Avishek Paul

Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the expression and localization of FGF and its functional receptors in the follicle of buffalo and the treatment of FGF2 on mRNA expression of CYP19A1 (aromatase), PCNA, and BAX (BCL-2 associated X protein) in cultured buffalo granulosa cells (GCs). Follicles were classified into four groups based on size and E2 level in follicular fluid (FF): F1, 4-6mm diameter, E2<0.5ng/ml of FF; F2, 7-9mm, E2=0.5-5ng/ml; F3, 10-13mm, E2=5-40ng/ml; F4, >14mm, E2>180ng/ml. The qPCR studies revealed that the mRNA expression of FGF1, FGF2 and FGF7 were maximum (P<0.05) in theca interna (TI) whereas the transcripts of FGFR1, FGFR2, FGFR2IIIB and FGFR2IIIC were up-regulated (P<0.05) in GCs of F4 follicles. Protein expression of most members were maximum (P<0.05) in F4 follicles except FGFR3 and FGFR4. All members were localized in GC and TI with a stage specific immunoreactivity. Primary culture of GCs with treatment of FGF2 at different dose-time combinations revealed that the mRNA expression and immunoreactivity of CYP19A1 and PCNA were maximum (P<0.05) whereas BAX was minimum (P<0.05) with 200ng/ml at 72h of incubation. The findings indicate that FGF family members are expressed in a regulated manner in buffalo ovarian follicles during different stages of development where FGF2 may promote steroidogenesis and GC survival through autocrine and paracrine manner.

A comparative study on the expression profile of MCTs and HSPs in Ghungroo and Large White Yorkshire breeds of pigs during different seasons.

Thermal stress has a significant adverse effect on commercial swine production but it is not easy to measure. Animals may adapt to stress conditions by an alteration in the expression of stress-related genes such as heat shock proteins (HSPs) and monocarboxylate transporters (MCTs). The present study presents a comparative analysis of seasonally varied effects on the expression profiles of HSPs (27, 70, and 90) and MCTs (1, 2, and 4) transcripts in thigh muscle and colon tissue of Ghungroo and Large White Yorkshire (LWY) breeds of pig. By real-time polymerase chain reaction, the mRNA expression of HSP27 and HSP90 genes was found to be higher in both thigh muscle and colon tissue in Ghungroo compared to Large White Yorkshire pigs during the summer. However, the relative expression of HSP70 was significantly higher (P < 0.01) in Ghungroo compared to Large White Yorkshire pigs during both seasons in both thigh muscle and colon tissue. The expression of HSP90 was higher in Ghungroo when compared to LWY though the variation was non-significant (P > 0.05) in the colon during different seasons. However, in Ghungroo, the mRNA expression of MCT1 was found to be significantly (P < 0.05) higher in thigh muscle and colon regions during the summer compared to LWY, whereas MCT2 was expressed more in the colon in LWY compared to Ghungroo during the summer. The relative expression of mRNA of MCT4 was found to be significantly (P < 0.05) higher in thigh region in both summer and winter in Ghungroo compared with LWY. Thus, the study demonstrated that both HSPs and MCTs gene expression during thermal stress suggests the possible involvement of these genes in reducing the deleterious effect of thermal stress, thus maintaining cellular integrity and homeostasis in pigs. These genes could be used as suitable markers for the assessment of stress in pigs.

Combined treatment with atorvastatin and imipenem improves survival and vascular functions in mouse model of sepsis

