MATERIALS AND METHODS

This study was conducted from 2005 to 2007 in Wuhan, China and ovaries of cattle were collected from local slaughter house.

Recipient Oocytes Collection and in vitro Maturation
Ovaries were transported to the laboratory within 1-3 h at 30°C in Dulbecco`s Phosphate-Buffered Saline (DPBS) containing 100 IU mL<sup>-1</sup> streptomycin and 100 IU mL<sup>-1</sup> penicillin from local slaughterhouse. Cumulus-Oocyte Complexes (COCs) were recovered from 3-8 mm follicles in diameter with a 12 mL disposable syringe fitted with an 18-gauge needle. The COCs with evenly granulated ooplasm surrounded by several layers (three or more dense layers) of compact cumulus cells were selected for use. Groups of 10-15 oocytes were placed in 100 μL droplets of IVM medium under mineral oil (Sigma). Briefly, the maturation medium was TCM 199 with Earle`s salts supplemented with 10% (v/v) fetal calf serum (FCS, Sigma), 25 mM Hepes (Sigma), 0.25 mM sodium pyruvate, 10 μg mL^-1 FSH, 10 μg mL^-1 LH, 1 μg mL^-1 estradiol, 100 IU mL^-1 streptomycin and 100 IU mL^-1 penicillin. The COCs were matured for 19 h at 38.5°C in an atmosphere of 5% CO₂.

Preparation of Somatic Donor Cells
Adult granulosa cells were isolated from follicular fluid after removal of the COCs. Granulosa cells were washed twice by centrifugation at 300xg for 2 min in Dulbecco`s modified Eagle`s medium (DMEM) and subsequently seeded onto 100 mm plastic culture dishes at a density of 100,000 cm^-2. Seeded cells were subsequently cultured in DMEM with 10% (v/v) FCS and 2 mM glutamine in 5% CO₂ in humidified air at 38.5°C. Once a granulosa cell layer was established (2-3 day), cells were grown in 100 mm culture plates until they reached 95% confluency. After three subpassages, cells were trypsinized with 1% (v/v) trypsin-EDTA solution and then exposed to a cryoprotectant solution consisting of DMEM, 20% calf serum and 5% dimethylsulfoxide (DMSO) in liquid nitrogen. To prepare adult fibroblasts as donors for nuclear transfer, the preserved cells were thawed and cultured in 100 mm culture plates in 10 mL DMEM containing 10% (v/v) FCS until the cells reached confluency. Granulosa cells were used as nuclear donors at passage three of culture. Before using the cells in nuclear transfer experiments, granulose cells were induced into quiescence by serum starvation (0.05% FCS) for 2-3 days.

Enucleation and Injection
Oocytes were enucleated after 19 h of maturation culture. Cumulus cells were removed mechanically by gentle pipetting in 1% (w/v) hyaluronidase solution and only oocytes with excellent morphology and a visible polar body were selected for this experiment. Prior to enucleation, oocytes were placed for 15 min in Hepes-buffered M199 with 7.5 μg mL^-1 cytochalasin B (sigma) and 5 μg mL^-1 Hoechst 33342 (sigma). Groups of oocytes were enucleated in Hepes-buffered M199 with 7.5 μg mL^-1 cytochalasin B using pipette with a sharp, beveled tip (inner diameter 20 mm). Only oocytes in which the removal of both the polar body and metaphase nucleus was confirmed by observation under UV light were included in the experiment. Granulosa cells were trypsinized, washed by centrifugation and than resuspended in 0.075 M KCl lower osmotic pressure medium for 30, 60, 90 or 180 sec after granulosa cells were thawed and cultured to confluency. After washing three times in M199 by centrifugation, Granulosa cells were resuspended in injection medium of TL-Hepes containing 10% (w/v) polyvinylpyrrolidone. Whole-cell injection was conducted using the procedure similar to the intracytoplasmic sperm injection (ICSI). Granulosa cells were combined with enucleated oocytes in 7.5 μg mL^-1 cytochalasin B in Hepes-buffered M 199 and injected into an enucleated oocytes via the injection pipette (inner diameter 10-13 mm) with a sharp and beveled tip. Reconstructed Embryos were then returned to M199+10% FCS for 2 h at 38.5°C under 5% CO₂ in air.

