In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.     

Vitreous Cryopreservation of Human Preantral Follicles Encapsulated in Alginate Beads with Mini Mesh Cups

To completely avoid ice crystal formation and thus get a higher survival rate, vitrification methods have been commonly used for cryopreservation of oocytes and embryos. However, currently used vitrification methods for oocytes and embryos are not suitable for the cryopreservation of preantral follicles (PFs). In the present study, stainless steel mesh was fabricated into mini mesh cups to vitrify isolated PFs. Moreover, isolated follicles were encapsulated and then subjected to vitreous cryopreservation to facilitate in vitro culture/maturation of follicles after warming. The results showed that the percentages of viable follicles did not differ significantly between the vitrification group and fresh group soon after warming (81.25% vs. 85.29%, P>0.05) and after a 7-day culture period (77.78% vs. 83.33%, P>0.05). No difference in mean follicular diameter was observed between cryopreserved and fresh follicles when cultured in vitro. Transmission electron microscopic analysis revealed that vitreous cryopreservation could maintain the ultrastructure of follicles in alginate beads. In conclusion, the present vitrification method could efficiently cryopreserve isolated human ovarian follicles encapsulated by calcium alginate, which could be put into immediate use (in vitro culture/ maturation) after warming. However, more follicles and some detailed biochemical analyses are required to further investigate the effects of vitrification on the long-term growth of human encapsulated PFs.     

Effect of 7,8-Dihydroxyflavone as an Antioxidant on In Vitro Maturation of Oocytes and Development of Parthenogenetic Embryos in Pigs

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 μM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 μM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.     

Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes

Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.     

Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

L‐carnitine improves hydrogen peroxide‐induced impairment of nuclear maturation in porcine oocytes

We investigated the effect of oxidative stress induced by hydrogen peroxide (H₂O₂) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre‐culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H₂O₂, and intra‐oocyte H₂O₂ and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H₂O₂ supplementation, the DO group showed severe accumulation of H₂O₂ and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H₂O₂ stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H₂O₂ with L‐carnitine, a H₂O₂ scavenger. L‐carnitine decreased H₂O₂ and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L‐carnitine is useful for alleviating H₂O₂‐induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.     

胰岛素和白血病抑制因子对猪卵母细胞体外成熟和孤雌激活胚胎的影响

为探讨胰岛素(Insulin)和白血病抑制因子(Leukemia inhibit factor,LIF)对猪卵母细胞体外成熟(IVM)和猪孤雌激活胚胎(PAEs)的影响,在卵母细胞体外成熟或者胚胎培养基中添加Insulin和LIF,研究卵裂率和囊胚率的变化。结果:添加了5μg/mL Insulin后猪卵母细胞体外成熟效果显著提高,但成熟后孤雌激活发育能力与非添加组相近;而胚胎培养基中添加Insulin对孤雌胚的卵裂和囊胚的形成也没有明显促进作用;添加1 000 U/mL的LIF后,卵母细胞核成熟率没有明显提高,反而孤雌激活后囊胚率急剧下降,但对卵裂率以及囊胚总细胞数影响不大;在胚胎培养基中添加LIF后,孤雌胚的卵裂和囊胚形成并没有明显的提高。表明:Insulin对卵母细胞体外成熟有益,但是对孤雌胚胎的最佳处理程序还需要摸索;本文所采用的LIF处理对猪卵体外成熟以及孤雌胚胎体外发育没有帮助,还需要进一步研究其他浓度和处理程序对猪卵母细胞体外成熟和孤雌激活胚胎发育能力的影响。     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation. After oocyte transfer, embryonic development was diagnosed at 15 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested, HTF:BME, SOFaa, and TCM 199. In experiment II, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 (±9.6), 23.4 (±8.4), and 13.5 (±12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 ± 22.4) or equine (25.8 ± 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 ± 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.     

Effects of N‐acetyl‐cysteine and N‐acetyl‐cysteine‐amide Supplementation on In Vitro Matured Porcine Oocytes

This study was conducted to evaluate the effects of different concentrations of the antioxidant N‐acetyl‐cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC‐amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm ) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post‐fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.     

Expression and Activation of Mitogen-activated Protein Kinases in Matured Porcine Oocytes under Thermal Stress

In this study, we determined the expression and activation of p38 MAPK in matured porcine oocytes subjected to heat shock (HS). When MII oocytes were heated, only the phosphorylated p38 levels relative to the total p38 levels decreased (P < 0.01) after HS, but no clear relationship with HS treatments was observed in the ERK, JNK and p90^rsk expressions of matured oocytes. To confirm p38 activation in matured oocytes, immunocytochemical staining was performed to localize its expression and distribution in the ooplasm, and the results were largely consistent with previous Western blot analyses. Moreover, when matured oocytes were co-cultured with a P38 MAPK inhibitor, SB203580, for 4 h at 41.5 C, the activation of its immediate downstream substrate MAPKAPK-2 was not inhibited within any of the treatment groups. It appears that the MAPKAPK2 levels increased only under prolonged culture (HS4h and C4h) compared with the control group. In conclusion, p38 activity in porcine oocytes was decreased after exposure to HS and prolonged culture. These alterations of p38 and activation of MAPKAPK2 may be associated with porcine oocyte viability under HS conditions, and a potential cross-talk between p38 MAPK and other signaling cascades may exist, which warrants additional investigation.     

Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Prolonged exposure to hyaluronidase decreases the fertilization and development rates of fresh and cryopreserved mouse oocytes

Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.