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PubMed2003

Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): a comparison between five methods.

The purpose of this study was the comparison of the diagnostic sensitivity between buffy coat (BC), peripheral blood (PB), lymph node (LN), bone marrow (BM) and short-term culture (P-D) cytology that has been based on the detection of Ehrlichia canis morulae, in the acute phase of canine monocytic ehrlichiosis (CME). Their cellular localization, total numbers and microscopic differentials were also investigated. The highest sensitivities were achieved after evaluating 1000 oil immersion fields (OIFs) in BC (66%) and an equal number in LN (60.9%) smears, separately or together (74%). The morulae were more often detected into lymphocytes than monocytes. The highest total number of morulae (n=143) were found in P-D smears. Finally, to avoid false positive diagnoses, platelets, lymphocytic azurophilic granules, lymphoglandular bodies and phagocytosed nuclear material should not be confused with the morulae.

Animals↗
PubMed1998

Development of embryos produced by intracytoplasmic sperm injection of cat oocytes.

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.

Animals↗
PubMed2006

Metabolic regulation of in-vitro-produced bovine embryos. I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls.

The toxic and/or beneficial effects of four metabolic regulators on embryo development were evaluated. In-vitro-produced compact morulae were cultured for 3 days in a chemically defined medium + bovine serum albumin (BSA; CDM-2) plus regulators (4991 total embryos). Phenazine ethosulfate (PES), phloretin (PL), pyrroline-5-carboxylate (P5C), and sodium azide (NaN3) were evaluated at four doses each in factorial combinations with four concentrations of glucose: 0, 0.5, 2, and 8 mm. Phenazine ethosulfate at 0.9 microm resulted in poorer development than lower or no PES. Phloretin was, in general, detrimental for embryo development, but most markedly at the highest dose (270 microm). Pyrroline-5-carboxylate had little effect on post-compaction embryos at the doses studied, 9 to 81 microm. Sodium azide at the concentrations used (3, 9, and 27 microm) had little effect on embryo development compared with controls. Concentrations of glucose had little effect on development of embryos. A fifth metabolic regulator, 2,4-dinitrophenol (DNP), was studied at various doses at pre-morula or morula-blastocyst stages cultured in 2 mm glucose. Embryos (2189 total) cultured in 90 microm DNP developed more slowly and were darker than embryos cultured at lower doses. Embryos cultured in 30 microm DNP had a higher blastocyst rate (48.3%) than controls (34.9%). In the last experiment using G1.2/G2.2 media, DNP (30 microm) resulted in a marked decrease in embryonic development when embryos were exposed at the zygote to 8- to 16-cell stages but had little effect when morulae were exposed for 2 days. The dose-response information for these metabolic regulators is crucial for designing future experiments.

2,4-Dinitrophenol↗
PubMed1991

Antibodies to sperm surface antigens and the c-myc proto-oncogene product inhibit early embryonic development in mice.

The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab' antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab' identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab' did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development.

Animals↗
PubMed1993

Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro.

In this study we examined the role of glucose, and the use of fructose as a replacement, in embryo culture medium. Three embryo culture media were used: a routine embryo culture medium (M16), M16 without glucose (M16-G) and M16-G supplemented with fructose (M16F-G). Their effect on fertilization, rate of cleavage, and embryo viability were examined in both an outbred (OF1) and an inbred (C57Bl) mouse strain. Of the three media, only M16 was found to support fertilization. In vitro-fertilized embryos from OF1 oocytes and C57Bl oocytes and OF1 sperm were placed in the different media at the early 2-cell stage. In 65% of OF1 embryos cultured in M16, development was blocked at the 2-cell stage, whereas in M16-G and M16F-G embryos, only 22% and 32%, respectively, were blocked. M16F-G medium also produced morulae and blastocysts with higher cell numbers than M16-G. In vitro-fertilized C57Bl 2-cell embryos cultured in M16 displayed retarded cleavage to the 4-cell stage compared to embryos cultured in M16-G and M16F-G. In contrast, the morulae and blastocyst cell numbers were significantly lower in M16-G compared to M16 and M16F-G. The viability of morulae and blastocysts obtained from OF1 and C57Bl embryos cultured in M16-G and M16F-G was lower compared to control C57Bl morulae and blastocysts cultured in M16. The results show that although morphologically normal embryos could be obtained in M16-G and M16F-G, inherent anomalies existed that limited viability.

Animals↗
PubMed1994

In vivo embryogenesis, embryo migration, and embryonic mortality in the domestic cat.

In vivo embryogenesis, embryo migration, and survival were studied in the domestic cat. Queens were naturally mated (three times daily) on the second and third days of behavioral estrus and, if ovulation occurred, ovariohysterectomized at 64 (n = 8), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), and 480 h (n = 8) after first copulation. Of 52 cats mated, 48 (92.3%) ovulated (as evidenced by the presence of ovarian CL), and of these, 38 (79.2%) either produced good-quality embryos or had implantation sites. From the remaining cats, only unfertilized oocytes (n = 5), degenerating embryos (n = 4), or no oocytes/embryos (n = 1) were recovered. Embryos at 64, 76, 100, and 124 h after the first copulation typically were 1 to 4 cells (17 of 20; 85.0%), 5 to 8 cells (18 of 28; 64.3%), 9 to 16 cells (14 of 24; 58.3%), and morulae (15 of 21; 71.4%), respectively; all were within the oviducts. At 148 h, embryos primarily were compact morulae or early blastocysts (15 of 18; 83.3%), and all were within the uterus. For the preimplantation groups, the overall recovery of embryos plus oocytes per CL was 80.6%, and the mean (+/- SEM) numbers of CL and embryos were 64 h, 4.8 +/- 0.3, 3.1 +/- 0.8; 76 h, 4.7 +/- 0.3, 3.9 +/- 0.6; 100 h, 5.8 +/- 0.5, 3.3 +/- 0.8; 124 h, 4.4 +/- 0.5, 4.0 +/- 0.6; and 148 h, 6.5 +/- 1.1, 3.7 +/- 0.7, respectively. Cats in the 480-h group produced a mean of 5.6 +/- 0.5 CL and 3.9 +/- 0.5 implantation sites. In six of eight cats in this group, there was a disparity between CL number on a given ovary and number of implantation sites in the ipsilateral horn, supporting the concept of transuterine embryo migration. In summary, results indicated that 1) more than 90% of cats ovulated following this multiple mating regimen, but approximately 21% of these failed to produce any fertilized or viable embryos; 2) embryo developmental rate in vivo was biphasic, with a rapid cleavage rate to the 5- to 8-cell stage followed by a slower cleavage rate to the morula stage; 3) cat embryos entered the uterus as compact morulae or early blastocysts approximately 5.5 days after the first copulation; and 4) on the basis of implantation/CL ratio, approximately 30% of all ovulated cat oocytes underwent either fertilization failure or preimplantation embryonic mortality.

Animals↗
PubMed2006

Apoptosis-independent poor morphology of bovine embryos produced by multiple ovulation.

In multiple ovulation and embryo transfer (MOET) programmes in cattle, a considerable number of morphologically poor-quality embryos continue to be produced; this is one of the limiting factors of the technique. Apoptosis has often been implicated in developmental arrest and fragmentation; these are regarded as poor traits of embryonic quality in mammalian pre-implantation embryos. In the present study, apoptosis was assessed in morphologically poor-quality embryos in comparison with good-quality embryos that were recovered from a MOET programme. Retarded embryos (two to 16 cell stage), morulae with severe fragmentation and morphologically good-quality morulae recovered from superstimulated cows at day 7 post-insemination were subjected to TdT-mediated dUTP nick-end labelling (TUNEL) and Hoechst staining. Cell nuclei that showed both TUNEL staining and apoptotic morphology were considered to be apoptotic. Apoptotic index (AI) was calculated as the percentage of apoptotic cells per embryo. Fifteen of 17 retarded embryos and 10 of 15 morphologically poor-quality morulae did not show signs of apoptosis. The mean AIs in the morphologically poor-quality embryos (two to 16 cell stage, 2.2%; poor morulae, 1.3%) were as low as that in the good-quality embryos (2.9%). These results suggest that another mode of developmental arrest and/or fragmentation that is independent of apoptosis occurs in morphologically poor-quality embryos recovered from MOET programmes.

Animals↗
PubMed1997

Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform.

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-), differing in the presence or absence of an 80 residue alpha domain were reported. Here, the temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA was present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also showed synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ was only apparent from the blastocyst stage. In addition, 33P-labelling showed both isoforms to be phosphorylated at the early blastocyst stage. The pattern and timing of membrane assembly of the two isoforms was also distinct. ZO-1alpha- was initially seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the membrane until they completely surrounded each cell with a zonular belt by the late morula stage. ZO-1alpha+ however, was first seen as perinuclear foci in late morulae before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurred during the 32-cell stage and was zonular just prior to the early blastocyst stage. Immunostaining indicative of both isoforms was restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation were sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. In addition, the tight junction transmembrane protein occludin co-localised with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. Moreover, the late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo.

Alternative Splicing↗
PubMed2003

Effects of BSA and fetal bovine serum in culture medium on development of rat embryos.

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.

Animals↗
PubMed1981

Effects of various cryoprotective agents on the survival of unfrozen and frozen mouse embryos.

The effects of DMSO, ethylene glycol, glycerol, erythritol, dimethylformamide and sucrose on the survival of unfrozen and frozen mouse morulae were examined. All the agents had a deleterious effect on survival of the unfrozen morulae at 20 degrees C. The harmful effect of erythritol was lower at 0 degrees C and sucrose had a protective effect at that temperature. Dimethylformamide and DMSO were more harmful than ethylene glycol and glycerol at both temperatures. Higher proportions of the morulae frozen in DMSO developed into expanded blastocysts when slowly frozen samples were thawed slowly (82%) than when they were thawed rapidly (85%) than when they were thawed slowly (4%). When the samples were frozen in the presence of ethylene glycol or glycerol, higher survival rates were obtained after slow (80--94%) than rapid (1--59%) freezing. When erythritol was used, only embryos thawed rapidly developed (31--50%) in subsequent cultures. Sucrose and dimethylformamide did not afford cryoprotection. Morulae frozen--thawed rapidly in the presence of ethylene glycol, glycerol or erythritol and transferred to recipients developed into normal young.

