Effect of Kisspeptin on the Developmental Competence and Early Transcript Expression in Porcine Oocytes Parthenogenetically Activated with Different Methods.

Recent studies showed the modulatory effect of kisspeptin (KP) on calcium waves through the cell membrane and inside the cell. Spermatozoon can induce similar ooplasmic calcium oscillations at fertilization to trigger meiosis II. Here, we evaluated the effect of KP supplementation with 6-dimethylaminopurine (6-DMAP) for 4 h on embryonic development after oocyte activation with single electric pulse, 5 µM ionomycin, or 8% ethanol. Compared to control nonsupplemented groups, KP significantly improved embryo developmental competence electric- and ethanol-activated oocytes in terms of cleavage (75.3% and 58.6% versus 64% and 48%, respectively, p < 0.05) and blastocyst development (31.3% and 10% versus 19.3% and 4%, respectively, p < 0.05). MOS expression was increased in electrically activated oocytes in presence of KP while it significantly reduced CCNB1 expression. In ionomycin treated group, both MOS and CCNB1 showed significant increase with no difference between KP and control groups. In ethanol-treated group, KP significantly reduced CCNB1 but no effect was observed on MOS expression. The early alterations in MOS and CCNB1 mRNA transcripts caused by KP may explain the significant differences in the developmental competence between the experimental groups. Kisspeptin supplementation may be adopted in protocols for porcine oocyte activation through electric current and ethanol to improve embryonic developmental competence.

The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture.

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

trans-10, cis-12 conjugated linoleic acid enhances in vitro maturation of porcine oocytes.

The aim of this study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) supplementation on oocyte maturation and embryo development in pigs. Compared with the control, supplementation of 50 µM t10c12 CLA to in vitro maturation (IVM) medium significantly increased the proportion of oocytes at the metaphase-II (MII) stage and subsequent parthenogenetic embryo development in terms of cleavage rate, blastocyst formation rate, and cell numbers in blastocysts. The t10c12 CLA-treated oocytes resumed meiotic maturation and progressed to the MII stage significantly faster than those of control. The expression of phosphorylated mitogen-activated protein kinase 3/1 (p-MAPK3/1) and cyclooxygenase-2 (COX2) in cumulus oocyte complexes (COCs) at 5, 10, and 22 hr of IVM were significantly increased in the t10c12 CLA-treatment group. The level of p-MAPK3/1 in t10c12 CLA-treated MII oocytes was also higher (P < 0.05) than that of control. Moreover, t10c12 CLA supplementation partially overcame the negative effects of U0126 on cumulus expansion and nuclear maturation, and completely recovered COX2 protein levels in the presence of U0126. Treatment of COCs with NS398 also significantly suppressed cumulus expansion and nuclear maturation, which was overcome by t10c12 CLA. Yet, this simulatory effect of t10c12 CLA was blocked in the presence of both U0126 and NS398. The t10c12 CLA treatment significantly reduced reactive oxygen species level and increased glutathione concentrations in MII oocyte. In conclusion, supplementation of t10c12 CLA during porcine oocyte maturation exerts its beneficial effects on nuclear and cytoplasmic maturation, which contributes to enhancing subsequent embryo development.

Effects of salinity and ultraviolet radiation on the bioaccumulation of mycosporine-like amino acids in Artemia from Lake Urmia (Iran).

We investigated the effects of salinity and artificial UV radiation on the accumulation of mycosporine-like amino acids (MAAs) in sexual and parthenogenetic Artemia from Lake Urmia. The nauplii hatched from the cysts were cultured until adulthood under two salinities (150 and 250 g L(-1) ) and two light treatments (PAR and PAR+UVR) in the laboratory. Finally, the Artemia were analyzed for their concentration of MAAs. In most of the cases, the higher salinity level applied was found to increase the MAA concentrations in both Artemia populations significantly. The acquisition efficiency of MAAs in both Artemia populations increased under exposure to UVR-supplemented photosynthetically active radiation (PAR) compared to those raised under PAR, except for Porphyra-334. It was observed that combination of UV radiation and elevated salinity significantly increased the bioaccumulation of MAAs. Thus, the presence of these compounds in these populations of Artemia may increase their adaptability for living in high-UV and high-salinity conditions prevailing in Lake Urmia. Higher concentrations of MAAs in the parthenogenetic population of Artemia could be probably attributed to its mono sex nature and higher adaptation capacities to extreme environmental conditions.

