Effects of Curcumin Supplementation on Viability and Antioxidant Capacity of Buffalo Granulosa Cells under In Vitro Culture Conditions

Ghanem, Nasser; Amin, Ahmed; Saeed, Ahmed; Abdelhamid, Sherif M.; El-Sayed, Ashraf S. A.; Ahmed-Farid, Omar A.; Dessouki, Sherif Mohamed; Faheem, Marwa

doi:10.36380/scil.2020.wvj19

Cited by 1 publication

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“…The collection of GCs was done according to Ghanem et al (2020a). Ovaries were collected from local slaughtered houses in physiological saline supplemented with gentamycin and kept at approximately 37°C.…”

Section: Collection Of Ovaries and Granulosa Cells Preparationmentioning

confidence: 99%

“…The trypsinization of GCs was performed according to Ghanem et al (2020a). The medium was aspirated from each well slowly and the cell layer was washed twice with sterilized phosphate buffer saline (PBS).…”

Section: Granulosa Cells Trypsinizationmentioning

confidence: 99%

“…Cells viability was determined using trypan blue (0.4%) according to Ghanem et al (2020a). A total volume of 10 μl of cell suspension was mixed with 10 μl of trypan blue and incubated at room temperature for 1-2 minutes.…”

Section: Granulosa Cells Viabilitymentioning

confidence: 99%

“…Intracellular reactive oxygen species (ROS) were detected by 6-carboxy-2′, 7′-dichlorodihydro fluorescein diacetate (H2DCFDA; life technologies, California, USA) according to the protocol described by the manufacturer with some modifications according to Ghanem et al (2020a). Granulosa cells from each group were incubated with 985 μl of 15 μl MH2DCFDA mixed with 970 μl PBS at 38.5°C for 45 minutes.…”

Section: Intracellular Reactive Oxygen Species Detectionmentioning

confidence: 99%

“…Mitochondrial activity of buffalo GCs was determined using MitoTracker® Green FM (M7514, life technologies) according to the manufacturer's instructions with some modifications according to Ghanem et al (2020a). The GCs from each group were incubated with 200 µl MitoTracker green dye to 800 µl PBS for 45 minutes, followed by washing with PBS.…”

Section: Mitochondrial Activitymentioning

confidence: 99%

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Transcriptional, Mitochondrial Activity, and Viability of Egyptian Buffalo’s Granulosa Cells In Vitro Cultured under Heat Elevation

Ghanem¹,

Faheem²,

Samy³

et al. 2021

WVJ

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It is documented that heat stress caused impairment on the reproductive performance of dairy animals. However, there are few reports that have focused on the molecular and intracellular responses of in vitro cultured buffalo granulosa cells during heat elevation. The present study was conducted to investigate the effect of heat elevation during in vitro culture of buffalo granulosa cells on their viability, quality, mitochondrial activity, and transcriptional activity. Granulosa cells were harvested after aspiration of cumulus-oocytes complexes that were collected from abattoir ovaries. The granulosa cells were cultured in vitro either at a normal physiological temperature suitable for oocyte maturation and embryo development (38.5°C) or exposed to the elevated temperature of 40.5°C on day 3 of culture (the first two days were for confluence) for two hours of culture then continued at 38.5°C up to day 7 of culture. The viability of granulosa cells was measured using trypan blue and quality was estimated by measuring the level of intracellular reactive oxygen species (ROS) on day 7. Moreover, metabolic activity was performed by measuring the fluorescent intensity of mitochondria. Moreover, transcriptional activity was done by profiling four selected candidate genes using quantitative real-time PCR. The results indicated that the granulosa cells viability rate significantly decreased in the heat stress group (25.1 ± 3.7), compared to the control group (36.6 ± 5.3) on confluence day (day 3). In addition, the viability rate on the last day of culture (day 7) decreased in heat stress, compared to control (83.7 ± 4.5 and 97.4 ± 0.4, respectively). On the other hand, there was a nonsignificant difference in ROS profile between the control (21.7*104 ± 1.3) and the heat-stressed group (15.7 ± 0.7) on day 7 of culture. However, the mitochondrial fluorescent intensity was higher in the control (21.9 ± 1.9) than in the heat-stressed group (15.4 ± 0.8) on day 7 of culture. The expression of cellular defense (HSF1) and apoptosis-inducing gene (P53) were significantly up-regulated in granulosa cells exposed to heat elevation, compared to the control group. On the other hand, the steroidogenesis-regulating gene (StAR) was down-regulated in granulosa cells cultured under heat shock, compared to the control group. In conclusion, heat stress reduced the viability of granulosa cells by inducing the expression of an apoptosis-related gene (P53) and compromised expression of genes regulating the steroid biosynthesis, which resulted in up-regulation of cell defense gene (HSF1) in an attempt to ameliorate the deleterious effect of heat stress on the biological activity of the granulosa cells.

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Section: Collection Of Ovaries and Granulosa Cells Preparationmentioning

confidence: 99%

Section: Granulosa Cells Trypsinizationmentioning

confidence: 99%

Section: Granulosa Cells Viabilitymentioning

confidence: 99%

Section: Intracellular Reactive Oxygen Species Detectionmentioning

confidence: 99%

Section: Mitochondrial Activitymentioning

confidence: 99%

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