Meiosis In Rat Oocytes Research Articles

Effects of Fullerenol Nanoparticles on Rat Oocyte Meiosis Resumption.

The excellent biocompatibility and biological effects of fullerenol and its derivatives make their biomedical application promising. The potential effects of fullerenol in mammals have been extensively studied, but little is known about its effects on female reproduction. Using canonical oocyte-granulosa cell complexes (OGCs) in vitro maturation culture model, we investigated the effect of fullerenol on the first oocyte meiotic resumption. In the surrounding granulosa cells, fullerenol nanoparticles occluded the extracellular domain of the epidermal growth factor receptor (EGFR) to reduce EGFR-ligand binding and subsequent extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation, which involved the regulation of connexin 43 (CX43) expression and internalization. Downregulation of CX43 expression and the retraction of transzonal projections (TZPs) interrupted the gap junction channel and TZPs based mass transportation. This effect decreased cyclic adenosine monophosphate (cAMP) levels in the oocyte and thereby accelerated rat oocyte meiosis resumption. Moreover, perinuclear distribution of CX43 and EGFR was observed in granulosa cells, which could further exacerbate the effects. Fullerenol nanoparticles interfered with the strict process of oocyte meiosis resumption, which likely reduced the oocyte quality.

Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis.

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism.

Changes in signal molecules and maturation promoting factor levels associate with spontaneous resumption of meiosis in rat oocytes.

The present study was aimed to find out changes in signal molecules and maturation promoting factor (MPF) levels during meiotic cell cycle progression from diplotene and metaphase-II (M-II) arrest, a period during which oocyte achieves meiotic competency. Data suggest that high levels of adenosine 3'-5'-cyclic monophosphate (cAMP), guanosine 3'-5'-cyclic monophosphate (cGMP), and nitric oxide (NO) are associated with diplotene arrest, while reduction in their levels correlates with reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increased intracellular NO, calcium (Ca(2+) ) as well as hydrogen peroxide (H2 O2 ) levels correlate with decreased cAMP, Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 levels. The decreased Thr-161 phosphorylated Cdk1 and cyclin B1 level reduce MPF level leading to exit from M-II arrest in oocytes cultured in vitro. These data suggest that the decrease of cAMP level and increase of cytosolic free Ca(2+) as well as H2 O2 levels associate with the reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increase of NO, cGMP, Ca(2+) as well as H2 O2 levels are associated with reduced MPF and spontaneous exit from M-II arrest in rat oocytes cultured in vitro.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3′,5′-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Roles of Microtubules and Microfilaments in Spindle Movements During Rat Oocyte Meiosis

Spindle movements, including spindle migration from the center to the cortex of oocytes during first meiosis and spindle rotation during second meiosis, are required for asymmetric meiotic divisions in many species. However, little is currently known in relation to the rat oocyte. To explore how spindles move and the mechanism controlling spindle movements in rat oocytes, we observed the spindle dynamics during the two meiotic divisions in the rat oocyte by confocal microscopy. Drugs that depolymerize microtubules or microfilaments were employed to further determine the roles of these two cytoskeletons in spindle movements. The results showed that peripheral spindle migration took place during first meiosis and spindle rotation took place during second meiosis in the rat oocytes. Microfilament inhibitor inhibited both spindle migration and spindle rotation, and depolymerization of microtubules inhibited spindle rotation. Severe depolymerization of microtubules inhibited spindle migration, while migration was achieved by partial but not complete depolymerization of microtubules. We thus conclude that microfilaments are important for both spindle migration and spindle rotation and that spindle microtubules are essential for spindle movements in rat oocytes.

Cytoplasmic polyadenylation controls cdc25B mRNA translation in rat oocytes resuming meiosis

Resumption of meiosis in oocytes represents the entry into M-phase of the cell cycle and is regulated by the maturation-promoting factor (MPF). Activation of MPF is catalyzed by the dual specificity phosphatase, cdc25. In mammals, cdc25 is represented by a multigene family consisting of three isoforms: A, B and C. A recent report that female mice lacking cdc25B exhibit impaired fertility suggests a role for this isoform in regulating the G2- to M-transition in mammalian oocytes. Supporting the above-mentioned observation, we demonstrate herein that microinjection of neutralizing antibodies against cdc25B interfered with the ability of rat oocytes to undergo germinal vesicle breakdown (GVB). We also show accumulation of cdc25B in GVB oocytes and a transient reduction in its amount at metaphase I of meiosis. The accumulation of cdc25B was associated with its mRNA cytoplasmatic polyadenylation and was prevented by the protein synthesis inhibitor cyclohexamide as well as by the polyadenylation inhibitor cordycepin. Immunofluorescence staining revealed translocation of cdc25B to the metaphase II spindle apparatus. Taken together, our findings provide evidence that cdc25B is involved in resumption of meiosis in rat oocytes. We further demonstrate for the first time, a periodic accumulation of cdc25B throughout meiosis that is translationally regulated and involves cdc25B mRNA polyadenylation.

