RESUMO
This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells' regulation and potential applications.
Assuntos
Búfalos , Testículo , Masculino , Animais , Testículo/metabolismo , Búfalos/genética , Espermatogônias/metabolismo , Células Cultivadas , Expressão GênicaRESUMO
The reproductive efficiency in buffalo is highly influenced by seasonal variability. Angiogenesis in the reproductive cycle is important for optimal physiological functioning of uterus. Estrogen receptor-α (ERα), vascular endothelial growth factor (VEGF) and reduced nicotinamide adenine dinucleotide phosphatase diaphorase (NADPH-d) are vital indicators for the uterine angiogenic process. This study was conducted to see the effect of season on the expression of different uterine angiogenic factors. Season wise (winter and summer) and phase wise (follicular and luteal), immune staining intensity of buffalo uterus was measured by calculating the optical density value (OD) for ERα and VEGF. Percentage of immuno-positive cell count for ERα was done. Histoenzymic NADPH-d expression was analysed. Expression of all these factors increased during follicular phase of oestrous cycle in order to support the angiogenesis; however, the expression was significantly lower (p ≤ 0.05) in term of OD value as well as percentage count of immuno-positive cells during summer season indicating lower angiogenic activity that subsequently affected reproduction in buffalo.
Assuntos
Búfalos , Ciclo Estral , Estações do Ano , Útero , Fator A de Crescimento do Endotélio Vascular , Animais , Búfalos/metabolismo , Feminino , Útero/metabolismo , Ciclo Estral/metabolismo , Ciclo Estral/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Neovascularização Fisiológica/fisiologiaRESUMO
A visible-light-induced efficient methodology has been developed for the C-H selenylation of pyrazolo[1,5-a]pyrimidine derivatives employing erythrosine B as the photocatalyst. This is the first report on the regioselective selenylation of pyrazolo[1,5-a]pyrimidines. The efficiency of this methodology for the selenylation of different electron-rich heterocycles like pyrazole, indole, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazole, and 4-(phenylamino)-2H-chromen-2-one has been also demonstrated. The exploration of erythrosine B as a photocatalyst with a simple and mild procedure, wide substrate scope, and practical applicability and the employment of eco-friendly energy, oxidant, and solvent are the attractive characteristics of this methodology.
Assuntos
Eritrosina , Pirimidinas , Pirimidinas/farmacologia , SolventesRESUMO
Arsenic exposure causes immense health distress by increasing risk of cardiovascular abnormalities, diabetes mellitus, neurotoxicity, and nephrotoxicity. The present study explored the role of inducible nitric oxide synthase (iNOS) inhibitors against sodium arsenite-induced renal and hepatic dysfunction in rats. Female Sprague Dawley rats were subjected to arsenic toxicity by administering sodium arsenite (5 mg/kg/day, oral) for 4 weeks. The iNOS inhibitors, S-methylisothiourea (10 mg/kg, i.p.) and aminoguanidine (100 mg/kg, i.p.) were given one hour before sodium arsenite administration in rats for 4 weeks. Sodium arsenite led rise in serum creatinine, urea, uric acid, electrolytes (potassium, fractional excretion of sodium), microproteinuria, and decreased creatinine clearance (p < 0.001) indicated renal dysfunction in rats. Arsenic-intoxication resulted in significant oxidative stress in rat kidneys, which was measured in terms of increase in lipid peroxides, superoxide anion generation and decrease in reduced glutathione (p < 0.001) levels. A threefold increase in renal hydroxyproline level in arsenic intoxicated rats indicated fibrosis. Hematoxylin-eosin staining indicated tubular damage, whereas picrosirius red staining highlighted collagen deposition in rat kidneys. S-methylisothiourea and aminoguanidine improved renal function and attenuated arsenic led renal oxidative stress, fibrosis, and decreased the kidney injury score. Additionally, arsenite-intoxication resulted in significant rise in hepatic parameters (serum aspartate aminotransferase, alanine transferase, alkaline phosphatase, and bilirubin (p < 0.001) along with multi-fold increase in oxidative stress, fibrosis and liver injury score in rats, which was significantly (p < 0.001) attenuated by concurrent administration of iNOS inhibitors). Hence, it is concluded that iNOS inhibitors attenuate sodium arsenite-induced renal and hepatic dysfunction in rats.
