Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.     

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.     

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly (P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher (P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group.In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.     

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca²⁺ chelator and mitochondrial Ca²⁺ uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P < 0.05) than those in the other vitrified groups (10.3–12.3% and 6.2–10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7–21.6% and 8.5–155%, respectively). When BAPTA-AM, an intracellular Ca²⁺ chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5–36.2% and 25.0–26.9%, respectively) were significantly higher (P < 0.05) than those in the absent vitrified groups (17.2–24.2% and 12.9–19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca²⁺ uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8–38.4% and 14.4–24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization.These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.     

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes

The effects of the presence or absence of cumulus cells and equilibration times (1 and 4 min) with cryoprotectant, and Taxol treatment before vitrification (0.5-5.0 microM) on development of in vitro matured porcine oocytes after vitrification were examined. Ethylene glycol (30%) and sucrose (0.5M) was used as a vitrification solution (39 degrees C), and cryotop was used for cryo-container. There was a significant relationship (F value: 6.077, P<0.05) in the rate of morphologically normal oocytes after vitrification between the equilibration times and the presence or absence of cumulus cells. The blastocyst rates were not significantly different between Taxol (1.4-5.5%) and non-treated control (8.8%). The results show that the optimal exposure time to achieve survival after vitrification depends on the presence or absence of cumulus cells, and that Taxol has no positive effect on the developmental capacity of vitrified, in vitro matured porcine oocytes.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.     

Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage

The susceptibility of in vitro matured (IVM) porcine oocytes to be fertilized in vitro after vitrification was investigated. IVM oocytes were cryopreserved by solid surface vitrification (SSV) or treated with cryoprotectants (toxicity control, TC). Control oocytes were not treated or vitrified. Live oocytes in the three groups were in vitro fertilized (IVF) and then cultured (IVC) for 6 days. In vitro maturation and IVC were performed under 5% or 20% O(2) tension. The percentage of live oocytes in the SSV group was lower than those in the control and TC groups. Fertilization rates after SSV were significantly lower than in the control group. Significantly fewer penetrated oocytes formed male pronuclei in the SSV group than in the control and TC groups. Cleavage rates were significantly lower in the SSV group than in the control and TC groups. Blastocyst formation rates in the control and TC groups were similar, whereas only a single embryo developed to the blastocyst stage from 113 oocytes after vitrification. Blastocyst formation rates in the control group and in the TC group were significantly higher under 5% O(2) IVC than under 20% O(2) IVC. Oxygen tension during IVM had no effect on embryo development. The glutathione (GSH) content of vitrified oocytes was significantly lower than in the controls. In contrast, the H(2)O(2) level was higher in vitrified oocytes than in control oocytes. Vitrification caused parthenogenetic activation in 44.9% of unfertilized oocytes. This significant increase in parthenogenetic activation along with significantly dropped GSH level in vitrified oocytes may explain the decreased ability of the SSV group to form male pronuclei. These factors might have contributed to the poor developmental competence of vitrified oocytes.     

Impact of anisosmotic conditions on structural and functional integrity of cumulus-oocyte complexes at the germinal vesicle stage in the domestic cat

During cryopreservation, the immature oocyte is subjected to anisosmotic conditions potentially impairing subsequent nuclear and cytoplasmic maturation in vitro. In preparation for cryopreservation protocols and to characterize osmotic tolerance, cat cumulus-oocyte complexes (COC) at the germinal vesicle (GV) stage were exposed for 15 min to sucrose solutions ranging from 100 to 2,000 mOsm and then examined for structural integrity and developmental competence in vitro. Osmolarities > or =200 and < or =750 mOsm had no effect on incidence of oocyte nuclear maturation, fertilization success, and blastocyst formation compared to control COC (exposed to 290 mOsm). This relatively high osmotic tolerance of the immature cat oocyte appeared to arise from a remarkable stability of the GV chromatin structure as well as plasticity in mitochondrial distribution, membrane integrity, and ability to maintain cumulus-oocyte communications. Osmolarities <200 mOsm only damaged cumulus cell membrane integrity, which contributed to poor nuclear maturation but ultimately had no adverse effect on blastocyst formation in vitro. Osmolarities >750 mOsm compromised nuclear maturation and blastocyst formation in vitro via disruption of cumulus-oocyte communications, an effect that could be mitigated through 1,500 mOsm by adding cytochalasin B to the hyperosmotic solutions. These results (1) demonstrate, for the first time, the expansive osmotic tolerance of the immature cat oocyte, (2) characterize the fundamental role of cumulus-oocyte communications when tolerance limits are exceeded, and (3) reveal an interesting hyperosmotic tolerance of the immature oocyte that can be increased two-fold by supplementation with cytochalasin B.