Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.

Xenooplasmic transfer between buffalo and bovine enables development of homoplasmic offspring.

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Assessment of stage T1 (TNM 1997) for renal cell carcinoma: is recommended the subdivision in T1a and T1b?

INTRODUCTION: Classification TNM 1997 defines renal cell carcinoma smaller than 7 cm and confined to the kidney as stage T1. Our goal is to discuss if tumors smaller than 4 cm have the same behavior characteristics then tumors between 4 and 7 cm, to compose the same stage of the disease. MATERIALS AND METHODS: Retrospective assessment of 138 patients in stage T1 (TNM - 97), divided into 2 groups; group-1: composed of 65 patients (47 percent) with tumors < 4 cm, and group-2: composed of 73 patients (53 percent) with tumors between 4 and 7 cm. The following prognostic factors were assessed in the recurrence of the disease and survival of patients: nuclear degree, microvascular invasion, sarcomatous degeneration, and involved lymph nodes. Statistical evaluation has been accomplished through the log rank test, chi-square test, and Fishers exact text. RESULTS: Average tumor size was 2.5 cm for group-1, and 5.3 cm for group-2. In group-2, there was the predominance of worse prognostic factors, with high-grade tumors (p = 0.01) and presence of microvascular invasion (p = 0.001). Sarcomatous tumors and involvement of lymph nodes did only happen in group-2. Disease-free survival for group-1, analyzed in the median period of 36 months, was 100 percent, and for group 2, in the median period of 31 months, was 81 percent (p = 0.008). CONCLUSION: The results obtained allow the conclusion that the present stage T1 for renal cell carcinoma gathers tumors of different evolution, being therefore recommendable the stratification in T1a for tumors smaller than 4 cm, and T1b for tumors between 4 and 7 cm

Assessment of stage T1 (TNM 1997) for renal cell carcinoma: is recommended the subdivision in T1a and T1b?

INTRODUCTION: Classification TNM 1997 defines renal cell carcinoma smaller than 7 cm and confined to the kidney as stage T1. Our goal is to discuss if tumors smaller than 4 cm have the same behavior characteristics then tumors between 4 and 7 cm, to compose the same stage of the disease. MATERIALS AND METHODS: Retrospective assessment of 138 patients in stage T1 (TNM - 97), divided into 2 groups; group-1: composed of 65 patients (47%) with tumors < 4 cm, and group-2: composed of 73 patients (53%) with tumors between 4 and 7 cm. The following prognostic factors were assessed in the recurrence of the disease and survival of patients: nuclear degree, microvascular invasion, sarcomatous degeneration, and involved lymph nodes. Statistical evaluation has been accomplished through the log rank test, chi-square test, and Fisher's exact text. RESULTS: Average tumor size was 2.5 cm for group-1, and 5.3 cm for group-2. In group-2, there was the predominance of worse prognostic factors, with high-grade tumors (p = 0.01) and presence of microvascular invasion (p = 0.001). Sarcomatous tumors and involvement of lymph nodes did only happen in group-2. Disease-free survival for group-1, analyzed in the median period of 36 months, was 100%, and for group 2, in the median period of 31 months, was 81% (p = 0.008). CONCLUSION: The results obtained allow the conclusion that the present stage T1 for renal cell carcinoma gathers tumors of different evolution, being therefore recommendable the stratification in T1a for tumors smaller than 4 cm, and T1b for tumors between 4 and 7 cm.