Assuntos
Arsênio , Arsenitos , Animais , Arsênio/metabolismo , Arsenitos/metabolismo , Arsenitos/toxicidade , Feminino , Fibrose , Rim/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Compostos de SódioRESUMO
Exposure to higher levels of arsenic is a serious threat affecting human health worldwide. We investigated the protective role of betaine (N,N,N-trimethylglycine) against sodium arsenite-induced renal dysfunction in rats. Sodium arsenite (5 mg/kg, oral) was given to rats for 4 weeks to induce nephrotoxicity. Betaine (125 and 250 mg/kg, oral) was administered in rats for 4 weeks along with sodium-arsenite feeding. Arsenic-induced renal dysfunction was demonstrated by measuring serum creatinine, creatinine clearance, urea, uric acid, potassium, fractional excretion of sodium, and microproteinuria. Oxidative stress in rat kidneys was determined by assaying thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione levels. Furthermore, hydroxyproline assay was done to assess renal fibrosis in arsenic intoxicated rats. Hematoxylin-eosin and picrosirius red staining revealed pathological alterations in rat kidneys. Renal endothelial nitric oxide synthase (eNOS) expression was determined by immuno-histochemistry. Concurrent administration of betaine abrogated arsenic-induced renal biochemical and histological changes in rats. Betaine treatment significantly attenuated arsenic-induced decrease in renal eNOS expression. In conclusion, betaine is protective against sodium arsenite-induced renal dysfunction, which may be attributed to its anti-oxidant activity and modulation of renal eNOS expression in rat kidneys.
Assuntos
Arsênio , Arsenitos , Nefropatias , Animais , Ratos , Antioxidantes/metabolismo , Arsenitos/toxicidade , Betaína/farmacologia , Creatinina , Glutationa/metabolismo , Hidroxiprolina/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Potássio , Ratos Wistar , Sódio , Superóxidos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ureia , Ácido ÚricoRESUMO
Cryopreservation of testicular cells and tissues is useful for the preservation and restoration of fertility in pre-pubertal males expecting gonadotoxic treatment for cancer and genetic diseases causing impaired spermatogenesis. A number of freezing and vitrification protocols have thus been tried and variable results have been reported in terms of cell viability spermatogenesis progression and the production of fertile spermatozoa. A few studies have also reported the production of live offspring from cryopreserved testicular stem cells and tissues in rodents but their replication in large animals and human have been lacking. Advancement in in vitro spermatogenesis system has improved the possibility of producing fertile spermatozoa from the cryopreserved testis and has reduced the dependency on transplantation. This review provides an update on various cryopreservation strategies for fertility preservation in males expecting gonadotoxic treatment. It also discusses various methods of assessing and ameliorating cryoinjuries. Newer developments on in vitro spermatogenesis and testicular tissue engineering for in vitro sperm production from cryopreserved SSCs and testicular tissue are also discussed.
Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Infertilidade Masculina/prevenção & controle , Infertilidade Masculina/terapia , Neoplasias/terapia , Sêmen/fisiologia , Testículo/fisiopatologia , Animais , Humanos , MasculinoRESUMO
Polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) are the two most investigated biopolymers for various tissue engineering applications. However, their poor tensile strength renders them unsuitable for cardiac tissue engineering (CTE). In this study, we developed and evaluated PVA-PVP-based patches, plasticized with glycerol or propylene glycol (0.1%-0.4%; v:v), for their application in CTE. The cardiac patches were evaluated for their physico-chemical (weight, thickness, folding endurance, FT-IR, and swelling behavior) and mechanical properties. The optimized patches were characterized for their ability to support in vitro attachment, viability, proliferation, and beating behavior of neonatal mouse cardiomyocytes (CMs). In vivo evaluation of the cardiac patches was done under the subcutaneous skin pouch and heart of rat models. Results showed that the optimized molar ratio of PVA:PVP with plasticizers (0.3%; v-v) resulted in cardiac patches, which were dry at room temperature and had desirable folding endurance of at least 300, a tensile strength of 6-23 MPa and, percentage elongation at break of more than 250%. Upon contact with phosphate-buffered saline, these PVA-PVP patches formed hydrogel patches having the tensile strength of 1.3-3.0 MPa. The patches supported the attachment, viability, and proliferation of primary neonatal mouse CMs and were nonirritant and noncorrosive to cardiac cells. In vivo transplantation of cardiac patches into a subcutaneous pouch and on the heart of rat models revealed them to be biodegradable, biocompatible, and safe for use in CTE applications.
