New Markers of Human Cumulus Oophorus Cells Cultured in Vitro – Transcriptomic Profile

Medical Journal of Cell Biology 2020 DOI:Brązert 10.2478/acb-2020-0007 et al. Received: 25.02.2020 Accepted: 3.04.2020

New markers of human cumulus oophorus cells cultured in vitro – transcriptomic profile

1 2, Karol Jopek3, Bartosz Kempisty2,3,4,5, Leszek Pawelczyk1

Maciej Brązert , Wiesława Kranc Abstract The presence of CCs around the oocyte after ovulation is one of the key elements contributing to oocyte developmental competence. In the presented study, we used CCs from 12 patients aged 18-40 diagnosed with infertility. After harvesting cells on day 1, 7, 15 and 30 of culture, total RNA was isolated and transcriptomic analysis was performed. The DAVID software indicated the following GO BP terms: “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility”. Of the genes belonging to all ontological groups, the most downregulated were: SLC7A8, DFNB31, COL1A1, CDC42SE1, TGFBR3, HMGB1, with the most upregulated genes being: ANXA3, KIAA1199, HTR2B, VCAM1, DKK1. While many studies focus on attempts to obtain fully competent oocytes, scientists still have difficulty attaining adequate results in vitro. Lack of adequate knowledge often results in low in vitro fertilization efficiency. Therefore, our research focuses on CCs cells, thanks to which the oocyte most likely acquires developmental competence. The main purpose of the study was to identify the potential molecular markers responsible for cell junction organization, migration, differentiation, morphogenesis and motility.

Running title: New markers of human cumulus oophorus cells cultured in vitro

Keywords: cumulus ophorus cells, microarrays, human reproduction

2Division of Infertility and Reproductive Endocrinology, Department of Gynecology, Obstetrics and Gynecological Oncology, Poznań University of Medical3 Sciences, 33 Polna St., 60-535 Poznań, Poland 4Department of Anatomy, Poznań University of Medical Sciences, 6 Ś�więcickiego St., 60-781 Poznań, Poland 5Department of Histology and Embryology, Poznań University of Medical Sciences, 6 Ś�wiecickiego St., 60-781 Poznań, Poland *Department Correspondence: of Obstetrics e-mail: and [email protected] Gynecology, University Hospital and Masaryk University, 20 Jihlavská St., 625 00 Brno, Czech Republic FullDepartment list of author of Veterinary information Surgery, is available Institute at of the Veterinary end of article Medicine, Nicolaus Copernicus University in Toruń, 1 Lwowska St., 87-100 Toruń, Poland 61 Medical Journal of Cell Biology (2020)

