Nucleoplasmin facilitates reprogramming and in vivo development of bovine nuclear transfer embryos

Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.     

Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.     

Defined media optimization for in vitro culture of bovine somatic cell nuclear transfer (SCNT) embryos

The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos.     

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow‐derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear‐transfer (BMSC‐NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC‐NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF‐NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC‐NT embryos (30.0%, 9.8%, respectively) than those in FF‐NT embryos (34.2%, 28.0%, respectively). We also found that BMSC‐NT embryos had more H3K9Ac and less H3K9me3 and 5‐methylcytosine than FF‐NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.     

Nuclear reprogramming in embryos generated by the transfer of yak (Bos grunniens) nuclei into bovine oocytes and comparison with bovine-bovine SCNT and bovine IVF embryos

Although inter-species SCNT may be useful for increasing and preserving populations of endangered species, there are many reports that inter-species nuclear transfer embryos only develop to the blastocyst stage. In this study, yak-bovine SCNT blastocysts were successfully implanted in the surrogate bovine uterus but failed to develop to term or aborted. To clarify the reasons, we examined yak-bovine SCNT blastocyst development, total cell number, inner cell mass (ICM) number, trophoblast (TE) cell number and relative gene expression in yak fibroblast cells and yak-bovine SCNT embryos at various stages. The potential for development of yak-bovine SCNT embryos to blastocysts was 30+/-5.7% (mean+/-S.E.M.); the total cell number was 85.3+/-16.3, fewer than in IVF bovine embryos (106.2+/-18.2) but within the reported range (60-300). The yak-bovine SCNT blastocysts had a lower ratio of TE cells to total cells (43.9+/-8.7%) than bovine IVF embryos (59.4+/-3.4%; P<0.05) or bovine-bovine SCNT (69.5+/-5.4%; P<0.05). Also, several yak-bovine SCNT embryos had abnormal initiation of expression of both Mash2 and IL6. However, expression of vimentin, collagen, Cx43 and PSMC3 were normal in yak fibroblast cells and yak-bovine SCNT embryos. In conclusion, we inferred that the normal allocation of ICM and TE cells in yak-bovine SCNT embryos and embryo-specific gene reprogramming may be important for successful inter-species animal cloning.     

Interleukin-1 beta induces autophagy of mouse preimplantation embryos and improves blastocyst quality

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.     

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Retinol improves bovine embryonic development in vitro

Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

Nuclear reprogramming in nuclear transplant rabbit embryos

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.     

Progesterone enhances in vitro development of bovine embryos

Increased pregnancy rates in cattle given progesterone (P₄) prior to 5 d after breeding have recently been reported. The objective was to determine if this increase in pregnancy rate could be attributed to a direct positive effect of P₄ on the developing embryo. In Experiment 1, 280 bovine oocytes were inseminated in vitro and at Day 3 (insemination = Day 0), good quality 8 cell embryos (n = 206) were randomly allocated to be cultured in either CR1aa+serum with 0 or ∼15 ng/mL (n = 102 and n = 104, respectively). In Experiment 2, 881 bovine oocytes were used; on Day 3, good quality 8 cell embryos (n = 511) were randomly allocated to either the control (CR1aa+FCS, n = 168), vehicle (CR1aa + FCS + ethanol, n = 170), or P₄ treatment (CR1aa + FCS + ∼15 ng/mL P₄ in ethanol, n = 173). On Day 7, in both experiments, there were increased numbers of blastocysts developing in the P₄ group (Experiment 1, 59% and Experiment 2, 71%) compared to the vehicle (Experiment 2, 53%) or control (40 and 62% in Experiments 1 and 2, respectively). The addition of P₄ (8%) stimulated the rate of embryo development (early blastocysts or more advanced stages on Day 6) compared to vehicle (3%) and control (0%) and the P₄ group had more hatched or hatching blastocysts (33%) on Day 9 compared to the control or vehicle group (21 or 22%). Additionally, the P₄ group had greater embryo diameter and significantly more Grade 1 blastocysts on Day 7. In conclusion, P₄ had a direct, positive effect on developing bovine embryos cultured in vitro.     

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.     

Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro

Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to demecolcine at various times post-activation. Onset and duration of demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in demecolcine-induced enucleation.     

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.     

Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O(2), 5% CO(2), and 90% N(2). In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 microM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 microM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 microM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm(2) vs. 0.71 mm(2) surface area; n = 27, P < 0.04).     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.