Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification

The purpose of this study was to determine the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the open pulled Straw method (OPS). We evaluated the effects of pre-treating the oocytes with 1 microM Taxol on chromosome organization, spindle morphology, cortical granule distribution and the ability of fertilized oocytes to develop to the blastocyst stage. After calf or cow oocyte vitrification without Taxol, significantly higher proportions of spindle abnormalities in the form of abnormal spindle structures or dispersed or decondensed chromosomes were observed compared to fresh control oocytes. In contrast, when we compared calf oocytes pre-treated with Taxol before vitrification with control calf oocytes, similar percentages of oocytes showing a normal spindle morphology were observed. The percentages of oocytes with a peripheral cortical granule (CG) distribution increased when the oocytes were pretreated with Taxol and vitrified, while oocytes vitrified without Taxol pre-treatment gave rise to higher cortical distribution percentages. Cleavage and blastocyst rates were significantly lower for vitrified versus untreated oocytes, both in cow and calf oocytes. Significantly higher cleavage rates were obtained when calf and cow oocytes were vitrified with Taxol. Pre-treatment with Taxol before cow oocyte vitrification yielded significantly higher blastocyst rates. Calf oocytes, however, were unable to develop to the blastocyst stage, irrespective of previous Taxol treatment. These results indicate that the pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by the cryopreservation process, and potentially improves the subsequent development of vitrified bovine oocytes. Summary sentence: Pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and potentially improves the development of vitrified bovine oocytes.     

Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000°C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over −180°C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction. Mol. Reprod. Dev. 51:53–58, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.Key words: oocyte, MII, vitrification, ultrastructure, TEM, human.     

Successful vitrification of bovine blastocysts on paper container

Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P < 0.05) than those obtained from non-vitrified blastocysts (38.0 and 32.7%) but were comparable (P > 0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos.     

Cryopreservation of immature bovine oocytes by vitrification in straws

The aim of this study was to cryopreserve by vitrification by ethylene glycol (EG) and dimethyl sulfoxide (DMSO) immature bovine oocytes in straws and to investigate the effects of vitrification on post-thaw oocyte maturation. A total of 575 cumulus oocyte complexes were obtained by follicle aspiration from 238 ovaries of cows slaughtered at a local abattoir. Following selection, oocytes with compacted cumulus cells and evenly granulated ooplasm were vitrified using one of the three different solutions with a non-vitrified group served as control. The first step vitrification solution contained 20% EG while the second step solution contained 40% EG+1M sucrose in a basic media used in group EG. Oocytes were matured in N-2-hidroxyethyl piperazine-N-2-ethanosulfonic acid (HEPES) buffered tissue culture medium (TCM) 199 for 24h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes were fixed following evaluation for polar body formation, stained with Giemsa solution and nuclear maturation was examined. The numbers of oocytes which were observed at Metaphase II (MII) stage were 41 (34.1%), 17 (14.9%), 29 (20.7%) and 78 (79.6%) in groups EG, DMSO, Mix and Control, respectively. Maturation rate distribution in group Mix was not statistically different when compared to maturation rate distributions in groups EG and DMSO (p>0.05). Differences between other groups were significant (p<0.001). However, better results were obtained in EG group compared to DMSO and mix groups. Maturation rates were lower in all treatment groups than the control group. The lowest maturation result was obtained in DMSO group. Maturation rate in group Mix was between maturation rates of EG and DMSO groups. Immature bovine oocytes can be vitrified in straws, but maturation success differs with the cryoprotectant and it seems that to obtain better maturation rates, new cryopreservation techniques specific for immature bovine oocytes are needed.     

Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol-based vitrification solution in unfertilized mouse oocytes

We investigated the characteristics of the changes in intracellular calcium (Ca2+) concentration ([Ca2+](i)) and the viability of the unfertilized mouse oocytes exposed to various concentrations of ethylene glycol (EG)-containing solutions or vitrification solutions. Oocytes exposed to EG (1, 5, 10, 20, and 40% (v/v)) exhibited a rapid and dose-dependent increase in [Ca2+](i). The survival rate was 100% when oocytes were exposed to the EG concentration up to 5% through 5 min, while all oocytes were dead within 3 min when exposed to 10, 20, or 40% EG. When extracellular Ca2+ was removed, increase in [Ca2+](i) at 10 and 20% EG was less than that at the same concentrations of EG with extracellular Ca2+. The survival rates of the oocytes exposed to 10, 20, and 40% EG at 3 min were 100, 97, and 0%, respectively. In the presence of 20 microM 1,2-bis(o-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator, a small increase in [Ca2+](i) exposed to 10, 20, and 40% EG was observed until 4 min. Subsequently prolonged elevation of the [Ca2+](i) was observed in the oocytes exposed to 40% EG but not with 10 and 20% EG. The survival rate of the oocytes, in the presence of 20 microM BAPTA-AM, exposed to 10 and 20% EG was 100% throughout 5 min, while the oocytes exposed to 40% EG were alive only for 3 min. Treatment by the vitrification solution with various concentrations of EG (10, 20, and 40%) caused a smaller increase in [Ca2+](i), while the survival rates were higher compared to those without vitrification solution at the same concentrations of EG. These data suggested that the sustained [Ca2+](i) rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore, the lowering of [Ca2+](i) in the oocytes exposed to the cryoprotectant may improve the viability of cryopreserved unfertilized oocytes.     

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.     

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.     

