Unique hyaluronan structure of expanded oocyte-cumulus extracellular matrix in ovarian follicles.

In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation.

A Systematic Analysis of the Clinical Outcome Associated with Multiple Reclassified Desmosomal Gene Variants in Arrhythmogenic Right Ventricular Cardiomyopathy Patients.

The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331).

Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles: Its Remodeling and Proteolytic Degradation.

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process.

Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations.

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts.

Versican G1 Fragment Establishes a Strongly Stabilized Interaction with Hyaluronan-Rich Expanding Matrix during Oocyte Maturation.

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle.

On the critical evaluation and confirmation of germline sequence variants identified using massively parallel sequencing.

Although massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes.

Targeted next-generation sequencing in Slovak cardiomyopathy patients.

Objectives: For the first time we used targeted next-generation sequencing to detect candidate pathogenic variants in Slovak cardiomyopathy patients.

Background: Targeted next-generation sequencing is considered to be the best practice in genetic diagnostics of cardiomyopathies. However, in Slovakia, with high cardiomyopathies prevalence of 1/440, the current diagnostic tests are still based on Sanger sequencing of a few genes.

Effect of bone morphogenetic protein-15 on gonadotropin-stimulated synthesis of hyaluronan and progesterone in porcine ovarian follicle.

Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures.

The Biological Role of Hyaluronan-Rich Oocyte-Cumulus Extracellular Matrix in Female Reproduction.

Fertilization of the mammalian oocyte requires interactions between spermatozoa and expanded cumulus extracellular matrix (ECM) that surrounds the oocyte. This review focuses on key molecules that play an important role in the formation of the cumulus ECM, generated by the oocyte-cumulus complex. In particular, the specific inhibitors (AG1478, lapatinib, indomethacin and MG132) and progesterone receptor antagonist (RU486) exerting their effects through the remodeling of the ECM of the cumulus cells surrounding the oocyte have been described.

Increased expression of pentraxin 3 after in vivo and in vitro stimulation with gonadotropins in porcine oocyte-cumulus complexes and granulosa cells.

It has been previously shown that multimeric pentraxin 3 (PTX3) is a key component of the cumulus oophorus extracellular matrix (ECM) in mice. In response to the ovulatory LH surge, the cumulus cells assemble a unique ECM that envelopes the oocyte and cumulus cell complex. Importantly, cumuli from PTX3(-/-) mice were defective in their ECM organization and their fertility was impaired.

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance.

Objectives: The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods.

Methods: Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation.

Organization of the expanded cumulus-extracellular matrix in preovulatory follicles: a role for inter-alpha-trypsin inhibitor.

It has been shown that following endogenous gonadotropin surge, oocyte-cumulus complexes (OCC) synthesize hyaluronan (HA) in a process called cumulus expansion. During this process, HA associates with proteins and proteoglycans to form the expanded HA-rich oocyte-cumulus extracellular matrix (ECM), where the heavy chains of the serum derived inter-α-trypsin inhibitor family (IαI) bind covalently to HA. No study has been performed on the occurrence and regulation of this process during oocyte maturation in species other than mouse and pig, although, the heavy chains (of IαI)-HA complex was purified from human amniotic membrane.

Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis.

Objectives: Different applications of high-resolution melting (HRM) analysis have been adopted for a wide range of research and clinical applications. This study compares the performance of selected DNA binding fluorescent dyes for their possible application in HRM.

Design And Methods: We compared twelve dyes with basic properties considered relevant for PCR amplification and melting curve analysis.

Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes.

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin.

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium.

Objective: To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC).

Design: Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling.

Setting: A reproductive biology laboratory.

Regulation of cumulus expansion and hyaluronan synthesis in porcine oocyte-cumulus complexes during in vitro maturation.

Unlabelled: This review deals with molecular mechanisms controlling three important ovarian follicular processes: 1) expansion of the cumulus, 2) synthesis of the hyaluronan (HA), and 3) production of the progesterone in oocyte cumulus complexes (OCCs). The expansion of the mice cumuli induced by FSH or 8-bromo cAMP is dependent upon a specific factor(s) secreted by the oocyte (called "cumulus expansion enabling factor", CEEF). The porcine oocytes produce at least two factors that have influence on the formation and stability of the preovulatory extracellular cumulus matrix (ECM), although oocytectomy does not alter the ability of the cumulus cells to respond to FSH and forskolin by increased cAMP content, HA synthesis, and subsequent cumulus expansion, The net synthesis of HA, during the FSH-stimulated expansion of the OCCs in the presence of serum, correlates directly with accumulation of glycosaminoglycans in the ECM.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes.

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA.

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis.

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix.

Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins.

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively.

Development of functional LH Receptors on pig cumulus-oocyte complexes cultured in vitro by a novel two-step culture system.

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH.

Expression of tumor necrosis factor alpha-induced protein 6 messenger RNA in porcine preovulatory ovarian follicles.

It has been shown previously that tumor necrosis factor alpha-induced protein 6 (TNFAIP6) is essential for formation of the cumulus extracellular matrix and female fertility. Therefore, we studied the expression of TNFAIP6 mRNA in porcine preovulatory follicles. In addition, we asked whether the expression of TNFAIP6 mRNA changes in mural granulosa cells (MGCs) during the periovulatory period or after culture of oocyte-cumulus complexes (OCCs) or MGCs in vitro.

Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, synthesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes.

In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100pM-100microM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro.

Synthesis of tumor necrosis factor alpha-induced protein 6 in porcine preovulatory follicles: a study with A38 antibody.

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum.

Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro.

The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP).