Amar Nath

Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle.

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10(-3), 10(-5), 10(-7) and 10(-9) M sodium nitroprusside (SNP). PFs were further cultured with 10(-5) M SNP + 1.0 mM N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10(-3), 10(-5), 10(-7) M) inhibited (P < 0.05) while, lower dose of SNP (10(-9) M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

Molecular and cellular characterization of buffalo bone marrow-derived mesenchymal stem cells.

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow-derived MSCs (BM-MSCs) at molecular and cellular level. Buffalo BM-MSCs were isolated by Ficoll density gradient method and cultured in Dulbeccos modified Eagles medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony-forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM-MSCs have the capacity to form plastic adherent clusters of fibroblast-like cells and were successfully maintained up to 16(th) passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT-PCR analysis and protein localization study showed that buffalo BM-MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM-MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM-MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development.

Expression and quantification of Oct-4 gene in blastocyst and embryonic stem cells derived from in vitro produced buffalo embryos

The POU-domain transcription factor Pou5f1 (Oct-4) is involved in transcriptional regulation during early embryonic development and cell differentiation. Despite highly conserved genomic organization of Oct-4 gene in mammals, expression pattern of Oct-4 is highly variable in different species. In the present study, expression pattern of Oct-4 in buffalo blastocyst, trophoectoderm (TE), and embryonic stem cells (ESCs) was investigated. For the derivation and characterization of buffalo ESCs, inner cell masses (ICMs) were isolated from 18 hatched and 21 expanded in vitro produced buffalo blastocyst and cultured over mitomycin-C-treated buffalo fetal fibroblast feeder layer. Alkaline phosphatase (AP) activity, SSEA-1 and 4, TRA 1–60 and 1–81, and Oct-4 proteins were localized in ICM, TE, and ESCs. Quantification of Oct-4 was done by amplifying a transcript of 125 base pairs by real-time polymerase chain reaction. Primary cell colony formation was higher (P < 0.05) in hatched blastocyst (83.33%, 15/18) compared to mechanically isolated ICMs from expanded blastocyst (52.38%, 11/21). Undifferentiated buffalo ESCs were positive for AP and expressed Oct-4, SSEA-1 and 4, TRA-1–60, and TRA-1–81 proteins. Oct-4 transcripts and proteins were detected in the ICM, TE cells and were invariably present in ESCs; however, expression level of Oct-4 transcript were significantly higher in ICM and ESCs as compared to TE cells. In conclusion, expression of Oct-4 is not only restricted to the ICM and ESCs but its expression was also detected in TE cells suggesting that instead of using Oct-4 as a single marker, it is better to have other flanking molecular markers for the identification of buffalo pluripotent embryonic stem cells.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.

Expression of mRNA Encoding IGF-I, IGF-II, Type-I, and II IGF-Receptors and IGF-Binding Proteins-1-4 during Ovarian Follicular Development in Buffalo (Bubalus bubalis)

The present study was designed to investigate the expression pattern of IGF-I, IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in different stages of buffalo ovarian preantral follicles (PFs), antral follicles (AFs), ovulatory follicles (OFs), and immature (IM) and in vitro matured (MO) oocytes. Buffalo ovaries were collected from local abattoir, PFs (200-250 µm), AFs (1-3 mm), and OFs (5-8 mm) were isolated by mechanical method. PFs, AFs, OFs, and oocytes were lysed to release mRNA, reverse transcribed, and then subjected to RT-PCR, whereas protein were localized through immunohistochemistry. Relative expression of mRNA transcripts was clearly seen for IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in all the stages of developing follicles and oocytes. We were unable to detect mRNA and protein expression of IGF-1 in any of the oocytes or follicles at any stage of the development. IGF-II and both IGF receptors mRNA expression were found higher (P < 0.05) in PFs compared to AFs and OFs. Expression of IGFBP-1 and 2 in PFs, as well as IGFBP-3 and 4 in AFs, was found with higher (P < 0.05) levels. The expression results were further confirmed by localization of IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 proteins. In conclusion, IGF-II appears to be the only ligand that is endogenously expressed by all the follicular stages and oocytes, which may act in an autocrine manner through the Type-1 IGF receptor. Expression of IGFBP-1-4 and IGF-II suggests the possible role of these genes in recruitment, growth, proliferation, and steroidogenic responses during developmental phases of buffalo ovarian follicles.

Comparative study on characterization and wound healing potential of goat (Capra hircus) mesenchymal stem cells derived from fetal origin amniotic fluid and adult bone marrow

Caprine amniotic fluid (cAF) and bone marrow cells (cBM) were isolated, expanded and phenotypically characterized by mesenchymal stem cells (MSCs) specific cell surface markers. Both cell types were compared for multilineage differentiation potential by flow cytometry using specific antibodies against lineage specific markers. Furthermore, in vitro expanded cAF-MSCs showed higher expression of trophic factors viz. VEGF and TGF-β1 as compared to cBM-MSCs. Full-skin thickness excisional wounds created on either side of the dorsal midline (thoracolumbar) of New Zealand White rabbits were randomly assigned to subcutaneous injection of either fetal origin cAF-MSCs (n=4) or adult cBM-MSCs (n=4) or sterile PBS (control, n=4). The rate of wound closure was found faster (p<0.05) in cAF-MSCs treated wounds as compared with cBM-MSCs and PBS treated wounds especially on 21st day post-skin excision. Histomorphological examination of the healing tissue showed that wound healing was improved (p<0.05) by greater epithelialization, neovascularization and collagen development in cAF-MSCs as compared to cBM-MSCs and PBS treated wounds.