The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro

The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied. The effects of the compounds were studied using a 3³ factorial experimental arrangement. The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed. Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl. The intracellular contents of potassium and sodium have also been measured in single zygotes using X-ray electron probe spectrometry. When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases. These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium. Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM. These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration. © 1993 Wiley-Liss, Inc.     

Purines inhibit the development of mouse embryos in vitro

The first cleavage of embryos derived from random-bred, inbred, and hybrid-inbred female mice was not arrested by purines at concentrations as high as 30 microM. Development after the first or second cleavage was arrested by hypoxanthine, adenosine or inosine, but not guanosine. In agreement with previous results, the purine-induced block was reversed when arrested embryos were transferred to purine-free media after 24 h in culture. The cleavage arrest was not due to elevations of cAMP as a result of inhibition of phosphodiesterase activity since similar concentrations of phosphodiesterase inhibitors or dibutyryl cAMP did not block development. Treatment with inhibitors of enzymes that convert IMP to AMP or to GMP did not reverse the hypoxanthine-induced block, thus demonstrating that mitotic arrest is mediated by a mechanism different from the hypoxanthine arrest of meiosis. Thymidine incorporation studies showed that the block did not prevent the onset of DNA synthesis. The results reveal a profound sensitivity to purine inhibition of a cell process that occurs during the first 30 h of mouse embryo development and is necessary for progession through the G2 or M phases of the second or third cleavage.     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

In this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2-4, 5-7 or 8-10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p>0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p>0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p<0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.     

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.     

Effect of culture media on in vitro development of cloned mouse embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.     

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.     

Effects of human diabetic serum on the in vitro development of mouse preimplantation embryos

The effects of sera from different types of human diabetes (type I with and without ketoacidosis; type II treated with insulin or Daonil or untreated) on the in vitro development of early preimplantation mouse embryos were studied. In controls, 20% of blastocysts failed to develop successfully when grown for 72 h in RPMI medium supplemented with 10% fetal bovine serum and 50% nondiabetic human serum. In experiments using 50% diabetic serum, the highest embryotoxic effect was found in type-I diabetes with and without ketoacidosis: The percents of undeveloped embryos were 66 and 58, respectively. In type-II diabetes, embryotoxic effects were found among all studied types: The percent of undeveloped blastocysts varied from 36% in insulin-treated type-II diabetes to 44% in untreated type-II diabetes. A high correlation was found between the number of undeveloped embryos and the blood concentrations of metabolic diabetic factors: glucose (r = .53-.64 in type-I diabetes), B-HOB (r = .7-.77 in type-II diabetes untreated or treated with Daonil), acetoacetate (r = .66 in insulin-treated type-II diabetes), and HbA1c (r = .89 in insulin-treated type-II diabetes or .99 in Daonil-treated type-II diabetes). A concentration of 80% serum was embryo-toxic when obtained from nondiabetic or from diabetic human. The possible role of diabetic metabolic factors in causing increased risk of spontaneous abortions and infertility among diabetic women is discussed.     

The teratogenic effects of 6-mercaptopurine on chick embryos in ovo.

Fertilized eggs of single-combed White Leghorn hens were each injected through a shell window directly beneath the embryo at 70 or 96 h of incubation with 2 mg of the sodium salt of 6-MP dissolved in 0.1 M phosphate buffer, and at 11 days of incubation the embryos were examined for gross morphological abnormalities. Various gross malformations and growth retardation were found, the most frequently and severely affected structures being limbs, beak, and eyes. Treatment at 70 h caused more severe abnormalities than at 96 h, but the spectrum of defects was not very different.     

Effect of gossypol and its metabolite on in vitro early development of mouse embryos

The purpose of this study was to examine the effect of gossypol and its metabolite on early in vitro mouse embryo development. One hundred and thirty-eight excellent quality mouse blastocysts were randomly assigned to five different treatments. Culture media were supplemented with 10% (V/N) normal steer serum. The embryos were cultured at 37 degrees C with an atmosphere of 5% O(2), 5% CO(2) and 90% N(2), and embryo development was examined and recorded at 12-h intervals for 72 h. The percentage of embryos that developed to expanded blastocyst (92%), hatching blastocyst (84%), and hatched blastocyst (76%) stages in control Ham's F-10 media was not different from that of embryos cultured in media containing 0.1 and 5 mug of gossypol; however, none of the embryos treated with 265 ng of gossypol metabolite (GM) developed beyond the blastocyst stage. A substantial decrease in the percentage of embryos reaching hatching blastocyst (29%) and hatched blastocyst (29%) stages was observed in the embryos cultured with 5.3 ng of GM. At both light and electron microscopic levels, the embryos appeared to be affected even by a lower concentration of GM in vitro. Our results suggest that GM has a much greater potency than the parent gossypol in inhibiting the early development of mouse embryos in vitro.     

Positive effect of taurine on preimplantation development of mouse embryos in vitro.

The effect of various taurine concentrations in modified Tyrode's medium on in vitro fertilization of mouse oocytes was examined. No significant difference in fertilization rate was found at concentrations of 0, 0.1, 1, 5, 10 and 20 mM taurine. In a second series of experiments, the effect of taurine on preimplantation embryonic development after fertilization in vitro was studied. At concentrations of 1, 5, 10 and 20 mM taurine, significantly more two-cell embryos reached the blastocyst stage compared with medium without taurine. Culture in the presence of 5 mM or 10 mM taurine resulted in blastocysts with the highest mean number of cells. The positive effect of taurine on embryonic development was found to be more pronounced both in a second medium (human tubal fluid medium) which has a higher potassium concentration than Tyrode's medium, and in a modified Tyrode's medium with an increased potassium concentration. In addition to these in vitro studies, it is reported that taurine comprised about 59% of the total free amino acid content in mouse oviduct flushings, compared with 17% in mouse serum.     

Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

The objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.     

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.