Animals↗
PubMed1994

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium.

Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three-four-cell, five-eight-cell, 9-16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11-62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula-blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three-four-cell and five-eight-cell), the higher the probability that they developed into morula-blastocyst: 70% of the embryos reaching the two-cell stage before 30-31 h after insemination developed into morula-blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.

Animals↗
PubMed1991

Cryopreservation of mouse zona-free embryos and embryos exposed to biopsy and reconstruction by vitrification in microdrops.

Zona-free zygotes, 2-cell, 4-cell, and 8-cell embryos, morulae, blastocysts and embryos exposed to biopsy and reconstruction were cryopreserved in microdrops of vitrification medium expelled directly into liquid nitrogen. While none of the zygotes and 2-cell embryos survived storage in liquid nitrogen, 54% 4-cell and 97% 8-cell embryos, 99% morulae, 88% blastocysts, 98% biopsied and 100% reconstructed embryos were intact one hour after thawing. Developmental potential of cryopreserved zona-free embryos evaluated after 30 h of in vitro culture was high. Of the frozen embryos, 49% 4-cell and 92% 8-cell embryos, 95% morulae, 79% blastocysts, 84% biopsied and 90% reconstructed embryos were capable of further cleavage. After the transfer of zona-free 8-cell embryos, morulae, blastocysts, biopsied and reconstructed embryos into day-1 recipients - 4 (16%), 14 (56%), 3 (12%), 0 and 8 (32%) live foetuses were recorded, respectively.

Animals↗
PubMed1982

Turnover of embryonic messenger RNA in preimplantation mouse embryos.

We have estimated the average half-life of embryonic messenger RNA in mouse embryos at two stages of preimplantation development. Embryos were collected at 48 and 75 h post-hCG and cultured overnight in the presence of [3H]uridine. Beginning at 65--68 h (morulae) or 92--94 h (early blastocysts), the label was withdrawn and replaced with unlabelled uridine, and samples were taken at intervals thereafter for RNA isolation. Label in cytoplasmic, poly(A)-containing RNA was measured after binding to oligo(dT)-cellulose, and was normalized to label in 28S and 18S ribosomal RNA, separated on sucrose gradients. The stability of rRNA in both stages was verified directly, as was the integrity and purity of the isolated mRNA. With morulae, the mRNA decay curve was monophasic, with an average half-life of 9.5 +/- 0.9 h. In three experiments with early blastocysts the decay curve appeared to be biphasic, consisting of short-lived (less than 6 h) and long-lived (30--50 h) components; in two other experiments a short-lived component was not evident. In all cases, however, the overall average half-life of mRNA in early blastocysts, determined by linear regression assuming monophasic kinetics, was greater than that in morulae. Our data indicate that the stability of embryonic mRNA increases by at least twofold during the morula-to-blastocyst transition. The results are considered in terms of the transcriptional dependency of early mouse embryos and the regulation of maternal and embryonic mRNA.

Animals↗
PubMed1981

Factors influencing the results of transfers of rabbit embryos stored at -196 degrees C.

Rabbit embryos at the 8-cell and morula stages were frozen and stored at -196 degrees C for 2-200 days. After thawing the embryos were examined for their viability in vitro and in vivo. In vitro, 62.5% of frozen 8-cell embryos and 81.4% of frozen morulae developed to blastocysts. In the control group of unfrozen embryos, 93.2% 8-cell embryos and 92.4% morulae developed to the blastocyst stage. Culture permitted a more reliable elimination of the embryos damaged during freezing and thawing. Embryos were transferred into the reproductive tracts of the recipients either directly after thawing or after 24 h in culture. Synchronous transfers of frozen rabbit embryos were not successful. After asynchronous transfers of morulae and blastocysts into the oviducts, implantation was 31.8% and 42.9%, respectively. After transfer of blastocysts into the uterine horns of the recipients, 47.6% embryos implanted.

Animals↗
PubMed1996

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 > or = 1 ng/ml 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.

Activins↗
PubMed1996

Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES.

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O2:5% CO2:90% N2. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's 1X NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8-16-cell stage embryos past the morula stage. In experiment 3, the addition of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P < 0.05) and 86, 77, 77, 78, and 69% on Day 5 (P > 0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when alpha-amanitin (20 microM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the alpha-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of alpha-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development.

Amanitins↗
PubMed1995

Expression of X-linked genes in androgenetic, gynogenetic, and normal mouse preimplantation embryos.

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplantation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androgenetic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blastocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both androgenones and gynogenones at the blastocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, whereas the developmental defects of gynogenones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those farthest away from the X inactivation center.

Animals↗