Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro

The effects of recombinant human activin A on the development of bovine one-cell embryos matured and fertilized in vitro were investigated. In experiment 1, one-cell embryos were cultured in a chemically-defined medium, of modified synthetic oviduct fluid supplemented with 1 mg/ml polyvinyl alcohol (mSOF-PVA), containing different concentrations of activin (0, 0.1, 1, 10, and 100 ng/ml) until 240 hr after in vitro fertilization. The addition of -1 ng/m activin to mSOF-PVA improved development to the blastocyst stage (14.5–17.1%), compared with no addition of activin (5.6%). However, there was no significant difference in hatching rate of embryos among treatments. In experiments 2 and 3, the embryos were also cultured in MSOF-PVA at various periods of exposure to 10 ng/ml activin to evaluate (development to the morula and blastocyst stages, respectively. The proportion of morulae was significantly higher in culture with activin at 20–120 hr postinsemination (37.2%) than with control (25.7%). Total number of cells in morulae at 120 hr postinsemination significantly increased by the addition of activin at 20–72 hr (26.1 cells) and 20–120 hr (24.2 cells) postinsemination, compared with control (20.1 cells). When activin was added to the medium during 20–120 hr and 20–192 hr postinsemination, the percentages of blastocysts (18.0% and 18.7%, respectively) were significantly higher than in the control (9.6%). However, the total number of cells in blastocysts was not significantly different. These results demonstrate that activin stimulates the development of bovine one-cell embryos to the morula and blastocyst stages in vitro. © 1996 Wiley-Liss, Inc.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.     

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.     

Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media

Data from other laboratories have shown that speed of bovine blastocyst development is higher when Ménézo B2 is used for coculture compared to TCM199. It was our purpose to investigate whether this early blastocyst formation was also indicative of embryo quality by studying the allocation of inner cells in embryos generated by B2-coculture and by TCM199-coculture. For this purpose, a differential staining technique was used. General embryo development was similar for TCM199- and B2-embryos expressed as rate of cleavage at day 3 and morula-blastocyst formation at day 8 (P > 0.05), but significantly different when expressed as number of eight-cell stages at day 3 and expanded or hatched blastocysts at day 8 (P < 0.01). B2-embryos cultured until day 5, 6, and 7 post insemination, had total cell numbers of 24, 65, and 109 respectively, which was significantly higher than the cell number of TCM199 embryos cultured over the same time period (18, 41, and 71 respectively, P < 0.001). Morphological differentiation was significantly more advanced for B2-embryos at day 7 and 8 (P < 0.0001 and P < 0.001, respectively). First presumptive inner cells appeared in eight- to 16-cell stages at day 3. Because the determination of inner cells by differential staining is depending upon the presence of functional tight junctions, we concluded that the establishment of the tight junction seal in B2-embryos differed from that in TCM199-embryos: Inner cells appeared 0.56 cell cycle later in B2-embryos (P < 0.001) and a larger variation existed in the number of ICM-cells in B2-blastocysts (P < 0.001). The higher total cell number of B2-expanded blastocysts was mainly acquired by trophectoderm growth (P < 0.06). These data indicate that the apparent better quality of B2-embryos (faster cleavage, earlier blastocyst formation) is not reflected in a reliable number of inner cells of B2-blastocysts. © 1996 Wiley-Liss, Inc.     

Genomic DNA methylation patterns in bovine preim-plantation embryos derived from in vitro fertilization

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos. Supported by the National Natural Science Foundation of China (Grant No. 30270956) and High-Tech Research & Development Program of China (Grant No. 2002AA206311)     

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 ± 3.9, 8.8 ± 1.6, 10.8 ± 4.7 and 47.7 ± 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-,4- and 8-cell embryos cleaved asynchronously with <1, 2.6 ± 2.5 and 9.2 ± 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 ± 10.7 and 145.8 ± 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16-cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer.     

Genomic DNA methylation patterns in bovine preimplantation embryos derived from <Emphasis Type="Italic">in vitro</Emphasis> fertilization

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.     

