OBJECTIVETo study the expression of gene associated with retinoid-interferon-induced mortality-19(GRIM-19) in preimplantation embryo of mice and explore its role in embryonic development.

METHODSThe protein and mRNA expressions of GRIM-19 in 2-cell, 4-cell, 8-cell, morula, and blastocyst phases of mice preimplantation embryo were detected by Western blot analysis and Real-time polymerase chain reaction (PCR).

RESULTSGRIM-19 was continuously expressed in every stage of preimplantation embryo of mice. Western blot analysis and Real-time PCR demonstrated a gradual increase of GRIM-19 expression from 2-cell, which reached a peak in 8-cell phase and then decreased progressively.

CONCLUSIONSThe expression of GRIM-19 in mouse preimplantation embryos changes as at different developmental phases. GRIM-19 may play an important role during embryonic development.

Animals ; Blastocyst ; drug effects ; metabolism ; Female ; Interferons ; pharmacology ; Mice ; NADH, NADPH Oxidoreductases ; genetics ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; Tretinoin ; pharmacology

Mouse blastocysts were exposed to doses of 0, 1 and 10 mumol/L retinoic acid (RA) for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10 mumol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.
Antigens, CD95/*biosynthesis ; Apoptosis/*drug effects ; Blastocyst/cytology ; Blastocyst/*metabolism ; Cell Culture Techniques/methods ; Cells, Cultured ; Gene Expression Regulation/*drug effects ; In Situ Nick-End Labeling ; RNA, Messenger/metabolism ; Tretinoin/*pharmacology

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.
Animal ; Blastocyst/secretion ; Blastocyst/enzymology* ; Female ; Fertilization in Vitro/methods ; Gonadotropins, Chorionic/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Pregnancy ; Serine Endopeptidases/secretion ; Serine Endopeptidases/metabolism* ; Zona Pellucida/physiology* ; Zona Pellucida/drug effects

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.
Animals ; Blastocyst/*drug effects ; Embryo, Mammalian/drug effects/*embryology/physiology/ultrastructure ; Fertilization in Vitro/veterinary ; Gonadotropins/administration & dosage/*metabolism ; In Vitro Oocyte Maturation Techniques/*methods/veterinary ; Parthenogenesis/*drug effects ; Sus scrofa/*embryology

In this study, we examined the effects of a two-step culture system, which involves the use of different culture media for early cleavage and later stage embryos, on the in vitro development of bovine embryos. We also investigated the effect of glucose, phosphate and citrate on the in vitro early developmental period of bovine embryos in a two-step culture system. Moreover, the supplementation of different protein sources (BSA-V, BSA-FAF and FBS) during IVC did not affect the frequency of blastocyst development. Using two-step culture, embryos were cultured in protein-free media for an initial 5 days. This was then followed by the same culture media or an FBS supplemented media. The developmental rates of blastocysts in the FBS containing group were significantly higher than in the replaced with no serum containing group. Embryos cultured in mSOF supplemented with 1.5 mM glucose plus 1.2 mM phosphate were significantly inhibited. The inhibition of developmental competence by glucose plus phosphate was consistent with the existence of 0.5 mM sodium citrate. This study indicates that a two-step culture system, which applies different conditions for early cleavage embryos, i.e., serum-free media, vs. later stage embryos, with serum containing media, may be effective for in vitro production systems. In addition, the developmental competence of bovine embryos was depressed in the presence of glucose plus phosphate as compared to either alone or the absence of both. Therefore, the avoidance of this negative effect should allow more optimal conditions to be developed for in vitro production.
Animals ; Blastocyst/drug effects/metabolism ; Cattle/*embryology ; Citric Acid/pharmacology ; Culture Media/*chemistry ; Culture Techniques/*methods ; Ectogenesis/drug effects ; Embryo/*drug effects/embryology/metabolism ; Embryonic and Fetal Development/drug effects ; *Energy Metabolism ; Female ; Fertilization in Vitro ; Glucose/pharmacology ; Male ; Phosphates/pharmacology ; Proteins/*pharmacology ; Zygote/drug effects/metabolism

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine