Lian Cai

Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbeccos modified Eagles medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 μL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 × 10(4) CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation.

Supplementation of zinc on oocyte in vitro maturation improves preimplatation embryonic development in pigs

We investigated the effects of zinc supplementation during the IVM of porcine oocytes. Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, subsequent embryonic development, and gene expression were evaluated. Zinc concentrations in porcine plasma and follicular fluid were 0.82 and 0.84 μg/mL, respectively. Zinc was not detected in IVM medium. After treatment with various zinc concentrations (0.0, 0.4, 0.8, 1.2, and 1.6 μg/mL), no significant difference in IVM was observed among groups (85.7%, 88.7%, 90.4%, 90.3%, and 87.2%, respectively). The effects of different zinc concentrations on porcine oocyte intracellular GSH and ROS levels were examined in mature oocytes. Intracellular GSH levels were significantly higher in the 0.8-, 1.2-, and 1.6-μg/mL groups than in the control (P < 0.05). Intracellular ROS levels of oocytes matured with 0.8, 1.2, and 1.6 μg/mL were reduced significantly (P < 0.05) compared with the control and 0.4-μg/mL groups. The developmental competence of oocytes matured with different zinc concentrations was evaluated after parthenogenetic activation (PA) and in vitro fertilization (IVF). Oocytes treated with zinc during IVM showed no significant difference in cleavage rate after PA. Oocytes treated with 0.8 and 1.2 μg/mL zinc during IVM had significantly higher blastocyst formation rates after PA (41.5% and 41.1%, respectively) than the control (27.2%). IVF embryos showed similar results. The blastocyst formation rate was significantly higher (28.2%) in the 0.8-μg/mL group. TNFAIP2 and Bax were decreased in zinc-treated cumulus cells. Increased POU5F1 and decreased Bax transcript levels were observed in zinc-treated oocytes. POU5F1 and Bcl-2 transcript levels were significantly higher in zinc-treated IVF blastocysts. These results indicate that treatment with adequate zinc concentrations during IVM improved the developmental potential of porcine embryos by regulating the intracellular GSH concentration, the ROS level, and transcription factor expression.

The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.

Effect of ganglioside GT1b on the in vitro maturation of porcine oocytes and embryonic development.

Ganglioside is an acidic glycosphingolipid with sialic acids residues. This study was performed to investigate the effect and mechanism of ganglioside GT1b in porcine oocytes in the process of in vitro maturation (IVM) and preimplantation development. Metaphase II (MII) rates were significantly (P < 0.05) different between the control group and the 5 nM GT1b treatment group. Intracellular glutathione (GSH) levels in oocytes matured with 5 nM and 20 nM and GT1b decreased significantly (P < 0.05). The 10 nM group showed a significant (P < 0.05) decrease in intracellular reactive oxygen species (ROS) levels compared with the control group. Subsequently, the level of intracellular Ca2+ in oocytes treated with different concentrations of GT1b was measured. Intracellular Ca2+ was significantly (P < 0.05) increased with a higher concentration of GT1b in a dose-dependent manner. Real-time PCR was performed and showed that the expression of bradykinin 2 receptor (B2R) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) in cumulus cells was significantly (P < 0.05) decreased in the 20 nM GT1b treatment group. Treatment with 5 nM GT1b significantly (P < 0.05) decreased the expression of CaMKIIδ. In oocytes, treatment with 5 nM GT1b significantly (P < 0.05) decreased CaMKIIγ and POU5F1 (POU domain, class 5, transcription factor 1). However, treatment with 20 nM GT1b significantly (P < 0.05) increased the expression of POU5F1. Finally, embryonic developmental data showed no significant differences in the two experiments (parthenogenesis and in vitro fertilization). In conclusion, the results of the present study indicated that GT1b plays an important role in increasing the nuclear maturation rate and decreasing the intracellular ROS levels during IVM. However, GT1b inhibited maturation of the cytoplasm by maintaining intracellular Ca2+ in the process of oocyte maturation regardless of the cell cycle stage. Therefore, GT1b is thought to act on another mechanism that controls intracellular Ca2+.

87 EFFECTS OF HUMAN RECOMBINATION GRANULOCYTE–COLONY STIMULATING FACTOR (hrG-CSF) ON IN VITRO CULTURE OF PORCINE CLONED EMBRYOS DERIVED FROM THIN CUMULUS CELL LAYER OF OOCYTES MATURED IN VITRO

Although several cloned pigs have been successfully produced, the developmental competence of cloned embryos in vitro is still very low. Granulocyte colony-stimulating factor receptor (G-CSFR) was founded in the human trophoblastic cell line that is implicated in regulation and proliferation of trophoblast. In the present study, the somatic cell NT embryos derived from oocytes that have more than 3 cumulus cells layer were cultured and supplemented with various concentrations of hrG-CSF (0, 10, 50, and 100ngmL-1, respectively). Although there were no significant effects on the various concentration of hrG-CSF treatment groups compared with control, the somatic cell NT blastocysts formation tended to increase after 10ngmL-1 hrG-CSF treatment (24.19±2.90%) compared with control (21.37±2.98%). Moreover, we investigated the effects of 10ngmL-1 hrG-CSF on in vitro culture of porcine cloned embryos derived from oocytes that were categorized into grade A (cumulus cell layer >10), grade B (10>cumulus cell layer ≥ 3), and grade C (cumulus cell layer <3). After supplementation of 10ngmL-1 hrG-CSF on in vitro-culture of different groups, the developmental competence, blastocyst quality, and gene transcript levels were observed. The results showed that 10ngmL-1 hrG-CSF has no beneficial effects on cloned embryos derived from grade A oocytes (10ngmL-1 hrG-CSF 25.35±2.53% v. control 25.00±2.66%), but it significantly increased blastocyst formation of embryos derived from grade B oocytes (22.09±2.10%) compared with grade B control (12.09±2.31%, P<0.05). There were obvious increases in blastocyst formation derived from grade C oocytes after 10ngmL-1 hrG-SCF treatment (25.74±1.65%) compared with grade C control (16.82±2.30%, P<0.05). However, there were no significantly differences in cleavage rate and total cell number of blastocysts among each group. Otherwise, the PCNA, POU5F1, Dnmt1, Bcl2, and Bax transcript levels were significantly increased in blastocysts that were derived from grade C oocytes after 10ngmL-1 hrG-SCF treatment compared with grade C control. In conclusion, supplementation of 10ngmL-1 hrG-CSF in in vitro-cultured porcine embryos increased blastocyst formation of embryos derived from thin cumulus layer of oocytes by reducing apoptosis while increasing cell proliferation and nuclear reprogramming. These results provide an experimental basis for the use of poor quality oocytes for agricultural production.

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Zinc supplementation during in vitro maturation increases the production efficiency of cloned pigs

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.