Brązert et al. Introduction ment of the mature ovarian follicle, and all process- Cumulus cells (CCs) are differ significantly from es occurring during follicle growth in the ovarian granulosa cells (GCs), resting on the basal lamina microenvironment [14]. of ovarian follicle. CCs are in close physical contact CCs also produce antioxidant compounds that re- with the oocyte, forming a cumulus-oocyte com- duce the level of oxidative stress caused by reactive plex (COC). The oocyte controls the differentiation oxygen species (ROS). These compounds, mainly and expansion of CCs, which in turn are involved in superoxide dismutase (SOD) [15] and Glutathione the metabolism of pyruvate and glucose consumed transferase S theta 1 (GSTT1) protect the oocyte during energy production in the oocyte [1,2]. Due to against oxidative stress [16,17]. this proximity, CCs are influenced by regulatory fac- Knowledge about the expression of individual tors produced by the oocyte. These regulatory fac- genes, as well as the presence of individual com- tors include primarily: growth differentiation factor ponents of the intracellular metabolic pathways, 9 (GDF9), bone morphogenetic protein 15 (BMP15) or the CCs apoptosis index can become a source of and fibroblast growth factor 8 (FGF8). They are pri- valuable molecular markers determining the devel- marily responsible for suppressing expression of opmental competence of oocytes. It is known that the LH receptor and genes responsible for steroid the expression of amphiregulin (AREG) and epiregulin (EREG) (epidermal growth factor (EGF) -like ruptured follicle, CCs are “ejected” from the rup- factors) depends on the dose of LH administered turedproduction. follicle Unlike during the ovulation GCs, which [3]. CCs “remain” also produce in the during ovarian stimulation. Therefore, it is sug- many inflammatory factors and cytokines that are gested that AREG and EREG are part of the signal released during ovulation. Cumulus cell-oocyte transduction pathway, which leads to the release of complex (COC) released from the ovary is there- the oocyte from the mature ovarian follicle and the fore a kind of a microenvironment [4–6]. Increased luteinization process in women [18]. Recent stud- concentration of gonadotropins in the female body ies described of new properties of CCs. CCs produce causes increased production of hyaluronic acid by a large amount of hyaluronan, which targets CD44 CCs, which expands the spaces between these cells. (marker of cancer cells). Culture of pancreatic can- Correct ovulation requires the production of pros- cer cells in a medium conditioned with CCs activat- taglandins. GDF9 (a member of the TGF-beta super- ed pro-apoptotic genes in these cancer cells [19]. family) plays a major role in the induction of Ptgs2 The mechanisms of communication between the oo- expression in CCs through increased luteinizing cyte and CCs cells through gap connections are well hormone (LH) concentration. Lack of GDF9 blocks known. It is known, however, that this communica- the development and growth of follicles, thus lead- tion also takes place by means of paracrine signaling to infertility [7]. ing. Less known methods of communication are the In most mammal species, including humans, CCs transfer of non-coding RNAs through exosomes from cells surround the oocyte at conception. It is be- the cumulus to the oocyte [20]. Coenzyme Q10 (CoQ) lieved that CCs interact with the oocyte and sperm, is another factor that has a significant impact on the and thus are involved in promoting the fertilization quality of oocyte and CCs cells. This relationship is process and oocyte developmental competence important for the proper functioning of mitochon- [8,9]. It has been proven many years ago that CCs dria, not only in the oocyte but also in CCs cells. A are involved in maintaining myocyte retention in decrease in mitochondrial activity is associated with the oocyte. Oocytes removed from the follicle, lack- aging of oocytes. Therefore, reduced CoQ production ing CCs, resume meiotic division, which should be in older women translates into reduced fertility and a completed only after fertilization [10]. In addition, higher risk of birth defects for embryos [21]. CCs facilitate the capture of COC by ciliated oviduct cells [11]. Glycodelin-C, a derivative of glycodelin, is regulation of gene expression in CCs may reflect isolated from the matrix of CCs cells. This substance processesUnderstanding in the oocyte. the molecular Changes mechanismsoccurring at andthe stimulates the binding of spermatozoa to the zona molecular level may be a signpost for identifying pellucida of oocyte [9]. It has also been reported oocyte quality, oocyte acquisition of developmental that the rate of CCs associated with morphological- competences, and quality of obtained blastocyst. ly abnormal oocytes or immature oocytes is much The main purpose of the research was to identify higher than in CCs surrounding morphologically the potential molecular markers responsible for cell normal, mature oocytes. The increase in CC apopto- junction organization, migration, differentiation, sis has a negative impact on a number of processes morphogenesis and motility. related to the correct fertilization and development of blastocyst and reduces the effectiveness of in vi- Material & Methods tro fertilization (IVF) [12,13]. Patients The expression of genes associated with the CCs were obtained from patients undergoing proper function of CCs results from a number of fac- IVF. The study used CCs from 12 patients aged 18- tors produced by the oocyte, but also the environ- 40 diagnosed with infertility. The factors excluding 62 Medical Journal of Cell Biology (2020)

