Elsevier

Theriogenology

Volume 59, Issues 3–4, February 2003, Pages 719-734
Theriogenology

Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig

https://doi.org/10.1016/S0093-691X(02)01085-3Get rights and content

Abstract

The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids. Oocyte–cumulus–granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h. The mature oocytes were subjected to a single electro-stimulation (El-St; 100 μs, 1500 V/cm), treated with 5.0 μg/ml Cytochalasin B for 4 h and then cultured under various conditions as described below. In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whitten’s medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water. In Experiment 2, the diploids were cultured in the five media used in Experiment 1 for the first 48 h, and then in mWM256 until 168 h after El-St. In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 h after El-St. In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 until 168 h after El-St. In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after El-St. All media were supplemented with 0.5 mg/ml hyaluronic acid and 4.0 mg/ml bovine serum albumin. The results obtained in Experiments 1–5 indicate that the osmolarity of a medium, but not the Na+/K+ ratio, exerts effects on the development of diploids to the blastocyst stage. The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids. The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280–320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220–270 mOsmol, respectively.

Introduction

Osmolarity of culture media is one of the important factors affecting in vitro development of preimplantation mammalian embryos to the blastocyst stage. However, the optimal osmolarity for embryo development differs among species. The concentration of NaCl should not be higher than 95 mM, and the osmolarity of media should be between 250 and 270 mOsmol for successful development of in vitro-matured and -fertilized eggs in rabbits [1] and cattle [2]. Both osmolarity and NaCl concentration affect embryo development, and zygotes are much more sensitive to these effects than 2-cell embryos in rabbits [3] and mice [4]. Rat embryos have shown a pronounced enhancement of ability to develop to the blastocyst stage after fertilization in a medium at 310 mOsmol and culture in the same medium with decreased osmolarity to 246 mOsmol [5]. It has also been shown that high proportions of rat 1-cell embryos can develop to the blastocyst stage in a chemically defined medium with the osmolarity adjusted to 246 mOsmol [6], [7]. Mouse zygotes and embryos at the early cleaving-stage are sensitive to increased osmolarity [8]. In a study in which fertilized 2-cell mouse embryos were cultured in media with various osmolarities (200–339 mOsmol) throughout the culture period, the best result was obtained in a medium with 276 mOsmol [9]. Furthermore, increased NaCl concentration impairs the ability of porcine embryos to develop to the morula and blastocyst stages [10]. Nonetheless, there have been few investigations into osmotic effects on the early development of porcine embryos, or on the effects of temporal changes in the osmolarity of media during early development of 1-cell embryos to the blastocyst stage. One of main reasons is the high incidence of polyspermy in in vitro fertilization of porcine eggs, which results in a low frequency of development to the blastocyst stage. Our previous reports have shown that electro-activated porcine oocytes that had been matured in vitro developed to the blastocyst stage in vitro, furthermore, the characteristics of development of porcine parthenogenetic diploids until the blastocyst stage resembled in vitro-fertilized eggs [11], [12], [13]. It was of interest that porcine parthenogenetic diploids showed a higher developmental ability up to the blastocyst stage in modified Whitten’s medium (mWM) than in modified Krebs and Ringer’s bicarbonate-buffered solution (mKRB) [12]. The two most marked differences between the media were the concentration of NaCl (119.4 mM in mKRB versus 68.5 mM in mWM), which affects their osmolarity (309 mOs in mKRB versus 256 mOs in mWM; mOs=mOsmol/l; Table 1), and the Na+/K+ ratio. It is, therefore, postulated that these differences in osmolarity and NaCl concentration between mKRB and mWM might partially explain the effects of the media on the development of parthenogenetic diploids. The present study was carried out to determine the effects of osmolarity, Na+/K+ ratio and temporal changes in the osmolarity of a culture medium on the development of porcine parthenogenetic diploids to the blastocyst stage.

Section snippets

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) acetylmethylammonium bromide (Wako Pure Chemical Industries Ltd., Osaka, Japan) and twice with Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS) containing 0.1% (w/v) polyvinyl alcohol (PBS–PVA; Sigma). Follicles 4–6 mm in diameter were dissected with two scalpels in PBS–PVA. Those that had a round form, clear follicular

Experiment 1: Effects of osmolarity and the ratio of Na+/K+ of culture media on the development of porcine parthenogenetic diploids

The frequency of development of diploids to the 2-cell stage was significantly higher in mKRB (67%), mWM-Na (78%), mWM-Man (71%), and mWM-DW (73%), i.e. in isotonic media than in hypotonic mWM256 medium (45%, P<0.05) at 24 h after El-St (Fig. 1). At 48 h after El-St, the percentage of diploids at the 3–4-cell stage was also significantly higher in the four isotonic media (64–70%) than in mWM256 (42%, P<0.05). There were no significant differences in the percentage of diploids at the 2-cell and

Discussion

This is the first study to show that a change of osmotic milieu from isotonic to hypotonic is effective for the efficient formation of blastocysts in porcine parthenogenetic diploids, and that this promoting effect is not related to the Na+/K+ ratio in media. As shown in Experiment 2, a medium change from 309 mOs and various Na+/K+ ratios to mWM256 at the first 48 h of culture after El-St significantly promoted the development of diploids thereafter.

From the results in Experiment 1, it is obvious

Acknowledgements

This study was supported by a Grant-in-Aid from JSPS (No. RFTF97L00905), a Grant-in-Aid for Scientific Research (B-2, No. 09460129) from the Ministry of Education, Science, Sports, and Culture of Japan, and a Grant-in-Aid (Bio Design Program) from the Ministry of Agriculture, Forestry and Fisheries (BDP-02-III-1).

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