Jyh-Cherng Ju

Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 μg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation (IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1 (BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01) expression (P<0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.

Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage

The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho(+)-Hd⁻) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho⁻-Hd(+)) or non-heated (Ho⁻-Hd⁻) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6-32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9-33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.

Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos

This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 μg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 μg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 μg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.

Trichostatin A and Ascorbic Acid Assist in the Development of Porcine Handmade Cloned Embryos via Different Physiologic Pathways

The objective was to determine the effects of ascorbic acid (AA), trichostatin A (TSA), and their combined treatment (TA) on reprogramming and development of cloned porcine embryos. Embryos treated with AA (50 and 100 µg/mL) had a higher blastocyst rate than controls (49.6% and 44.0% vs 30.7%, P < .05). Blastocyst rates of handmade cloned (HMC) embryos were nearly 60% in both the 30 and 40 nmol/L TSA treatment groups, which were higher (P < .05) than the control (29.4%). The TA treatment groups had a higher blastocyst rate compared with the AA treatment alone (58.9% vs 43.5%, P < .05). Histone acetylation was much higher in the TSA and TA treatments (primarily in 2- and 4-celled embryos) but was not significantly different between AA-treated and untreated embryos. Both AA and TA treatments reduced apoptotic rates of blastocysts. In conclusion, AA supplementation improved blastocyst development in porcine HMC embryos mainly by a traditional antioxidant pathway rather than by cellular reprogramming.

Transgenic cloned mice expressing enhanced green fluorescent protein generated by activation stimuli combined with 6-dimethylaminopurine.

Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6-DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71-72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6-DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 mug/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1-8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)-expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM-AA, MEM-alpha and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM-alpha medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6-DMAP following embryo transfer. In conclusion, treatment with 6-DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full-term development of the transgenic SCNT cloned embryos.