Oogenesis in Women: From Molecular Regulatory Pathways and Maternal Age to Stem Cells.

It is a well-known fact that the reproductive organs in women, especially oocytes, are exposed to numerous regulatory pathways and environmental stimuli. The maternal age is one cornerstone that influences the process of oocyte fertilization. More precisely, the longer a given oocyte is in the waiting-line to be ovulated from menarche to menopause, the longer the duration from oogenesis to fertilization, and therefore, the lower the chances of success to form a viable embryo. The age of menarche in girls ranges from 10 to 16 years, and the age of menopause in women ranges from approximately 45 to 55 years. Researchers are paying attention to the regulatory pathways that are impacting the oocyte at the very beginning during oogenesis in fetal life to discover genes and proteins that could be crucial for the oocyte's lifespan. Due to the general trend in industrialized countries in the last three decades, women are giving birth to their first child in their thirties. Therefore, maternal age has become an important factor impacting oocytes developmental competence, since the higher a woman's age, the higher the chances of miscarriage due to several causes, such as aneuploidy. Meiotic failures during oogenesis, such as, for instance, chromosome segregation failures or chromosomal non-disjunction, are influencing the latter-mentioned aging-related phenomenon too. These errors early in life of women can lead to sub- or infertility. It cannot be neglected that oogenesis is a precisely orchestrated process, during which the oogonia and primary oocytes are formed, and RNA synthesis takes place. These RNAs are crucial for oocyte growth and maturation. In this review, we intend to describe the relevance of regulatory pathways during the oogenesis in women. Furthermore, we focus on molecular pathways of oocyte developmental competence with regard to maternal effects during embryogenesis. On the background of transcriptional mechanisms that enable the transition from a silenced oocyte to a transcriptionally active embryo, we will briefly discuss the potential of induced pluripotent stem cells.

Expression Profiles of AQP3 and AQP4 in Lung Adenocarcinoma Samples Generated via Bronchoscopic Biopsies.

Aquaporins (AQPs) are highly conserved channel proteins which are mainly responsible for the exchange of water and small molecules and have shown to play a pivotal role in the development and progression of cancer. Lung adenocarcinoma is the most common primary lung cancer seen in patients in Europe and the United States. However, in patients it is often not diagnosed until the advanced tumor stage is present. Previous studies provided strong evidence that some members of the AQP family could serve as clinical biomarkers for different diseases. Therefore, we aimed to investigate how AQP3 and AQP4 protein expression in lung adenocarcinoma (ADC) biopsy samples correlate with clinical and pathological parameters. The protein expression of AQP3 and AQP4 was analyzed based on immunohistochemical staining. AQP3 protein was observed in the cytoplasmic membrane of cancer tissue in 82% of lung samples. Significant differences in relative protein expression of AQP3 were noted between advanced age patients compared to younger counterparts (p = 0.017). A high expression of AQP3 was significant in cancer tissue when compared to the control group (p < 0.001), whereas a low AQP4 membrane expression was noted as significantly common in cancer tissue compared to non-neoplastic lung tissue (p < 0.001). Moreover, a low AQP4 membrane expression was positively correlated with a more advanced disease status, e.g., lymph node metastases (p = 0.046). Based on our findings, AQP3 and AQP4 could be used as biomarkers in ADC patients.

Relevance of Vitamin D and Its Deficiency for the Ovarian Follicle and the Oocyte: An Update.

For many years, vitamin D (VD) has been known to be an essential micronutrient with important relevance not only for the skeletal system, but also for numerous other mammalian organ systems. Low levels of VD result in a VD deficiency, which is a global health problem. Moreover, VD deficiencies are linked to several pathologies, for instance, diseases of the cardiovascular system, diabetes mellitus, or sub- and infertility. In the past two decades, an increasing body of evidence has shown that adequate physiological levels of VD are crucial for the female gamete and its microenvironment, and VD deficiency has been associated with decreased live birth rates among women undergoing in vitro fertilization (IVF). With regard to the female reproductive tract, VD receptors (VDRs) have been detected in the ovary, endometrium, and the placenta. Although it has been reported that VD seems to be relevant for both calcium-dependent and independent pathways, its relevance for the oocyte's developmental competence and life span remains elusive. Therefore, herein, we aim to provide an update on the importance of VD and VD deficiency for the oocyte and the follicular microenvironment.

The Interplay between Telomeres, Mitochondria, and Chronic Stress Exposure in the Aging Egg.

While at the organismal level, biological aging can be estimated by telomere length and DNA methylation signatures, reliable biomarkers that can predict reproductive age are much needed to gauge the quality of an oocyte. Reproductive medicine and fertility centers often merely quantitate the ovarian reserve to predict the likelihood of fertilization and pregnancy in women of advanced reproductive age. It is highly important to address the level of age-related decline in oocyte quality since it leads to an increased risk of miscarriages and aneuploidy. Conversely, the pathways behind oocyte aging remain, in large part, elusive. Telomere shortening upon chronic stress exposure regulates mitochondria function and biogenesis by various pathways; therefore, establishing a link between these two important players and extrapolating them for the aging of oocytes will be the purpose of our commentary.

