Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P < 0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P < 0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P < 0.05) after 10 days of heat exposure (corresponds to chronic heat stress) as compared to the first 5 days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

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.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.

Expression and localization of angiopoietin family in buffalo ovarian follicles during different stages of development and modulatory role of angiopoietins on steroidogenesis and survival of cultured buffalo granulosa cells.

The present study investigated the expression and localization of angiopoietin (ANPT) family members in buffalo ovarian follicles of different size. It also looked at the role of ANPTs in estradiol secretion and mRNA expression of phosphoinositide-3-kinase-protein kinase B signaling pathway cellular proliferation (phosphoinositide-dependant kinase and protein kinase B [AKT]) and proapoptotic (BAD) factors with caspase 3 in cultured buffalo granulosa cells (GCs). The mRNA and protein expression of ANPT-1 was greatest (P < 0.05), whereas ANPT-2 was reduced (P < 0.05) in preovulatory follicles as compared to F1 follicle. Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 transcripts and protein expression did not change in all follicular groups, whereas tyrosine kinase with immunoglobulin-like and EGF-like domains 2 mRNA was highest (P < 0.05) in theca interna but not GC layer of preovulatory follicle. All members of ANPT family were localized in GC and theca interna showing a stage specific immunoreactivity. Cultured GCs were treated with ANPT-1 and ANPT-2 separately at doses of 1, 10, and 100 ng/mL and in combination at 100 ng/mL for three incubation periods (24, 48, and 72 hours). Estradiol secretion was highest (P < 0.05) at 100 ng/mL at 72 hours of incubation when GCs were treated with either protein alone. The mRNA expression of phosphoinositide-dependant kinase and AKT was highest (P < 0.05), and BAD with caspase 3 was lowest (P < 0.05) at 100 ng/mL at 72 hours of incubation, when cultured GCs were treated separately with each protein or in combination. The immuoreactivity of AKT, pAKT, and pBAD were maximal, whereas BAD was minimal with 100 ng/mL at 72 hours when cultured GCs treated with either protein alone. The findings indicate that ANPTs are expressed in a regulated manner in buffalo ovarian follicle during different stages of development where they may promote steroidogenesis and GC survival through autocrine and paracrine actions.

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.

Expression and localization of insulin-like growth factor system in corpus luteum during different stages of estrous cycle in water buffaloes (Bubalus bubalis) and the effect of insulin-like growth factor I on production of vascular endothelial growth factor and progesterone in luteal cells cultured in vitro.

This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were significantly higher (P < 0.05) when the culture medium was supplemented with 100 ng/mL of IGF-I for 72 hours. Moreover, IGF-I at a dose of 100 ng/mL increased P4 and VEGF production (P < 0.05). It can be concluded that IGF family members via their autocrine and paracrine effect play significant roles in promoting angiogenesis through the production of VEGF in luteal cells and steroid synthesis through the production of key steroidogenic factors.

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on progesterone secretion and viability of cultured bubaline luteal cells.

We evaluated the temporal (24, 48 and 72 hours) and dose-dependent (5, 10, and 100 ng/mL of LH, IGF-1, and EGF, respectively) production and secretion of progesterone (P4) in cultured luteal cells from different stages of estrous cycle as well as the expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (HSD3B), anti-apoptotic gene PCNA, and pro-apoptotic gene BAX in luteal cells of mid-luteal phase in buffalo. Samples from early luteal phase (ELP; Day 1 to 4; n = 4), mid-luteal phase (MLP; Day 5 to 10; n = 4), and late luteal phase (LLP; Day 11 to 16; n = 4) of estrous cycle were collected. Progesterone was assayed by RIA, whereas mRNA expression was determined by quantitative real-time polymerase chain reaction. Results depicted that highest dose (100 ng/mL) of LH, IGF-1, and EGF and longer duration of time brought about a (P < 0.05) rise in P4 level and expression of steroidogenic enzymes and PCNA compared with the lower level(s) and control while, all treatments (P < 0.05) inhibited BAX expression in a time dependent-manner. Analysis of interaction between stage and treatments revealed that LH treatment (P < 0.05) increased P4 production compared with IGF-1 and EGF in ELP and MLP. However in LLP, treatment with IGF-1 and EGF significantly (P < 0.05) increased P4 production compared with LH treatment. Summarizing, our study explores the steroidogenic potential of LH and growth factors across different luteal stages in buffalo, which on promoting steroidogenic enzyme expression and cell viability culminated in enhanced P4 production in luteal cells.

