ABSTRACT
This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8-71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6-46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus-oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.
Subject(s)
Buffaloes , Oocytes , Animals , Blastocyst , Embryonic Development , Fertilization in Vitro , Indicators and Reagents , ParthenogenesisABSTRACT
Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.
Subject(s)
Apoptosis , Blastocyst/physiology , Buffaloes/physiology , Epigenesis, Genetic , Fertilization in Vitro , MicroRNAs/antagonists & inhibitors , Nuclear Transfer Techniques/veterinary , Acetylation , Animals , Blastocyst/metabolism , Embryo Culture Techniques/veterinary , Female , Gene Expression Regulation, Developmental , Histones/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , PregnancyABSTRACT
Hyperoxaluria-associated deposition of calcium oxalate crystals results from oxalate-induced renal injury and inflammation. The present study was designed to evaluate the effect of 4-Phenyl butyric acid (4-PBA), a chemical chaperone, in ethylene glycol-induced hyperoxaluria and compare its effect with antioxidant, N-acetyl cysteine (NAC). Male Sprague-Dawley rats were given ethylene glycol in drinking water for 28 days to induce hyperoxaluria. 4-PBA and NAC were given by oral gavage. Effect of 4-PBA was analyzed in both prophylactic and curative regimens. After every 7 days, 24-h urine samples were analyzed for kidney injury and inflammation markers. Increased amounts of kidney injury markers like Kidney injury molecule-1, Lactate dehydrogenase, and N-acetyl-ß-glucoseaminidase were found in the urine of hyperoxaluric rats which were significantly reduced by 4-PBA treatment in both prophylactic and curative regimens. Inflammatory markers IL-1ß, IL-6, and MCP-1 were also raised in the urine of hyperoxaluric rats which were significantly decreased by 4-PBA treatment. Hyperoxaluria was accompanied with renal oxidative stress as reflected by decreased glutathione redox status and increased reactive oxygen species which was significantly reduced by 4-PBA treatment. Histological study with H&E and Pizzolato staining showed numerous calcium oxalate crystal deposits in the renal tissues of hyperoxaluric rats. However, no significant crystal deposits were seen in the 4-PBA-treated hyperoxaluric rats. N-acetyl cysteine treatment effectively decreased renal oxidative stress but did not alter the production of inflammatory markers. Collectively, the present study suggested the potential protective effect of 4-PBA in hyperoxaluria-induced renal injury and inflammation.
Subject(s)
Antineoplastic Agents/pharmacology , Hyperoxaluria/complications , Inflammation/drug therapy , Kidney Diseases/drug therapy , Oxidative Stress/drug effects , Phenylbutyrates/pharmacology , Animals , Biomarkers/analysis , Inflammation/etiology , Inflammation/metabolism , Inflammation/pathology , Kidney Diseases/etiology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 µM) for 10 hr from the start of reconstruction till activation. At 10 µM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 µM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 µM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 µM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.
Subject(s)
Apoptosis/drug effects , Buffaloes/embryology , Cinnamates/pharmacology , Cloning, Organism , Embryo Culture Techniques/veterinary , Embryo, Mammalian/drug effects , Animals , Cinnamates/administration & dosage , Dose-Response Relationship, Drug , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Developmental/drug effectsABSTRACT
Myocardial Abscess (MA) is a rare life threatening condition mostly occurring secondary to underlying infective endocarditis. But it can also be found secondary to septicemia with some unknown focus of sepsis in the body. Development of MA as a consequence of left ventricular aneurysm (LVA) secondary to myocardial infarction is very rare. We report the case of 56 year old male, who presented with high grade fever and leukocytosis. On detailed evaluation, he was found to have a myocardial abscess with underlying LVA.
Subject(s)
Abscess/etiology , Heart Aneurysm/etiology , Heart Ventricles , Myocardial Infarction/complications , Abscess/diagnosis , Heart Aneurysm/diagnosis , Humans , Male , Middle AgedABSTRACT
We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.
Subject(s)
Blastocyst/drug effects , Buffaloes , Cloning, Organism/veterinary , Ectogenesis/drug effects , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Developmental/drug effects , Acetylation/drug effects , Animals , Apoptosis/drug effects , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Blastocyst/enzymology , Blastocyst/metabolism , Cells, Cultured , Cloning, Organism/methods , DNA Methylation/drug effects , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , Decitabine , Embryo Culture Techniques/veterinary , Female , Histone Deacetylase Inhibitors/pharmacology , Histones/metabolism , Hydroxamic Acids/pharmacology , India , Methylation/drug effects , Protein Processing, Post-Translational/drug effectsABSTRACT
The purpose of the present study was to evaluate the nephro-protective potential of N-acetylcysteine against hyperoxaluria-induced renal mitochondrial dysfunction in rats. Nine days dosing of 0.4 % ethylene glycol +1 % ammonium chloride, developed hyperoxaluria in male wistar rats which resulted in renal injury and dysfunction as supported by increased level of urinary lactate dehydrogenase, calcium, and decreased creatinine clearance. Mitochondrial oxidative strain in hyperoxaluric animals was evident by decreased levels of superoxide dismutase, glutathione peroxidase, glutathione reductase, reduced glutathione, and an increased lipid peroxidation. Declined activities of respiratory chain enzymes and tricarboxylic acid cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. N-acetylcysteine (50 mg/kg, i.p.), by virtue of its -SH reviving power, was able to increase the glutathione levels and thus decrease the oxidative stress in renal mitochondria. Hence, mitochondrial damage is, evidently, an essential event in ethylene glycol-induced hyperoxaluria and N-acetylcysteine presented itself as a safe and effective remedy in combating nephrolithiasis.
Subject(s)
Acetylcysteine/pharmacology , Hyperoxaluria/physiopathology , Mitochondria/drug effects , Mitochondrial Diseases/drug therapy , Protective Agents/pharmacology , Animals , Calcium/urine , Citric Acid Cycle/drug effects , Creatinine/metabolism , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Hyperoxaluria/metabolism , Hyperoxaluria/urine , Kidney/metabolism , Kidney/physiopathology , L-Lactate Dehydrogenase/urine , Lipid Peroxidation/drug effects , Male , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/physiopathology , Mitochondrial Diseases/urine , Nephrolithiasis/metabolism , Nephrolithiasis/physiopathology , Nephrolithiasis/urine , Oxidative Stress/drug effects , Rats , Rats, Wistar , Superoxide Dismutase/metabolismABSTRACT
Following IVF, embryos which cleave early have been shown to have higher developmental competence and quality than those that cleave relatively later across many species. We investigated the effect of time of cleavage on the developmental competence, quality, epigenetic status and gene expression in buffalo embryos produced by handmade cloning (HMC). Following classification of embryos as early cleaving (EC) or late cleaving (LC) based on whether they had cleaved or not at 24 h post in vitro culture, 54% (164/303) were found to be EC and the rest to be LC. The blastocyst rate (58.1 ± 3.4 vs 36.9 ± 1.6%, p < 0.01) and the total cell number (285.5 ± 41.9 vs 141.4 ± 36.1, p < 0.05) were higher, whereas the apoptotic index (3.6 ± 0.6 vs 12.2 ± 1.7, p < 0.01) and the global level of H3K9ac and H3K27me3 were lower (p < 0.05) in the blastocysts produced from EC than in those produced from LC embryos. The relative transcript level of CASPASE3, CASPASE7, DNMT1, DNMT3a and CDX2 was higher (p < 0.05) and that of SOX2 was lower (p < 0.05) in blastocysts produced from LC than in those produced from EC embryos, whereas the expression level of CASPASE6, P53, P21, HDAC1, OCT4 and NANOG was not significantly different between the two groups. These results show that (i) following HMC, blastocysts produced from embryos that cleave early differ from those produced from late cleaving embryos in terms of epigenetic status and expression level of many important apoptosis-, pluripotency-, trophectoderm- and epigenetics-related genes, and (ii) EC embryos are superior to LC embryos in view of their higher developmental competence and quality.