Brązert et al. patients from these studies were: polycystic ovary agent®, Sigma; Merck KGaA). Next, chloroform was syndrome (PCOS), AMH less than 0.7 ng/ml, antral added to separate the phases during centrifugation. follicle count less than 9, day 2‑3 FSH serum level The upper aqueous phase, containing isolated RNA, in vi- was collected. RNA was extracted with 2‑propanol tro fertilization procedures were conducted in the (Sigma; Merck KGaA, catalog number I9516), add- Departmenthigher than 15of mU/mlInfertility and and endometriosis. Reproductive All Endo - ed in an amount adequate for 1 ml of TRI‑reagent. Finally, RNA was washed with 75% ethanol, dried, The IVF procedure was based on adapted and resuspended in 20 µl of pure water and measured. controlledcrinology, Poznan ovarian Medical hyperstimulation University, Poland. protocol. FSH (Gonal‑F, Merck Serono) and highly purified Microarray expression analysis and statistics hMG‑HP (Menopur, Ferring) were used for ovarian Total RNA (100ng) was converted to dou- stimulation. Additionally, to stop pituitary func- ble-stranded cDNA. In the next step, labeled comple- tions, Cetrorelix Acetate (Cetrotide, Merck Serono) mentary RNA (cRNA) was synthesized and ampli- injections at the right dose were performed. Ovula- fied by in vitro transcription of the double-stranded - cDNA template (GeneChipTM relle, Merck-Serono). Kit, Applied BiosystemsTM tion was induced by injection of 6500 U hCG (Ovit Obtained cRNA was fragmentated 3’IVT by PLUS divalent Reagent cat- complexes (COCs) have been selected by an embry- ions and elevated temperature., Foster Fragmentated City, CA, USA). and ologistAfter for oocyte further pick-up IVF procedure. (OPU), In oocyte-cumulus the next step of IVF routine, the obtained COCs were denuded. Corona radiata cells and cumulus oophorus somatic Biosystemslabeled cRNATM (7.5 μg) were hybridized to Human- cells forming COCs have been removed during de- croarraysGenome U219were Arraywashed Strip and (45°C/16stained according h, Applied to the technical , protocol Foster City, using CA, Affymetrix USA). Then, GeneAtlas the mi obtained in this way from multiple follicles of one Fluidics Station. Subsequently, the array strips were patientnudation were process pooled (800 and IU/mL transferred of HYASE-10X). to cell culture CCs scanned by Imaging Station of the GeneAtlas Sys- laboratory for further analysis. tem. The preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Oper- Condition of long-term primary in vitro culture ating Software. The quality of gene expression data CCs were collected after denudation of oocytes. Af- was checked according to quality control criteria terwards, they were washed twice with basal culture provided by the software. Obtained CEL files were medium and centrifuged at RT (200 x g for 10 min). imported into downstream data analysis software. Basal culture medium consisted of DMEM (Dulbec- All of presented analyses and graphs were per- co’s Modified Eagle’s Medium, Sigma; Merck KGaA, formed by Bioconductor and R programming lan- Darmstadt, Germany) supplemented with 10 mg/ guage. Each CEL file was merged with a description ml gentamicin (Invitrogen; Thermo Fisher Scientific, file. In order to correct background, normalize and Inc.), 2% fetal bovine serum FBS (FBS; Sigma; Merck summarize results, we used the Robust Multiarray KGaA), 4 mM L‑glutamine (stock 200 mM, Invitrogen; Averaging (RMA) algorithm. Statistical significance of the analyzed genes was - performed by moderated t-statistics from the empiri- cinThermo (Invitrogen; Fisher Thermo Scientific, Fisher Inc., Scientific, Waltham, Inc.) MA, [22]. USA), cal Bayes method. Obtained p-value was corrected for 10,000 U/ml penicillin and 10,000 μg/ml streptomy CCs were cultured at 37°C in 5% CO2 and humid multiple comparisons using the Benjamini and Hoch- atmosphere. After attaining 90% confluence, the berg’s false discovery rate. The selection of signifi- cells in the culture were detached from the bot- cantly changed gene expression was based on p-val- tom of the 6-well plate by 1-2 min incubation with ue beneath 0.05 and expression fold higher than 2. 0.05% trypsin-EDTA (Invitrogen; Thermo Fisher Differentially expressed genes were subjected to the Scientific, Inc.). Later, the cells were counted using selection of genes involved in cellular morphogene- the ADAM Cell Counter and Viability Analyzer (Bull- sis, junction and migration. Differentially expressed dog Bio). CCs were cultured fir 30 days. Medium gene list was uploaded to the DAVID software (Da- was changed every 72-75 hours of culture. Cells for tabase for Annotation, Visualization and Integrated the analysis were harvested on day 1, 7, 15 and 30 Discovery), where “cell junction organization”, “cell of in vitro culture. migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility” GO Total RNA isolation BP terms were obtained. Expression data of these After harvesting cells on the 1st, 7th, 15th and 30th genes were subjected to hierarchical clustering pro- day of culture, total RNA was isolated. The process of cedure and presented as a heatmap graph. Detailed RNA isolation was performed according to modified analysis of genes belonging to selected GO BP terms - were presented as plots using “GOplot” library [24]. tained CCs were suspended in 1 ml of monophasic Moreover, the list of differentially expressed genes guanidinemethod of thiocyanateChomczyński and and phenol Sacchi solution [23]. Briefly, (TRI Reob- from selected GO BP terms was uploaded to the 63 Medical Journal of Cell Biology (2020)

