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PubMed1994

The effective culture method of zona-free rabbit 1-, 2- and 4-cell embryos.

The effect of modified incubation systems on the development capacity of the zona-free rabbit embryos was examined. Embryos at 1-, 2- and 4-cell stages were used. The removal of the zona pellucida was accomplished by the enzymic-mechanical technique. Denuded rabbit embryos were cultured using 3 incubation systems. In the first and the second system the embryos were cultured in microdrops. The difference between these first 2 systems concerned the volume of the microdrops and the kind of paraffin oil used. In the first system the embryos were cultured in 5mul microdrops covered with light or heavy paraffin oil; in the second system embryos were cultured in 40-mul microdrops under light paraffin oil. The third traditional system involved the incubation of embryos in glass capillaries into separated columns of medium. The percentage of blastocysts obtained from 1-cell embryos cultured in the first incubation system was 6.1% with heavy paraffin oil as the covering layer and 29.0% with light paraffin oil. In the second and third incubation systems blastocyst yield was 30.8 and 59.6%, respectively. The percentage of blastocysts obtained from 2-cell and 4-cell stage embryos with heavy paraffin oil was 18.7 and 25.0%, respectively; with light paraffin oil these figures were 40.0 and 50.0%, respectively. In the second incubation system these figures were 49.3 and 72.3%; and in the third incubation system they were 72.9 and 78.3%, respectively. The results of the experiment showed that culture into glass capillaries is undoubtedly an effecient method of culturing of the zona-free rabbit embryos.

Journal Article↗
PubMed1994

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium.

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P=0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P or=0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P=0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P<0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P=0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P>or=0.535).

Journal Article↗
PubMed1994

Successful in vitro and in vivo development of in vitro fertilized two- to four-cell cat embryos following cryopreservation, culture and transfer.

In vitro and in vivo survival of in vitro-derived 2- to 4-cell cat embryos following cryopreservation was examined. Prefreeze 1- vs 2-step cryoprotectant exposure (Experiment 1) and warming method (Experiment 2) on zona pellucida damage and development in vitro were compared. To determine viability in vivo, frozen/thawed embryos were cultured in vitro to the morula/early blastocyst stage and transferred to synchronous recipients (Experiment 3). At 24 to 26 h after IVF, embryos were cryopreserved in 1.4 M propanediol (Pr)+0.125 M sucrose (Su) by cooling at 0.3 degrees C/min from -6 degrees C to -30 degrees C and storing in liquid nitrogen. Autologous embryos were cultured in vitro for 7 d. After warming for 5 sec in air and 10 sec at 37 degrees C in water (Experiments 1 to 3), or at room temperature air (22 degrees C; Experiment 2), the cryoprotectant was removed and embryos were cultured in vitro for 6 d (Experiments 1 and 2). Development was assessed after staining by counting cell numbers/embryo and determining the percentages at the 2- to 4-cell (nonsurvivor), pre (5 to 15), early (16 to 32), mid (33 to 50), late (>50) morula or blastocyst stages. Post-thaw development to late morula/blastocyst after 1-step exposure (68%, 15 min Pr+Su) was higher (P<0.05) than that after 2-step exposure (36%, 15 min Pr and 15 min Pr+Su). Both warming methods produced similar percentages of embryos with damaged zonae (13 to 15%) and equivalent development to morula/blastocyst (64 to 69%). Development in vitro to early morula/blastocyst of frozen embryos with intact zonae was similar to that of nonfrozen embryos. Following cryopreservation, most 2- to 4-cell cat embryos retained their capability for in vitro development to morula/blastocyst, and in vivo viability was demonstrated by the birth of 3 live kittens to 2 of 4 recipients following the transfer of 58 embryos.

Journal Article↗
PubMed1995

Cryopreservation of oyster (Crassostrea gigas) embryos.

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.

Journal Article↗
PubMed1991

Serum corticosterone concentrations in developing shell-less and shelled turkey embryos.

1. Turkey embryos grown in shell-less culture (EO) display normal development to day 14 of incubation, after which growth rate is reduced and mortality increases, compared to age-matched in ovo (IO) embryos (Richards 1982. Long term shell-less culture of turkey embryos. Poultry Sci. 61, 2089-2096). 2. In this study serum corticosterone concentrations were monitored in normally incubated embryos and embryos maintained in shell-less culture. 3. On days 14 and 16 of incubation, corticosterone levels were greater (P less than 0.05) in EO than IO embryos, whereas during the later stages corticosterone increased dramatically in the shelled embryos, and remained relatively constant in the EO embryos. 4. EO embryos do not exhibit a pattern of adrenal hormonal secretion that would indicated a stressful condition. 5. The absence of a normal increase in corticosterone in shell-less embryos may contribute to the abnormal embryological development in the later stages of incubation.

Animals↗
PubMed1996

Cryopreservation of mammalian embryos and oocytes: recent advances.

The cryopreservation of embryos of most domestic species has become a routine procedure in embryo transfer, and recently, advances have been made in the cold storage of mammalian oocytes. The ability to sustain viable oocytes and embryos from mammalian species at low temperature for prolonged periods of time has important implications to basic and applied biotechnology. Recent advances in the study of physico-chemical behaviour of different cryoprotectants, use of various macromolecule additives in cryoprotective solutions and isolation and use of proteins of plant and animal origin with antifreeze activity offers many new options for cryopreservation of oocytes and embryos of animal and human origin. At the same time rapidly developing methods of oocyte/embryo manipulation such as in vitro embryo production, embryo splitting, embryo biopsying for gene and sex determination, embryo cloning and the isolation of individual blastomers, create new challenges in cryopreservation. Very recent advances in the cryopreservation of mammalian oocytes, in vivo- and in vitro-derived embryos, and micromanipulated embryos are reviewed in this manuscript.

Journal Article↗
PubMedDate not supplied

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent teratogen in several animal species, especially during the period of organogenesis. The purpose of this study was to test the hypothesis that TCDD has direct effects on the earliest stages of murine embryonic development, namely the preimplantation stages. Three endpoints were measured: 1) embryo cell number, a measure of embryo viability, 2) competitive embryonic cell proliferation utilizing chimeric embryos, another measure of embryo viability, and 3) cavitation rate, a functional measure of trophectoderm differentiation. Neither embryo cell numbers nor competitive embryonic cell proliferation (chimera assay) were affected by TCDD, either when the mother was dosed in vivo (prior to fertilization), or when 2-cell embryos were dosed in vitro. However, cavitation rates of in vitro-dosed embryos, in the presence of 10% fetal bovine serum, were significantly higher for TCDD than controls, suggesting that TCDD accelerated differentiation of murine preimplantation embryos. Taken together, these results demonstrate that: 1) TCDD can act directly on the murine preimplantation embryo, and 2) TCDD's actions are primarily on accelerated differentiation and not on embryo viability. To our knowledge, this is the earliest stage of mammalian development during which TCDD has been shown to exert an effect.

Animals↗
PubMed2005

Relationships between maternal hormone secretion and embryo development on day 5 of pregnancy in dairy cows.

In cattle, increasing early embryonic losses are associated with inadequate progesterone concentrations within the first three weeks of pregnancy. The aim of this study was to investigate the complex relationship between early maternal progesterone concentration and embryo development early within the first week of pregnancy, specifically, on day 5 post-oestrus in dairy cows. Twenty Holstein-Friesian cows at the end of lactation were inseminated at oestrus (day 0) and on day 5 post-oestrus cows were slaughtered and the reproductive tract flushed to determine the presence and stage of embryo development. Three cows that had failed to synchronise correctly were excluded from analysis while in the remaining 17 cows 11 (65%) were pregnant with embryos at the morula (n = 3), 9-16 (n = 3) and 8-cell (n = 5) stages of development. No differences in day 5 plasma progesterone concentrations or corpus luteum (CL) size or progesterone content were observed between pregnant (n = 11) and non-pregnant (n = 6) cows. In cows with embryos beyond the 8-cell stage of development (n = 6) plasma progesterone concentration (P < 0.001) and CL weight (P < 0.01) were higher and plasma insulin concentrations lower (P < 0.001) than in cows with 8-cell embryos (n = 5). In addition there was a negative relationship between plasma progesterone and plasma insulin in pregnant cows (R(2) = 0.65; P < 0.005). In cows with an embryo present in the oviduct, oviductal glucose concentrations were lower (P < 0.05) than in cows with no embryo present. These results confirm progesterone is not only directly associated with embryo development, but that it may indirectly modulate embryo development via changes in the oviductal environment. In summary, the association between maternal progesterone concentration and embryo development exists as early as day 5 of pregnancy in dairy cows.

Animals↗
PubMed2006

The role of oxygen in ruminant preimplantation embryo development and metabolism.

The long-term effects of in vitro embryo culture on animal health are presently unknown, however, current knowledge directs investigations toward understanding the mechanisms involved in regulating embryo development. In vitro culture is known to have short-term effects, particularly on gene expression and metabolism at the blastocyst stage, while large offspring syndrome is commonly observed following transfer of in vitro produced bovine embryos. Indeed, it is likely that the environment surrounding the early embryo, prior to implantation, may program later development. Regulation of gene expression and metabolism, through gene activation, is mediated by transcription factors, which are themselves controlled by internal and external factors. Alterations in the surrounding environment during preimplantation embryo development, such as that which occurs with inadequate developmental 'support' during in vitro culture, may modify the activation, or inactivation, of several transcription factors, and may therefore have long-term consequences for the developing offspring. In vitro culture deviates from in vivo conditions in many respects, but one of the critical factors that is generally not considered is the oxygen tension under which embryos are cultured. Numerous studies have demonstrated that atmospheric oxygen conditions during culture have detrimental effects on embryo development. While it is generally believed that this arises from the production of reactive oxygen species, this presents an over-simplistic view of the role of oxygen during development. The hypoxia-inducible factor transcription factor family is involved in the responses of cells to alterations in external oxygen concentrations, regulating the expression of numerous genes. Alterations in expression of some of these genes have been highlighted by recent studies in the bovine embryo, implicating oxygen as a regulator of several cellular and metabolic pathways. While it is clear that oxygen plays a role during embryo development, further work to investigate interactions between oxygen and other signaling pathways such as pH and Ca(2+), mitochondria and metabolism is required, as well as exposure of embryos at different time points, to determine the mechanisms that control preimplantation development, the interactions of a range of stimuli and to establish culture procedures that support optimal development and minimize risks to health. This review focuses largely on work undertaken in ruminant models, with brief references to other species.

Animals↗
PubMed2004

Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage.

OBJECTIVE: To quantify the mitochondrial DNA contents in human embryos with good or poor quality at the 8-cell stage. DESIGN: Prospective study. SETTING: Private infertility clinic. PATIENT(S): Five women aged 24 to 34 years. INTERVENTION(S): Embryos obtained in standard superovulation and embryo culture procedures. MAIN OUTCOME MEASURE(S): Mitochondrial DNA (mtDNA) copy numbers in human embryos at cleavage stage were quantified by real-time polymerase chain reaction, in an effort to correlate with morphology. RESULT(S): The grade 8A embryos contained a mean mtDNA copy number at 1163937 (n = 8, from three patients); grade 8B embryos, at 939345 (n = 5, from two patients); grade 8C(+) embryos, at 637872 (n = 12, from 5 patients); and grade 8C(+) embryos derived from 3PN zygotes, at 300429 (n = 3, from a single patient). CONCLUSION(S): Great variations were found among blastomeres from a single embryo and among embryos from a single patient. The native variations of mtDNA copy number may affect developmental ability irrespective of morphology.

Adult↗
PubMed2006

Insulin-like growth factor-II (IGF-II), IGF-binding protein-3 (IGFBP-3), and IGFBP-4 in follicular fluid are associated with oocyte maturation and embryo development.

