Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long‑term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem‑like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P‑value <0.05 and expression higher than two‑fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse‑transcription‑quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long‑term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

• MeSH
• Cell Differentiation * MeSH
• Antigens, Differentiation biosynthesis   MeSH
• Adult MeSH
• Granulosa Cells metabolism   pathology   MeSH
• Humans MeSH
• Adolescent MeSH
• Osteoblasts metabolism   pathology   MeSH
• Gene Expression Regulation * MeSH
• Oligonucleotide Array Sequence Analysis * MeSH
• Gene Expression Profiling * MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Differentiation MeSH

PURPOSE: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15. METHODS: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed. RESULTS: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes. CONCLUSIONS: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.

• Keywords
• Follicular fluid, Follicular granulosa cells, Growth/differentiation factor-15, IVF,
• MeSH
• Cell Differentiation genetics   MeSH
• Adult MeSH
• Fertilization in Vitro MeSH
• Granulosa Cells metabolism   MeSH
• Follicular Fluid metabolism   MeSH
• Humans MeSH
• Oocyte Retrieval MeSH
• Oocytes metabolism   MeSH
• Growth Differentiation Factor 15 genetics   isolation & purification   MeSH
• Gene Expression Regulation, Developmental MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• GDF15 protein, human MeSH Browser
• Growth Differentiation Factor 15 MeSH

PURPOSE: The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs). METHODS: GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors. RESULTS: The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions. CONCLUSION: Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

• MeSH
• Cell Culture Techniques MeSH
• Time Factors MeSH
• Phenotype * MeSH
• Granulosa Cells cytology   MeSH
• Follicular Fluid MeSH
• Karyotyping MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Cell Proliferation * MeSH
• Flow Cytometry MeSH
• Telomere MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH