MI Stage Research Articles

P-706: Do late maturing oocytes enhance the ICSI outcome?

To optimize the chances of normal fertilization, MI stage oocytes at time of cumulus cell removal are reassessed within a few hours of in vitro incubation for PB extrusion, following which they are injected. While such oocytes exhibiting this delay in maturation are generally considered as equivalent to the original MII pool, specific data on the outcome of their use is limited.

Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer

Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-alpha-tubulin antibody for microtubules and a rabbit anti-gamma-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of gamma-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of gamma-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.

349 DEVELOPMENTAL COMPETENCE AFTER IVF OF CUMULUS-ENCLOSED OOCYTES FROM A ICR MOUSE STRAIN CULTURED WITH A LOW CONCENTRATION OF HYPOXANTHINE DURING IVM

Hypoxanthine (HX, 4 mM) is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of cumulus-enclosed oocytes (CEOs) cultured in vitro by causing accumulation of cAMP within the oocytes. Recently, it was found that when CEOs at the GV stage from the ICR mouse strain were treated with a small amount of HX (200 �M) during in vitro maturation (IVM) culture, cortical granule migration was much improved. The aim of this study was to examine the effects of treatment of ICR mouse CEOs with 200 �M HX during IVM culture on the in vitro development of oocytes after in vitro fertilization (IVF). CEOs were isolated from eCG-primed 3-week-old ICR mice by rupturing preovulatory follicles with needles in M16 medium containing 5% FCS, 1 IU mL-1 FSH, and essential and nonessential amino acids (basal medium). IVM media were basal medium without (control) or with 200 �M HX, 100 �M dibutyryl cyclic adenosine monophosphate (dbcAMP), or 10 IU mL-1 FSH. Denuded oocytes (DOs) were prepared by treating CEOs for 30 min with 0.1% hyaluronidase and 0.1 mM EDTA, followed by repeated pipetting. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h. After IVM, CEOs and DOs were transferred to TYH medium (modified Krebs-Ringer bicarbonate) containing 0.4% BSA, and inseminated with capacitated sperm. After 6 h of IVF, zygotes were freed from cumulus cells by gently pipetting, and cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was determined at 24 h and 120 h after IVF, respectively. All experiments included 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's PLSD test. When CEOs matured in basal medium (controls), although almost all of the oocytes reached the MII stage, the rates of their post-fertilization development to the 2-cell and blastocyst stages were very low (21.8% and 8.9%, respectively), whereas those of CEOs matured with 200 �M HX were significantly increased (63.4% and 52.2%, respectively; P < 0.05). Next, CEOs were cultured with 100 �M dbcAMP or a high concentration (10 IU mL-1) of FSH during IVM. The rates of the 2-cell and blastocyst stages of CEOs cultured with 100 �M dbcAMP were 53.3% and 41.3%, respectively, and those of CEOs cultured with 10 IU mL-1 FSH were 55.5% and 27.6%, respectively. Although the blastocyst rate was significantly lower in FSH-treated oocytes than in HX- or dbcAMP-treated oocytes (P < 0.05), this result suggests that increasing concentration of intracellular cAMP is important for improving the developmental competence of mouse CEOs after IVF. Finally, when DOs were cultured with 200 �M HX, although most of the oocytes were arrested at the MI stage, 6.3% of the oocytes developed into 2-cell embryos after IVF but not into blastocysts. Therefore, it is suggested that some accumulation of cAMP in cumulus cells by 200 �M HX enhances fertility and post-fertilization development of oocytes in the ICR mouse strain.

