Syed Mohmad Shah

WNT3A signalling pathway in buffalo (Bubalus bubalis) embryonic stem cells.

The aim of this study was to investigate the transcriptional profile and role of WNT3A signalling in maintaining buffalo embryonic stem (ES) cells in a pluripotent state and in the induction of their differentiation. ES cells were derived from embryos produced by in vitro fertilisation (iESC), parthenogenesis (pESC) and hand-made cloning (cESC). The expression of WNT3A, its receptors and intermediate signalling pathways were found to be conserved in ES cells derived from the three different sources. WNT3A was expressed in ES cells but not in embryoid bodies derived from iESC or in buffalo fetal fibroblast cells. It was revealed by real-time polymerase chain reaction analysis that following supplementation of culture medium with WNT3A (100, 200 or 400ngmL(-1)) a significant increase (P<0.05) was observed in the expression level of β-CATENIN, which indicated the activation of the canonical WNT pathway. WNT3A, in combination with exogenous fibroblast growth factor-2 and leukaemia inhibitory factor, induced proliferation of undifferentiated ES cells. Differentiation studies showed that WNT3A caused formation of scaffold-like structures and inhibition of differentiation into neuron-like cells. In conclusion, the WNT3A signalling pathway is necessary both for maintaining undifferentiated buffalo ES cells as well as for directing their differentiation.

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.

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.

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus-oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus-oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus-oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.

Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa). Out of the series of RA doses (2, 4, 8, 16, 20 and 30μM), 16μM RA for 8day culture interval was found to induce highest expression of PGC- and meiosis-associated genes like DAZL, VASA, SYCP3, MLH1, TNP1/2 and PRM2, while mature germ cell genes like BOULE and TEKT1 (Spermatocyte markers), GDF9 and ZP2 (Oocyte markers) showed higher expression at 2μM RA dose, suggesting functional concentration-gradient of RA activity. Immunocytochemistry revealed expression of germ lineage-specific markers like: c-KIT, DAZL and VASA (PGC-markers); SYCP3, MLH1 and PROTAMINE1 (Meiotic-markers); ACROSIN and HAPRIN (Spermatocyte-markers); and GDF9 and ZP4 (Oocyte-markers) in optimally differentiated embryoid bodies (EBs) and adherent cultures. We observed significantly reduced (p<0.05) concentration of 5-methyl-2-deoxycytidine in RA-differentiated EBs which is suggestive of the occurrence of methylation erasure. FACS analysis of optimally differentiated cultures detected 3.07% haploid cell population, indicating completion of meiosis. Oocyte-like structures (OLS) were obtained in adherent differentiated cultures. They had a big nucleus and a zona pellucida (ZP4) coat. They showed progression through 2-cell, 4-cell, 8-cell, morula and blastocyst-like structures upon extended culture beyond 14days.

Spontaneous differentiation of buffalo (Bubalus bubalis) embryonic stem cells towards germ cell lineage

Buffalo ES cells were subjected to differentiation in 15% KoSRand 15% FBSbased spontaneous differentiation media in static suspension cultures. The embryoid bodies (EBs) so formed, were analyzed for germline-specific gene expression on day 4, 8 and 14 in order to identify the optimum differentiation strategy. Immunocytochemical analysis was performed for detection of germ lineage specific markers in EBs differentiated under the identified optimum conditions. Global DNA methylation analysis was performed to assay methylation erasure in the optimized differentiation cultures. We observed a significantly (p<0.05) increased expression of all germ lineage genes like DAZL, VASA and PLZF (PGC-markers); SYCP3, PRM2, TNP1/2 and MLH1 (Meiotic markers); BOULE and TEKT1 (Spermatocyte-markers); GDF9 and ZP2/3 (Oocyte-markers) upon spontaneous differentiation in comparison to undifferentiated ES cells. EBs collected from FBSbased medium showed significantly (p<0.05) higher expression of early germ lineage genes (DAZL, TNP1, PRM2), while those from KoSR-based medium showed greater expression of meiotic and developmental genes (SYCP3, MLH1, TEKT1, GDF9 and ZP2). Immunocytochemistry revealed expression of c-Kit, Dazl, Vasa (PGC-markers); Sycp3, Mlh1, Protamine1 (Meiotic markers); Acrosin and Haprin (Spermatocyte-makers); and Gdf9 and Zp4 (Oocyte-markers) in day 14 EBs collected from both the cultures. However, only PGC-specific markers were detected in 14 day monolayer cultures differentiated in KoSR-based media. The levels of 5-methyl-2-deoxycytidine were not significantly (p<0.05) different between EBs collected from the two media formulations. However, EBs collected from KoSRbased medium showed lower concentration of 5-Methyl-2-deoxycytidine, indicating comparatively greater methylation erasure. Correspondence to: Syed Mohmad Shah, Embryo Biotechnology Lab, Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India, E-mail: syedalhyderi14@gmail.com Manmohan Singh Chauhan, Embryo Biotechnology Lab, Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India, E-mail: chauhanabtc@gmail.com