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.

Department of Anatomy Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland

Department of Histology and Embryology Poznan University of Medical Sciences 6 Święcickiego St 60 781 Poznań Poland

Department of Histology and Embryology Wroclaw Medical University Chałubińskiego St 50 368 Wrocław Poland

Department of Obstetrics and Gynecology University Hospital and Masaryk University 20 Jihlavská St 625 00 Brno Czech Republic

Department of Toxicology Poznan University of Medical Sciences 30 Dojazd St 60 631 Poznań Poland

Division of Anatomy and Histology University of Zielona Gora 28 Zyty St 65 046 Zielona Góra Poland

Division of Infertility and Reproductive Endocrinology Department of Gynecology Obstetrics and Gynecological Oncology Poznan University of Medical Sciences 33 Polna St 60 535 Poznań Poland

Physiology Graduate Program North Carolina State University Campus Box 7608 Raleigh NC 27695 7608 USA

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Chermuła B., Brązert M., Iżycki D., Ciesiółka S., Kranc W., Celichowski P., Ożegowska K., Nawrocki M.J., Jankowski M., Jeseta M., et al. New Gene Markers of Angiogenesis and Blood Vessels Development in Porcine Ovarian Granulosa Cells during Short-Term Primary Culture In Vitro. Biomed Res. Int. 2019;2019:6545210. doi: 10.1155/2019/6545210. PubMed DOI PMC

Rybska M., Knap S., Jankowski M., Jeseta M., Bukowska D., Antosik P., Nowicki M., Zabel M., Kempisty B., Jaśkowski J.M. Characteristic of factors influencing the proper course of folliculogenesis in mammals. Med. J. Cell Biol. 2018;6:33–38. doi: 10.2478/acb-2018-0006. DOI

Rybska M., Knap S., Jankowski M., Jeseta M., Bukowska D. Cytoplasmic and nuclear maturation of oocytes in mammals–living in the shadow of cells developmental capability. Med. J. Cell Biol. 2018;1 doi: 10.2478/acb-2018-0003. DOI

Kossowska-Tomaszczuk K., De Geyter C. Cells with stem cell characteristics in somatic compartments of the ovary. Biomed Res. Int. 2013;2013:310859. doi: 10.1155/2013/310859. PubMed DOI PMC

Kossowska-Tomaszczuk K., Pelczar P., Güven S., Kowalski J., Volpi E., De Geyter C., Scherberich A. A novel three-dimensional culture system allows prolonged culture of functional human granulosa cells and mimics the ovarian environment. Tissue Eng. Part A. 2010;16:2063–2073. doi: 10.1089/ten.tea.2009.0684. PubMed DOI

Kossowska-Tomaszczuk K., De Geyter C., De Geyter M., Martin I., Holzgreve W., Scherberich A., Zhang H. The Multipotency of Luteinizing Granulosa Cells Collected from Mature Ovarian Follicles. Stem Cells. 2009;27:210–219. doi: 10.1634/stemcells.2008-0233. PubMed DOI

Moncrieff L., Mozdziak P., Jeseta M., Machatkova M., Kranc W., Kempisty B. Ovarian follicular cells–living in the shadow of stemness cellular competence. Med. J. Cell Biol. 2019;7:134–140. doi: 10.2478/acb-2019-0018. DOI

Kranc W., Brązert M., Budna J., Celichowski P., Bryja A., Nawrocki M.J., Ożegowska K., Jankowski M., Chermuła B., Dyszkiewicz-Konwińska M., et al. Genes responsible for proliferation, differentiation, and junction adhesion are significantly up-regulated in human ovarian granulosa cells during a long-term primary in vitro culture. Histochem. Cell Biol. 2019;151:125–143. doi: 10.1007/s00418-018-1750-1. PubMed DOI PMC

Bryja A., Dyszkiewicz-Konwińska M., Jankowski M., Celichowski P., Stefańska K., Chamier-Gliszczyńska A., Popis M., Mehr K., Bukowska D., Antosik P., et al. Ion homeostasis and transport are regulated by genes differentially expressed in porcine buccal pouch mucosal cells during long-term culture in vitro-a microarray approach. Bryja al. Med. J. Cell Biol. 2018 doi: 10.2478/acb-2018-0013. DOI

Nawrocki M.J., Celichowski P., Jankowski M., Kranc W., Bryja A., Borys-Wójcik S., Jeseta M., Antosik P., Bukowska D., Bruska M., et al. Ontology groups representing angiogenesis and blood vessels development are highly up-regulated during porcine oviductal epithelial cells long-term real-time proliferation—A a primary cell culture approach. Med. J. Cell Biol. 2018;6:186–194. doi: 10.2478/acb-2018-0029. DOI

