Effect of prooxidant agents added at the morula/blastocyst stage on bovine embryo development,cell death and glutathione content

Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.     

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos.     

Intracytoplasmic glutathione concentration and the role of beta-mercaptoethanol in preimplantation development of bovine embryos

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of beta-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 microM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 microM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 x 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.     

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.     

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.     

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.     

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.     

Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes

The low number of embryos produced from in vitro matured, fertilized, and cultured (IVM-IVF-IVC) oocytes of prepubertal goat is mainly due to a low incidence of sperm head decondensation at fertilization (Martino et al., 1995: Theriogenology 43:473-485; Mogas et al., 1997: Theriogenology 48:815-829). Thiol compounds stimulate glutathione (GSH) synthesis and improve the rates of male pronucleus (MPN) formation and embryo development. The present study was carried out to determine whether supplementation of the IVM medium with 100 microM of cysteamine, 100 microM of beta-mercaptoethanol, 0.57 mM of cysteine, and 0.57 mM cystine might improve the embryo development and intracellular GSH level of prepubertal goat oocytes. After 27 hr post IVM, a sample of oocytes was frozen and the intracytoplasmic GSH content was evaluated by spectrophotometry. IVM-oocytes were inseminated with fresh semen and cultured in SOF medium. Only the addition of cysteamine to IVM media significantly improved the percentage of the morula plus blastocyst yield compared to the control group (oocytes matured in absence of thiol compounds) (22.2 vs. 6.4%, respectively; P < 0.05). The percentage of expanded blastocysts in cysteamine and control groups was 13.0 and 2.6%, respectively, and the mean cell number per blastocyst was 86.8 and 60.5, respectively. None of the other thiol compounds studied significantly improved the percentage of embryos obtained. It has been demonstrated that prepubertal goat oocytes synthesize GSH during IVM and that thiol compounds increase this GSH synthesis. In conclusion, only the addition of 100 microM of cysteamine to the maturation medium improves embryo development from prepubertal goat oocytes although all the thiol compounds used in this study increased intracellular GSH content.     

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.     

Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 microM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three features were observed from the 8-cell stage in treated embryos, and blastomeres with apoptotic features appeared more numerous in treated than in untreated embryos. Ultrastructural evidence of apoptosis occurred with a comparable distribution pattern as apoptotic features detected by fluorescence microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre-implantation embryos.     

Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin

In preliminary experiments, the treatment of donor somatic cells with beta-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitro-development of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P < 0.05) effect was found after the combined treatment of cloned embryos with Hb (1 microg/ml) and ME (10 microM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142-154 vs. 123 cells/embryo), inner cell mass (ICM) (39-41 vs. 27), and trophectoderm (TE) (103-114 vs. 98), and the ratio of ICM to TE cell number (0.26-0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056-0.069). When embryos cloned with ME-treated cells were cultured in Hb + ME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with Hb + ME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in Hb + ME-containing medium yielded the optimal results for promoting the production of blastocysts with improved quality.     

Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.     

Improved survival of vitrified porcine embryos after partial delipation through chemically stimulated lipolysis and inhibition of apoptosis

Mechanical removal of intracellular lipids has been the most effective approach to increase the cryosurvival of porcine embryos. In this experiment, we tested the hypotheses that the cryosurvival of porcine embryos can be improved after partial delipation through chemically stimulated lipolysis and that the survival can be further improved by inhibition of apoptosis. Porcine embryos were produced in vitro using sow oocytes. On Day 5 of embryonic development, embryos were cultured in the presence of 10 microM forskolin for 24h. On Day 6 blastocysts were vitrified using an open pulled straw (OPS) method and warmed blastocysts were cultured 18 h for them to recover. A caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used at 20 microM during vitrification and subsequent culture to inhibit apoptosis. A 2 x 2 x 2 factorial design experiment was conducted to examine the effect of chemical delipation, vitrification and apoptosis inhibition. We also measured the lipolytic activity of porcine embryos cultured with or without forskolin. Chemical delipation increased the cryosurvival of porcine embryos compared to the controls (71.2+/-2.8% versus 37.1+/-5.1%). Apoptosis inhibition increased the ability of blastocysts to fully recover (23.8+/-3.1% versus 14.6+/-4.3%). However, there was no interaction between chemical delipation and apoptosis inhibition. Lipolytic agent treatment increased the lipolytic activity of porcine blastocysts. In conclusion, cryosurvival of porcine embryos was improved by partial delipation through chemical stimulation of lipolysis or apoptosis inhibition.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Promoting effect of beta-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos

The effects of beta-mercaptoethanol (beta-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O(2) supplemented with beta-ME. Addition of beta-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O(2), to almost the same rates when they were cultured in 5% O(2). To investigate whether the growth-promoting effect of beta-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without beta-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without beta-ME. In contrast, addition of beta-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by beta-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of beta-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that beta-ME has a protective effect in embryo development against oxidative stress and that the effect of beta-ME is associated with the promotion of cystine uptake of low availability in embryos.     

Presence of beta-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization

The present study examined the effect of beta-mercaptoethanol (BME) during in vitro maturation (IVM) of pig oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration, subsequent embryo development and blastocyst cell numbers. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU)-23 medium containing porcine follicular fluid, cysteine, hormonal supplements and 0 to 50 microM BME for 20 to 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20 to 22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5 to 6 h. Putative embryos were transferred to NCSU-23 containing 0.4% BSA and cultured for 144 h (Experiment 1). In comparisons between the presence or absence of BME, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, compared with no addition (26%), the presence of 12.5 and 25 microM BME increased (P < 0.05) the proportion of blastocysts in a dose-dependent manner (34 and 41%). Further increase from 25 to 50 microM BME reduced (P < 0.05) the blastocyst development rate. Blastocysts derived from oocytes matured with 25 microM BME had the highest (P < 0.05) trophectoderm (TE) and total cell numbers. No difference was found in inner cell mass (ICM) cells among treatments. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P < 0.01) level of GSH was found in oocytes matured with 25 microM BME. Compared with 25 microM BME, GSH was low (P < 0.05) at 50 microM BME. The results show that at certain concentrations BME in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in pig oocytes.