PKB/AKT is involved in resumption of meiosis in mouse oocytes

BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

Mitochondrial reorganization during resumption of arrested meiosis in the mouse oocyte

Correlated nuclear and cytoplasmic reorganizations during the 14 hr of reactivated meiosis in vivo and in vitro were examined in the laboratory mouse. Observations of living oocytes by differential interference contrast microscopy, and by fluorescent microscopy with nontoxic mitochondrial and DNA-specific probes, enabled us to determine that the major cytoplasmic reorganization involved two mitochondrial translocations associated with two stages of nuclear maturation. These observations were confirmed at the fine structural level by parallel transmission electron microscopy. Mitochondria translocate to the perinuclear region during formation of the first metaphase spindle and subsequently disperse during abstriction of the first polar body. Determinations of frequency of maturation in more than 2,900 normal oocytes, and in more than 1,100 oocytes in which germinal vesicle breakdown was reversibly inhibited, indicated that mitochondrial redistributions are a normal and probably necessary feature of reactivated meiosis in the laboratory mouse. We suggest that these two rapid translocations serve to concentrate mitochondria for localized activities that require elevated levels of adenosine triphosphate.     

Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.     

Specialization of CDK1 and cyclin B paralog functions in a coenocystic mode of oogenic meiosis

Oogenesis in the urochordate, Oikopleura dioica, occurs in a large coenocyst in which vitellogenesis precedes oocyte selection in order to adapt oocyte production to nutrient conditions. The animal has expanded Cyclin-Dependant Kinase 1 (CDK1) and Cyclin B paralog complements, with several expressed during oogenesis. Here, we addressed functional redundancy and specialization of CDK1 and cyclin B paralogs during oogenesis and early embryogenesis through spatiotemporal analyses and knockdown assays. CDK1a translocated from organizing centres (OCs) to selected meiotic nuclei at the beginning of the P4 phase of oogenesis, and its knockdown impaired vitellogenesis, nurse nuclear dumping, and entry of nurse nuclei into apoptosis. CDK1d-Cyclin Ba translocated from OCs to selected meiotic nuclei in P4, drove meiosis resumption and promoted nuclear envelope breakdown (NEBD). CDK1d-Cyclin Ba was also involved in histone H3S28 phosphorylation on centromeres and meiotic spindle assembly through regulating Aurora B localization to centromeres during prometaphase I. In other studied species, Cyclin B3 commonly promotes anaphase entry, but we found O. dioica Cyclin B3a to be non-essential for anaphase entry during oogenic meiosis. Instead, Cyclin B3a contributed to meiotic spindle assembly though its loss could be compensated by Cyclin Ba.     

Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes

Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with ³H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of ³H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG. During coculture, cattle cumulus cells were closely associated with the basement membrane, but no gap junctions were formed among heterologous granulosa cells. These results suggest that an inhibitory factor secreted by pig granulosa cells is not species specific and it can act in vitro without the mediation of gap junctions. © 1993 Wiley-Liss, Inc.     

Role of divalent cations in the resumption of meiosis of rat oocytes.

Resumption of meiotic maturation was induced in follicle-enclosed rat-oocytes by treatment with the divalent cationophore A23187 (10(-5)M). However, the same effect was attained by incubation in Ca++-deficient medium, even in the presence of EDTA or EGTA (1mM). The stability of the first polar body was increased under Ca++-deficient conditions. Neither the ionophore nor Ca++-deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.     

Towards a new understanding on the regulation of mammalian oocyte meiosis resumption

Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in granulosa cells in oocyte meiosis resumption. Unresolved questions and future directions on research regarding signaling changes in follicle cells and oocytes as well their communication in response to the gonadotropin surge are addressed in this review.     

Spatiotemporal pattern of calmodulin and [Ca2+]i is related to resumption of meiosis in mouse oocytes

During meiotic maturation, mammalian oocytes undergo a series of morphological and physiological changes that prepare them for fertilization. Calcium-initiated signaling is thought to trigger these processes. In this study, we examine the spatio-temporal pattern of calcium and calmodulin (CaM), its downstream receptor, in order to investigate their association with meiotic maturation. Intracellular free calcium and activated CaM levels were measured using the fluorescent probes Calcium Green-1 and TA-CaM, respectively. The distribution patterns were examined using confocal microscopy. Both calcium and activated CaM showed a dynamic spatiotemporal distribution during meiotic maturation. After release from IBMX buffer, calcium was found to periodically translocate from the perinuclear region to the germinal vesicle (GV) in 90 s intervals. After 90 min, calcium stopped oscillating and became concentrated within the GV. After a further 60 min, the GV broke down and calcium dispersed into the ooplasm, but calcium levels were slightly lower here than in the original nuclear region. Activated CaM also showed a dynamic patterning process similar to calcium. Taking the data from calcium chelation and CaM inhibition together, our results suggest that the dynamic distribution patterns of calcium and activated CaM are crucial for oocyte maturation.     

MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes

Resumption of meiosis of mammalian oocytes is facilitated by the maturation promoting factor (MPF) and accompanied by activation of mitogen activated protein kinases (MAPK) which are phosphorylated by the MAPK kinase (MEK). In this study we examined the effects of PD 98059, which inhibits the activity of MEK, on in vitro maturation of pig oocytes. Cumulus-oocyte complexes (COCs) were cultured in the presence or absence of the drug (50 microM) for various time periods. To elucidate the influence of cumulus cells, COCs were first cultured in inhibitor-free medium, subsequently denuded, and incubated further in PD 98059 supplemented medium. Reversibility of drug action as tested following PD 98059 treatment of COCs by transferring them to drug-free medium. Culture of COCs in medium supplemented with PD 98059 prevents resumption of nuclear maturation in the majority of COCs. This inhibition was reversible and accompanied by a non-activation of both MAP and MPF. Addition of the MEK inhibitor to extracts of in vitro matured oocytes revealed that the kinase activities were not directly influenced by the inhibitor, suggesting a link between MAP and MPF kinases. Preincubation of COCs in inhibitor-free medium for 6 h followed by further culture of COCs or denuded oocytes in the presence of PD 98059 for various periods resulted in elevated MAP and MPF kinase activities, indicating an early and transient MEK signalling in the oocyte itself. These results support the idea that MAP and MPF are involved in the induction of germinal vesicle breakdown in porcine oocytes.     

The cytoskeleton organizes germ nuclei with divergent fates and asynchronous cycles in a common cytoplasm during oogenesis in the chordate Oikopleura

Germline cysts are conserved structures in which cells initiating meiosis are interconnected by ring canals. In many species, the cyst phase is of limited duration, but the chordate, Oikopleura, maintains it throughout prophase I as a unique cell, the coenocyst. We show that despite sharing one common cytoplasm with meiotic and nurse nuclei evenly distributed in a 1:1 ratio, both entry into meiosis and subsequent endocycles of nurse nuclei were asynchronous. Coenocyst cytoskeletal elements played central roles as oogenesis progressed from a syncytial state of indistinguishable germ nuclei, to a final arrangement where the common cytoplasm had been equally partitioned into resolved, mature oocytes. During chromosomal bouquet formation in zygotene, nuclear pore complexes clustered and anchored meiotic nuclei to the coenocyst F-actin network opposite ring canals, polarizing oocytes early in prophase I. F-actin synthesis was required for oocyte growth but movement of cytoplasmic organelles into oocytes did not require cargo transport along colchicine-sensitive microtubules. Instead, microtubules maintained nurse nuclei on the F-actin scaffold and prevented their entry into growing oocytes. Finally, it was possible to both decouple meiotic progression from cellular mechanisms governing oocyte growth, and to advance the timing of oocyte growth in response to external cues.     

Effect of cumulus and granulosa cells on meiosis resumption in murine oocytes in vitro

Effects of different follicular cell types on resumption of meiosis were studied. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), cumulus cells (CCs) and mural granulosa cells (GCs) were used. Oocytes were obtained from mature gonadotrophin-stimulated and unstimulated mice. The resumption of meiosis was assessed by the germinal vesicle breakdown (GVBD) at the end of cultivation. It has been shown that GCs produced a meiosis activating substance due to gonadotrophin stimulation; for meiosis resumption connections between CCs and the oocyte were not necessary, but the very production of the meiosis activating substance, was, however, dependent on the initial connection between CCs and the oocyte. The presence of oocyte was necessary for stimulating CCs to produce a diffusible heat stable meiosis activating substance; gonadotrophins induced CCs to produce a diffusible thermostable meiosis activating substance. This substance induced, in a paracrine fashion, resumption of meiosis directly. It is proposed that the heat stable meiosis activating component of the used media from gonadotrophins-stimulated CEO may belong to a kind of meiosis activating sterols, previously isolated from human follicular fluid and from adult bull testes.     

Lead cations affect the control of both meiosis arrest and meiosis resumption of the mouse oocyte in vitro at least via the PKC pathway

The aim of this study was to determine in vitro whether lead has a direct cytotoxic effect on the female gamete or through its surrounding somatic cells. We had previously demonstrated that it partly accumulates in the mouse ovary and induces follicle and oocyte apoptosis. The data reported here demonstrate for the first time that low levels of Pb(NO3)2 (相似文献