Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.     

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles.

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.     

Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.     

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

Summary Bovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5μg/ml insulin, and 0.5μg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

基因表达谱分析牛早期胚胎发育过程的调控机理

早期胚胎的死亡会给畜牧业的发展带来巨大的经济损失, 特别是对于母牛生产来说更是如此, 因此研究早期胚胎发育过程具有极为重要的价值。文章从公共数据库GEO中选取了关于牛早期胚胎发育过程的一套基因表达谱数据, 通过显著性分析及聚类分析来研究牛早期胚胎发育过程中不同时期的基因组表达模式。结果表明: 整个牛早期胚胎发育过程大致可划分为3组不同的基因组表达模式阶段; 同时, 差显基因在不同时期表达量的波动情况表明8-细胞期和16-细胞期在牛的整个早期胚胎发育过程的重要性。另外, 通过进一步的功能注释和通路分析表明在牛胚胎早期发育不同阶段时期存在着一些重要因子及相关通路。     

Development of early bovine embryos in co-culture with KSOM and taurine, superoxide dismutase or insulin

Three experiments, utilizing 2578 embryos, were designed to test the effects of media, taurine, Superoxide dismutase and insulin on the development of embryos produced by in vitro maturation and in vitro fertilization (IVM/IVF). Embryos showing at least 1 cleavage during culture for 40 to 44 h after IVM/IVF were selected for further culture under various conditions for 6 d at 39 degrees C in 5% C0(2):95% air. A Buffalo rat liver (BRL) cell co-culture was used in all 3 experiments. Experiment 1 was a 3 x 2 factorial arrangement with KSOM (a high potassium simplex optimization-derived medium containing only 12 ingredients), Menezo B(2) and TCM-199 media with or without 7 mM taurine. Blastocyst production in the 3 media, respectively, was 48, 36 and 29% (P<0.05). Addition of 7 mM taurine increased the percentage of blastocysts from 34 to 42 (P<0.05). In Experiment 2, Superoxide dismutase (SOD) did not improve blastocyst development (P>0.05). In Experiment 3, insulin (75 ng/ml) added to KSOM resulted in 46% morulae plus blastocysts compared with 35% for the control (P<0.05). These results indicate that the co-culture of embryos in KSOM with taurine or insulin added is superior to commonly used complex media for efficient production of blastocysts following IVM/IVF.     

Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro–produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.     

LPS/CD14 activation triggers SGLT-1-mediated glucose uptake and cell rescue in intestinal epithelial cells via early apoptotic signals upstream of caspase-3

Recent findings indicate that enhanced glucose uptake protects enterocytes from excessive apoptosis and barrier defects induced by LPS exposure. The aim of this study was to characterize the mechanisms responsible for increased sodium-dependent glucose cotransporter (SGLT)-1 activity in enterocytes challenged with LPS. SGLT-1-transfected Caco-2 cells were incubated with LPS in high glucose media. LPS increased SGLT-1 activity in dose- and time-dependent fashion, and is due to increased V(max) of the cotransporter. Elevated apical expression of SGLT-1 was also demonstrated. This LPS-induced effect was colchicine-inhibitable, suggesting microtubule-dependent translocation of SGLT-1 onto apical surface. Immunofluorescence staining showed expression of CD14 on the apical surface, but no TLR-4, on these cells. Neutralizing anti-CD14 decreased the LPS-induced upregulation of SGLT-1 activity, whereas anti-TLR-4 had no effect. Pharmacological studies indicated that signaling for LPS-mediated SGLT-1 glucose uptake depends on caspase-8 and -9 activation, but occurs independently of caspase-3. The findings describe a novel feedback mechanism within the apoptotic signaling pathway for SGLT-1-dependent cytoprotection. The observation suggests a new function for CD14 on enterocytes, involving the induction of the caspase-dependent SGLT-1 activity, which ultimately leads to cell rescue. The understanding of these signaling events may shed light on enterocytic cytoprotection and homeostasis mechanism upon pro-apoptotic challenges.     

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.This study was supported by grants from the China National Natural Science Foundation (nos. 30430530 and 30571337) and the “973” Project of the Chinese Science and Technology Ministry (no. G200016108).     

Developmental competence and mRNA expression of preimplantation in vitro-produced embryos from prepubertal and postpubertal cattle and their relationship with apoptosis after intraovarian administration of IGF-1

Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1-treated preP group (11.2 ± 2.4%), the control preP group (10.4 ± 3.0%), and in the IGF-1 postP group (10.9 ± 2.3%). These were lower (P ≤ 0.01) compared with the control postP group (21.2 ± 3.8%) and with cows (23 ± 3.7%). The expression profile of the six genes was partly affected by age and IGF-1 treatment. Apoptosis was correlated with the age of the oocyte donors and was increased in blastocysts derived from prepubertal heifers. Results show that apoptosis is a critical feature of the acquisition of developmental competence of oocytes from prepubertal cattle and that IGF-1 did not beneficially affect oocyte developmental competence.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.