU of porcine follicle-stimulating hormone and porcine luteinising hormone (pFSH-LH; Pluset, Laboratorios Calier, Barcelona, Spain) supplemented with 25 ng/ml EGF, 25 ml/ml insulin–transferrin– sodium selenite (ITS) and 1.2 mmol/l L-cysteine in a 5% CO2 incubator at 38.5 °C for 24 h [25]. After this 24-h period, oocytes were evaluated at a stereomicroscope for survival, and those showing cytoplasmic degeneration were discarded. Fresh and vitrified in vitro matured COC were immediately in vitro fertilised (Control 2 and Experimental 2, respectively). Sperm collection, in vitro fertilisation and culture In vitro matured COC were in vitro fertilised with fresh epididymal spermatozoa recovered from epididymides obtained in the ASL hospital of Naples after orchiectomy of 12 mixed-breed domestic cats (Felis catus). Each collected testicular–epididymis complex was immediately plunged in Dulbecco’s PBS with 0.0036% (w/v) Na-pyruvate, 0.1% (w/v) glucose, 0.0066% (w/v) Na-benzylpenicillin, 0.01% (w/v) streptomycin sulphate and 4 mg/ml BSA (fraction V, Sigma) in a Dewar vessel at 20–24 °C and transported to the laboratory within 3 h of removal. The epididymides were separated from the testes, and visible blood vessels were removed to isolate epididymides and proximal deferent
Fig. 1. COCs stained with cFDA/Trypan blue to assess viability: viable COCs (a); non-viable COCs (b); non-viable COCs with non-viable cumulus cells and viable oocytes (c); non-viable COCs with viable cumulus cells and non-viable oocytes (d).
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ducts. The caudal portion of each epididymis was placed in 1 ml Tris extender (3.025% Tris(hydroxymethyl)aminomethane, 1.7% citric acid, 1.25% fructose, 0.06% Sodium Benzyl penicillin, 0.1% streptomycin sulphate) in a Petri dish (35 10 mm; Falcon) and was dissected with a scalpel blade to release the spermatozoa. After 10 min of incubation at 38 °C, the epididymal tissue was removed with forceps, and the medium was collected in a Falcon tube and centrifuged at 700g for 6 min. The supernatant was removed, and the pellet concentration was evaluated by phase-contrast microscopy (400) using a Bürker chamber. Then, the pellet was resuspended in an adequate volume of fresh IVF medium consisting of SOFaaBSA (6 mg/ml) to obtain a final concentration of 10 106 spermatozoa/ml. Motile sperm were selected by a swim-up assay performed in a 15-ml Falcon tube for 2 h in a 5% CO2 incubator. After incubation, the supernatant was collected, the concentration was re-evaluated, the volume was adjusted to obtain a final concentration of 1 106 motile spermatozoa/ml, and the mixture was supplemented with 20 mg/ml penicillamine–hypotaurine–epinephrine (PHE) and 10 mg/ml heparin. For IVF, 30–40 COCs were transferred into a 4WD containing 500 ml of IVF suspension and cultured for 18 h at 38.5 °C in 5% CO2, 5% O2 and 90% N2. After co-culturing with sperm, cumulus cells were removed into a 0.25% trypsin solution for 60 s. Oocytes were washed once in HSOF containing 10% FBS to inactivate trypsin and then washed twice in HSOF. Denuded oocytes were evaluated again to exclude from the culture degenerated oocytes that were not detected after thawing because of the presence of cumulus cells. Presumptive zygotes were cultured (20–30/500 ml) in SOFaaBSA (16 mg/ml) at 38.5 °C in 5% CO2, 5% O2 and 90% N2. Cleavage was determined after 24 h of IVC, and embryos were cultured in the same medium supplemented with 10% FBS until day 8. The medium was refreshed every 4 days. Experimental design and statistical analysis The experiment was performed ten times in a 16-week period. The collected oocytes were divided into vitrified (564 COC) and control (328 COC) groups. The oocytes were then randomly divided and immediately stained with CFDA/Trypan blue (Control 1 and Experimental 1, 100 and 150 COC, respectively) or incubated in IVM medium in the same conditions for 24 h (Control 2 and Experimental 2, 228 and 414 COC, respectively). Subsequently, fresh and vitrified in vitro matured COC were immediately in vitro fertilised. The percentage of degenerated COC after vitrification was defined as the number of COC with non-viable cumulus cells or non-viable oocytes, as identified by cFDA/Trypan blue staining. The percentage of degenerated oocytes in the control group was determined in the same manner. The cleavage rate was calculated as the total number of in vitro fertilised COC, and the blastocyst rate on day 8 was calculated as the total number of in vitro fertilised COC and total number of cleaved embryos. Developmental competence was defined as the number of blastocysts produced relative to the total number of cleaved embryos. Data were compared using a chi-square test performed using Graphpad in Stat Version for Windows XP and Medcalc version 7.3.01. Probabilities of less than 0.05 were considered statistically significant. Results Vitrified GV oocytes revealed a high incidence of cumulus cell damage (Table 1). The embryo developmental efficiency was assessed after IVM and IVF with fresh epididymal sperm, evaluating
Table 1 Viability of cat oocytes vitrified in OPS at GV stage, cFDA/Tripan blue staining. Total Viable COCs COCs COCs (group)
N°
Fresh (Control 1) 100 Vitrified/thawing 150 (Experimental 1)
Non-viable COCs
Viable COCs Non-viable N° (%) COCs N° (%) 89(89) A 5(5) A 68(45.3) B 28(18.6) B
Non-viable cumulus N° (%) 2(2) A 43(28.6) B
Non-viable oocytes N° (%) 4(4) 11(7.3)
A, B; values with different superscripts within columns are significantly different (p < 0.05).