348 INACTIVATION OF MAPK AFFECTS CENTROSOME ASSEMBLY, BUT NOT ACTIN FILAMENT ASSEMBLY, IN MOUSE OOCYTES MATURING IN VITRO

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, γ-tubulin and NuMA distribution, and actin filament assembly in mouse oocytes maturing in vitro. Significant differences between control and treatment were determined using Tukey's multiple range test; P < 0.05 was considered significant. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 h after the initiation of maturation cultures, was fully active at 6 h, and remained active until metaphase II (MII). When GV oocytes were cultured in medium containing 20 �M U0126 for 9 h, the oocytes underwent geminal vesicle breakdown (GVBD), but were significantly inhibited in progression to the MI stage as compared to controls (control: 76.7 � 5.66%; 10 �M U0126: 29.0 � 7.35%; 20 �M U0126: 26.4 � 5.74%). Polar body extrusion was observed in most oocytes when cultured in the presence of 20 �M U0126 from 4 h after initiation of in vitro oocyte maturation to 14 h into the in vitro maturation incubation (control: 76.6 � 4.21%; 20 �M U0126: 70.7 � 4.61%). However, there was a decrease in normal MII-stage oocytes when cultured for 4 to 14 h (control: 87.7 � 5.12%; 20 �M U0126: 32.8 � 3.79%); abnormality included, for example, a polar body lacking a nucleus, as well as an abnormal spindle or metaphase plate. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of γ-tubulin and NuMA, but did not affect actin filament assembly. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.

Effects of culture media and energy sources on the inhibition of nuclear maturation in bovine oocytes

The influence of the culture medium and energy sources on spontaneous nuclear maturation and inhibition of maturation in bovine cumulus-enclosed oocytes (CEO) was examined. CEO were cultured in Medium 199, minimum essential medium, M16, or synthetic oviduct fluid (SOF), all containing 3 mg/mL bovine serum albumin (BSA), and SOF without BSA, alone or supplemented with hypoxanthine (HYPO, 4 mM) or forskolin (FSK, 100 μM) for 21 h. More CEO remained at the GV stage in M16 compared to other media ( P < 0.05). Supplementation with HYPO increased and FSK reduced the percentage of CEO remaining at the GV stage ( P < 0.05) only in M16. The effects of energy sources, in the absence or presence of HYPO or FSK, were examined in CEO cultured in M16 salts + PVA. Glucose (0.5 and 5.5 mM), pyruvate (0.32 and 3.2 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of CEO remaining at the GV stage compared to M16 salts alone; only glutamine significantly increased the percentage of CEO at the MII stage compared to M16 salts. In M16 salts + HYPO, glucose (0.5 mM), pyruvate (0.32 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of GV and degenerate oocytes and increased the percentage of CEO at the MI stage. In M16 salts + FSK, the energy sources significantly decreased the percentage of oocytes with condensed chromosomes and increased the percentage of CEO reaching metaphase I. In conclusion, meiotic inhibitors had different effects in different culture media and glucose, pyruvate, lactate and glutamine were stimulatory to nuclear maturation. It was noteworthy that some of the results obtained were contrary to previous findings in mouse oocytes.

355 IMPROVEMENT OF CUMULUS-FREE IN VITRO MATURATION AND ITS APPLICATION TO MICROINSEMINATION WITH PRIMARY SPERMATOCYTES IN MICE

Manipulation of immature oocytes requires removal of cumulus cells, which may compromise ooplasmic maturation. This problem can be overcome by cytoplasm exchange at the MII stage, but this requires a second micromanipulative step. This study was undertaken to determine whether cumulus-free IVM can be improved by modifying IVM medium. We assessed the quality of oocytes by several cytochemical parameters, and by the subsequent development of embryos constructed by IVF, parthenogenetic activation, or microinsemination using primary spermatocytes. GV-stage oocytes were collected from superovulated B6D2F1 females. After freeing the oocytes from cumulus cells, they were subjected to IVM in either �MEM or TYH media, or a 1:1 mixture of the two (termed TaM). IVM was performed under chemically semidefined conditions by using BSA as the only protein source. Oocytes that reached the MII stage were analyzed for distribution of mitochondria, activity of the catalytic p34cdc2 kinase subunit of the maturation promoting factor (MPF), and chromosomal alignment within the MII spindle. Microinsemination with primary spermatocytes was performed in oocytes at the MI stage as described previously (Ogura et al. 1998 PNAS 95, 5611-5615), but without MII chromosome exchange. TYH and �MEM media had opposing effects on several parameters. TYH resulted in a better maturation rate (181/196, 92.3%) than �MEM (184/257, 71.6%). In contrast, �MEM supported better embryo development to the morula/blastocyst stage than TYH following IVF (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse in �MEM, but aggregated in TYH. MPF activity in MII oocytes was significantly higher in TYH than in �MEM (P &lt; 0.05). Oocytes derived from TaM had intermediate characteristics between TYH- and �MEM-oocytes and essentially resembled in vivo-matured oocytes, with the mitochondrial distribution pattern being most typically intermediate. By using TaM, 23.8% of GV oocytes developed into full-term fetuses following IVF and embryo transfer (compared to TYH: 6.3%, and �MEM: 18.2%). This IVM system was then applied to oocytes to be injected with spermatocytes when in the MI stage. Approximately 60% of the oocytes survived injection. After 127 two-cell embryos were transfered, two normal-looking offspring were obtained. This is the first successful birth of pups derived from fertilization with spermatocytes by single injection, without serial nuclear transfer. Chromosomal analysis at MII revealed that the low developmental efficiency after embryo transfer could be attributed mainly to premature sister chromatid separation, as reported previously (Ogura et al. 1998). These results demonstrate that optimization of IVM media can improve the quality of cumulus-free IVM oocytes in mice, although it cannot overcome defects in the kinetics of injected spermatocyte chromosomes.

