Joanna Budna

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.

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.

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.

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.

Does Porcine Oocytes Maturation in Vitro is Regulated by Genes Involved in Transforming Growth Factor Beta Receptor Signaling Pathway

Summary The oocyte growth and development in follicular environment are substantially accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). During these processes, the mammalian gametes reach full maturational stage and may be further successfully fertilized by single spermatozoon. These unique mechanisms are regulated by expression of clusters of genes and their biochemical signaling pathways. In this article we described differential expression pattern of transforming growth factor beta (TGFB) gene superfamily in porcine oocytes before and after in vitro maturation (IVM). We performed Affymetrix® microarray assays to investigate the TGFB-related genes expression profile in porcine immature oocytes and gametes cultured for 44h in vitro. In results we found 419 different genes, 379 genes with lower expression, and 40 genes characterized by increased RNA profile. Moreover, significant up-regulation of 6 genes belonging to TGFB signaling pathway such as: TGFBR3, SMAD4, FOS, KLF10, ID1, MAP3K1 in immature porcine oocytes (before IVM), was also observed. It may be suggested that genes involved in TGFB-related signaling pathway are substantially regulated before IVM. Furthermore, these genes may play a significant role during early stages of nuclear and/or cytoplasmic porcine oocytes maturation. The investigated transcripts may be also recommended as the markers of oocytes maturational capability in pigs.

Positive Regulation of Macromolecule Metabolic Process Belongs to the Main Mechanisms Crucial for Porcine Oocytes Maturation

Summary The mammalian oocytes maturation is the compound process that involves morphological and molecular changes. These modifications include storage of macromolecules, which are crucial for proteins biosynthesis during periimplantation stages of embryo development. This study was aimed to investigate the genes expression profile encoding macromolecules important for regulation of proper porcine oocytes maturation. The porcine oocytes were collected from large ovarian follicles and analyzed both before and after in vitro maturation (IVM). Additionally, to check the developmental competence status, brilliant crezyl blue test (BCB) was performed. The obtained cDNA was used for biotin labeling and fragmentation by AffymetrixGeneChip® WT Terminal Labeling and Hybridization (Affymetrix). The preliminary analysis of the scanned chips was performed using AffymetrixGeneAtlasTM Operating Software. The created CEL files were imported into downstream data analysis software. In results, we found expression of 419 different genes, 379 genes were down-regulated and 40 genes were up-regulated in relation to the oocyte transcriptome before in vitro procedure. We observed up-regulation of all genes involved in “positive regulation of macromolecule metabolic process” before IVM as compared to transcriptional profile analyzed after IVM. In conclusion, we suggested that genes encoding proteins involved in macromolecule metabolism are important for achieving of porcine oocytes maturational stage. Moreover, the “activity of macromolecules metabolism” is much more increased in immature oocytes.

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

BackgroundThe 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.MethodsOocytes 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.ResultsThe 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 (TGFβR3), decapentaplegic homolog 4 (SMAD4), and inhibitor of DNA binding 1 (ID1).ConclusionsIncreased expression of CHRDL1, FST, TGFβR3, 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.

Analysis of fructose and mannose – regulatory peptides signaling pathway in porcine epithelial oviductal cells (OECs) primary cultured long-term in vitro

Abstract The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the compound process responsible for proper oocytes transport and successful fertilization. However, the main mechanisms which regulated this process are still not entirely known. Moreover, the OECs metabolism, which may be identified as the “cellular activity” marker, is poorly recognized. In this study we investigated the fructose and mannose metabolic pathway in porcine OECs primary long-term cultured in vitro. In our study, we employ a primary long term in vitro culture (IVC) and microarray approach (the Affymetrix microarray were used for analysis of transcriptomic profile of OECs) for expression levels analysis. We found that from the whole analyzed transcriptome, 1537 genes were upregulated and 995 were down regulated after 7 days of culture, 1471 genes were upregulated and 1061 were downregulated after 15 days of culture and 1329 genes were upregulated and 1203 were downregulated after 30 days of culture. Moreover, the differential expression of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 at 24 hours, 7 day, 15 day and 30 day, was also observed. We suggested that fructose and mannose metabolism may be important molecular bio-marker of porcine OECs capability in in vitro model. The metabolic profile is significantly accompanied by cells proliferation in vitro. The transcriptomic profile of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 expression may be identified as “fingerprint” of fructose and mannose metabolism in OECs as well as involved in cellular in vitro developmental capacity in pigs.

The blood vessels development, morphogenesis and blood circulation are three ontologic groups highly up-regulated in porcine oocytes before in vitro maturation

Abstract The mammalian oocytes undergo significant biochemical and structural modifications during maturation both in vitro and in vivo. These changes involve chromatin reorganization and modification within metabolic status of cytoplasmic organelles. After oocytes’ successful maturation the substantially increased storage of RNA was observed. Moreover, the early embryo interaction with maternal endometrial tissue after fertilization is up to now considered as the main marker of proper embryo implantation and early growth. In this study, we first investigated the expression profile of genes involved in blood vessel formation and blood circulation in porcine oocytes before and after in vitro maturation. The cumulus-oocyte complexes were collected from pubertal Landrace gilts and classified as before in vitro maturation (in Vivo) or after in vitro maturation (in Vitro). The RNA was isolated from these two experimental groups and analyzed using Affymetrix microarrays. We found an increased expression of genes involved in ontological groups such as “blood circulation” (TPM1, ECE1, ACTA2, EPHX2, EDNRA, NPR2, MYOF, TACR3, VEGFA, GUCY1B3), “blood vessel development” (ANGPTL4, CYR61, SEMA5A, ID1, RHOB, RTN4, IHH, ANGPT2, EDNRA, TGFBR3, MYO1E, MMP14), and “blood vessels morphogenesis” (ANGPT2, as well as other common transcripts) in in Vivo group as compared to decreased expression of these genes in in Vitro group of oocytes. It has been suggested that investigated genes undergo significant expression before in vitro maturation, when enhanced storage of large amount of RNA takes place. Creating templates for synthesis of proteins is required for formation of fully mature gametes and early embryo growth. Therefore we hypothesized that the processes of vascularization and/or angiogenesis reach a high activity in immature oocytes and are distinct from achievement of maturational stage by oocytes in pigs.

Amino acids metabolism and degradation is regulated during porcine oviductal epithelial cells (OECs) primary culture in vitro – a signaling pathways activation approach

Abstract The ovary is part of the reproductive system, possessing very important functions in the reproduction process (ovum and embryo transfer, providing a suitable environment for sperm capacitation, etc.). There are two types of cells in the fallopian tubes: alveolar and secretive cells. These study shows the metabolic processes in pig oviductal epithelial cells associated with the activation of signaling pathways of amino acids metabolism and degradation during long-term in vitro culture. Oviductal epithelial cells from 45 colonies in the anestrous phase of the estrous cycle have been utilized in this study. RNA extract from the OEC primary cultures was pooled after 24h, 7days, 15 days and 30 days from the beginning of culture and the transcriptome investigated by Affymetrix® Porcine Gene 1.1 ST. From the whole transcript that consisted of 2009 different genes, 1537 were upregulated and 995 were downregulated after 7 days of culture, 1471 were upregulated and 1061 were downregulated after 15 days of culture and 1329 were upregulated and 1203 were downregulated after 30 days of culture. The results of these studies provide, for the first time, information on the activation of metabolic pathways of amino acids such as valine, leucine, isoleucine, cysteine, and methionine in the investigated tissue. They also indicate genes that may be OECs-specific genetic markers that are expressed or upregulated during long-term in vitro culture.