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.

Recombinant human gonadotropins stimulate steroid and inhibin production in human granulosa cells

1997 ◽  
Vol 136 (6) ◽  
pp. 617-623 ◽  
Author(s):  
Christina Bergh ◽  
Ulrika Selleskog ◽  
Torbjörn Hillensjö
Keyword(s):  
Granulosa Cells ◽  
Fetal Bovine Serum ◽  
Experimental Studies ◽  
Oocyte Retrieval ◽  
Human Granulosa Cells ◽  
Fetal Bovine ◽  
Recombinant Technology ◽  
Human Ovarian Follicles ◽  
Natural Cycles

Abstract Objective: Recently pure gonadotropins have become available through recombinant technology. In parallel with ongoing clinical trials it is important to examine the effects of these new gonadotropin preparations in experimental studies in human granulosa cells. In the present study the effects of recombinant FSH (rFSH) and LH (rLH) on steroid and inhibin production were examined in human granulosa cells in culture. Patients and methods: Granulosa cells were obtained during the follicular phase of the menstrual cycle in seven women undergoing gynecological laparotomy and from follicles in stimulated cycles in women undergoing oocyte retrieval in connection with in vitro fertilization/embryo transfer. The granulosa cells were cultured in modified Medium 199 containing 1% fetal bovine serum for 4–8 days with and without hormones. Media were changed on alternate days and stored at −20°C until analyzed for estradiol, progesterone and inhibin. Results: Granulosa cells from natural cycles were highly responsive to rFSH which caused a dose-related (rFSH 0·1 to 100 ng/ml) increase in estradiol and progesterone accumulation. The maximal stimulatory effect was reached with a concentration of rFSH between 1 and 10 ng/ml. Granulosa cells from stimulated cycles responded highly to rLH in terms of increased progesterone production during the whole culture period. A maximal stimulatory effect was observed with rLH at a concentration of 0·1 ng/ml. Both types of granulosa cells responded to recombinant gonadotropins in terms of increased inhibin production. Conclusions: The present study demonstrates that granulosa cells from human ovarian follicles are highly responsive to recombinant gonadotropins as demonstrated by increased steroid and inhibin production. European Journal of Endocrinology 136 617–623

scholarly journals Expression of genes involved in the inflammatory response in human granulosa cells in short-term in vitro culture

2020 ◽  
Vol 8 (4) ◽  
pp. 190-195
Author(s):  
Sandra Kałużna ◽  
Rut Bryl ◽  
Błażej Chermuła ◽  
Rafał Sibiak ◽  
Katarzyna Stefańska ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Primary Culture ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Proper Function ◽  
Short Term ◽  
Human Granulosa Cells ◽  
Expression Of Genes ◽  
The Difference

AbstractThe essential function of granulosa cells is to maintain the proper course of oogenesis and folliculogenesis.The immune system is an additional local regulator of ovarian function, with cytokines necessary for the proper function of the ovaries, including the secretion of steroid hormonesThis study aimed to analyze the expression of genes in human GCs in short-term primary culture and define the difference in the expression of IL1β, IL6, and TNFα genes at 48h and 72h of culture compared to the 24h control. Total RNA was isolated using the Chomczyński and Sacchi protocol. RNA samples were treated with DNase I and reverse transcribed (RT) into cDNA. The determination of transcript levels of the mentioned genes was performed using the Light Cycler® 96 Real-Time PCR kit, Roche Diagnostics GmbH (Mannheim, Germany).The present study proved that granulosa cells in a short-term primary in vitro culture express IL-1β, IL-6, and TNFα. The tested genes show a decrease in expression at 24h of culture and a subsequent slight increase at 72h, not exceeding the initial levels. The expression changes the most for IL1β and the least for TNFα.The fluctuations in the amount of transcript may be influenced by factors stored in granulosa cells before the IVM procedure, the procedure of in vitro fertilization, as well as factors related to the process of primary culture. More research is needed to understand the details of these occurrences.Running title: The inflammatory response in human granulosa cells

