Manoj Kumar Jena

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line.

Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

ISOLATION AND CHARACTERIZATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO PRODUCED GOAT (Capra hircus) EMBRYOS

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P < 0.01) for hatched blastocysts (77.14%) than early/expanded blastocysts (54%) or morula (14%). When ICMs were isolated mechanically the primary colony formation for hatched blastocysts (90%) as well as blastocysts (66%) were significantly more than when ICMs were isolated by enzymatic digestion (60% and 30%, respectively). The colonies were disaggregated either mechanically or by enzymatic digestion for further subculture. When mechanical method was followed, the colonies remained undifferentiated up to 15 passages and three ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). However, enzymatic disaggregation resulted in differentiation. The undifferentiated cells showed stem cell like morphological features, normal karyotype, and expressed stem cell specific surface markers like alkaline phosphatase, TRA-1-61, TRA-1-81, and intracellular markers Oct4, Sox2, and Nanog. Following prolonged culture of the ES cell-like cells were differentiated into several types of cells including neuron like and epithelium-like cells. In conclusion, goat embryonic stem cell-like cells can be isolated from in vitro produced goat embryos and can be maintained for long periods in culture.

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.

Hand-Made Cloned Goat (Capra hircus) Embryos—A Comparison of Different Donor Cells and Culture Systems

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): Lactating vis-a-vis heifer

UNLABELLED Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

Cardiomyocytes rhythmically beating generated from goat embryonic stem cell

The aim of present investigation was isolation, characterization and differentiation into cardiomyocytes of putative goat embryonic stem cells produced from in vitro fertilized goat embryos. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) techniques. The ICMs isolated from IVF blastocysts were cultured on 10 μl/ml mitomycin-C inactivated fetal fibroblast feeder layer with LIF. The putative ES colonies were characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The ES cells were successfully subcultured up to 22nd passage with feeder layer and LIF and up to 12th passage without feeder layer with LIF only. They exhibited normal karyotyping (20th passage) and maintained the expression of specific surface markers like alkaline phosphatase, SSEA-4, TRA-1-61, TRA-1-81 and intracellular markers Oct4, Sox2 and Nanog. The embryoid bodies (EBs) were generated from goat ES cells of 20th passage and were analyzed with markers like Gata4, BMP4 and Nestin. Differentiation was induced by medium containing 100 ng/ml Activin-A, 10 ng/ml FGF-2 and 100 ng/ml BMP-4. The embryoid bodies were analyzed with markers like Gata4, BMP4 and Nestin. The rhythmic beating of cardiomyocytes was observed after 30 d and the beating was still continuing even after 160 d of culturing. Similarly, 2nd and 3rd batches of EBs were also beating and the beating continues after 75 d and on. The beating cells were observed positive for cardiac specific markers like α Actinin, C-Troponin and α-Myosin heavy chain. Histological studies also revealed morphology similar to cardiomyocytes. Prominent contractions typical of cardiac tissue have been maintained in the differentiated cells up to 160 d and still continuing beating at the rate of 30 beats/min. It could be concluded that ES cells generated from goat embryos were maintained undifferentiated up to 22nd passage on feeder layer and to 12th passage without feed layer using LIF and that the differentiation protocol induced rhythmic beating cells.

Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus).

The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

MicroRNAs in the development and neoplasia of the mammary gland

Study on the role of microRNAs (miRs) as regulators of gene expression through posttranscriptional gene silencing is currently gaining much interest,due to their wide involvement in different physiological processes. Understanding mammary gland development, lactation, and neoplasia in relation to miRs is essential. miR expression profiling of the mammary gland from different species in various developmental stages shows their role as critical regulators of development. miRs such as miR-126, miR-150, and miR-145 have been shown to be involved in lipid metabolism during lactation. In addition, lactogenic hormones influence miR expression as evidenced by overexpression of miR-148a in cow mammary epithelial cells, leading to enhanced lactation. Similarly, the miR-29 family modulates lactation-related gene expression by regulating DNA methylation of their promoters. Besides their role in development, lactation and involution, miRs are responsible for breast cancer development. Perturbed estrogen (E2) signaling is one of the major causes of breast cancer. Increased E2 levels cause altered expression of ERα, and ERα-miR cross-talk promotes tumour progression. miRs, such as miR-206, miR-34a, miR-17-5p, and miR-125 a/b are found to be tumour suppressors; whereas miR-21, miR-10B, and miR-155 are oncogenes. Oncogenic miRs like miR-21, miR-221, and miR-210 are overexpressed in triple negative breast cancer cases which can be diagnostic biomarker for this subtype of cancer. This review focuses on the recent findings concerning the role of miRs in developmental stages of the mammary gland (mainly lactation and involution stages) and their involvement in breast cancer progression. Further studies in this area will help us to understand the molecular details of mammary gland biology, as well as miRs that could be therapeutic targets of breast cancer.