EFFECT OF Murraya koenigii AND MELATONIN ON MODULATION OF BUFFALO GRANULOSA CELL FUNCTION IN HEAT STRESS CONDITION
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Date
2022
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ICAR-NDRI, KARNAL
Abstract
This study aimed to establish the buffalo granulosa cells (GCs) culture model mimicking the intra-follicular environment in order to access the effect of heat stress on cell viability and functional characteristics of buffalo GCs and its amelioration by treatment of melatonin and Murraya koengii plant extracts. Buffalo GCs isolated from small antral follicles were cultured for 6 days in 3D-like by using gelatine pre-coated plates and a 3D culture model by using the hanging drop (HD) method. Three different cells seeding densities (2, 3, and 4X105/mL) were compared and found that seeding density of 4X105/mL was optimum to develop GCs of preovulatory phenotype after 96 hrs of culture based on morphology and functional attributes (CYP19, FSHR, RUNX, and PCNA) in both the culture models. Based on structural and functional characteristics, a 3D culture model using the hanging drop method was selected to access the effect of heat stress by exposing GCs to higher temperatures (40.5˚C & 41.5˚C) and the functional attributes of cells were studied. Exposure to acute heat stress for 2-24 hrs after attaining preovulatory phenotype showed a significant difference in morphology, growth characteristics, and functional attributes. Chronic heat stress exposure for 24-120 hrs after 24 hrs of culture affected the GCs growth and cells didn’t form compact spheroids and started to lose their phenotype earlier (after 72 hrs) than in the control group (120 hrs). Further, heat stress exposure affected the function of GCs by reducing the expression of PCNA, CYP19, FSHR, and RUNX and increasing the expression of HSP70 after 72 hrs. Subsequently, the GCs in the 3D culture model were treated with melatonin and Murraya koenigii plant extracts to study their ameliorating effect against heat stress on GCs functional attributes. Total phenolics (TP), Total flavonoid content (TFC), and reducing power of Murraya koenigii leaves were 459.67±9.1 μg GAE/mg, 788.83±5.49 μg QE/mg, and 16556.81±1445.2 μmol TE/mg in methanolic extract respectively and 417±69.9 μg GAE/mg, 52.21±14. μg QE/mg and 20042.95±1866.32 μmol TE/mg in aqueous extract respectively. Cytotoxicity assay revealed maximum cell proliferation at concentrations of 0.390 mg/mL, 3.125 mg/mL, and 300 ng/mL for methanolic extract, aqueous extract, and melatonin, respectively. Melatonin & plant extracts (aqueous and methanolic) treatment to GCs has shown an ameliorative response against heat stress by the maintenance of their structural & functional phenotype evidenced by significantly higher expression (p<0.05) of PCNA, CYP19, FSHR, antioxidant genes (CAT and SOD2) & reduced expression (p<0.05) of HSP70 & BAX transcript under acute & chronic heat stress condition. The estradiol-17-β (E2) analysis in the spent culture medium revealed reduced basal production of estradiol in heat stress conditions (acute- 6.48 pg/μg of cellular protein; chronic- 6.76 pg/μg of cellular protein) compared to the control (acute-7.52 pg/μg of cellular protein and chronic-8.26 pg/μg of cellular protein) whereas, melatonin treatment improved basal production of estradiol under control (acute-9.06 pg/μg of cellular protein and chronic-8.07 pg/μg of cellular protein) and heat stress condition (acute-8.48 pg/μg of cellular protein and chronic-7.27 pg/μg of cellular protein). It could be concluded that the hanging drop method with a seeding density of 4X105 cells/ mL is one of the best strategies to culture buffalo GCs mimicking the inta-follicular environment. Heat stress exposure to GCs hampers the growth, differentiation, and steroidogenesis which can be ameliorated by the use of Murraya koenigii leaves extracts and melatonin.