Activation of Reconstructed Embryos
Chemical activation was performed within 2 h of injection by a 4 min incubation in 5 μM ionomycin (sigma) and then the reconstructed embryos were cultured in M199 containing 2 mM 6-dimethylaminopurine (DMAP, sigma) for 3 h. After washing in M199, the reconstructed embryos were cultured in M199 + 5% FCS for 20 h.

In vitro Culture of Reconstructed Embryos
After activation, the reconstructed embryos were subsequently transferred to development culture drops of M199 and co-cultured with granulosa cells for 7 days. Embryos were culture at 38.5°C under 5% CO₂ in air. The rates of cleaved embryos and blastocysts development were examined at day 2 and 7, respectively.

Detection of Apoptosis by the TUNEL Assay
Apoptosis in blastocysts was detected by the TUNEL assay using a in situ Apoptosis Detection Kit (Takara Bio Inc., Shiga-ken, Japan) (Jang et al., 2004). Blastocysts were fixed in PBS containing 4% (v/v) paraformaldehyde for 30 min at 39°C and placed in a Permeabilization Buffer from the Kit for 10 min at 4°C. After washing in PBS containing 1 mg mL^-1 BSA, blastocysts were treated with a terminal deoxynucleotidyl transferase-labeling buffer for 2 h at 39°C. The blastocystes were then placed in 70 μL fluorescein isothiocyanate solution for 60 min at 39°C and treated with 50 μg mL^-1 propidium iodide for 30 min at room temperature. Blastocysts were mounted on glass slides after being treated with 0.2 M diazabicyclo-ctane in PBS supplemented with 50% (v/v) glycerol and were examined under an epifluorescence inverted microscope. Apoptotic blastomeres appeared as yellow, fragmented and condensed nuclei, while normal blastomeres as red color.

Experimental Design
Experiment 1: The objective of this experiment was to investigate the effect of duration of 0.075 M KCl to bovine granulosa cells on reconstructed embryos formation and subsequent reconstructed embryos developmental potential. Enucleated oocytes were divided into five groups according to different durations of 0.075 M KCl to granulosa cells: control (No. 0.075 M KCl), 30, 60, 90 and 180 sec. There were 242, 236, 227, 236 and 241 enucleated oocytes in the treatment of 30, 60, 90, 180 sec and control, respectively.

Experiment 2: The objective of this experiment was to investigate the effect of 0.075 M KCl to bovine granulosa cells on the apoptosis in blastocysts. There were 12, 12, 12, 7 and 12 blastocysts in the treatment of 30, 60, 90, 180 sec and control, respectively.

Statistical Analysis
The results were pooled and then tested by Chi-square analysis in experiment 1 and 2. Differences at a probability p<0.05 were considered significant. Each experiment was repeated three or four times.

RESULTS AND DISCUSSION

A total of 1182 bovine oocytes were enucleated and 431 were successfully reconstructed. There were significant differences between different holding time of 0.075 M KCl and control in reconstructed embryos formation (p<0.05) and control had low rate of oocytes reconstructed (27.4%). The 180 sec experiment group had significant low rate of cleaved embryos and blastocysts than other groups (23.5 and 8.2%, respectively, p<0.05) (Table 1).

The 90 and 180 sec groups had higher rate of apoptotic blastomeres than other groups and they were 10.4 and 11.6%, respectively. The differences were significant as shown in Table 2.

Table 1:	The effects of duration of 0.075 M KCl to bovine granulosa cells on subsequent reconstructed embryos developmental potential
a,bSuperscripts indicate that the mean values in the same column are significantly different (p<0.05), cbased on the number of enucleated oocytes, dBased on the number of reconstructed embryos

Table 2:	The effect of duration of 0.075 M KCl to bovine granulosa cell on apoptosis in blastocysts
Within columns, values with different superscripts are significantly different (p<0.05)

Formation the couplet of oocyte cytoplast with the donor cell is crucial not only to electrofusion but also to intraplasmic injection. Although electrofusion was widely used for inducing oocytes activation (Dominko et al., 1999), there were many unsolved problem (Zimmermann and Vienken, 1982). In contrast to the method of electrofusion, intraplasmic injection can avoid their disadvantageous aspects. Studies of nuclear transfer were successful by intraplasmic injection (Wakayama et al., 1998; Onishi et al., 2000; Lee et al., 2003; Choi et al., 2002), supporting the view that whole-cell injection was conducted using the procedure similar to ICSI. However, litter work was done in intraplasmic injection because of concerns that incomplete breakage of donor cell membrane may persist in the oocytes resulting in failure of transfer. Therefore, it is important to make membrane breakdown and pore formation before whole-cell injection and assess the effects of breaking the membrane on the efficiency of nuclear transfer by intraplasmic injection.