Pleiotropic Effects of IGF1 on the Oocyte.

A woman's endocrine system plays a crucial role in orchestrating cellular interactions throughout her life. The growth hormone (GH) and insulin-like growth factor (IGF) system appears to impact crucial reproductive events and cell types of the ovary, such as granulosa cells, theca cells, and oocytes. Further, IGF1 is a cornerstone during embryonic development and influences predominantly developing and pre-antral follicles. In this commentary, we will emphasize the pleiotropic effects of IGF1 on physiological processes inside the egg. Herein, we will provide a brief overview on IGF1 related cell signal transduction pathways during the maturation and aging of oocytes. We aim to elucidate from a molecular and biochemical point of view if IGF1 in women with metabolic imbalances such as obesity or diabetes could be used in clinics as a novel, reliable estimator for the developmental competence of an oocyte.

Pannexins and Connexins: Their Relevance for Oocyte Developmental Competence.

The oocyte is the major determinant of embryo developmental competence in all mammalian species. Although fundamental advances have been generated in the field of reproductive medicine and assisted reproductive technologies in the past three decades, researchers and clinicians are still trying to elucidate molecular factors and pathways, which could be pivotal for the oocyte's developmental competence. The cell-to-cell and cell-to-matrix communications are crucial not only for oocytes but also for multicellular organisms in general. This latter mentioned communication is among others possibly due to the Connexin and Pannexin families of large-pore forming channels. Pannexins belong to a protein group of ATP-release channels, therefore of high importance for the oocyte due to its requirements of high energy supply. An increasing body of studies on Pannexins provided evidence that these channels not only play a role during physiological processes of an oocyte but also during pathological circumstances which could lead to the development of diseases or infertility. Connexins are proteins that form membrane channels and gap-junctions, and more precisely, these proteins enable the exchange of some ions and molecules, and therefore they do play a fundamental role in the communication between the oocyte and accompanying cells. Herein, the role of Pannexins and Connexins for the processes of oogenesis, folliculogenesis, oocyte maturation and fertilization will be discussed and, at the end of this review, Pannexin and Connexin related pathologies and their impact on the developmental competence of oocytes will be provided.

The In Vitro Effect of Steroid Hormones, Arachidonic Acid, and Kinases Inhibitors on Aquaporin 1, 2, 5, and 7 Gene Expression in the Porcine Uterine Luminal Epithelial Cells during the Estrous Cycle.

Aquaporins (AQPs) are integral membrane proteins, which play an important role in water homeostasis in the uterus. According to the literature, the expression of aquaporins in reproductive structures depends on the local hormonal milieu. The current study investigated the effect of selected PKA kinase inhibitor H89 and MAPK kinase inhibitor PD98059, on the expression of AQP1, 2, 5, and 7, and steroid hormones (E2), progesterone (P4), and arachidonic acid (AA) in the porcine endometrium on days 18-20 and 2-4 of the estrous cycle (the follicular phase where estrogen and follicle-stimulating hormone (FSH) are secreted increasingly in preparation for estrus and the luteal phase where the ovarian follicles begin the process of luteinization with the formation of the corpus luteum and progesterone secretion, respectively). The luminal epithelial cells were incubated in vitro in the presence of the aforementioned factors. The expression of mRNA was determined by the quantitative real-time PCR technique. In general, in Experiment 1, steroid hormones significantly increased expression of AQP1, 2, and 5 while arachidonic acid increased expression of AQP2 and AQP7. On the other hand, MAPK kinase inhibitor significantly decreased the expression of AQP1 and 5. In Experiment 2, E2, P4, or AA combined with kinase inhibitors differentially affected on AQPs expression. E2 in combination with PKA inhibitor significantly decreased expression of AQP1 but E2 or P4 combined with this inhibitor increased the expression of AQP5 and 7. On the contrary, E2 with PD98059 significantly increased AQP5 and AQP7 expression. Progesterone in combination with MAPK kinase inhibitor significantly downregulated the expression of AQP5 and upregulated AQP7. Arachidonic acid mixed with H89 or PD98059 caused a decrease in the expression of AQP5 and an increase of AQP7. The obtained results indicate that estradiol, progesterone, and arachidonic acid through PKA and MAPK signaling pathways regulate the expression of AQP1 and AQP5 in the porcine luminal epithelial cells in the periovulatory period.

The Relevance of Aquaporins for the Physiology, Pathology, and Aging of the Female Reproductive System in Mammals.

Aquaporins constitute a group of water channel proteins located in numerous cell types. These are pore-forming transmembrane proteins, which mediate the specific passage of water molecules through membranes. It is well-known that water homeostasis plays a crucial role in different reproductive processes, e.g., oocyte transport, hormonal secretion, completion of successful fertilization, blastocyst formation, pregnancy, and birth. Further, aquaporins are involved in the process of spermatogenesis, and they have been reported to be involved during the storage of spermatozoa. It is noteworthy that aquaporins are relevant for the physiological function of specific parts in the female reproductive system, which will be presented in detail in the first section of this review. Moreover, they are relevant in different pathologies in the female reproductive system. The contribution of aquaporins in selected reproductive disorders and aging will be summarized in the second section of this review, followed by a section dedicated to aquaporin-related proteins. Since the relevance of aquaporins for the male reproductive system has been reviewed several times in the recent past, this review aims to provide an update on the distribution and impact of aquaporins only in the female reproductive system. Therefore, this paper seeks to determine the physiological and patho-physiological relevance of aquaporins on female reproduction, and female reproductive aging.