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FIGURE 1 Heatmaps presenting differentially expressed genes involved in “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility” based on GO BP terms. Each row onSTRING the Y axis software represents (Search a single Tool transcript. for Retrieval The red of color Inter indicates- cumulus downregulated oophorus genes cells. while This the methodgreen are allowedupregulated us acting Genes/Proteins) for interaction prediction. to study the gene expression of 22,480 transcripts Finally, we used ReactomeFIViz app from the Cy- at 1, 7, 15 and 30 days of in vitro cumulus oopho- toscape software for creating the Reactome Func- rus cell culture. We selected genes with more than tional Interaction (FI) network from the set of dif- 2- fold changes and corrected p-values less than ferentially expressed genes. 0.05 for downstream analysis. A total of 4773 differentially expressed genes (DEGs) were identified Ethical approval according to the above criteria. We started the mi- - croarray gene expression analysis with subjecting versity of Medical Sciences Bioethical Committee the list of DEGs to DAVID software, which showed withThis 1290/18 research resolution. has been approved by Poznań Uni that the genes can be assigned to 775 GO BP, 33 GO MF and 125 GO CC gene ontology terms. This paper Results focused on the genes involved cellular morphogenesis, junction and migration. The DAVID software in- the microarray gene expression analysis of human dicated the following GO BP terms, which cover the We used Human Genome U219 Array Strip for 64 Medical Journal of Cell Biology (2020)

Brązert et al. TABLE 1 The 10 most significantly upregulated and all of the downregulated genes involved cellular morphogenesis, junction and migration

FIGURE 2 The circular scatter plots of differentially expressed genes involved in “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility” GO BP terms. Each dot represents a single gene. The z-scores were presented as segments of inner circles above processes: “cell junction organization”, “cell sis (after 7, 15 and 30 days of in vitro culture). The 10 migration”, “cell morphogenesis involved in differen- most significantly upregulated and all of the down- tiation”, “cell morphogenesis” and “cell motility”. The regulated genes, their symbols, fold changes and cor- 150 genes involved in those processes were clustered rected p- values are shown in table 1. using hierarchical clustering and presented as heat- In the next part of analysis, we focused on the maps (Fig. 1). It is worth mentioning that 144 genes z-scores, which tell us whether the molecular func- were upregulated, which is the greater part of the list tion is more likely to be decreased (negative value) of genes used for hierarchical clustering. The down- or increased (positive value). The z-scores were pre- regulated genes are: SLC7A8- solute carrier family 7 sented as segments of inner circles in the figure 2. As (amino acid transporter light chain, L system), mem- can be seen from the figure, expression of most genes ber 8; DFNB31- deafness, autosomal recessive 31; was increased (green dots) in all ontological groups. COL1A1- collagen, type I, alpha 1; CDC42SE1- CDC42 The z-scores of above-mentioned GO BP terms had small effector 1; TGFBR3- transforming growth fac- positive values, so the processes described by these tor, beta receptor III; HMGB1- high mobility group GO BP terms were upregulated. The expression box 1. The direction of expression change (upregula- pattern did not change at any of the analyzed time tion or downregulation) was maintained in cumulus points. Considering the above, the subsequent analy- oophorus cell culture in subsequent points of analysis was based only on 7D/1D comparison. 65 Medical Journal of Cell Biology (2020)

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FIGURE 3 The dendrogram of differentially expressed genes involved in “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility” GO BP terms. The DEGs were clustered based on their logFC values