Rapid detection of male-specific DNA sequence in bovine embryos using fluorescence in situ Hybridization

An accurate, reliable, and quick (less than an hour) method for determining the sex of bovine embryos was developed using a fluorescence in situ hybridization (FISH), with a probe designed from a bovine Y chromosome specific DNA (BC1.2). First, to improve a protocol of FISH and evaluate an accuracy of the method, lymphocyte nuclei prepared from three bulls, two cows, and one freemartin were tested. We found that 5 min was enough for hybridization. The washing solution adequate for posthybridization was 0.5× SSC at 72°C for 5 min. The whole procedure for FISH can be accomplished in less than an hour. A male-specific signal was detected, on average, as 97, 0.5, and 83%, respectively, of lymphocytes in males, females, and a freemartin. Using the rapid FISH protocol developed, 28 embryos were divided. According to the presence of the digoxigenin signal, 16 embryos (57.1%) were predicted as male, and 12 embryos (42.9%), predicted as female. Mol. Reprod. Dev. 51:390–394, 1998. © 1998 Wiley-Liss, Inc.     

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32/46) in Bull A and 54.2% (58/107) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 °C; (II) 30 to 80%, 10 °C; (III) 30 to 80% 20 °C; (IV) 30 to 90%, 20 °C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV)_66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.     

Pyruvate prevents peroxide‐induced injury of in vitro preimplantation bovine embryos

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H₂O₂ as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H₂O₂ degrading ability. Essential and nonessential amino acids also significantly reduced the H₂O₂ concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9–16‐cell stage embryos and blastocysts to 0 M; 10⁻⁷ M ; 10⁻⁶ M, and 10⁻⁵ M H₂O₂ in pyruvate‐free mSOF was evaluated. Developmental rates of the H₂O₂‐treated zygotes to the 5–8‐cell or blastocyst stages and survival of H₂O₂‐treated blastocysts were reduced in a dose‐dependent manner whereas the 9–16‐cell embryos were unaffected by those treatments. Blastocysts treated with H₂O₂ also tended to have lower numbers of bisbenzimide‐stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10⁻⁵ M H₂O₂ pulse highly reduced the H₂O₂ concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H₂O₂, the 9–16‐cell embryos being more resistant than zygotes and blastocysts and that H₂O₂ and its toxic effects can be attenuated by including pyruvate in the medium. Mol. Reprod. Dev. 52:149–157, 1999. © 1999 Wiley‐Liss, Inc.     

A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Successful implantation of in vitro cultured rabbit embryos after uterine transfer: A role for mucin

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 ± 0.2 μm of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 ± 5.5 μm. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20–40 μm thick mucin layer. No live young were obtained from 57 embryos with less than a 20 μm thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos. © 1996 Wiley-Liss, Inc.     

In vitro culture of Arabidopsis embryos within their ovules

Embryogenesis of flowering plants establishes a basic body plan with apical-basal, radial and bilateral patterns from the single-celled zygote. Arabidopsis embryogenesis exhibits a nearly invariant cell division pattern and therefore is an ideal system for studies of early plant development. However, plant embryos are difficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here we present a method for the culture of zygotic Arabidopsis embryos in vitro. The technique omits excision of the embryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables external manipulation of embryo development and culture from the earliest developmental stages up to maturity. Administration of various chemical treatments as well as the use of different molecular markers is demonstrated together with standard techniques for visualizing gene expression and protein localization in in vitro cultivated embryos. The presented set of techniques allows for so far unavailable molecular physiology approaches in the study of early plant development.     

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (beta-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06 +/- 3.63 mmol/L), D3UF (10.54 +/- 5.16 mmol/L), or D5UF (10.23 +/- 6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89 +/- 1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3 +/- 7.9%) or D5UF (PZM3-D5UF, 14.3 +/- 10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5 +/- 15.1), 72 (25.6 +/- 10.4), and 96 (18.7 +/- 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8 +/- 9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6 +/- 8.3) was increased compared to PZM3-SAA (40.5 +/- 3.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 318 +/- 8, 320 +/- 32, 321, and 293 +/- 8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360 mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.3 +/- 3.4%), the blastocyst rate was higher than original PZM3 (21.2 +/- 2.2%) (288 mOsM).     

Production of bovine tetrapolid embryos by electrofusion and their developmental capability in vitro

Optimal conditions of electrofusion for blastomeres of two-cell bovine embryos to produce tetraploid embryos were investigated. The high fusion rate (73–95%), viability, and develop mental capacity were obtained under a field strength of 1.0 kV/cm with direct current pulses of 10 or 25 μsec duration applied twice. Cytological study showed that 78.6% (11/14 embryos) of embryos exposed to electrofusion had tetraploid chromosome sets and the others were diploid or hexaploid. The tetraploid embryos had the capability to develop up to morulae stage in vitro.