In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.     

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8‐cell (demi‐embryo) parthenogenetic and four 8‐cell stage (demi‐embryo) IVF‐derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8‐cell stage) and a whole IVF‐derived embryo (8‐cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF‐derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF‐derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8‐cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8‐cell parthenogenetic and two 4‐cell IVF‐derived blastomeres or eight 16‐cell parthenogenetic and four 8‐cell IVF‐derived blastomeres were constructed. Parthenogenetic demi‐embryos were aggregated with sexed (male) IVF demi‐embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona‐free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi‐embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF‐derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4‐cell and four 8‐cell chimeric embryos compared with 10% for intact 8‐cell parthenogenetic embryos and 15% for intact 16‐cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona‐free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically‐ chimeric viable male calf was born. We conclude that the IVF‐derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Telomerase activity in bovine embryos during early development

The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.     

Telomerase activity and telomere detection during early bovine development

The ends of mammalian chromosomes are composed of repeated DNA sequences of (TTAGGG)(n) known as telomeres. Telomerase is a ribonucleoprotein that synthesizes telomeric DNA to replenish the 50-200 bp lost during cell replication. Cellular aging and senescence are associated with a lack of telomerase activity and a critical shortening of the telomere. The objectives of this study were to confirm the presence of TTAGGG repeats on the chromosomes of bovine embryos using in situ hybridization and assess the relative amounts of telomerase activity using a telomeric repeat amplification protocol (TRAP) during oocyte maturation and early embryo development. Applying a telomere DNA probe to the chromosomes of blastocysts and adult fibroblasts, telomeres were identified on the terminal ends of the p and q arms of chromosomes in all cells examined. Immature oocytes, matured oocytes, zygotes, 2- to 5-cell embryos, 6- to 8-cell embryos, morulae, and blastocysts were lysed in NP-40 lysis buffer and telomerase activity was assayed using the TRAP assay. Telomerase activity was detected in all developmental stages examined. Relative telomerase activity (based on telomerase internal standards and positive controls) appeared to decrease during oocyte maturation and subsequent development to the 8-cell stage but significantly increased (P < 0.05) by approximately 40-fold at the morula and blastocyst stages. It was concluded that the telomeres of bovine chromosomes contain TTAGGG repeats and that telomerase activity is up-regulated in morulae and blastocysts.     

The localization of LAP2beta in bovine oocytes after in vitro activation and fertilization

The present study was designed to clarify the localization of LAP2beta and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2beta was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2beta was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2beta on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2beta first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2beta staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2beta from the PN-stage oocyte to the blastocyst. The assembly of LAP2beta was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2beta at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2beta was constantly expressed in the nuclear membrane in in vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of in vitro fertilized embryos.     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.     

The effect of activation of Mammalian oocytes on remodeling of donor nuclei after nuclear transfer

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization is essential both for intracytoplasmic sperm injection and for nuclear transfer (NT). Therefore, with the goal of producing haploid activated oocytes, we evaluated whether butyrolactone I and bohemine, either alone or in combination with ionomycin, are able to activate young matured mammalian oocytes. Furthermore, the effect on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of cyclin-dependent kinases (CDKs) in metaphase II bovine oocytes induces parthenogenetic activation in a dose dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). The effect of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis, and development to blastocyst of embryos produced by somatic nuclear transfer were studied. Pronuclear formation was significantly higher when activation lasted 5 h compared to 3 h for both ethanol-cycloheximide and ionomycin-bohemine treatment. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12-h labeling 100% of embryos synthesized DNA in both groups. Analysis of two-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy, independent of the activation protocol.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.     

Differential acetylation of histone H4 lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Abnormal histone H3K9 dimethylation but normal dimethyltransferase EHMT2 expression in cloned sheep embryos

The objective was to investigate the relationship between histone H3 lysine 9 (H3K9) dimethylation (me2) and the histone methyltransferase EHMT2 (also known as G9A) in ovine embryos cloned by somatic cell nuclear transfer (SCNT). Levels of H3K9me2 or EHMT2 were detected (with immunostaining) and compared between SCNT and IVF-derived preimplantation embryos. In one-cell embryos, SCNT zygotes had significantly higher levels of H3K9me2 and EHMT2 than IVF zygotes. In cloned embryos, H3K9me2 remained hypermethylated relative to IVF embryos at two-cell and late developmental stages (morula and blastocyst), with no difference (P > 0.05) between IVF and SCNT embryos in EHMT2 levels from two-cell to blastocyst stages. The EHMT2-specific inhibitor, BIX01294, reduced global H3K9me2 levels in cultured ovine cells or SCNT embryos, but it was not appropriate for somatic cell nuclear transfer because of its high cellular toxicity. We inferred that abnormal H3K9me2 hypermethylation in SCNT embryos may not completely arise from EHMT2 expression error.     

Heat shock of porcine zygotes immediately after oocyte activation increases viability

We evaluated the effect of different timing variations of an applied heat shock on parthenogenetically activated (PA) porcine embryos. PA embryos were heat shocked for 9 hr at 42 degrees C from either 0-9 hr post activation (hpa; 09HS), 13-22 hpa (1322HS), or 22-31 hpa (2231HS). Analysis of 24-hr cleavage rates (P < 0.0001), day 5 cell numbers (P < 0.005), day 7 blastocyst rates (P < 0.0001), and day 7 cell numbers (P < 0.05) showed that 09HS embryos developed more successfully in vitro than did all other treated and control embryos. In vitro fertilized (IVF) embryos were exposed to similar heat treatments as described for PA embryos, and embryos derived from somatic cell nuclear transfer (SCNT) were exposed only to the control and 09HS treatments to assess the effects of the different heat treatments on the timing of first cleavage and development to blastocyst. Embryos derived from both IVF and SCNT showed higher proportions of cleaved embryos on day 1 of development when heat shocked immediately after fertilization or fusion/activation as compared to NHS controls (P < 0.05). Blastocyst rates however, showed only modest (IVF; P = 0.089) or no (SCNT; P > 0.1) improvement as compared with control embryos. In summary, exposing PA embryos to elevated temperatures immediately after oocyte activation results in dramatically enhanced developmental potential. A thorough characterization of this phenomenon may yield findings that can serve to increase the efficiency with which PA, IVF, and SCNT embryos are produced in vitro.