Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Pesquisa Veterinária Brasileira

Medeiros, Amanda M.²  Silva, Leandro S.²  Jimenez, Carolina R.³  Penitente-Filho, Jurandy M.¹  Torres, Ciro A.A.¹ 

[1] Universidade Federal de Viçosa, Viçosa, Brazil;UFV, Viçosa, Brazil;Universidade de São Paulo, Piracicaba, Brazil

关键词:  Antioxidants;    dimethylsulfoxide;    cryopreservation;    cryoprotectant;    oocyte;    granulosa cells;   

DOI  :  10.1590/S0100-736X2016000300010

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

:The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.Index Terms: Antioxidants; dimethylsulfoxide; cryopreservation; cryoprotectant; oocyte; granulosa cellsResumo:Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C). Após uma semana os fragmentos foram aquecidos e analisados. No experimento I, folículos pré-antrais foram observados morfologicamente para avaliação histológica (normal, degenerados e estádio de desenvolvimento). No experimento II, folículos pré-antrais foram mecanicamente isolados do tecido ovariano e examinados com o azul de trypan, observando mortos e vivos corados e não corados respetivamente. Os tratamentos a DSm, DSms e DST10m foram eficazes na preservação da morfologia in situ. No entanto, a viabilidade de folículos pré-antrais isolados após a vitrificação manteve-se elevada apenas no tratamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.Termos de Indexação: Antioxidantes; dimetilsulfóxido; criopreservação; crioprotetor; oócito; células granulosasIntroductionThe main problem of cryopreservation of oocytes is associated to the movement of water and intra or extracellular cryoprotectant during cryopreservation. Alternatively, the preantral follicles surface area in relation to the volume is large, providing excellent exchange of substances. Besides being smaller than the mature oocytes, these oocytes are less differentiated, possess fewer organelles, lack pellucida zone and cortical granules, besides being less metabolically active (Campebell et al. 1999). All these features are potentially beneficial to cryopreservation (Oktay et al. 1998) and thus preantral follicles are regarded as an important target of cryopreservation programs.Generally, the cryopreservation protocols comprise the steps of exposure to the cryoprotectant, cooling, storage, thawing and removal of the cryoprotectants. Exposure to the cryoprotectants, also known as equilibrium period, is the time required for of cryoprotectants penetration or infusion into cells and,or, tissues (Ernst et al. 2010). This is a very important stage and is strongly influenced by cryoprotectants concentration, time and temperature which is performed the exposure (Rodrigues et al. 2010). The vitrification cooling rate is extremely rapid, ranging around 20000 to 40000°C/min (Lin et al. 2008), and as a result, the water goes from liquid to a vitreous state without the formation of intracellular ice crystals (Yeoman et al. 2005). Finished cooling, the sample is stored, usually in liquid nitrogen (-196°C) until the moment from the thawing. During thawing, undesirable changes may occur in the cells, due to chemical (oxidation) or physical (recrystallization, volume changes) (Hui et al. 2004). Thus, the thawing time, the substances and concentrations used for the removal of cryoprotectants are important steps in the process of cryopreservation.Different cryoprotectants have been used in combination, with the possibility of optimizing protocols. Rodrigues et al. (2014) demonstrated that ovarian tissue can be frozen using intracellular cryoprotectant as ethyleneglycol, propanediol or dimethylsulfoxide, and can be combined with intra or extracellular cryoprotectant as sucrose in protocols of female gametes, acting as a buffer against osmotic stress during removal of the intracellular cryoprotectant (Demirci et al. 2003). Moreover, in the process of cryopreservation may occur increased of the reactive oxygen species (ROS) that can affect the balance between pro- and anti-oxidant factors in a biological system, leading to lipidic peroxidation (Romero et al. 1998) and, consequently, cellular death. The α-tocopherol reduces generation of free radicals and removes them when formed (Souza et al. 1999). The most important biological function of α- tocopherol is to protect cell membrane from lipidic peroxidation, avoiding irreversible damages and cellular death (Wang et al. 2002). Thus, since oxidative stress can be one of the factors involved in apoptosis and, or, follicular atresia, the use of antioxidants may contribute for the success of vitrification of preantral follicles.Therefore, the purpose of this study was (1) to evaluate the vitrification technique in bovine ovarian tissue, testing