New gene markers involved in regulation of granulosa cells development and differentiation towards endodermal and epithelial tissues – a new insight into the stemness specificity of ovarian follicular cells

Abstract Maintaining of female fertility is strictly dependent on proper hormonal regulation. Granulosa cells (GCs) are components of ovarian follicles, and they are important in paracrine regulation of the ovary. Preovulatory follicle GCs are responsible for production of estrogens to the ovary microenvironment and lead to the LH surge. Proper functioning of GCs is necessary to ensure appropriate conditions for oocyte development, maturation, ovulation and its release to the oviduct. Long-term in vitro culture of GCs show significant stem-like characteristics. Understanding the molecular processes underlying GCs differentiation towards different cell lineages may reveal other possible stem cell markers. A transcriptomic analysis of short-term primary in vitro cultured GCs, which were isolated from porcine preovulatory follicles was the major focus of the study. The ontological groups herby considered are associated with endodermal and epithelial tissues. Results were and compare to freshly isolated GC cells. 6 the most reduced expression: HSD17B1, DAPL1, NEBL, MAL2, DAB1, ITM2A were chosen for analysis. These genes have been response for processes associated with GCs development and differentiation towards endodermal and epithelial tissues, which make them important for further consideration.

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Muscle Cell Morphogenesis, Structure, Development and Differentiation Processes Are Significantly Regulated during Human Ovarian Granulosa Cells In Vitro Cultivation

Journal of Clinical Medicine ◽

10.3390/jcm9062006 ◽

2020 ◽

Vol 9 (6) ◽

pp. 2006

Author(s):

Claudia Dompe ◽

Wiesława Kranc ◽

Karol Jopek ◽

Katarzyna Kowalska ◽

Sylwia Ciesiółka ◽

...

Keyword(s):

Growth Factor ◽

Muscle Cell ◽

Granulosa Cells ◽

Tissue Growth ◽

Cell Morphogenesis ◽

Structure Development ◽

Ovarian Granulosa Cells ◽

Development And Differentiation

Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, “muscle cell development”, “muscle cell differentiation”, “muscle contraction”, “muscle organ development”, “muscle organ morphogenesis”, “muscle structure development”, “muscle system process” and “muscle tissue development”. The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.

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Transcriptomic Profile of New Gene Markers Encoding Proteins Responsible for Structure of Porcine Ovarian Granulosa Cells

Biology ◽

10.3390/biology10111214 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1214

Author(s):

Jakub Kulus ◽

Magdalena Kulus ◽

Wiesława Kranc ◽

Karol Jopek ◽

Maciej Zdun ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Adhesion ◽

Granulosa Cells ◽

Ovarian Follicle ◽

Genetic Material ◽

Intercellular Signaling ◽

Gene Markers ◽

New Gene ◽

Cell Cell

The extracellular matrix (ECM) in granulosa cells is functionally very important, and it is involved in many processes related to ovarian follicle growth and ovulation. The aim of this study was to describe the expression profile of genes within granulosa cells that are associated with extracellular matrix formation, intercellular signaling, and cell–cell fusion. The material for this study was ovaries of sexually mature pigs obtained from a commercial slaughterhouse. Laboratory-derived granulosa cells (GCs) from ovarian follicles were cultured in a primary in vitro culture model. The extracted genetic material (0, 48, 96, and 144 h) were subjected to microarray expression analysis. Among 81 genes, 66 showed increased expression and only 15 showed decreased expression were assigned to 7 gene ontology groups “extracellular matrix binding”, “extracellular matrix structural constituent”, “binding, bridging”, “cadherin binding”, “cell adhesion molecule binding”, “collagen binding” and “cadherin binding involved in cell-cell adhesion”. The 10 genes with the highest expression (POSTN, ITGA2, FN1, LAMB1, ITGB3, CHI3L1, PCOLCE2, CAV1, DCN, COL14A1) and 10 of the most down-regulated (SPP1, IRS1, CNTLN, TMPO, PAICS, ANK2, ADAM23, ABI3BP, DNAJB1, IGF1) were selected for further analysis. The results were validated by RT-qPCR. The current results may serve as preliminary data for further analyses using in vitro granulosa cell cultures in assisted reproduction technologies, studies of pathological processes in the ovary as well as in the use of the stemness potential of GCs.

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Hormonal regulation of apoptosis in rabbit granulosa cells in vitro: evaluation by flow cytometric detection of plasma membrane phosphatidylserine externalization

Reproduction ◽

10.1530/rep.0.1230243 ◽

2002 ◽

pp. 243-251 ◽

Cited By ~ 16

Author(s):

G Maillet ◽

E Breard ◽

A Benhaim ◽

P Leymarie ◽

C Feral

Keyword(s):

Granulosa Cells ◽

Hormonal Regulation ◽

Apoptotic Cell ◽

Bivariate Analysis ◽

Dibutyryl Camp ◽

Isolated Cells ◽

Cellular Models ◽

Preovulatory Follicles ◽

Apoptotic Effect

Annexin V and propidium iodide bivariate analysis and the TUNEL method were used to quantify hormonal regulation of apoptosis in rabbit granulosa cells from preovulatory follicles in vitro. The aim of this study was to analyse comparatively the effects of gonadotrophins and their second messenger in the regulation of granulosa cell apoptosis in (i) cultured isolated granulosa cells and (ii) granulosa cells scraped from cultured follicles. The results showed that increasing doses of FSH had no effect on apoptosis of cultured isolated cells but caused a decrease in the number of apoptotic granulosa cells from preovulatory follicles cultured in serum-free conditions. Unlike FSH, addition of hCG did not modify apoptosis of granulosa cells significantly. In contrast, dibutyryl cAMP had an apoptotic effect in the two cellular models in the presence of serum. Moreover, a biphasic effect of dibutyryl cAMP in isolated granulosa cells was observed with an increase in the incorporation of [(3)H]thymidine into DNA at the lowest dose and an increase in apoptotic cell death at the highest dose. It was concluded that, in rabbits: (i) FSH requires follicle integrity to exert its anti-apoptotic effect in granulosa cells; (ii) dibutyryl cAMP induces a dose-dependent apoptotic effect in granulosa cells cultured alone or obtained from cultured preovulatory follicles; and (iii) cAMP signals induce opposite effects on growth and apoptosis in granulosa cells.

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The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

Reproductive Sciences ◽

10.1007/s43032-021-00691-3 ◽

2021 ◽

Author(s):

Er-Meng Gao ◽

Bongkoch Turathum ◽

Ling Wang ◽

Di Zhang ◽

Yu-Bing Liu ◽

...

Keyword(s):

Oocyte Maturation ◽

Granulosa Cells ◽

Quantitative Polymerase Chain Reaction ◽

Cumulus Cells ◽

Cholesterol Transport ◽

Vitro Fertilization ◽

Expression Of Genes ◽

Preovulatory Follicles ◽

Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

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Effects of Silver Nanoparticles on Proliferation and Apoptosis in Granulosa Cells of Chicken Preovulatory Follicles: An In Vitro Study

Animals ◽

10.3390/ani11061652 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1652

Author(s):

Dorota Katarzyńska-Banasik ◽

Anna Kozubek ◽

Małgorzata Grzesiak ◽

Andrzej Sechman

Keyword(s):