Chermuła B., Brązert M., Jeseta M., Ożegowska K., Kocherova I., Jankowski M., Celichowski P., Sujka-Kordowska P., Konwerska A., Piotrowska-Kempisty H., et al. Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence—A Transcriptomic Study. Int. J. Mol. Sci. 2019;20:2238. doi: 10.3390/ijms20092238. PubMed DOI PMC

Budna J., Bryja A., Celichowski P., Kranc W., Ciesiółka S., Borys S., Rybska M., Kolecka-Bednarczyk A., Jeseta M., Bukowska D., et al. “Bone Development” Is an Ontology Group Upregulated in Porcine Oocytes before In Vitro Maturation: A Microarray Approach. DNA Cell Biol. 2017;36:638–646. doi: 10.1089/dna.2017.3677. PubMed DOI

D’Aurora M., Sperduti S., Di Emidio G., Stuppia L., Artini P.G., Gatta V. Inside the granulosa transcriptome. Gynecol. Endocrinol. 2016;32:951–956. doi: 10.1080/09513590.2016.1223288. PubMed DOI

Ferraretti A.P., La Marca A., Fauser B.C.J.M., Tarlatzis B., Nargund G., Gianaroli L. ESHRE working group on Poor Ovarian Response Definition ESHRE consensus on the definition of “poor response” to ovarian stimulation for in vitro fertilization: The Bologna criteria. Hum. Reprod. 2011;26:1616–1624. doi: 10.1093/humrep/der092. PubMed DOI

Kranc W., Budna J., Kahan R., Chachuła A., Bryja A., Ciesiółka S., Borys S., Antosik M.P., Bukowska D., Brussow K.P., et al. Molecular basis of growth, proliferation, and differentiation of mammalian follicular granulosa cells. J. Biol. Regul. Homeost. Agents. 2017;31:1–8. PubMed

Kranc W., Brązert M., Celichowski P., Bryja A., Nawrocki M., Ożegowska K., Jankowski M., Jeseta M., Pawelczyk L., Bręborowicz A., et al. ‘Heart development and morphogenesis’ is a novel pathway for human ovarian granulosa cell differentiation during long‑term in vitro cultivation—A microarray approach. Mol. Med. Rep. 2019;19:1705–1715. doi: 10.3892/mmr.2019.9837. PubMed DOI PMC

Brązert M., Iżycki D., Kranc W., Borowiec B., Popis M., Ożegowska K., Bręborowicz A., Rachoń D., Nowicki M., Kempisty B. Genes involved in hormone metabolism and cellular response in human ovarian granulosa cells. J. Biol. Regul. Homeost. Agents. 2019;33:461–468. PubMed

Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–159. doi: 10.1016/0003-2697(87)90021-2. PubMed DOI

Kranc W., Brązert M., Ożegowska K., Nawrocki M.M.J., Budna J., Celichowski P., Dyszkiewicz-Konwińska M., Jankowski M., Jeseta M., Pawelczyk L., et al. Expression Profile of Genes Regulating Steroid Biosynthesis and Metabolism in Human Ovarian Granulosa Cells—A Primary Culture Approach. Int. J. Mol. Sci. 2017;18:2673. doi: 10.3390/ijms18122673. PubMed DOI PMC

Budna J., Celichowski P., Knap S., Jankowski M., Magas M., Nawrocki M.J., Ramlau P., Nowicki A., Rojewska M., Chermuła B., et al. Fatty Acids Related Genes Expression Undergo Substantial Changes in Porcine Oviductal Epithelial Cells During Long-Term Primary Culture. Med. J. Cell Biol. 2018;6:39–47. doi: 10.2478/acb-2018-0008. DOI

Kranc W., Brązert M., Ożegowska K., Budna-Tukan J., Celichowski P., Jankowski M., Bryja A., Nawrocki M.J., Popis M., Jeseta M., et al. Response to abiotic and organic substances stimulation belongs to ontologic groups significantly up-regulated in porcine immature oocytes. Med. J. Cell Biol. 2018;6:91–100. doi: 10.2478/acb-2018-0015. DOI