302 PROTEIN SUPPLEMENTATION TO IVM MEDIUM IN RELATION TO THE INCIDENCE OF APOPTOSIS IN BOVINE OOCYTES MATURED IN VITRO

Mammalian embryos derived from in vitro fertilization display lower developmental competence and quality when compared to their in vivo counterparts. The composition of culture media significantly contributes to this phenomenon. Media supplemented with FBS or the serum derivate BSA are described as biochemically undefined. Those macromolecules were shown to exert a wide range of effects on cultured embryos, dependent on batch-to-batch variability. Therefore, replacement of these protein sources with a synthetic macromolecule such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) provides a possibility to use a chemically defined culture medium (Ali et al. 2002 Biol. Reprod. 66, 901–905). Apoptosis as programmed cell death naturally occurs in mammalian oocytes and embryos; however, its incidence is significantly higher in vitro. The aim of this study was to investigate whether protein supplementation (FBS, fatty acid-free (faf)-BSA, PVP40) of IVM medium affects the incidence of apoptotic oocytes. In the present study, the IVM system previously described by Makarevich et al. (2002 Biol. Reprod. 66, 386–392) was used. Briefly, follicular oocytes aspirated from slaughterhouse ovaries were matured in vitro in one of three maturation media supplemented with FBS (10%), faf-BSA (6 mg mL−1) or PVP40 (4 mg mL−1). The terminal TUNEL assay kit was used to detect the DNA fragmentation in apoptotic cells (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA). The data were analyzed by chi-square test of independence. Altogether, 630 oocytes collected during 12 IVM experiments were subjected to the Tunel test, and 563 (89.4%) of them were successfully investigated: 426 after maturation in vitro and 137 follicular, non-matured. The remaining 67 cells were lost during manipulation. The rate of Tunel-positive cells differed (P &lt; 0.001) between matured (11.8%) and follicular oocytes (1.5%). Protein supplementation of IVM media did not significantly affect the rate of apoptotic oocyte occurrence, which was 9% in the faf-BSA group, 11.5% in the FBS group, and 15% in the PVP group. No differences were observed in the rate of Tunel-positive cells between oocytes at MII and MI stages. In conclusion, protein supplementation of IVM medium used in the present study did not affect the incidence of apoptotic oocytes after maturation in vitro. This research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-084 from 2003 to 2005 year.

230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring. This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).