Table 2 Embryo development of cat oocytes vitrified in OPS at GV stage using a association of EG and DMSO. COCs (group) Fresh (Control 2) Vitrified/thawing (Experimental 2)
COCs number 228 A 414 B
Cleaved N° (%) 110(48.2) A 77(18.6) B
Morule N° (%) 76(33.3) A 43(10.4) B
Blastocyst N° (%) 47(20.6) A 18(4.3) B
Cleavage/ blastocyst (%) (42.7) A (23.4) B
A, B; values with different superscripts within columns are significantly different (p < 0.05).
the percentage of cleavage and blastocyst production. The results showed that vitrified GV oocytes had a low incidence of in vitro embryo production (IVEP). The percentage of non-viable COC was significantly higher (P < 0.05) in vitrified oocytes than in control oocytes (Table 1), while the cleavage and morula blastocyst rates were significantly lower (P < 0.05) for vitrified oocytes than for controls (Table 2). The blastocyst rate (Fig. 2) was higher (P < 0.05) for control oocytes than for vitrified oocytes, and developmental competence was higher (P < 0.05) for non-vitrified oocytes (Table 2). Discussion Immature cat oocyte vitrification in OPSs allows researchers to obtain competent oocytes that are able to develop in vitro until the blastocyst stage. This is the first study in which this stage of development was reached in vitro using cat oocytes vitrified at the GV stage. Oocyte cryopreservation is still considered an experimental technique in all species, because adequate rates of survival, fertilisation and embryo development of frozen oocytes have yet to be determined. The percentage of surviving COC obtained in the present study (45.3%) after vitrification/thawing of immature cat oocytes using the OPS technique and the cleavage rate (18.6%) after IVF with fresh epididymal spermatozoa are lower than those reported by Merlo et al. [24] and Murakami et al. [26] after vitrification of in vitro matured cat oocytes in OPSs (50.2% viable and 32.2% cleaved) and in straws (50.8% viable and 29.7% cleaved), respectively. Our values are also lower than those found by Luvoni and Pellizzari [21] after slow freezing in EG (84.1% viable and 38.7% cleaved), but after IVF of these cryopreserved oocytes, it has not been possible to obtain embryos that are able to reach the blastocyst stage. Despite the low percentage of cleaved in vitro fertilised vitrified immature cat oocytes that develop to the blastocyst stage (23.4%) obtained in this study, these results should be sufficient to encourage new studies in order to optimise the vitrification procedure. The oocyte freezing efficiency depends from many factors, including cryoprotectant type, freezing method and cooling and thawing rates, each of which may be responsible for oocyte cryodamage [20].
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Fig. 2. Cat blastocysts developed from vitrified GV oocytes after in vitro maturation and in vitro fertilisation by fresh epididymal cat sperm.
In the present study, the efficiency of immature domestic cat oocyte vitrification in OPSs using a mixture of EG and Me2SO has been evaluated by the rate of COC cell survival and the embryo development rate after vitrification/thawing. The cooling speed is dependent on the vitrification solution volume, such that smaller sample volumes correspond to higher cooling rates. Direct contact with liquid nitrogen also increases the cooling rate. To reduce the sample volume in our experiment, the OPSs have been charged by a pulled glass pipette using stereomicroscopy. To avoid ice formation, the vitrification technique in this study uses high cryoprotectant (CPA) concentrations, which have been previously described as toxic to cells [9]. Evaluation of cryoprotectant solution toxicity is, therefore, very important for vitrification. COCs have some characteristics that are adverse to cryoprotectant equilibrium between intra- and extracellular environments. The oocyte is a large single cell with a small surface/volume ratio and is surrounded by several cumulus cell layers, which reduce cryoprotectant entry into the cell [23]. Factors that influence cryoprotectant passage velocity through the cell membrane and entry into the ooplasm are important for oocyte vitrification. These problems notwithstanding, an appropriate phased composition of CPA mitigates the toxic and osmotic consequences of highly concentrated CPAs. Thus, a mixture of CPAs can decrease individual specific toxicities. The most common mixture used is EG, Me2SO and sucrose. To optimise results, in addition to an appropriate selection of CPAs, these agents should be also used at the lowest possible concentration [17,39]. Luvoni and Pellizzari [21] demonstrated that exposing immature domestic cat oocytes to 1.5 M EG at room temperature (25 °C) has no adverse effect on the ability to resume nuclear maturation, but they observed poor fertilisation and developmental competence of the oocytes after thawing. Another common permeating CPA used for mammalian oocytes is 1,2-propanediol (PrOH), which has low toxicity and favourable ability to support maturation and fertilisation after thawing [18]. Comizzoli [4] recently demonstrated that EG is more detrimental to cat oocytes than PrOH at room temperature and 0 °C. In the present study, we used EG and Me2SO as a cryoprotectant combination for the following reasons: recent studies have demonstrated that short exposure time to a vitrification solution containing EG + Me2SO with different concentration gradients supports cryoprotectant equilibrium between intra- and extracellular environments in immature bovine oocytes [23]; EG is suitable for less permeable immature oocytes, as demonstrated in goats and cattle