We have recently reported that pre-treatment, but not the post-treatment with atorvastatin showed survival benefit and improved hemodynamic functions in cecal ligation and puncture (CLP) model of sepsis in mice. Here we examined whether combined treatment with atorvastatin and imipenem after onset of sepsis can prolong survival and improve vascular functions. At 6 and 18h after sepsis induction, treatment with atorvastatin plus imipenem, atorvastatin or imipenem alone or placebo was initiated. Ex vivo experiments were done on mouse aorta to examine the vascular reactivity to nor-adrenaline and acetylcholine and mRNA expressions of α1D AR, GRK2 and eNOS. Atorvastatin plus imipenem extended the survival time to 56.00±4.62h from 20.00±1.66h observed in CLP mice. The survival time with atorvastatin or imipenem alone was 20.50±1.89h and 27.00±4.09h, respectively. The combined treatment reversed the hyporeactivity to nor-adrenaline through preservation of α1D AR mRNA/protein expression and reversal of α1D AR desensitization mediated by GRK2/Gβγ pathway. The treatment also restored endothelium-dependent relaxation to ACh through restoration of aortic eNOS mRNA expression and NO availability. In conclusion, combined treatment with atorvastatin and imipenem exhibited survival benefit and improved vascular functions in septic mice.

Expression analysis of Toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure

Six male Tharparkar cattle of 2-3 years old were selected for the study. After 15 days acclimation at thermo neutral zone (TNZ) in psychrometric chamber, animals were exposed at 42°C for 6h up to 23 days followed by 12 days of recovery period. Blood samples were collected during control period at TNZ (day 1, 5 and 12), after heat stress exposure (day 1-10, Short Term Heat Stress Acclimation - STHSA; day 15-23, Long Term Heat Stress Acclimation - LTHSA) and recovery period (day 7 and 12) and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. Serum cortisol concentration was assessed by RIA. The mRNA and protein expression in PBMCs were determined by qPCR and western blot respectively. Samples at TNZ were taken as control. Serum cortisol concentration was increased (P<0.05) during STHSA and gradually declined during LTHSA. Toll like receptor 2 (TLR 2) expression was up regulated (P<0.05) during STHSA and declined to basal level during LTHSA and recovery phase. However, toll like receptor 4 (TLR 4) expression was up regulated (P<0.05) during STHSA and LTHSA while declined in recovery phase. Interleukin 2 (IL2) and interleukin 6 (IL 6) were up regulated (P<0.05) during STHSA and reduced to basal level during LTHSA. PBMCs culture study was conducted to study transcriptional abundance of TLR2/4 and IL2/6 at different temperature-time combinations. The present findings indicate that TLR 2/4 and IL 2/6 could possibly play a vital role in thermo tolerance in Tharparkar cattle during short term and long term heat stress exposure.

Localization of IGF proteins in various stages of ovarian follicular development and modulatory role of IGF-I on granulosa cell steroid production in water buffalo (Bubalus bubalis)

The present study aimed to determine the expression of insulin like growth factor (IGF) genes in the bubaline ovarian follicles and modulatory role of IGF-I on progesterone production from granulosa cells (GC) of pre-ovulatory follicle in vitro. According to size, follicles were classified into four groups: GI (small), GII (medium), GIII (large) and GIV (preovulatory). All IGF genes were expressed in both GC and theca interna (TI) cells. The relative expression of IGF-I and IGF receptor I (IGFR-I) genes increased with follicle size and was greatest in the pre-ovulatory follicle (P<0.05). Expression of IGF-II and IGFR-II genes was minimal in GC but was readily detected in TI cells. In TI cells, the gene expression was greater in medium and large as compared to small and pre-ovulatory follicles. The expression of all binding protein (IGFBP) genes was detected in both GC and TI cells. Expression of IGFBP-3 gene increased with follicle size and was greatest in pre-ovulatory follicles (P<0.05). The expression of IGFBP-2 and IGFBP-4 was less in pre-ovulatory follicles but expression of IGFBP-5 and IGFBP-6 genes were greater at this stage. The GC culture was conducted for three time durations and with three doses of IGF-I. Expression of steroidogenic genes (StAR, CYP11A1, HSD3B) and progesterone concentration were increased in a dose and time dependent fashion. The present study, therefore, provided evidence of an autocrine/paracrine role of IGFs in follicular development and a stimulatory role of IGF1 in steroid production in GC of preovulatory follicles in the bubaline species.