Assuntos
Miócitos Cardíacos/citologia , Plastificantes/química , Álcool de Polivinil/química , Povidona/química , Engenharia Tecidual , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Coração , Hidrogéis , Teste de Materiais , Camundongos , Ratos , Ratos Sprague-Dawley , Resistência à TraçãoRESUMO
Rhabdomyolysis is a clinical syndrome caused by damage to skeletal muscle, which consequently releases breakdown products into circulation and causes acute kidney injury (AKI) in humans. Intramuscular injection of glycerol mimics rhabdomyolysis and associated AKI. In this study, we explored the role of umbelliferone against glycerol-induced AKI in rats. Kidney function was assessed by measuring serum creatinine, urea, electrolytes, and microproteinuria. Renal oxidative stress was quantified using thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione assay. Renal histological changes were determined using periodic acid Schiff and hematoxylin-eosin staining, and immunohistology of apoptotic markers (Bax, Bcl-2) was done. Serum creatine kinase was quantified to assess glycerol-induced muscle damage. Umbelliferone attenuated glycerol-induced change in biochemical parameters, oxidative stress, histological alterations, and renal apoptosis. Pretreatment with bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist, attenuated umbelliferone-mediated protection. It is concluded that umbelliferone attenuates glycerol-induced AKI possibly through PPAR-γ agonism in rats.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Glicerol/toxicidade , Mioglobina/metabolismo , PPAR gama/agonistas , Umbeliferonas/farmacologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Rim/metabolismo , Rim/fisiopatologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Solid surface vitrification (SSV) is a cost effective and simple method for testis tissue preservation. Vitrified-warmed testis tissue was successfully cultured using various organ culture methods. In this study, we compared two culture methods viz. hanging drop (HD) and organ culture (OC) methods for in vitro spermatogenesis of goat testis tissue vitrified-warmed by SSV. It was observed that OC method was superior (p < 0.05) to HD method in terms of post-warming metabolic activity of testicular tissue, as measured by MTT assay on Day 7 and Day 14 of culture, respectively. The size of the tissue also played an important role in post-warming metabolic activity and viability (4 mm3: 72.7 ± 1.2% vs. 9 mm3: 62.7 ± 1.3% vs. 16 mm3: 40.5 ± 1.7%) of vitrified tissues with smaller tissue resulting in better result. The vitrification-induced ROS activity significantly decreased during their in vitro culture. Histology and scanning electron microscopy (SEM) showed the rupture of basal membrane, surface morphology and, cell loss due to vitrification. However, histology and immunohistochemistry showed the progression of in vitro spermatogenesis and formation of elongated spermatozoa in both fresh and vitrified-warmed testis tissue cultured by OC method. Taken together, our results suggest that OC method is superior to HD method for culturing goat testis tissue vitrified-warmed by SSV.
