Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs.

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.

Enhancing effects of serum-rich and cytokine-supplemented culture conditions on developing blastocysts and deriving porcine parthenogenetic embryonic stem cells.

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.

Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer.

The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the perivitelline space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes.

Treatment with the proteasome inhibitor MG132 during the end of oocyte maturation improves oocyte competence for development after fertilization in cattle.

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0-6 h or 16-22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM - effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16-22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.

L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression.

The objective of this study was to examine the effect of L-carnitine treatment during in vitro maturation (IVM) of immature pig (Sus scrofa) oocytes. Specifically, the effects of L-carnitine treatment on nuclear maturation and oocyte intracellular glutathione (GSH) levels, embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT pig embryos were determined. During IVM culture, immature oocytes were either treated or not treated with 10 mM L-carnitine. L-carnitine treatment did not improve the nuclear maturation of oocytes but significantly increased intracellular GSH levels, which led to a reduction of reactive oxygen species (ROS) levels in IVM oocytes. Oocytes treated with L-carnitine showed higher (P<0.05) rates of blastocyst formation after PA (39.4% vs. 27.1%) and SCNT (23.2% vs. 14.9%) compared with untreated oocytes. SCNT embryos that were derived from L-carnitine-treated oocytes showed increased (P<0.05) expression levels of DNMT1, PCNA, FGFR2, and POU5F1 mRNA compared with control embryos. Treatment of recipient oocytes with L-carnitine increased (P<0.05) the expression of both BAX and p-Bcl-xl mRNA in SCNT blastocysts. However, the increase was more prominent in BAX than in p-Bcl-xl mRNA. Our results demonstrate that L-carnitine treatment during IVM improves the developmental competence of SCNT embryos. This effect is probably due to increased intracellular GSH synthesis in recipient ooplasts, which reduces ROS levels, and the stimulation of nuclear reprogramming via increased expression of POU5F1 and transcription factors.

MG132 treatment during oocyte maturation improves embryonic development after somatic cell nuclear transfer and alters oocyte and embryo transcript abundance in pigs.

The objective of this study was to examine the effect of treating pig oocytes during in vitro maturation (IVM) with a proteasome inhibitor, MG132, on oocyte maturation and embryonic development. In one series of experiments, oocytes from medium-sized follicles (3-8 mm in diameter) were untreated (MCO) or treated with MG132 during 0-22 hr (M0-22) or 30-42 hr (M30-42) of IVM. There was no significant effect of MG132 on nuclear maturation or cytoplasmic maturation (as assessed by intracellular amounts of glutathione and p34cdc2 kinase activity). Blastocyst formation after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), however, was increased for M30-42 (65.2% and 27.7% for PA and SCNT, respectively) compared to MCO (42.6% and 13.6%, respectively) and M0-22 (45.3% and 19.5%, respectively; P<0.05). Expression of PCNA and ERK2 was increased in M30-42 for IVM oocytes while transcript abundance for POUF51, DNMT1, FGFR2, and PCNA was increased in M30-42 for 4-cell SCNT embryos. When oocytes derived from small follicles (<3 mm in diameter) were untreated (SCO) or treated with MG132 during 0-22 hr (S0-22), 30-42 hr (S30-42) of IVM, or 0-22 and 30-42 hr of IVM (S0-22/30-42), expression of POU5F1, DNMT1, FGFR2, and PCNA and blastocyst formation were increased for SCNT embryos derived from S30 to 42 (16.5%) and S0-22/30-42 oocytes (20.8%) as compared to embryos from SCO (8.7%) or S0-22 oocytes (8.8%; P<0.05). Results demonstrate that treatment of oocytes with MG132 during the later stage of IVM improves embryonic development and alters gene expression in pigs.

Prolonged interval between fusion and activation impairs embryonic development by inducing chromosome scattering and nuclear aneuploidy in pig somatic cell nuclear transfer.

