Cycloheximide Group Research Articles

Identification of thyroid hormone response genes in the remodeling of dorsal muscle during Microhyla fissipes metamorphosis.

Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle. In our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of Microhyla fissipes after TH, cycloheximide (CHX), and TH_CHX treatment. We first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in M. fissipes. Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.

Production of collared peccary (Pecari tajacu Linnaeus, 1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ± 1.2 vs. 100% ± 0.0), presence of first polar body (65.9% ± 1.2 vs. 70.5% ± 1.8), nuclear status in second metaphase (62.5% ± 11.6 vs. 68.4% ± 4.9), cytoplasmic maturation (100.0% ± 0.7 vs. 75.0% ± 0.7), reactive oxygen species levels (0.5 ± 0.2 vs. 0.3 ± 0.1), and mitochondrial membrane potential (1.1 ± 0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40V/mm, 20μs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION)+6-DMAP+CHX (12.07%) activation was higher than that of ION+6-DMAP (8.6%) activation or ION+CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION+6-DMAP+CHX and DC+6-DMAP+CHX groups. The blastocyst rate of ION+6-DMAP+CHX-activated oocytes in the basic rabbit culture medium (M-199)+10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199+10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

Preliminary study of estrogen effects on calcium free smooth muscle cells at the endometrial-myometrial interface in uteri with adenomyosis

To investigate the effect and mechanism of estrodial (E(2)) on intracellular free calcium in the endometrial-myometrial interface (EMI) smooth muscle cells from uteri with adenomyosis. From March 2011 to October 2011, 16 uterus specimens were collected from patients with adenomyosis undergoing hysterectomy in Beijing Obstetrics and Gynecology Hospital, which included 9 proliferative endometrium and 7 secretory endometrium. EMI smooth muscle cells from the uterus were cultured and loaded with calcium ion (Ca(2+)) fluorescent probe fluo-4/AM. The labeled cells were stimulated with the various concentration of E(2)(1×10(2), 1×10(3), 1×10(4), 1×10(5) pmol/L, respectively), then the changes of intracellular Ca(2+) fluorescence intensity were measured by laser scanning microscopy. The most suitable concentration of E(2) was selected, and the reaction difference between the EMI smooth muscle cells of two menstrual phases were also investigated; The changes of intracellular Ca(2+) fluorescence intensity were detected proliferative and secretory smooth muscle cells in E(2) conjugated to bovine serum albumin (17β-E(2)-BSA) group, cycloheximide (CHX) group, fulvestrant (ICI182780) group and pertussis toxin (PTX) group. (1) The cell viability of primary cultured EMI smooth muscle cells was well at 24 hours culture. (2) 1×10(2) - 1×10(5) pmol/L E(2) can rapidly increase the intracellular Ca(2+) fluorescence intensity within 1 min (P < 0.01);The increased amplitudes caused by 1×10(4) pmol/L and 1×10(5) pmol/L E(2) were the most significant, but there was no significant difference between them (P > 0.05). 1×10(4) pmol/L was the most suitable concentration. (3) With the 1×10(4) pmol/L E(2), the Ca(2+) fluorescence intensity changes showed no significant difference between the EMI smooth muscle cells from the proliferative phase and secretory phase uterus (P > 0.05). The Ca(2+) fluorescence intensity changes were 646 ± 32 in 17β-E(2)-BSA group and 602 ± 31 in CHX group, when compared with 513 ± 26 and 617 ± 35 in respective control group, no significant difference was observed (P > 0.05). The increased amplitude of 188 ± 20 in the PTX group and 302 ± 11 in ICI182780 group exhibited significant difference with 632 ± 33 and 635 ± 24 in respective control group (P < 0.01). E(2) could increase the intracellular Ca(2+) of EMI through a membrane receptor dependent and nongenomic mechanism of action.

Effects of Chemical Activation Treatment on Development of Swamp Buffalo (Bubalus bubalis) Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Injection

The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in-vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6-dimethylaminopurine (6-DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6-DMAP (48%) groups (p<0.01) whereas Io + 6-DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6-DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6-DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6-DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.

