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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004812
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Biology of Reproduction 67, 1688-1698 (2002)
DOI: 10.1095/biolreprod.102.004812 © 2002 Society for the Study of Reproduction, Inc.

Embryo

Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro1

Nguyen Van Thuana, Hiroshi Harayamaa, and Masashi Miyake2,a

a Department of Life Science, Graduate School of Science and Technology, Kobe University, Kobe City, Hyogo 657-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01–5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05–5.0 mg/ml of PVA (49%–53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%–20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3–5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 ± 2.7) and inner cell mass (12 ± 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.

developmental biology, early development, embryo


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The in vitro culture system is one of the most useful strategies for understanding the mechanisms of early development and establishing the optimum culture conditions. A "chemically defined medium" has meant that highly purified reagents were used without biological fluids or the partially purified products from them. Thus, a medium including fractionated BSA was conceived of as a chemically defined medium [1]. Because polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) have the ability to support the viability and development of mammalian gametes and embryos in vitro [2–8], it has become possible to establish chemically defined and semidefined culture systems for studying the sensitivities and requirements during early development of mammalian embryos.

Many researchers have reported that exogenous amino acids show beneficial effects on preimplantational development of cow [7, 9–14], mouse [15–21], sheep [22, 23], rat [24, 25], rabbit [26, 27], human [28, 29], and hamster [30–32] embryos. Bavister and Arlotto [33] have reported the effects of single amino acid on hamster embryos. Those authors suggested that some amino acids (glycine, cystine, and lysine) were stimulatory, whereas others (cystine, proline, serine, threonine, histidine, alanine, hydroxyproline, leucine, aspartic acid, and methionine) were neutral or inhibitory (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine). Lane and Gardner [18] reported that the amino acid requirements were dependent on the developmental stage in the mouse and that nonessential, but not essential, amino acids with glutamine stimulated the early cleaving embryo. Moreover, the presence of essential amino acids in culture medium during the early cleavage stages resulted in a decreased number of cells in the subsequent blastocysts and a decline of embryo viability following transfer [18]. Steeves and Gardner [13] showed the temporal and differential effects of amino acids on development of the bovine embryo from zygote to blastocyst.

Recently, successful in vitro development of both in vivo- and in vitro-fertilized porcine oocytes to the blastocyst stage has been reported [34–39]. Nonetheless, detailed effects of amino acids on the early development of porcine embryos to the blastocyst stage, especially during the early cleaving period, are still unclear. With in vitro fertilization, high developmental ability to the blastocyst stage is hardy expected because of the high incidence of polyspermy in the pig. Our previous reports have shown that electroactivated porcine oocytes that had been matured in vitro can develop to the blastocyst stage in vitro; furthermore, the characteristics of development of porcine parthenogenetic diploids to the blastocyst stage resembled those of in vitro fertilized eggs [40–43]. These results suggested that parthenogenetic diploids could be used as model embryos instead of fertilized oocytes for studies regarding the early development and establishment of culture systems in the pig. The present study was designed to investigate the stimulatory and inhibitory effects of amino acid components on the development of parthenogenetic diploids to the blastocyst stage to characterize early development in the pig.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte Collection and In Vitro Maturation

Porcine ovaries, which were collected at local abattoirs, were kept at 20–30°C and transported to the laboratory within 2 h. Ovaries were washed once with 0.2% (w/v) cetylmethylammonium bromide (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and twice with Ca2+- and Mg2+-free Dulbecco PBS containing 0.1% (w/v) polyvinyl alcohol (PBS-PVA; Sigma Chemical Co., St. Louis, MO). Follicles with a diameter of 4–6 mm were dissected out in PBS-PVA. Only those that had a round form and clear follicular fluid without detachment of granulosa cells and an oocyte were ruptured. Oocytes with a cumulus oophorus and a portion of parietal granulosa cells were removed from follicles in 25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethane sulfonic acid (Hepes; Dojindo Laboratory Co., Ltd., Kumamoto, Japan)-buffered TCM-199 medium (Earle salt; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 0.1% (w/v) PVA (Hepes-TCM). The oocyte-cumulus-granulosa cell complexes (OCGCs) were then washed with fresh Hepes-TCM, followed by transfer to a maturation medium. The maturation medium (mTCM) was based on bicarbonate-buffered TCM-199 containing 10% (v/v) heat-treated fetal calf serum (Biocell, Inc., Carson, CA), 0.1 mg/ml of sodium pyruvate (Nacalai Tesque, Inc., Kyoto, Japan), 0.08 mg/ml of kanamycin sulfate (Sigma), 2.2 mg/ml of sodium bicarbonate (Nacalai Tesque), and 0.1 IU/ml of human menopausal gonadotropin (Pergonal; Serono, Rome, Italy). Before maturation culture, OCGCs were morphologically assessed under a stereomicroscope, and only oocytes that had a homogeneous ooplasm with a compact and vivid cumulus oophorus were selected. Each group of fewer than 50 oocytes was transferred to 2.0 ml of the maturation medium in a polystyrene dish (35 x 10 mm; Becton Dickinson Labware, Lincoln Park, NJ). Two theca shells collected from healthy follicles (diameter, 4–6 mm) were added to the dish after removal of follicular fluid and granulosa cells using two pairs of fine forceps. The mixtures of OCGCs and follicle shells were cultured in a CO2 incubator at 38.5°C under humidified air containing 5% CO2 with gentle agitation for 48 h.

