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.

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Genes of cellular components of morphogenesis in porcine oocytes before and after IVM.

Proper oocyte maturation in mammals produces an oocyte capable of monospermic fertilization and embryo preimplantation. The cumulus-oocyte complexes (COCs), surrounding an oocyte, play a significant role in oocyte maturation. During this process, when the COCs undergo cumulus expansion wherein tightly compact cumulus cells (CCs) form a dispersed structure, permanent biochemical and molecular modifications occur in the maturing oocytes, indicating that the gene expression between immature and mature oocytes differs significantly. This study focuses on the genes responsible for the cellular components of morphogenesis within the developing oocyte. Brilliant cresyl blue (BCB) was used to determine the developmental capability of porcine oocytes. The immature oocytes (GV stage) were compared with matured oocytes (MII stage), using microarray and qRT-PCR analysis to track changes in the genetic expression profile of transcriptome genes. The data showed substantial upregulation of genes influencing oocyte's morphology, cellular migration and adhesion, intracellular communication, as well as plasticity of nervous system. Conversely, downregulation involved genes related to microtubule reorganization, regulation of adhesion, proliferation, migration and cell differentiation processes in oocytes. This suggests that most genes recruited in morphogenesis in porcine oocyte in vitro, may have cellular maturational capability, since they have a higher level of expression before the oocyte's matured form. It shows the process of oocyte maturation and developmental capacity is orchestrated by significant cellular modifications during morphogenesis.

Expression of genes associated with BMP signaling pathway in porcine oocytes before and after IVM - a microarray approach.

BACKGROUND: The full maturational capability of mammalian oocytes is accompanied by nuclear and cytoplasmic modifications, which are associated with proliferation and differentiation of surrounding cumulus cells. These events are regulated on molecular level by the expression of target genes involved in signal transduction pathways crucial for folliculogenesis and oogenesis. Transforming growth factor beta signaling includes several molecules that are involved in the regulation of oogenesis and embryo growth, including bone morphogenetic protein (BMP). However, the BMP-related gene expression profile in oocytes at different maturational stages requires further investigation. METHODS: Oocytes were isolated from pubertal crossbred Landrace gilts follicles, selected with a use of BCB staining test and analyzed before and after in vitro maturation. Gene expression profiles were examined using an Affymetrix microarray approach and validated by RT-qPCR. Database for Annotation, Visualization, and Integrated Discovery (DAVID) software was used for the extraction of the genes belonging to a BMP-signaling pathway ontology group. RESULTS: The assay revealed 12,258 different transcripts in porcine oocytes, among which 379 genes were down-regulated and 40 were up-regulated. The DAVID database indicated a "BMP signaling pathway" ontology group, which was significantly regulated in both groups of oocytes. We discovered five up-regulated genes in oocytes before versus after in vitro maturation (IVM): chordin-like 1 (CHRDL1), follistatin (FST), transforming growth factor-beta receptor-type III (TGFßR3), decapentaplegic homolog 4 (SMAD4), and inhibitor of DNA binding 1 (ID1). CONCLUSIONS: Increased expression of CHRDL1, FST, TGFßR3, SMAD4, and ID1 transcripts before IVM suggested a subordinate role of the BMP signaling pathway in porcine oocyte maturational competence. Conversely, it is postulated that these genes are involved in early stages of folliculogenesis and oogenesis regulation in pigs, since in oocytes before IVM increased expression was observed.

Morphogenesis-related gene-expression profile in porcine oocytes before and after in vitro maturation.

Mammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.

Significant Down-Regulation of "Biological Adhesion" Genes in Porcine Oocytes after IVM.

Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes' maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB⁺ oocytes were directly exposed to microarray assays and RT-qPCR ("before IVM" group), or first in vitro matured and then if classified as BCB⁺ passed to molecular analyses ("after IVM" group). As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes' successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte's achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.

Expression Profile of Genes Regulating Steroid Biosynthesis and Metabolism in Human Ovarian Granulosa Cells-A Primary Culture Approach.

Because of the deep involvement of granulosa cells in the processes surrounding the cycles of menstruation and reproduction, there is a great need for a deeper understanding of the ways in which they function during the various stages of those cycles. One of the main ways in which the granulosa cells influence the numerous sex associated processes is hormonal interaction. Expression of steroid sex hormones influences a range of both primary and secondary sexual characteristics, as well as regulate the processes of oogenesis, folliculogenesis, ovulation, and pregnancy. Understanding of the exact molecular mechanisms underlying those processes could not only provide us with deep insight into the regulation of the reproductive cycle, but also create new clinical advantages in detection and treatment of various diseases associated with sex hormone abnormalities. We have used the microarray approach validated by RT-qPCR, to analyze the patterns of gene expression in primary cultures of human granulosa cells at days 1, 7, 15, and 30 of said cultures. We have especially focused on genes belonging to ontology groups associated with steroid biosynthesis and metabolism, namely "Regulation of steroid biosynthesis process" and "Regulation of steroid metabolic process". Eleven genes have been chosen, as they exhibited major change under a culture condition. Out of those, ten genes, namely STAR, SCAP, POR, SREBF1, GFI1, SEC14L2, STARD4, INSIG1, DHCR7, and IL1B, belong to both groups. Patterns of expression of those genes were analyzed, along with brief description of their functions. That analysis helped us achieve a better understanding of the exact molecular processes underlying steroid biosynthesis and metabolism in human granulosa cells.

