Cumulus cell-conditioned medium supports embryonic stem cell differentiation to germ cell-like cells.

Cumulus cells provide cellular interactions and growth factors required for oogenesis. In vitro studies of oogenesis are limited primarily because of the paucity of their source, first trimester fetal gonads, and the small number of germ lineage precursor cells present within these tissues. In order to understand this obscure but vitally important process, the present study was designed to direct differentiation of embryonic stem (ES) cells into germ lineage cells. For this purpose, buffalo ES cells were differentiated, as embryoid bodies (EBs) and monolayer adherent cultures, in the presence of different concentrations of cumulus-conditioned medium (CCM; 10%, 20% and 40%) for different periods of culture (4, 8 and 14 days) to identify the optimum differentiation-inducing concentration and time. Quantitative polymerase chain reaction analysis revealed that 20%-40% CCM induced the highest expression of primordial germ cell-specific (deleted in Azoospermia- like (Dazl), dead (Asp-Glu-Ala-Asp) box polypeptide 4 (Vasa also known as DDX4) and promyelocytic leukemia zinc finger protein (Plzf)); meiotic (synaptonemal complex protein 3 (Sycp3), mutl homolog I (Mlh1), transition protein 1/2 (Tnp1/2) and protamine 2 (Prm2); spermatocyte-specific boule-like RNA binding protein (Boule) and tektin 1 (Tekt1)) and oocyte-specific growth differentiation factor 9 (Gdf9) and zona pellucida 2 /3 (Zp2/3)) genes over 8-14 days in culture. Immunocytochemical analysis revealed expression of primordial germ cell (c-KIT, DAZL and VASA), meiotic (SYCP3, MLH1 and PROTAMINE 1), spermatocyte (ACROSIN and HAPRIN) and oocyte (GDF9 and ZP4) markers in both EBs and monolayer differentiation cultures. Western blotting revealed germ lineage-specific protein expression in Day 14 EBs. The significantly lower (P<0.05) concentration of 5-methyl-2-deoxycytidine in differentiated EBs compared to undifferentiated EBs suggests that methylation erasure may have occurred. Oocyte-like structures obtained in monolayer differentiation stained positive for ZONA PELLUCIDA protein 4 and progressed through various embryo-like developmental stages in extended cultures.

Expression profiling of major heat shock protein genes during different seasons in cattle (Bos indicus) and buffalo (Bubalus bubalis) under tropical climatic condition.

Heat shock proteins consist of highly conserved stress proteins, expressed in response to stress and play crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and other HSPs and to evaluate their expression pattern in Sahiwal and Tharparkar breeds of zebu cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) with respect to different seasons. Quantitative real time polymerase chain reaction was performed to analyze the transcript variants of three HSP70 family genes (HSPA1A, HSPA1B, and HSPA8) and HSP10, HSP60, HSP90 and HSF1 in each breed. The major finding of this study was the higher abundance of all the studied HSP genes during summer and winter compared to spring season, but the magnitude of increase was higher during summer as compared to winter. HSPA1A and HSPA1B genes showed maximal induction (P<0.001) during summer and winter while HSP60 and HSP10 were found to be the second most abundantly expressed HSPs. The relative mRNA abundance of HSF1 significantly increased (P<0.001) in Murrah buffalo compared to Tharparkar and Sahiwal cattle during summer and winter. Expression pattern of heat shock protein genes indicated that amongst the breeds, the expression was higher in Murrah buffalo compared to Sahiwal and Tharparkar cattle, thereby indicating the more adaptive capacity of later during periods of stress. Hence, this study suggests that heat shock protein genes may be conveniently used as biomarkers for assessing stress response in cattle and buffalo and the expression is species and breed-specific. Furthermore, the variation in expression is associated with heat tolerance and adaptation to different climatic conditions.

Developmental competence and expression pattern of bubaline (Bubalus bubalis) oocytes subjected to elevated temperatures during meiotic maturation in vitro.

