Volume 81, Issue 4 pp. 453-460

ORIGINAL ARTICLE

Free Access

Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

Yamato MIZOBE,

Yamato MIZOBE

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Saori KURINO,

Saori KURINO

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Yoshiaki SATA,

Yoshiaki SATA

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Hironori MORI,

Hironori MORI

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Mitsutoshi YOSHIDA,

Mitsutoshi YOSHIDA

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Kazuchika MIYOSHI,

Kazuchika MIYOSHI

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Yamato MIZOBE,

Yamato MIZOBE

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Saori KURINO,

Saori KURINO

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Yoshiaki SATA,

Yoshiaki SATA

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Hironori MORI,

Hironori MORI

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Mitsutoshi YOSHIDA,

Mitsutoshi YOSHIDA

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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Kazuchika MIYOSHI,

Kazuchika MIYOSHI

Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

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First published: 21 July 2010

https://doi.org/10.1111/j.1740-0929.2010.00758.x

Citations: 5

Kazuchika Miyoshi, Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. (Email: [email protected])

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ABSTRACT

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium-3 (mPZM-3) with increased NaCl to 138 mmol/L (mPZM-3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM-3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM-3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM-3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM-3+NaCl for the first 2 days and then cultured in mPZM-3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM-3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.

INTRODUCTION

Successful development of cloned embryos produced using nuclear transfer depends on artificial stimulation-induced activation (Robl et al. 1992). Electric stimulation is the most common method of activation of embryos in somatic cell nuclear transfer (SCNT) studies that have succeeded in producing cloned piglets (Onishi et al. 2000; Polejaeva et al. 2000; Bondioli et al. 2001; Park et al. 2001, 2002; Boquest et al. 2002; Dai et al. 2002; De Sousa et al. 2002; Lai et al. 2002a,b). Recently, we have shown that ultrasound stimulation can induce nuclear activation and parthenogenetic development of pig oocytes matured in vitro (Sato et al. 2005). Ultrasound stimulation was also useful for inducing activation and in vitro development of cloned embryos derived from miniature pig somatic cells (Miyoshi et al. 2006). In addition, miniature pig SCNT embryos activated by ultrasound stimulation had the ability to develop into piglets after transfer to recipient females (Miyoshi et al. 2007). The advantage of the activation protocol using ultrasound is that it can treat more oocytes and embryos at one time compared with the activation protocol using electric pulses (Sato et al. 2005). This advantage is valuable in SCNT studies requiring activation of a lot of cloned embryos. Moreover, ultrasound is considered to be better artificial stimulation for activation of embryos than electric pulses in production of cloned miniature pigs because no viable piglets have been obtained from embryos activated by electric pulses in our laboratory (Miyoshi et al. 2007).

Establishment of culture systems to support the pre-implantation development of SCNT embryos is quite important not only for basic research to clarify the mechanism controlling their development, but also for the nonsurgical embryo transfer of them to recipient females at the morula and blastocyst stages (Martinez et al. 2004). Osmolarity of culture media is one of the factors affecting in vitro development of mammalian embryos and optimal osmolarity is species-specific (Miyoshi et al. 1996). Porcine zygote medium-3 (PZM-3) (Yoshioka et al. 2002) which is the most successful, compared to other media, for the culture of pig embryos derived in vivo and in vitro to the blastocyst stage is hypotonic (Li et al. 2007; Hwang et al. 2008). Recently, Nguyen et al. (2003) reported that the NaCl-induced change of osmolarity from 280–310 mOsm to 220–270 mOsm at 2–3 days after electric activation supports the parthenogenetic development into blastocysts of pig oocytes. In addition, the blastocyst formation rate of pig SCNT embryos activated by electric pulses was improved by culturing in PZM-3 with increased NaCl to 138 mmol/L (300–320 mOsm) for the first 2 days and then transferring into normal PZM-3 (260–270 mOsm) (Hwang et al. 2008). These reports suggest that the change of osmolarity during in vitro culture is necessary to improve the developmental capacity of pig oocytes and SCNT embryos activated by ultrasound. The present study was conducted to test this hypothesis.

MATERIALS AND METHODS

In vitro maturation of oocytes

Ovaries were collected from pre-pubertal gilts at a local slaughterhouse and transported to the laboratory in saline at 32–35°C. The follicular contents were recovered by aspiration from follicles (2–5 mm diameter) using an 18-gauge needle (Terumo, Tokyo, Japan) and a 5-mL disposable syringe (Nipro, Osaka, Japan). The cumulus-oocyte complexes (COCs) were gathered from the follicular contents and washed twice with HEPES (Nacalai Tesque, Kyoto, Japan)-buffered Tyrode-lactate-pyruvate-polyvinyl alcohol (PVA; Sigma-Aldrich Chemical, St. Louis, MO, USA) and the maturation medium, respectively. Only COCs possessing a compact cumulus mass and evenly granulated ooplasm were selected. COCs in groups of 30–60 were transferred to a droplet of the maturation medium (200 µL) under paraffin oil (Nacalai Tesque) in a 35-mm polystyrene dish (Becton Dickinson, Franklin Lakes, NJ, USA) and cultured at 38.5°C in an atmosphere of 5% CO₂ in air. The maturation medium consisted of 90% (v : v) TCM-199 with Earle's salts (Gibco BRL, Grand Island, NY, USA) supplemented with 0.91 mmol/L sodium pyruvate (Sigma), 3.05 mmol/L D-glucose (Wako Pure Chemical, Osaka, Japan), 0.57 mmol/L cysteine hydrochloride hydrate (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 IU/mL eCG (Teikoku-Zoki, Tokyo, Japan), 10 IU/mL hCG (Teikoku-Zoki), 100 µg/mL amikacin sulfate (Meiji Seika, Tokyo, Japan), 0.1% (w : v) PVA and 10% (v : v) pig follicular fluid. After 40–42 h of culture, cumulus cells were removed by pipetting with 0.1% (w : v) hyaluronidase (Sigma). Oocytes with a polar body were selected for the experiments.

Donor cells

Fetal fibroblasts were obtained from a female fetus of Clawn miniature pigs on Day 33 of pregnancy. Body tissues were cut into small pieces and cultured in a mixture of Dulbecco's modified Eagle medium and Ham's F-12 medium (Gibco) supplemented with 100 µg/mL amikacin sulfate and 10% (v : v) fetal calf serum (FCS) under 5% CO₂ in air at 37°C. After reaching confluence, cells were passaged. Passage 6 cells were trypsinized, suspended in Cell Banker 1 (Wako) and stored as frozen aliquots. After thawing, the cells were cultured in HEPES-buffered TCM-199 with Earle's salts (Gibco) supplemented with 100 µg/mL amikacin sulfate and 10% FCS, and used as donors for nuclear transfer between passages 7 and 15 of culture. The cells were allowed to grow to confluency and continued to culture for an additional 5–6 days without a change of medium. A single cell suspension was prepared by standard trypsinization immediately prior to nuclear transfer.

Nuclear transfer

HEPES-buffered TCM-199 with the osmolarity adjusted to 300 mOsm by adding sucrose supplemented with 10% FCS was used as the basic medium (BM) for nuclear transfer. In vitro-matured and denuded oocytes were cultured in 100 µL of BM supplemented with 0.2 µg/mL demecolcine (Sigma) and 20 mmol/L sucrose for 0.5–1 h (Miyoshi et al. 2008a). Oocytes with a protruding membrane were transferred into BM supplemented with 0.2 µg/mL demecolcine and 5 µg/mL cytochalasin B. The protrusion and the first polar body were removed by aspiration with a 15-µm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each enucleated oocyte using the same glass pipette. Cell-oocyte complexes were transferred to BM and kept in 5% CO₂ in air at 38.5°C until fusion.