Calves born after open pulled straw vitrification of immature bovine oocytes

The aim of this study was to evaluate the developmental capacity of immature bovine oocytes after vitrification with 20% ethylene glycol (EG)+20% dimethyl sulfoxide (Me(2)SO) and 0.5M sucrose (SUC), by open pulled straw (OPS) technology. The effect of treatment with cytochalasin D before vitrification was also examined. No differences were observed in cleavage and blastocyst rates among the group vitrified without cytochalasin D treatment (Vitri) (49.0% and 6.1%) and that with cytochalasin D treatment before vitrification (CDVitri) (46.4% and 3.6%), but both were lower (P<0.05) than the unvitrified control group (85.1 and 45.9%). Calves were obtained after transfer of fresh and vitrified blastocysts from the Vitri group and after transfer of vitrified blastocysts from the CDVitri group. Cytochalasin D treatment does not improve the development of immature bovine vitrified oocytes. The results show that a small proportion of immature oocytes vitrified with this technology are fully competent to produce blastocysts, which may be transferred immediately or vitrified before transfer, and go on to develop healthy offspring.     

Embryo development,oocyte morphology,and kinetics of meiotic maturation in bovine oocytes exposed to 6-dimethylaminopurine prior to in vitro maturation

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 ± 12% of the oocytes were at the germinal vesicle stage compared to 97 ± 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 ± 18% were at MII stage, compared with 93 ± 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 ± 8% vs 89 ± 7%; P < 0.01), oocyte activation higher (11 ± 5% vs 2 ± 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 ± 7% vs 4 ± 4%; P > 0.05), compared with the control group. Cleavage was lower (61 ± 13% vs 81 ± 6%; P < 0.001), as well as day 8 blastocyst formation (17 ± 7% vs 36 ± 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development. Mol. Reprod. Dev. 50:334–344, 1998. © 1998 Wiley-Liss, Inc.     

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140–170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.     

Ultrastructure of the Parabasalid Protist Holomastigotoides

ABSTRACT. The ultrastructure of two species of Holomastigotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber, axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.     

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.     

Nylon mesh cryodevice for bovine mature oocytes,easily removable excess vitrification solution

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-μm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-μm group (39%) was higher than that in the 77-μm group (28%), and the yield in the 57-μm group (31%) was the intermediate. The 37-μm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-μm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.     

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoproteclant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P <.05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P <.05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that (1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and (2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.     

Effects of cooling and warming rates during vitrification on fertilization of in vitro-matured bovine oocytes

The purpose of this study was to clarify the relationship of cooling rates (CR) and warming rates (WR) during vitrification with postwarming viability of in vitro-matured bovine oocytes. In Experiment 1, oocytes were vitrified in a solution containing 7.2 M ethylene glycol and 1.0 M sucrose by use of open-pulled glass capillaries with five different outer diameters and were warmed by placement of the capillaries into 0.25 M sucrose solution. The capillaries of 2000-, 1400-, 1000-, 630-, and 440-mm diameters provided CR of 2000, 3000, 5000, 8000, and 12,000 degrees C/min and WR of 5000, 8000, 17,000, 33,000, and 62,000 degrees C/min, respectively. In oocytes vitrified in capillaries of 1400-mm diameter (CR, 3000 degrees C/min; WR, 8000 degrees C/min), the morphological survival rate (86% of vitrified), penetration rate (79% of inseminated), and normal fertilization rate (69% of penetrated) were higher or tended to be higher than those in the other vitrification groups. In Experiment 2, oocytes cooled at 2000, 3000, or 12,000 degrees C/min were warmed at 8000 degrees C/min, and oocytes cooled at 3000 degrees C/min were warmed at 5000, 8000, or 33,000 degrees C/min. Among these CR-WR combinations, cooling of oocytes at 3000 degrees C/min regardless of the WR resulted in higher postwarming survival. These results indicate that survival of in vitro-matured bovine oocytes after vitrification and subsequent warming is improved by a slightly rapid cooling rate in open-pulled glass capillaries compared to that obtained in conventional straws.     

不同群系小鼠胚胎玻璃化冷冻保存技术的研究

利用 EFS40 ,二步法对近交系 C5 7BL/6、DBA/2和远交群 ICR小鼠囊胚玻璃化冷冻保存 ,并对冷冻后胚胎体内、外发育效果进行比较。结果表明 ,相同条件下鲜胚经培养 ,近交系 C5 7BL /6小鼠的囊胚发育率 ( 93% )与 ICR( 1 0 0 % )相比差异不显著 ( P>0 .0 5 ) ;而两近交系的囊胚孵化率明显低于 ICR( P<0 .0 1 )。 C5 7BL/6、DBA/2小鼠囊胚冷冻后发育率 ( 93% ,96% )和孵化率 ( 5 2 % ,46% )与各自对照组 ( 1 0 0 % ,1 0 0 %和 61 % ,62 % )相比均无显著差异 ( P>0 .0 5 ) ;并且与 ICR冷冻组发育率和孵化率 ( 94% ,5 3% )之间也无显著差异 ( P>0 .0 5 )。两近交系冻胚移植妊娠与各自对照组和 ICR冷冻组比较均无显著差异 ( P>0 .0 5 )。C5 7BL/6胚胎移植产仔率 ( 35 % )与对照组 ( 5 1 % )之间差异显著 ( P<0 .0 1 ) ,而 DBA/2胚胎移植产仔率 ( 4 7% )与对照组和 ICR冷冻组 ( 39% ,5 8% )相比差异不显著 ( P>0 .0 5 )。     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.