Changes in expression of monocarboxylate transporters, heat shock proteins and meat quality of Large White Yorkshire and Ghungroo pigs during hot summer period

Objective Present study explores the effect of hot summer period on the glycolytic rate of early post-mortem meat quality of Ghungroo and Large White Yorkshire (LWY) pig and comparative adaptability to high temperature between above breeds by shifting the expression of stress related genes like mono-carboxylate transporters (MCTs) and heat shock proteins (HSPs). Methods Healthy pigs of two different breeds, viz., LYW and Ghungroo (20 from each) were maintained during hot summer period (May to June) with a mean temperature of about 38°C. The pigs were slaughtered and meat samples from the longissimus dorsi (LD) muscles were analyzed for pH, glycogen and lactate content and mRNA expression. Following 24 h of chilling, LD muscle was also taken from the carcasses to evaluate protein solubility and different meat quality measurements. Results LWY exhibited significantly (p<0.01) higher plasma cortisol and lactate dehydrogenase concentration than Ghungroo indicating their higher sensitivity to high temperature. LD muscle from LWY pigs revealed lower initial and ultimate pH values and higher drip loss compared to Ghungroo, indicating a faster rate of pH fall. LD muscle of Ghungroo had significantly lower lactate content at 45 min postmortem indicating normal postmortem glycolysis and much slower glycolytic rate at early postmortem. LD muscle of LWY showed rapid postmortem glycolysis, higher drip loss and higher degrees of protein denaturation. Ghungroo exhibited slightly better water holding capacity, lower cooking loss and higher protein solubility. All HSPs (HSP27, HSP70, and HSP90) and MCTs (MCT1, MCT2, and MCT4) in the LD muscle of pigs inclined to increase more in Ghungroo than LWY when exposed to high temperature. Conclusion Effect of high temperature on the variation of HSPs and MCTs may play a crucial role in thermal tolerance and adaptation to different climatic conditions, pH regulation, muscle acidification, drip loss, protein denaturation and also in postmortem meat quality development.

Impact of oocyte-secreted factors on its developmental competence in buffalo

Oocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus-oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

Transcriptional and translational abundance of Bone morphogenetic protein (BMP) 2, 4, 6, 7 and their receptors BMPR1A, 1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells

BMPs and their receptors modulate the granulosa cell (GC) function in the follicle of domestic animals. Since little is known on BMPs in the buffalo, the present study was aimed to investigate the expression of BMP2, 4, 6, 7 and their receptors BMPR1A, BMPR1B, BMPR2 in the GC and theca cells (TC) of ovarian follicles and the role of BMP4 and BMP7 on buffalo GC. Follicles were classified into four groups based on size and E2 level in the follicular fluid as follows: (i) Group1(4-6 mm; <0.5 ng/mL) (ii) Group 2 (7-9 mm; 0.5-5 ng/mL) (iii) Group 3 (10-13 mm; 5-40 ng/mL) and (iv) Group 4 (dominant follicle) (>13 mm; >180 ng/mL). The results revealed that except BMP6, BMP2, 4 7 and receptors BMPR1A, BMPR1B and BMPR2 showed a minimum of 1.5-2 fold increase in mRNA expression in the GC of dominant follicle as compared to other follicle classes. In the dominant follicle, a two-fold increase in BMP4 and BMP7 expression was observed in the TC. At 100 ng/mL, the BMP4 and BMP7 either alone or in combination maximally down-regulated CASPASE3 and stimulated the transcripts of PCNA, FSHR and CYP19A1 that was supported by E2 secretion in the granulosa cell culture suggesting their role in cell survival and E2 production. In conclusion, GC and TC of dominant follicles express BMP 2, 4, 6, 7 and their receptors BMPR1A, BMPR1B and BMPR2. BMP4 and BMP7 stimulate E2 production and promote GC survival.

