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The growth and development of early mammalian embryos mainly take place in the fallopian tube, which not only provides nutrients for embryonic growth and development but also offers suitable mechanical conditions. The embryo culture system established in assisted reproductive technology mainly simulates the environment in which oocytes and embryos grow and develop in vivo. However, current in vitro embryo culture is mainly static and cannot completely mimic the mechanical environment in which embryos grow and develop in vivo. Therefore, to more accurately simulate the mechanical environment of embryos in the fallopian tube, we have developed a dynamic culture device to investigate the effects of mechanical stimulation on the in vitro maturation of immature oocytes and their parthenogenetic developmental potential. Immature mice oocytes were subjected to in vitro maturation by static culture and vibration (3 Hz, 6 Hz) with tilting for 15∼16 hours. The maturation of oocytes was observed after the culture period. The mature oocytes were activated by parthenogenesis and the rate of embryo compaction and formation of parthenogenetic blastocysts was analyzed. The results showed that using 3 Hz vibration and tilting can significantly improve the parthenogenetic development potential of immature mice oocytes.

Early cleavage (EC) influences the development of the pre-implantation and post-implantation embryo. Symmetric cleavage (Sym) and asymmetric cleavage (Asy) have been observed in EC, but its molecular mechanism remains unclear. This study was designed to pick out the key candidate genes and signaling pathway between Sym and Asy embryos by applying Smart-seq2 technique. In in-vitro fertilization (IVF) 2-cell embryos, Sym embryos and Asy embryos accounted for 62.55% and 37.45%, respectively. The 2-cell rate, blastocyst rate and total blastocyst cells of Sym group were significantly higher than those of Asy group (31.38% vs 18.79%, 47.55% vs 29.5%, 71.33 vs 33.67, P < 0.05). The 2-cell rate, blastocyst rate and total blastocyst cell number in parthenogenetic activation (PA) embryos in Sym group were significantly higher than those in Asy group (40.61% vs 23.64%, 63.15% vs 30.11%, 50.75 vs 40.5, P < 0.05). A total of 216 differentially expressed genes (DEGs) incorporating 147 genes up-regulated and 69 genes down-regulated genes were screened under the p-value <0.05 and |log2 (fold change)| ≥ 1 when compared with Sym group. Further Gene Ontology (GO) analysis showed that these DEGs were related to the regulation of metabolic process, cell cycle, chromosome segregation, centromeric region and microtubule cytoskeleton. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly enriched to oocyte meiosis, cell cycle, p53 and Hippo signaling pathways. We concluded that asymmetric cleavage is a consequence of altered gene expression. Atg4c, Sesn2, Stk11ip, Slc25a6, Cep19 and Cep55 associated with mitochondrial function and cytoskeletal structure were probably the key candidate genesto determine the zygote cleavage pattern., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.

Background: One goal of evolutionary developmental biology is to understand the role of development in the origin of phenotypic novelty and convergent evolution. Geckos are an ideal system to study this topic, as they are species-rich and exhibit a suite of diverse morphologies-many of which have independently evolved multiple times within geckos., Results: We characterized and discretized the embryonic development of Lepidodactylus lugubris-an all-female, parthenogenetic gecko species. We also used soft-tissue μCT to characterize the development of the brain and central nervous system, which is difficult to visualize using traditional microscopy techniques. Additionally, we sequenced and assembled a de novo transcriptome for a late-stage embryo as a resource for generating future developmental tools. Herein, we describe the derived and conserved patterns of L. lugubris development in the context of squamate evolution and development., Conclusions: This embryonic staging series, μCT data, and transcriptome together serve as critical enabling resources to study morphological evolution and development, the evolution and development of parthenogenesis, and other questions concerning vertebrate evolution and development in an emerging gecko model., (© 2019 Wiley Periodicals, Inc.)

Baicalin, a monomer of flavonoids extracted from dried roots of Scutellaria baicalensis, is used to treat female infertility. However, the effect of baicalin on oocyte maturation is unknown. In this study we investigated the effects of baicalin on the IVM of pig oocytes and subsequent embryo development following parthenogenetic activation (PA). We found that 0.1µgmL-1 baicalin significantly (P<0.05) increased the IVM rate of oocytes compared with the non-treatment (control) group by reducing levels of reactive oxygen species (ROS). In addition, the mRNA expression of genes related to nuclear maturation and cumulus cell expansion, mitochondrial membrane potential and ATP content was significantly (P<0.05) higher in baicalin-treated than control oocytes. To determine whether baicalin treatment during IVM of pig oocytes improves subsequent development of PA embryos, we measured the cleavage and blastocyst formation rates, as well as the number of cells per blastocyst. All these parameters were significantly (P<0.05) higher in the baicalin-treated than control group. In conclusion, this study demonstrates that baicalin improves pig oocyte maturation and subsequent embryo development invitro by inhibiting production of ROS and reducing apoptosis in oocytes.

