Buffalo Granulosa Cells Research Articles

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.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP), a light for mammalian transcript analysis in low-input laboratories.

Transcript analysis is usually performed by costly, time-consuming, and expertise intensive methods, like real time-PCR, microarray, etc. However, they are not much feasible in low-input laboratories. Therefore, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a means of mammalian transcript analysis. Particularly, RT-LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D-cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19-RT-LAMP assay rapidly identified the LPS-induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19-RT-LAMP assay provided a hope on the application of RT-LAMP for mammalian transcript analysis in low-input laboratories.

Curcumin primed exosomes reverses LPS-induced pro-inflammatory gene expression in buffalo granulosa cells.

Curcumin possesses anti-inflammatory properties and provides a promising treatment for inflammation. The aim of the study is to establish that buffalo granulosa cells when primed with curcumin (20 μM), release improved cellular contents through exosome that can mitigate granulosa cell dysfunction. Recently, we have shown that buffalo granulosa cells exposed to LPS (1 μg/mL) in serum free culture, transiently increased the pro-inflammatory cytokine genes (IL-1β, TNF-α, IL-6) expression followed by the inhibition of CYP19A1 gene expression and estradiol production. Therefore, LPS-treated granulosa cells were used as a model of inflammation and curcumin primed exosomes were utilized to check their potential for reducing granulosa cell dysfunction. Expression level of pro-inflammatory cytokines and CYP19A1 were detected by real time PCR while estradiol levels were measured by ELISA. Exosomes derived from curcumin-treated cells alleviated LPS mediated inflammation. In conclusion, our study potentiates the use of curcumin primed exosomes in mitigating granulosa cell dysfunction. Results show the therapeutic conservatories of curcumin via primed exosomes.

Three-dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage.

Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.

Endotoxin induced TLR4 signaling downregulates CYP19A1 expression through CEBPB in buffalo granulosa cells

Estrogen is essential for growth and development of ovarian follicles. Infections associated with E. coli or Endotoxin (LPS) suppress estradiol production by the downregulation of CYP19A1 expression. However, the molecular mechanism of its down regulation is not yet known. To elucidate the molecular mechanisms of LPS-mediated downregulation of CYP19A1 gene expression, we studied the effect of LPS and TLR4 signaling pathway inhibitor (OxPAPC, OxPAPC-Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) on CYP19A1 expression, and expression of CEBPB and CEBPB binding on CYP19A1 proximal promoter (CYP19A1 PII) in buffalo granulosa cells in vitro. The results showed that LPS (1μg/ml) significantly declined the expression of CYP19A1 gene. In further experiments, inhibitor studies confirmed the involvement of TLR4 in LPS induced CYP19A1 gene down regulation in buffalo granulosa cells. LPS promoted higher levels of CEBPB at cellular and nuclear level in granulosa cells. Chromatin immunoprecipitation results showed, that LPS induces higher amount of CEBPB binding on the CYP19A1 PII. Further, TLR4 inhibitor attenuated the LPS induced implications. In conclusion, our results demonstrated that CEBPB could be a potential regulator for LPS mediated downregulation of CYP19A1 and decline of 17-beta estradiol levels in buffalo granulosa cells.

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) synergistically promote steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E2) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E2 concentration in follicular fluid (FF): Small, 4–6mm diameter, E2<0.5ng/ml; Medium, 7–9mm, E2=0.5–5ng/ml; Large, 10–13mm, E2=5–40ng/ml; Preovulatory (PFs), >14mm, E2>180ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75–80% confluent. Then cultured GCs were treated with FGF2 (200ng/ml) and VEGF-A (100ng/ml) separately and in combination for three incubation periods (24, 48 and 72h). Estradiol secretion was greater in GCs treated with FGF2+VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P<0.05) at 72h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E2 secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P<0.05) was at 72h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs.

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 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.

The gene expression pattern induced by high plating density in cultured bovine and buffalo granulosa cells might be regulated by specific miRNA species.