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[1] (in fact, EG (MW = 62.07) penetrates better than PrOh (MW = 76.09)); a vitrification procedure with immediate plunging of material into liquid nitrogen achieves rapid cooling rates and a shorter CPA exposure time at room temperature and 0 °C, reducing the toxic effects, as observed by Comizzoli [4]. The obtained data demonstrated, for the first time, that is possible to obtain cat blastocysts from immature cat oocytes that were cryopreserved by exposure for 30 s in cryoprotectant solution with 16.5% ethylene glycol (2.94 M) and 16.5% Me2SO (2.32 M) followed by vitrification in OPSs. These results demonstrated that feline immature oocytes vitrified in OPSs develop to the blastocyst stage and confirm that the selection of an appropriate cryoprotectant mixture and sample volume reduction are two simple but important parameters in the study of a successful vitrification method for feline species. Immature oocytes at the germinal vesicle (GV) stage have lower membrane permeability and stability than mature oocytes [1,15]. Cryopreservation of oocytes at this stage could induce membrane injury of oocytes or cumulus cells [13,33], which plays an important role in oocyte maturation via the gap junction [2,6,8,11,25]. Cryobanking of immature oocytes could provide a high number of COC that are good sources for in vitro embryo production and transfer (IVEP-ET), assisted reproductive programmes and research studies. However, further studies are needed to assess other ultrastructural changes in cat oocytes and to optimise vitrification procedures. Furthermore, banking of immature oocyte could provide an adequate source of karyoplasts and cytoplasts for the application of GVT germinal vesicle transplantation [19]. The results that we have obtained in the domestic cat, which is a model for other felids, encourage studies investigating assisted reproduction programs for endangered exotic feline species. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cryobiol.2010.01.003. References [1] Y. Agca, J. Liu, A.T. Peter, E.S. Critser, J.K. Critser, Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics, Mol. Reprod. Dev. 49 (1998) 408–415. [2] E. Anderson, D.F. Albertini, Gap junctions between the oocytes and companion follicle cells in the mammalian ovary, J. Cell Biol. 71 (1976) 680–686. [3] A. Arav, D. Shehu, M. Mattioli, Osmotic and cytotoxic study of vitrification of immature bovine oocytes, J. Reprod. Fertil. 99 (1993) 353–358. [4] P. Comizzoli, D.E. Wildt, B.S. Pukazhenthi, Effect of 1,2-propanediol versus 1,2ethanediol on subsequent oocyte maturation, spindle integrity, fertilization, and embryo development in vitro in the domestic cat, Biol. Reprod. 71 (2004) 598–604. [5] P. Comizzoli, D.E. Wildt, B.S. Pukazhenthi, In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes, Reprod. Domest. Anim. 44 (Suppl. 2) (2009) 269–274. [6] J.J. Eppig, Oocyte–somatic cell communication in the ovarian follicles of mammals, Semin. Dev. Biol. 5 (1994) 51–59. [7] A. Eroglu, T.L. Toth, M. Toner, Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes, Fertil. Steril. 69 (1998) 944–957. [8] C.F. Fagbohun, S.M. Downs, Metabolic coupling and ligand stimulated meiotic maturation in the mouse oocyte cumulus cell complex, Biol. Reprod. 45 (1991) 851–859. [9] S. Fuller, S. Paynter, Fundamentals of cryobiology in reproductive medicine, Reprod. Biomed. Online 9 (2004) 680–691. [10] R.C. Ganassin, N.C. Bols, Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix, Methods Cell Sci. 22 (2–3) (2000) 147–152. [11] R.B. Gilchrist, L.J. Ritter, D.T. Armstrong, Oocyte–somatic cell interactions during follicle development in mammals, Anim. Reprod. Sci. 82 (2004) 431–446. [12] M.C. Gomez, C.E. Pope, A. Giraldo, L.A. Lyons, R.F. Harris, A.L. King, A. Cole, R.A. Godke, B.L. Dresser, Birth of African wildcat cloned kittens born from domestic cats, Cloning Stem Cells 6 (3) (2004) 247–258.
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[28] P. Pedro, E. Yokoyama, S.E. Zhu, N. Yoshida, D.M. Valdez Jr., M. Tanaka, K. Edashige, M. Kasai, Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants, J. Reprod. Dev. 51 (2005) 235– 246. [29] H.J. Phillips, J.E. Terryberry, Counting actively metabolizing tissue cultured cells, Exp. Cell. Res. 13 (1957) 341–347. [30] C.E. Pope, Embryo technology in conservation efforts for endangered felids, Theriogenology 53 (2000) 163–174. [31] C.E. Pope, M.A. McRae, B.R. Plair, G.L. Keller, B.L. Dresser, In vitro ed in vivo development of embryos produced by in vitro maturation and in vitro fertilization of cat oocytes, J. Reprod. Fertil. (Suppl. 51) (1997) 69–82. [32] C.E. Pope, M.C. Gomez, B.L. Dresser, In vitro embryo production and transfer in domestic and non-domestic cats, Theriogenology 66 (2006) 1518–1524. [33] B.S. Pukazhenthi, R. Spindler, D.E. Wildt, Short term exposure to room temperature disrupts the meiotic spindle of in vitro matured domestic cat oocytes, Biol. Reprod. 58 (Suppl. 1) (1998) 201. [34] C.J. Ruppert-Lingham, S.J. Paynter, J. Godfrey, B.J. Fuller, R.W. Shaw, Membrane integrity and development of immature murine cumulus-oocyte complexes following slow cooling to 60 °C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling, Cryobiology 52 (2) (2006) 219–227. [35] G.T. Sharma, K. Loganathasamy, Effect of meiotic stages during in vitro maturation on the survival of vitrified-warmed buffalo oocytes, Vet. Res. Commun. 31 (2007) 881–893. [36] R.E. Spindler, E.G. Crichton, Y. Agca, N. Loskutoff, J. Crister, D.K. Gardner, D.E. Wildt, Improved felid embryo development by group culture is maintained with heterospecific companions, Theriogenology 66 (1) (2006) 82–92. [37] G. Vajta, Vitrification of the oocytes and embryos of domestic animals, Anim. Reprod. Sci. 61 (2000) 357–364. [38] J. Van der Elst, Oocyte freezing: here to stay?, Hum Reprod. Update 9 (2003) 463–470. [39] N.A. Wani, A.K. Misra, S.N. Maurya, Maturation rates of vitrified-thawed immature buffalo (Bubalus bubalis) oocytes: effect of different types of cryoprotectants, Anim. Reprod. Sci. 84 (2004) 327–335. [40] B. Wu, J. Tong, S.P. Leibo, Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation, Mol. Reprod. Dev. 54 (1999) 388–395. [41] C. Yamada, R. Sim~oes, A.C. Nicacio, W.B. Feitosa, M.E. Ortiz D’A´ vila Assumpc, J.A. Visintin, Immature bovine oocyte cryopreservation: Comparison of different associations with ethylene, glycol, glycerol and dimethylsulfoxide, Anim. Reprod. Sci. 99 (2007) 384–388.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture q N. Cocchia a,*, F. Ciani b, M. Russo a, R. El Rass b, I. Rosapane a, L. Avallone b, G. Tortora a, R. Lorizio a a b
