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PubMed2002

Sex-chromosome linked gene expression in in-vitro produced bovine embryos.

The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT-PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT-PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8-16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8-16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.

Animals↗
PubMed1991

The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro.

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.

Animals↗
PubMed1977

[A Study On The Lipids Of Ascaris Suum Ova]

The present study was undertaken to observe the quality and quantity of lipid constituents by the developing Ascaris suum eggs. The collected eggs from the uterus of A. suum were classified into 3 groups, i.e., single cell stage, morula stage and embryonated eggs, and were subjected to analyse their lipid fractions. To obtain the morula stage eggs, 10 to 11 incubation days at 30 degree C were needed and for the embryonated eggs, 30 to 31 days were lasted. At the time of experiment, their indices of development by Hoffman were 285(morula stage) and 42 (embryonated stage) respectively. Lipid extraction was done by the methods of Folch et a1. (1957) and Kenny (1952), and then the extracted lipid fractions from the above 3 groups of eggs were separated by thin layer chromatography. Those fractions were also subjected to perform the quantitative analyses of fatty acids, glycerides, cholesterol and phospholipids. The results obtained were summarized as follows. 1. Total amount of fatty acid was decreased from 12.9 mg per gram of eggs (single cell stage) to 6.6 mg/gm (embryonated eggs), whereas the proportion of free fatty acid to total fatty acid was constantly increased from 77.5% to 89.4% during the period of egg development. 2. Total amount of glycerides was also increased from 33.0 mg/gm of single cell stage to 55.9 mg/gm of the embryonated eggs. The most abundant glyceride among 3 glycerides discovered from A. suum eggs was triglyceride, and the least was monoglyceride. 3. The amount of free cholesterol was much larger than that of ester form in general, and it reached maximum in the eggs of morula stage (4.6 mg/gm). The increase of total cholesterol was monitored during the development of A. suum eggs from 3.3 mg/gm to 5.4 mg/gm. 4. The following 8 phospholipids were detected in the embryonated eggs, i.e., lysophosphatidyl choline, phosphatidyl inositol, sphingomyelin, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine and unknown phospholipid. But in the single cell stage eggs, 4 kinds out of the above 8 phopholipids were not observed, and in the morula stage eggs, 2 kinds were absent among the 8 phospholipids.

Journal Article↗
PubMed1997

Changes in the relative abundance of various housekeeping gene transcripts in in vitro-produced early bovine embryos.

The relative abundances of transcripts of different origins and housekeeping functions were measured by Northern blot analysis of RNA samples derived from in vitro-matured oocytes and in vitro-produced bovine embryos at selected stages of early development. The gene products studied included: two mitochondrial transcripts, 12S rRNA and cytochrome b mRNA; two RNAs involved in the processing of other RNAs, U2 and U3 snRNA; and two nuclear-derived transcripts, beta-actin mRNA and histone H3 mRNA. Overall, the RNA levels for the various genes studied remained constant or decreased slightly from the mature oocyte to the 6- to 8-cell or morula stage and were greatly increased in blastocysts. Differences were observed in the degree to which the RNA levels increased and in the timing of the increase. For 12S rRNA, a major increase was not observed until the blastocyst stage where levels increased 7.1 times the amount detected in morulae. Cytochrome b mRNA levels started to increase at the 6- to 8-cell stage and reached levels in blastocysts that were 20 times more than the cytochrome b mRNA level in 2- to 4-cell embryos. U2 snRNA levels did not increase until the blastocyst stage where levels were 6.4 times the amount found in morulae. U3 snRNA and beta-actin mRNA levels started to increase at the morula stage and blastocysts contained 118 and 110 times more U3 snRNA and beta-actin mRNA, respectively, than 6- to 8-cell embryos. However, blastocysts contained only two times the amount of histone H3 mRNA present in 6- to 8-cell embryos.

Actins↗
PubMed1999

Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium.

The ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum-free medium (IVMD101) or in a serum-containing medium (TCM199+CS) was compared. Embryos up to the eight-cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight-cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well-developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome-like vesicles were partially filled with electron-dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome-like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome-like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)-cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome-like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum-free and serum-supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos.

Animals↗
PubMed2003

Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryos.

In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n = 188), 16-cell (n = 94), morula (n = 35), and blastocyst (n = 15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes.

Animals↗
PubMed1993

Presence and possible role of c-ras and nuclear (c-fos and c-jun) proto-oncogene products in preimplantation embryonic development in mice.

The presence and possible role of products of nuclear (c-fos and c-jun) and c-ras proto-oncogenes were investigated in preimplantation embryonic development in mice. Polyclonal antibodies to c-fos or c-jun proto-oncogene products did not affect development of in vitro-cultured embryos from two-cell to morula or from morula to late blastocyst stages. However, v-H-ras monoclonal antibody (mAb) to c-ras protein (p21), although it did not inhibit the development of in vitro-cultured embryos from two-cell to morula stages, it significantly (P < .001-.005) inhibited the development of morula to late blastocyst stages in a dose-dependent manner. The effects of v-H-ras mAb were specific, since immunoabsorption with synthetic ras peptide completely blocked inhibitory effects of v-H-ras mAb. Neither c-fos nor c-jun antibodies reacted with specific proteins corresponding to c-fos (62 kDa) and c-jun (39 kDa) products on the Western blots of various murine ova/embryos extracts. However, the c-fos and c-jun antibodies reacted with 62 and 39 kDa protein bands, respectively, on the blot of NIH 3T3 cells extract. The v-H-ras mAb specifically identified 21 +/- 3 kDa protein corresponding to c-ras p21 on the blots of early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any protein band on the blots of various ova/embryo extracts. The reactions of v-H-ras mAb on the Western blots of blastocyst extracts were specific, since immunoabsorbed antibody was unable to react with any specific band on blots of early or late blastocyst extract. These results were further confirmed by immunoprecipitation procedure utilizing v-H-ras mAb. Again, the v-H-ras mAb immunoprecipitated a 21 kDa band from early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any band corresponding to p21 in the immunoprecipitation procedure. These results suggest that the specific products of nuclear proto-oncogenes, the c-fos and c-jun, are not detected in murine ova and preimplantation embryos, and the respective antibodies do not inhibit embryogenesis, indicating that they may not play a major role in early embryonic development. On the other hand, the product of c-ras proto-oncogene is specifically expressed in the blastocyst-stage embryos and may have a possible role in preimplantation embryonic development in mice.

3T3 Cells↗
PubMed1994

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The finding that TEC-3 is not expressed before the onset of embryonic transcription, which occurs at the eight-cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC-03 antibody showed that the TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program.

Animals↗
PubMed2001

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.

Animals↗
PubMed1988

Viability of mouse half-embryos in vitro and in vivo.

Mouse embryos at the 2-, 4-, 8-cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2-cell (70%), 4-cell (75%), 8-cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2-cell, 4-cell, 8-cell, and morula stages, respectively, developed into live fetuses. Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.

Animals↗
PubMed2005

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos.

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.

Animals↗
PubMed1997

Effects of epidermal growth factor on preimplantation mouse embryos.

PURPOSE: Our purpose was to clarify the effects of epidermal growth factor (EGF) on mouse preimplantation embryos. METHODS: We examined the effect of EGF on two-cell and four-cell stage mouse embryos cultured in vitro. In preimplantation embryos, we analyzed the binding of 125I-EGF by autoradiography and EGF receptor mRNA by the reverse transcription-polymerase chain reaction. RESULTS: At more than 0.005 ng/ml, EGF relieved the two-cell block and regulated the differentiation of morula-stage embryos. These effects were negated by antiserum. EGF did not exhibit any marked effect on embryos between the four-cell and the morula stages. Specific binding for EGF and EGF receptor mRNA was detected during and after the morula stage. CONCLUSIONS: The effects of EGF on preimplantation mouse embryos differed according to the stage of development, promoting cleavage before the four-cell stage and regulating differentiation after the morula stage. This regulatory action is thought to be transmitted to the cells via EGF receptors.

Animals↗
PubMedDate not supplied

Embryotoxicity of polychlorinated biphenyls (PCBS) for preimplantation embryos.