In recent years, a great deal of attention has been focused on the permeability of biological membrane. A variety of factors and methods have been studied on membrane disruption (Karoonuthaisiri et al., 2003; Logisz et al., 2005) and lower osmotic pressure was one of many methods causing cell membrane disruption. In the study of the effect of 0.075 M KCl to somatic donor cells on the formation of reconstructed embryos and subsequent reconstructed embryos developmental potential, these data showed that 0.075 M KCl had important effect on the rate of reconstructed embryos. Higher rates of reconstructed embryos were observed in 30, 60, 90 and 180 sec treatments as compared to control. Based on this, we though that lower osmotic pressure played a crucial role in the formation of reconstructed embryos. Cells viability depends strongly on the osmotic pressure of the medium and plasma membrane breakdown and pore formation can be induced by lower osmotic pressure of the medium. In this study, 0.75 M KCl affected the formation of reconstructed embryos through breaking granulosa cells membrane. Cell membrane is capable of withstanding pressure to some extent and low rate of reconstructed embryos of control might be caused by incomplete breakage of cell membrane. However, there were no significant differences in the formation of reconstructed embryos among different holding time of lower osmotic pressure. It was more likely that, granulosa cell membrane could be broke easily with lower osmotic pressure and low lever of holding time of lower osmotic pressure was enough to membrane disruption. Experiment treatments (control, 30, 60 and 90 sec) had similar effects on the rates of reconstructed embryos developed to cleavage and blastocysts and 180 sec treatment had significant low rate of cleaved embryos and blastocysts than other groups. It suggested that low lever of holding time of lower osmotic pressure to granulosa cells had no directly effect on blastocysts formation and their blastocysts formation was mainly associated with the developmental competence of the reconstructed embryos. Besides accelerating the formation of reconstructed embryos, high lever of holding time of lower osmotic pressure had potential disadvantageous effects on reconstruced embryos developmental potential.

Besides the rate of blastocysts development, another approach to evaluate embryos developmental potential is to observe the apoptosis in blastocyst using the TUNEL assay (Brison and Schultz, 1997). In the present study, there were significant differences in the incidence of apoptosis in blastocysts between 90, 180 sec experiment groups and other groups. Apoptosis depends on the development stage of embryos and occurs at different frequencies in different strains. Apoptosis is also affected by a wide variety of non physiological stimuli of in vitro culture conditions (Kamjoo et al., 2002; Navarro et al., 2004). Present study and earlier studies showed that different holding time of lower osmotic pressure resulted in the difference of apoptosis in blastocyst and high level of apoptosis in blastocysts was coupled to long holding time of lower osmotic pressure. It was more likely that, besides breaking granulosa cells membrane, long time of lower osmotic pressure to granulosa cells had potential disadvantageous effect on subsequent reconstructed embryos developmental potential. In this process, long holding time of lower osmotic pressure might result in some disadvantageous alterability of cell nuclei.

In conclusion, these results suggested that lower osmotic pressure to bovine granulosa cells accelerated the formation of reconstructed embryos in nuclear transfer of intraplasmic injection and long holding time of lower osmotic pressure had disadvantageous effects on blastocysts through resulting in low blastocysts rate and high level of apoptosis in blastocysts. The outcome of this study should contribute to establish a steady and efficient cloning system of intraplasmic injection in the future.

ACKNOWLEDGMENTS

This research was sponsored by my Ph.D project and Science Foundation of Office of Science and Technology, Hubei Province (2006AA205A03). The author also would like to thank all the teachers and colleagues in Key Laboratory of Animal Breeding and Reproduction, Ministry of Education.