Department of Veterinary Clinic Sciences, University of Naples Federico II, Via F. Delpino, 1 – 80137 Naples, Italy Department of Biological Structures, Functions and Technologies, University of Naples Federico II, Via F. Delpino, 1 – 80137 Naples, Italy
a r t i c l e
i n f o
Article history: Received 30 July 2009 Accepted 12 January 2010 Available online 15 January 2010 Keywords: Oocyte Vitrification Germinal vesicle Cryoprotectant Dimethylsulfoxide Assisted reproductive techniques In vitro embryo production Blastocyst Domestic cat
a b s t r a c t Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P < 0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P < 0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P < 0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC. Ó 2010 Elsevier Inc. All rights reserved.
Introduction A great deal of progress has been made in recent years toward the development of assisted reproductive techniques (ART) for species conservation [32,31]. Most wild felid species are classified as rare, vulnerable or endangered due to poaching and habitat loss. It has been demonstrated that the domestic cat can serve as a successful recipient of embryos from closely related, small, non-domestic cats, as shown by the birth of Indian desert cat kittens and African wildcat kittens after IVF-derived embryo transfers in female domestic cats [12,31]. The domestic cat is often used as a model from which ART can be developed in felidae species [30,36]. Gamete cryopreservation represents an important tool for the development of efficient ART, and oocyte cryopreservation could facilitate the preservation of genetic resources in domestic and wild animals. Vitrification is an alternative procedure that has been investigated for the preser-
q Work supported by research funds of Veterinary Clinical department and research funds of Specialty School of Theriogenology, Naples University. * Corresponding author. Fax: +39 81 2536019. E-mail address: [email protected] (N. Cocchia).
0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.01.003
vation of mammalian embryos as well as oocytes. This procedure shortens the period of exposure to cryoprotectant solutions through immediate plunging of material into liquid nitrogen, achieving rapid cooling rates [37]. This process results in material solidification without ice crystal formation, minimising injury to cumulus-oocyte complexes (COC). Conflicting results have been obtained after oocyte cryopreservation at different maturation stages [7]. The freezing of immature oocytes could expand oocyte sources for ART programmes and experimental studies. Cryopreservation of oocytes at this stage could induce injury to oocyte membranes or cumulus cells [13,33], which play an important role in oocyte maturation via the gap junction [2,6,8,11,25]. Through gap junctions, cumulus cells facilitate the passage of nutrients, inhibitory substances and small molecules from follicle cells to the oocyte and are essential for oocyte growth and differentiation [4,21]. In addition, the presence of multiple layers of tightly compacted cumulus cells around immature oocytes may alter the rate and extent of dehydration, which, in turn, could worsen the degree of cytoplasmic damage [21]. Cryopreservation steps can be detrimental to the functionality of cumulus-oocyte communication in mouse [34], cow [22], buffalo [35] and porcine oocytes [40]. The immature cat oocyte
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shows an unusual resistance to osmotic stress can be used to investigate the fundamental role of cumulus-oocyte communications when tolerance limits are exceeded [5]. Cat oocytes have peculiar physical characteristics that increase the difficulty of developing successful cryopreservation methods compared to gametes of other species [20]. Domestic cat oocytes have high lipid droplet content in the ooplasm [14]; thus, oocyte permeability to cryoprotectant solutions may be lower than in oocytes of other species [19,23,27,38]. Only a few studies have investigated cat oocyte cryopreservation, and the few successes were only obtained for mature oocyte cryopreservation [20]. In the first study [21], mature and immature oocytes were cryopreserved by slow cooling, but no blastocysts were obtained after in vitro fertilisation (IVF). In the second study [26], matured cat oocytes were vitrified in straws and, after IVF with frozen–thawed epididymal spermatozoa, the first two blastocysts were obtained [26]. In a recent study, the first attached cat blastocysts were obtained from matured cat oocytes that were vitrified using a cryo-loop system [24]. Another very recent study reported blastocyst production from vitrified GV cat oocytes exposed to resveratrol (Res) in order to compact the decondensed chromatin contained in the large GV of cat oocytes [5]. Despite the importance of cryoprotectant penetration to avoid intracellular ice crystal formation, the greater cryoprotectant concentrations in vitrification solutions are toxic and may cause osmotic injury [3]. Suggestions to minimise the toxicity of vitrification solutions include the use of less toxic substances, association with different cryoprotectants, previous exposure to lesser concentrations of cryoprotectants and reduction of exposure time to vitrification solutions [16,37]. The major penetrating cryoprotectants for oocyte cryopreservation are ethylene glycol (EG), glycerol (GLY), dimethylsulfoxide (Me2SO), propylene glycol (PrOH) and acetamide [28]. Another common permeating CPA, 1,2-ethanediol (EG) [28], is also suitable for less permeable immature oocytes, as demonstrated in cattle [1]. A recent study investigating bovine oocyte vitrification demonstrated that a solution of EG + Me2SO is a favourable cryoprotectant combination, as the Me2SO (MW = 78.13) molecule is smaller and consequently more permeable than the glycerol molecule (MW = 92.1) [41]. The aim of this study was to assess the efficiency of immature domestic cat oocyte vitrification in OPSs (Open Pulled Straws) using a mixture of GE and Me2SO and evaluating the survival rate of COC cells and embryo development rates after vitrification/ thawing, IVM and IVF by fresh epididymal cat sperm. Materials and methods All chemicals were purchased from Sigma–Aldrich (Milan, Italy) unless otherwise stated.