DIRECT GONADOTROPHIC EFFECT OF GROWTH HORMONE ON OESTRADIOL PRODUCTION BY HUMAN GRANULOSA CELLS IN VITRO

1990 ◽  
Vol 126 (3) ◽  
pp. R1-R4 ◽  
Author(s):  
H D Mason ◽  
H Martikainen ◽  
R W Beard ◽  
V Anyaoku ◽  
S Franks
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Direct Action ◽  
Ovarian Tissue ◽  
Physiological Role ◽  
Fold Increase ◽  
Polycystic Ovaries ◽  
Human Granulosa Cells ◽  
Oestradiol Production

ABSTRACT In-vitro studies in both rodents and man suggest that GH can stimulate ovarian steroidogenesis, but it is not clear whether this effect is mediated by changes in circulating concentrations of insulin-like growth factor-1 (IGF-1) or whether it is a direct action on the ovary (or, indeed, both). In this study the effects of biosynthetic human GH (hGH) on the production of oestradiol and IGF-1 by human granulosa cells in culture were examined using ovarian tissue (from both normal and polycystic ovaries) which had not previously been exposed to exogenous gonadotrophin therapy. Addition of hGH (1 or 10ng/ml) to the incubation medium resulted in a significant (1.7 to 3.6 fold) increase in oestradiol accumulation after 48h in culture. Human GH also had a significant additive effect on the dose-related responsiveness of granulosa cell oestradiol production to hFSH. Concentrations of IGF-1 in the medium were undetectable in each of these experiments. These studies demonstrate that hGH has a potent, direct stimulatory effect on production of oestradiol by the human ovary which is independent of the effect of FSH. These findings have important implications for understanding the physiological role of hGH in human ovarian function as well as for therapeutic use of biosynthetic hGH for induction of ovulation.

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

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Absence of leptin expression and secretion by human luteinized granulosa cells

2003 ◽  
Vol 31 (1) ◽  
pp. 233-239 ◽  
Author(s):  
M Karamouti ◽  
P Kollia ◽  
E Karligiotou ◽  
A Kallitsaris ◽  
N Prapas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Messenger Rna ◽  
Limit Of Detection ◽  
Culture Media ◽  
Point Of View ◽  
In Vitro Fertilisation ◽  
Ob Gene ◽  
Human Granulosa Cells

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

Relationships between concentrations of steroids, igf-1 and igf binding proteins during follicular development in the ewe

1995 ◽  
Vol 1995 ◽  
pp. 56-56
Author(s):  
M. Khalid ◽  
W. Haresign
Keyword(s):  
Follicular Fluid ◽  
Biochemical Markers ◽  
Binding Proteins ◽  
Ovarian Function ◽  
Follicular Development ◽  
Physiological Status ◽  
Igf Binding Proteins ◽  
Ovarian Cells ◽  
Igf Binding

Insulin-like growth factor-1 (IGF-1) is one of the potential autocrine/paracrine regulators of ovarian function. Not only do relationships exist between follicular fluid concentrations of IGF-1 and various biochemical markers of follicular differentiation, but IGF-1 has also been shown to stimulate both proliferation and steroidogenesis in ovarian cells in vitro (Adashi et al., 1985). The actions of IGF-1 are thought to be modulated by IGF-binding proteins (IGFBPs). Indeed, follicular growth and atresia in the ewe have been reported to be determined more by changes in IGFBPs than by changes in IGF-1 (Monget et al., 1993). However, in mat particular study, stage of follicular development was determined by follicle size and by microscopic examination of the granulosa cells of individual follicles rather than by biochemical markers of follicle status. The objective of the present study was, therefore, to investigate changes in IGF-1 and IGFBPs levels in follicular fluid and to relate these to the physiological status as determined by steroidogenic content of follicular fluid.

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