Expression and modification of PKA and AKAPs during meiosis in rat oocytes

Meiosis in oocytes is initiated during fetal life, arrested around birth and resumed after puberty. Meiotic arrest is controlled by a cAMP-dependent protein kinase (PKA)-mediated cAMP action. We examined oocytes for the presence and modulation of the regulatory (R) subunits of PKA and the A-kinase anchoring proteins (AKAPs) that target PKA to specific subcellular locations. We found that rat oocytes express the two regulatory subunit isoforms, RI and RII of PKA. Immunocytochemistry revealed that the regulatory subunits underwent cellular translocation upon resumption of meiosis. We also demonstrated the presence of a novel 140 kDa AKAP, AKAP140 that exhibited a retarded electrophoretic motility at reinitiation of meiosis. The mobility shift of AKAP140 was susceptible to alkaline phosphatase and prevented by inhibition of p34cdc2 kinase. We conclude that rat oocytes express AKAP140 that is phosphorylated during meiosis. AKAP140 phosphorylation is sensitive to p34cdc2 kinase inhibitors. We hypothesize that AKAP140 and its phosphorylation state may influence the translocation of the R subunits of PKA throughout resumption of meiosis.

Role of phosphodiesterase type 3A in rat oocyte maturation.

It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P < 0.05) in PDE3 activity was measured in the oocytes after 30 min of culture (70 min after isolation) compared with immediately after collection (10 min after isolation). Conversely, no changes in activity were observed when denuded oocytes or cumulus cells were incubated for up to 130 min. Evaluation of oocyte maturation indicated that only 10% of oocytes had resumed meiosis at the peak of the PDE3 activity. A significant increase (P < 0.05) in PDE3 activity was measured in COCs when follicle-enclosed oocytes were cultured in the presence of hCG. Again, this increase preceded oocyte maturation. In conclusion, these data demonstrate that PDE3A is the major PDE form expressed in mammalian oocytes. PDE3A activity increases prior to resumption of meiosis in both spontaneous and gonadotropin-stimulated maturation. These findings strongly support the hypothesis that an increase in oocyte PDE3A activity is one of the intraoocyte mechanisms controlling resumption of meiosis in rat oocytes, at least in vitro.

The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.

The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.

Hormonally Regulated Expression and Alternative Splicing of Kit Ligand May Regulate Kit-Induced Inhibition of Meiosis in Rat Oocytes

Mutations in the genes encoding the Kit tyrosine kinase receptor or kit ligand (KL) cause numerous phenotypic defects, including sterility. In the postnatal ovary, Kit is expressed on the oocyte surface and KL is produced by the surrounding granulosa cells, but its function in these cells is still unknown. The purpose of this study was to determine the role KL/Kit interactions play in the regulation of oocyte meiosis. Here, we demonstrate that meiotically arrested rat oocytes that are microinjected with Kit antisense oligonucleotides have decreased Kit expression. This decreased expression is associated with an increased ability of these oocytes to resume meiosis compared with those microinjected with missense oligonucleotides or buffer alone. In addition, oocytes cultured in the presence of KL were delayed in their resumption of meiosis, but KL could not enhance the meiosis inhibitory effects of dibutyryl cAMP, suggesting that KL operates through a mechanism that is independent of cAMP. Human chorionic gonadotropin-induced meiotic resumption in oocytes was accompanied by a shift in follicular granulosa cell KL expression from membrane-bound to soluble forms and a loss of expression of both forms of KL in cumulus cells. Thus, KL-activated Kit inhibits meiotic progression, and thein vivoluteinizing hormone-stimulated resumption of meiosis may negate Kit activity by a localized decrease in KL expression and by altering the form of KL produced within the follicle.

Maintenance of Meiotic Arrest by a Phosphorylated p34cdc2 is Independent of Cyclic Adenosine 3′,5′-Monophosphate

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition and is associated with a drop in intraoocyte concentrations of cAMP. Recent studies imply that tyrosine dephosphorylation of p34cdc2 is a prerequisite for entry into the M-phase of the cell cycle. Our study was designed to test the involvement of protein tyrosine phosphatase (PTPase)-regulated dephosphorylation of p34cdc2 in resumption of meiosis in rat oocytes and to explore the possible control of this event by the intraoocyte concentrations of cAMP. Isolated rat oocytes undergoing meiotic maturation spontaneously in vitro served as our experimental model. We found that sodium metavanadate, an inhibitor of PTPase, reversibly blocked the spontaneous maturation in vitro of rat oocytes (ED50 = 0.26 mM). We further demonstrated that the vanadate-sensitive event is completed by 2 h after reinitiation of meiosis. Immunoblot analysis using specific antiphosphotyrosine antibodies revealed that vanadate caused accumulation of phosphotyrosine on a 34-kDa protein, also recognized by anti-p34cdc2 antibodies. The phosphorylated form of p34cdc2 was also detected in oocytes arrested in the G2 phase by the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX). Intraoocyte concentrations of cAMP in vanadate-inhibited oocytes were similar to those in oocytes that resumed meiosis spontaneously in vitro and lower than those in oocytes maintained in meiotic arrest by IBMX (0.73 +/- 0.08, 0.84 +/- 0.09, and 1.42 +/- 0.3 fmol/oocyte, respectively) [corrected]. We conclude that a PTPase that regulates the phosphorylation state of p34cdc2 participates in the control of meiosis in rat oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3‘,5’‐cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP

The aim of the present study was to measure the level of cyclic GMP (cGMP) compared with the level of cyclic AMP (cAMP) in rat oocytes during resumption of meiosis I (oocyte maturation) and to microinject these cyclic nucleotides into the oocyte to study their effects on oocyte maturation. Immature oocytes were obtained from prepubertal rats primed with pregnant mare's serum gonadotropin. OOcytes were isolated adn short-term cultured under conditions enabling spontaneous maturation. The levels of cGMP and cAMP decreased in the oocyte during spontaneous maturation (from 0.41 to 0.25 and from 0.64 to 0.42 fmol per oocyte respectively). The decrease was observed during the first hour of culture, and no further decline was seen after 2 h. Microinjection of cGMP or cAMP into isolated immature oocytes delayed the spontaneous maturation, cGMP being slightly more effective than cAMP, whereas 2-deoxy-cAMP (which does not stimulate protein kinase A) did not. These results demonstrate for the first time that the level of cGMP decreases in the oocyte parallel to spontaneous meiosis, as already shown for cAMP. This suggests that cGMP, as well as cAMP, may be of importance for regulating this process. This assumption is further supported by data demonstrating a delay in the maturation of oocytes injected with cGMP.

Human Recombinant Mullerian Inhibiting Substance Inhibition of Rat Oocyte Meiosis Is Reversed by Epidermal Growth Factorin Vitro

Mullerian inhibiting substance (MIS), a glycoprotein responsible for the regression of Mullerian duct in the male mammalian embryo, was recently localized not only in the fetal and newborn testis, but also in the older ovary throughout reproductive life. Bovine MIS purified from newborn testicular tissue inhibited spontaneous oocyte meiosis in vitro in the rat. These studies show that partially purified recombinant MIS produced from a human MIS genomic construct caused inhibition of oocyte meiosis, but when purified to homogeneity, the effect was lost. Addition of low concentrations of the detergent Nonidet P-40, used to maintain stability in the purified bovine preparations, however, restored the MIS inhibitory effect to the human preparation, which could, in turn, be blocked by a polyclonal antibody raised to human recombinant MIS; Nonidet P-40 itself caused no inhibition. Since we have shown that epidermal growth factor (EGF) and MIS are antagonistic in a number of other systems, we tested the effect of EGF on the ability of MIS to inhibit oocyte meiosis. EGF added to the medium at a dose that caused no effect on oocytes (25 ng/ml) blocked the MIS inhibitory action on spontaneous rat oocyte meiosis, while EGF had no effect on a known oocyte meiosis inhibitor, 3-isobutyl-1-methylxanthine. These data indicate that human recombinant MIS can inhibit oocyte meiosis in the ovary and that its regulatory effect can be modulated by EGF, which appears to be an antagonist of MIS.

Oocyte maturation as regulated by follicular factors.

Follicular fluid (FFl), obtained from 24 women treated with clomiphene/hCG in an in vitro fertilization program, was characterized with respect to steroid hormone levels and oocyte maturation inhibitor (OMI) activity. Three FFl samples apparently were derived from cystic follicles and contained low steroid levels and no OMI activity in an in vitro rat oocyte assay. The remaining 21 follicles contained normal preovulatory steroid levels and mature and generally fertilizable oocytes. In 7 of these follicles the FFl (at 50% concentration) significantly inhibited rat oocyte meiosis, while 14 exerted no OMI activity. The results confirm earlier work on porcine and human FFl, suggesting that the putative OMI activity declines with follicular maturation.

Stimulation and inhibition of rat oocyte meiosis by forskolin.

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 microM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.

In vitro inhibition of rat oocyte meiosis by human follicular fluid fractions.

We studied the effect of low molecular weight fractions of human follicular fluid (FF1) on rat oocyte meiosis and progesterone secretion by the granulosa cells. Steroid-free FF1 extracts were filtered through Sephadex G50 gel. One highly retarded fraction was obtained (GF-3), which was either used for testing or further purified by filtration on Sephadex G10, the G10-3 fraction of which was used for experiments. The GF-3 and G10-3 fractions of FF1 inhibited (in a dose-dependent and reversible manner) meiosis of isolated rat oocytes during a 4-h culture. Similarly treated fractions of serum had no effect. The inhibition was not abolished by ether extraction, trypsin treatment, heating to 56 degrees C for 1 h or boiling for 5 min, whereas heating to 105 degrees C for 18 h decreased the effect. Gonadotropin-stimulated progesterone secretion by cumulus and mural granulosa cells was also dose-dependently and reversibly inhibited by the GF-3 fractions. Our results confirm those obtained in earlier studies on porcine oocyte maturation inhibitor (OMI).