In the next section, we checked the interaction be- In the gene ontology database, single genes may tween selected ontological groups. One of the most belong to many ontological terms. For this reason, visually appealing way of presenting such interaction we used plots with visualization of logFC values is dendrogram (Fig. 3). Clusters contain functionally and relationship between genes and selected GO related genes based on their expression pattern. The BP terms (Fig. 4). The relationship was also pre- middle circle represents a logarithm of fold change sented as a heatmap (Fig. 5). The strongest upreg- (logFC) of differentially expressed genes assigned to ulated genes from examined GO BP terms includ- the studied GO terms. The GO terms are shown as the ed, among others: ANXA3- annexin A3, KIAA1199, outer ring. The genes whose expression is downreg- HTR2B- 5-hydroxytryptamine (serotonin) receptor ulated form clusters marked by blue part of the mid- 2B, G protein-coupled, VCAM1- vascular cell adhe- dle circle and analogously, red indicates upregulated sion molecule 1 and DKK1-dickkopf WNT signaling genes. Clusters of the same color over the entire width pathway inhibitor 1. of the outer circle represent genes that are unique for In the next part of analysis, we focused on the a specific GO term. Clusters of different colors on the interaction between proteins encoded by DEGs cross section of outer circle show sets of genes which belonging to studied GO BP terms. Firstly, we used are likely to be functionally related. The dendrogram STRING software for the interaction prediction. The showed that many genes belong simultaneously to number of genes used to create STRING interaction “cell motility” and “cell migration” or to “cell morpho- network was limited to 50 most changed DEGs for genesis” and “cell morphogenesis involved in differ- readability (Fig. 6). entiation”. The genes that are unique for a specific GO Finally, we used ReactomeFIViz app for investiga- term belong mainly to “cell morphogenesis”. tion of functional interactions between proteins en- 66 Medical Journal of Cell Biology (2020)

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FIGURE 4 Analysis of enriched gene ontological groups involved in cellular morphogenesis, junction and migration. The network plot presenting the linkages of genes and GO BP terms

FIGURE 5 Heatmap presenting the relationship between genes and selected GO BP terms. The yellow color of tiles indicates the absence of logFC values coded by DEGs belonging to selected GO BP terms. Discussion Among the most significantly enriched functional It is known that CCs are necessary in the process interaction networks were FI networks for “Cell mi- of acquiring developmental competence by the oo- gration” and “Positive regulation of cell migration” cyte, including enabling the resumption of meio- (Figs 7 and 8). sis and the transition to the meiosis metaphase II 67 Medical Journal of Cell Biology (2020)

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FIGURE 6 Interaction network of proteins encoded by 50 most changed DEGs belonging to “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility” GO BP terms. The network was generated by STRING software. Network nodes represent proteins. Empty nodes indicate proteins of unknown 3D structure

[25]. Thanks to the gap connections, it is possible to one place to another; 2) “cell junction organization”, transport molecules between CCs cells and the oocyte “cell morphogenesis involved in differentiation”, “cell [26]. Proper oocyte maturation without CCs is practi- morphogenesis “ are responsible for organizing the cally impossible, and the effectiveness of fertilization components of connections between two cells but of such an oocyte and obtaining a correct blastocyst also between the cell and the extracellular matrix. drastically decreases [27]. Furthermore, metabolomic In addition, a second group of selected ontologi- studies of the spent culture medium obtained show cal groups describes the genes responsible for cell that CCs are secreted into the external environment movement from one site to the destination directed and allow oocyte maturation [28,29]. Due to the close- by molecular information. Moreover, genes involved ness and interaction of CCs and oocyte, they have be- in shaping cells and changing their form, shape and come an interesting research model in embryology. size that occur when non-specialized cells acquire In the presented studies, during the transcrip- the specialized structural features of a cell popula- tome analysis of CCs cells maintained in long-term tion characteristic of an organ. primary in vitro culture, groups of genes were select- The presented research results indicate that the ed, responsible primarily for processes associated strongest upregulated genes from examined GO with “cell junction organization”, “cell morphogene- BP terms included, among others: DKK1, ANXA3, sis involved in differentiation”, cell morphogenesis”, KIAA1199, VCAM1, HTR2B. Only 6 genes demon- “cell motility”, “cell migration”. For the purposes of strated downregulation relative to the reference the presented research, these selected ontological value, which was the gene expression shown on day groups can be conventionally divided into two group: 1 of primary culture in vitro. 1) “cell motility”, “cell migration” - here, genes are The factor conditioning correct embryo implan- primarily responsible for the movement of cells from tation is activation of canonical WNT signaling. The 68 Medical Journal of Cell Biology (2020)

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FIGURE 7 Reactome FI network for “Cell migration”. “--->” indicates activating/catalyzing, “-“ FIs extracted from complexes or inputs and “---” predicted FIs

FIGURE 8 Reactome FI network for “Positive regulation of cell migration”. “--->” indicates activating/catalyzing, “-“ FIs extracted from complexes or inputs and “---” predicted FIs

WNT signaling pathway is regulated by steroids pregnancy or estrus in other mammals [34,35]. It [30,31]. WNT activation in the embryo may or may is believed, that the proper development of the em- not affect the correct implantation of the embryo, bryo and the proper implantation depend on a num- but is responsible for regulating pluripotency [32]. ber of regulatory factors secreted by the mother’s DKK1 is an antagonist of the WNT signaling path- reproductive system [36]. DDK1 is also potentially way [33], as well as the protein produced by en- involved in mother-embryo communication [37]. dometrial cells during the menstrual cycle, early Studies on bovine embryos indicate that DKK1 facil- 69 Medical Journal of Cell Biology (2020)

Brązert et al. itates TE and hypoblast differentiation. DKK1 plays strated in mouse granular cells after dehydroepi- one of the key roles in the proper functioning of the androsterone (DHEA) androgenization. The result reproductive system of most mammals, including of the increase in VCAM1 expression was an exac- humans [37–41]. In the light of our research and erbation of the symptoms of polycystic ovarian syn- existing knowledge about the role of CCs in commu- drome in mice (PCOS). It is suggested that VCAM1 nication with the oocyte, high DKK1 expression may expression knockout is one therapeutic option for also affect the quality of obtained blastocyst and its PCOS [56]. Expression of the VCAM1 protein was implantability [42]. Studies suggest that adding CCs not detected in mouse embryos prior to implanta- cells to the embryo environment improves embryo tion. However, it is known that VCAM1 is present quality and pregnancy rates.[43]. Perhaps the high in oocytes and early human embryos [57,58]. The expression of this gene in CCs cells may also affect presented research results suggest that CCs cells in the acquisition of oocyte developmental compe- in-vitro culture medium show significant increase tences and then having sufficiently high pluripotent in expression in the following days of the in vitro properties. Therefore, our research suggests that primary culture. Perhaps the lack of interaction the presence of CCs cells in the oocyte environment with the oocyte determines this growth. during fertilization may result in improved in vitro Only 6 genes showed a decrease in expression fertilization efficiency. relative to the control. In the presented article we The genes also responsible for the proper de- focused on two genes the most downregulated (TG- velopment of blastocyst include the ANXA3 gene. FBR3 and HMGB1). The protein encoded by this gene is responsible for In the presented studies, it seems surprising binding and maintaining primarily cell membranes. that the expression of TGFBR3 gene shows a de- ANX are proteins found in a variety of tissues, from crease. This gene codes for the receptor protein for the respiratory and urinary epithelium to the pres- TGF-beta. The transforming growth factor family ence in peripheral nerves. Proteins from the anexin (TGF-beta) are multifunctional molecules involved family due to their expression in individual tissues in a number of processes primarily associated with most likely take part in the processes of equalization tissue development, cell differentiation process, cell of individual tissues, and are also partly responsible migration and adhesion, as well as the production for their physiological properties [44]. The present- of extracellular matrix. Receptor types I, II and III ed studies suggest an increase in ANXA3 gene ex- are involved in the action of TGF-beta. TGFBR3 is a pression in the following days of primary culture in TGF-beta binding glycoprotein that can have a mem- vitro in the absence of oocyte. To date, the presence brane-bound form as well as a dissolved form [59]. of the ANXA3 gene has been reported in the morula This receptor may also exist as an inhibin co-re- and blastocyst stages during preimplantation devel- ceptor [60]. TGFBR3 expression in CCs cells during opment [45]. The ANXA3 protein is part of human long-term culture decreases. This may be associat- neutrophils and promotes tight connections be- ed with a lack of communication with the oocyte tween membranes of individual neutrophils, which and a change in the natural environment for CCs causes their aggregation [46–48]. cells. The expression of TGFBR3 decreases during As mentioned in the introduction, hyaluronan the development of breast cancer [61]. In studies on (HA), or hyaluronic acid, plays an important role the effect of age on the quality of oocytes, TGFBR3 in the reproductive system. It is produced primar- was found to be overexpressed in a group of women ily by the oocyte, embryo and other elements of over 37 years of age [2]. It turns out that changing the reproductive system, depending on the spe- the environment to extracorporeal can have a great cies of the mammal (fallopian tube, uterus, cervix) impact on obtaining oocyte developmental compe- [49,50]. In addition, HA is produced by CCs and GCs tences, as well as the development of the embryo cells [51,52]. The KIAA1199 (CEMIP; HYBID) gene and its proper implantation after transfer to the showed a significant change in expression. The pro- mother’s uterus [62]. This factor can have a signifi- tein encoded by this gene plays a key role in depo- cant impact on the success and effectiveness of the lymerization of HA molecules. Microarray analysis in vitro fertilization procedure. The decrease in TG- by Abe et al. it identified the expression of this gene FBR3 expression is associated with reduced oocyte in the cochlea, as well as in the inner ear [53]. Other developmental competence [63]. On the other hand, studies claim that this gene is responsible for pro- studies in mice prove that the Tgfbr3 knockout was moting the process of apoptosis, cancer progres- associated with their high fertility and increased sion. In addition, he is responsible for regulating folliculogenesis [64]. cell proliferation, adhesion, motility and epitheli- HMGB1 is another gene that showed a decrease al-mesenchymal transition cells of cancer. [54]. in expression. Melatonin has shown increased ex- The gene closely related to cell adhesion and expression of this gene during oocyte maturation tracellular matrix modeling is VCAM1. It shows in- [65]. High expression of HMBG1 reduces blastocyst creased expression in GCs and CCs cells [55]. High demand and thus increases the chances of embryo expansion of the VCAM1 gene has also been demon- survival [66,67]. Moreover, studies show that there 70 Medical Journal of Cell Biology (2020)

Brązert et al. is a correlation with the amount of HMGB1 protein 7. Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocri- in follicular fluid (FF) and pregnancy and endome- nol. 1999;13(6):1035–48; DOI:10.1210/mend.13.6.0310. trial thickness. There are scientific reports confirm- 8. Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A. Minire- ing the fact that the protein encoded by the present view: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev. 2002;61(3):414–24; gene can be an important indicator of the success DOI:10.1002/mrd.10102. of in vitro fertilization [68]. 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Russell DL, Gilchrist RB, Brown HM, Thompson JG. Bidirectional communication between cumulus cells and the oocyte: Old hands and are primarily responsible for processes related to new players? Theriogenology. 2016;86(1):62–8; DOI:10.1016/j. communication with the oocyte as well as the prop- theriogenology.2016.04.019. er development of the embryo and its implanta- 15. Matos L, Stevenson D, Gomes F, Silva-Carvalho JL, Almeida H. Superoxide dismutase expression in human cumulus oophorus cells. Mol Hum Re- tion. The obtained results indicate that in in vitro prod. 2009;15(7):411–9; DOI:10.1093/molehr/gap034. cultured CCs do not lose their properties or, on the 16. contrary, their gene expression decreases due to the Reproductive aging: accelerated ovarian follicular development associa- tedKlein with NA, a Battagliamonotropic DE, follicle-stimulating Fujimoto VY, Davis hormone GS, Bremner rise inWJ, normal Soules older MR. loss of contact with the oocyte. women. J Clin Endocrinol Metab. 1996;81(3):1038–45; DOI:10.1210/ jcem.81.3.8772573. 17. Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H. Eight- Acknowledgements hydroxy-2’-deoxyguanosine in granulosa cells is correlated with the This research was funded by National Science Centre, quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril. 2002;77(6):1184–90; DOI:10.1016/ s0015-0282(02)03103-5. CorrespondingUMO-2018/31/B/NZ5/02475. author 18. Freimann S, Ben-Ami I, Dantes A, Ron-El R, Amsterdam A. EGF-like factor Bartosz Kempisty Ph.D., Department of Histology and Embryology, epiregulin and amphiregulin expression is regulated by gonadotropins/ cAMP in human ovarian follicular cells. Biochem Biophys Res Commun. 2004;324(2):829–34; DOI:10.1016/j.bbrc.2004.09.129. Department of Anatomy, Poznań[email protected]. University of Medical Sciences, 6 19. Ś�więcickiego St., 60-781 Poznań, Poland Tel./Fax: +48 61 8546567 F, et al. 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