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Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

ABSTRACT: The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

SUBMITTER: Kulus M

PROVIDER: S-EPMC7299926 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

Kulus Magdalena M Kranc Wiesława W Sujka-Kordowska Patrycja P Celichowski Piotr P Konwerska Aneta A Jankowski Maurycy M Jeseta Michal M Skowroński Mariusz T MT Skowroński Mariusz T MT Piotrowska-Kempisty Hanna H Bukowska Dorota D Zabel Maciej M Bruska Małgorzata M Mozdziak Paul P Kempisty Bartosz B Antosik Paweł P

Histochemistry and cell biology 20200310 6

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycl ...[more]

PMID: 32157392

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Project description:The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.

Project description:Study questionCan cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?Summary answerUnder gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.What is known alreadyStudies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.Study design, size, durationCultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.Participants/materials, setting, methodsMurine and human follicles were cultured in alginate hydrogel and hormone production [17β-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.Main results and the role of chanceWhen grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.Limitations, reasons for cautionThis was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.Wider implications of the findingsThe eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.Study funding/competing interestsThis work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Project description:Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

Dataset Information

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

Publications

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

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