Stimulatory effect of vascular endothelial growth factor on progesterone production and survivability of cultured bubaline luteal cells.

The objectives of the present study were to investigate the effects of vascular endothelial growth factor (VEGF) on progesterone (P4) synthesis in cultured luteal cells from different stages of the estrous cycle and on expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side chain cleavage (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B), antiapoptotic gene PCNA, and proapoptotic gene BAX in luteal cells obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Corpus luteum samples from the early luteal phase (ELP; day 1st-4th; n=4), MLP (day 5th-10th; n=4), and the late luteal phase (LLP; day 11th-16th; n=4) of oestrous cycle were obtained from a slaughterhouse. Luteal cell cultures were treated with VEGF (0, 1, 10 and 100 ng/ml) for 24, 48 and 72h. Progesterone was assessed by RIA, while mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Results indicated a dose- and time-dependent stimulatory effect of VEGF on P4 synthesis and expression of steroidogenic enzymes. Moreover, VEGF treatment led to an increase in PCNA expression and decrease in BAX expression. In summary, these findings suggest that VEGF acts locally in the bubaline CL to modulate steroid hormone synthesis and cell survivability, which indicates that this factor has an important role as a regulator of CL development and function in buffalo.

Expression and localization of ghrelin and its functional receptor in corpus luteum during different stages of estrous cycle and the modulatory role of ghrelin on progesterone production in cultured luteal cells in buffalo.

Evidence obtained during recent years provided has insight into the regulation of corpus luteum (CL) development, function, and regression by locally produced ghrelin. The present study was carried out to evaluate the expression and localization of ghrelin and its receptor (GHS-R1a) in bubaline CL during different stages of the estrous cycle and investigate the role of ghrelin on progesterone (P4) production along with messenger RNA (mRNA) expression of P4 synthesis intermediates. The mRNA and protein expression of ghrelin and GHS-R1a was significantly greater in mid- and late luteal phases. Both factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of ghrelin and GHS-R1a was greater during mid- and late luteal phases. Luteal cells were cultured in vitro and treated with ghrelin each at 1, 10, and 100 ng/mL concentrations for 48 h after obtaining 75% to 80% confluence. At a dose of 1 ng/mL, there was no significant difference in P4 secretion between control and treatment group. At 10 and 100 ng/mL, there was a decrease (P < 0.05) in P4 concentration, cytochrome P45011A1 (CYP11A1), and 3-beta-hydroxysteroid dehydrogenase mRNA expression and localization. There was no difference in mRNA expression of steroidogenic acute regulatory protein between control and treatment group. In summary, the present study provided evidence that ghrelin and its receptor are expressed in bubaline CL and are localized exclusively in the cell cytoplasm and ghrelin has an inhibitory effect on P4 production in buffalo.

Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells.

The objective of this study was to characterize in vitro expression and secretion of vascular endothelial growth factor (VEGF) in bubaline granulosa cells (GC), grown in serum containing media supplemented with luteinizing hormone (LH), insulin like growth factor-1 (IGF-1), and epidermal growth factor (EGF) at three different doses and time durations. GCs were collected from ovarian follicles of varying diameters [Gp-I (small), 4-6 mm; Gp-II (medium), 7-9 mm; Gp-III (large), 10-13 mm; Gp-IV (pre-ovulatory), >13 mm]. In general, each of the three treatments resulted in a dose as well as time dependent increase in the mRNA expression and secretion of VEGF in the cultured GCs of Gp-IV follicles. These results were well supported by our observations on immunocytochemistry in Gp IV granulosa cell culture (GCC). We also looked into the expression dynamics of an anti-apoptotic factor--proliferating cellular antigen (PCNA) and a pro-apoptotic factor--Bcl-2-associated X protein (BAX) in GCs of Gp IV follicles on treatments with LH, IGF-1, and EGF to evaluate their cytoprotective/anti-apoptotic property. Relative expressions of PCNA and BAX showed a mutually opposite trend with the PCNA expression increasing and BAX expression decreasing with increase in dose and time to reach the zenith (P<0.05) and nadir (P<0.05) at the highest dose(s) at the maximum time duration (72 h) for PCNA and BAX respectively on treatment with all the three factors. Thus, it can be concluded that LH, IGF-1, and EGF treatments have a cytoprotective/anti-apoptotic effect and stimulate VEGF production in granulosa cells of bubaline pre-ovulatory follicles.