Subject(s)
Blastocyst/physiology , Buffaloes/embryology , Cloning, Organism/veterinary , Embryo, Mammalian/physiology , Embryonic Development/physiology , Animals , Blastocyst/cytology , Cloning, Organism/methods , Embryo, Mammalian/cytology , FemaleABSTRACT
This study was conducted to identify and analyse the expression of gametogenesis-associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis-associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p < 0.05) increased expression of primordial germ cell-specific, meiotic as well as genes associated with oocyte maturation and development in foetal ovaries as compared to the adult ones. However, significantly (p < 0.05) increased expression of proteins associated with oocyte maturation like GDF9 and ZP4 was found in adult ovaries, indicating temporal regulation of mRNA translation during oogenesis. Meiotic genes showed significantly (p < 0.05) increased expression in adult testes as compared to foetal testes and ovaries, indicating onset of meiosis at a later stage in spermatogenesis. In general, the expression of primordial germ cell-associated as well as meiotic genes was higher in adult testes, indicating the increased biological activity in the organ. Immunohistochemistry revealed localized expression of gametogenesis-associated proteins in ovarian follicles and seminiferous tubules of testes, while the surrounding somatic tissues were devoid of these proteins. The study gives an understanding of the sequential and temporal events of gene expression as well as mRNA translation during male and female gametogenesis. It could also be concluded that follicles and seminiferous tubules are the functional units of the female and male gonads, respectively, and their function could be enhanced by appropriate chemical and genetic intervention of the somatic tissue immediately surrounding them. This assumes importance in the context that buffalo attains sexual maturity at an older age of 2-3 years and have smaller ovaries with lesser number of primordial follicles in comparison with cattle, which is suggested to be the main reason of their poor breeding performance.
Subject(s)
Buffaloes/embryology , Fetus/metabolism , Gametogenesis/physiology , Gene Expression Regulation, Developmental/physiology , Ovary/embryology , Testis/embryology , Animals , Blotting, Western , Buffaloes/growth & development , Female , Immunohistochemistry , Male , Ovary/metabolism , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Testis/metabolismABSTRACT
PURPOSE: For most cases, urolithiasis is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate (CaOx) deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE) of Terminalia arjuna (T. arjuna). MATERIALS AND METHODS: The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software 'Image-Pro Plus 7.0' of Olympus upright microscope (BX53). Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro. RESULTS: Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals with an IC50 at 13.1µg/mL. CONCLUSIONS: The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.
Subject(s)
Antioxidants/pharmacology , Calcium Oxalate/chemistry , Plant Extracts/pharmacology , Terminalia/chemistry , Urinary Calculi/prevention & control , Analysis of Variance , Biphenyl Compounds/chemistry , Crystallization , Free Radical Scavengers/pharmacology , Phytotherapy , Picrates/chemistry , Reference Values , Reproducibility of Results , Urinary Calculi/chemistryABSTRACT
The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.
Subject(s)
Buffaloes/embryology , Buffaloes/genetics , Cloning, Organism/veterinary , Nuclear Transfer Techniques/veterinary , Oocytes/physiology , Animals , Apoptosis , Cloning, Organism/methods , Embryo Culture Techniques , Embryo, Mammalian , Embryonic Development , Female , Species SpecificityABSTRACT
Role of herbal drugs and medicinal plant extracts in the successful treatment of urolithiasis, classified as the third most common urinary tract diseases is well documented. Ayurvedic plants and their components mediate antilithogenic effects by altering ionic composition of urine, being diuretic, antioxidant or having antimicrobial activity. Therapeutic peptides and proteins have unique place in pharmaceutical biotechnology due to their critical roles in cell biology. The innovation in antilithiatic proteins is that they are anionic, rich in acidic amino acids which make oxalate unavailable by interacting with calcium and have EF Hand domain which is a characteristic feature of various calcium binding protein like calgranulin, osteopontin. The review provides a background on the pathogenesis of urolithiasis and medical treatments. It focusses on the present research evaluating the scientific basis of antilithiatic potential of various plants and role of plant proteins as therapeutic agents thus opening new vista in the management of urolithiasis. Further investigations are required to fully decipher the mode of action of the potent biomolecules so as to exploit their preventive and therapeutic potential.
Subject(s)
Medicine, Ayurvedic , Phytotherapy , Plant Extracts/therapeutic use , Urolithiasis/drug therapy , Humans , Leukocyte L1 Antigen Complex/therapeutic use , Osteopontin/therapeutic use , Plant Extracts/chemistry , Urolithiasis/pathologyABSTRACT
This study was carried out to compare the post-thaw cryosurvival rate and the level of apoptosis in vitro produced zona-free cloned buffalo blastocysts subjected to slow freezing or vitrification in open-pulled straws (OPS). Zona-free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re-expansion rate following post-thaw culture for 22-24 h. The post-thaw re-expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen-thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non-cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non-cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona-free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.
Subject(s)
Blastocyst/physiology , Buffaloes/embryology , Cloning, Organism/veterinary , Cryopreservation/veterinary , Animals , Apoptosis , Blastocyst/cytology , Cell Count/veterinary , Cryopreservation/instrumentation , Cryopreservation/methods , Freezing , Hot Temperature , In Situ Nick-End LabelingABSTRACT
For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8-cell, 16-cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8-16-cell and blastocyst stages, relative mRNA abundance of stress-related genes HSP 70.1 and HSP 70.2 and pro-apoptotic genes CASPASE-3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress-related gene HSF1. Expression level of anti-apoptotic genes BCL-XL and MCL-1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8-16-cell and blastocyst stages. Among the genes related to embryonic development, at 8-16-cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR-1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress-, apoptosis- and development-related genes.
Subject(s)
Apoptosis/physiology , Buffaloes/embryology , Embryo, Mammalian/metabolism , Fertilization in Vitro/veterinary , Gene Expression Regulation, Developmental/physiology , Hot Temperature/adverse effects , Animals , Embryo Culture Techniques/veterinary , Embryo, Mammalian/radiation effects , RNA, Messenger/physiology , Stress, PhysiologicalABSTRACT
When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.
Subject(s)
Biomarkers , Buffaloes , Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Muscle, Skeletal/physiology , Animals , Cell Culture Techniques/veterinary , Embryonic Stem Cells/physiology , Feeder Cells , Fibroblasts/cytology , Fibroblasts/physiology , Gene Expression Regulation, Developmental/physiology , Muscle, Skeletal/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
PURPOSE: Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established. Thus, the present study was proposed to evaluate the effect of coconut water as a prophylactic agent in experimentally induced nephrolithiasis in a rat model. MATERIALS AND METHODS: The male Wistar rats were divided randomly into three groups. Animals of group I (control) were fed standard rat diet. In group II, the animals were administrated 0.75% ethylene glycol in drinking water for the induction of nephrolithiasis. Group III animals were administrated coconut water in addition to ethylene glycol. All the treatments were continued for a total duration of seven weeks. RESULTS AND CONCLUSION: Treatment with coconut water inhibited crystal deposition in renal tissue as well as reduced the number of crystals in urine. Furthermore, coconut water also protected against impaired renal function and development of oxidative stress in the kidneys. The results indicate that coconut water could be a potential candidate for phytotherapy against urolithiasis.
Subject(s)
Cocos , Nephrocalcinosis/drug therapy , Phytotherapy , Animals , Creatinine/blood , Ethylene Glycol , Kidney/drug effects , Male , Nephrocalcinosis/chemically induced , Nephrocalcinosis/prevention & control , Random Allocation , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Treatment Outcome , Urea/blood , Urolithiasis/drug therapy , Urolithiasis/prevention & control , WaterABSTRACT
PURPOSE: Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo. METHODS: The spermatogonial cells were isolated from testes of 3-7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20-65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro. RESULTS: Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7-10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1. CONCLUSION: Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.
Subject(s)
Cell Culture Techniques , Cell Lineage , Spermatogonia/cytology , Stem Cells/cytology , Animals , Buffaloes , Cattle , Cell Differentiation , Cells, Cultured , Male , Sertoli Cells/cytology , Sertoli Cells/metabolism , Testis/cytology , Testis/metabolismABSTRACT
This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two-, four- and eight-cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM-199 + 10% FBS + 5 µg/ml pFSH + 1 µg/ml estradiol-17ß + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 µg/ml gentamycin sulphate for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2-4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross-reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi-quantitative expression of NOS genes by RT-PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre-implantation stage embryos.
Subject(s)
Buffaloes/physiology , Embryo, Mammalian/enzymology , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Enzymologic/physiology , Nitric Oxide Synthase/metabolism , Oocytes/enzymology , Animals , Embryo Culture Techniques , In Vitro Oocyte Maturation Techniques , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/immunology , RNA, Messenger/metabolismABSTRACT
Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.
Subject(s)
Buffaloes/embryology , Cloning, Organism/veterinary , Milk/cytology , Nuclear Transfer Techniques/veterinary , Animals , Blastocyst/physiology , Cell Separation/veterinary , Cells, Cultured , Cloning, Organism/methods , Ear , Embryo Culture Techniques/veterinary , Embryonic Development , Female , Fibroblasts/ultrastructureABSTRACT
This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 µm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.