Neha Saini

Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis.

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis, gradual dose-dependent single exposure pediocin-resistant (Pedr) mutants of E. faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Pedr mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in PedrE. faecalis. Decreased level of branched chain fatty acids in Pedr mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Pedr mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Pedr mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC50 of CAMPs for wild-type and Pedr mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs.

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.

151 EFFECT OF ASCORBIC ACID ON OXIDATIVE STRESS AND ITS THERMOPROTECTANT ROLE ON IN VITRO EMBRYONIC DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

The most important factors that lead to stress in farm animals are oxidative and thermal stress, leading to reduced reproductive efficiency. Oxidative stress leads to an increase in proportion of reactive oxygen species, whereas heat stress affects the physiology of animals, which lowers the conception rates of dairy cattle. In vitro culture systems have been enhanced by manipulating media with various supplements such as vitamins, growth factors, and antioxidants that have overcome these problems. Ascorbic acid has been shown to play an antioxidant role in many species such as sheep, goat, and pigs. Keeping this in mind, this study was conducted to investigate the effect of supplementation of in vitro-matured (IVM) and/or in vitro-cultured (IVC) media with ascorbic acid and evaluate its antioxidant role in in vitro development of buffalo embryos. Immature oocytes were collected from visible surface follicles (2 to 8mm in diameter) in slaughterhouse buffalo ovaries and subjected to IVM, IVF, and IVC in a humidified CO2 incubator at 38.5°C. Ascorbic acid was supplemented to IVM [TCM-199+10% featl bovine serum (FBS)+1µgmL-1 oestradiol-17β+5µgmL-1 pFSH+0.81mM sodium pyruvate+0.68mM l-glutamine+50µgmL-1 gentamicin sulfate] at 50 or 100µM or IVC (mCR2aa+0.6% BSA+10% FBS+ 50µgmL-1 gentamicin sulfate) at 50µM or both IVM and IVC media at 50µM. Oocytes without ascorbic acid were treated as the control group. Cleavage and blastocyst rate was improved when 50µM (66.67±2.27; 16.67±1.26%) ascorbic acid was supplemented to IVM medium but no significant difference (P<0.05) was observed at 100µM (54.04±2.20; 6.16±0.37%) as compared with the control (62.77±2.71; 10.67±0.24%), respectively. When 50µM ascorbic acid was supplemented in IVM, IVC, or both media, though cleavage rate (66.67±2.27; 69.09±3.22; 66.67±2.23%) was similar in 3 groups, a significant increase was observed in blastocyst rate (16.67±1.26; 20.18±0.86; 28.57±0.37%) when both media were supplemented, respectively. To evaluate the thermoprotectant effect, 4 groups were taken: group 1 without and group 2 with ascorbic acid supplementation, oocytes were given heat treatment at 39.5°C initially for 12h during IVM; group 3 without and group 4 with ascorbic acid supplementation, oocytes were given heat treatment at 40.5°C initially for 12h during IVM. No significant difference in developmental rate was observed at elevated temperature of 39.5°C or 40.5°C as compared with the control. Relative mRNA abundance of heat stress-related genes, HSP 70.1 and HSP 70.2, was nonsignificantly higher in oocytes matured at 39.5°C or 40.5°C after supplementation with ascorbic acid as compared to control. Relative mRNA abundance of BAX decreased at 50µM and increased at 100µM ascorbic acid compared with control, whereas BID showed similar results between control and treatment. Regarding anti-apoptotic gene expression, significantly higher expression was observed in MCL1 for 50µM and lower for 100µM ascorbic acid, and a similar nonsignificant trend was observed for BCL-XL. Developmental genes GDF9 and BMP15 showed a nonsignificant increase in 50µM, and a nonsignificant decrease in the 100µM supplemented group as compared with the control. Oxidative stress-related genes SOD and GPX showed a nonsignificant decrease in treated groups as compared to control. From above results, it was concluded that ascorbic acid had an anti-oxidant as well as thermoprotectant role in developmental competence that increased the potential for generation of large domestic animal in vitro embryos for research and applied technologies such as cloning and transgenesis.

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