scholarly journals The Cultivation of Human Granulosa Cells

2008 ◽  
Vol 51 (3) ◽  
pp. 165-172 ◽  
Author(s):  
Lenka Brůčková ◽  
Tomáš Soukup ◽  
Jiří Moos ◽  
Martina Moosová ◽  
Jana Pavelková ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Doubling Time ◽  
Ovarian Follicle ◽  
Vitro Fertilization ◽  
Thecal Cells ◽  
Cell Doubling Time

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals Involvement of miRNAs in equine follicle development

Reproduction ◽  
2013 ◽  
Vol 146 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S N Schauer ◽  
S D Sontakke ◽  
E D Watson ◽  
C L Esteves ◽  
F X Donadeu
Keyword(s):  
Cell Survival ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Follicle Development ◽  
Previous Evidence ◽  
Ovarian Follicle Development ◽  
Fluid Levels ◽  
Follicle Selection

Previous evidence fromin vitrostudies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels ofCYP19A1andLHCGR(P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets,PTEN,RASA1, andSMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

scholarly journals sRAGE Downregulates the VEGF Expression in PCOS Ovarian Granulosa Cells

2020 ◽  
Author(s):  
Jingyan Wang ◽  
Yichun Guan ◽  
Yi Liu ◽  
Liang Wang ◽  
Zhan Zhang ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Rt Pcr ◽  
Vegf Expression ◽  
Vitro Fertilization ◽  
Protein Levels ◽  
Ovarian Granulosa Cells

Abstract Objective High expression of VEGF in ovarian tissue, serum and follicular fluid of PCOS women is involved in the physiological and pathogenesis processes of PCOS. Our objective was to investigate the effect of sRAGE on VEGF expression and EGF-like growth factor in PCOS ovarian granulosa cells.Methods We collected ovarian granulosa cells of PCOS patients who underwent in vitro fertilization (IVF). Then treatment ovarian granulosa cells with different concentrations of sRAGE. Levels of VEGF, AREG, BTC and EREG mRNA were examined by quantitative RT-PCR. The protein levels of VEGF, AREG, BTC and EREG were measured by ELISA.Results Treatment with sRAGE decrease the production of VEGF, and the effects were dependent on the concentrations of sRAGE (P < 0.05). Simultaneously, the expression of the EGF-like growth factors AREG, BTC and EREG were decreased, and the expression were dependent on the concentrations of sRAGE (P < 0.05).Conclusions sRAGE may downregulate VEGF expression in PCOS ovarian granulosa cells,and EGF-like growth factor pathway may be involved in this process.

scholarly journals Higher Follicular Fluid Progesterone Down-regulated HPGD and COX2 in Granulosa Cells via Suppressing NF-кB Signaling in Endometriosis

2021 ◽  
Author(s):  
Jing-Yi Li ◽  
Jian-Peng Chen ◽  
Yu-Li Qian ◽  
Jun-Yan Ma ◽  
Fei-Da Ni ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Chromatin Immunoprecipitation ◽  
Cell Model ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Progesterone ◽  
Vitro Fertilization ◽  
Cox 2

Abstract Background: Luteinized unruptured follicular follicle syndrome (LUFS) is a special type of ovulatory dysfunction and a common cause of infertility. It is estimated that its prevalence is 13% ~ 73% in endometriosis patients. Increasing evidences prove that LUFS is one of the reasons for endometriosis-related infertility. Any alteration in FF components and GCs in endometriosis may influence the developing oocyte and ovulation. This study aimed to explore the effect of local elevated progesterone in follicular fluid (FF) on ovulation in endometriosis patients.Methods: A Prospective study with matched pairs design was conducted at a reproductive medicine center between July 2017 and January 2018 in patients undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection treatment (ICSI), while granulosa tumor-like cell line KGN (Bena culture collection, China) was used as in vitro cell model. Alterations in follicular and peritoneal fluid (PF) components identified with metabolomics analyses; Differentially expressed genes in GCs identified with transcriptome analysis; Polymerase chain reaction (PCR), western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were used to determine the expression of progesterone, NF-кB related genes, HPGD and COX-2; NF-кB binding identified with chromatin immunoprecipitation (ChIP).Results: Patients with endometriosis exhibited a significantly higher basal serum progesterone level, higher serum level of progesterone on trigger day and higher progesterone expression level in FF and PF. GCs from endometriosis patients revealed decreased expression of HPGD, COX-2 and suppressed NF-кB signaling, as manifested by decreased expressions of IL1R1 and IRAK3. Similarly, progesterone treatment in vitro down-regulated HPGD and COX2 expression and suppressed NF-кB signaling in KGN cells in a dose dependent manner, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIкBα/IкBα ratio and nucleus translocation of p65. TNF-α, by contrast, increased expression of IL1R1, IRAK3, pIкBα, p65 and HPGD in KGN cells. Furthermore, one potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation.Conclusion: Endometriosis showed repression of NF-кB pathways and down-regulation of HPGD and COX2, which play important roles in the process of ovulation by participating in the metabolism of prostaglandin E2 (PGE2), in granulosa cells (GCs) due to elevated progesterone in FF.

Estradiol-17β and androgen secretion by isolated porcine ovarian follicular cells in vitro

1982 ◽  
Vol 60 (8) ◽  
pp. 1112-1118 ◽  
Author(s):  
B. K. Tsang ◽  
Y. S. Moon ◽  
D. T. Armstrong
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Adenosine Monophosphate ◽  
Defined Medium ◽  
Medium Size ◽  
Prostaglandin E ◽  
Thecal Cells ◽  
Androgen Secretion ◽  
Estradiol 17Β

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4–6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17β (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH, 0.25 μg/mL) or luteinizing hormone (LH, 1 μg/mL). Addition of testosterone or androstenedione (0.5 μM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3′,5′-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.

scholarly journals LRH-1 High Expression in the Ovarian Granulosa Cells of PCOS Patients

2021 ◽  
Author(s):  
Xiao Yang ◽  
Qiumin Wang ◽  
Ying Wang ◽  
Tian Song ◽  
Yanjun Zheng ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Polycystic Ovary ◽  
Pearson Correlation ◽  
Statistical Significance ◽  
Control Group ◽  
Vitro Fertilization ◽  
Real Time Quantitative Pcr ◽  
Ovarian Granulosa Cells

Abstract Purpose: Polycystic ovary syndrome (PCOS) is considered as one of the most common endocrine disorder with heterogeneity. There are also reports that liver receptor homolog 1 [LRH-1 or nuclear receptor subfamily 5 group A member 2 (NR5A2)] plays an important role in the reproductive system. But up to now, there are no reports related to the link with PCOS and LRH-1. In this study, we aimed to detect the LRH-1 expression in the ovarian granulosa cell of PCOS patients and explore the potential relationship between LRH-1 and PCOS. Methods: 146 follicular fluid sample were collected in this study, including 72 from PCOS patients and 74 from control patients who underwent intracytoplasmic sperm injection (ICSI) or in vitro fertilization-embryo transfer (IVF-ET). The ovarian granulosa cells were extracted from the patient's follicular fluid by magnetic-activated cell sorting (MACS) method, and the real-time quantitative PCR (qRT-PCR) was used to measure the expression of LRH-1 in ovarian granulosa cells. Then we analyzed the correlation between the expression level of LRH-1 and the clinical characteristics of patient by using Pearson Correlation analysis. Results: The expression of LRH-1 was significantly higher in PCOS patients ovarian granulosa cells than that in the control patients [vs(1.38±0.47)vs(1.03±0.32), t=5.327, p<0.0001], and it was positively correlated with antral follicles counting (AFC) (r=0.3607, p<0.0001)and the serum AMH(r=0.2662, p=0.0012)\LH(r=0.2518, p=0.0022)\T(r=0.2516, p=0.0022) in all patients. No statistical significance between LRH-1 and BMI, FSH, HOMA-IR, DHE-S, progesterone. Conclusions: Compared with the control group, we found that LRH-1 was highly expressed in the ovarian granulosa cells of PCOS patients. Our study has revealed the relationship between the LRH-1 expression and PCOS, which suggested that LRH-1 may play an important role in ovulation disorders. While this finding provided new ideas for the study of the pathogenesis, it also provided a theoretical basis for the clinical diagnosis and treatment for PCOS.

Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage

2015 ◽  
Vol 104 (4) ◽  
pp. 1037-1046.e1 ◽  
Author(s):  
Juan Manuel Moreno ◽  
María José Núñez ◽  
Alicia Quiñonero ◽  
Sebastian Martínez ◽  
Marina de la Orden ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Maturation Stage ◽  
Vitro Fertilization

scholarly journals Evidence for the Presence of Angiogenin in Human Follicular Fluid and the Up-Regulation of Its Production by Human Chorionic Gonadotropin and Hypoxia*

2000 ◽  
Vol 85 (9) ◽  
pp. 3352-3355 ◽  
Author(s):  
Kaori Koga ◽  
Yutaka Osuga ◽  
Osamu Tsutsumi ◽  
Mikio Momoeda ◽  
Akihiko Suenaga ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Angiogenic Factor ◽  
Regulatory Mechanisms ◽  
Human Ovary ◽  
Human Follicular Fluid ◽  
Potent Inducer ◽  
Vitro Fertilization

Abstract Angiogenesis is an essential event during the development of the ovarian follicle and ensuing formation of the corpus luteum. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human ovary. Follicular fluid (FF) and granulosa cells (GCs) were collected from women undergoing in vitro fertilization and embryo transfer. The presence of angiogenin in FF and GCs was demonstrated by Western blot analysis. The production of angiogenin by cultured GCs was stimulated with the addition of human CG or cAMP or under the hypoxic milieu. Concentrations of angiogenin in FF from an individual follicle were positively correlated with those of progesterone, but not estradiol and testosterone. Given the presence of angiogenin in FF and up-regulation of its production by human CG and hypoxia, it seems logical to assume that angiogenin may play a role as a local angiogenic factor in the human ovary.

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