The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents.

We hypothesized that oocyte morphology may be associated with the accumulation of specific mRNAs encoding proteins responsible for the gamete fertilization ability. Therefore, the aim of the study was to evaluate the transcript levels of porcine zona pellucida (pZP1, pZP2, pZP3 and pZP4) glycoproteins in oocytes classified by a four-grade morphological scale (I-IV) accounting for either a homogeneous cytoplasm and a complete cumulus oophorus (grade I) or a heterogenous cytoplasm and decreased number of cumulus layers in the other grades (II, III and IV). We observed a significant increase of all investigated pZP glycoprotein mRNAs in grade I oocytes as compared to other grades (p<0.05). Our observations suggest that porcine oocyte morphology is associated with pZP transcript contents and may be related to an increased fertilization ability of higher quality oocytes.

Enhanced Suppressive Activity of Regulatory T Cells in the Microenvironment of Malignant Pleural Effusions.

Cancer metastatic spread to serous cavity causes malignant pleural effusions (MPEs), indicating dismal prognosis. Tumor microenvironment can implement suppressive activity on host immune responses. Thus, we investigated the prevalence of Tregs and the relationship between them and TGF-ß and IL-10 concentrations and measured expression of FOXP3, CTLA-4, CD28, and GITR genes, as well as protein expression of selected genes in benign effusions and MPEs. The percentage of Tregs was determined by means of multicolor flow cytometry system. TGF-ß and IL-10 concentrations were measured using human TGF-ß1 and IL-10 ELISA kit. Relative mRNA expression of studied genes was analyzed by real-time PCR. The frequency of Tregs was significantly higher in MPEs compared to benign effusions; however, the level of TGF-ß and IL-10 in analyzed groups was comparable, and no correlation between concentrations of TGF-ß and IL-10 and percentage of Tregs was observed. Relative mRNA expression of all the genes was higher in CD4+CD25+ compared to CD4+CD25- cells. In CD4+CD25+ cells from MPEs, relative mRNA expression of FOXP3, CTLA-4, and CD28 genes was significantly higher than in benign effusions; however, the level of CD4+CD25+CTLA-4+ cells in analyzed groups showed no significant differences. We found numerous genes correlations in an entire CD4+CD25+ cell subset and CD4+CD25+ cells from MPEs. Enhanced suppressive activity of Tregs is observed in the microenvironment of MPEs. Understanding of relations between cellular and cytokine immunosuppressive factors in tumor microenvironment may determine success of anticancer response.

"Positive Regulation of RNA Metabolic Process" Ontology Group Highly Regulated in Porcine Oocytes Matured In Vitro: A Microarray Approach.

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in "oocytes RNA synthesis" in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to "positive regulation of RNA metabolic process" ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group "positive regulation of RNA metabolic process" involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of "RNA metabolic process" related genes before IVM indicated that they might be recognized as important markers and specific "transcriptomic fingerprint" of RNA template accumulation and storage for further porcine embryos growth and development.

Threatened preterm labour - analysis of the cytokine profile and progesterone treatment efficiency.

OBJECTIVE: Preterm labour is defined as the onset of labour between weeks 23 and before 37 of gestation. The purpose of this study was to compare the cytokine profile in the blood serum of patients experiencing threatened premature labour, along with the time of delivery and the foetus body mass at birth. METHODS: The study included 89 women hospitalised at the Obstetrics and Gynaecology Clinical Hospital in the period 2011-2013. Group 1 comprised 31 patients diagnosed with threatened preterm labour, prior to tocolytic treatment. Group 2 comprised 32 patients with tocolytic treatment. Group 3 constituted the control group. RESULTS: Groups 1, 2 and 3 differed considerably in terms of the week of delivery of pregnancy (p = 0.006). Analogous results were obtained by analysing the body mass at birth, where a statistically significant difference in body mass at birth was found between Groups 1, 2 and 3 (p < 0.001). Our analysis focussed on the cytokine profile of the women included in the study, but no considerable changes in cytokine concentration were observed. CONCLUSIONS: No statistically significant differences were found in the cytokine profile among those patients in normally progressing pregnancy or in threatened preterm labour. No suitability of progesterone treatment in threatened preterm labour was observed.

"Cell Migration" Is the Ontology Group Differentially Expressed in Porcine Oocytes Before and After In Vitro Maturation: A Microarray Approach.

Maturation of cumulus-oocyte complexes (COCs) is crucial for further successful monospermic fertilization, embryo growth, and implantation. All these events are accompanied by proliferation and differentiation of cumulus cells. The migration of COCs to the oviduct after ovulation and the interaction between female gametes and/or embryos with maternal tissues are still poorly recognized on the molecular level. This study was aimed to first demonstrate the mRNA expression profile of cell migration markers during different stages of porcine oocytes maturation and developmental capability in vitro. The COCs were collected from a total of 45 pubertal crossbred Landrace gilts, brilliant cresyl blue (BCB) stained, and analyzed before (n = 150) or after (n = 150) in vitro maturation (IVM). Using the Affymetrix® Porcine Gene 1.1 ST Array, the expression profile of 12,258 porcine transcripts was examined. We found nine genes involved in cell migration mechanisms, that is, PLD1, KIT, LAMA2, MAP3K1, VEGFA, TGFBR3, INSR, TPM1, and RTN4. These genes were upregulated in porcine oocytes before IVM as compared with post-IVM expression analysis. Moreover, important mechanisms of biological interaction between VEGFA-KIT and VEGFA-INSR were also observed. The upregulation and/or downregulation of selected mRNAs expression after microarray assays was checked and approved by real-time quantitative polymerase chain reaction. We suggest that several genes, including LAMA2 or TPM1, encode proteins participating in the formation of the oocyte's protein architecture such as microtubules and kinetochore reorganization. As the expression of all "migration regulatory genes" investigated in this study was significantly upregulated in oocytes before IVM, we conclude that they may contribute to the maturational capability of porcine oocytes. However, migration potency of COCs is not accompanied by achievement of the MII stage by porcine oocytes in vitro. The investigated genes such as PLD1, KIT, LAMA2, MAP3K1, VEGFA, TGFBR3, INSR, TPM1, and RTN4 may be recognized as a new marker of porcine oocytes maturational competence during in vitro culture.

Time- and Dose-Dependent Effects of 17 Beta-Estradiol on Short-Term, Real-Time Proliferation and Gene Expression in Porcine Granulosa Cells.

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 µM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs.

"Bone Development" Is an Ontology Group Upregulated in Porcine Oocytes Before In Vitro Maturation: A Microarray Approach.

Mammalian cumulus-oocyte complexes (COCs) reach full developmental capability during folliculogenesis and oogenesis. It is well recognized that only gametes achieving MII stage after in vivo or in vitro maturation (IVM) are successfully fertilized by a single spermatozoon. Although the process of oocyte nuclear and/or cytoplasmic maturation in pigs is well determined, there exist many differences that promote these processes in vivo and in vitro. Therefore, this study aimed to investigate the differences in RNA expression profiles between porcine oocytes before and after IVM using microarray and real-time quantitative polymerase chain reaction (RT-qPCR) assays. Experiments were performed on oocytes isolated from 55 pubertal crossbred Landrace gilts. The oocytes were analyzed both before and after IVM and only Brilliant Cresyl Blue (BCB)-positive gametes were used for subsequent microarray analysis (Affymetrix) and RT-qPCR analysis. The microarray assay, which measures expression of 12,258 transcripts, revealed 419 differentially expressed transcripts in porcine oocytes, from which 379 were downregulated and 40 were upregulated before IVM compared to those analyzed after IVM. After DAVID analysis, we found eight different transcripts, including IHH, BMP1, WWTR1, CHRDL1, KLF10, EIF2AK3, MMP14, and STC1. Their expression is related to the "bone development" ontology group and was further subjected to hierarchical clusterization. Using RT-qPCR analysis, we confirmed the results of the microarray assay, showing increased expression of the eight genes in oocytes before IVM compared to oocytes after maturation in vitro. It has been suggested that "bone development" belongs to one ontological group involving genes substantially upregulated in porcine oocytes before IVM. We suggest that the gamete mRNA expression profile before IVM may comprise stored transcripts, which are templates for protein biosynthesis following fertilization. We also hypothesize that these mRNAs may be a specific "fingerprint" of folliculogenesis and oogenesis in pigs.

Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation.

Progesterone (P4) and estradiol (E2) play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs) and/or granulosa cells (GCs) might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form) (pgr), progesterone receptor membrane component 1 (pgrmc1), and estrogen-related receptor beta 3 (esrrb3) in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

Silane-modified surfaces in specific antibody-mediated cell recognition.

The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.

Follicular size is associated with the levels of transcripts and proteins of selected molecules responsible for the fertilization ability of oocytes of puberal gilts.

The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (BCB) test, real-time quantitative PCR reaction methods, western-blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts.