Xi-Jun Yin

Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts

Embryonic stem (ES)‐like cells were isolated from in vivo‐produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin‐C‐treated cat embryonic fibroblast feeder layers in medium supplemented with knockout™ Serum Replacement (KSR‐medium) or fetal bovine serum (FBS‐medium). Putative ES‐like cell colonies developed in both KSR‐ and FBS‐medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES‐like cell colony formation were significantly higher in KSR‐medium, but subsequent cell proliferation was significantly lower than in FBS‐medium. For passaging, 32 and 18 colonies in KSR‐ and FBS‐medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES‐like cell lines that continued to grow for up to eight passages in FBS‐medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell‐markers Oct‐4, stage‐specific embryonic antigen‐1 (SSEA‐1), SSEA‐3, and SSEA‐4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium‐like and neuron‐like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha‐actinin. These observations indicate that cat ES‐like cells were successfully isolated and characterized from in vivo‐produced blastocysts. Mol. Reprod. Dev. 75: 1426–1432, 2008.

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.

Chromatin, microtubule and microfilament configurations in the canine oocyte

In the present study, we observed chromatin, microtubule and microfilament distribution in canine oocytes. The germinal vesicle (GV) chromatin of canine oocytes was classified into four configurations (GV-I, -II, -III and -IV) based on the degree of chromatin separation and condensation. Oocytes recovered from follicular phase ovaries had a greater amount (68%, P < 0.05) of GV-III or GV-IV chromatin than did those from non-follicular phase ovaries (35%). The majority (86.7%) of in vivo ovulated oocytes were at GV-IV. The rates of development to GV breakdown/metaphase I/metaphase II were higher in oocytes recovered from follicular ovaries than from non-follicular ovaries. Immunostaining results revealed cytoplasmic microtubules present in all GV-stage oocytes. Following GV breakdown, microtubular asters were produced from condensed chromatin. The asters appeared to be elongated, and encompassed condensed chromatin particles to form meiotic metaphase chromatin. Microfilaments were located in the cortex and around the GV. During meiotic maturation, a microfilament-rich area, in which the chromatin is allocated, was observed in the oocyte. Our results indicate that oocytes recovered from follicular ovaries were in an advanced stage of GV, and were more competent to complete maturation compared to those from non-follicular phase ovaries. Both microtubules and microfilaments are closely associated with reconstruction of chromatin during meiotic maturation in canine oocytes.

Effect of crude canine pituitary extract (cCPE) on the in vitro production of progesterone and nuclear maturation of canine oocytes

We examined the effect of crude canine pituitary extract (cCPE) on the in vitro nuclear maturation of canine oocytes and production of progesterone by cumulus cells. cCPE was extracted from canine pituitaries and the concentrations of canine follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined. Cumulus oocyte complexes (COCs) were harvested from anestrus cycle ovaries and matured in NCSU-37 supplemented with 10% estrus bitch serum, 50 µg/ml gentamycin and 0, 40 or 400 µg/ml cCPE at 38°C in a humidified atmosphere of 5% CO2 for 72 h. The nuclear maturation of the oocytes and the level of progesterone in the culture medium were evaluated. Development to metaphase I (MI) - metaphase II (MII) of canine oocytes in 400 µg/ml cCPE (15.4%) was significantly higher than in 0 and 40 µg/ml cCPE (4.3 and 8.7%), respectively. Treatment with 40 and 400 µg/ml cCPE also generated 0.33 and 0.65 ng/ml progesterone in the culture medium, respectively. Thus, the addition of cCPE to the culture medium promotes the nuclear maturation of canine oocytes and elevates the production of progesterone by cumulus cells.