Electrostimulation of In Vitro-Matured Oocytes and Treatment with Cytochalasin B

After maturation culture, 200 µl of 0.1% (w/v) hyaluronidase (Type I-S; Sigma) in PBS-PVA were added to the maturation medium. Several minutes later, oocytes were transferred to 100-µl drops of mTCM, which had been previously covered with paraffin oil (analytical grade for analysis of amino acids; Nacalai Tesque), in a polystyrene culture dish (60 x 15 mm; Becton Dickinson Labware). They were freed from almost all cumulus cells by gentle pipetting. Denuded oocytes were transferred to new drops of mTCM to rinse off the hyaluronidase solution. Extrusion of the first polar body was inspected under an inverted microscope to assess oocyte maturation. Oocytes with the first polar body were washed three times in a field solution composed of 0.3 mM mannitol, 0.1 mM MgSO4, and 0.05 mM CaCl2 [44] and supplemented with 0.01% (w/v) PVA. Each group of less than 20 oocytes was transferred to 100 µl of the field solution between two parallel stainless-steel electrodes in a chamber (FTC-03; Shimadzu Co., Ltd., Kyoto, Japan). A single direct-current pulse of 1500 V/cm for 100 µsec was supplied from an electrocell manipulator (ECM 2000; BTX, Inc., San Diego, CA). Electrostimulated oocytes were immediately transferred to fresh mTCM and washed three times. For development, 10–15 activated oocytes were washed four times in each media described in culture methods, then cultured for 4 h in their respective culture media, which contained 5.0 µg/ml of cytochalasin B (Sigma), to produce parthenogenetic diploids.

Media for Culture and Experimental Designs

The basic culture medium was Whitten medium [45] containing 0.5 mg/ml of hyaluronic acid (mWM; from rooster comb; provided by Seikagaku Co., Tokyo, Japan) [40, 46]. The osmolarity of the medium was 290 mOsmol (mWM290) for the first 48 h of culture after electrostimulation (El-St) and 256 mOsmol (mWM256) from 48 h to the end of culture in all experiments except for experiment 2 [47]. Osmolarity of mWM290 was adjusted by the concentration of sodium chloride. In all experiments except for experiment 6, in which mWM290 for the first 48 h and mWM256 from 48 h to the end of culture was used, control media for both culture periods were supplemented with 4 mg/ml of BSA (BSA-mWMs; Intergen, New York, NY). The experimental designs of the present study are summarized in Figure 1.



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  		FIG. 1. Schematic explanation of culture methods

Essential amino acids without glutamine (E-AA) for Eagle basal medium (BME) and nonessential amino acids (NE-AA) (Table 1) for minimum essential medium were supplied by Sigma. Each E-AA used in experiments 4 and 5 was purchased from Sigma and was added to PVA-mWM at the same concentration of BME.


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  		TABLE 1. Components of E-AA for Eagle basal medium and NE-AA for minumum essential medium

Nuclear Staining and Differential Staining of Inner Cell Mass and Trophectoderm Cells

In experiment 1, all blastocysts were washed three times with PBS-PVA at the end of culture, placed in 300 µl of 2.5% (w/v) paraformaldehyde in PBS-PVA for 30 min, and then washed three times with PBS-PVA. Blastocysts were stained with 2 µg/ml of bisbenzimide (Hoechst 33342; Polysciences, Inc., Warrington, PA) in PBS-PVA for 15–20 min, then washed three times with PBS-PVA. Next, they were put on a vaseline-spotted slide glass with a drop of the dye solution and whole-mounted with a cover slip. The number of nuclei in each blastocyst was counted under an epifluorescence microscope (Optiphoto; Nikon, Tokyo, Japan).