Ascorbic acid improves the developmental competence of porcine oocytes after parthenogenetic activation and somatic cell nuclear transplantation.

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.

Functional characterization of an apple apomixis-related MhFIE gene in reproduction development.

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple.

In vitro maturation and artificial activation of donkey oocytes.

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 µl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.

Zinc availability regulates exit from meiosis in maturing mammalian oocytes.

Cellular metal ion fluxes are known in alkali and alkaline earth metals but are not well documented in transition metals. Here we describe major changes in the zinc physiology of the mammalian oocyte as it matures and initiates embryonic development. Single-cell elemental analysis of mouse oocytes by synchrotron-based X-ray fluorescence microscopy (XFM) revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. Perturbation of zinc homeostasis with a cell-permeable small-molecule chelator blocked meiotic progression past telophase I. Zinc supplementation rescued this phenotype when administered before this meiotic block. However, after telophase arrest, zinc triggered parthenogenesis, suggesting that exit from this meiotic step is tightly regulated by the availability of a zinc-dependent signal. These results implicate the zinc bolus acquired during meiotic maturation as an important part of the maternal legacy to the embryo.

Effects of L-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine cumulus cells free oocytes.

The maturation and developmental potential on cumulus-cell-free oocytes is of great importance theoretically and practically. The present study was to investigate the effects of l-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine denuded oocytes (DOs). Porcine DOs were cultured in maturation medium supplemented with vitamin C (0, 50, 100, 250, 500, 750 microM) and vitamin E (0, 10, 20, 50, 100, 250 microm), respectively. And they were also co-cultured with dispersed cumulus cells (group CCscoculture), intact cumulus cells oocyte complexes (COCs) (group COCscoculture), and COCs whose oocytes were removed (group OOXcoculture), respectively. After 44 h incubation, the maturation rates, cleavage rates and blastocyst rates after parthenogenetic activation in three experiments mentioned above were collected and analysed, respectively. L-Ascorbic acid promoted porcine DOs in vitro maturation and blastocyt development after parthenogenetic activation while alpha-tocopherol did not increase the in vitro maturation rates, but improved the blastocyst rate. None of the three co-culture manner promoted the in vitro maturation and the cleavage of porcine DOs after parthenogenetic activation, but all the co-culture manners improved the blastocyst rates. Both Vitamin C and E enhance the in vitro developmental potential of porcine DOs. Co-culture increases the developmental potential of porcine DOs.

Calmodulin-dependent protein kinase gamma 3 (CamKIIgamma3) mediates the cell cycle resumption of metaphase II eggs in mouse.

Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca(2+) signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIgamma3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca(2+) stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIgamma3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIgamma3 plays a necessary and sufficient role in transducing the oscillatory Ca(2+) signal into cell cycle resumption, but not into cortical granule release.

Stage-dependent effect of leptin on development of porcine embryos derived from parthenogenetic activation and transgenic somatic cell nuclear transfer.

Accumulating evidence suggests that leptin may play important roles in preimplantation embryonic development, although this remain controversial, and little is known about whether leptin has a stage-dependent regulatory effect on development of porcine embryos derived by parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). The objective of this study was to investigate the effects of addition of leptin to in vitro culture (IVC) medium on development of porcine embryos derived by PA and SCNT. We found that addition of 50 ng/ml human recombinant leptin improved the rate of PA embryos reaching the blastocyst stage and increased the total cell number of blastocysts compared with the control group. The maximal blastocyst rate of SCNT embryos was achieved at 50 ng/ml, and the total cell number of blasocysts was increased significantly at 500 ng/ml leptin concentration. However, the ratio of the inner cell mass (ICM) to total cell number was not affected in any of the groups. Supplementation of leptin (50 ng/ml) from day 3, approximately the 4-8-cell stage, as in the case of the positive control, significantly increased the blatocyst rate of PA embryos compared with the negative control and inhibited cell apoptosis. There were no beneficial effects on embryonic development when 50 ng/ml leptin was added to the culture medium from day 1 to day 3 or from day 4 to day 6. These results indicate that leptin could improve the development and the quality of PA and SCNT embryos; and 50 ng/ml leptin performs its primary stimulatory effect at 4-8-cell stage and that leptin may have no effect on the maternal-zygote transition (MZT) of porcine PA and SCNT embryos.