New Gene Markers Involved in Molecular Processes of Tissue Repair, Response to Wounding and Regeneration Are Differently Expressed in Fibroblasts from Porcine Oral Mucosa during Long-Term Primary Culture.

The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

Stemness Potency of Human Gingival Cells-Application in Anticancer Therapies and Clinical Trials.

Gingivae, as the part of periodontium, are involved in tooth support and possess the ability to heal rapidly, without scar formation. Recently, dental tissues have been identified as a potential source of mesenchymal stem cells (MSCs) and several populations of MSCs were isolated from the orofacial region, including gingival mesenchymal stem cells (GMSCs). GMSCs exhibit robust immunomodulatory and differentiation potential and are easily obtainable, which make them promising candidates for cellular therapies. Apart from being tested for application in immunologic- and inflammatory-related disorders and various tissue regeneration, GMSCs promise to be a valuable tool in cancer treatment, especially in tongue squamous cell carcinoma (TSCC) with the use of targeted therapy, since GMSCs are able to selectively migrate towards the cancerous cells both in vitro and in vivo. In addition to their ability to uptake and release anti-neoplastic drugs, GMSCs may be transduced with apoptosis-inducing factors and used for cancer growth inhibition. Moreover, GMSCs, as most mammalian cells, secrete exosomes, which are a subset of extracellular vesicles with a diameter of 40-160 nm, containing DNA, RNA, lipids, metabolites, and proteins. Such GMSCs-derived exosomes may be useful therapeutic tool in cell-free therapy, as well as their culture medium. GMSCs exhibit molecular and stem-cell properties that make them well suited in preclinical and clinical studies.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Changes in Aquaporin 1, 5 and 9 Gene Expression in the Porcine Oviduct According to Estrous Cycle and Early Pregnancy.

Aquaporins (AQPs) are a group of small, integral membrane proteins which play an important role in fluid homeostasis in the reproductive system. In our previous study, we demonstrated AQP1, 5 and 9 protein expression and localization in the porcine oviduct. The presence of these isoforms could suggest their role in the transport of the ovum to the uterus by influencing the epithelial cells' production of oviductal fluid. The aim of this study was to evaluate the expression of AQP1, AQP5 and AQP9 in the infundibulum, ampulla and isthmus in the porcine oviduct during the estrous cycle (early luteal phase, days 2-4, medium luteal phase, days 10-12, late luteal phase days 14-16, follicular phase days 18-20) and pregnancy (period before implantation, days 14-16 and after the implantation, days 30-32) using the Real-Time PCR technique. As clearly demonstrated for the first time, AQP1, 5, and 9 gene expression is influenced by the estrus cycle and pregnancy. Furthermore, expression of AQPs in the porcine oviduct may provide the physiological medium that sustains and enhances fertilization and early cleavage-stage embryonic development. Overall, our study provides a characterization of oviduct AQPs, increasing our understanding of fluid homeostasis in the porcine oviduct to successfully establish and maintain pregnancy.

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

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.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Pituitary Hormones (FSH, LH, PRL, and GH) Differentially Regulate AQP5 Expression in Porcine Ovarian Follicular Cells.

This study aimed to examine the effect of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) on Aquaporin 5 (AQP5) expression in granulosa (Gc) and theca cells (Tc) from medium (MF) and large (LF) ovarian follicles of pigs. The results showed that GH significantly decreased the expression of AQP5 in Gc from MF in relation to the control. In the Gc of large follicles, PRL stimulated the expression of AQP5. However, the increased expression of AQP5 in the Tc of LF was indicated by GH and PRL in relation to the control. A significantly higher expression of the AQP5 protein in the Gc from MF and LF was indicated by FSH and PRL. In co-cultures, an increased expression of AQP5 was observed in the Gc from LF incubated with LH, PRL, and GH. A significantly increased expression of AQP5 was also observed in co-cultures of Tc from all type of follicles incubated with LH, whereas PRL stimulated the expression of AQP5 in Tc from MF. Moreover, AQP5 protein expression increased in the co-culture isolated from MF and LF after treatment with FSH, LH, PRL, and GH. AQP5 immunoreactivity was observed in the cytoplasm, mainly in the perinuclear region and endosomes, as well as in the cell membranes of Gc and Tc from the LF and MF.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence-A Transcriptomic Study.

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

Genes Involved in the Processes of Cell Proliferation, Migration, Adhesion, and Tissue Development as New Potential Markers of Porcine Granulosa Cellular Processes In Vitro: A Microarray Approach.

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.

New Gene Markers of Angiogenesis and Blood Vessels Development in Porcine Ovarian Granulosa Cells during Short-Term Primary Culture In Vitro.

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.