Cycloheximide speeds the porcine oocyte nuclear kinetics and retains embryonic developmental potential in the presence of gonadotrophins

Animal embryo engineering requires large amounts of synchronized mature oocytes in vitro. However, porcine cumulus-oocyte complexes aspirated from 3-8 mm follicles are at different germinal vesicle stages. They reach metaphase II stages asynchronously when cultured in vitro. In this study, we examined the effects of pretreatment with or without cycloheximide (CHX), equine chorionic gonadotrophin (eCG), human chorionic gonadotrophin (hCG), and their combinations on meiotic synchronization and the developmental competence of porcine oocytes in vitro following electrical activation. The COCs were pretreated for 12 h with either control medium (TCM 199), CHX (TCM 199 + CHX), eCG/hCG (TCM 199 + eCG/hCG) or eCG/hCG + CHX (TCM 199 + CHX + eCG/hCG), and then cultured for up to 32 h with TCM199 + eCG/hCG. After 12 h pretreatment, the rates of germinal vesicle breakdown (GVBD) were lower (P &lt; 0.05) in the CHX (8.4%) and eCG/hCG + CHX (1.5%) groups compared with control (55.4%) and eCG/hCG (27.2%) groups. After removal of CHX and culture for an additional 12 h in vitro, the majority of the oocytes were synchronized at the GVBD stage in CHX (75.6%) and eCG/hCG + CHX (65.0%) groups. At additional 32 h of culture, the rate of oocytes in metaphase II in eCG/hCG + CHX group (68.3%) was significantly (P &lt; 0.05) higher than the eCG/hCG group (54.8%), but did not differ from other groups (control: 61.3%, CHX: 58.8%). After electrical activation, the cleavage and blastocyst formation rates in the CHX group (80.3%; 19.5%) were significantly (P &lt; 0.05) lower than those in the control group (95.5%; 45.3%), while no difference was found between eCG/hCG + CHX (82.2%; 34.4%) and control groups. Our data, hence, demonstrate pretreatment with CHX hastened nuclear kinetics of porcine oocytes cultured in vitro; however, embryo development potential was retained only when gonadotrophins is present in the in vitro maturation (IVM) medium. Thus, CHX should be used in the two-step culture systems in combination with gonadotrophins. Key words: Oocyte meiosis, synchronization, cycloheximide, embryo development, pig

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte's molecular programming and so does not significantly improve the efficiency of producing cloned pig births.

Neutralization of IL-10 Exacerbates Cycloheximide-Induced Hepatocellular Apoptosis and Necrosis

Cycloheximide (CHX)-induced liver injury in rats has been characterized by hepatocellular apoptosis and necrosis. We previously reported that Kupffer cell inactivation causes a reduction of IL-10 production, resulting in the exacerbation of CHX-induced liver injury. In this study, we directly evaluate the role of IL-10 in liver injury by a pretreatment with anti-IL-10 neutralizing antibody (IL-10Ab). Rats were given goat IgG or IL-10Ab before being treated with CHX (CHX group or IL-10Ab/CHX group). In the CHX group, the CHX treatment markedly induced hepatic mRNA and serum protein levels of IL-10. The up-regulation of IL-10 was significantly suppressed in the IL-10Ab/CHX group. Blocking IL-10 in the IL-10Ab/CHX group led to greater increases in hepatic mRNA and serum levels of proinflammatory cytokines, such as TNF-alpha and IL-6. The IL-10Ab/CHX group developed more severe hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes compared with the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in the livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results demonstrate that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines, such as a TNF signaling cascade, and in attenuating the CHX-induced liver injury.

215 DISTRIBUTION OF DIMETHYLATION OF HISTONE H3K9 IS NOT AFFECTED BY DNA, MESSENGER RNA, AND PROTEIN SYNTHESIS IN PRONUCLEAR-STAGE PORCINE EMBRYOS

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked with repression of transcription. Dimethylated H3K9 adopts a strict asymmetrical distribution in murine zygotes, with dimethylated H3K9 detectable only on maternally derived chromatin. In contrast, both male and female pronuclei in porcine zygotes can possess dimethylated H3K9; however, some asymmetry in H3K9 dimethylation exists between individual pronuclei, particularly in polyspermic embryos. The objective of this study was to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes. We hypothesized that the distribution of dimethylated H3K9 between individual pronuclei would not depend on alternations in chromatin structure induced by DNA or mRNA synthesis but would be affected by protein synthesis. To test this hypothesis, in vitro-matured porcine oocytes were fertilized in vitro, cultured in porcine zygote medium-3 containing 3 mg mL–1 of BSA, and allocated to 1 of 4 treatment groups: (1) incubation with 25 μg mL–1 of α-amanitin (α-AM), (2) incubation with 3 μg mL–1 of aphidicolin (APH), (3) incubation with 50 μg mL–1 of cycloheximide (CYC), and (4) nontreated controls. Embryos were removed from each treatment group at 10, 15, 20, and 25 h post gamete mixing, fixed, and processed to detect dimethylated H3K9 immunocytochemically. For monospermic embryos in the control group, 24% (7/29), 31% (8/26), 30% (7/24), and 20% (4/20) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the control group, 82% (32/39), 78% (31/40), 74% (28/38), and 65% (24/37) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the α-AM group, 29% (4/14), 14% (2/14), 8% (1/12), and 11% (1/9) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the α-AM group, 71% (15/21), 63% (12/19), 55% (10/18), and 47% (8/17) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the APH group, 31% (4/13), 23% (3/13), 23% (3/13), and 18% (2/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the APH group, 75% (15/20), 67% (12/18), 63% (12/19), and 56% (10/18) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the CYC group, 33% (5/15), 25% (4/16), 14% (2/14), and 9% (1/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the CYC group, 78% (18/23), 67% (16/24), 58% (14/24), and 59% (13/22) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. These results suggest that the distribution of dimethylated H3K9 between pronuclei is not affected by DNA, mRNA, or protein synthesis (P &gt; 0.05), but is affected by the age of the pronuclei (P &lt; 0.05).