Kaempferol attenuates acute lung injury in caecal ligation and puncture model of sepsis in mice

ABSTRACT Aim of the study: Kaempferol is a flavonoid and important part of the diet. Kaempferol has shown antioxidant, antiinflammatory and antidiabetic activities in various studies. However, protective potential of kaempferol in acute lung injury induced by sepsis and its mechanism remains unclear. The present study was undertaken to evaluate the effect of kaempferol in sepsis-induced acute lung injury in mice and its possible mechanism of action. Materials and methods: Acute lung injury was induced by CLP surgery in mice. Kaempferol (100 mg/kg bw) was administered orally one hour before caecal ligation and puncture surgery in mice. Mice were divided into four groups sham, KEM+sham, sepsis (CLP), and KEM+sepsis. Assessment of lung injury was done by estimation of protein content in lung tissue, lung edema, proinflammatory cytokines in plasma and lung tissue, oxidative stress, antioxidant enzymes, nitrite production, and histopathology. Results: Kaempferol pretreated mice showed significant (P < 0.001) decrease in water content in lungs. Kaempferol pretreatment showed reduction in cytokines IL-6, IL-1β, and TNF-α in plasma as well as in lung tissue in comparison with septic mice without pretreatment. Pretreatment with kaempferol did not show any reduction in MDA level in comparison with septic mice. Antioxidant enzymes SOD and catalase and nonenzymatic antioxidant GSH activities were also increased with kaempferol pretreatment in septic mice. Further, kaempferol pretreatment reduced the lung tissue nitrite level (P < 0.01) and iNOS (P < 0.05) level in septic mice. A significant (P < 0.01) downregulation of mRNA expression of ICAM-1 and iNOS was observed with this pretreatment. Kaempferol pretreatment did not decrease bacterial load in septic mice. Mice pretreated with kaempferol followed by sepsis showed lesser infiltration of cells and more arranged alveolar structure in histopathological analysis. Conclusions: The study suggests that kaempferol showed attenuation in sepsis-induced acute lung injury in mice through suppression of oxidative stress, iNOS, and ICAM-1 pathways.

Luteolin attenuates acute lung injury in experimental mouse model of sepsis

HighlightsLuteolin pretreatment attenuated lung edema in septic mice.Cytokines levels were reduced with luteolin pretreatment in septic mice.Antioxidant enzymes activities and lipid peroxidation were improved by luteolin.Luteolin reduced acute lung injury through suppression in ICAM‐1/NF‐kappa B/partially iNOS pathways. &NA; The present investigation was undertaken to assess the result of pretreatment of luteolin in sepsis‐induced acute lung injury in mice and its mechanism of action. Luteolin was administered intraperitoneally one hour before caecal ligation and puncture (CLP) surgery in mice. Acute lung injury was assessed by estimation of different parameters like lung edema, protein content, cytokines level, oxidative stress, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)‐1 expression and histopathology. Pretreatment of mice with luteolin showed decrease lung edema and protein content in tissue and bronchoalveolar lavage fluid (BALF). However, mice pretreated with luteolin showed reduction (p = 0.92) in blood and lung tissue bacterial counts however it was non significant. Further, luteolin showed significant reduction in interleukin (IL)‐6 and IL‐1&bgr; in lung tissue which are the proinflammatory cytokines. However, plasma IL‐1&bgr; and tissue tumor necrosis factor (TNF)‐&agr; level decrease (p = 0.24; p = 0.19) with this pretreatment. Further, ICAM‐1 mRNA expression and nuclear factor (NF)‐kappa B protein expression were significantly (p < 0.01) decreased in luteolin pretreated septic mice. The lung iNOS level, iNOS mRNA and protein expressions were markedly (p = 0.25; p = 0.50; p = 0.06) altered with luteolin pretreatment, respectively. Also, significant reduction in lipid peroxidation and increase in the activity of antioxidant enzymes like superoxide dismutase (SOD) and catalase was noted with luteolin pretreatment. However, luteolin did not alter (p = 0.36) the non enzymatic antioxidant GSH activity in septic mice. Histopathology of lung tissue showed reduction in lung injury with the luteolin pretreatment in septic mice. The study suggests that luteolin showed attenuation in sepsis‐induced acute lung injury in mice through suppression in ICAM‐1, NF‐kappa B, oxidative stress and partially iNOS pathways.