The chamber for fusion was a 60-mm dish filled with 7 mL of fusion medium composed of 250.3 mmol/L sorbitol, 0.5 mmol/L Mg(CH₃COO)₂, 0.3 mmol/L HEPES and 0.2% (w : v) BSA. Two stainless-steel wires (100 µm diameter) were used as electrodes and they were attached to micromanipulators. The single cell-oocyte complex was sandwiched between the electrodes and oriented with the contact surface between the cytoplast and the donor cell perpendicular to the electrodes. Membrane fusion was induced by applying a single direct-current pulse of 25 V for a duration of 20 µs with a prepulse of alternating-current field of 5 V, 1 MHz for 2 s using an LF 101 Fusion Machine (Nepa Gene, Chiba, Japan). Following the fusion pulse, the complexes were cultured for 2 h in 100 µL of modified PZM-3 (mPZM-3) (Sato et al. 2005) until activation. Fusion was determined by microscopic examination at 1 h after applying the pulse.

Activation and culture of oocytes and embryos

In vitro-matured and denuded oocytes or fused embryos in groups of 30–60 were washed twice in activation medium composed of 250.3 mmol/L sorbitol, 0.5 mmol/L Ca(CH₃COO)₂, 0.5 mmol/L Mg(CH₃COO)₂ and 0.1% BSA (Miyoshi et al. 2008b) and then transferred to a well of a 4-well plate (Nunc, Roskilde, Denmark) containing 800 µL of the same medium. The ultrasound probe (8 mm diameter) of a KTAC-3000 Sonopore (Nepa Gene) was inserted directly into the activation medium and the oocytes were exposed to 2872-kHz ultrasound at an intensity of 45 V for 30 s with 10 Hz burst rate and 30% duty cycle. The embryos were exposed to the same ultrasound except that the intensity and duty cycle were changed to 65 V and 10%, respectively. A miniature stirrer was placed within the well and spun at 300 rpm during ultrasound exposure. After exposure, the oocytes or embryos were cultured in 50 µL of mPZM-3 containing different concentrations of NaCl or sucrose under 5% CO₂, 5% O₂ and 90% N₂ at 38.5°C. During the first 2 h of the culture, 2.2 µg/mL cytochalasin B was added to each medium to prevent extrusion of a second polar body or polar body-like structure. The oocytes or embryos were assessed for cleavage and blastocyst formation at 2 and 7 days of culture, respectively. At the end of culture, blastocysts were placed on slides with a drop of mounting medium consisting of glycerol and PBS (9:1) containing 100 µg/mL Hoechst 33342 (Sigma). A cover slip was placed on top of the blastocysts and the edge was sealed with nail polish. The number of nuclei was counted under ultraviolet light.

Experimental designs

In experiment 1, effects of high osmolarity induced by increasing NaCl concentration at the early culture stage on the development of oocytes were examined. Oocytes were cultured in mPZM-3 with increased NaCl to 123, 138 or 153 mmol/L for 2 days and then transferred into normal mPZM-3 containing 108 mmol/L NaCl. Control oocytes were cultured in normal mPZM-3 for 7 days. The osmolarities of mPZM-3 containing 108, 123, 138 and 153 mmol/L NaCl were 273, 299, 326 and 361 mOsm, respectively, when measured using a vapor pressure osmometer (Wescor, Logan, UT, USA).

In experiment 2, effects of culture durations in a medium with NaCl-induced high osmolarity on the development of oocytes were examined. Oocytes were cultured in mPZM-3 with increased NaCl to 138 mmol/L (mPZM-3+NaCl) for 1, 2 or 3 days and then transferred into normal mPZM-3 containing 108 mmol/L NaCl. Control oocytes were cultured in normal mPZM-3 for 7 days.

In experiment 3, effects of timing of return into a medium with NaCl-induced high osmolarity from one with low osmolarity on the development of oocytes were examined. Oocytes were cultured in mPZM-3+NaCl for 2 days and then transferred into mPZM-3 for an additional 1, 2, 3 or 5 days of culture. The oocytes cultured in mPZM-3 for 1, 2 or 3 days were returned into mPZM-3+NaCl and cultured for an additional 4, 3 or 2 days, respectively. Some oocytes were cultured in mPZM-3 or mPZM-3+NaCl for 7 days.