Stefańska K., Chamier-Gliszczyńska A., Jankowski M., Celichowski P., Kulus M., Rojewska M., Antosik P., Bukowska D., Bruska M., Nowicki M., et al. Epithelium morphogenesis and oviduct development are regulated by significant increase of expression of genes after long-term in vitro primary culture—A microarray assays. Med. J. Cell Biol. 2018;6:195–204. doi: 10.2478/acb-2018-0030. DOI

Stefańska K., Kocherova I., Knap S., Kulus M., Celichowski P., Jeseta M. The genes regulating maintenance of cellular protein location are differentially expressed in porcine epithelial oviductal cells during longterm in vitro cultivation. Med. J. Cell Biol. 2019;7:77–85. doi: 10.2478/acb-2019-0010. DOI

Huang D.W., Sherman B.T., Tan Q., Kir J., Liu D., Bryant D., Guo Y., Stephens R., Baseler M.W., Lane H.C., et al. DAVID Bioinformatics Resources: Expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res. 2007;35:W169–W175. doi: 10.1093/nar/gkm415. PubMed DOI PMC

Von Mering C., Jensen L.J., Snel B., Hooper S.D., Krupp M., Foglierini M., Jouffre N., Huynen M.A., Bork P. STRING: Known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res. 2004;33:D433–D437. doi: 10.1093/nar/gki005. PubMed DOI PMC

Walter W., Sánchez-Cabo F., Ricote M. GOplot: An R package for visually combining expression data with functional analysis. Bioinformatics. 2015;31:2912–2914. doi: 10.1093/bioinformatics/btv300. PubMed DOI

Chamier-Gliszczyńska A., Brązert M., Sujka-Kordowska P., Popis M., Ożegowska K., Stefańska K., Kocherova I., Celichowski P., Kulus M., Bukowska D., et al. Genes involved in angiogenesis and circulatory system development are differentially expressed in porcine epithelial oviductal cells during long-term primary in vitro culture—A transcriptomic study. Med. J. Cell Biol. 2018;6:163–173. doi: 10.2478/acb-2018-0026. DOI

Kocherova I., Kulus M., Dompe C., Antosik P., Bukowska D. Biochemical properties of cofactor and coenzyme metabolism in porcine oviductal epithelial cells —A microarray study. 2019 doi: 10.2478/acb-2019-0017. DOI

Morey J.S., Ryan J.C., Van Dolah F.M. Microarray validation: Factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol. Proced. Online. 2006;8:175–193. doi: 10.1251/bpo126. PubMed DOI PMC

Thompson W.E., Branch A., Whittaker J.A., Lyn D., Zilberstein M., Mayo K.E., Thomas K. Characterization of Prohibitin in a Newly Established Rat Ovarian Granulosa Cell Line. Endocrinology. 2001;142:4076–4085. doi: 10.1210/endo.142.9.8354. PubMed DOI

Rotmensch S., Dor J., Furman A., Rudak M.E., Mashiach S., Amsterdam A. Ultrastructural characterization of human granulosa cells in stimulated cycles: Correlation with oocyte fertilizability. Fertil. Steril. 1986;45:671–679. doi: 10.1016/S0015-0282(16)49340-4. PubMed DOI

Nottola S.A., Heyn R., Camboni A., Correr S., Macchiarelli G. Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization. Microsc. Res. Tech. 2006;69:508–516. doi: 10.1002/jemt.20309. PubMed DOI

Archangelo L.F., Greif P.A., Maucuer A., Manceau V., Koneru N., Bigarella C.L., Niemann F., dos Santos M.T., Kobarg J., Bohlander S.K., et al. The CATS (FAM64A) protein is a substrate of the Kinase Interacting Stathmin (KIS) Biochim. Biophys. Acta Mol. Cell Res. 2013;1833:1269–1279. doi: 10.1016/j.bbamcr.2013.02.004. PubMed DOI

Zhang C., Han Y., Huang H., Min L., Qu L., Shou C. Integrated analysis of expression profiling data identifies three genes in correlation with poor prognosis of triple-negative breast cancer. Int. J. Oncol. 2014;44:2025–2033. doi: 10.3892/ijo.2014.2352. PubMed DOI

Piekny A.J., Maddox A.S. The myriad roles of Anillin during cytokinesis. Semin. Cell Dev. Biol. 2010;21:881–891. doi: 10.1016/j.semcdb.2010.08.002. PubMed DOI

Wang D., Chadha G.K., Feygin A., Ivanov A.I. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells. Cell. Mol. Life Sci. 2015;72:3185–3200. doi: 10.1007/s00018-015-1890-6. PubMed DOI PMC

Hall P.A., Todd C.B., Hyland P.L., McDade S.S., Grabsch H., Dattani M., Hillan K.J., Russell S.E.H. The septin-binding protein anillin is overexpressed in diverse human tumors. Clin. Cancer Res. 2005;11:6780–6786. doi: 10.1158/1078-0432.CCR-05-0997. PubMed DOI

Ghisoni E., Maggiorotto F., Borella F., Mittica G., Genta S., Giannone G., Katsaros D., Sciarrillo A., Ferrero A., Sarotto I., et al. TOP2A as marker of response to pegylated lyposomal doxorubicin (PLD) in epithelial ovarian cancers. J. Ovarian Res. 2019;12:17. doi: 10.1186/s13048-019-0492-6. PubMed DOI PMC

Miles G.D., Seiler M., Rodriguez L., Rajagopal G., Bhanot G. Identifying microRNA/mRNA dysregulations in ovarian cancer. BMC Res. Notes. 2012;5:164. doi: 10.1186/1756-0500-5-164. PubMed DOI PMC

Wandji S.A., Gadsby J.E., Barber J.A., Hammond J.M. Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinology. 2000;141:2648–2657. doi: 10.1210/endo.141.7.7576. PubMed DOI

Cheng J.-C., Chang H.-M., Fang L., Sun Y.-P., Leung P.C.K. TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways. PLoS ONE. 2015;10:e0126532. doi: 10.1371/journal.pone.0126532. PubMed DOI PMC

Kalimutho M., Sinha D., Jeffery J., Nones K., Srihari S., Fernando W.C., Duijf P.H., Vennin C., Raninga P., Nanayakkara D., et al. CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer. EMBO Mol. Med. 2018;10:e8566. doi: 10.15252/emmm.201708566. PubMed DOI PMC

Brodská B., Otevřelová P., Holoubek A. Decitabine and SAHA-induced apoptosis is accompanied by survivin downregulation and potentiated by ATRA in p53-deficient cells. Oxid. Med. Cell. Longev. 2014;2014:165303. doi: 10.1155/2014/165303. PubMed DOI PMC

Guo J., Zhang T., Guo Y., Sun T., Li H., Zhang X., Yin H., Cao G., Yin Y., Wang H., et al. Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice. Proc. Natl. Acad. Sci. USA. 2018;115:E5326–E5333. doi: 10.1073/pnas.1800352115. PubMed DOI PMC

Zheng Q., Zhou F., Cui X., Liu M., Li Y., Liu S., Tan J., Yan Q. Novel Serum Biomarkers Detected by Protein Array in Polycystic Ovary Syndrome with Low Progesterone Level. Cell. Physiol. Biochem. 2018;46:2297–2310. doi: 10.1159/000489619. PubMed DOI

Yin C., Liu W., hua, Liu Y., Wang L., Xiao Y. PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/PKA pathway activation. PLoS ONE. 2019;14:e0214606. doi: 10.1371/journal.pone.0214606. PubMed DOI PMC

Kong D., Guan Q., Li G., Xin W., Qi X., Guo Y., Zhao J., Xu J., Sun S., Gao L. Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes. Biochem. Biophys. Res. Commun. 2018;495:587–593. doi: 10.1016/j.bbrc.2017.11.053. PubMed DOI

Gatla H.R., Zou Y., Uddin M.M., Singha B., Bu P., Vancura A., Vancurova I. Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells. J. Biol. Chem. 2017;292:5043–5054. doi: 10.1074/jbc.M116.771014. PubMed DOI PMC

O’Neill R.S., Clark D.V. Partial Functional Diversification of Drosophila melanogaster Septin Genes Sep2 and Sep5. G3 (Bethesda) 2016;6:1947–1957. doi: 10.1534/g3.116.028886. PubMed DOI PMC

Carletti M.Z., Christenson L.K. Rapid effects of LH on gene expression in the mural granulosa cells of mouse periovulatory follicles. Reproduction. 2009;137:843–855. doi: 10.1530/REP-08-0457. PubMed DOI PMC

Jin P., Song Y., Yu G. The Role of Abnormal Methylation of Wnt5a Gene Promoter Regions in Human Epithelial Ovarian Cancer: A Clinical and Experimental Study. Anal. Cell. Pathol. 2018;2018:1–9. doi: 10.1155/2018/6567081. PubMed DOI PMC

Lu B., Shi H. An In-Depth Look at Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT): Clinical Implications from Recent Molecular Findings. J. Cancer. 2019;10:223–237. doi: 10.7150/jca.26978. PubMed DOI PMC