The aim of the present study was to examine the effect of various intervals between electrofusion and activation (FA interval) on the nuclear remodelling and development of somatic cell nuclear transfer (SCNT) embryos in pigs. Reconstructed oocytes were activated at 0 (simultaneous fusion and activation; SFA), 1, 2 and 3 h (delayed activation) after electrofusion; these groups were designated as DA1, DA2 and DA3, respectively. When oocyte nuclear status was examined at 0.5, 1, 2 and 3 h after electrofusion, the incidence of chromosome scattering was increased (P < 0.01) as the FA interval was extended (0.0%, 12.0%, 77.3% and 78.0%, respectively). Extending the FA interval led to an increase (P < 0.01) in the percentage of oocytes containing multiple (>or=3) pseudopronuclei (PPN) (0.0% of SFA; 5.3% of DA1; 21.7% of DA2; and 33.5% of DA3). The development of SCNT embryos to the blastocyst stage was decreased (P < 0.05) in DA2 (5.7%) and DA3 (5.0%) compared with SFA (18.1%) and DA1 (19.5%). Our results demonstrate that extending the FA interval impairs the development of SCNT pig embryos by inducing chromosome scattering and the formation of multiple PPN, which may result in increased nuclear aneuploidy.

Anthocyanin stimulates in vitro development of cloned pig embryos by increasing the intracellular glutathione level and inhibiting reactive oxygen species.

The objective was to examine the nuclear maturation of oocytes, embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression in SCNT embryos in pigs (Sus scrofa) when anthocyanin was added to oocytes during maturation and in vitro culture (IVC) of embryos. Immature oocytes were untreated or treated with 0.1 microg/mL anthocyanin during in vitro maturation (IVM). Next, PA and SCNT embryos were produced from oocytes and cultured in medium supplemented with or without 0.1 microg/mL anthocyanin for 7 d. Anthocyanin treatment during IVM did not improve the nuclear maturation of oocytes, but significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). Oocytes treated with anthocyanin during IVM had higher (P < 0.05) rates of blastocyst formation after PA (55.7 vs. 44.9 %) and SCNT (32.2 vs. 16.1%) compared to untreated oocytes. In PA and SCNT embryos, anthocyanin treatment during IVM or IVC significantly increased the intracellular GSH level, which led to the reduced ROS level. Somatic cell nuclear transfer embryos derived from anthocyanin-treated oocytes had increased (P < 0.05) expression of DNMT1, PCNA, FGFR2, and POU5F1 mRNA compared to control embryos. In conclusion, anthocyanin treatment during IVM improved developmental competence of SCNT embryos, most likely by increasing intracellular GSH synthesis, reducing ROS level, and stimulating nuclear reprogramming via increased transcription factor expression.

Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig.

The objective of this study was to assess the practical usefulness of morphologically poor oocytes (MPCOCs) in relation to follicular size and oocyte diameter. Oocytes collected from medium (3-8 mm in diameter) and small (<3 mm) follicles were classified into five categories of morphologically good oocytes (MGCOCs) from medium follicles (MA, control), MPCOCs with larger and smaller diameters from medium follicles (ML and MS, respectively), and those from small follicles (SL and SS, respectively). The oocytes were examined for maturation and developmental competence after parthenogenesis and somatic cell nuclear transfer (SCNT). Nuclear maturation of ML oocytes (91%) was similar to that of control oocytes (94%), but higher than MS (80%), SL (79%), and SS (63%) oocytes. This pattern was also observed in the intracellular glutathione level, p34(cdc2) kinase activity, and gene (CDK1, PCNA, and ERK2) expression levels in in vitro-matured oocytes. ML oocytes showed a similar proportion of blastocyst formation (20%) after SCNT to control oocytes (21%). In addition, the use of ML oocytes resulted in a 50% farrowing rate with 1.8% efficiency of piglet production after SCNT embryo transfer, while control oocytes showed a 60% farrowing rate with 2.4% production efficiency. Our results demonstrate that MPCOCs, if appropriately selected, have a comparable ability to MGCOCs in supporting not only in vitro blastocyst formation, but also development to term in vivo after SCNT. These oocytes can be used as a source for in vitro production of embryos with normal in vivo viability in pigs.