Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 microM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 micros) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5-81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2-32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.     

The effects of various antioxidants on the development of parthenogenetic porcine embryos

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, β-mercaptoethanol (β-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 μM β-ME; 17.2% and 17.5% for 50 and 100 μM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, β-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P?<?0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P?<?0.05). When the reactive oxygen species levels in two-cell embryos after 1 μM β-ME and 100 μM α-tocopherol treatment were examined, those were lower than control group (P?<?0.05). In conclusion, it was found that the three antioxidants, β-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

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.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.     

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.     

Heat stress-induced apoptosis in porcine in vitro fertilized and parthenogenetic preimplantation-stage embryos

Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Developmental competence of porcine parthenogenetic embryos relative to embryonic chromosomal abnormalities

Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM3) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 +/- 7.4 vs. 28.7 +/- 3.7; 10.9 +/- 2.8 vs. 21.5 +/- 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 +/- 1.8 vs. 29.7 +/- 0.8; 29.7 +/- 3.3 vs. 32.0 +/- 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 +/- 12.8 vs. 16.4 +/- 7.4 on day 6), and increased with culture time (71.9 +/- 12.1 vs. 10.9 +/- 2.8. on day 7,and 75.0 +/- 22.6 vs. 12.1 +/- 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20-39, >40) and into three groups according to the morphological diameter (<150, 150-180, >180 microm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 microm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro.     

Effects of leptin supplementation in in vitro maturation medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs

The objective of the present study was to investigate the effects of leptin addition in in vitro maturation (IVM) medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs. In experiment 1, oocytes were matured in North Carolina State University 23 (NCSU-23) medium supplemented with various concentrations of leptin: 0, 1, 10 and 100 ng/ml. IVM medium added with 10 or 100 ng/ml leptin significantly increased the rate of oocytes reaching metaphase II compared to the control (76.8% and 73.8% versus 61.7%). In experiment 2, the influence of the timing of leptin addition in IVM medium on meiotic maturation of porcine oocytes was assessed, and maximum maturation rate of oocytes developing to metaphase II was achieved when supplemented during the first half (0-22 h), the latter half (22-44 h) or the entire maturation period (0-44 h) compared to the control (80.5%, 84.7% and 78.1% versus 70.4%). In experiment 3, leptin strikingly increased the blastocyst rate of parthenogenetic embryos at the concentration of 10 ng/ml (37.5% versus 21.7%) and this increase was independent of the addition timing (0-44, 0-22, 22-44 h) compared to the control (32.5%, 34.6% and 31.5% versus 16.2%). Moreover, total cell number per blastocyst of parthenogenetic embryos was obviously increased in the 10 and 100 ng/ml leptin treatments as compared with the control (36, 38 versus 28). In experiment 4, 10 ng/ml leptin treatment significantly increased the rate of cleavage (72% versus 56%) of cloned embryos. Meanwhile, the rate of blastocyst formation was also improved although no significant difference was found (12.8% versus 7.1%). Collectively, our results indicate that leptin supplementation in IVM medium may be beneficial not only for developmental potential of oocytes but for subsequent developmental competence of embryos produced by parthenogenetic activation and the cleavage of embryos derived by somatic cell nuclear transfer (SCNT).     

Oct4基因在猪孤雌激活和体外受精早期胚胎中的表达特征

目的研究植入前胚胎发育重要基因Oct4在猪孤雌和体外受精胚胎中的表达特征。方法收集成熟卵母细胞、孤雌和体外受精2细胞、4细胞、8细胞胚胎和囊胚,做荧光即时定量PCR检测,以体外成熟的猪卵母细胞做对照分析相对表达量。结果孤雌组和体外受精组胚胎在8细胞期Oct4表达量均最高(P<0.05),在孤雌和体外受精组囊胚相对于其他时期Oct4表达量最低(P<0.05)。在同一时期孤雌和体外受精胚胎上Oct4表达并没有差异。结论多能性基因Oct4在卵裂发育时期表达量动态变化,孤雌胚胎在一定程度上可作为体外胚胎基因表达的模型,且不同的胚胎培养条件可能导致基因表达的差异。     

Developmental Potential of Haploid-derived Parthenogenetic Cells in Mouse Chimeric Embryos1

Studies were made on the contribution of haploid-derived parthenogenetic cells to haploid parthenogenetic ? fertilized chimeric embryos on day 9 and 10 of pregnancy. In most cases, the contribution of haploid-derived parthenogenetic cells to embryonic tissues was higher than that to extraembryonic tissues. The contribution of haploid-derived cells to embryonic tissues of some chimeras was more than 90%. Chromosomal analysis showed that actively dividing cells in most chimeric embryos contained about 40 chromosomes, indicating that they were diploidized, as haploid parthenogenetic blastocysts have about 20 chromosomes. Results suggested that haploid-derived parthehogenetic cells in chimeric embryos diploidized spontaneously after the blastocyst stage. These cells were capable of differentiating into most cell types of embryonic tissues, but scarcely differentiated into extraembryonic tissues of day 9 embryos. The fate of haploid-derived parthenogenetic cells during postimplantational development was similar to that of diploid parthenogenetic cells that had been diploidized experimentally in the one-cell stage.     

Heat shock of porcine zygotes immediately after oocyte activation increases viability

We evaluated the effect of different timing variations of an applied heat shock on parthenogenetically activated (PA) porcine embryos. PA embryos were heat shocked for 9 hr at 42 degrees C from either 0-9 hr post activation (hpa; 09HS), 13-22 hpa (1322HS), or 22-31 hpa (2231HS). Analysis of 24-hr cleavage rates (P < 0.0001), day 5 cell numbers (P < 0.005), day 7 blastocyst rates (P < 0.0001), and day 7 cell numbers (P < 0.05) showed that 09HS embryos developed more successfully in vitro than did all other treated and control embryos. In vitro fertilized (IVF) embryos were exposed to similar heat treatments as described for PA embryos, and embryos derived from somatic cell nuclear transfer (SCNT) were exposed only to the control and 09HS treatments to assess the effects of the different heat treatments on the timing of first cleavage and development to blastocyst. Embryos derived from both IVF and SCNT showed higher proportions of cleaved embryos on day 1 of development when heat shocked immediately after fertilization or fusion/activation as compared to NHS controls (P < 0.05). Blastocyst rates however, showed only modest (IVF; P = 0.089) or no (SCNT; P > 0.1) improvement as compared with control embryos. In summary, exposing PA embryos to elevated temperatures immediately after oocyte activation results in dramatically enhanced developmental potential. A thorough characterization of this phenomenon may yield findings that can serve to increase the efficiency with which PA, IVF, and SCNT embryos are produced in vitro.