the effects of dimethylsulfoxide, sucrose and α-tocopherol and (2) to evaluate the effect the thawing with minimal essential medium with or without sucrose.Materials and MethodsCollection of ovaries.Ovaries (n=117) were collected in a slaughterhouse. In the experiment I, 66 ovaries were used to study the morphology of preantral follicles in situ. For Experiment II, 51 ovaries were used to evaluate the viability of isolated preantral follicles. All collected ovaries were washed in 70% alcohol for 10s and then rinsed twice in saline solution (NaCl 0.9%; Sigma/vetec® Brazil), supplemented with streptomycin sulfate (50mg/L; Sigma/Vetec® Brazil). Thereafter, ovaries were transported to the laboratory at 4°C, cut into fragments of approximately 9mm3 and assigned to nine treatments: a fresh control and eight vitrification treatments (Table 1). Table 1: Designation of treatments, fresh control and vitrified and thawed fragments * Vitrification solution in uppercase. Thawed solution in lowercase. Dm = Dimethylsulfoxide thawed with minimal essential medium (MEM); Dms = Dimethylsulfoxide thawed with MEM + sucrose; DSm = Dimethylsulfoxide + sucrose thawed with MEM; DSms = Dimethylsulfoxide + sucrose thawed with MEM + sucrose; DST5m = Dimethylsulfoxide + sucrose + α-tocopherol 5mmol/L thawed with MEM; DST5ms = Dimethylsulfoxide + sucrose + α-tocopherol 5mmol/L thawed with MEM + sucrose; DST10m = Dimethylsulfoxide + sucrose + α-tocopherol 10mmol/L thawed with MEM; DST10ms = Dimethylsulfoxide + sucrose + α-tocopherol 10mmol/L thawed with MEM + sucrose. Vitrification and thawing. Ovarian fragments were placed in cryotubes with 1.8 mL of vitrification solution, followed by an equilibration period of 5 min at room temperature, time sufficient for ovarian fragments are kept in touch with all components of diluent without causing toxicity. Then the tubes were immersed vertically in liquid nitrogen (-196°C) and stored for one week. After of one cryostorage week, all cryotubes were removed, exposed to room temperature (25°C) for 1 min, and then immersed in a water bath (37°C) until the vitrification solution was completely thawed. The cryoprotectant was removed from the ovarian cortex fragments in three washings with MEM or MEM with decreasing sucrose concentrations (0.50, 0.25, 0.0mol/L of sucrose), for 5 min each. Then, the fragments for histology were fixated in 4 % paraformaldehyde for 12 h.Histology. After fixation, the fragments were dehydrated in increasing concentrations of alcohol, diaphanized in xylene, embedded in paraffin and cut into sections (5μm thickness). The slides were stained with hematoxylin and eosin (Martinez-Madrid et al. 2004) and a total of 50 preantral follicles per slide were examined under an optical microscope (400x; Olympus BX60, Japan) in 30 replications (1500 preantral follicles per treatment).Classification of preantral follicles. Ovarian fragments (fresh control and vitrification treatments) were analyzed and classified as primordial follicles with spherical or ovoid oocytes completely surrounded by a single layer of flattened and some cuboid-shaped granulosa cells (GCs); the primary follicles were described as spherical oocytes with a single layer of cuboid-shaped GCs; and the secondary follicles as spherical oocytes with two or more layers of cuboid-shaped GCs. The qualitative evaluation of the follicles was based on the integrity of the oocyte and the GCs. The follicles were classified morphologically as: normal - follicles containing intact oocytes and GCs; type I degenerated follicles - follicles with cytoplasmic shrinkage and/or a pyknotic oocyte nucleus; type II degenerated follicles - follicles with oocyte degeneration accompanied by GCs disorganization (Basso & Esper 2002).Mechanical isolation and viability analysis. Ovarian fragments were finely cut with a scalpel blade and placed in 50mL tubes with 10mL of MEM plus 5% bovine serum albumin and mechanically dissociated by repeated pipetting (1000 and 500μL). Thereafter, the suspension was filtered twice through nylon mesh (300 and 106μm, respectively). In each 100μL of suspension medium containing isolated preantral follicles 5μL of trypan blue (0.4%; Sigma/Vetec®, Brazil) was added. Follicles were classified as viable or non-viable, when non-stained or blue-stained, respectively, under an inverted microscope (400x; Olympus, Japan). Fifty preantral follicles were observed per treatment with 30 replicates (1,500 preantral follicles per treatment).Statistical analysis.The data of the morphological (normal, type I and II degenerated) of preantral follicles were subjected to analysis of variance (f test, P<0.05), and means were compared by Tu

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