In experiment 6, the number of nuclei in inner cell mass (ICM) and trophectoderm (TE) cells of the diploids at the blastocyst stage were determined by using double-staining methods [48]. Blastocysts were treated with 0.5% protease (Sigma) in Hepes-buffered Tyrode albumin lactate pyruvate (TALP-Hepes) for 1 min to remove the zona pellucida. Zonae pellucidae of hatching blastocysts were removed by pipetting. Zona pellucida-free diploid blastocysts were incubated on ice for 10–15 min in 50 µl of TALP-Hepes supplemented with 10 mM trinitrobenzen sulfonic acid (Sigma) and 4 mg/ml of PVP. Subsequently, blastocysts were quickly washed in TALP-Hepes and then incubated in TALP-Hepes supplemented with 0.1 mg/ml of anti-pig whole serum (Sigma) for 10 min at 37°C. After washing, blastocysts were incubated for 10–15 min in 50 µl of TALP-Hepes medium supplemented with 10 µg/ml of propidium iodide (Sigma) and 10% (v/v) guinea pig serum (Sigma). They were quickly washed in TALP-Hepes, then transferred to 50 µM bisbenzimide (Sigma) in absolute alcohol and kept overnight at 4°C. The stained blastocysts were carefully mounted in glycerol on a microscopic slide and observed under an epifluorescence microscope. Red- to pink-colored nuclei were classified as TE cells, and blue nuclei were classified as ICM cells.

Statistical Analysis

The frequencies at which diploids developed to each stage were subjected to an arcsine transformation for each replication. The transformed values were analyzed using one-way ANOVA followed by the Tukey test for multiple comparisons. Numbers of nuclei in blastocysts and in the ICM were also analyzed by the Tukey multiple-comparison test following one-way ANOVA. A probability of less than 0.05 was considered to indicate statistical significance. Values are reported as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Effects of the Concentration of PVA Used as a Substitute for BSA on Development of Porcine Parthenogenetic Diploids

The initial experiment was designed to examine the optimum concentration of PVA as a substitute for BSA in the culture medium on development of porcine parthenogenetic diploids to the blastocyst stage. Diploids were cultured in mWM290 including 0.01, 0.05, 0.1, 0.5, 1.0, or 5.0 mg/ml of PVA for the first 48 h and then in mWM256 including the same concentration of PVA as used for the first culture, and the ability of media to support the development of diploids to the blastocyst stage was compared with that of BSA-mWMs.

The frequencies at which diploids developed to the 2-cell and to the 3- to 4-cell stages at 24 and 48 h after El-St were not significantly different among experimental and control groups (data not shown). At 72 h after El-St, the frequency of development to the 5- to 8-cell stage was lower in 0.01 mg/ml of PVA (8%) than in the control BSA (26%, P < 0.05) (Fig. 2). At 120 h, the frequency of diploids developing to the morula stage was significantly lower in 0.01 mg/ml of PVA (33%) than in 0.05, 0.1, 0.5, 1.0, and 5.0 mg/ml of PVA or in the control (50%–65%, P < 0.05). At 144 h, the frequency with which diploids reached the blastocyst stage was lower in 0.01 (15%), 0.05 (30%), and 0.1 mg/ml of PVA (28%) than in 0.5, 1.0, and 5.0 mg/ml of PVA or the control (47%–58%, P < 0.05). The frequency of abnormal diploids was significantly higher in 0.01 mg/ml of PVA than in 0.05 to 5 mg/ml of PVA at 168 h after El-St. The total frequencies with which diploids developed to the blastocyst and expanded-blastocyst stages were similar among the control and groups with more than 0.5 mg/ml of PVA. The frequency with which diploids developed to the expanded-blastocyst stage in all concentrations of PVA, however, was significantly lower (5%–20%) than that in control BSA (39%, P < 0.05) (Figs. 2 and 3).



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  		FIG. 2. Effects of the concentration of PVA used as a substitute for BSA on development of porcine parthenogenetic diploids. Diploids were cultured in mWM at 290 mOsmol until 48 h and subsequently cultured in mWM at 256 mOsmol in each additive. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. The number at the top of each column at 72 h is the number of oocytes evaluated. Values among different letters within each developmental stage are significantly different (P < 0.05).



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  		FIG. 3. Porcine parthenogenetic diploid blastocysts 168 h after El-St. A) Blastocysts derived from electrostimulated porcine oocytes that had been cultured in isotonic mWM290 medium until 48 h and subsequently cultured in hypotonic mWM256 up to 168 h after El-St under the presence of 0.5 mg/ml of PVA instead of 4.0 mg/ml of BSA. B) Blastocysts derived from electrostimulated porcine oocytes that had been cultured in isotonic mWM290 medium until 48 h and subsequently cultured in hypotonic mWM256 up to 168 h after El-St under the presence of 4.0 mg/ml of BSA.

