Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.     

Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.     

Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement

Cytoplasmic dynein is a family of cytoskeletal motor proteins that move towards the minus-end of the microtubules to perform functions in a variety of mitotic processes such as cargo transport, organelle positioning, chromosome movement and centrosome assembly. However, its specific roles during mammalian oocyte meiosis have not been fully defined. Herein, we investigated the critical events during porcine oocyte meiotic maturation after inhibition of dynein by Ciliobrevin D treatment. We found that oocyte meiotic progression was arrested when inhibited of dynein by showing the poor expansion of cumulus cells and decreased rate of polar body extrusion. Meanwhile, the spindle assembly and chromosome alignment were disrupted, accompanied by the reduced level of acetylated α-tubulin, indicative of weakened microtubule stability. Defective actin polymerization on the plasma membrane was also observed in dynein-inhibited oocytes. In addition, inhibition of dynein caused the abnormal distribution of cortical granules and precocious exocytosis of ovastacin, a cortical granule component, which predicts that ZP2, the sperm binding site in the zona pellucida, might be prematurely cleaved in the unfertilized dynein-inhibited oocytes, potentially leading to the fertilization failure. Collectively, our findings reveal that dynein plays a part in porcine oocyte meiotic progression by regulating the cytoskeleton dynamics including microtubule stability, spindle assembly, chromosome alignment and actin polymerization. We also find that dynein mediates the normal cortical granule distribution and exocytosis timing of ovastacin in unfertilized eggs which are the essential for the successful fertilization.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Effects of spermatozoa during in vitro meiosis progression in the porcine germinal vesicle oocytes

We have investigated the effect of co-culture with porcine spermatozoa on in vitro maturation of porcine germinal vesicle (GV) oocytes before fertilization. Most oocytes were arrested at the first prophase of meiosis when oocytes were cultured in TCM 199 alone, but the proportion of oocytes that reached metaphase II was significantly elevated by co-incubation with spermatozoa in vitro. The oocyte maturation effect was observed with intact and parts of spermatozoa (head and tail) collected from adult swine (regardless of source). However, gonocytes from the newborn porcine testis were not able to enhance in vitro maturation of porcine germinal vesicle oocytes. Interestingly, the oocyte maturation effect by spermatozoa was not decreased with heat treatment, but the maturation effect of oocyte treatment disappeared with exposure to detergent in sperm suspension. Porcine spermatozoa were also observed to stimulate meiosis of oocytes, which was maintained at meiotic arrest using dibutyryl cyclic AMP or forskolin. The study suggests that (i) membrane of porcine spermatozoa contains a substance(s) that can enhance in vitro maturation of oocytes prior to fertilization, (ii) the putative meiosis-enhancing substance(s) of spermatozoa from adult testes retains the oocyte maturation effect during transportation of spermatozoa through epididymis, and (iii) the putative meiosis-enhancing substance(s) is able to overcome the inhibitory effect of dibutyryl cyclic AMP or forskolin by inducing germinal vesicle breakdown of porcine cumulus-oocyte complexes maintained in meiotic arrest.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes

Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes

During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, we used confocal immunofluorescence microscopy to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibited microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promoted MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupted the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA.     

The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis

Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.     

Effect of microtubule reactive drugs on steroid- and centrifugation-induced germinal vesicle migration during goldfish oocyte meiosis

During the process of progestogen-induced meiotic maturation in the goldfish oocyte, the oocyte nucleus (germinal vesicle, GV) migrates to the sperm entry site or micropyle at the animal pole. Following GV migration (GVM) to the micropyle, the nuclear membrane undergoes dissolution (GVD) and the cell enters metaphase I in preparation to generate the first polar body. Microtubule destabilizing drugs including colcemid, nocodazole and vinblastine were found to elicit GVM, mimicking the process which occurs just prior to the prophase I-metaphase I transition during steroid induced oocyte meiotic maturation. In addition, these drugs enhanced the induction of GVM by 17 alpha, 20 beta dihydroxy-4-pregnen-3-one, a potent, naturally occurring meiotogenic steroid in this species. By contrast, taxol, a microtubule stabilizing drug, was found to inhibit steroid induced GVM. A new assay for centrifugation induced GVM was applied to the goldfish oocyte in order to assess effects of steroids and drugs on GVM, without the complication of GVD or the restrictions imposed by the slow time course of naturally occurring GVM. The effective centrifugal force (ECF) required to elicit GVM in 50% of the oocytes (ECF50) decreased significantly after short incubations (1-5 hr) of oocytes with either 17 alpha,20 beta dihydroxy-4-pregnen-3-one or microtubule disrupting drugs (i.e., colcemid, nocodazole, or vinblastine). A working hypothesis, modeled after the effects of microtubule disrupting agents on intermediate filament arrays in somatic cells, is proposed in which a small number of microtubules or other polymeric tubulin units are responsible for maintaining a cytoskeletal array.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.     

The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.     

哺乳动物卵母细胞生发泡移植

生发泡(GV)移植是指将GV期卵母细胞的GV移入到去核的受体细胞(GV期卵母细胞、MII期卵母细胞或受精卵)透明带下,经融合形成一个重组卵的过程。GV移植对研究卵母细胞的细胞周期调控、成熟及受精时细胞核与细胞质之间的相互作用非常重要,可用于研究卵母细胞减数分裂异常和与年龄相关变化之间的关系及细胞质衰老与卵母细胞非整倍性之间的关系。现简要介绍了GV移植的基本程序,GV核体与胞质体的融合,重组卵的培养条件,重组卵成熟后的受精、人工激活和胚胎发育能力以及GV移植的意义。     

The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm

The chromatin of Xenopus laevis sperm nuclei was induced to decondense, swell and form mitotic chromosomes following its injection into mature Rana pipiens oocytes. In contrast, the sperm chromatin did not decondense or form mitotic chromosomes when injected into oocytes from which the germinal vesicle (GV) was removed prior to the initiation of maturation. Injection into enucleated oocytes of the material extracted from manually-isolated GVs restored their ability to decondense sperm nuclei. This soluble GV material was stable at 18 °C for 16 h but was inactivated by heating to 80 °C for 10 min. We examined the distribution of this GV material in a cytoplasmic preparation from activated eggs which can induce sperm pronuclear formation in vitro. The cytoplasmic preparation was separated into soluble and particulate fractions by centrifugation and then each fraction was injected into enucleated eggs to determine whether or not it restored the ability to decondense sperm nuclei. We found that the soluble, but not the particulate fraction could restore the ability to decondense sperm nuclei to enucleated oocytes. This result clearly indicates that the soluble fraction contains most of the GV material required for chromatin decondensation. However, since the soluble fraction fails to decondense sperm chromatin in vitro in the absence of material from the paticulate fraction, sperm pronuclear formation appears to require both the soluble material derived from the GV and particulate material which can develop in the oocyte cytoplasm in the absence of the GV.     

Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

Establishment of animal-vegetal polarity during maturation in ascidian oocytes

Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal-vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal-vegetal (a-v) axis to establish the embryonic (D-V, A-P) axes. To understand how this radial a-v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a-v axis occurs after maturation in several steps which take 3-4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians.     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.