Oocyte vitrification After collection, COC were washed twice in HSOF and vitrified after a three-step exposure to vitrification solution. The vitrification solution used in this study was Holding medium (HM: HSOF with 6 mg/ml BSA) and Sucrose medium (SM: 1 M sucrose dissolved in HM) with the following cryoprotectant mixture: dimethylsulfoxide, Me2SO (Sigma D2650) (16.5% – 2.32 M final concentration) and ethylene glycol, EG (Sigma E9129) (16.5% – 2.94 M final concentration). Cryoprotectants were added at room temperature. The holding and vitrification solutions were placed into a 4-well dish as follows: 800 ll HM in wells 1 and 2, 850 ll HM with 75 ll Me2SO and 75 ll EG (Vitrification Solution 1; VS1) in well 3 and 670 ll SM with 165 ll EG and 165 ll Me2SO (Vitrification Solution 2; VS2) in well 4. The 4-well dish (4WD) was warmed for an additional 5–10 min. OPSs were marked (date, code, and other pertinent information) at their thick ends. The oocytes were placed (up to 30) into well 1 and were transferred after approximately 1 min to well 2. This well was used for oocyte storage during stepwise vitrification. A 20-ll droplet from well 4 was transferred close to the centre right side of the bottom of the 4WD. A set of two oocytes was transferred to well 3 with a minimal volume of medium [37]. Subsequently, the oocytes were transferred in the smallest possible volume into the 20-ll droplet of medium prepared in the centre of the 4WD. Rapidly, within 30 s, the oocytes were recovered into a glass pipette and transferred into an OPS [3]. The straws were immersed into LN2 with a continuous rapid movement, passing through the vapour very quickly. The straws were placed into 15-ml blue-cap Falcon centrifuge tubes that were punctured by an 18G needle to ensure LN2 penetration into the small holder, allowing them to float vertically. Before each new vitrification cycle, the medium in the 20-ll droplet was changed [37]. Oocytes were preserved at least 4 weeks before warming. Oocytes were warmed by immersing OPSs in well 1 containing 800 ll HM and 400 ll SM for 30 s and then washing three times in well 2 containing 800 ll HM and 400 ll SM, in well 3 containing 800 ll HM and 200 ll SM and in well 4 containing 800 ll HM. OPSs were taken out of LN2 after approximately 30 s and rapidly immersed into well 1 at an angle of 30–45° (measured from the horizontal position), remaining completely submerged in the vitrified liquid. Immediately after immersion, oocytes were transferred into wells 2, 3 and 4, with 5-min incubations in each well. All oocytes were placed into the appropriate dish, randomly divided and immediately stained with cFDA/Trypan blue (Experimental 1) or incubated in IVM medium in the same conditions for 24 h (Experimental 2).
Oocyte collection Viability staining of COC cells (CFDA/Trypan blue staining) Cat oocytes were recovered from ovaries obtained in the ASL hospital of Naples after ovariectomies of 45 mixed-breed domestic queens (Felis catus), between 1 and 8 years of age, from September 2008 to April 2009. Ovaries were kept in Dulbecco’s PBS supplemented with 75 lg/ml kanamycin at room temperature until oocyte collection. Within 3 h of excision, ovaries were minced with a scalpel blade in a 35 0.7-mm Petri dish, flushed by HEPES synthetic oviductal fluid (HSOF), and cumulus-oocyte complexes (COC) were collected. Grade I and II oocytes [20] were washed three times in HSOF and then randomly divided into 3 groups: (1) immediately stained with CFDA + Trypan blue, (2) subjected to IVM and (3) vitrified immediately (see below method).