In experiment 4, effects of high osmolarity induced by adding sucrose at the early culture stage on the development of oocytes were examined. Oocytes were cultured in mPZM-3 with 25, 50 or 75 mmol/L sucrose for 2 days and then transferred into normal mPZM-3 containing 0 mmol/L sucrose. Control oocytes were cultured in normal mPZM-3 for 7 days. The osmolarities of mPZM-3 with 0, 25, 50 and 75 mmol/L sucrose were 273, 296, 318 and 344 mOsm, respectively.

In experiment 5, effects of the change of osmolarity and medium exchange at 2 days of culture on the development of oocytes were examined. Oocytes were cultured in mPZM-3, mPZM-3+NaCl or mPZM-3 with 50 mmol/L sucrose (mPZM-3+sucrose). Some oocytes of each treatment were transferred into mPZM-3 at 2 days of culture.

In experiment 6, effects of high osmolarity induced by increasing NaCl concentration at the early culture stage on the development of SCNT embryos were examined. SCNT embryos were cultured in mPZM-3 or mPZM-3+NaCl. The embryos cultured in mPZM-3+NaCl were transferred into mPZM-3 at 2 days of culture.

Statistical analysis

All percentage data were subjected to an arcsin transformation in each replicate. The transformed values and numbers of cells in blastocysts were analyzed by one-way or two-way analysis of variance followed by Fisher's protected least significant difference test. All data were expressed as mean ± SEM and a probability of P < 0.05 was considered statistically significant.

RESULTS

Experiment 1

As shown in Table 1, there were no significant differences in the cleavage rates (29.7–63.2%) of oocytes among different concentrations of NaCl. However, the blastocyst formation rates (30.3–32.0%) of oocytes at 138–153 mmol/L NaCl were significantly (P < 0.01) higher than that (8.0%) of oocytes at 108 mmol/L NaCl. The mean numbers of cells (56.6–72.4 cells) in the blastocysts were not affected by different concentrations of NaCl.

Table 1. Parthenogenetic development of pig oocytes cultured in mPZM-3 containing different concentrations of NaCl for the first 2 days†

Concentration of NaCl (mmol/L)‡	No. of oocytes cultured	Mean % ± SEM§ of oocytes developed to		Mean no. ± SEM of cells in blastocysts
Concentration of NaCl (mmol/L)‡	No. of oocytes cultured	≥2-cell (2)¶	Blastocyst (7)¶	Mean no. ± SEM of cells in blastocysts
108	64	29.7 ± 7.8	8.0 ± 1.8^a	72.4 ± 6.7
123	66	46.1 ± 8.1	17.9 ± 4.1^a,b	56.6 ± 3.8
138	66	63.2 ± 8.1	32.0 ± 5.5^b	60.9 ± 4.0
153	65	60.0 ± 11.6	30.3 ± 5.2^b	57.7 ± 5.0

† Experiments were repeated three times.
‡ Oocytes were cultured in mPZM-3 with increased NaCl to 123, 138 or 153 mmol/L for 2 days and then transferred into normal mPZM-3 containing 108 mmol/L NaCl. Control oocytes were cultured in normal mPZM-3 for 7 days.
§ §Percentage per oocytes cultured.
¶ ¶Numbers in parentheses indicate the time of examination (days of culture).
^a,b Values with different superscripts are significantly different (at least P < 0.05).

Experiment 2

The cleavage rates (54.3–63.6%) of oocytes cultured in mPZM-3+NaCl for 1–3 days were significantly (P < 0.05) higher than that (28.3%) of oocytes cultured in normal mPZM-3 (Table 2). Moreover, the blastocyst formation rate (31.1%) of oocytes cultured in mPZM-3+NaCl for 2 days was significantly (P < 0.01) higher than that (9.3%) of oocytes cultured in normal mPZM-3. There were no significant differences in the mean numbers of cells (50.0–67.6 cells) in the blastocysts among different durations of culture in mPZM-3+NaCl.

