Development of a novel protocol for isolation and purification of human granulosa cells

Chilvers, Rebecca A.; Bodenburg, Yvonne H.; Denner, Larry; Urban, Randall J.

doi:10.1007/s10815-012-9739-5

Cited by 23 publications

(13 citation statements)

References 32 publications

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“…Normal granulosa cell function is therefore essential for oocyte development [ 42 ]. The in vitro culture model of pGCs purified from human follicles is widely used in reproductive research [ 43 , 44 ], but the viability and status of pGCs also gradually decreased with longer culture time; thus, it is a potential aging cell model. We found that TF3 could delay granulosa cell apoptosis in vitro and significantly reduce ROS-induced apoptosis and oxidative stress injury in the H 2 O 2 -induced oxidative stress model.…”

Section: Discussionmentioning

confidence: 99%

“…Normal granulosa cell function is therefore essential for oocyte development [42]. The in vitro culture model of pGCs purified from human follicles is widely used in reproductive research [43,44], but the viability and status of pGCs also gradually decreased with longer culture time; Figure 6: TF3 may affect granulosa cell function by regulating autophagy through the mTOR pathway. pGCs were routinely cultured for 24 h and then starved in a serum-free culture medium for 24 h. TF3 (10 μM) or control treatment was given for 6 h. Cells were collected for transcriptome sequencing, and differentially expressed genes were subjected to GO analysis related to cell biological processes (a) and MCODE analysis related to signaling pathways (b).…”

Section: Discussionmentioning

confidence: 99%

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Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Yao

et al. 2021

Oxidative Medicine and Cellular Longevity

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Ovarian aging refers to the gradual decline of ovarian function with increasing physiological age, manifested as decreased ovarian reserve, elevated aging-related markers, and reduced oocyte quality. With a declining female fertility and a growing aging population, it is urgent to delay ovarian aging to maintain fertility and improve the life quality of women. Theaflavin 3, 3 ′ -digallate (TF3) is a naturally bioactive polyphenol compound extracted from black tea, and its antioxidant properties play an important role in maintaining human health and delaying aging; however, the effects of TF3 on female reproduction and ovarian function are not yet clear. Here, we show that TF3 can preserve primordial follicle pool, partially restore the estrous cycle, and increase the offspring number of aged mice. Meanwhile, TF3 gavage increased the number of oocytes retrieved, decreased the level of reactive oxygen species, increased the level of glutathione, and decreased the abnormal rate of oocyte spindle after ovulation induction. Moreover, TF3 inhibited human granulosa cell apoptosis and improved their antioxidative stress ability. High-throughput sequencing and small-molecule-targeted pharmacological prediction show that TF3 affects multiple pathways and gene expression levels, mainly involved in reproductive and developmental processes. It may also affect cellular function by targeting mTOR to regulate the autophagic pathway, thereby delaying the process of ovarian aging. This study shows that TF3 can be used as a potential dietary supplement to protect ovary function from aging and thereby improving the life quality of advanced-age women.

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Section: Discussionmentioning

confidence: 99%

“…Normal granulosa cell function is therefore essential for oocyte development [42]. The in vitro culture model of pGCs purified from human follicles is widely used in reproductive research [43,44], but the viability and status of pGCs also gradually decreased with longer culture time; Figure 6: TF3 may affect granulosa cell function by regulating autophagy through the mTOR pathway. pGCs were routinely cultured for 24 h and then starved in a serum-free culture medium for 24 h. TF3 (10 μM) or control treatment was given for 6 h. Cells were collected for transcriptome sequencing, and differentially expressed genes were subjected to GO analysis related to cell biological processes (a) and MCODE analysis related to signaling pathways (b).…”

Section: Discussionmentioning

confidence: 99%

Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Yao

et al. 2021

Oxidative Medicine and Cellular Longevity

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“…After removing the supernatant, samples were resuspended in phosphate-buffered saline (PBS). To isolate GCs from FF, PBS-resuspended samples were run through a 50% Percoll gradient (Sigma, St. Louis, USA), followed by centrifugation at 1000 g for 20 min at 4 °C as previously described [19, 20], with minor modifications. GCs were collected from the second layer of the liquid.…”

Section: Methodsmentioning

confidence: 99%

Circular RNA expression profiling of granulosa cells in women of reproductive age with polycystic ovary syndrome

Zhang

Liu

Lai

et al. 2019

Arch Gynecol Obstet

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Purpose This study aimed to explore the expression profiles of circRNA in granulosa cells of women of reproductive age with polycystic ovary syndrome (PCOS). Methods Total RNA was isolated from granulosa cells of 15 women with PCOS and 15 body mass index- and age-matched healthy women (control). RNA sequencing was conducted on ribosomal-depleted RNA for circRNA expression profiling. The differential expression of circRNA between women with PCOS and controls was compared and visualized using hierarchical clustering heat maps and Volcano plots. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to determine the role of the differential expression of circRNAs. The expression rates of circRNAs were confirmed using quantitative real-time PCR (qRT-PCR) using divergent primers. Results A total of 4258 and 7395 candidate circRNAs were predicted in PCOS and controls, respectively, based on the RNA-sequencing data. Differences were noted in the expression patterns of circRNA between the two groups. Analysis of the expression profiles revealed that four circRNAs were upregulated, whereas 23 were downregulated in the women with PCOS. GO analysis suggested that the 27 differentially expressed circRNAs were mainly distributed in biological process pathways, particularly in pathways involving inflammation, proliferation, and the vascular endothelial growth factor-related signaling pathway. Six circRNAs were identified in PCOS-affected women using divergent primers. qRT-PCR confirmed that hsa_circ_0001577 was significantly upregulated and hsa_circ_0020093 was downregulated in the women with PCOS. Conclusions Several circRNAs were differentially expressed in women of reproductive age with PCOS, suggesting the involvement of these circRNAs in the development of PCOS and the potential clinical implications of their use as PCOS biomarkers. Electronic supplementary material The online version of this article (10.1007/s00404-019-05129-5) contains supplementary material, which is available to authorized users.

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“…Granulosa cells were isolated from both the follicular fluid aspirate and the subsequent flush of the same follicle, as previously described (Chilvers, Bodenburg, Denner, & Urban, ; Quinn et al, ). Samples were centrifuged at 2,000 g for 5 min at 4°C and the supernatant (follicular fluid or flush media) was removed.…”

Section: Methodsmentioning

confidence: 99%

Expression of granulosa cell microRNAs, AVEN and ATRX are associated with human blastocyst development

O’Doherty

O’Brien

Browne

et al. 2018

Molecular Reproduction Devel

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A greater understanding of the key molecules associated with embryo development during human-assisted reproduction is imperative for the development of advanced diagnostics. Previous studies have shown that follicular microRNAs (miRNAs) are reliable markers of the polycystic ovarian syndrome (PCOS). Leveraging the utility of miRNAs in PCOS, the aim of this study was to identify miRNAs in human granulosa cells that may be indicative of blastocyst development. Granulosa cells and oocytes were collected from the first follicle aspirated from patients undergoing oocyte retrieval for in vitro fertilization or intracytoplasmic sperm injection. The development of isolated oocytes was recorded, and granulosa cell samples in this study were separated as follows. Group 1-BLAST: granulosa cells from follicles containing an oocyte that fertilized and developed into a blastocyst, and Group 2-FERT: granulosa cells from oocytes that fertilized but failed to reach blastocyst. A panel of 84 miRNAs, related to development and cellular differentiation, was assessed between the two groups using a miScript PCR array. Fourteen miRNAs and one snoRNA were differentially expressed between the groups. In addition, two downstream candidate protein biomarkers, ATRX and AVEN, were also found to be differentially expressed between the groups. The findings of this pilot study reveal follicular abnormalities on a molecular level, which may affect oocyte competence and its potential to develop successfully as an embryo. We encourage additional studies to confirm and expand on our findings and to determine the usefulness of granulosa-borne miRNAs, ATRX, and AVEN as biomarkers.

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Development of a novel protocol for isolation and purification of human granulosa cells

Abstract: A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.

Cited by 23 publications

References 32 publications

Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Circular RNA expression profiling of granulosa cells in women of reproductive age with polycystic ovary syndrome

Expression of granulosa cell microRNAs, AVEN and ATRX are associated with human blastocyst development

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Development of a novel protocol for isolation and purification of human granulosa cells

Abstract: A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.

Cited by 23 publications

References 32 publications

Theaflavin 3, 3 ′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Theaflavin 3, 3 ′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Circular RNA expression profiling of granulosa cells in women of reproductive age with polycystic ovary syndrome

Expression of granulosa cell microRNAs, AVEN and ATRX are associated with human blastocyst development

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Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Theaflavin 3, 3 ^′‐Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function