Assuntos
Criopreservação , Testículo , Criopreservação/métodos , Humanos , Masculino , Técnicas de Cultura de Órgãos , Espermatogênese , VitrificaçãoRESUMO
We explored the potential role of peroxisome proliferator activated receptor-γ (PPAR-γ) in stevioside-mediated renoprotection using rhabdomyolysis-induced acute kidney injury (AKI) model in rats. Rhabdomyolysis refers to intense skeletal muscle damage, which further causes AKI. Glycerol (50% w/v, 8 ml/kg) was injected intramuscularly in rats to induce rhabdomyolysis. After 24 hr, AKI was demonstrated by quantifying serum creatinine, urea, creatinine clearance, microproteinuria, and electrolytes in rats. Further, oxidative stress was measured by assaying thiobarbituric acid reactive substances, generation of superoxide anion, and reduced glutathione levels. Additionally, serum creatine kinase (CK) level was assayed to determine glycerol-induced muscle damage in rats. Pathological changes in rat kidneys were studied using hematoxylin-eosin and periodic acid Schiff staining. Moreover, the expression of apoptotic markers (Bcl-2, Bax) in rat kidneys was demonstrated by immunohistochemistry. Stevioside (10, 25, and 50 mg/kg) was administered to rats, prior to the induction of AKI. In a separate group, bisphenol A diglycidyl ether (BADGE, 30 mg/kg), a PPAR-γ receptor antagonist was given prior to stevioside administration, which was followed by rhabdomyolysis-induced AKI in rats. The significant alteration in biochemical and histological parameters in rats indicated AKI, which was attenuated by stevioside treatment. Pretreatment with BADGE abrogated stevioside-mediated renoprotection, which is suggestive of the involvement of PPAR-γ in its renoprotective effect. In conclusion, stevioside protects against rhabdomyolysis-induced AKI, which may be attributed to modulation of PPAR-γ expression.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Diterpenos do Tipo Caurano/uso terapêutico , Glucosídeos/uso terapêutico , PPAR gama/agonistas , Substâncias Protetoras/uso terapêutico , Rabdomiólise/tratamento farmacológico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Diterpenos do Tipo Caurano/farmacologia , Glucosídeos/farmacologia , Glutationa/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Rabdomiólise/complicações , Rabdomiólise/metabolismo , Rabdomiólise/patologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Ischemia/reperfusion (I/R) is one of the common reasons for acute kidney injury (AKI) and we need to develop effective therapies for treating AKI. We investigated the role of fenofibrate against I/R-induced AKI and associated hepatic dysfunction in rats. In male wistar albino rats, renal pedicle occlusion for 40 min and 24 h reperfusion resulted in AKI. I/R-induced AKI was demonstrated by measuring serum creatinine, creatinine clearance, urea, uric acid, potassium, fractional excretion of sodium and urinary microproteins. Oxidative stress in rat kidneys was quantified by assaying superoxide anion generation, thiobarbituric acid reactive substances, and reduced glutathione levels. AKI-induced hepatic damage was quantified by assaying serum aminotransferases, alkaline phosphatase and bilirubin levels. Moreover, serum cholesterol, high density lipoprotein and triglycerides were quantified. Hematoxylin-eosin staining of renal and hepatic tissues was done and the kidney and liver injury scores were determined. Immunohistology of endothelial nitric oxide synthase (eNOS) was done in rat kidneys. Fenofibrate was administered for 1 week before subjecting rats to AKI. In separate group, the nitric oxide synthase inhibitor, L-nitroarginine methyl ester (L-NAME) was administered prior to fenofibrate treatment. In I/R group, significant alteration in the serum/urine parameters indicated AKI and hepatic dysfunction along with marked increase in kidney and liver injury scores. Treatment with fenofibrate attenuated AKI and associated hepatic dysfunction. Moreover, I/R-induced decrease in renal eNOS expression was abrogated by fenofibrate. Pre-treatment with L-NAME abolished fenofibrate mediated reno- and hepato-protective effects. In conclusion, fenofibrate attenuates I/R-induced AKI and associated hepatic dysfunction putatively through modulation of eNOS expression.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Fenofibrato/farmacologia , Hepatopatias/tratamento farmacológico , Hepatopatias/etiologia , Traumatismo por Reperfusão/complicações , Animais , Biomarcadores/metabolismo , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