Table 2. Parthenogenetic development of pig oocytes cultured in mPZM-3 with increased NaCl to 138 mmol/L (mPZM-3+NaCl) for the first 1, 2 or 3 days†

Duration of culture in mPZM-3+NaCl (days)‡	No. of oocytes cultured	Mean % ± SEM§ of oocytes developed to		Mean no. ± SEM of cells in blastocysts
Duration of culture in mPZM-3+NaCl (days)‡	No. of oocytes cultured	≥2-cell (2)¶	Blastocyst (7)¶	Mean no. ± SEM of cells in blastocysts
0	80	28.3 ± 5.1^a	9.3 ± 2.9^a	67.6 ± 6.8
1	78	55.3 ± 3.8^b	22.2 ± 1.8^a,b	64.9 ± 6.2
2	78	54.3 ± 2.1^b	31.1 ± 6.5^b	62.4 ± 4.4
3	76	63.6 ± 10.4^b	20.0 ± 3.6^a,b	50.0 ± 3.9

† Experiments were repeated four times.
‡ Oocytes were cultured in mPZM-3+NaCl for 1, 2 or 3 days and then transferred into normal mPZM-3 containing 108 mmol/L NaCl. Control oocytes were cultured in normal mPZM-3 for 7 days.
§ §Percentage per oocytes cultured.
¶ ¶Numbers in parentheses indicate the time of examination (days of culture).
^a,b Values with different superscripts within each column are significantly different (at least P < 0.05).

Experiment 3

As shown in Table 3, there were no significant differences in the cleavage rates (42.5–82.2%) of oocytes among different media and culture durations. When oocytes were returned into mPZM-3+NaCl after culture in mPZM-3 for 1 or 2 days, the blastocyst formation rates (16.2–17.3%) did not differ from those (12.1–12.2%) of oocytes cultured in mPZM-3 or mPZM-3+NaCl for 7 days. However, the blastocysts formation rate (30.2%) of oocytes returned into mPZM-3+NaCl after culture in mPZM-3 for 3 days was significantly (P < 0.05) higher than those (12.1–12.2%) of oocytes cultured in mPZM-3 or mPZM-3+NaCl for 7 days and did not differ from that (36.1%) of oocytes cultured in mPZM-3 for 5 days after transfer from mPZM-3+NaCl. There were no significant differences in the mean numbers of cells (39.4–53.6 cells) in the blastocysts among different media and culture durations.

Table 3. Effects of timing of return into mPZM-3 with increased NaCl to 138 mmol/L (mPZM-3+NaCl) from mPZM-3 on parthenogenetic development of pig oocytes that were cultured in mPZM-3+NaCl for the first 2 days and then transferred into mPZM-3†

Medium and culture duration (days)	No. of oocytes cultured	Mean % ± SEM‡ of oocytes developed to		Mean no. ± SEM of cells in blastocysts
Medium and culture duration (days)	No. of oocytes cultured	≥2-cell (2)§	Blastocyst (7)§	Mean no. ± SEM of cells in blastocysts
mPZM-3 (7)	61	42.5 ± 7.5	12.2 ± 3.3^a	53.6 ± 10.3
mPZM-3+NaCl (2)→mPZM-3 (1)→mPZM-3+NaCl (4)	63	75.2 ± 7.2	16.2 ± 1.9^a	43.4 ± 4.3
mPZM-3+NaCl (2)→mPZM-3 (2)→mPZM-3+NaCl (3)	61	62.1 ± 6.8	17.3 ± 4.7^a,b	48.3 ± 2.5
mPZM-3+NaCl (2)→mPZM-3 (3)→mPZM-3+NaCl (2)	63	68.8 ± 10.2	30.2 ± 6.9^b,c	40.5 ± 1.8
mPZM-3+NaCl (2)→mPZM-3 (5)	61	82.2 ± 8.9	36.1 ± 2.2^c	42.0 ± 1.7
mPZM-3+NaCl (7)	61	72.2 ± 9.5	12.1 ± 4.2^a	39.4 ± 3.3

† Experiments were repeated three times.
‡ ‡Percentage per oocytes cultured.
§ §Numbers in parentheses indicate the time of examination (days of culture).
^a–c Values with different superscripts are significantly different (at least P < 0.05).