Precise regulation of cell type-specific gene expression profiles precedes the profound morphological reorganization of somatic cell layers during folliculogenesis, ovulation and luteinization. Cell culture models are essential to the study of corresponding molecular mechanisms of gene regulation. In a recent study, it was shown that an increased cell plating density can largely change gene expression profiles of cultured bovine granulosa cells. In our present study, we comparatively analyzed cell plating density effects on cultured bovine and buffalo granulosa cells. Cells were isolated from small- to medium-sized follicles (2–6 mm) and cultured under serum-free conditions at different plating densities. The abundance of selected marker transcripts and associated miRNA candidates was determined by quantitative real-time RT-PCR. We found in both species that the abundance of CYP19A1, CCNE1 and PCNA transcripts was remarkably lower at a high plating density, whereas VNN2 and RGS2 transcripts significantly increased. In contrast, putative regulators of CYP19A1, miR-378, miR-106a and let-7f were significantly higher in both species or only in buffalo, respectively. Also miR-15a, a regulator of CCNE1, was upregulated in both species. Thus, increased plating density induced similar changes of mRNA and miRNA expression in granulosa cells from buffalo and cattle. From these data, we conclude that specific miRNA species might be involved in the observed density-induced gene regulation.

IGF-1 attenuates LPS induced pro-inflammatory cytokines expression in buffalo (Bubalus bubalis) granulosa cells

Interaction between immune and endocrine system is a diverse process influencing cellular function and homeostasis in animals. Negative energy balance (NEB) during postpartum period in dairy animals usually suppresses these systems resulting in reproductive tract infection and infertility. These negative effects could be due to competition among endocrine and immune signaling pathways for common signaling molecules. The present work studied the effect of IGF-1 (50ng/ml) on LPS (1μg/ml) mediated pro-inflammatory cytokine expression (IL-1β, TNF-α, IL-6) and aromatase (CYP19A1) genes’ expressions as well as proliferation of buffalo granulosa cells. The crosstalk between LPS and IGF-1 was also demonstrated through studying the activities of downstream signaling molecules (ERK1/2, Akt, NF-κB) by western blot and immunostaining. Gene expression analysis showed that IGF-1 significantly reduced the LPS induced expression of IL-1β, TNF-α and IL-6. LPS alone inhibited the CYP19A1 expression. However, co-treatment with IGF-1 reversed the inhibitory effect of LPS on CYP19A1 expression. LPS alone did not affect granulosa cell proliferation, but co-treatment with IGF-1, and IGF-1 alone enhanced the proliferation. Western blot results demonstrated that LPS caused the nuclear translocation of the NF-κB and increased the phosphorylation of ERK1/2 and Akt maximum at 15min and 60min, respectively. Nonetheless, co-treatment with IGF-1 delayed LPS induced phosphorylation of ERK1/2 (peak at 120min), while promoting early Akt phosphorylation (peak at 5min) with no effect on NF-κB translocation. Overall, IGF-1 delayed and reversed the effects of LPS, suggesting that high IGF-1 levels may combat infection during critical periods like NEB in postpartum dairy animals.

Nuclear receptor peroxisomal proliferators activated receptors-gamma ligands decrease histone acetylation and attenuate CYP19 gene expression by chromatin remodeling in buffalo (Bubalis bubalis) granulosa cells

