Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates

2006 ◽  
Vol 18 (5) ◽  
pp. 501 ◽  
Author(s):  
M. C. J. Quinn ◽  
S. B. McGregor ◽  
J. L. Stanton ◽  
P. A. Hessian ◽  
W. R. Gillett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cell ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Blood Cells ◽  
Cell Aggregates ◽  
Percoll Gradient ◽  
Human Granulosa Cells ◽  
Selection Of

Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.

scholarly journals Two pathways for prostaglandin F2α synthesis by the primate periovulatory follicle

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Brandy L Dozier ◽  
Kikuko Watanabe ◽  
Diane M Duffy
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Cell Conversion ◽  
Gonadotropin Surge ◽  
Cell Expression

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2α levels in primate periovulatory follicles. To better understand the role of PGF2α in ovulation, pathways utilized for PGF2α synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34–36 h after hCG) from in vitro fertilization patients. PGF2α can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24–36 h after hCG administration. Human granulosa cells converted PGD2 into 11β-PGF2α, confirming that these cells possess AKR1C3 activity. PGF2α can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0–12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [3H]PGE2 into [3H]PGF2α was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2α can be synthesized via two pathways during the periovulatory interval.

Regulatory effect of vasopressin and its analogs on the steroidogenesis of human granulosa cells (GC) in vitro

1989 ◽  
Vol 120 (3_Suppl) ◽  
pp. S183-S185
Author(s):  
H. MUELLER ◽  
T. RABE ◽  
B. HAUFF ◽  
L. KIESEL ◽  
B. RUNNEBAUM
Keyword(s):  
Granulosa Cells ◽  
Regulatory Effect ◽  
Human Granulosa Cells

scholarly journals A rapid and robust method for the cryopreservation of human granulosa cells

2021 ◽  
Author(s):  
Sarah Beschta ◽  
Katja Eubler ◽  
Nancy Bohne ◽  
Ignasi Forne ◽  
Dieter Berg ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Large Scale ◽  
Ovarian Function ◽  
Oocyte Retrieval ◽  
Cell Contact ◽  
Cellular Model ◽  
Preovulatory Follicle ◽  
Human Granulosa Cells ◽  
Ovarian Cells

AbstractHuman primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

scholarly journals The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jennifer L Juengel ◽  
Lisa J Haydon ◽  
Brigitta Mester ◽  
Brian P Thomson ◽  
Michael Beaumont ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
Antral Follicle ◽  
Brushtail Possum ◽  
Follicular Growth ◽  
Theca Cells ◽  
Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

scholarly journals AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 73-80 ◽  
Author(s):  
JongYeob Choi ◽  
MinWha Jo ◽  
EunYoung Lee ◽  
DooSeok Choi
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Apoptotic Cell ◽  
Follicular Development ◽  
Negative Regulator ◽  
Metabolic Clearance ◽  
Akt Inhibitor ◽  
Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

P–229 Oxidative stress and Metformin; an in-vitro study on serum and primary human granulosa cell cultures

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Alam ◽  
R Rehman ◽  
N Farooqui ◽  
F Jehan ◽  
S H Abidi
Keyword(s):  
Oxidative Stress ◽  
Sample Size ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Metformin Treatment ◽  
Serum Samples ◽  
Human Granulosa Cells ◽  
Human Granulosa Cell ◽  
Infertile Patients

Abstract Study question What is the effect of administration of Metformin on the oxidative stress (OS) levels in serum and primary human granulosa cell cultures of infertile females? Summary answer Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment. What is known already Oxidative stress (OS) is a resultant of mitochondrial dysfunction when it either fails to fight against the oxidants or the expression of the antioxidants is not sufficient. Cellular damage including DNA damage is a common resultant of oxidative stress. OS effects the oocyte maturation and moreover, the cleavage phase in the early embryonic stage. The raised levels of OS makers are hypothesized to compromise the nuclear maturation and the mitotic spindles of the maturing oocytes. Metformin seemed to decrease oxidative stress and improve insulin resistance, dyslipidaemia and endothelial dysfunction in PCOS patients Study design, size, duration This cross-sectional study was conducted from August 2017 – July 2019, at Aga Khan Hospital in collaboration with Australian Concept Infertility Medical Centre (ACIMC) on ten infertile patients undergoing egg retrieval after ethical approval from of Aga Khan Hospital (AKU-ERC–2018–0557–601). Participants/materials, setting, methods Serum samples were obtained and analysed. Follicular fluid of these subjects was collected for establishment of primary cell culture model of normal human granulosa cells (hGCs). Serum and hGC cultures were grouped as; a) control: treatment, b) Test1: H2O2 induced OS, and c) Test2: H2O2 induced OS treated with metformin. OS was estimated in all groups by Mishra method. The two Test groups were assessed for SIRT1 levels using quantitative PCR employing SIRT1 specific primers Main results and the role of chance With mean age of 32.04 ± 2.29 years the mean BMI was 27.61 ± 2.15 kg/m2. OS was induced and measured by an increase in optical density (OD) in hGC Test samples which showed 0.28 (0.16–0.40) OD when compared with control hGC samples 0.153 (0.09–0.23). There was a significant reduction in ODs after metformin treatment in the stress induced cells 0.182 (0.05–0.30). A similar pattern was observed in the serum samples in ODs; control: 0.105 (0.09–0.15), stress induced samples: 0.199 (0.19–0.20). and stress induced serum sample with metformin treatment: 0.1415 (0.06–0.18). The Ct values obtained to express the effect of metformin on SIRT1 levels, for OS induced (Test1) and OS induced metformin treated (Test2) cells were found to be 29.12 and 26.42, respectively. We also observed a significant (85%) difference in the fold change of SIRT1 expression between metformin treated and untreated cells. Limitations, reasons for caution Small sample size is the limitation of this study. The impact of metformin on cell cultures due to different causes of infertility could not be ascertained Wider implications of the findings: Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment, therefore, metformin may be considered as a treatment of oxidative stress in infertile patients. Randomized control trial with large sample size is recommended to confirm the cause and effect relationship. Trial registration number Not applicable

scholarly journals MiRNA-143 mediates the proliferative signaling pathway of FSH and regulates estradiol production

2017 ◽  
Vol 234 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Li Zhang ◽  
XiaoXin Zhang ◽  
Xuejing Zhang ◽  
Yu Lu ◽  
Lei Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Loss Of Function ◽  
Cellular Processes ◽  
Cell Functions ◽  
Genes Expression ◽  
Underlying Mechanisms

MicroRNAs (MiRNAs) play important regulatory roles in many cellular processes. MiR-143 is highly enriched in the mouse ovary, but its roles and underlying mechanisms are not well understood. In the current study, we show that miR-143 is located in granulosa cells of primary, secondary and antral follicles. To explore the specific functions of miR-143, we transfected miR-143 inhibitor into primary cultured granulosa cells to study the loss of function of miR-143 and the results showed that miR-143 silencing significantly increased estradiol production and steroidogenesis-related gene expression. Moreover, our in vivo and in vitro studies showed that follicular stimulating hormone (FSH) significantly decreased miR-143 expression. This function of miR-143 is accomplished by its binding to the 3’-UTR of KRAS mRNA. Furthermore, our results demonstrated that miR-143 acts as a negative regulating molecule mediating the signaling pathway of FSH and affecting estradiol production by targeting KRAS. MiR-143 also negatively acts in regulating granulosa cells proliferation and cell cycle-related genes expression. These findings indicate that miR-143 plays vital roles in FSH-induced estradiol production and granulosa cell proliferation, providing a novel mechanism that involves miRNA in regulating granulosa cell functions.

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