In a previous study oral administration of a commercial PCB mixture (Aroclor 1,260) to female rabbits (4 mg/kg b.wt. for 14 weeks) resulted in a significant accumulation of PCBs in 6-d-old blastocysts and increased preimplantation embryo mortality (1). In the present study, the direct toxicity of PCBs on preimplantation rabbit embryos was investigated during in vitro culture. One-day-old cleavage stages and 3-d-old rabbit morulae were cultured in BSM II medium supplemented with 1.5% BSA in a reduced oxygen concentration of 5% O2. They were exposed to 50, 5, or 0.5 microgram Aroclor 1,260/mL culture medium for 24 h. PCB (50 micrograms) led to a complete degeneration of the exposed embryos. Following exposure to 5 micrograms only 16% of the morulae developed into blastocysts. The others were either arrested in the morulae stage or were degenerated. Cell proliferation of the nondegenerated embryos was approximately 20% of that of corresponding control embryos. Compared with nonexposed controls, addition of 0.5 microgram PCB/mL showed either no or only a slight impairment of development. Preliminary embryo transfer experiments showed that morulae exposed to 5 micrograms PCB with clear signs of degeneration after 24 h in vitro culture were able to implant. Aroclor 1,260 is embryotoxic in a dose-dependent manner in cultured rabbit preimplantation embryos.

Animals↗
PubMed2005

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

Animals↗
PubMed2003

Coculture of human oviductal cells maintains mitochondrial function and decreases caspase activity of cleavage-stage mouse embryos.

OBJECTIVE: To investigate the mitochondrial function and caspase activity in mouse embryos after human oviductal cell coculture. DESIGN: Experimental laboratory study. SETTING: University gynecology unit. ANIMAL(S): MF-1 (female); BALB/c (male) mice. INTERVENTION(S): Mouse embryos were cocultured with human oviductal cells. MAIN OUTCOME MEASURE(S): Mitochondrial transmembrane potential (Delta psi(m)) and caspase activity. RESULT(S): Compared to embryos after coculture in Chatot-Ziomek-Bavister (CZB) medium supplemented with 0.5 mg/mL of BSA (CZB), Delta psi m of embryos cultured in CZB was significantly lower at the two-cell (CZB, 2.04 +/- 0.412; coculture, 4.34 +/- 0.563) and morula (CZB, 6.06 +/- 0.548; coculture, 7.12 +/- 0.568) stages. Cocultured embryos and in vivo developed embryos had comparable Delta psi m. Caspase activity was not detected in unfragmented cleavage-stage embryos and morula developed in vivo. In vitro cultured morula possessed caspase activity. The activity was significantly reduced in the cocultured morula. CONCLUSION(S): Human oviductal cells maintained the mitochondria function in terms of mitochondrial transmembrane potential and decreased the caspase activity to improve the development of mouse embryo.

Animals↗
PubMed1988

Improved pronuclear mouse embryo development over an extended pH range in Ham's F-10 medium without protein.

Pronuclear mouse embryos were cultured at four different pHs (7.17, 7.27, 7.37, and 7.50) in Ham's F-10 medium (Gibco, Grand Island, NY) alone and in Ham's F-10 medium supplemented with 10% human cord serum (v/v) to assess the effects of pH and serum on preimplantation embryo development. Media used in these experiments was stored both without (old protocol) or with triple gas equilibration (new protocol). Development was assessed by the percent of embryos developing to the 2-cell stage and on to the morula and blastocyst stages. Triple gas equilibration prior to media storage improved development to the morula and blastocyst stages. Development to the 2-cell and morula stages was not adversely affected by the presence of serum, but development to the blastocyst stage was significantly higher in Ham's F-10 medium without serum at all pHs tested. In conditions without serum, percent development at all stages examined was similar over the pH range of 7.17 to 7.37, with a significant decrease in development to the morula and blastocyst stage at pH 7.50. Embryos cultured in the presence of serum demonstrated a greater decrease in the percent development to blastocyst at both the low, 7.17 and 7.27, and high, 7.50, pHs, with an apparent pH optimum at 7.37. In summary, the complex media Ham's F-10 medium supports mouse embryo development without the need for serum. Serum inhibits pronuclear embryo development to the blastocyst stage. Mouse embryo culture in the absence of serum can tolerate a pH as low as 7.17 (8.4% CO2) without any detrimental effects.

Animals↗
PubMed2003

Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development.

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.

Acetylcysteine↗
PubMed1999

Vitrification of mouse embryos in two cryoprotectant solutions.

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM.

Animals↗