29 COMPARISON OF DNA METHYLATION RATES BETWEEN CLONED CATS AND DOMESTIC CATS

Many mammalian species have recently been successfully cloned using somatic cells. We have produced cloned kittens by somatic cell nuclear transfer (SCNT). The cloning of mammals by SCNT requires epigenetic reprogramming of the differentiated state of donor cells. We recently observed that feline catus satellite DNA regions exhibit DNA methylation in the donor and somatic cells of a cloned cat. The cause of variation could lie in alterations occurring at different steps of the cloning procedure or incomplete reestablishment of DNA methylation patterns. This study was conducted to determine the methylation status of the feline catus satellite DNA region in the somatic cells of cloned cats and domestic normal cats by using a bisulfite sequencing method. Satellite sequences are heavily methylated in somatic cell chromosomes and are associated with dense heterochromatin where bundles of silent genes are wrapped up together. For the analysis of the cat satellite sequence, a 400-bp segment of the satellite genomic region, which has highly conserved 22 CpG sites, was amplified by PCR from bisulfite-treated genomic DNA, and the resulting PCR products were individually cloned and sequenced. For methylation analysis, genomic DNA was first isolated from somatic and placental cells of cloned and domestic normal cats. The DNA methylation status of the satellite DNA region was not significantly different among donor (using NT) cells (70.6, 77.0, and 79.1% in Donors A, B, and C, respectively), somatic cells of cloned cats (88.9, 82.2, and 85.9% in cloned A-1, 2, and 3; 78.0% in cloned B-1; 88.1, 78.9, 80.3, 83.2, 74.6, and 82.3% in cloned C-1, 2, 3, 4, 5, and 6, all respectively), and domestic cats (88.0, 81.6, and 81.8% in NC 1, 2, and 3, respectively). Also, the DNA methylation status of satellite DNA regions in the placenta was significantly different between cloned cats (80.5, 76.7, and 76.8% in cloned C-2, 5, and 7, respectively) and normal domestic cats (64.2, 66.7, and 74.9% in NC 1, 2, and 3, respectively). In conclusion, all of the somatic cells were highly methylated. The methylation status of the somatic cells was not different among groups, but that of placental cells was significantly different. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

223 COMPARISON OF NEURAL DIFFERENTIATION BETWEEN RAT AND CAT MESENCHYMAL STEM CELLS UNDER CHALLENGE BY THE SAME NEUROINDUCTION AGENT

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes. Moreover, MSCs have the capacity to differentiate into neural lineages. The present study aimed to investigate the differences between the expression profiles during neural differentiation of rat and cat MSCs. MSCs in the 4th passage, which were isolated from the femurs and tibias by standard methodology, were used in our study. Culture media was divided into pre-induction medium, which consisted of DMEM-HG, 10% fetal bovine serum, 10 ng mL-1 of basic fibroblast growth factor (bFGF), and 1 mM ²-mercaptoethanol, for 24 h, and neuron induction medium, which was composed of DMEM-HG, 2% DMSO, 200 µM BHA, 25 mMKCl, 10 µM forskolin, 5 µg mL-1 insulin-transferrin-sodium selenite, and 20 ng mL-1 bFGF, for 24 h. Thereafter, cell morphology and growth traits were determined by light microscopy imaging and by examining the cell-surface antigen profile and differentiation repertoire by immunocytochemistry, respectively. Regarding the expression of 3 MSC-related surface antigens, rat cells were positive for CD18 and CD44, whereas cat cells expressed CD9 and CD44. Under proneurogenic conditions, rat neuron-like cells progressively increased at 30 min post-induction, peaked at 1 h, and gradually declined after 12 h. At 24 h after neural induction, there were still some neuron-like cells. Meanwhile, cat cells were expressed increasingly during the first hour, peaked at 2 to 3 h, were sustained for 8 h after neural induction, and then gradually declined.A few neuron-like cells remained until 24 h post-induction. In neural differentiation, rat MSCs were positive for ²III-tubulin, NF-L, NF-M, NF-H, trkA, and vimentin; cat MSCs were negative for ²III-tubulin and NF-H but positive for NF-L, NF-M, trkA, and vimentin. Both MSCs were negative for oligodendrocyte markers. Our results revealed that there is variation in neural differentiation sensitivity due to species type. Rat cells were more sensitive in response to neuroinduction agents, maintained their morphology for a longer time, and expressed relatively mature neuronal markers. On the contrary, the sensitivity of cat cells was weaker, and survival time was shorter compared to that of the rat cells. The cat cells expressed immature neuronal markers. The present data suggest a significant aspect of the culture of MSCs from higher-grade species. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

334 Effect of insulin on in vitro maturation of canine oocytes.

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 ¼m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 ¼g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5°C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 ¼g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.