ObjectiveRole of peroxisomal proliferator activated receptors-gamma (PPARγ) in regulating fertility establishes it as novel signal for the integration of energy balance and reproduction. PPARγ ligands are known to down regulate CYP19 gene expression, a candidate gene encoding rate-limiting enzyme aromatase involved in estradiol-17β biosynthesis. It has been well established that CYP19 gene is regulated epigenetically during folliculogenesis and luteinization. In the present study, we investigated if PPARγ ligands epigenetically regulate CYP19 gene. MethodsThe total acetylation of histone H3 (K9/14) and difference in enrichment of acetylated histone H3 (K9/14) on CYP19 gene promoter were analyzed by western analysis and ChIP assay, respectively. ResultsResult showed that acetylated histone H3 (K9/14) is down-regulated in granulosa cells treated with PPARγ ligands, whereas its expression was reversed when cells were treated with PPARγ antagonist. To validate further, analysis of histone modification under basal and treated conditions using a ChIP assay revealed that the CYP19 gene proximal promoter (PII, known to be ovary-specific promoter) was 450 and 550 fold more enriched with acetylated histone H3 (K9/14) in control and antagonist (GW9662) treated cells, respectively, than the ligand treated cells. The present study demonstrated that CYP19 gene proximal promoter (PII) was more accessible to transcription in control than treated cells. ConclusionIn conclusion, the present findings provide a novel mechanistic insight into nuclear receptor PPARγ mediated decrease in acetylated histone H3 (K9/14) which in turn remodel chromatin through histone modification and regulate key steroidogenic gene in buffalo granulosa cells.

Rosiglitazone suppresses FSH and IGF-1 induced downstream PI3K/Akt pathway mediated granulosa cell function involving PPARg and PTEN: Potential targets of fertility regulation

Nuclear receptor peroxisome proliferative activated receptor gamma (PPARg) is a prominent targeted molecule in ovarian syndrome. Rosiglitazone, a potent drug administered during Polycystic ovarian syndrome (PCOS), executes its effects through this receptor. However, the exact mechanism through which Rosiglitazone induces PPARg and mediates its action in ovary is not well documented. In the present study, we delineated the signaling pathway for Rosiglitazone action in granulosa cell functioning by using buffalo granulosa cell model. Experiments were performed in serum free primary buffalo granulosa cell culture when cells were follicle stimulating hormone (FSH) responsive with pre-ovulatory phenotype. The cells were incubated with Rosiglitazone alone or in combination with FSH (25 ng/ml) and IGF-1(50 ng/ml). Results showed that Rosiglitazone (25 µM) inhibited FSH and insulin like growth factor-1 (IGF-1) induced granulosa cell proliferation, aromatase, GATA-4, IGF-1 mRNA and oestradiol-17β production. Western blot analysis of total cell lysates revealed that Rosiglitazone intervene with the FSH and IGF-1 signaling by decreasing p-Akt. In addition, Rosiglitazone was found to up-regulate PPAR g and (PTEN) in granulosa cells. PPARg antagonist (GW9662) had no effect on p-Akt level, as well as on steroidogenesis. PTEN inhibitor bpV(pic) revived Akt phosphorylation to the level of control. In conclusion, the present study demonstrated that Rosiglitazone attenuated FSH and IGF-1 induced downstream PI3K/Akt pathway mediated granulosa cell function ( proliferation and steroidogenesis) involving PPARg and PTEN . The elucidated signaling pathway and participating molecules, PPARg and PTEN, could be potential targets while developing novel molecules in PCOS management and fertility regulation

Conjugated linoleic acids attenuate LPS-induced pro-inflammatory gene expression by inhibiting the NF-κB translocation through PPARγ in buffalo granulosa cells.

In granulosa cells, TLR4-mediated LPS-induced immune response interferes with ovarian granulosa cell function. LPS-induced pro-inflammatory gene expression was monitored by real-time PCR and NF-κB was determined by the immuno-blotting and immuno-staining in granulosa cells in vitro. The LPS (1 μg/mL) increased pro-inflammatory gene expression which was reverted back by treatment with TLR4 signaling inhibitor. Cotreatment of CLA (10 μm) with LPS did not show any effect on LPS-induced pro-inflammatory gene expression but granulosa cells pre-treated with CLA for 24 hr, attenuated LPS-induced pro-inflammatory gene expression and nuclear NF-κB. GW9662, a PPARγ-inhibitor, further increased the expression of pro-inflammatory genes. The present findings reiterated that pre-treatment with CLA can prevent LPS-induced granulosa cells dysfunction.

