V.P. Yadav

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-17β (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.

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.

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.

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 3β-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.

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.

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 3β-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.

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.