scholarly journals Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells

2020 ◽  
Vol 510 ◽  
pp. 110816
Author(s):  
Kati Hensen ◽  
Martin Pook ◽  
Anu Sikut ◽  
Tõnis Org ◽  
Toivo Maimets ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
In Vitro Cultivation ◽  
Serum Free ◽  
Human Granulosa Cells

Effect of noradrenaline and dopamine on progesterone and estradiol secretion of human granulosa cells

1993 ◽  
Vol 129 (2) ◽  
pp. 165-168 ◽  
Author(s):  
József Bódis ◽  
Hans R Tinneberg ◽  
Attila Török ◽  
Philippe Cledon ◽  
Volker Hanf ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Direct Action ◽  
Serum Free Medium ◽  
Free Medium ◽  
Vitro Fertilization ◽  
Serum Free ◽  
Human Granulosa Cells ◽  
Estradiol Secretion ◽  
Cells Cultured

The aim of this study was to explore the direct action of noradrenaline and dopamine on progesterone and estradiol secretion of human granulosa cells cultured in serum-free medium. Progesterone and estradiol production was measured in the presence and absence of noradrenaline, dopamine or propranolol using radioimmunoassays; statistical analysis was performed by analysis of variance and Newman-Keul's multiple range test. Twenty-six women aged 31±3 years undergoing in vitro fertilization and embryo transfer for infertility treatment at University Women's Hospital, University of Tübingen, Germany, took part in this study. Noradrenaline significantly inhibited progesterone production by human granulosa cells in a dose-related manner at a concentration of 10−4–10−6 mol/l. Dopamine significantly stimulated estradiol secretion by granulosa cells in an inverse dose-related manner. Both effects were blocked by propranolol. The results suggest that catecholaminergic actions switch over the steroid production of human granulosa cells cultured in serum-free medium from progesterone to estradiol.

OXYTOCIN BIOSYNTHESIS IN SERUM-FREE CULTURES OF HUMAN GRANULOSA CELLS

1990 ◽  
Vol 124 (2) ◽  
pp. R5-R8 ◽  
Author(s):  
I. plevrakis ◽  
C. Clamagirand ◽  
G. Pontonnier
Keyword(s):  
Granulosa Cells ◽  
High Performance ◽  
Serum Free Medium ◽  
Vitro Fertilization ◽  
Serum Free ◽  
Human Granulosa Cells ◽  
Cell Extracts ◽  
Preovulatory Follicles ◽  
Follicular Puncture

ABSTRACT Human granulosa cells were collected from preovulatory follicles during follicular puncture for in-vitro fertilization. They were cultured in serum-free medium supplemented with ascorbic acid. Using a combination of high-performance liquid chromatography and radioimmunoassay, the oxytocin material present in the cell extracts and secreted into the medium was identified. When cells were deprived of ascorbate, intermediary forms resulting of the post-translational processing of pro-oxytocin/neurophysin were detected. These data demonstrate that oxytocin biosynthesis occurs in human granulosa cells.

scholarly journals Human Ovarian Granulosa Cells Isolated during an IVF Procedure Exhibit Differential Expression of Genes Regulating Cell Division and Mitotic Spindle Formation

2019 ◽  
Vol 8 (12) ◽  
pp. 2026
Author(s):  
Maciej Brązert ◽  
Wiesława Kranc ◽  
Błażej Chermuła ◽  
Katarzyna Kowalska ◽  
Maurycy Jankowski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Granulosa Cells ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
In Vitro Cultivation ◽  
Cellular Ultrastructure ◽  
Mitotic Spindle Assembly

Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups (“cell cycle arrest”, “cell cycle process”, “mitotic spindle organization”, “mitotic spindle assembly checkpoint”, “mitotic spindle assembly”, and “mitotic spindle checkpoint”) were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics.

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 effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model

2020 ◽  
Author(s):  
Julia Fernández-Pérez ◽  
Peter W. Madden ◽  
Robert Thomas Brady ◽  
Peter F. Nowlan ◽  
Mark Ahearne
Keyword(s):  
Stromal Cells ◽  
Scar Tissue ◽  
Dendritic Morphology ◽  
Lamellar Keratoplasty ◽  
Human Cornea ◽  
Serum Free ◽  
Anterior Lamellar Keratoplasty ◽  
Potential Alternative

AbstractDecellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 µm thick decellularized lenticule against one that had been recellularized with human stromal cells. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. We conclude that recellularization with keratocytes alone may not be sufficiently beneficial to justify introducing allogeneic cells without concurrent treatment to further manage keratocyte phenotype.

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