Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and $CoCl_2$ exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with $CoCl_2$. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.
This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.
Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-$\beta$, EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-$\beta$ gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.
Hur, Yong Soo;Ryu, Eun Kyung;Yoon, San Hyun;Lim, Kyung Sil;Lee, Won Don;Lim, Jin Ho
Clinical and Experimental Reproductive Medicine
/
v.43
no.3
/
pp.146-151
/
2016
Objective: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface. Methods: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development. Results: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups. Conclusion: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos.
One of the biggest problems involved in transgenic animal production is lack of appropriate market genes. To overcome this problem, we tested whether the genes of SEAP (secreted alkaline phosphatase) and GFP (green fluorescence protein) on our retrovirus vectors can be applicable to the transgenic animal production. The main advantage of these marker genes over other generally mainpulation can be selected without sacrificing viability. The results obtained in this study are summarized as follows: 1. Removal of zona pellucida from the mouse zygotes did not affect embryo developments to blastocysts. 2. Co-culture of zona-free embryos with virus-producing cells for 6 hours also did not affect embryo developments to blastocysts. 3. Among 58 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells, SEAP expression was observed from the 6 blastocysts. 4. Expression of the GFP gene was detected from the virus- producing cells but no embryo expressing the gene was counted among 50 blastocysts developed from the zona-free zygotes co-cultured with the virus-producing cells.
The ultrasound-guided oocytes cllection (ovum pick-up ; OPU) has become a substitution for superovlation in cattle. The objective of this study was to examine the effect of OPU frequency on the in vitro production of embryos in Hanwoo cattle. Six cycling Hanwoo cows were distributed into two groups for either once or twice weekly OPU sessions. Oocytes were collected by ultrasound-guided follicle aspiration(SA600) using a 6.5HMz transducer and attached with 18 gauge needle, with vacuum pressure of 40 mmHg. The cumulus-oocyte complexes (COCs) collected from each donor were matured in TCM 199 supplemented with 10% fetal bovine serum at 5% CO2 in air at 38.5$^{\circ}C$ for 22h and in vitro matured oocytes were co-incubated with sperm(separated by Percoll gradient) for 6h. The zygotes were co-cultured on cumulus cell monolayer in 10ul droplets in the same culture medium and conditions used for IVM for 7 days. On Day 7 of culture, development to blastocysts was examined. Although the number of oocytes collected was variable depending on individuals, overall embryo production in the twice per week OPU sessions was better that in the once per week sessions(6~21 vs 2~7 blastocysts produced, respectively). Two cows(E, A) were good oocyte donors and embryo production was superior in cow C ; however, cow F was a poor donor as compared to the others. In conclusion, these results suggest that for embryo production, twice weekly OPU sessions were better than once per week for producing embryos in vitro from Hanwoo cattle.
This experiment was conducted to investigate the effects of length of storage period under low temperature, $CO_2$ enrichment and addition of plant growth regulators in Murashige and Skoog medium on the plant regeneration of Korean ginseng (Panax ginseng C. A. Meyer). Seeds were treated for 60 and 80 days respectively under $5^{\circ}C$ environment. 2500ppm of $CO_2$ was enriched by ventilation. Three plant growth regulators added to the medium were Indolbutyric acid, Benzyladenin and Gibberellic acid (GA3). The result indicated that : The capacity of differentiation was higher in the aged cotyledons from the seeds treated for 80 days under low temperature condition than in those treated for 60 days. $CO_2$ enrichment had stimulating effects on the growth and development of shoot primordium significantly but less effects on the formation of adventitious buds. From one zygotic embryo hundreds of plantlets were differentiated. $CO_2$ enrichment had effects on the formation of both indirect somatic embryo and direct somatic embryo. Indirect somatic embryo showed little growth and differentiation, being undifferentiated vascular stele and epicotyl. Direct somatic embryos were formed on the epidermis of backside basal part of cotyledon. Those embryos developed to whole plant having latent bud.
Lee, Hae-Lee;Park, Jae-Hee;Kim, Yong-Su;Kim, Jong Gug
Journal of Embryo Transfer
/
v.28
no.1
/
pp.41-48
/
2013
This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.
The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized(IVM /IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements. A total of 1,386 IVM /IVF oocytes were used to compare a simple defined medium(KSOM) with more complex culture conditions used successfully for culture of bovine embryos but do not permit study of specific requirements. All experiments were extensively replicated factorials. In Experiment 1, KSOM was superior to Menezo B$_2$ medium in producing morulae plus blastocysts from IVM /IVF oocytes(33 vs 20%, P<0.()5). The yield of morulae plus blastocysts with KSOM was 22% and with BRLC added was 30%. In Experiment 2, (a 2x2 factorial of KSOM with or without BRLC and 0, 1 ng /ml of platelet derived growth factor, PDGF) more morulae plus blastocysts (40%) were produced in KSOM-BRLC co-culture containing 1 ng /ml PDGF than in the control KSOM(12%). In Experiment 3, there was no dose response when 0, 1 and 5 ng /ml of PDGF were added. The results with simple defined KSOM medium are sufficiently promising to indicate that specific requirements of the embryo may be examined in future studies with KSOM as a base.
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