OBJECTIVE: The objective of this study was to investigate the association between follicular fluid (FF) levels of insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and pregnancy-associated plasma protein-A (PAPP-A), which is a protease for IGFBP-4, and the quality of subsequent embryo development from in vitro fertilized oocytes aspirated from the same follicle. DESIGN: Prospective study. SETTING: University infertility clinic and academic research laboratory. PATIENT(S): One hundred sixty-two infertile women undergoing controlled ovarian hyperstimulation for IVF and embryo transfer were recruited in a university hospital. INTERVENTION(S): During oocyte retrieval, samples of 225 FFs and matched mature oocytes were collected and studied. MAIN OUTCOME MEASURE(S): Concentrations of FF IGF-I, IGF-II, IGFBP-1, IGFBP-3, IGFBP-4, and PAPP-A were determined using ELISA. Progesterone secretion by cultured granulosa cells (GC) was measured by RIA. RESULT(S): Levels of IGF-II, IGFBP-3, and IGFBP-4 in FF on the day of oocyte retrieval were significantly correlated with embryo scores on day 3 (72 hours after oocyte retrieval). Levels of IGF-II, IGFBP-3, and IGFBP-4 in FF from follicles in which oocytes developed into day 2 embryos (48 hours after oocyte retrieval; 20 >or= embryo score >or= 6) after fertilization were significantly higher than those from follicles in which oocytes were unable to be fertilized and were arrested in embryo development on day 2, whereas the levels of PAPP-A were significantly lower in the former than the latter group. Using multiple regression analysis, we found that high levels of IGFBP-3 and IGFBP-4 combined with low levels of PAPP-A in FF were significantly correlated with successful fertilization and early development into day 2 embryos. In contrast, high FF IGFBP-1 and IGFBP-4 in combination with low FF IGF-I were significantly correlated with a later (day 2-day 3) embryo development. A significant stimulation of P secretion in cultured GCs by the combination of recombinant IGF-II, IGFBP-3, and IGFBP-4 further strengthened these proteins' functional roles in promoting late follicular development. CONCLUSION(S): High IGF-II, IGFBP-3, IGFBP-4, and low PAPP-A levels in FF at the time of oocyte retrieval suggest better oocyte maturation and early embryo development (within 48 hours after oocyte retrieval), whereas high IGFBP-1, IGFBP-4, and low FF IGF-I levels may favor later embryo development (between 48 and 72 hours after oocyte retrieval).

Cells, Cultured↗
PubMed2005

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides.

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.

Bioreactors↗
PubMed2005

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.

Ascorbic Acid↗
PubMed2004

In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres.

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.

Animals↗
PubMed2004

Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos.

After in vitro maturation and fertilization of porcine oocytes, the fertilized embryos were cultured under 5 or 20% oxygen (O2) for 7 days. In embryos cultured under 5% O2 versus 20% O2, development to the blastocyst stage was higher (36.3% versus 22.5%, P < 0.05); the hydrogen peroxide (H2O2) content as a reactive oxygen species was lower (92 pixels versus 111 pixels, P < 0.05); and fragmentation of DNA in 8- to 16-cell stage embryos (estimated by the comet assay) resulted in a shorter (P < 0.05) DNA tail (36 microm versus 141 microm). Antioxidants such as beta-mercaptoethanol (beta-ME) and Vitamin-E (Vit-E) suppressed oxidative damage in the embryos and improved their developmental ability. For embryos cultured under 20% O2, there were the following differences (P < 0.05) between embryos exposed to 0 microM versus 50 microM beta-ME: 28% versus 57% developed to the blastocyst stage; 125 pixels versus 98 pixels per embryo in H2O2 content; and a DNA tail of 209 microm versus 105 microm. In addition, for embryos cultured under 20% O2, there were also differences (P < 0.05) between those exposed to 0 microM versus 50 microM of Vit-E: 28% versus 40% rate of development to the blastocyst stage; 28.9 cells versus 35.9 cells in the expanded blastocyst; 122 pixels versus 95 pixels per embryo (H2O2 content); and 215 microm versus 97 microm length of the DNA tail. Therefore, a low O2 concentration during in vitro culture of porcine embryos decreased the H2O2 content and, as a consequence, reduced DNA fragmentation, and, thereby, improved developmental ability.

Animals↗
PubMed2001

The incidence of cytoplasmic fragmentation in mouse embryos in vitro is not affected by inhibition of caspase activity.