Silver Nanoparticles ◽

Granulosa Cells ◽

Caspase 3 ◽

In Vitro Study ◽

Poultry Production ◽

Cell Death Pathways ◽

Preovulatory Follicles ◽

Proliferation And Apoptosis ◽

Apoptosis Process

The continuous development of poultry production related to the growing demand for eggs and chicken meat makes it necessary to use modern technologies. An answer to this demand may be the use of nanotechnology in poultry farming. One of the promising nanomaterials in this field are silver nanoparticles (AgNPs), which are used as disinfectants, reducing microbial pollution and the amounts of greenhouse gases released. This study aimed to evaluate the effect of AgNPs on the proliferation and apoptosis process in the granulosa cells of chicken preovulatory follicles. The in vitro culture experiment revealed that both 13 nm and 50 nm AgNPs inhibited the proliferation of the granulosa cells. However, a faster action was observed in 50 nm AgNPs than in 13 nm ones. A size-dependent effect of AgNP was also demonstrated for the caspase-3 activity. AgNPs 13 nm in size increased the caspase-3 activity in granulosa cells, while 50 nm AgNPs did not exert an effect, which may indicate the induction of distinct cell death pathways by AgNPs. In conclusion, our study reveals that AgNPs in vitro inhibit granulosa cell proliferation and stimulate their apoptosis. These results suggest that AgNPs may disrupt the final stage of preovulatory follicle maturation and ovulation.

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Gonadotropin-Induced Expression of Messenger Ribonucleic Acid for Cyclooxygenase-2 and Production of Prostaglandins E and F2α in Bovine Preovulatory Follicles Are Regulated by the Progesterone Receptor

Endocrinology ◽

10.1210/en.2005-1575 ◽

2006 ◽

Vol 147 (10) ◽

pp. 4713-4722 ◽

Cited By ~ 43

Author(s):

P. J. Bridges ◽

C. M. Komar ◽

J. E. Fortune

Keyword(s):

Granulosa Cells ◽

Medroxyprogesterone Acetate ◽

Cyclooxygenase 2 ◽

Induced Expression ◽

Messenger Ribonucleic Acid ◽

Preovulatory Follicles ◽

Gonadotropin Surge ◽

Induced Changes

Follicular production of prostaglandins (PGs) is essential for ovulation, but the factors mediating gonadotropin-induced secretion of PGE and PGF2α remain largely unknown. We tested the hypothesis that gonadotropin-induced changes in progesterone and its receptor (PR) mediate the increase in periovulatory PGs. Heifers were treated with PGF2α and GnRH to induce luteolysis and the LH/FSH surge (ovulation occurs ∼30 h after GnRH). Because there are two increases in intrafollicular progesterone/PR mRNA during the bovine periovulatory period, we first examined the temporal pattern of PG production by follicles collected at 0, 3.5, 6, 12, 18, and 24 h after GnRH. Although PGs did not increase in the follicular fluid until 24 h after GnRH, acute secretion of PGs by follicle wall (theca + granulosa cells) was initiated by 18 h and had increased manyfold by 24 h after GnRH. In vitro, FSH and LH induced dramatic transient increases in PG production by follicle wall and granulosa, but not theca, cells isolated from preovulatory follicles (0 h after GnRH). PG accumulation peaked on d 2 of culture, mimicking the secretion pattern after a gonadotropin surge in vivo. In cultures of follicle wall and granulosa cells, the PR antagonist mifepristone (MIFE, 1 μm) inhibited LH-induced PG secretion and the progestin medroxyprogesterone acetate (1 or 10 μm), but not the glucocorticoid dexamethasone (1 or 10 μm), overcame the effect of MIFE on PGs. Semiquantitative RT-PCR revealed that MIFE inhibited LH-induced expression of cyclooxygenase-2 mRNA in granulosa cells in vitro. Again, treatment with medroxyprogesterone acetate overcame the effect of MIFE. Together these results provide strong evidence that periovulatory increases in cyclooxygenase-2 mRNA, PGE, and PGF2α are mediated by gonadotropin-induced increases in progesterone/PR, indicating that in some species there is an important functional relationship between these pathways in the ovulatory cascade.

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Tokishakuyakusan Stimulates Progesterone and Estradiol-17 β Production by Rat Granulosa Cells and Progesterone, Testosterone and Estradiol-17 β by the Residual Portion of the Follicle in Vitro

The American Journal of Chinese Medicine ◽

10.1142/s0192415x91000223 ◽

1991 ◽

Vol 19 (02) ◽

pp. 155-161 ◽

Cited By ~ 4

Author(s):

Satoshi Usuki

Keyword(s):

Granulosa Cells ◽

Cell Level ◽

Testosterone Production ◽

Progesterone Production ◽

Preovulatory Follicles ◽

Immature Rats ◽

Serum Gonadotropin ◽

Tissue Compartments ◽

Stimulation Of

To examine the possible effects of Tokishakuyakusan (TS) on steroidogenesis by preovulatory follicles at the cell level, the expressed granulosa cells and remaining portion of follicles from pregnant mare's serum gonadotropin (PMS)-treated immature rats were incubated in vitro with increasing concentrations of TS for 3 h. TS significantly stimulated progesterone and estradiol-17 b production, with a predominant stimulation of progesterone, by the expressed granulosa cells, while testosterone production was not stimulated. In the remaining portion of the follicle, TS also significantly stimulated progesterone, testosterone and estradiol-17 b production. Similar to the effect produced by granulosa cells, the stimulatory effect of TS was stronger on progesterone than on testosterone and estradiol-17 b production. These results suggest that TS has a potent, direct stimulatory effect on steroidogenesis, especially progesterone production, by constituent tissue compartments of rat preovulatory follicles in vitro.

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Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Acta Endocrinologica ◽

10.1530/acta.0.1100401 ◽

1985 ◽

Vol 110 (3) ◽

pp. 401-407 ◽

Cited By ~ 13

Author(s):

T. Hillensjö ◽

A. Sjögren ◽

B. Strander ◽

L. Nilsson ◽

M. Wikland ◽

...

Keyword(s):

Granulosa Cells ◽

Maximal Effect ◽

Tubal Infertility ◽

Vitro Fertilization ◽

Follicular Maturation ◽

Progesterone Secretion ◽

Preovulatory Follicles ◽

Ultrasound Guided Puncture

Abstract. Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34–36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6–8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2–6. The β-adrenergic agonist isoproteronol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.

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Oocyte-secreted factros regulate granulosa cell steroidogenesis

Zygote ◽

10.1017/s0967199400003324 ◽

1996 ◽

Vol 4 (04) ◽

pp. 317-321 ◽

Cited By ~ 12

Author(s):

Barbara C. Vanderhyden

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Granulosa Cell ◽

Granulosa Cells ◽

Follicular Fluid ◽

Follicular Development ◽

Inhibitory Factor ◽

Preovulatory Follicles ◽

Loss Of Contact

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.

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Coenzyme and cofactor metabolism belongs to biochemical processes significantly regulated in human granulosa cells collected after IVF during long-term primary in vitro culture

Medical Journal of Cell Biology ◽

10.2478/acb-2019-0021 ◽

2019 ◽

Vol 7 (4) ◽

pp. 152-160

Author(s):

Mariusz J. Nawrocki ◽

Rafał Sibiak ◽

Maciej Brązert ◽

Piotr Celichowski ◽

Leszek Pawelczyk ◽

...

Keyword(s):

Granulosa Cells ◽

Metabolic Process ◽

Enzymatic Reactions ◽

Biochemical Processes ◽

Affymetrix Microarrays ◽

Enzymatic Regulation ◽

Cofactor Metabolism ◽

Gene Ontology Biological Process

AbstractGranulosa cells (GCs) provide the microenvironment necessary for the development of the follicle and the maturation of the oocyte. GCs are associated with reproductive system function and the maintenance of pregnancy by participating in the synthesis of steroid hormones. Many authors point to new ways of using GCs in regenerative medicine and indicate the significant plasticity of this cell population, suggesting that GCs can undergo a transdifferentiation process. Employing primary in vitro cell cultures and high-throughput transcriptome analysis via Affymetrix microarrays, this study describes groups of genes associated with enzymatic reactions. 52 genes were identified belonging to four gene ontology biological process terms (GO BP): “coenzyme biosynthetic process”, “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process”. All identified genes showed reduction in the level of mRNA expression during long-term in vitro cultivation. Significanthe transcriptomic profile variability was exhibited for the genes (ELOVL5, ELOVL6 and GPAM) involved in enzymatic regulation of fatty acid metabolism.Running title: Enzymatic regulation in granulosa cells

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