235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P &gt; 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P &lt; 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.

Influence of Kupffer cell inactivation on cycloheximide-induced hepatic injury

In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl 3) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl 3 before being treated with the same dose of CHX (GdCl 3/CHX group). The necrotic change in the GdCl 3/CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl 3/CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl 3/CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl 3/CHX group. Thus, Kupffer cell inactivation by the GdCl 3 treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl 3/CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment.

25 THE EFFECTS OF OOCYTE SOURCES AND ACTIVATION PROTOCOLS ON THE DEVELOPMENT OF CLONED GOAT EMBRYOS

The objective of this study was to compare the development to the morula and blastocyst stages, after either cycloheximide (CHX) or ethanol (ETOH) activation, in somatic nuclear transfer (NT) goat embryos derived from 2 sources of oocytes. In vivo- and in vitro-matured oocytes were obtained from FSH-stimulated goats (Native, Saanen, and Native-Saanen crossbred goats). Gonadotropin treatment was performed with a modified program of a previous report (Reggio et al. 2001 Biol. Reprod. 64, 849-856). In vivo-matured oocytes were flushed from the oviduct of donor goats by exposing the reproductive tract via a small ventral laparotomy incision. In vitro-matured oocytes were aspirated and cultured in maturation medium (M199 + 10% FCS, 10 �g mL-1 FSH, 10 �g mL-1 LH, and 1 �g mL-1 17�-estradiol) for 22 h, at 38.5�C in 5% CO2 and air. Donor cells were prepared from ear skin fibroblasts of a female goat (Native breed). Cells, at passage 3-9, starved by culturing in 0.5% FCS for 4-8 days, were used for NT. Matured oocytes were enucleated, and cell-cytoplast couplets (n = 162 in vivo-, and n = 190 in vitro-matured oocyte groups, respectively) were fused by applying 2 DC pulses of 2.2 kV cm-1 for 30 �s. One to 2 h after fusion, fused embryos were either incubated in 10 �g mL-1 cycloheximide plus 5 �g mL-1 cytochalasin B for 5 h (CHX treatment) or in 7% ethanol for 5 min followed by a 4-h incubation in 2 mM 6-dimethylaminopurine plus 5 �g mL-1 cytochalasin B (ETOH treatment). NT embryos were then cultured in B2 medium supplemented with 5% FCS and Vero cells for 9 days. At the end of the culture period, the NT embryos were fixed and stained with Hoechst 33342 (Begin et al. 2003 Theriogenology 59, 1839-1850). The numbers of nuclei were counted under ultraviolet light. Fusion, cleavage, and development rates were compared using chi-square test or Fisher&amp;apos;s exact test. For the in vivo-matured oocyte group, there were no significant differences in fusion rates (78.1% vs. 68.7%), cleavage rates (87.7% vs. 87.0%, based on the numbers of embryos fused) between the CHX and ETOH treatment groups, respectively (P &amp;gt; 0.05). However, the development rates to morula and blastocyst stages of NT embryos derived from either in vivo- or in vitro-matured oocytes were significantly higher in the ETOH group than in the CHX group (in vivo: 15.2% vs. 0%, and in vitro: 7.1% vs. 0%, for ETOH and CHX groups, respectively; P &amp;lt; 0.05). For the in vitro-matured oocyte group, no significant differences were found between the CHX and ETOH groups in fusion rates (78.6% vs. 83.6%; P &amp;gt; 0.05), cleavage rates (80.5% vs. 83.9%: P &amp;gt; 0.05, based on the numbers of embryos fused). NT embryos from the CHX treatment group derived from in vivo- or in vitro-matured oocytes did not develop beyond the 16-cell stage. These results demonstrate that activation with CHX plus cytochalasin B treatment affects the development to the blastocyst stage of cloned goat embryos whether derived from in vivo- or in vitro-matured oocytes. This work was supported by the RGJ PhD program, Thailand Research Fund, and the Bureau of Biotechnology in Animal Production, Department of Livestock Development.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P &lt; 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

26 ASSESSMENT OF CHROMOSOME ABNORMALITIES IN SHEEP PARTHENOGENETIC AND NUCLEAR TRANSFER EMBRYOS: EFFECT OF 6-DMAP AND CYCLOHEXIMIDE ON PLOIDY

In current somatic cell nuclear transfer (NT) protocols, the reconstructed embryos are activated by incorporation of secondary oocyte activation compounds such as 6-DMAP or cycloheximide (CHX). The effects of these compounds on the chromosome complement of sheep NT embryos have not been studied in detail. Therefore, the aim of this study was to assess the chromosome abnormalities using sex chromosome specific probes of Day 6 blastocyst-stage sheep embryos produced from parthenogenetic activation and NT. Following 20–22 h of IVM, the oocytes were activated by electric pulsing followed by 30-min culture in cytochelasin B. They were reactivated using ionomycin (5 min) followed by 2-h culture in 6-DMAP or CHX. In contrast, NT embryos were produced using standard NT procedures using male sheep fetal fibroblasts. Reconstructed embryos were activated using the same methods described earlier. The embryos (compact morulae and blastocysts) were fixed and subjected to FISH analysis using cattle X and Y chromosome painting probes. The data were analyzed using Fisher's exact test. Of the parthenogenetic embryos (6-DMAP, n = 28; CHX, n = 32) analyzed, none of the embryos was totally haploid (X) or totally polyploid. When all of the nuclei per embryo were considered, normal (XX) genotype embryos were 6.2% and 0.0% in CHX and 6-DMAP groups, respectively. The rest of the embryos were abnormal due to mixoploidy (100% vs. 93.8%, P &lt; 0.05) in 6-DMAP and CHX treatment groups, respectively. The abnormal nuclei per embryo ranged from 7.3% to 72.2%. The mean total cell number of parthenogenetic blastocysts was 91.2 ± 4.3 and 81.8 ± 6.2 (mean ± SE) in 6-DMAP and CHX, respectively. Among NT embryos analyzed, (6-DMAP, n = 30; CHX, n = 32) only 40.0% and 43.8% of embryos were completely normal for XY chromosomes in 6-DMAP- and CHX-treated groups, respectively. The rest of the embryos were abnormal due to mixoploidy (60.0% vs. 56.2%, P &gt; 0.05) in 6-DMAP and CHX groups, respectively. Monosomy (XO or OY), trisomy (XXY), and tetrasomy (XXYY) were the common abnormalities detected in mixoploid embryos. The abnormal cells per embryo ranged from 3.8% to 41.8% in both treatment groups. The mean total cell number of NT blastocysts was 71.2 ± 9.8 and 63.8 ± 8.4, in 6-DMAP and CHX treatment groups, respectively. In conclusion, the 6-DMAP-treated embryos derived from parthenogenetic activation had significantly higher chromosomal abnormalities than CHX-treated embryo groups (P &lt; 0.05). In contrast, the NT embryos derived from either 6-DMAP or CHX treatment did not show any significant difference in producing chromosomally abnormal embryos at the blastocyst stage. This study also highlights the feasibility of using bovine chromosome painting probes on ovine embryo spreads. This work was supported by NSERC, OMAFRA, and ICCS.

Optimization of parthenogenetic activation protocol in porcine.

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.

Surface Ultrastructural Alterations of Bovine Oocytes after Parthenogenetic Activation

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.

Effects of d-amphetamine and strychnine on cycloheximide- and diethyldithiocarbamate-induced amnesia in mice.

Groups of C57BL/6J mice were administred cycloheximide (CYC) 30 min before or immediately after training on a passive avoidance task and tested 72 hr later. Some CYC-pretreated groups were given strychnine or d-amphetamine (d-amp) immediately after training and others were given d-amp 1 hr after training. Other groups were given diethyldithiocarbamate (DDC) at various times before or after training. Some DDC-pretreated groups were gived-amp or strychnine as described above for CYC groups. Immediate posttraining administration of 5 mg/kg d-amp, but not strychnine, prevented amnesia in CYC-pretreated mice. The DDC induced an apparent amnesia when administered from 30 min before training to 3 hr after training. Posttraining administration of d-amp or strychnine did not prevent DDC-induced amnesia. These results are discussed in relation to previous suggestions that CYC- and DDC-induced amnesia may be the result of a functional impairment of catecholamine neurotransmitter systems by these drugs.