The compound 5-carboxyfluorescein diacetate (cFDA) (Sigma Chemical Co.; C4916) is an esterase substrate that can be converted by non-specific esterases of living cells from a non-polar, non-fluorescent substance to a polar, fluorescent dye (CF). The cFDA molecule passes through cell membranes passively. The presence of CF in cells indicates plasma membrane integrity and can be used as general indicator of cell viability. In contrast, the Trypan blue molecule exclusively stains cells with broken plasma membranes. Trypan blue has been used in vital staining of various tissues. The reactivity of this dye is based on the fact that the chromophore is negatively charged and does not react with cells that have any damaged membrane. Staining facilitates the visualisation of cell
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morphology. Live (viable) cells do not take up the Trypan blue dye, while dead (non-viable) cells do take up this dye [29]. The assay was performed as previously described [10]. Fresh (Control 1) and vitrified/thawing oocytes (Experimental 1) were stained with cFDA by immersion into SOFaaBSA (Synthetic Oviductal Fluid with 10 ll/ml essential and 10 ll/ml non-essential amino acids and 6 mg/ml bovine serum albumin) with 11 mg/ml of cFDA for 15 min at 38.5 °C. After being washed with H–SOF, the oocytes were placed on a slide under a cover slip mounted with paraffin at a concentration of 5–10 oocytes per slide. Since the Trypan blue staining solution (Sigma Chemical Co.; T8154) was perfused onto the slide, excess dye was removed using a sterile paper. The slides were evaluated using an optical microscope. Blue staining of cells following CFDA/Trypan blue staining indicates that the cells were damaged. COC were classified as viable COC (viable oocytes and cumulus cells, CFDA stained) (Fig 1a and b) and non-viable COC (non-viable oocytes and cumulus cells, Trypan blue stained (Fig. 1c) or non-viable oocytes or cumulus cells, Trypan blue stained with viable, CFDA stained counterpart (Fig. 1d and e). In vitro maturation Fresh (Control 2) and vitrified/thawing oocytes (Experimental 2) were cultured (25–50 oocytes/ml) in synthetic oviductal fluid
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with amino acids and 6 mg/ml BSA (SOFaaBSA) containing 0.1 IU of porcine follicle-stimulating hormone and porcine luteinising hormone (pFSH-LH; Pluset, Laboratorios Calier, Barcelona, Spain) supplemented with 25 ng/ml EGF, 25 ml/ml insulin–transferrin– sodium selenite (ITS) and 1.2 mmol/l L-cysteine in a 5% CO2 incubator at 38.5 °C for 24 h [25]. After this 24-h period, oocytes were evaluated at a stereomicroscope for survival, and those showing cytoplasmic degeneration were discarded. Fresh and vitrified in vitro matured COC were immediately in vitro fertilised (Control 2 and Experimental 2, respectively). Sperm collection, in vitro fertilisation and culture In vitro matured COC were in vitro fertilised with fresh epididymal spermatozoa recovered from epididymides obtained in the ASL hospital of Naples after orchiectomy of 12 mixed-breed domestic cats (Felis catus). Each collected testicular–epididymis complex was immediately plunged in Dulbecco’s PBS with 0.0036% (w/v) Na-pyruvate, 0.1% (w/v) glucose, 0.0066% (w/v) Na-benzylpenicillin, 0.01% (w/v) streptomycin sulphate and 4 mg/ml BSA (fraction V, Sigma) in a Dewar vessel at 20–24 °C and transported to the laboratory within 3 h of removal. The epididymides were separated from the testes, and visible blood vessels were removed to isolate epididymides and proximal deferent
Fig. 1. COCs stained with cFDA/Trypan blue to assess viability: viable COCs (a); non-viable COCs (b); non-viable COCs with non-viable cumulus cells and viable oocytes (c); non-viable COCs with viable cumulus cells and non-viable oocytes (d).
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ducts. The caudal portion of each epididymis was placed in 1 ml Tris extender (3.025% Tris(hydroxymethyl)aminomethane, 1.7% citric acid, 1.25% fructose, 0.06% Sodium Benzyl penicillin, 0.1% streptomycin sulphate) in a Petri dish (35 10 mm; Falcon) and was dissected with a scalpel blade to release the spermatozoa. After 10 min of incubation at 38 °C, the epididymal tissue was removed with forceps, and the medium was collected in a Falcon tube and centrifuged at 700g for 6 min. The supernatant was removed, and the pellet concentration was evaluated by phase-contrast microscopy (400) using a Bürker chamber. Then, the pellet was resuspended in an adequate volume of fresh IVF medium consisting of SOFaaBSA (6 mg/ml) to obtain a final concentration of 10 106 spermatozoa/ml. Motile sperm were selected by a swim-up assay performed in a 15-ml Falcon tube for 2 h in a 5% CO2 incubator. After incubation, the supernatant was collected, the concentration was re-evaluated, the volume was adjusted to obtain a final concentration of 1 106 motile spermatozoa/ml, and the mixture was supplemented with 20 mg/ml penicillamine–hypotaurine–epinephrine (PHE) and 10 mg/ml heparin. For IVF, 30–40 COCs were transferred into a 4WD containing 500 ml of IVF suspension and cultured for 18 h at 38.5 °C in 5% CO2, 5% O2 and 90% N2. After co-culturing with sperm, cumulus cells were removed into a 0.25% trypsin solution for 60 s. Oocytes were washed once in HSOF containing 10% FBS to inactivate trypsin and then washed twice in HSOF. Denuded oocytes were evaluated again to exclude from the culture degenerated oocytes that were not detected after thawing because of the presence of cumulus cells. Presumptive zygotes were cultured (20–30/500 ml) in SOFaaBSA (16 mg/ml) at 38.5 °C in 5% CO2, 5% O2 and 90% N2. Cleavage was determined after 24 h of IVC, and embryos were cultured in the same medium supplemented with 10% FBS until day 8. The medium was refreshed every 4 days. Experimental design and statistical analysis The experiment was performed ten times in a 16-week period. The collected oocytes were divided into vitrified (564 COC) and control (328 COC) groups. The oocytes were then randomly divided and immediately stained with CFDA/Trypan blue (Control 1 and Experimental 1, 100 and 150 COC, respectively) or incubated in IVM medium in the same conditions for 24 h (Control 2 and Experimental 2, 228 and 414 COC, respectively). Subsequently, fresh and vitrified in vitro matured COC were immediately in vitro fertilised. The percentage of degenerated COC after vitrification was defined as the number of COC with non-viable cumulus cells or non-viable oocytes, as identified by cFDA/Trypan blue staining. The percentage of degenerated oocytes in the control group was determined in the same manner. The cleavage rate was calculated as the total number of in vitro fertilised COC, and the blastocyst rate on day 8 was calculated as the total number of in vitro fertilised COC and total number of cleaved embryos. Developmental competence was defined as the number of blastocysts produced relative to the total number of cleaved embryos. Data were compared using a chi-square test performed using Graphpad in Stat Version for Windows XP and Medcalc version 7.3.01. Probabilities of less than 0.05 were considered statistically significant. Results Vitrified GV oocytes revealed a high incidence of cumulus cell damage (Table 1). The embryo developmental efficiency was assessed after IVM and IVF with fresh epididymal sperm, evaluating
Table 1 Viability of cat oocytes vitrified in OPS at GV stage, cFDA/Tripan blue staining. Total Viable COCs COCs COCs (group)
N°
Fresh (Control 1) 100 Vitrified/thawing 150 (Experimental 1)
Non-viable COCs
Viable COCs Non-viable N° (%) COCs N° (%) 89(89) A 5(5) A 68(45.3) B 28(18.6) B
Non-viable cumulus N° (%) 2(2) A 43(28.6) B
Non-viable oocytes N° (%) 4(4) 11(7.3)
A, B; values with different superscripts within columns are significantly different (p < 0.05).
Table 2 Embryo development of cat oocytes vitrified in OPS at GV stage using a association of EG and DMSO. COCs (group) Fresh (Control 2) Vitrified/thawing (Experimental 2)
COCs number 228 A 414 B
Cleaved N° (%) 110(48.2) A 77(18.6) B
Morule N° (%) 76(33.3) A 43(10.4) B
Blastocyst N° (%) 47(20.6) A 18(4.3) B
Cleavage/ blastocyst (%) (42.7) A (23.4) B
A, B; values with different superscripts within columns are significantly different (p < 0.05).
the percentage of cleavage and blastocyst production. The results showed that vitrified GV oocytes had a low incidence of in vitro embryo production (IVEP). The percentage of non-viable COC was significantly higher (P < 0.05) in vitrified oocytes than in control oocytes (Table 1), while the cleavage and morula blastocyst rates were significantly lower (P < 0.05) for vitrified oocytes than for controls (Table 2). The blastocyst rate (Fig. 2) was higher (P < 0.05) for control oocytes than for vitrified oocytes, and developmental competence was higher (P < 0.05) for non-vitrified oocytes (Table 2). Discussion Immature cat oocyte vitrification in OPSs allows researchers to obtain competent oocytes that are able to develop in vitro until the blastocyst stage. This is the first study in which this stage of development was reached in vitro using cat oocytes vitrified at the GV stage. Oocyte cryopreservation is still considered an experimental technique in all species, because adequate rates of survival, fertilisation and embryo development of frozen oocytes have yet to be determined. The percentage of surviving COC obtained in the present study (45.3%) after vitrification/thawing of immature cat oocytes using the OPS technique and the cleavage rate (18.6%) after IVF with fresh epididymal spermatozoa are lower than those reported by Merlo et al. [24] and Murakami et al. [26] after vitrification of in vitro matured cat oocytes in OPSs (50.2% viable and 32.2% cleaved) and in straws (50.8% viable and 29.7% cleaved), respectively. Our values are also lower than those found by Luvoni and Pellizzari [21] after slow freezing in EG (84.1% viable and 38.7% cleaved), but after IVF of these cryopreserved oocytes, it has not been possible to obtain embryos that are able to reach the blastocyst stage. Despite the low percentage of cleaved in vitro fertilised vitrified immature cat oocytes that develop to the blastocyst stage (23.4%) obtained in this study, these results should be sufficient to encourage new studies in order to optimise the vitrification procedure. The oocyte freezing efficiency depends from many factors, including cryoprotectant type, freezing method and cooling and thawing rates, each of which may be responsible for oocyte cryodamage [20].
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Fig. 2. Cat blastocysts developed from vitrified GV oocytes after in vitro maturation and in vitro fertilisation by fresh epididymal cat sperm.