OBJECTIVE: To determine the direct effect of physiologically relevant high temperatures (40.5 and 41.5 °C) for two time periods (12 and 24 h) on bubaline oocytes during in vitro maturation. METHOD: The control group oocytes were cultured at 38.5 °C for 24 h. The treatment 1 (T1) and 3 (T3) group oocytes were cultured at 40.5 and 41.5 °C respectively, for the first 12 h and at 38.5 °C for rest of the 12 h. However, treatment 2 (T2) and 4 (T4) group oocytes were cultured at 40.5 and 41.5 °C for complete 24 h. RESULTS: Development of oocytes to blastocyst was severely compromised (p < 0.001) when matured at 40.5 and 41.5 °C for both exposure periods (12 h and 24 h). It was found that the cleavage rates, blastocyst yield and mean cell number decreased remarkably (p < 0.001) in the treatment groups compared to control. The relative mRNA expression of heat shock protein (Hsp 70.1, 70.2, 70.8, 60, 10 and HSF1), pro-apoptotic (caspases-3, -7, -8, Bid and Bax) and oxidative stress (iNOS) related genes was significantly higher (p < 0.05) in all the treatment groups compared to control. However, mRNA abundance of anti-apoptotic (Bcl-2, Mcl-1, Bcl-xl), glucose transport (Glut1, Glut3 and IGF1R), developmental competence (ZAR1 and BMP15) and oxidative stress (MnSOD) related genes was significantly decreased (p < 0.05) in the treatment groups compared to control. CONCLUSION: The present study clearly establishes that physiologically relevant elevated temperatures during in vitro meiotic maturation reduce developmental competence of bubaline oocytes.

Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method.

DNA is the prerequisite for life's inception that transfers hereditary information, past several years; various types of commercial kits are made available which vary depending on the type of the biological sample being used. The present study is focused on developing an improvised methodology for the isolation of genomic DNA from stored bovine blood samples. DNA was isolated by using the conventional Phenol: Chloroform: Isoamyl alcohol (PCI) method and Detergent method. The aim of the study was to make a comparative analysis and evaluation of these two methods to identify the one that gives a superior quality and quantity of genomic DNA. Total (n=48) each duplicate blood samples from three different buffalo(Bubalus bubalis) breeds Banni, Surti, Murrah, three zebu cattle (Bos indicus) breeds Kankerj, Gir, Sahiwal were collected from the jugular vein. The quantity, purity of the genomic DNA was assessed based on the total DNA yield, purity ratios, spectral profile, agarose gel electrophoresis analysis and polymerase chain reaction amplification of MC1R gene product without any inhibitors. The results of our study suggest that detergent method is also suitable for extraction of genomic DNA from the bovine blood and results were significant (*P>0.05). The total mean yield was found to be 329.05±11 µg/5ml for all six breeds while the PCI method was employed. The total mean yield of the gDNA for all six breeds was 406.6±43 µg/5ml of blood when the detergent method was used. One way ANOVA test showed that the total DNA yield varied depending on the isolation method used. The DNA yield obtained from the DG method was (***P< 0.001) significant as compared to the PCI method (**P<0.01).

Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear transferred blastocysts.

We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

RETRACTED: Bone morphogenetic protein 4 (BMP4) induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into germ cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Problems related to images published in this paper in Figure 12 were brought to the authors' attention. Unfortunately this figure contains duplicate images for ESC controls for VASA, GDF9, and ZP4, which display identical patterns superimposed on varying intensities of background. Therefore, the authors retract the paper with the agreement of the editors and deeply regret this situation and apologize for any inconvenience to the editors and readers of Biochimie.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro-Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos.

We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro-fertilized, somatic cell nuclear-transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro-produced blastocysts. Most of the ICMs (45-55%) resulted in formation of primary colonies that were subcultured after 8-10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture-derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture-derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.