Experiment 4

As shown in Table 4, there were no significant differences in the cleavage rates (32.6–62.3%) of oocytes among different concentrations of sucrose. However, the blastocyst formation rate (33.3%) of oocytes at 50 mmol/L sucrose was significantly (P < 0.01) higher than that (10.5%) of oocytes at 0 mmol/L sucrose. The mean numbers of cells (49.0–61.4 cells) in the blastocysts were not affected by different concentrations of sucrose.

Table 4. Parthenogenetic development of pig oocytes cultured in mPZM-3 containing different concentrations of sucrose for the first 2 days†

Concentration of sucrose (mmol/L)‡	No. of oocytes cultured	Mean % ± SEM§ of oocytes developed to		Mean no. ± SEM of cells in blastocysts
Concentration of sucrose (mmol/L)‡	No. of oocytes cultured	≥2-cell (2)¶	Blastocyst (7)¶	Mean no. ± SEM of cells in blastocysts
0	98	32.6 ± 8.3	10.5 ± 3.0^a	58.6 ± 6.5
25	97	48.2 ± 9.1	17.1 ± 4.4^a	61.4 ± 6.5
50	97	62.3 ± 10.7	33.3 ± 5.7^b	54.3 ± 3.4
75	95	61.4 ± 10.2	23.4 ± 4.8^a,b	49.0 ± 2.9

† Experiments were repeated five times.
‡ Oocytes were cultured in mPZM-3 with 25, 50 or 75 mmol/L sucrose for 2 days and then transferred into normal mPZM-3 containing 0 mmol/L sucrose. Control oocytes were cultured in normal mPZM-3 for 7 days.
§ §Percentage per oocytes cultured.
¶ ¶Numbers in parentheses indicate the time of examination (days of culture).
^a,b Values with different superscripts are significantly different (at least P < 0.05).

Experiment 5

The cleavage rates (76.3–88.7%) of oocytes cultured in mPZM-3+NaCl or mPZM-3+sucrose were significantly (P < 0.01) higher than those (39.9–43.6%) of oocytes cultured in mPZM-3 (Table 5). There were no significant differences in the blastocyst formation rates (7.7–16.4%) of oocytes among different media when they were cultured for 7 days without the exchange of medium. The blastocyst formation rates (16.4–21.1%) of oocytes cultured in mPZM-3 were not affected by the exchange of medium at 2 days of culture. However, when oocytes were cultured in mPZM-3+NaCl or mPZM-3+sucrose for 2 days and then transferred into mPZM-3, their blastocyst formation rates (45.2–54.1%) were significantly (P < 0.05) higher than those (7.7–16.4%) of oocytes cultured in mPZM-3, mPZM-3+NaCl or mPZM-3+sucrose consecutively. The mean numbers of cells (41.4–52.6 cells) in the blastocysts were not affected by different media and the exchange of medium.

Table 5. Effects of change of osmolarity and medium change at 2 days of culture on parthenogenetic development of pig oocytes†

Medium‡	Transfer into mPZM-3 at 2 days of culture	No. of oocytes cultured	Mean % ± SEM§ of oocytes developed to		Mean no. ± SEM of cells in blastocysts
Medium‡	Transfer into mPZM-3 at 2 days of culture	No. of oocytes cultured	≥2-cell (2)¶	Blastocyst (7)¶	Mean no. ± SEM of cells in blastocysts
mPZM-3	–	65	43.6 ± 7.6^a	16.4 ± 4.4^a,b	46.9 ± 3.8
mPZM-3	+	65	39.9 ± 0.9^a	21.1 ± 2.4^a	42.9 ± 2.2
mPZM-3+NaCl	–	63	87.4 ± 2.5^b	12.6 ± 0.4^a,b	52.6 ± 6.0
mPZM-3+NaCl	+	65	83.3 ± 2.1^b	54.1 ± 6.8^c	43.3 ± 1.5
mPZM-3+sucrose	–	62	88.7 ± 0.9^b	7.7 ± 2.4^b	48.0 ± 5.9
mPZM-3+sucrose	+	66	76.3 ± 7.1^b	45.2 ± 2.9^c	41.4 ± 1.2