Aldehyde dehydrogenase 1 (ALDH1) and hepatocyte nuclear factor 4A (HNF4A) are the putative mammary stem cell markers. Tissue necrosis factor alpha (TNFA) is involved in inflammation-associated carcinogenesis and cell proliferation. In this study, the gene expression profile of ALDH1, HNF4A and TNFA of buffalo mammary tissue using real-time quantitative PCR (RT-qPCR). Analysis of RT-qPCR data revealed that the relative expression (log2 fold change) of ALDH1 and TNFA during mastitis (vs. lactation) was increased (P < .05) by 2.98 and 4.71, respectively. The relative expression (log2 fold change; -7.39) of stem cell marker, HNF4A was decreased (P < .05) during mastitis. Histological analysis of mammary tissue during mastitis showed thickening of stroma and occasionally hyperplasia, predominantly in prepubertal and non-lactating animals. Although, the level of expression of these genes may vary, depending upon the physiological stage of the animals, however expression of ALDH1 and TNFA was high during mastitis. A systematic study on large samples of buffalo mammary tissue with appropriate comparisons needs to be evaluated with these markers for prognosis of buffalo mammary health.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Glândulas Mamárias Animais , Mastite Bovina , Fator de Necrose Tumoral alfa/metabolismo , Família Aldeído Desidrogenase 1/genética , Animais , Búfalos/genética , Búfalos/metabolismo , Bovinos , Fator 4 Nuclear de Hepatócito/genética , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/enzimologia , Mastite Bovina/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Tumour necrosis factor-α (TNF-α) is a cytokine that plays multiple important roles in corpus luteum (CL). Immunolocalization of expression of TNF-α in CL of buffalo was studied in different stages of its development and regression. Corpus luteum of healthy buffaloes (24) was collected from local slaughterhouses and categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal (stage III, 12-16 days, n = 6) and regressing phase (stage IV, 17-20 days, n = 6). In earliest phase of cyclic CL, per cent immunoexpression of TNF-α was significantly (p < .05) lower as compared to all phases with its expression being restricted to few developing luteal cells, usually in neutrophils. A significantly (p < .05) higher number of neutrophils with TNF-α immunoexpression were observed as compared to mid-luteal phase that indicated its role in initiation of angiogenesis at this stage. TNF-α immunoexpression almost doubled in mid-luteal phase, but the number of neutrophils exhibiting TNF-α was significantly (p < .05) lower with respect to all phases of CL. Immunoexpression percentage in late luteal phase increased sharply being significantly (p < .05) higher than earlier two phases of CL. In regressing phase, per cent immunostaining was maximum with highly significant (p < .05) difference as compared to all other stages, observed in all degrading luteal cells, abundant immune cells, that is neutrophils and macrophages which finally led to apoptosis and phagocytosis. Immunoexpression of TNF-α in early luteal phases served its role in initiation of angiogenesis, and its intense expression in regressing phase of CL suggested a shift in its role to apoptosis and structural luteal regression signifying both luteotropic and luteolytic roles in buffalo. This is probably the first study of its kind in buffaloes.
Assuntos
Corpo Lúteo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Búfalos/fisiologia , Corpo Lúteo/irrigação sanguínea , Ciclo Estral/fisiologia , Feminino , Imuno-Histoquímica/veterinária , Neutrófilos/metabolismoRESUMO
Strict environmental regulations as well as requirement of conservation of natural resources compelled the researchers to recycle the metal values from secondary resources. The scrap magnets found to be a potential alternative resource to extract rare earth metal, Nd. Present paper reports a novel process flow-sheet for the recycling of scrap Nd-Fe-B magnets to recover Nd as marketable salt and other valuable by-products. The Nd-Fe-B magnets were demagnetized, crushed and charged to atmospheric leaching resulting in ~99.99% recovery of REMs (Nd, Pr, Dy) and Fe using 1 M H2SO4 solution at room temperature for 90 min and pulp density 50 g/L. The obtained leach liquor was subjected to acid extraction procedure by mixing the liquor with 70% TEHA diluted in kerosene for 10 min, which requires five stages for complete extraction of acid from the liquor having O/A ratio 2:1. Ammonia solution was used to increase the pH of acid free leach liquor to 1.65 for the precipitation of Nd (with minor amount of Pr and Dy) and above that to precipitate Fe. Further, processing of these valuables make them industrially applicable. The leached residue was checked using Toxicity Characteristics Leachability Procedure (TCLP) test and found the remained metals within the permissible limit. This process offers a simple and efficient means to reduce the toxicological effect by recovering Nd from magnets of computer hard disks.
Assuntos
Metais Terras Raras , Neodímio , Imãs , Metais , ReciclagemRESUMO
Although water buffaloes are the main milk-producing animals in Indian subcontinent, only limited attempts have been made to identify canonical pathways and gene regulatory networks operating within the mammary glands of these animals. Such information is important for identifying unique transcriptome signatures in the mammary glands of diseased animals. In this report, we analyzed the transcription profile of 3 prepubertal buffalo mammary glands and identified common genes (mean FPKM > 0.2 in all samples) operating in the glands. Among 19,994 protein coding genes, 14,678 genes expressed and 5316 unique genes did not express in prepubertal buffalo mammary glands. Of these 14,678 expressed genes, 79% comprised a ubiquitous transcriptome that was dominated by very lowly expressed genes (51%). The percentage of rarely, moderately, and abundantly expressed genes was 25%, 2%, and 1%, respectively. Gene Ontology (GO) terms reflected in the expression of common genes (mean FPKM > 5.0) for molecular function were related to binding and catalytic activity. Products of these genes were involved in metabolic and cellular processes and belong to nucleic acid binding proteins. The canonical pathways for growth of mammary glands included integrin signaling, inflammation, GnRH and Wnt pathways. KEGG enriched pathways revealed many pathways of cancer including ribosome, splisosome, endocytosis, and ubiquitin-mediated proteolysis, pathways for viral infection, and bacterial invasion of epithelial. Highly expressed genes (mean FPKM > 500 included beta-actin (ACTB), beta-2 microglobulin (B2M), caseins (CSN2, CNS3), collagens (COL1A1, COL3A1), translation elongation factors (EEF1A1, EEF1G, EEF2), keratins (KRT15, KRT19), major histocompatibility complex genes (CD74, JSP.1), vimentin (VIM), and osteopontin (SPP1). Interestingly, expression of milk protein genes in prepubertal glands opens possible roles of these genes in development of mammary glands. We report the whole transcriptomic signature of prepubertal buffalo mammary gland and indicated its molecular signature is similar to cancer type.
Assuntos
Búfalos/genética , Glândulas Mamárias Animais/metabolismo , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/crescimento & desenvolvimentoRESUMO
We investigated the involvement of peroxisome proliferator activated receptor-γ (PPAR-γ)/endothelial nitric oxide synthase (eNOS) pathway in estradiol mediated protection against ischemia reperfusion (I/R)-induced acute kidney injury (AKI) in rats. To induce AKI, rats underwent 40 min of bilateral renal ischemia followed by 24 h of reperfusion. I/R-induced kidney damage was quantified by measuring serum creatinine, creatinine clearance, urea nitrogen, uric acid, potassium, fractional excretion of sodium, microproteinuria, and renal oxidative stress (thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione). Hematoxylin eosin stain demonstrated renal histology, while renal expression of apoptotic markers (Bcl-2, Bax), PPAR-γ and eNOS were quantified by immunohistochemistry. Estradiol (1 mg/kg, i.p.) was administered 30 min before I/R in rats. In separate groups, PPAR-γ antagonist, BADGE (30 mg/kg, i.p.), and NOS inhibitor, L-NAME (20 mg/kg, i.p.) were administered prior to estradiol treatment, which was followed by I/R in rats. I/R caused significant renal damage as demonstrated by biochemical (serum/urine), renal oxidative stress and histological changes alongwith increased expression of Bax and decreased levels of Bcl-2, PPAR-γ and eNOS, which were prevented by estradiol. Pre-treatment with BADGE and L-NAME abolished estradiol mediated renoprotection. Notably, I/R + estradiol + BADGE group revealed decreased expression of PPAR-γ and eNOS in renal tissues. In I/R + estradiol + L-NAME group, eNOS expression was reduced while PPAR-γ levels remained unchanged. These results suggest that estradiol modulates PPAR-γ which consequently regulates eNOS expression in rat kidneys. We conclude that estradiol protects against I/R-induced AKI through PPAR-γ stimulated eNOS activation in rats.
Assuntos
Injúria Renal Aguda , Estradiol/farmacocinética , Óxido Nítrico Sintase Tipo III/metabolismo , PPAR gama/metabolismo , Traumatismo por Reperfusão , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Ativação Enzimática/efeitos dos fármacos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.
Assuntos
Búfalos/metabolismo , Receptor alfa de Estrogênio/metabolismo , Ciclo Estral/fisiologia , Tubas Uterinas/fisiologia , Fase Luteal/fisiologia , Folículo Ovariano/fisiologia , Animais , Feminino , Humanos , Imuno-Histoquímica , Oviductos , Progesterona/sangueRESUMO
This study examined the hypothesis that xanthosine (XS) treatment would promote mammary-specific gene expression and stem cell transcripts and have a positive influence on milk yield of dairy goats. Seven primiparous Beetal goats were assigned to the study. Five days after kidding, one gland (either left or right) was infused with XS (TRT) twice daily for 3 d and the other gland with no XS infusion served as a control (CON). Mammary biopsies were collected at 10 d and RNA was isolated. Gene expression analysis of milk synthesis genes, mammary stem/progenitor cell markers, cell proliferation and differentiation markers were performed using real time quantitative PCR (RT-qPCR). Results showed that the transcripts of milk synthesis genes (BLG4, CSN2, LALBA, FABP3, CD36) and mammary stem/progenitor cell markers (ALDH1 and NR5A2) were increased in as a result of XS treatment. Average milk yield in TRT glands was increased marginally (approximately ~2% P = 0·05, paired t-test) per gland relative to CON gland until 7 wk. After 7 wk, milk yield of TRT and CON glands did not differ. Analysis of milk composition revealed that protein, lactose, fat and solids-not-fat percentages remained the same in TRT and CON glands. These results suggest that XS increases expression of milk synthesis genes, mammary stem/progenitor cells and has a small effect on milk yield.
Assuntos
Expressão Gênica/efeitos dos fármacos , Cabras , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Ribonucleosídeos/farmacologia , Animais , Biomarcadores/análise , Diferenciação Celular/genética , Proliferação de Células/genética , Feminino , Lactação/efeitos dos fármacos , Lactação/fisiologia , Glândulas Mamárias Animais/citologia , Leite/química , Proteínas do Leite/análise , Proteínas do Leite/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Células-Tronco/fisiologia , XantinasRESUMO
BACKGROUND: Sildenafil is a phosphodiesterase inhibitor used clinically for treating erectile dysfunction. Few studies suggest sildenafil to be a renoprotective agent. The present study investigated the involvement of peroxisome proliferator-activated receptor γ (PPAR-γ) in sildenafil-mediated protection against ischemia-reperfusion-induced acute kidney injury (AKI) in rats. MATERIALS AND METHODS: The rats were subjected to ischemia-reperfusion injury (IRI) with 40 min of bilateral renal ischemia followed by reperfusion for 24 h. The renal damage was assessed by measuring creatinine clearance, blood urea nitrogen, plasma uric acid, electrolytes, and microproteinuria in rats. The thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione levels were measured to assess oxidative stress in renal tissues. The hematoxylin-eosin staining was carried out to demonstrate histopathologic changes in renal tissues. Sildenafil (0.5 and 1.0 mg/kg, intraperitoneal) was administered 1 h before subjecting the rats to renal IRI. In a separate group, bisphenol A diglycidyl ether (30 mg/kg, intraperitoneal), a PPAR-γ receptor antagonist, was given before sildenafil administration followed by IRI. RESULTS: The ischemia-reperfusion demonstrated marked AKI with significant changes in serum and urinary parameters, enhanced oxidative stress, and histopathologic changes in renal tissues. The administration of sildenafil demonstrated significant protection against ischemia-reperfusion-induced AKI. The prior treatment with bisphenol A diglycidyl ether abolished sildenafil-mediated renal protection, thereby confirming involvement of PPAR-γ agonism in the sildenafil-mediated renoprotective effect. CONCLUSIONS: It is concluded that sildenafil protects against ischemia-reperfusion-induced AKI through PPAR-γ agonism in rats.
Assuntos
Injúria Renal Aguda/prevenção & controle , PPAR gama/agonistas , Inibidores da Fosfodiesterase 5/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Citrato de Sildenafila/uso terapêutico , Injúria Renal Aguda/patologia , Animais , Compostos Benzidrílicos , Avaliação Pré-Clínica de Medicamentos , Compostos de Epóxi , Rim/patologia , Testes de Função Renal , Masculino , Estresse Oxidativo , Inibidores da Fosfodiesterase 5/farmacologia , Proteinúria/prevenção & controle , Distribuição Aleatória , Ratos Wistar , Traumatismo por Reperfusão/patologia , Citrato de Sildenafila/farmacologiaRESUMO
Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that ( 1 ) HNF4A identifies putative buffalo mammary stem/progenitor cells and ( 2 ) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5-10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4-4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify putative mammary stem/progenitor cells was confirmed but the second hypothesis that the number of mammary stem/progenitor cells decreases during mastitis was unsupported. This is the first report outlining the expression of HNF4A and identification of putative mammary stem/progenitor cells in buffalo mammary gland.