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

Yadav, Monica; Agrawal, Himanshu; Pandey, Mamta; Singh, Dheer; Onteru, Suneel Kumar

doi:10.1002/jcp.25909

Cited by 11 publications

(21 citation statements)

References 18 publications

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“…Three‐dimensional in vitro culture of the buffalo granulosa cells was carried out under serum free conditions. Typical spheroids of granulosa cells were observed in hanging drop method (Figure ) as reported earlier . After 144 h, these spheroids were treated with LPS for 2 h, 6 h, 12 h and, 24 h to study the effect of LPS on CYP19 gene expression.…”

Section: Resultsmentioning

confidence: 90%

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

Pandey

Singh

Onteru

2018

J of Cellular Biochemistry

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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.

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Section: Resultsmentioning

confidence: 90%

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

Pandey

Singh

Onteru

2018

J of Cellular Biochemistry

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“…It is well known that FSH and IGF1 induced steroidogenesis and proliferation of GC is obligatory for ovarian follicle development in many mammals, including cows 25,26 . Therefore supplementation of FSH and IGF1 is universally practiced for GC cultures, especially for attaining an "estradiol active" phenotype [27][28][29][30][31] . In the present study, treatment of GC with physiologically relevant doses of FSH (20 ng/ml) and IGF1 (25 and 50 ng/ml) independently induced the mRNA expression of HIF1A.…”

Section: Discussionmentioning

confidence: 99%

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

Baddela

Sharma

Michaelis

et al. 2020

Sci Rep

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Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A). In the present study, we analyzed the HIF1 driven transcriptional activity in bovine granulosa cells (GC). Treatment of GC with FSH (follicle stimulating hormone) and IGF1 (insulin-like growth factor 1) resulted in the upregulation of HIF1A mRNA expression under normoxia. Immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A in the GC layer of different staged ovarian follicles. Suppression of HIF1 using echinomycin and gene knockdown procedures revealed that HIF1 transcriptionally regulates the genes associated with steroidogenesis (STAR, HSD3B and CYP19A1) and proliferation (CCND2 and PCNA) of GC. Further, our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis. Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles.

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“…Therefore the culture duration for 1 week was somehow long enough to maintain cells and marker gene expression in a higher fold change (Hatzirodos et al, 2017). Furthermore, these data indicated that the cultured GCs did not transform into luteinizing cells, because GCs cultured from all groups still expressed a considerable amount of FSHR and CYP19 mRNAs (Portela et al, 2010;Monga et al, 2011;Yadav et al, 2018). Profiling of differently sized in vitro cultured and in vivo buffalo GCs…”

Section: Discussionmentioning

confidence: 93%

Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart

Sosa

Ibrahim

Mahmoud

et al. 2020

Zygote

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SummaryThis study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

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

Cited by 11 publications

References 18 publications

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

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

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart

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