Influence of gonadotropins on steroidogenesis by large follicle buffalo granulosa cells under in vitro serum-free conditions

The study was made to examine the effects of FSH and LH on estradiol–17β and progesterone production by large follicle derived buffalo granulosa cells cultured under completely serum-free conditions. Granulosa cells (3×105) from large (&gt;9 mm diameter) follicles were cultured for 24 h in 48-well plates coated with 3.3μg/cm2 fibronectin in DMEM: Nutrient mixture F-12 Ham (1:1 ratio) supplemented with 10-7 M androstenedione, 5 mg/ml human apo-transferrin and 0.1% BSA, in the presence or absence of FSH or LH (1 to 64 ng/ml each). FSH had no effect on estradiol-17β output but at 4 and 8 ng/ml increased progesterone secretion by granulosa cells. All doses of LH tested had stimulatory effect on estradiol-17β while progesterone secretion by granulosa cells was unaffected.

Conjugated linoleic acids attenuate FSH- and IGF1-stimulated cell proliferation; IGF1, GATA4, and aromatase expression; and estradiol-17β production in buffalo granulosa cells involving PPARγ, PTEN, and PI3K/Akt

Conjugated linoleic acid (CLA) has drawn much interest in last two decades in the area ranging from anticancer activity to obesity. A number of research papers have been published recently with regard to CLA's additional biological functions as reproductive benefits. However, not much is known how this mixture of isomeric compounds mediates its beneficial effects particularly on fertility. In this study, we demonstrated the cross talk between downstream signaling of CLA and important hormone regulators of endocrine system, i.e. FSH and IGF1, on buffalo granulosa cell function (proliferation and steroidogenesis). Experiments were performed in primary serum-free buffalo granulosa cell culture, where cells were incubated with CLA in combination with FSH (25 ng/ml) and IGF1 (50 ng/ml). Results showed that 10 μM CLA inhibits FSH- and IGF1-induced granulosa cell proliferation; aromatase, GATA4, and IGF1 mRNA; and estradiol-17β production. Western blot analysis of total cell lysates revealed that CLA intervenes the IGF1 signaling by decreasing p-Akt. In addition, CLA was found to upregulate peroxisome proliferator-activated receptor-gamma (PPARG) and phosphatase and tensin homolog (PTEN) level in granulosa cells. Further study using PPARG- and PTEN-specific inhibitors supports the potential role of CLA in granulosa cell proliferation and steroidogenesis involving PPARG, PTEN, and PI3K/Akt pathway.

Ovary-specific novel peroxisome proliferator activated receptors-gamma transcripts in buffalo

In the present study, we describe the isolation and characterization of the transcripts encoding peroxisome proliferator-activated receptor gamma (PPARγ1 and PPARγ2) in buffalo ovary. 5′ RACE experiments and sequence analysis showed that these transcripts (PPARγ1a, PPARγ1b and PPARγ2) were transcribed by the different promoter usage and alternative splicing of terminal 5′-exon. The distribution of these isoforms of PPARγ transcripts in different tissues (ovary, mammary gland, spleen, liver, lung, adipose tissue) was investigated using quantitative real time analysis. Tissue- and transcript-specific expression analyses showed that a transcript, transcribed from distal promoter, not only expressed preferentially in ovary but contributes predominantly to PPAR gamma expression in ovary. Western blot analysis of both, in vivo and in vitro, experiments also supported that PPARγ1 predominantly expressed in ovary. In buffalo granulosa cells culture, the isolated transcripts were found to be up-regulated by both natural (CLA) and synthetic (Rosiglitazone) ligands and effect was reversed by PPARγ antagonist GW9662. In conclusion, the present study identified an ovary-specific novel transcript, transcribed by distal promoter, predominantly expressed in ovary which could have functional relevance in buffalo ovary.