† Experiments were repeated three times.
‡ mPZM-3+NaCl, mPZM-3 with increased NaCl to 138 mmol/L; mPZM-3+sucrose, mPZM-3 with 50 mmol/L sucrose.
§ §Percentage per oocytes cultured.
¶ ¶Numbers in parentheses indicate the time of examination (days of culture).
^a–c Values with different superscripts within each column are significantly different (at least P < 0.05).

Experiment 6

As shown in Table 6, the cleavage rate (62.6%) of embryos cultured in mPZM-3+NaCl was significantly (P < 0.01) higher than that (39.8%) of embryos cultured in mPZM-3. When embryos were cultured in mPZM-3+NaCl for 2 days and then transferred into mPZM-3, their blastocyst formation rate (18.1%) was significantly (P < 0.05) higher than that (10.9%) of embryos cultured in mPZM-3. There was no significant difference in the mean numbers of cells (38.4–42.5 cells) in the blastocysts between different culture conditions.

Table 6. Development of miniature pig somatic cell nuclear transfer embryos cultured in mPZM-3 containing different concentrations of NaCl for the first 2 days†

Medium and culture duration (days)‡	No. of embryos cultured	Mean % ± SEM§ of embryos developed to		Mean no. ± SEM of cells in blastocysts
Medium and culture duration (days)‡	No. of embryos cultured	≥2-cell (2)¶	Blastocyst (7)¶	Mean no. ± SEM of cells in blastocysts
mPZM-3 (7)	95	39.8 ± 3.7^a	10.9 ± 0.9^a	42.5 ± 3.6
mPZM-3+NaCl (2)→mPZM-3 (5)	94	62.6 ± 2.7^b	18.1 ± 2.2^b	38.4 ± 1.1

† Experiments were repeated five times.
‡ mPZM-3+NaCl, mPZM-3 with increased NaCl to 138 mmol/L.
§ §Percentage per embryos cultured.
¶ ¶Numbers in parentheses indicate the time of examination (days of culture).
^a,b Values with different superscripts within each column are significantly different (at least P < 0.05).

DISCUSSION

The results of the present study show that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound stimulation.

In the present study, both the NaCl- and sucrose-induced changes of osmolarity from 318–326 mOsm to 273 mOsm at 2 days after activation by ultrasound supported the parthenogenetic development into blastocysts of pig oocytes. In addition, the blastocyst formation rate of oocytes was not affected by the exchange of medium at 2 days of culture. These results indicate the change of osmolarity, and not the change of NaCl concentration or medium exchange, is responsible for maintaining the development of pig oocytes. The optimal osmolarity of a medium for early embryonic development is species-specific and depends on the developmental stages of the embryos. For example, the development of rabbit 2-cell embryos occurs at 230–339 mOsm (Naglee et al. 1969) and the possible development of hamster 2-cell and 8-cell embryos has also been reported over a wide range of osmolarities, 250–325 mOsm (McKiernan & Bavister 1990) and 225–300 mOsm (Bavister et al. 1983), respectively. In contrast, more limited ranges of osmolarity are reported for the optimal development of mouse 1-cell (250–280 mOsm) (Whitten 1971) and 2-cell (272–280 mOsm) (Biggers & Brinster 1965; Brinster 1965) embryos. Similarly, rat and rabbit 1-cell embryos are sensitive to the osmolarity of the culture medium, showing optimal development at 244–264 mOsm (Miyoshi et al. 1994, 1995) and 250–270 mOsm (Li & Foote 1995), respectively. Cattle and pig in vitro-matured and -fertilized oocytes also successfully develop in the medium with the osmolarity adjusted to 250–270 mOsm (Liu & Foote 1996) and 270–290 mOsm (Li et al. 2007), respectively. On the basis of the reports described above, it was considered that mammalian 1-cell embryos prefer hypotonic media to isotonic or hypertonic media. In the case of pigs, PZM-3 with the osmolarity adjusted to 260–280 mOsm (Li et al. 2007; Hwang et al. 2008) has been the most common culture medium not only for 1-cell embryos produced in vivo and in vitro but also for artificially activated oocytes and cloned embryos produced by nuclear transfer. This osmolarity is clearly lower than the physiological one because the osmolarity of pig oviductal fluid was reported to be 318–321 mOsm (Li et al. 2007). A similarly high osmolarity (304–333 mOsm) has been determined for Clawn miniature pig oviductal fluid (Mizobe Y. et al., 2009, unpublished data). Therefore, the medium with the osmolarity adjusted to 318–326 mOsm should be close to physiological conditions compared to that with 273 mOsm. Recent reports have indicated that pig in vitro-fertilized embryos (Li et al. 2007), activated oocytes (Nguyen et al. 2003; Im et al. 2005) and cloned embryos produced by nuclear transfer (Im et al. 2005; Hwang et al. 2008) prefer media with physiological osmolarity to hypotonic media during the early developmental stages. The results of the present study are consistent with these reports. Culture in a medium with high osmolarity during the first 2 days improved the development of electrically activated pig oocytes and SCNT embryos by reducing their fragmentation rate (Im et al. 2005). The same culture condition reduced the apoptosis of pig SCNT embryos activated by electric stimulation, thus resulting in improved development (Hwang et al. 2008). The development of pig oocytes and miniature pig SCNT embryos cultured under the same condition after activation by ultrasound stimulation in the present study may be improved by these reasons.

The results of the present study indicated that pig oocytes must be transferred into a hypotonic medium at 2 days of culture and continued to culture in the medium until 5 days of culture for maintaining their parthenogenetic development, suggesting that they are sensitive to high osmolarity at the 2-cell to early blastocyst stages. It is considered that there are amino acids serving as organic osmolytes, such as glycine (Dawson & Baltz 1997), glutamine (Biggers et al. 1993) and β-alanine (Hammer & Baltz 2003), in the female reproductive tract and they protect embryos from high physiological osmolarity. The concentrations of glycine in the oviductal fluid at 3 days after estrus and uterine fluid at 5 days after estrus of mated gilts are 41 and 38 times higher, respectively, than that in mPZM-3 (Li et al. 2007). Addition of these amino acids at adequate concentrations during 2–5 days of culture probably improves the parthenogenetic development of pig oocytes in mPZM-3 with the osmolarity adjusted to 318–326 mOsm.

In conclusion, we have established an effective culture system to support the in vitro development of miniature pig SCNT embryos activated by ultrasound stimulation. This system would be useful not only for basic research to clarify the mechanism controlling the development of SCNT embryos, but also for the nonsurgical embryo transfer of them to recipient females at the morula and blastocyst stages.

ACKNOWLEDGMENTS

We express gratitude to the staff of the Kagoshima City Meat Inspection Office and Meat Center Kagoshima (Kagoshima, Japan) for supplying pig ovaries. The present study was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 17100007 to MY and 19580328 to KM).

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Citing Literature

Volume81, Issue4

August 2010

Pages 453-460

This article also appears in:

Embryo Technologies in Livestock

Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

In vitro maturation of oocytes

Donor cells

Nuclear transfer

Activation and culture of oocytes and embryos

Experimental designs

Statistical analysis

RESULTS

Experiment 1

Experiment 2

Experiment 3

Experiment 4

Experiment 5

Experiment 6

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Citing Literature

References

Information

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Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

In vitro maturation of oocytes

Donor cells

Nuclear transfer

Activation and culture of oocytes and embryos

Experimental designs

Statistical analysis

RESULTS

Experiment 1

Experiment 2

Experiment 3

Experiment 4

Experiment 5

Experiment 6

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

Citing Literature

References

Related

Information