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The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles ( 3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

The zona pellucida (ZP) of mature pig oocytes is believed to consist of a dense filamentous meshwork, less compact on the inner and outer faces. The uneven surface of the ZP is made of unordered and stretched fibrils surrounding deep funnels which are the openings of the radial canaliculi. The topography of the ZP surface may contribute to the initial interplay between male and female gametes. Using cytochemical techniques for transmission electron microscopy (TEM), such as tannic acid and ruthenium red treatments, we found that the ZP of pig oocytes was essentially made of bundles of fibrils distributed in concentric layers (except in the innermost and outer parts). A correlation appears between the dense structure of the core layer of the ZP and its texture: it is constituted of superposed layers of fibril bundles, whereas only a random meshwork is found in a very thin innermost and in the outer layer. The fascicular configuration may control the permeability of the ZP, giving its semi-rigidity and elasticity, and may facilitate sperm penetration. The liquid crystal-like design of the core layer of the ZP is similar to textures found in the the vitelline envelope (zona radiata) of other vertebrates and possibly of all the deuterostomes. Such texture is probably related to the unique ZP protein composition and to a coordinated synthesis.

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2-3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

The relationship between cytoskeleton and morphology of cumulus granulosa cells in expanding bovine oocyte-cumulus complexes (OCCs) cultured in vitro has been investigated by the means of indirect immunofluorescence and transmission electron microscopy. The round-shaped cells in unstimulated control OCCs displayed a homogeneous distribution of cytoskeletal networks and cytoplasmic organelles. Luteinizing hormone (LH) stimulation caused the redistribution of microfilaments (MFs), accelerated the development of Golgi apparatus, and led to the generation of lipid droplets in cumulus cells. These changes culminated in the elongation and polarization of cumulus cells and in the extension of the cytoplasmic networks of microtubules (MTs) and intermediate filaments (IFs) into the newly formed cytoplasmic projections. The culture of OCCs in the presence of microfilament disruptor cytochalasin B prevented cumulus expansion, formation of cellular projections and cell elongation and suppressed the development of the Golgi apparatus. On the contrary, cytochalasin had no effect on the abundance and distribution of lipid droplets and on the integrity of IFs and MTs. The present data support the hypothesis that the response of cumulus granulosa cells to LH is partially mediated by F-actin.

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

The development of fertilized ova in explanted parts of oviducts was studied from 12 to 36 hours after fertilization, and from 36 to 84 hours after fertilization. No egg mortality during cultivation, nor any slowing of development could be detected in either experiment. It may be presumed that the mortality of mouse ova during the period before implantation has no essential effect on the resulting fertility. During cultivation of oviducts for 144 hours, there occurred in most blastocysts herniation of their walls resulting in a formation similar to the blastocyst, and a migration of a disorganized mass of cells, some of which resembled the giant trophoblastic cells. After transplantation of ova fertilized in vitro to recipients, in four cases there occurred nidation and subsequent normal development of the embryo.