Validation of Gene Silencing Using RNA Interference in Buffalo Granulosa Cells

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes. (Key Words : RNAi, Transfection, CYP19 Gene, 17β- Estradiol, Buffalo, Transcript Abundance)

Characterization of serum-free buffalo granulosa cell culture and analysis of genes involved in terminal differentiation from FSH- to LH-responsive phenotype

In the present study, buffalo granulosa cells were cultured under serum-free conditions and characterized to study the changes in gene expression associated with the transition of granulosa cells from estrogen- to progesterone-secreting phenotype. The cells were cultured in vitro under completely serum-free conditions for 8 d. Gene expression and hormone analysis showed that on day 4 granulosa cells exhibit FSH responsiveness with preovulatory phenotype having highest CYP19 gene expression and 17β-estradiol production, whereas a significant increase in transcript abundance of STAR, CYP11, and HSD3B genes accompanied with an increase in progesterone production was observed on day 8. Cells treated with LH on day 4 followed by gene expression analysis at 1, 2, 4, 6, 18, and 24 h showed significant increase in transcripts of LH-responsive genes. In conclusion, culture condition used in the present study showed that granulosa cells were FSH responsive and attained attributes of granulosa cells of dominant follicles at day 4 with highest CYP19 and LHR gene expression beyond which they acquired the ability to luteinize and thus were more LH responsive. In addition, after LH treatment, analysis of early LH-responsive genes ( EGR2, RUNX1, and NR4A1) on day 4 showed that granulosa cells at this stage in culture exhibits phenotype similar to that of preovulatory follicles before LH surge in vivo and corresponds to the in vivo transition of well-orchestrated gene expression profile after LH surge. The characterized culture conditions represent a suitable in vitro model for analysis of genes involved in terminal differentiation of granulosa cells from FSH- to LH-responsive phenotype during folliculogenesis in buffalo.

Influence of Nitric Oxide on Steroid Synthesis, Growth and Apoptosis of Buffalo (Bubalus bubalis) Granulosa Cells In vitro

Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor on steroid synthesis, growth and apoptosis of buffalo granulosa cells (GCs) in vitro. Follicular fluid of antral follicles (3-5 mm diameter) was aspirated and GCs were cultured in 0 (control), , , , of SNP for 48 h. To evaluate whether this effect was reversible, GCs were cultured in presence of SNP+1.0 mM -nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, ) as NO scavenger. Nitrate/nitrite concentration was evaluated by Griess method, progesterone and estradiol concentrations by RIA and apoptosis by TUNEL assay. SNP (, , ) significantly (p SNP induced the progesterone synthesis and stimulated GCs to develop into a uniform monolayer. Combination of SNP M+L-NAME strengthened the inhibitory effect while, SNP+Hb together reversed these inhibitory effects. In conclusion, SNP at greater concentrations (, and ) has a cytotoxic effect and it may lead to cell death whereas, at a lower concentration () induced progesterone synthesis and growth of GCs. These findings have important implications that NOS derived NO are involved at physiological level during growth and development of buffalo GCs which regulates the steroidogenesis, growth and apoptosis.

Molecular Cloning and Characterization of cDNA Encoding Buffalo (Bubalus bubalis) Cytochrome P450 Aromatase in the Ovary

One of the predominant causes of poor reproduction in buffaloes is low levels of ovarian estrogens. A rate limiting enzyme in estrogen biosynthesis is cytochrome P450 aromatase (P450 AROM), the product of CYP19 gene. In the present study CYP19 cDNA was cloned and its 5′UTR was characterized by 5′RACE in granulosa cells of large follicles. CYP19 transcripts with four different 5′UTRs (206, 114, 90 and 3 bases) were found in buffalo granulosa cells of large ovarian follicles. Interestingly, a predominant aromatase transcript with short 5′UTR (3 nucleotides) was found. Further studies are required to understand the relevance of these transcripts and their translational efficiency in granulosa cells of large follicles during folliculogenesis of buffalo ovary.