Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.     

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.     

Telomerase Activity in Swamp Buffalo (Bubalus bubalis) Oocytes and Embryos Derived from In Vitro Fertilization, Somatic Cell Nuclear Transfer and Parthenogenetic Activation

The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and pre-implantation stage embryos derived from in vitro fertilization (IVF), somatic cell nuclear transfer (NT) and parthenogenetic activation (PA). Immature and mature oocytes, and embryos at the 2-4 cell, 8-16 cell, morula and blastocyst stages produced by IVF, NT and PA were collected and the telomerase activity was assayed by using a Telomerase PCR ELISA kit. Telomerase activity was detected in all developmental stages evaluated from immature oocytes to blastocyst stage embryos. Telomerase activity was detected in higher amounts in immature as compared with mature oocytes (p < 0.05). Embryos derived from NT showed a profile of telomerase activity similar to that of IVF. In IVF and NT embryos, telomerase activity was low in the 2-4 cell and 8-16 cell stages, but the activity significantly increased (p < 0.05) at the morula stage, reaching its highest level at the blastocyst stage. In PA embryos, low levels of telomerase activity were detected from the 2-4 cell to the morula stage, and the highest level of telomerase activity was found at the blastocyst stage. Telomerase activity in NT blastocysts is higher than that derived from IVF and the activity is highest in PA blastocysts. These results suggest that the successful reprogramming of telomerase activity in buffalo NT embryos follow a pattern similar to that in embryos derived from IVF and PA.     

Timing and uniformity of embryonic gene activation affect subsequent pre-implantation development of cloned bovine embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.     

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

In vitro developmental potential of bovine nuclear transfer embryos derived from primary cultured cumulus cells

The in vitro development and the quality of blastocysts produced from the nuclear transfer (NT) embryos reconstituted from primary cultured cumulus cells (NT-cumulus) were examined compared to in vitro fertilized embryos (IVF) and NT embryos reconstituted from the embryonic blastomeres (NT-blastomere). The cleavage rate, and the development to blastocyst were the same for all three sets of embryos. The time required for blastocoel formation starting from the time of the initial cleavage was shorter for NT embryo groups than IVF ones. All experimental groups produced morphologically similar and normal blastocysts containing the same cell number. The percentage of the blastocysts with normal chromosomal complements were the same for NT-cumulus and IVF.     

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.     

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.     

Suberoylanilide hydroxamic acid,a novel histone deacetylase inhibitor,improves the development and acetylation level of miniature porcine handmade cloning embryos

Histone deacetylase inhibitors (HDACis) can change the histone acetylation and significantly enhance the developmental competence of the pre‐implantation SCNT embryo. To select a proper histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of miniature porcine, we compared the effect of two histone deacetylase inhibitors (SAHA vs. VPA) on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and number of blastocyst cells of HMC embryos treated with SAHA (SAHA‐HMC) or VPA (VPA‐HMC) were significantly higher than those of control (Control‐HMC), respectively, but there were no significant difference between SAHA‐HMC and VPA‐HMC groups. In addition, the acetylation level (AcH4K8) of Control‐HMC and VPA‐HMC embryos at the blastocyst stage, respectively, was significantly lower than that of in vitro fertilized (IVF) and SAHA‐HMC embryos. However, the acetylation H4K8 of the blastocysts had no significant difference between SAHA‐HMC and the IVF groups. The SAHA‐HMC blastocysts indicated comparative expression levels of Oct4 and HDAC1 (histone deacetyltransferase gene) with those of IVF blastocysts. In contrast, the expression levels of Oct4 were lower and those of HDAC1 were higher in the VPA‐HMC and Control‐HMC blastocysts, respectively, compared to those of the IVF blastocysts. Our results demonstrated that the HMC embryos treated by SAHA could promote the pre‐implantation development and increase the levels of histone H4K8 acetylation and the expression of the OCT4 gene, yet decrease the expression of the HDAC1 gene to the comparable level of the IVF embryos. Our results proved that SAHA may be a better histone deacetylase inhibitor for porcine HMC compared to VPA, and furthermore, it may indicate that SAHA can effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full‐term developmental potential of the HMC embryos after embryo transplantation.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.     

Expression of Interferon-tau mRNA in Bovine Embryos Derived from Different Procedures

Interferon-tau (IFN-τ) is a secreted conceptus protein which plays a critical role in the establishment of ruminant pregnancy by its antiluteolytic and antiviral effects. In the present study, we hypothesized that IFN-τ expression was temporally and spatially regulated in different pre-implantation embryos and the levels of IFN-τ expression were different among bovine embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). By using in situ hybridization with Digoxingenin (DIG)-labelled IFN-τ cDNA as a probe, we detected IFN-τ mRNA in bovine embryos from days 3 to 9 in culture. However, the timing of the initiation of IFN-τ mRNA expression was different among PA, IVF and SCNT embryos. Interferon-τ mRNA was first expressed in 16-cell stage IVF embryos on day 4, in SCNT morula on day 5 and early PA blastocyst on day 6. Semi-quantitative RT-PCR analysis showed that the expression levels of IFN-τ mRNA did not differ significantly among IVF, SCNT and PA embryos on day 7. In addition, freezing and thawing did not have a major impact either on IFN-τ mRNA expression in IVF or in vivo -produced bovine blastocysts.