Exposure of oocytes to the Fusarium toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal embryo development in pigs

Fungi of the Fusarium species can infect food and feed commodities and produce the mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte maturation were examined. Pig oocytes were matured in the presence of ZEA (a mycotoxin with estrogenlike activity), 17beta-estradiol, and DON (all 3.12 micromol/L). Zearalenone, 17beta-estradiol, and DON inhibited oocyte maturation and caused approximately 34% of the oocytes to form an aberrant spindle. Different ratios of ZEA:DON did not lead to a more severe inhibition of oocyte maturation. Both mycotoxins caused abnormal formation of the meiotic spindle. The developmental competence of oocytes matured in the presence of mycotoxins was further investigated after in vitro fertilization. Presence of ZEA (3.12 micromol/L) during maturation reduced the percentages of oocytes that cleaved and formed a blastocyst to about 12%, compared with 25% of control oocytes. Maturation in the presence of equimolar concentrations of DON was not compatible with development. The ploidy of blastomeres from blastocysts derived from mycotoxin-exposed oocytes was analyzed with fluorescent in situ hybridization. All blastocysts, even those from the control group, contained at least one blastomere with abnormal ploidy, but the variation in the percentages of aneuploid blastomeres was significantly larger in embryos from oocytes exposed to mycotoxins. It is concluded that ZEA and DON can lead to abnormal spindle formation, leading to less fertile oocytes and embryos with abnormal ploidy, and that the effects of ZEA and DON are not synergistic.     

Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.     

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Spindle Dynamics during Meiosis in Drosophila Oocytes

Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro–activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.     

Use of polarized light microscopy in porcine reproductive technologies

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) could be useful for a non-invasive evaluation of the meiotic spindle and may allow removal of nuclear structures without fluorochrome staining and ultraviolet exposure. In this study, PLM was used to assess its potential application in porcine reproductive technologies. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine its effects on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM; and to assess the efficiency oocyte enucleation assisted with PLM. In the first experiment, the presence of microtubule-polymerized protein was assessed and confirmed in oocytes (n = 117) by immunostaining and chromatin detection. In the second experiment, oocytes (n = 160) were exposed or not (controls) to PLM for 10 minutes, and then parthenogenetically activated and cultured in vitro. In the third experiment, development competence of oocytes with a positive or negative signal to PLM was analyzed after in vitro fertilization. Finally, oocytes (n = 54) were enucleated using PLM as a tool to remove the meiotic spindle. A positive PLM signal was detected in 98.2 % of the oocytes, which strongly correlated (r = 1; p < 0.0001) with the presence of microtubule-polymerized protein as confirmed by immunostaining. Oocytes exposed to PLM did not differ significantly from controls on cleavage, total blastocyst, expanded blastocyst rates and total cell numbers. The percentage of oocytes at the MII stage and blastocyst formation rate in the negative PLM group significantly differed from control and PLM positive groups. Overall efficiency of spindle removal using the PLM-Oosight system was 92.6%. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured porcine oocytes and does not exert detrimental effects on porcine oocyte developmental competence. Selecting oocytes by the presence of a PLM signal provides limited improvement on IVF results. Finally, PLM appears as an efficient method to enucleate porcine oocytes.     

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.     

Role of AMPK throughout meiotic maturation in the mouse oocyte: Evidence for promotion of polar body formation and suppression of premature activation

This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell‐enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK‐activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK‐activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C‐induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles, and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. Mol. Reprod. Dev. 77:888–899, 2010. © 2010 Wiley‐Liss, Inc.     

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.     

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes

The effects of the pesticide carbendazim (MBC) on the in vitro meiotic maturation of mouse oocytes were evaluated using conventional and confocal fluorescence microscopy. The response of oocytes exposed to 0, 3, 10, or 30 μM MBC during meiotic maturation was analyzed with respect to chromosome organization, meiotic spindle microtubules, and cortical actin using fluorescent labels for each of these structures. Continuous exposure to MBC during the resumption of meiosis resulted in a dose-dependent inhibition of meiotic cell cycle progression at metaphase of meiosis-1. Drug exposure at the metaphase-anaphase transition of meiosis-1 did not interfere with cell cycle progression to metaphase-2 except at high concentrations (30 μM). At the level of spindle microtubule organization, MBC caused a loss of nonacetylated microtubules and a decrease in spindle size at 3 or 10 μM concentrations. Thirty μM MBC prevented spindle assembly when added at the beginning of meiotic maturation or caused spindle pole disruption and fragmentation when added to preformed spindles. Spindle disruption involved a loss of phosphoprotein epitopes, as monitored by MPM-2 staining, and resulted in the appearance of dispersed chromosomes that retained a metaphase-plate location on spindle fragments associated with the oocyte cortex. Polar body extrusion was impaired by MBC, and abnormal polar bodies were observed in most treated oocytes. The results suggest that MBC disrupts cell cycle progression in mouse oocytes by altering meiotic spindle microtubule stability and spindle pole integrity. Mol. Reprod. Dev. 46:351–362, 1997. © 1997 Wiley-Liss, Inc.     

CDC20 downregulation impairs spindle morphology and causes reduced first polar body emission during bovine oocyte maturation

The cell division cycle protein 20 (CDC20) is an essential regulator of cell division, encoded by the CDC20 gene. However, the role of CDC20 in bovine oocyte maturation is unknown. In this study, CDC20 morpholino antisense oligonucleotides (MOs) were microinjected into the cytoplasm of bovine oocytes to block the translation of CDC20 mRNA. CDC20 downregulation significantly reduced the rate of first polar body emission (PB1). Further analysis indicated that oocytes treated with CDC20 MO arrested before or at meiotic stage I with abnormal spindles. To further confirm the functions of CDC20 during oocyte meiotic division, CDC20 MOs were microinjected into oocytes together with a supplementary PB1. The results showed that newly synthesized CDC20 was not necessary at the meiosis II-to-anaphase II transition. Our data suggest that CDC20 is required for spindle assembly, chromosomal segregation, and PB1 extrusion during bovine oocyte maturation.     

The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts

The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons—summer (S: January to March) and winter (W: June to August)—and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus–oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding estrous cycle.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.     

Cytoplasmic microtubular dynamics and chromatin organization during mammalian oogenesis and oocyte maturation.

A chronological series of coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. Timely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nucleation capacity of centrosomes, underscore key nuclear events during the progressive stages of meiosis I and II. The regulation of these transitional states during meiosis is discussed with respect to hormonal influences imparted to the oocyte within the follicular microenvironment, and the possible ways in which environmental perturbations may result in defective chromosomal partitioning during meiosis.