Transcriptional analysis of buffalo (Bubalus bubalis) oocytes during in vitro maturation using bovine cDNA microarray.

The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation.

Gene expression in the in vitro-produced preimplantation bovine embryos.

Recent studies have demonstrated the relevance of a gene expression profile as a clinically important key feature determining embryo quality during the in vitro preimplantation period. Although the oocyte origin can play a crucial role in blastocyst yield, the postfertilization culture period has a profound effect in determining the blastocyst quality with particular regard to the relative abundance of many developmentally and clinically important candidate genes. During the preimplantation period, the embryo undergoes several morphogenetic developmental events including oocyte maturation, minor and major forms of embryonic genome activation and transition of transcription from maternal to embryonic control. The effect of an altered gene expression pattern on the in vitro-produced bovine embryos, particularly when cultured under suboptimal conditions, was reflected by the occurrence of clinically important phenomena like apoptosis and the large offspring syndrome. This review attempts to focus on the morphogenetic embryo development and gene expression profile in the in vitro-produced bovine embryos, with special emphasis on the different parameters that may alter gene expression pattern during the critical period of in vitro culture. The effect of the in vitro system, as reflected by some clinically important phenomena like apoptosis, is also discussed.

Morphology of dromedary camel oocytes and their ability to spontaneous and chemical parthenogenetic activation.

The present work was conducted to examine (1) the morphology of dromedary cumulus-oocytes complexes (COCs), (2) to study the incidence of spontaneous development of oocytes in vivo and (3) to assess the ability of in vitro matured dromedary oocytes to chemical parthenogenetic activation compared with in vitro fertilized (IVF) oocytes. COCs were recovered from dromedary ovaries classified according to their morphology into six categories. Oocyte diameter was measured using eye piece micrometer. For chemical activation, COCs with at least three layers of cumulus-cells were in vitro matured (IVM) in TCM 199 + 10 microg/ml FSH + 10 IU hCG/ml + 10% FCS + 50 microg/ml gentamycin. COCs were incubated for 40 h at 38.5 degrees C under 5% CO2 in humidified air. After IVM, matured oocytes with first polar body (first Pb) were divided into two groups. Group 1: activated in 7% ethanol (E) for 5 min followed by culture in 2 mM 6-dimethylaminopurin (6-DMAP, E D, subgroup 1) or 10 microg/ml cycloheximide (CHX, E CHX, subgroup 2) for 3.5 h at 38.5 degrees C under 5% CO2. In group 2, oocytes were activated using 50 microM Ca A23187 (Ca A) for 5 min followed by culture in 2 mM 6-DMAP (Ca D, subgroup 3) or 10 microg/ml CHX(Ca CHX, subgroup 4) for 3.5 h at 38.5 degrees C under 5% CO2. For control group, IVM oocytes were fertilized using frozen-thawed camel spermatozoa separated by swim-up method then suspended in Fert-TALP medium supplemented with 6 mg/ml BSA (FAF) + 10 microg/ml heparin. In all groups, oocytes were in vitro cultured in SOFaa medium + 5% FCS and 5 microg/ml insulin + 50 microg/ml gentamycin. Cleavage rate and embryo development were checked on Days 2, 5 and 8. An average of 11.3 +/- 0.3 COCs were recovered/dromedary ovary. Categories 1 and 2 represented 33.1% and 34.8%, respectively, and were significantly higher (p < 0.01) than the other categories (19.1, 9.2 and 2.6% for categories 3-5, respectively). Category 6 (embryo-like structures) represented 1.2% of the recovered oocytes, staining of these embryo-like structures with orcien dye indicated the presence of divided cells with condensed nuclei. Dromedary oocytes averaged 166.2 +/- 2.6 microm in diameter with black cytoplasm. Chemical activation of IVM dromedary oocyte with first Pb in 7% ethanol or 50 microM Ca A followed by culture in 2 mM 6-DMAP showed significantly higher (p < 0.01) cleavage and developmental rates to the morula stage than oocytes activated using 7% ethanol or 50 microM Ca A followed by 10 microg/ml CHX or in vitro fertilized control group. Higher (p < 0.01) proportion of oocytes sequentially cultured in 10 microg/ml CHX or that in vitro fertilized were arrested at the 2-4-cell stage compared with that cultured in 6-DMAP.