Table 2 shows the numbers of nuclei of blastocysts at the end of culture. The number of cells was significantly greater in the blastocysts cultured in the control group than in those cultured in any concentration of PVA (P < 0.05).


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  		TABLE 2. Effects of the concentration of PVA as substitutes for BSA in a medium on the number of nuclei in blastocyst at 168 h after El-St.a

Experiment 2: Effects of the Timing of E-AA and NE-AA Addition on Development of Porcine Parthenogenetic Diploids

Based on the results of experiment 1, diploids were cultured in mWM290 containing 0.5 mg/ml of PVA (PVA-mWM290) for 0, 48, or 72 h after El-St, then transferred to 0.5 mg/ml of PVA-mWM256 supplemented with the recommended concentration of E-AA and NE-AA (E+NE-AA). The frequency of diploids developing to the 2-cell stage was not significantly different among the experimental and control groups (data not shown) at 24 h after El-St. At 48 h, a higher percentage of diploids in the 0-h group remained at the 2-cell stage (44%, P < 0.05), whereas almost all diploids in the 48-h, 72-h, and BSA groups had reached the 3- to 4-cell stage (88%, 84%, and 90%, respectively) (Fig. 4). The frequency with which diploids developed to the 3- to 4-cell stage was also significantly lower in the 0-h group (32%) than in the 48-h (62%), 72-h (57%), or control group (71%) at 72 h after El-St (P < 0.05). The frequencies of degenerated diploids were always significantly higher in the 0-h group than in the 48-h, 72-h, or control groups at later than 72 h after El-St (P < 0.05). The frequencies of diploids that developed beyond the morula stage were always significantly lower in the 0-h group than in the other groups at later than 120 h (P < 0.05).



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  		FIG. 4. Effects of the timing of E+NE-AA addition on development of porcine parthenogenetic diploids. Diploids were cultured in mWM containing 0.5 mg/ml of PVA-mWM for 0, 48, or 72 h after El-St, then transferred to their respective group in PVA-mWM supplemented with E+NE-AA up to 168 h after El-St. The number at the top of each column at 48 h indicates the number of oocytes evaluated. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. Values among different letters within each developmental stage are significantly different (P < 0.05)

At 168 h after El-St, the total frequencies of diploids developed to the blastocyst stage and the rates of expanded blastocyst were significantly lower in the 0-h group (22% and 7%, respectively; P < 0.05) than in the 48-h (61% and 39%, respectively), 72-h (57% and 35%, respectively), or control group (60% and 39%, respectively).

Experiment 3: Effects of E-AA and/or NE-AA for the First 48 h of Culture on Development of Porcine Parthenogenetic Diploids

From the results of experiment 2, it was found that the existence of E+NE-AA during the first 48 h of culture after El-St significantly decreased the frequency of development of porcine parthenogenetic diploids to the blastocyst stage. Experiment 3 examined whether the presence of E-AA and/or NE-AA during the first 48 h after El-St caused a decrease in development to the blastocyst stage. Diploids were cultured for the first 48 h after El-St in PVA-mWM290 supplemented with E-AA, NE-AA, E+NE-AA, or without amino acids. Then, all diploids of each group were transferred to PVA-mWM256 including E+NE-AA and were cultured up to 168 h after El-St (Fig. 1).

The frequencies of diploids at the 2-cell and the 3- to 4-cell stages were extremely lower in E-AA (6% and 19%, respectively) than in PVA only (93% and 90%, respectively), NE-AA (94% and 83%, respectively), E+NE-AA (91% and 80%, respectively), and BSA (94% and 86%, respectively; P < 0.05) (Fig. 5). At 72 h, the frequency of 2-cell diploids was still higher in E-AA (42%) than in NE-AA, E+NE-AA, and BSA (8%, 11%, and 5%, respectively; P < 0.05), and the frequency of diploids that developed to the 5- to 8-cell stage was significantly higher in NE-AA than in the other groups (P < 0.05). The frequencies of diploids at the most advanced stage were always lower in groups cultured under the existence of E-AA (Fig. 5, E-AA and E+NE-AA) during the first 48 h than in those cultured with PVA only, NE-AA, or BSA at later than 96 h after El-St. Finally, only a small number of diploids developed to the blastocyst stage in E-AA and E+NE-AA (3% and 20%, respectively) at 168 h. Most of the diploids in E-AA and E+NE-AA had stopped developing at the 4-cell stage, where they remained even at 96 and 120 h after El-St. On the other hand, the frequencies with which diploids developed to the blastocyst stage 168 h after El-St were higher in PVA only, NE-AA, and BSA (63%, 67%, and 68%, respectively) than in E-AA and E+NE-AA (P < 0.05). The frequency of development to the expanded-blastocyst stage was significantly higher in diploids cultured for the first 48 h in PVA, in BSA, and especially in NE-AA (39%, 36%, and 52%, respectively) than in those cultured in E-AA or E+NE-AA (0% and 4%, respectively; P < 0.05).



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  		FIG. 5. Effects of the existence of E-AA and/or NE-AA for the first 48 h of culture on development of porcine parthenogenetic diploids. Diploids were cultured for the first 48 h after El-St in PVA-mWM290 supplemented with E-AA, NE-AA, E+NE-AA, or without amino acids. Then, all diploids of each group were transferred to PVA-mWM256 including E+NE-AA and cultured up to 168 h after El-St. The number at the top of each column at 24 h indicates the number of oocytes evaluated. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. Values among different letters within each developmental stage are significantly different (P < 0.05)

Experiment 4: Effects of Basic, Polar, and Nonpolar E-AA for the First 48 h of Culture after El-St on Development of Porcine Parthenogenetic Diploids

Diploids were cultured for the first 48 h after El-St in PVA-mWM290 supplemented with basic, polar, or nonpolar E-AA at the same concentration of E-AA in BME. Then, all diploids of each group were subsequently cultured in PVA-mWM256 including E+NE-AA up to 168 h after El-St.

The frequency with which diploids developed to the 2-cell stage was extremely lower in nonpolar E-AA (12%) than in basic E-AA (94%), polar E-AA (91%), and BSA (93%) at 24 h after El-St (P < 0.05) (Fig. 6). The same results were also observed for frequencies of the most advanced stages at all periods of observation after 48 h, as was the case in E-AA and E+NE-AA in experiment 3 (Figs. 5 and 6). The total frequencies beyond the blastocyst stage and of the expanded-blastocyst stage did not affect development in diploids that were cultured under the existence of nonpolar E-AA (20% and 11%, respectively) than in those cultured under the existence of basic E-AA (60% and 34%, respectively), polar E-AA (59% and 35%, respectively), and BSA (61% and 36%, respectively) in mWM290 during the first 48 h after El-St. No significant differences were observed in the frequencies beyond the blastocyst stage or of the expanded-blastocyst stage among basic E-AA, polar E-AA, and BSA at 168 h after El-St.



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  		FIG. 6. Effects of the existence of basic, polar, and nonpolar E-AA for the first 48 h culture after El-St on development of porcine parthenogenetic diploids to the blastocyst stage. Diploids were cultured for the first 48 h after El-St in PVA-mWM290 supplemented with basic, polar, or nonpolar E-AA at the same concentration of E-AA in BME. Then, all diploids of each group were subsequently cultured in PVA-mWM256 including E+NE-AA up to 168 h after El-St. Basic indicates basic E-AA (arginine, histidine, and lysine). Polar indicates polar E-AA (tyrosine and threonine). Nonpolar indicates nonpolar E-AA (cystine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and valine). The number at the top of each column at 24 h indicates the number of oocytes evaluated. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. Values among different letters within each developmental stage are significantly different (P < 0.05)

Experiment 5: Effects of a Single Nonpolar E-AA for the First 48 h of Culture after El-St on Development of Porcine Parthenogenetic Diploids

Diploids were cultured for the first 48 h in PVA-mWM290 containing cystine (Cys), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp), or valine (Val) at the same concentration of E-AA in BME. The PVA-mWM290 without amino acids was used as a negative control for the first 48 h of culture. Then, diploids were transferred in their respective groups to PVA-mWM256 supplemented with E+NE-AA for culture up to 168 h after El-St.

At 24 and 48 h after El-St, the addition of Val, Leu, Ile, and Met significantly reduced the development of diploids to the 2-cell (40%, 45%, 69%, and 79%, respectively) and 4-cell stages (48%, 49%, 59%, and 64%, respectively) compared to PVA only (94% and 90%, respectively) and to BSA (94% and 91%, respectively) (Fig. 7). At 120 h after El-St, the frequency of diploids developed to the morula and blastocyst stages was also significantly lower in the presence of Val, Leu, Ile, and Met (morula: 33%, 36%, 42%, and 43%, respectively; blastocyst: 5%, 7%, 5%, and 5%, respectively) than in the presence of PVA only (58% and 14%, respectively; P < 0.05). Finally, the frequencies of totally yielded blastocysts and of expanded blastocysts at 168 h from diploids that were cultured in the presence of Val (27% and 7%, respectively), Leu (34% and 16%, respectively), Ile (38% and 17%, respectively), Met (40% and 21%, respectively), and Phe (47% and 24%, respectively) for the first 48 h after El-St were significantly lower than those in PVA (61% and 39%, respectively) or BSA (67% and 41%, respectively). However, Trp (60% and 31%, respectively) and Cys (58% and 35%, respectively) did not suppress development.