Temporal changes in pregnancy-associated glycoproteins across different stages of gestation in the Barbari goat.

The objective of this study was to characterize the temporal profile of pregnancy-associated glycoproteins (PAGs; isoforms 1-11) across different stages of gestation in the Barbari goat. Placentae were collected from local abattoir, classified according to crown rump length of the corresponding foetus into five groups (0-30, 31-60, 61-90, 91-120, and 121-150 days of gestation), and used for relative quantification of mRNA expression by Pfaffl method. In addition, adult female goats (pregnant, n = 7; non-pregnant, n = 5) were used to estimate weekly plasma PAG and progesterone (P4) concentrations. The relative mRNA expression of PAGs was greater (p<0.05) during 31-60 days of gestation, which correlated well with the temporal changes in plasma PAG concentrations. Relative expression of PAGs decreased steadily as gestation advanced with minimum expression observed just before parturition, except for PAG-4 and PAG-8 that showed constantly higher expression throughout pregnancy. Plasma PAG and P4 concentrations showed a distinct temporal pattern with a significant increase beginning at 2 weeks and return to basal levels by 20 weeks of gestation. However, PAG concentrations reached a peak earlier in gestation (8 weeks) than P4 (10-14 weeks). Correlation analysis indicated a strong positive association (r = 0.748, p<0.01) between plasma PAG and P4 concentrations. In conclusion, results of this study indicate a distinct temporal pattern of PAG expression and secretion during gestation in the Barbari goat. The temporal changes in PAGs and the positive association with P4 are suggestive of their role in maintenance of pregnancy and progressive foetal development.

Expression and localization of vascular endothelial growth factor and its receptors in the corpus luteum during oestrous cycle in water buffaloes (Bubalus bubalis).

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid-luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non-angiogenic function.

Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis).

The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17ß (E2) in follicular fluid (FF): Group I (small), 4-6mm diameter, E2>0.5ng/ml of FF; Group II (medium), 7-9mm, E2=0.5-5ng/ml; Group III (large), 10-13mm, E2=5-40ng/ml; Group IV(pre-ovulatory), >13mm, E2>180ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P<0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P<0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms.

Expression of leptin and its receptor in corpus luteum during estrous cycle in buffalo (Bubalus bubalis).

Leptin is supposed to play a crucial role in ovarian luteal dynamics. The present study was aimed to investigate the importance of leptin and its receptors in buffalo corpus luteum (CL) obtained from different stages of the estrous cycle. Real-time RT-PCR (qPCR), western blot and immunohistochemistry techniques were applied to investigate mRNA expression, protein expression and localization of examined factors. Additionally to assess the contribution of leptin in progesterone production the expression profiles of StAR, P450scc and HSD were also investigated. In general, we demonstrated presence of leptin and its receptors in buffalo CL during the estrous cycle. The mRNA levels of leptin and its receptors were significantly up regulated in (P<0.05) in all the stages and highest levels were observed in mid and late luteal stages consistent with in vivo luteinization of buffalo CL and declined coincidental to luteal regression. The expression of StAR, P450scc and HSD factors maintained low in early luteal phase, after that level of expression increased steadily to show a significant rise (P<0.05) in mid luteal phase followed by gradual decline in late luteal phase and regressed CL and this correlates well with the Ob and ObR receptor activity, verifying their key role in progesterone and other steroids production in functional CL. As revealed by immunohistochemistry, leptin protein was localized predominantly in large luteal cells however leptin receptor (Ob-R) was localized in large luteal cells as well as in endothelial cells. It can be concluded from our study that leptin via its autocrine/paracrine effects play a significant role in promoting angiogenesis, steroidogenesis and also acts as key survival factor in bubaline CL.