OBJECTIVE: To investigate the relationship between cytoplasmic fragmentation and caspase activity in the mouse embryo. DESIGN: Experimental laboratory study. SETTING: University gynacology unit. ANIMAL(S): One-cell zygote of mouse (MF1 x BALB/c). INTERVENTION(S): Mouse embryos were treated with caspase inhibitors: benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-fmk). MAIN OUTCOME MEASURE(S): Morphological development of the embryo, proportion of fragmented embryos, caspase-3-like activity, DNA breakage, and phosphatidylserine exposure in blastomeres. RESULT(S): The proportion of embryo reaching two-cell, three- to four-cell, and morula stage at 48, 72, and 96 hours after hCG administration, respectively, were comparable between the control embryos and those treated with either z-VAD-fmk or z-DEVD-fmk, at three concentrations (10 microM, 50 microM, and 200 microM). Although the inhibitors suppressed the caspase-3-like activity in the embryo fragment before compaction and decreased DNA breakages, there was no statistically significant difference in the percentage of fragmented embryo between the control and those treated with caspase inhibitors. The inhibitors did not affect the incidence of phosphatidylserine exposure in the blastomere of the treated embryos. CONCLUSION(S): Cytoplasmic fragmentation in precompaction mouse embryos is not a consequence of caspase-related apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human preimplantation embryos.

OBJECTIVE: To examine human embryos at various stages of preimplantation development for simultaneous expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs). DESIGN: mRNAs of specific MMPs and TIMPs were examined in single human embryos, at different stages of preimplantation development, by reverse transcription-polymerase chain reaction (RT-PCR). Single embryo immunohistochemistry was applied to examine the protein expression. SETTING: University-affiliated IVF-ET program. PATIENT(S): Couples, attending the university-affiliated IVF-ET program, electing to donate poor prognosis embryos with anomalous morphology. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in preimplantation embryos. RESULT(S): The MMP-2 mRNA was expressed consistently during development from one-cell to blastocyst stage. The TIMP-1 and TIMP-3 mRNAs were detected in embryos at all stages; however, in the later preimplantation developmental stages, an increasing proportion of embryos expressing TIMP-1 and TIMP-3 mRNA were noted. The MMP-1, MMP-9, and TIMP-2 mRNAs were detected in only a minority of human embryos studied. Immunohistochemistry showed MMP-1 and TIMP-1 protein expression in preimplantation embryos. CONCLUSION(S): The existence of MMP and TIMP mRNA expression in human preimplantation embryos argues for a role for these metalloproteinases and their inhibitors during the process of implantation in humans.

Blastocyst↗
PubMed1986

Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro.

Mouse preimplantation embryos were used to test the effects of three culture parameters on development of 2-cell stage embryos to the late morula stage (40 to 43 hours of culture, 65 to 80 hours after fertilization): concentration of potassium (K)--0.6 mM, 1.4 mM, and 6.0 mM; type of protein supplement, bovine serum albumin (BSA), and bovine serum immunoglobulins (BSIG); and number of embryos (2 or 20) cultured per 10- to 12-microliter drop of T6 culture medium under paraffin oil. Cell number per embryo of cultured late morulae was compared with that of same-age embryos developing in vivo. The developmental rate to late morula stage with 20 embryos per drop of medium was media containing 0.6- and 1.4-mM K, 0.15% BSA, 0.15% BSIG greater than medium containing 1.4-mM K, 0.3% BSA greater than all media containing 6.0-mM K (P less than 0.01 comparing media containing 0.6- or 1.4-mM K with media containing 6.0-mM K). For a given medium, developmental rate was significantly slower for 2 embryos per drop than for 20 embryos per drop of medium with those media containing either 0.3% BSA (for all concentrations of K tested) or 6.0-mM K (for 0.3% BSA and for 0.15% BSA + 0.15% BSIG). Finally, cell number per embryo was: in vivo-developed embryos greater than medium containing 1.4-mM K, 0.15% BSA, 0.15% BSIG much greater than 6.0- or 1.4-mM K, 0.3% BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