BACKGROUND: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles. METHODS: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed. RESULTS: The maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF. CONCLUSIONS: This work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

• Klíčová slova
• Chromatin configuration, Developmental potential, Follicle size, Gene expression, Growth factors, MAPK activation, Oocyte maturation,
• MeSH
• chromatin metabolismus   MeSH
• fibroblastový růstový faktor 2 * farmakologie   metabolismus   MeSH
• IVM techniky * MeSH
• meióza MeSH
• oocyty metabolismus   MeSH
• ovariální folikul MeSH
• prasata MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• fibroblastový růstový faktor 2 * MeSH

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. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte-cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion-related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

• Klíčová slova
• extracellular matrix, hyaluronan, oocyte–cumulus complex, proteasome,
• MeSH
• buněčné mikroprostředí fyziologie   MeSH
• extracelulární matrix fyziologie   MeSH
• lidé MeSH
• oocyty fyziologie   MeSH
• ovariální folikul fyziologie   MeSH
• proteolýza MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: The gonadotropin-induced resumption of oocyte meiosis in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. Both the gonadotropins and the EGF-like peptides possess the capacity to stimulate resumption of oocyte meiosis in vitro via activation of a broad signaling network in cumulus cells. To better understand the rapid genomic actions of gonadotropins (FSH) and EGF-like peptides, we analyzed transcriptomes of cumulus cells at 3 h after their stimulation. METHODS: We hybridized aRNA from cumulus cells to a pig oligonucleotide microarray and compared the transcriptomes of FSH- and AREG/EREG-stimulated cumulus cells with untreated control cells and vice versa. The identified over- and underexpressed genes were subjected to functional genomic analysis according to their molecular and cellular functions. The expression pattern of 50 selected genes with a known or potential function in ovarian development was verified by real-time qRT-PCR. RESULTS: Both FSH and AREG/EREG increased the expression of genes associated with regulation of cell proliferation, cell migration, blood coagulation and extracellular matrix remodeling. FSH alone induced the expression of genes involved in inflammatory response and in the response to reactive oxygen species. Moreover, FSH stimulated the expression of genes closely related to some ovulatory events either exclusively or significantly more than AREG/EREG (AREG, ADAMTS1, HAS2, TNFAIP6, PLAUR, PLAT, and HSD17B7). In contrast to AREG/EREG, FSH also increased the expression of genes coding for key transcription factors (CEBPB, FOS, ID1/3, and NR5A2), which may contribute to the differing expression profiles of FSH- and AREG/EREG-treated cumulus cells. CONCLUSIONS: The impact of FSH on cumulus cell gene transcription was higher than the impact of EGF-like factors in terms of the number of cell functions affected as well as the number of over- and underexpressed genes. Both FSH and EGF-like factors overexpressed genes involved in the post-ovulatory switch in steroidogenesis and tissue remodelling. However, FSH was remarkably more efficient in the up-regulation of several specific genes essential for ovulation of matured oocytes and also genes that been reported to play an important role in maturation of cumulus-enclosed oocytes in vitro.

• MeSH
• amfiregulin farmakologie   fyziologie   MeSH
• epidermální růstový faktor farmakologie   fyziologie   MeSH
• epiregulin farmakologie   fyziologie   MeSH
• folikuly stimulující hormon farmakologie   fyziologie   MeSH
• kultivované buňky MeSH
• kumulární buňky účinky léků   metabolismus   MeSH
• oocyty účinky léků   metabolismus   MeSH
• prasata MeSH
• regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfiregulin MeSH
• epidermální růstový faktor MeSH
• epiregulin MeSH
• folikuly stimulující hormon MeSH