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos.

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes.

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.

Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo.

Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes.

Human factors facilitating the spread of a parasitic honey bee in South Africa.

Workers of the honey bee subspecies Apis mellifera capensis (Eschscholtz) produce female offspring by thelytokous parthenogenesis and can parasitize colonies of other subspecies. In 1990, translocation of 400 colonies of A. m. capensis into the distribution area of A. m. scutellata by a commercial beekeeper triggered a dramatic parasitic phenomenon. Parasitized colonies died within a few months of infestation, and this resulted in the loss of tens of thousands of colonies by commercial beekeepers in the A. m. scutellata range in South Africa. To deal with the problem and to identify methods that would limit the impact of the social parasite, we investigated the link between beekeeping management and severity of parasitic infestations in terms of colony mortality and productivity. We demonstrate that colonies from apiaries subjected to migrations are very susceptible to infestation and consequently show dramatic mortality. Their productivity is also inferior to sedentary colonies and those in isolated apiaries in terms of honey yield and brood quantity. Furthermore, by concentrating hives in small areas and often in the vicinity of other beekeepers, cross-infestations can easily occur. This can undermine previously parasite-free beekeeping businesses. As a result of our surveys, we propose beekeeping practices based on locally trapped bees, reduced migration, and better control of parasite spread, thus promoting the conservation of these pollinators. If followed by all the South African beekeepers, these measures should limit the spread of the parasite until it is eliminated within a few years, after which full migratory beekeeping practices could resume.

The effect of oxygen tension on porcine embryonic development is dependent on embryo type.

Reducing oxygen concentration from atmospheric levels during in vitro culture generally, but not invariably, improves embryonic development across a range of species. Since the few published reports of such an action in the pig are contradictory--perhaps a consequence of the derivation of the embryos prior to culture--a study was performed to examine the effect of O2 tension during culture on three different types of porcine embryos, namely: in vivo flushed embryos, and in vitro matured oocytes either fertilized in vitro or parthenogenetically activated. In vivo embryos (n=208) were flushed at the 2-8 cell stage. Cumulus oocyte complexes (COCs) destined for IVF or parthenogenetic activation were derived from 2 to 6 mm, post-pubertal ovarian follicles and matured for 48 h in TCM-199. Parthenogenones were generated by activating denuded oocytes (n=573) with 10 mM calcium ionophore, followed by 2 mM DMAP prior to culture. The IVF embryos (n=971) were produced by fertilizing COCs (day 0) with fresh ejaculated semen in modified tris-based medium for 6 h before cumulus removal. All embryos were cultured in BECM-3 containing 12 mg/mL fatty-acid-free BSA up to day 4, followed by BECM-3 supplemented with 10% calf serum until day 7. The gas environment for IVM/IVF was 5% CO2 in air, while that for IVC was either 5% CO2 in air or 5% O2, 5% CO2 and 90% N2. Low O2 tension increased both day 7 blastocyst rates (high versus low O2, respectively; 9.3+/-2.9%: 26/280; 23.9+/-4.2%: 71/293; P<0.001) and total cell numbers (39.3+/-2.9, n=24 versus 61.2+/-7.7, n=61; P=0.01) of parthenogenetically activated embryos. In contrast, such a treatment neither affected blastocyst rates (89.3+/-6.9 versus 87.8+/-7.5) nor cell numbers (87.4+/-4.5 versus 87.7+/-4.8) of in vivo flushed embryos. The effect of reduced O2 concentration on IVF embryos was intermediate, since only cell numbers were improved (69.8+/-3.5, range=17-204, n=49; 88.5+/-5.8, range=28-216; n=66; P<0.01), equivalent to that recorded in in vivo flushed embryos. However, blastocyst rates were unaffected (10.7+/-1.4%: 51/486; 12.9+/-2.2%: 67/485). The effect, when present, of reducing O2 concentration from 20 to 5% was beneficial for pig in vitro embryonic development. The responses are apparently dependent on firstly, the manner by which the embryonic cell cycle is activated and secondly, the derivation of the tissue prior to placement into culture, if the observed resilience of in vivo embryos is independent of treatment duration.