Postinfarction gene therapy against transforming growth factor-β signal modulates infarct tissue dynamics and attenuates left ventricular remodeling and heart failure

We examined effects of transforming growth factor-β (TGF-β) signal inhibition on post-myocardial infarction (MI) ventricular remodeling and dysfunction. MI was made by left coronary ligation in mice, and adenovirus encoding soluble TGF-β type II receptor (sTβRII), a competitive inhibitor of TGF-β, was injected into the hindlimb muscles on the 3rd day of MI (control, LacZ). Post-MI survival was significantly improved in the sTβRII mice (96% vs. control, 71%, p < 0.05). The sTβRII gene treatment resulted in a significant attenuation in ventricular dilatation and dysfunction 4 weeks post-MI, where a reduction in ventricular fibrosis was histologically confirmed. Although the MI size was not different, the MI thickness was greater and the MI circumference was smaller in the sTβRII group; in the infarct area, α-smooth muscle actin-positive cells were abundant, which might have contributed for infarct contraction. Myofibroblast apoptosis in granulation tissue during the subacute stage (10 days old MI) was less frequent in the sTβRII group, and an in vitro experiment revealed an apoptosis-inhibitory effect of sTβRII on cultured myofibroblasts. Finally, treatment of MI bearing mice with sTβRII was ineffective when started at the chronic stage (scar phase) of MI. In conclusion, the postinfarction gene therapy against TGF-β signal improved cardiac remodeling through affecting infarct tissue dynamics (apoptosis inhibition and infarct contraction) in addition to cardiac fibrosis.

Delayed fertilization of oocytes following in vitro culture results in increased embryo aneuploidy

Rescue ICSI (rICSI), the re-insemination of oocytes after failed fertilization or delayed ICSI (dICSI), the fertilization of oocytes following in vitro maturation from the GV or MI stage, are controversial assisted reproduction techniques. While some laboratories have reported pregnancies from rICSI and dICSI, it is generally accepted that the pregnancy rates are lower than traditional ART procedures and there may be an increased risk of chromosomal abnormalities. The intent of this study was to identify if dICSI results in an increased embryonic aneuploidy status.

Fertilization and InsP 3-induced Ca 2+ release stimulate a persistent increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca 2+. This increase in Ca 2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca 2+ can activate the destruction machinery during MI. We have investigated Ca 2+-induced cyclin destruction in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca 2+. We find no evidence for a role for Ca 2+ in MI since oocytes progress through MI in the absence of detectable Ca 2+ transients. Furthermore, Ca 2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca 2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca 2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. We conclude that increases in cytosolic Ca 2+ induce transient changes in autofluorescence and that the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca 2+-induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca 2+ acts to release the CSF-mediated brake on cyclin B1 destruction.

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.

Study of newly synthesized proteins during bovine oocyte maturation in vitro using image analysis of two-dimensional gel electrophoresis.

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).

339CORRELATION BETWEEN FOLLICLE SIZE AND QUALITY OF OOCYTES FROM THE SUPEROVULATED CYNOMOLGUS MONKEY

Variability in the superovulatory response continues to be one of the most frustrating problems with the application of assisted reproductive technologies in non-human primates. Superstimulation of donor animals with equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) is widely used, but individual responses to these hormones vary widely. In human in vitro fertilization, follicle size is commonly used as a marker to determine the timing of hCG treatment in order to acquire oocytes matured in vivo. Incorporation of techniques designed to control follicular size in humans may improve superstimulatory response in non-human primates. In this study, we measured follicle size and examined oocyte quality in Cynomologus Monkeys superstimulated as described below. On the first day of spontaneous menses, monkeys were treated with long-acting GnRH (Luprin: Takeda Pharm, Co. Ltd., Osaka, Japan;; Day 0). A dose of 25IU/kg/day eCG (Serotoropin;; Teikokuzoki Pharm, Co. Ltd., Tokyo, Japan) was administered i.m. from Day 1 to Day 9, followed by 200IUkg−1 hCG (Puberogen;; Sankyo Pharm, Co. Ltd., Tokyo, Japan) i.m. on Day 10. Dominant follicle sizes were measured on Days 7 and 9 by ultrasonography. Oocytes were collected by laparoscopy in anesthetized monkeys 40h after the hCG injection. Oocytes were classified by nuclear status. Immature oocytes at the stages of germinal vesicles (GV) and metaphase (MI) were cultured until reaching the stage of Metaphase II (MII). Matured oocytes (MII) were fertilized by ICSI and cultured for 7 days. At the end of culture, the developmental stage of oocytes was examined. The ovaries with different-sized follicles on Day 7 were divided into two groups;; ovaries with large follicles (&amp;gt;4.5mm) were in the first group and ovaries with small follicles (&amp;lt;4.0mm) were in the second group. On Day 9, follicles in first group grew to more than 5.0mm and follicles in second group remained less than 5.0mm. Sixty-two percent of oocytes from follicles in first group were at MI or MII stage, while only 15% of oocytes in second group reached the MI or MII stage. After ICSI, 42% of MII oocytes from first group developed to the blastocyst stage, while no blastocyts were observed in second group. These results suggest that the size of dominant follicle was a limiting factor for the developmental ability of oocytes in vitro. For production of Cynomolgus monkey blastocysts derived from ICSI, the diameter of dominant follicle was required to be at least 5mm before hCG in order to collect MI and MII oocytes. Incorporation of hormonal treatments designed to optimize follicular size probably reduced the variability in quality of oocytes. Therefore, we expect that an adjustment of dose and duration of eCG and hCG treatment may improve developmental ability of oocytes from follicles that had not reached 5mm.