In the present study, the efficiency of immature domestic cat oocyte vitrification in OPSs using a mixture of EG and Me2SO has been evaluated by the rate of COC cell survival and the embryo development rate after vitrification/thawing. The cooling speed is dependent on the vitrification solution volume, such that smaller sample volumes correspond to higher cooling rates. Direct contact with liquid nitrogen also increases the cooling rate. To reduce the sample volume in our experiment, the OPSs have been charged by a pulled glass pipette using stereomicroscopy. To avoid ice formation, the vitrification technique in this study uses high cryoprotectant (CPA) concentrations, which have been previously described as toxic to cells [9]. Evaluation of cryoprotectant solution toxicity is, therefore, very important for vitrification. COCs have some characteristics that are adverse to cryoprotectant equilibrium between intra- and extracellular environments. The oocyte is a large single cell with a small surface/volume ratio and is surrounded by several cumulus cell layers, which reduce cryoprotectant entry into the cell [23]. Factors that influence cryoprotectant passage velocity through the cell membrane and entry into the ooplasm are important for oocyte vitrification. These problems notwithstanding, an appropriate phased composition of CPA mitigates the toxic and osmotic consequences of highly concentrated CPAs. Thus, a mixture of CPAs can decrease individual specific toxicities. The most common mixture used is EG, Me2SO and sucrose. To optimise results, in addition to an appropriate selection of CPAs, these agents should be also used at the lowest possible concentration [17,39]. Luvoni and Pellizzari [21] demonstrated that exposing immature domestic cat oocytes to 1.5 M EG at room temperature (25 °C) has no adverse effect on the ability to resume nuclear maturation, but they observed poor fertilisation and developmental competence of the oocytes after thawing. Another common permeating CPA used for mammalian oocytes is 1,2-propanediol (PrOH), which has low toxicity and favourable ability to support maturation and fertilisation after thawing [18]. Comizzoli [4] recently demonstrated that EG is more detrimental to cat oocytes than PrOH at room temperature and 0 °C. In the present study, we used EG and Me2SO as a cryoprotectant combination for the following reasons: recent studies have demonstrated that short exposure time to a vitrification solution containing EG + Me2SO with different concentration gradients supports cryoprotectant equilibrium between intra- and extracellular environments in immature bovine oocytes [23]; EG is suitable for less permeable immature oocytes, as demonstrated in goats and cattle
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[1] (in fact, EG (MW = 62.07) penetrates better than PrOh (MW = 76.09)); a vitrification procedure with immediate plunging of material into liquid nitrogen achieves rapid cooling rates and a shorter CPA exposure time at room temperature and 0 °C, reducing the toxic effects, as observed by Comizzoli [4]. The obtained data demonstrated, for the first time, that is possible to obtain cat blastocysts from immature cat oocytes that were cryopreserved by exposure for 30 s in cryoprotectant solution with 16.5% ethylene glycol (2.94 M) and 16.5% Me2SO (2.32 M) followed by vitrification in OPSs. These results demonstrated that feline immature oocytes vitrified in OPSs develop to the blastocyst stage and confirm that the selection of an appropriate cryoprotectant mixture and sample volume reduction are two simple but important parameters in the study of a successful vitrification method for feline species. Immature oocytes at the germinal vesicle (GV) stage have lower membrane permeability and stability than mature oocytes [1,15]. Cryopreservation of oocytes at this stage could induce membrane injury of oocytes or cumulus cells [13,33], which plays an important role in oocyte maturation via the gap junction [2,6,8,11,25]. Cryobanking of immature oocytes could provide a high number of COC that are good sources for in vitro embryo production and transfer (IVEP-ET), assisted reproductive programmes and research studies. However, further studies are needed to assess other ultrastructural changes in cat oocytes and to optimise vitrification procedures. Furthermore, banking of immature oocyte could provide an adequate source of karyoplasts and cytoplasts for the application of GVT germinal vesicle transplantation [19]. The results that we have obtained in the domestic cat, which is a model for other felids, encourage studies investigating assisted reproduction programs for endangered exotic feline species. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cryobiol.2010.01.003. References [1] Y. Agca, J. Liu, A.T. Peter, E.S. Critser, J.K. Critser, Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics, Mol. Reprod. Dev. 49 (1998) 408–415. [2] E. Anderson, D.F. Albertini, Gap junctions between the oocytes and companion follicle cells in the mammalian ovary, J. Cell Biol. 71 (1976) 680–686. [3] A. Arav, D. Shehu, M. Mattioli, Osmotic and cytotoxic study of vitrification of immature bovine oocytes, J. Reprod. Fertil. 99 (1993) 353–358. [4] P. Comizzoli, D.E. Wildt, B.S. Pukazhenthi, Effect of 1,2-propanediol versus 1,2ethanediol on subsequent oocyte maturation, spindle integrity, fertilization, and embryo development in vitro in the domestic cat, Biol. Reprod. 71 (2004) 598–604. [5] P. Comizzoli, D.E. Wildt, B.S. Pukazhenthi, In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes, Reprod. Domest. Anim. 44 (Suppl. 2) (2009) 269–274. [6] J.J. Eppig, Oocyte–somatic cell communication in the ovarian follicles of mammals, Semin. Dev. Biol. 5 (1994) 51–59. [7] A. Eroglu, T.L. Toth, M. Toner, Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes, Fertil. Steril. 69 (1998) 944–957. [8] C.F. Fagbohun, S.M. Downs, Metabolic coupling and ligand stimulated meiotic maturation in the mouse oocyte cumulus cell complex, Biol. Reprod. 45 (1991) 851–859. [9] S. Fuller, S. Paynter, Fundamentals of cryobiology in reproductive medicine, Reprod. Biomed. Online 9 (2004) 680–691. [10] R.C. Ganassin, N.C. Bols, Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix, Methods Cell Sci. 22 (2–3) (2000) 147–152. [11] R.B. Gilchrist, L.J. Ritter, D.T. Armstrong, Oocyte–somatic cell interactions during follicle development in mammals, Anim. Reprod. Sci. 82 (2004) 431–446. [12] M.C. Gomez, C.E. Pope, A. Giraldo, L.A. Lyons, R.F. Harris, A.L. King, A. Cole, R.A. Godke, B.L. Dresser, Birth of African wildcat cloned kittens born from domestic cats, Cloning Stem Cells 6 (3) (2004) 247–258.
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