Effect of protein kinase C activator on mitogen-activated protein kinase and p34(cdc2) kinase activity during parthenogenetic activation of porcine oocytes by calcium ionophore.

The objective of this study was to elucidate the role of a [Ca2+]i rise and protein kinase C (PKC) activation on decreases of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase activity during parthenogenetic activation of porcine oocytes. In oocytes treated with 50 microM Ca2+ ionophore, degradations of both p34(cdc2) kinase and MAP kinase activity were observed and half of these oocytes formed pronuclei. However, a supplement of PKC inhibitor, calphostin C, after 50 microM Ca2+ ionophore treatment, was sufficient to inhibit the inactivation of MAP kinase and pronuclear formation in the oocytes. These results showed that PKC played an important role in Ca2+-induced oocyte activation. On the other hand, 10 microM Ca2+ ionophore treatment could not affect the MAP kinase activity but induced a transient decrease of p34(cdc2) kinase activity, which resulted in recovery of p34(cdc2) kinase activity and progression to meiotic metaphase III stage. To investigate the effects of PKC activator on oocytes treated with 10 microM Ca2+ ionophore, matured oocytes were cultured with phorbol 12-myriatate 13-acetate (PMA), after 10 microM Ca2+ ionophore treatment. The additional treatment suppressed the recovery of p34(cdc2) kinase activity and rapidly induced a decrease of MAP kinase activity, and these low activities were maintained until 12-h cultivation. As a result, a significantly higher percentage of these oocytes (67%) had pronuclei at 12-h cultivation. Moreover, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not p34(cdc2) kinase activity in matured porcine oocytes. In conclusion, the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34(cdc2) kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes.

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation.

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 degrees C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8-15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents--CaA, ethanol, 6-DMAP, or cycloheximide--could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78-82% versus 3-13%) and blastocyst development (31-40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide-treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro-fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes.

Effects of cumulus cells and follicle-stimulating hormone during in vitro maturation on parthenogenetic activation of bovine oocytes.

The response to parthenogenetic activation (calcium ionophore A23187) of bovine oocytes after 30 hr of culture in different maturation conditions was evaluated. The activation rates of oocytes in response to 10 mu M A23187 were similar (86% +/- 4 vs. 89% +/- 8) when the oocytes matured with or without cumulus cells were cultured in TCM-199 + 10% fetal calf serum (maturation medium [MM]) alone. However, the activation rates were significantly lower in oocytes matured with than without cumulus cells (48 +/- 10 vs. 79% +/- 16; P < 0.05) when MM was supplemented with 0.5 microgram/ml follicle-stimulating hormone (FSH), 50 micrograms/ml luteinizing hormone (LH), and 1.0 microgram/ml estradiol-1 beta (E2). When oocytes with cumulus cells were cultured in different media, MM along, MM + 0.5 microgram/ml FSH, MM + 50 micrograms/ml LH, MM + 1.0 microgram/ml E2, and MM + all three hormones, the percentages of activated oocytes were 91% +/- 3.52% +/- 14.83% +/- 7, 83% +/- 13, and 54% +/- 7, respectively. Oocytes with or without cumulus cells during culture began to form a pronucleus 5 hr after activation (5% +/- 1 and 7% +/- 1, respectively). Finally, the proteins synthesized by oocytes matured with or without cumulus cells before (3 hr) and after (10 hr) activation was labelled with [35S]methionine for analysis. Before activation, a 47 kD protein complex was produced by cumulus cell-intact oocytes. An active protein synthesis of 60 kD was characteristic for the oocytes with cumulus cells cultured in MM containing 0.5 micrograms/ml FSH. After activation, there was an appearance of proteins at 75 kD and 47 kD in the oocytes with cumulus cells but not in the oocytes without cumulus cells during the maturation period. These results indicate that bovine oocytes matured in vitro have different capacities for parthenogenetic activation depending on the presence or absence of cumulus cells and the presence of FSH in MM during culture.