Characterization of aurora-a in porcine oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization and cleavage

Aurora-A is a serine/threonine protein kinase that plays important regulatory roles during mitotic cell cycle progression. In this study, Aurora-A expression, subcellular localization, and possible functions during porcine oocyte meiotic maturation, fertilization and early embryonic cleavage were studied by using Western blot, confocal microscopy and drug treatments. The quantity of Aurora-A protein remained stable during porcine oocyte meiotic maturation. Confocal microscopy revealed that Aurora-A distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, Aurora-A concentrated around the condensed chromosomes and the metaphase I spindle, and finally, Aurora-A was associated with spindle poles during the formation of the metaphase II spindle. Aurora-A concentrated in the pronuclei in fertilized eggs. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. In conclusion, Aurora-A may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.     

Cadherins expression during gamete maturation and fertilization in the rat

A role for adhesion molecules in gamete fusion, preceding fertilization, has been previously suggested. We investigated the presence of cadherins, Ca(2+) dependent cell-cell adhesion molecules, in rat oocytes and spermatozoa using an anti-pan-cadherin antibody and specific antibodies against the 3 classical cadherins: E- (epithelial), P- (placental), and N- (neural) cadherins. Electrophoretic separation was performed on samples of lysed oocytes of different stages: germinal vesicle oocytes, metaphase II eggs, newly fertilized and 2-cell embryos, as well as spermatozoa from testes, caput and cauda epididymis and ejaculate. Localization of cadherins was determined on intact, gametes by immunocytochemistry, using confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a major band of approximately 120 kD in all oocyte and sperm extracts. Oocytes presented E-cadherin at appropriate molecular weight but N-cadherin only as a specific 40 kD band. In sperm lysate, at all stages, both E- and N-cadherin were demonstrated as major protein bands but a series of lower molecular weight proteins (that may represent protein degradation) were also detected. Immunohistochemical evaluation showed that E- and N-cadherins are already present on the plasma membrane of immature unfertilized oocytes, although their concentration increases after fertilization in early cleavage stage embryos. Cadherin localization on spermatozoa changed during maturation from a dispersed pattern over the entire head plasma membrane of testicular spermatozoa to a restricted equatorial and post-acrosomal plasma membrane staining in ejaculated spermatozoa. These findings suggest a specific cadherin organization at the fusogenic domains of both gametes.     

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.     

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.     

Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage

RhoA, a small GTPase, plays versatile roles in many aspects of cell function such as stress fiber formation, cytokinesis, and cell polarization. In this study, we investigated the subcellular localization of RhoA and its possible roles during oocyte maturation and fertilization. RhoA was localized in the cytoplasm of eggs from the germinal vesicle (GV) stage to 2-cell stage, especially concentrating in the midbody of telophase spindle when oocyte extruded PB1 and PB2. The RhoA kinases (ROCKs) specific inhibitor Y-27632 blocked GV breakdown (GVBD) and first polar body extrusion, but did not affect apparatus formation and anaphase/telophase I entry. Anti-RhoA antibody microinjection into the oocytes showed similar results. RhoA inhibitor caused abnormal organization of microfilaments, failure of spindle rotation, PB2 extrusion as well as cleavage furrow formation, while sister chromatid separation was not affected. Microinjection of RhoA antibody also blocked PB2 emission. Our findings indicate that RhoA, by regulating microfilament organization, regulates several important events including GVBD, polar body emission, spindle rotation, and cleavage.     

Timing of meiotic progression in bovine oocytes and its effect on early embryo development

This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest. Mol. Reprod. Dev. 47:456–467, 1997. © 1997 Wiley-Liss, Inc.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

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.     

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.     

Function and regulation mechanism of Chk1 during meiotic maturation in porcine oocytes

Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study, we used porcine oocyte as a model to investigate the function of Chk1 during porcine oocyte maturation. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages, mainly localized in the cytoplasm at GV stage and moved to the spindle after germinal vesicle breakdown (GVBD). Chk1 depletion not only induced oocytes to be arrested at MI stage with abnormal chromosomes arrangement, but also inhibited the degradation of Cyclin B1 and decreased the expression of Mitotic Arrest Deficient 2-Like 1 (Mad2L1), one of spindle assembly checkpoint (SAC) proteins, and cadherin 1 (Cdh1), one of coactivation for anaphase-promoting complex/cyclosome (APC/C). Moreover, Chk1 overexpression delayed GVBD. These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at anaphase I (AI) and maintained the expression of Mad2L1 and Cdh1, which ensured that all chromosomes were accurately located in a line, and then oocytes passed metaphase I (MI) and AI and exited from the first meiotic division successfully. In addition, we proved that Chk1 had not function on GVBD of porcine oocytes, which suggested that maturation of porcine oocytes did not need the DNA damage checkpoint, which was different from the mouse oocyte maturation.     

MEK1/2 is a critical regulator of microtubule assembly and spindle organization during rat oocyte meiotic maturation

MEK (MAPK kinase) is an upstream protein kinase of MAPK in the MOS/MEK/MAPK/p90rsk signaling pathway. We previously reported the function and regulation of MAPK during rat oocyte maturation. In this study, we further investigated the localization and possible roles of MEK1/2. First, immunofluorescent staining revealed that p-MEK1/2 was restricted to the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), p-MEK1/2 condensed in the vicinity of chromosomes and then translocated to the spindle poles at metaphase I, while spindle microtubules stained faintly. When the oocyte went through anaphase I and telophase I, p-MEK1/2 disappeared from spindle poles and became associated with the midbody. By metaphase II, p-MEK1/2 was again localized to the spindle poles. Second, p-MEK1/2 was localized to the centers of cytoplasmic microtubule asters induced by taxol. Third, p-MEK1/2 co-localized with gamma-tubulin in microtubule-organizing centers (MTOCs). Forth, treatment with U0126, a non-competitive MEK1/2 inhibitor, did not affect germinal vesicle breakdown, but caused chromosome mis-alignment in all MI oocytes examined and abnormal spindle organization as well as small cytoplasmic spindle-like structure formation in MII oocytes. Finally, U0126 reduced the number of cytoplasmic asters induced by taxol. Our data suggest that MEK1/2 has regulatory functions in microtubule assembly and spindle organization during rat oocyte meiotic maturation.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

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.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。