308MITOCHONDRIA RELOCATION, MICROTUBULE ASSEMBLING AND PARTHENOGENETIC DEVELOPMENTAL COMPETENCE OF PIG OOCYTES

Developmental competence of in vitro-produced porcine embryos appears to be limited by specific maternally inherited cytoplasmic factors. We previously reported a relationship between mitochondria distribution during IVM, energy status, and oocyte developmental ability after parthenogenetic activation. The aim of the present study was to investigate the timing of mitochondria relocation during meiosis and the possible relationship with cytoskeleton organization in high and low competence oocytes. To this purpose, homogeneous groups of oocytes were matured in vitro (IVM) with 25% or 0% porcine follicular fluid (pff) to obtain different cytoplasmic competence (high and low, respectively) but similar nuclear maturation (Brevini et al., 2003, Theriogenology, 59, 440). After maturation, oocytes were parthenogenetically activated and cultured as previously described by Grupen et al., 2002 (Mol. Reprod. Dev., 62, 387–396). Active mitochondria were stained with MitoTracker® Orange CMTM Ros (Molecular Probes, Leiden, The Netherlands) at GV, MI and MII meiotic stages. At the same time microtubule organization was determined by immuno-cytochemistry using an antibody raised against α-tubulin (Sigma, St. Louis, MO, USA). Meiotic stages were assessed with DAPI. Specimens were examined with a Leica TCS-NT confocal microscope through an equatorial optical section. Nuclear maturation rate was comparable in the two groups at the end of IVM (46h). Mitochondria relocation from the periphery to the center of the oocyte was evident as early as 20h IVM in the 25% pff group (high competence), while 0% pff oocytes (low competence) did not show any mitochondria relocation at this time point. In agreement with the literature, α-tubulin was not detectable in GV oocytes, while at the end of IVM, α-tubulin was associated with the DNA, forming the meiotic spindle both in high and low competence oocytes. However, oocytes in the 25% pff group displayed a cytoplasmic microtubular organization that co-localized with mitochondria at 20 to 28h IVM. Conversely, α-tubulin was not detected in the cytoplasm of 0% pff oocytes at the same time points and 71% of these oocytes did not undergo any mitochondria relocation at all by the end of IVM. Altogether the present results show that mitochondria relocation takes place at a well-defined time during IVM and is temporally associated with the formation of the microtubule mesh in the oocyte cytoplasm. Low-developmental-competence oocytes display an altered mitochondria distribution and microtubule arrangement or seem to lack the temporal coupling of the two phenomena. We speculate that a tightly linked timing of mitochondria relocation and cytoskeleton microtubule formation in the cytoplasm of the oocyte during IVM may represent a key cytoplasmic factor regulating pig embryo parthenogenetic development.

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes.

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.

ACE inhibitor reduced mortality following myocardial infarction by attenuating the inflammatory response during early stage of MI

ACE inhibitor reduced mortality following myocardial infarction by attenuating the inflammatory response during early stage of MI

Fertilization and blastocyst progression in in vitro matured oocytes in stimulated cycles

Objective: The maturation of immature oocytes retrieved in unstimulated cycle has been shown to be successful in PCOS patients, but fertilization and pregnancy rates are poor in stimulated cycle. Approximately 10–14 % of retrieved oocytes are in GV and 10–12 % in MI stage in our assisted fertilization program. The aim of this study was to evaluate fertilization and progression to blastocyst stage of immature oocytes retrieved in stimulated cycles. Design: Prospective study. Materials and Methods: All patients were stimulated with a long GnRH protocol and either with human menopausal gonadotropin or pure urinary FSH. All oocytes were kept in culture medium for 2 hours prior to denudation and subsequently they evaluated for maturation. After the removal of cumulus-oocyte complex MII oocytes were injected with spermatozoa and MI and GV oocytes were incubated for in vitro maturation. Maturation medium consists of TCM 199 supplemented with 10% HSA, 0,075 IU r-FSH/ml (Gonal-F, Serono, Switzerland), 0.5 IU/ml hCG (Profasi, Serono, Switzerland), 10 IU/ml r-LH/ml, 0,29 mM pyruvate, Streptomisine and Penicilline. We kept denuded GV and MI oocytes in maturation medium in 30μl drops for 24 to 48 hours and maturation was assessed by the presence of polar body. Metaphase II oocytes were fertilized using ICSI and they were kept in G1 medium until day 2, then they transferred into G2 medium. Embryos at four-cell stage at day 2 were transferred into G2 medium in microdrops and monitored for progression to blastocyst stage. Results: Totally 2136 oocytes were retrieved from 178 patients undergoing their first ICSI cycle. Three hundred and five (14%) of them were in GV and 236 of them were in MI stage. As shown in figure I, 24 hours after incubation 82 of GV oocytes progressed to MI stage and 97 of remained as GV, 48 hours after incubation 22 of 97 GV oocytes and 28 of 82 MI oocytes progressed to MII stage. In overall 176 of 305 GV and 156 of 236 MI oocytes progressed to M-II stage after 48 hours of incubation. For MI oocytes 147 out of 236 of them progressed to MII stage after 24 hours and another 9 oocytes after 48 hours. Table I. Progression to MII oocytes in in vitro matured GV and MI oocytes Oocytes 24 hours 48 hours GV (n=305) 126 (41.3%) 176 (57.7%) MI (n=236) 147 (62.1%) 156 (66.1%) Fertilization rate in MII oocytes developed from GV oocytes after 24 hours was 48% (60/126) and 25% (15/60) of them were 3PN. In GV oocytes reached to MII-stage after 48 hours ferilization rate was 32% (16/50) and 5 of them (31.25%) were 3PN. Overall fertilization rate in all in vitro matured GV oocytes after 48 hours was 43.2%(76/176) and 20 of them were 3PN (26.3%). Blastocyst progression rate was 12%. Fertilization rate in MII oocytes developed from MI oocytes after 24 hours was 55%(81/147) and 21%(17/81) of them were 3PN. In those reached to MII-stage after 48 hours fertilization rate was 22% (2/9). Overall fertilization rate in all in vitro matured MI oocytes was 53.2% (83/156) and blastocyst progression rate was 23%. All abnormally fertilized oocytes did not cleave or arrested at 2–4 cell stage. Conclusion: Fertilization and blastocyst progression rate is low and abnormal fertilization rate is higher than normal in both GV and MI oocytes retrieved in stimulated cycles. This may be due to intrinsic abnormalities in these oocytes. Although 24 hours of in-vitro maturation seems to be enough for MI oocytes, GV oocytes can be incubated for 48 hours.

Haploidization of metaphase somatic chromosomes using enucleated MI ooplast

Objective: During haploidization the somatic nuclei would be enforced to transform from interphase to premature condensed chromosomes (PCC) when MII cytoplasts are used. Recently, it has been reported unequal (or non-equilibrated) distribution of genetic materials during this process (using MII ooplasts). Therefore, the objective of the present study was to test the possibility of haploidization without the occurrence of PCC when the somatic metaphase chromosomes interact with meiotic metaphase I cytoplasm instead of metaphase II cytoplasm.Design: Nuclear transfer of somatic cell at the metaphase to the cytoplasm of enucleated MI oocyte followed by successive observation of meiotic spindle formation and chromosome alignment.Materials and Methods: To obtain somatic cells at metaphase, mouse skin fibroblast cells were cultured with 0.125 μg/ml nocodazole for 3 hr in DMEM with 10 % FCS. After gentle pipetting, fibroblast cells floating in the medium were at metaphase and collectedfor nuclear transfer. The mitotic nuclei were transferred and electrofused to enucleated MI-stage mouse oocytes and in vitro matured with HTF and 20% FCS. The reconstructed oocytes were fixed and stained with propidium iodide and FITC-conjugated anti-α-tubulin antibody to investigate the chromosomes and microtubule networks using laser scanning confocal microscopy.Results: A total 10 out of 47 reconstructed oocytes fused successfully and were examined. After 12 h of in vitro maturation, 6 out of the 10 fused oocytes extruded polar bodies (PB) at the expected MII stage. Among these 4 had assembled meiotic II spindles while all 6 had misalignment of somatic chromosomes. The remaining 4 reconstructed oocytes (without PB extrusion) were likely remained at MI stage. All the expected MI arrest oocytes had bipolar spindles (4/4) while the somatic chromosomes were not aligned consistently on the metaphase plate of the meiotic spindles (4/4).Conclusion: This study demonstrates that enucleated MI ooplasts are able to coordinate the microtubule networks to form the first and second meiotic spindle and to induce mitotic chromosomes to undergo artificial meiosis. However, high frequency of alterations in the somatic chromosome alignment was also found during this novel haploidization procedure, indicating the deficiency of attachment between microtubule and somatic chromosomes even without PCC. Objective: During haploidization the somatic nuclei would be enforced to transform from interphase to premature condensed chromosomes (PCC) when MII cytoplasts are used. Recently, it has been reported unequal (or non-equilibrated) distribution of genetic materials during this process (using MII ooplasts). Therefore, the objective of the present study was to test the possibility of haploidization without the occurrence of PCC when the somatic metaphase chromosomes interact with meiotic metaphase I cytoplasm instead of metaphase II cytoplasm. Design: Nuclear transfer of somatic cell at the metaphase to the cytoplasm of enucleated MI oocyte followed by successive observation of meiotic spindle formation and chromosome alignment. Materials and Methods: To obtain somatic cells at metaphase, mouse skin fibroblast cells were cultured with 0.125 μg/ml nocodazole for 3 hr in DMEM with 10 % FCS. After gentle pipetting, fibroblast cells floating in the medium were at metaphase and collectedfor nuclear transfer. The mitotic nuclei were transferred and electrofused to enucleated MI-stage mouse oocytes and in vitro matured with HTF and 20% FCS. The reconstructed oocytes were fixed and stained with propidium iodide and FITC-conjugated anti-α-tubulin antibody to investigate the chromosomes and microtubule networks using laser scanning confocal microscopy. Results: A total 10 out of 47 reconstructed oocytes fused successfully and were examined. After 12 h of in vitro maturation, 6 out of the 10 fused oocytes extruded polar bodies (PB) at the expected MII stage. Among these 4 had assembled meiotic II spindles while all 6 had misalignment of somatic chromosomes. The remaining 4 reconstructed oocytes (without PB extrusion) were likely remained at MI stage. All the expected MI arrest oocytes had bipolar spindles (4/4) while the somatic chromosomes were not aligned consistently on the metaphase plate of the meiotic spindles (4/4). Conclusion: This study demonstrates that enucleated MI ooplasts are able to coordinate the microtubule networks to form the first and second meiotic spindle and to induce mitotic chromosomes to undergo artificial meiosis. However, high frequency of alterations in the somatic chromosome alignment was also found during this novel haploidization procedure, indicating the deficiency of attachment between microtubule and somatic chromosomes even without PCC.

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.