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.     

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment

Mitotic spindles assemble from two centrosomes, which are major microtubule‐organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an “inside‐out” mechanism, ending with establishment of the poles. We used HSET (kinesin‐14) as a tool to shift meiotic spindle assembly toward a mitotic “outside‐in” mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic‐like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique “inside‐out” mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.     

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.     

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.     

Loss of HDAC3 contributes to meiotic defects in aged oocytes

Maternal age‐related decline in oocyte quality is associated with meiotic defects, but the underlying mechanisms remain to be explored. Histone deacetylase 3 (HDAC3) has been shown to govern multiple cellular events via deacetylating diverse substrates. We previously found that HDAC3 could promote meiotic apparatus assembly in mouse oocytes. In the present study, we identified a substantial reduction in HDAC3 protein in oocytes from old mice. Importantly, overexpression of HDAC3 in old oocytes not only partially prevents spindle/chromosome disorganization, but also significantly lowers the incidence of aneuploidy. Meanwhile, we noticed the elevated acetylation level of α‐tubulin in oocytes derived from old mice. By employing site‐directed mutagenesis, we showed that acetylation‐mimetic mutant tubulin‐K40Q disrupts the kinetochore–microtubule attachments and results in the assembly failure of meiotic apparatus in mouse oocytes. Importantly, forced expression of tubulin‐K40R (nonacetylatable‐mimetic mutant) was capable of alleviating the defective phenotypes of oocytes from aged mice. To sum up, this study uncovers that loss of HDAC3 represents one potential mechanism mediating the effects of advanced maternal age on oocyte quality.     

Hypoxanthine (HX) inhibition of in vitro meiotic resumption in goat oocytes

To improve in vitro maturation and to understand the mechanism for meiotic resumption of oocytes, meiotic progression, and its control by hypoxanthine (HX) were studied in goat oocytes. Ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) and follicular fluid were collected from follicles of different surface diameters (SDs). The meiotic competence and progression of oocytes were observed, and the concentration of HX in the follicular fluid and culture media was measured by high-performance liquid chromatography (HPLC). Full meiotic competence of goat oocytes was acquired in follicles of >/=1.5 mm in SD with 90% of the oocytes developing to metaphase II (MII) stage after 24 hr in culture. The HX concentration in follicular fluid decreased with follicle development, from the highest level of 1.16 mM in /=5 mm follicles. HX inhibited meiotic resumption of goat oocytes in a concentration-related manner but this inhibitory effect declined gradually. When we renewed the medium at 4 hr of HX-199 (TCM-199 supplemented with 4 mM HX) culture, the percentage of oocytes with intact germinal vesicle (GV) did not increase but decreased significantly instead. HPLC measurement of HX in the HX-199 culture drops indicated that the HX concentration declined from 0 hr to 4 hr of culture and after medium renewal at 4 hr of culture. By adding dibutyryl cAMP (db-cAMP) at medium renewal, we found that db-cAMP held up the decline of GV percentages. Together, these results were consistent with the possibility that the decline of HX inhibitory effect was not due to HX depletion but rather due to the negative feedback of the metabolites on its further uptake by oocytes. Goat oocytes were capable of normal nuclear maturation and activation after temporal arrest by HX, but prolonged exposure to HX induced spontaneous activation.     

Estradiol synergism and inhibition of insulin-induced meiotic maturation in Rana pipiens oocytes in vitro

Estradiol 17-β (E2) was found to either inhibit or synergize Na-insulin (Ins)-induced meiotic maturation of Rana oocytes. Inhibition of Ins activity occurred in the presence of the follicular investments of the oocyte; synergism with Ins occurred in oocytes denuded of the follicle wall. Similarly, co-incubation of E2 with frog pituitary homogenate (FPH) or pregnenolone (Pe) significantly decreased meiotic reinitiation as determined by germinal vesicle dissolution (GVD) in follicle-enclosed oocytes. By contrast, E2 had no consistently significant effect on progesterone (P)-induced meiosis in follicle-enclosed oocytes. Furthermore, E2 had no significant effect, either inhibitory or synergistic, on Pe- or P-induced GVD of denuded oocytes. Thus, of the meiotogens tested (Ins, P, Pe, FPH), all but P were consistently inhibited by E2 in the presence of the follicle wall. Na-insulin was the only meiotogen tested (Ins, P, Pe) which was potentiated by E2 in denuded oocytes, However, when E2 and Ins were spatially separated on the surface of individual intact follicles, the result was synergism of Ins-induced GVD rather than inhibition. These results suggest that Ins acts to induce GVD in the denuded oocyte through a mechanism distinct from that used by P (ie, Ins mechanism allows E2 synergism while the P mechanism does not). The E2 inhibitory effect on Ins-induced GVD appears to be dependent upon simultaneous exposure of follicle wall tissue to mixtures of E2 and Ins. The synergistic effect of E2 on Ins-induced GVD is dependent upon the simultaneous exposure of the oocyte surface to Ins and E2, either as a homogenous mixture in the case of denuded oocytes or as single substances at independent sites, for follicle-enclosed oocytes.     

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

The maize desynaptic1 mutation disrupts meiotic chromosome synapsis

In most eukaryotes, homologous chromosomes undergo synapsis during the first meiotic prophase. A consequence of mutations that interfere with the fidelity or completeness of synapsis can be failure in the formation or maintenance of bivalents, resulting in univalent formation at diakinesis and production of unbalanced spores or gametes. Such mutations, termed desynaptic mutations, can result in complete or partial sterility. We have examined the effect of the maize desynaptic1-9101 mutation on synapsis, using the nuclear spread technique and electron microscopy to examine microsporocytes ranging from early pachytene until the diplotene stage of prophase I. Throughout the pachytene stage, there was an average of about 10 sites of lateral element divergence (indicating nonhomologous synapsis), and during middle and late pachytene, an average of two and three sites of foldback (intrachromosomal) synapsis, per mutant nucleus, respectively. By the diplotene stage, the number of sites of lateral element divergence had decreased to seven, and there was an average of one foldback synapsis site per nucleus. Lateral element divergence and foldback synapsis were not found in spread pachytene nuclei from normal plants. These results imply that the normal expression of the dsy1 gene is essential for the restriction of chromosome synapsis to homologues. The abundance of nonhomologous synapsis and the persistence of extended stretches of unsynapsed axial elements throughout the pachytene stage of dsy1–9101 meiocytes suggests that this mutation disrupts both the fidelity of homology search and the forward course of the synaptic process. This mutation may identify a maize mismatch repair gene. Dev. Genet. 21:146–159, 1997. © 1997 Wiley-Liss, Inc.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro

Follicular oocytes collected prior to the expected time of the LH surge from PMSG-treated immature rats were incubated cummulus-intact (with or without LH) or cumulus-free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm-related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm-related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.     

New evidence that SAC can tolerate misaligned chromosomes in mouse oocytes

Comment on: Sebestova J, et al. Cell Cycle 2012; 11:3011-8.     

Age‐dependent aneuploidy in mammalian oocytes instigated at the second meiotic division

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.     

Activated proline‐rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte

Proline‐rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p‐PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p‐PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p‐PYK2 colocalizes with γ‐tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p‐PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.     

Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes

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