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  		FIG. 7. Effects of the existence of singe nonpolar E-AA for the first 48 h culture after El-St on development of porcine parthenogenetic diploids to the blastocyst stage. Diploids were cultured for the first 48 h in PVA-mWM290 containing each nonpolar E-AA—cystine (Cys), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and valine (Val)—at the same concentration in BME solution or without amino acids. Then, diploids were transferred in their respective groups to PVA-mWM256 supplemented with E+NE-AA for culture up to 168 h after El-St. The number at the top of each column at 24 h indicates the number of oocytes evaluated. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. Values among different letters within each developmental stage are significantly different (P < 0.05)

Experiment 6: Effects of E-AA and/or NE-AA from 48 to 168 h after El-St on Development of 4-Cell Diploids to the Blastocyst Stage

To examine the effects of the existence of amino acids after the 4-cell stage on the development of parthenogenetic diploids, diploids were cultured in PVA-mWM290 including NE-AA for the first 48 h, but then only those at the 3- to 4-cell stage were divided into four groups. Each group of diploids was subsequently cultured in PVA-mWM256 supplemented with E-AA, NE-AA, E+NE-AA, or without amino acids up to 168 after El-St.

At 72 h after El-St (24 h after medium change), the frequencies with which diploids cleaved to the 5- to 8-cell stage were significantly higher in NE-AA and E+NE-AA (41% and 39%, respectively) than in E-AA or PVA only (19% and 14%, respectively; P < 0.05) (Fig. 8). The frequency of diploids developed to the morula stage, however, was higher in E-AA (36%) and E+NE-AA (34%) than in NE-AA (14%) or PVA only (9%) at 96 h after El-St (P < 0.05). The frequency with which diploids reached the blastocyst stage was higher in E-AA (33%) and E+NE-AA (31%) than in NE-AA (14%) or PVA (8%) at 120 h (P < 0.05). The frequency with which diploids reached the expanded-blastocyst stage was also higher in NE-AA (20%) and E+NE-AA (22%) than in E-AA (3%) or PVA (0%) at 144 h after El-St (P < 0.05). At 168 h after El-St, the frequencies of final yields of expanded blastocysts were higher in NE-AA (33%) and E+NE-AA (41%) than in E-AA (17%) or PVA (3%, P < 0.05), and the difference in the frequency of expanded blastocysts between E-AA (17%) and PVA (3%) was significant (P < 0.05).



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  		FIG. 8. Effects of the existence of E-AA and/or NE-AA during culture from 48 to 168 h after El-St on development of the 4-cell diploids to the blastocyst stage. A total of 296 activated oocytes were cultured in PVA-mWM290 including NE-AA for the first 48 h after El-St. Of these, 273 (92.2%) and 256 (86.5%) diploids were at the 2- and the 4-cell embryo stage at 24 and 48 hours after El-St, respectively. Only diploids at the 3- to 4-cell stage were divided into four groups. Each group of diploids was subsequently cultured in PVA-mWM256 supplemented with E-AA, NE-AA, E+NE-AA, or without amino acids up to 168 after El-St. The number at the top of each column at 72 h indicates the number of oocytes evaluated. Abnormal indicates parthenogenetic oocytes with features of degeneration and/or development delay. Values among different letters within each developmental stage are significantly different (P < 0.05)

Table 3 shows the number of nuclei in blastocysts and in ICM at the end of culture. The mean number of nuclei in blastocysts was significantly higher in those that had been cultured under the presence of E-AA (53.7) and E+NE-AA (63.2) than in those cultured in NE-AA (41.6) or PVA only (33.6, P < 0.05). The mean number of ICM nuclei was significantly higher in diploids cultured under the presence of E-AA and E+NE-AA from the 3- to 4-cell stage (12.0 and 13.1, respectively) than in those cultured under the presence of NE-AA (9.7) and the absence of amino acids (7.2, P < 0.05). The difference in the number of nuclei in ICM between NE-AA and PVA was also significant (P < 0.05).


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  		TABLE 3. Effects of E-AA and/or NE-AA from 48 to 168 h after El-St on the mean number of cells in diploids blastocyst and ICM.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that PVA efficiently supports the development of porcine parthenogenetic diploids to the blastocyst stage before expansion. It is clearly shown that the existence of nonpolar E-AA, especially for Val, Leu, Ile, and Met, during the first 48 h after El-St results in a severe delay of first cell division, so that the majority of diploids are trapped in the 4-cell block in the early development of parthenogenetic diploids in the pig. In addition, it is shown that the addition of E-AA from 48 h after El-St (after the 4-cell stage) is effective for increasing the number of cells in the whole blastocysts and ICM, whereas the addition of NE-AA is effective for the expansion of blastocysts in the porcine parthenogenetic diploids. It is also shown that the addition of E+NE-AA from 48 h after El-St promotes the developmental ability of both expansion and number of cells in the blastocysts.

Results of experiment 1 clearly demonstrate that porcine parthenogenetic diploids can develop up to the blastocyst stage in a protein-free medium including 0.05–5.0 mg/ml of PVA as a substitute for BSA, but that the replacement of BSA with PVA alone could not support the expansion of blastocysts or their cell proliferation (Figs. 2 and 3 and Table 2). These results strongly suggest that the presence of amino acids or polypeptides plays an important role in the expansion of the blastocoele. In fact, BSA may provide amino acids to diploids at the morula and/or early blastocyst stage for the synthesis of proteins that are necessary for expansion. It has been discussed that BSA provides free amino acids as fixed nitrogen sources to mouse embryos [2].

The supplementation of amino acids to a culture medium significantly improved the development of hamster [49], mouse [50–52], and cow embryos [7, 9, 10]. However, the addition of a mixture of E-AA was found to inhibit embryo development of mouse [16] and bovine embryos [11]. Recently, Steeves and Gardner [13] reported the temporal and differential effects of amino acids that occur during development of the bovine embryo from zygote to blastocyst. In the present study, the results of experiment 2 clearly showed that the addition of E+NE-AA to a protein-free simple medium including PVA from 48 or 72 h after El-St (after the 4-cell stage) supported the expansion of blastocysts as well as BSA did. The addition of E+NE-AA at the beginning of culture, however, severely impaired the development of diploids to the morula and blastocyst stages (Fig. 4).

The presence of E-AA in a medium during the first 48 h resulted in a severe delay of first cell division, and the majority of diploids ceased development at the 4-cell stage. Meanwhile, NE-AA supported the development of diploids to the blastocyst and expanded-blastocyst stages as well as BSA did. Furthermore, NE-AA partially neutralized the suppression of development by the addition of E-AA.

Because these results of experiment 3 obviously demonstrated that some or all components of E-AA strongly inhibited the early cleavage of parthenogenetic diploids, we investigated which components of E-AA inhibited early cleavage. The results of experiment 4 clearly showed that inhibition of the first and second cleavages was mainly caused by the existence of nonpolar E-AA. It was clearly demonstrated that Val, Leu, Ile, and Met inhibited the early cleavage of diploids in experiment 5. Because only a single nonpolar amino acid was added at the same concentration of E-AA in BME solution, the blocking effect was less obvious with Phe, Trp, and Cys. That single amino acids were less inhibitory than all the E-AA in combination indicates that the E-AA cooperatively inhibit the development of parthenogenetic diploids.

Kim et al. [7] reported that the culture of 1-cell bovine embryos in modified TLP-PVA containing 19 amino acids (without glutamine) resulted in complete inhibition of the first cleavage. Some porcine diploids, however, could develop beyond the 4-cell stage with normal morphology, although normal development was dramatically suppressed in later periods (experiment 2). These inhibiting effects of E-AA on preimplantation development have also been reported in the mouse. The number of implantation sites and living fetuses decreased when mouse zygotes were cultured for 48 h before transfer in a medium supplemented with E-AA with or without glutamine, but not in that supplemented with NE-AA [50]. The presence of E-AA in the culture medium also reduced the beneficial effects of the NE-AA in the mouse [52].

McKiernan et al. [53] reported that the development of hamster embryos and the mean number of cells in the blastocyst were significantly decreased by the presence of Leu, Tyr, Val, Ile, Phe, Arg, Met, or Cys at the 0.5 mM level. Most of those amino acids belong to nonpolar E-AA. Moreover, the presence of Cys, Tyr, Val, Leu, Phe, Arg, Pro, and Trp were found to arrest development of fertilized oocytes beyond the 8-cell stage in the hamster. In the present study, the presence of nonpolar E-AA (more accurately, of Val, Leu, Ile, and Met) inhibited the development of parthenogenetic diploids to the blastocyst in the pig. In the study by McKiernan et al. [53], however, fertilized hamster oocytes were cultured in the same medium, including one of each of the amino acids throughout the culture period, so that the stimulating effects of those amino acids were not recognized during the later stages. In the present study, the effects of the existence of amino acids in a culture medium on the development of porcine diploids were examined at different stages (i.e., the first 48 h and the period from 48 to 168 h after El-St). It was revealed that E-AA inhibited early cleavage before the 4-cell stage in contrast to its effect during the second culture period (after the 4-cell stage), in which it stimulated compaction, blastocoele formation, and proliferation of cells in the blastocyst derived from parthenogenetic diploids in the pig.

Lee and Fukui [54] reported that bovine morulae and blastocysts were well developed in a medium including alanine and glycine. Bavister and Arlotto [33] showed that glycine, Cys, and lysine were stimulatory influences, whereas other amino acids (Cys, proline, serine, threonine, histidine, alanine, hydroxyproline, Leu, aspartic acid, and Met) were neutral influences on hamster embryo development. Most of those amino acids belong to NE-AA, or basic and polar E-AA. In accord with those previous findings, the present study revealed that these amino acids did not show inhibitory effects. Lane and Gardner [52] reported that E-AA did not confer any benefit for development before the 8-cell stage, whereas NE-AA and glutamine significantly increased cleavage rates during the first four cell cycles in the mouse. Thus, it is strongly suggested that inhibitory effects of E-AA, especially nonpolar amino acids, on the early cleaving stages and stimulatory effects of NE-AA on the preimplantation development may be a common phenomenon in mammals.

The results of the present study also demonstrate that NE-AA played important roles in the expansion of blastocysts during the second culture period, from 48 to 168 h after El-St, during the in vitro development of porcine parthenogenetic diploids. On the other hand, the presence of only E-AA during the second culture period showed no effect on the expansion of the blastocysts (Fig. 8). The results of experiment 6 demonstrate, however, that E-AA decreased the time of resumption of compaction and blastoration and increased the numbers of cells in the whole blastocyst and ICM (Table 3). The presence of E+NE-AA supported both the expansion of blastocysts and the increase in the number of cells in the whole blastocysts and ICM, indicating that E+NE-AA acted cooperatively on the development of blastocysts during the second culture period. The same tendency was observed on the development of mouse embryos [52]. The best result of development to the blastocyst stage in vitro was achieved when mouse fertilized oocytes were cultured with NE-AA and glutamine to the 8-cell stage, followed by culture in the presence of E+NE-AA and glutamine. Bovine embryos required E+NE-AA including glutamine later than 4 days post-insemination, and the combination of E+NE-AA and glutamine stimulated the development to the blastocyst stage and the number of cells in the trophectoderm and ICM [13]. Blastocysts that were derived from in vivo-fertilized oocytes and cultured in the presence of E+NE-AA without glutamine showed the highest number of cells in the mouse [19] and human embryos [29]. The numbers of whole cells in in vivo blastocysts and expanded blastocysts reportedly ranged from 9 to 64 (mostly from 33 to 64) and from 17 to 240 (mostly from 65 to 120), respectively, and the number of ICM from 12 to 16 in the pig [48]. In the present study, the mean numbers of cells in blastocysts and ICM were 63.2 ± 6.4 and 13.1 ± 2.4 after culture of porcine diploids at the 3- to 4-cell stage under the presence of E+NE-AA (Table 3). These numbers are within the range of in vivo-developed blastocysts, though a slight delay of development was observed.

In conclusion, the presence of nonpolar E-AA, especially Val, Leu, Ile, and Met, before the 4-cell stage resulted in the 4-cell block and clearly impaired the development of diploids to the morula and blastocyst stages. The presence of NE-AA during the first 48 h of culture (before the 4-cell stage) stimulated the expansion of the blastocysts during the later period. The addition of E+NE-AA stimulated the development of diploids from the 4-cell stage to the expanded-blastocyst stage. The NE-AA supported the early cleavage and the expansion of the blastocyst in porcine parthenogenetic diploids, whereas E-AA induced an increase in the number of cells in the whole blastocyst and ICM. Further studies are required to elucidate the mechanism of 4-cell block in porcine parthenogenetic diploids induced by the presence of nonpolar amino acids during the first and the second diploid division.


   FOOTNOTES
 
1 Supported by a Research for the Future Program grant from the Japan Society for the Promotion of Science (JSPS-RFTF97L00905). Back

2 Correspondence and current address: Masashi Miyake, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho Nada-ku, Kobe City, Hyogo 657-8501, Japan. FAX: 81 78 803 5807; miyake{at}ans.kobe-u.ac.jp Back

Received: 20 February 2002.

First decision: 21 March 2002.

Accepted: 2 July 2002.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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