This study was conducted to evaluate whether the poor developmental capacity of pig embryos after vitrification was related to the occurrence of apoptosis. Parthenogenetic blastocysts were used as the research material. The blastocoel recovery rate, mitochondrial membrane potential (ΔΨm), amount of early apoptosis, activities of several caspases, and relative abundance of mRNA of apoptosis-related genes involved in mitochondria and death receptor apoptotic pathways were detected before or after vitrification. The results indicate that the blastocoel recovery rate (31.0%) and total cells (31.8) of vitrified blastocysts were less than those of fresh blastocysts (100% and 38.2, P <  0.05). The ΔΨm of vitrified blastocysts was 0.46, which was less than that of fresh blastocysts (1.02, P <  0.05). The rate of apoptotic cells in vitrified blastocysts (8.1%) after TUNEL (TdT-mediated dUTP Nick-End Labeling) assay was markedly greater than that in fresh blastocysts (3.9%, P <  0.05). The pan-caspase, caspase-3, caspase-8 and caspase-9 activities of vitrified blastocysts (20.7, 20.6, 17.6 and 19.9) were markedly greater than those of fresh blastocysts (7.4, 6.5, 5.5 and 6.3, P <  0.05). The real-time PCR results indicated that relative abundance of caspase-8 and TNF-α mRNA from death receptor apoptotic pathway and caspase-9 for the mitochondrial apoptotic pathway genes in the vitrified group were greater than those in the fresh group P <  0.05). The relative abundance of Bcl-2 and SOD-1 mRNA for the mitochondrial pathway genes in the vitrified group was less than those in the fresh group (P <  0.05). In conclusion, the poor developmental capacity of vitrified parthenogenetic pig blastocysts was closely related with apoptosis. Both mitochondria and death receptor-mediated apoptotic pathways participated the occurrence of this apoptosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Already at first embryo cleavage subsequent blastocyst formation can be predicted based on morphology but the finer morphological details can be difficult to determine due to the presence of the zona pellucida (ZP). Therefore, we monitored zona-free porcine parthenogenetically activated (PA) embryos in a time-lapse system to: (1) describe and characterise the morphological activity of the cytoplasmic membrane and the distribution to the two nuclei during first cleavage and (2) determine the relationship between specific morphological activities and subsequent embryonic development. After ZP removal the membrane surface activities were clearly visible, so all cleaved embryos could be divided into two groups depending on the surface activity during first cleavage: regular morphology (MN) or irregular morphology with 'bumps' (MB). The two nuclei were more unequal in MB embryos in both nucleus size and DNA quantity. After first cleavage, MB embryos could be further divided into three types of irregularities (MB1, MB2, MB3) based on their subsequent behaviour. Clear differences in developmental patterns were found between MN and MB embryos, such as delayed first cleavage, compromised blastocyst formation and total cell number. The predictive value of these new types of morphological events was comparable to the more traditionally used time of first cleavage. In conclusion, zona-free embryos allow visualisation of finer morphological details that can provide an early prediction of embryo developmental potential, but further studies are needed on other type of embryos.

DNA methylation catalyzed by DNA methyltransferase (DNMT) family plays an important role during mammal preimplanted embryo development. However, the effects of RG108, a DNMT inhibitor (DNMTi), on DNMT in the development of bovine preimplanted embryos are not fully elucidated. In this study, we investigated the role of RG108 on the development, dynamics of gene-specific DNA methylation and transcription of bovine parthenogenetic preimplantation embryos. We found that Dnmt1 and Dnmt3b showed highly transcription in parthenogenetic 2-cell embryos, and then the transcription levels decreased during the following development stages, whereas Dnmt3a was always maintained at a lower transcription level during bovine parthenogenetic preimplantation embryo development. Treatment with RG108 blocked the development of bovine parthenogenetic preimplantation embryos and induced hypomethylation in the embryos. RG108 decreased the methylation level of the Nanog gene promoter region, which caused activation of the Nanog gene in 8-cell embryos and increased the transcription level. RG108 also induced the hypomethylation of the repeat elements (satellite I and α-satellite), which may cause genome instability, increasing the number of apoptotic cells in the blastocysts and also the transcription level of the apoptotic gene Bax. These results indicate that RG108, a DNMT inhibitor (DNMTi), inhibits the development of bovine parthenogenetic preimplantation embryos, suggesting that the DNMT is necessary for bovine parthenogenetic preimplanatation embryo development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Parthenote embryos offer multiple opportunities in biotechnological research, so it is important to analyse the possibilities for their cryopreservation in order to establish a biobank. The aim of this experiment was to determine the effect of culture conditions and vitrification on rabbit parthenogenetic embryos. Parthenotes were cultured under in vivo and in vitro conditions until day 3 (late morula/early blastocyst), when they were vitrified. Immediately after warming, they were newly cultured under in vivo and in vitro conditions till day 6 (blastocyst stage). Both culture conditions showed similar late morula/early blastocyst (0.39±0.056 vs. 0.46±0.043, for in vivo and in vitro, respectively) and blastocyst rates (0.12±0.068 vs. 0.13±0.070, for in vivo and in vitro, respectively). However, no parthenote was recovered when a combination of culture conditions was performed. To our best knowledge, this is the first demonstration of the ability of rabbit parthenogenetic embryos to develop after vitrification, with similar embryo development after in vivo or in vitro culture. Nevertheless, our results highlight the importance of culture conditions on the morphology of parthenote embryos. Therefore, we have described that special attention should be paid on culture conditions to generate parthenote embryos, with a view to their subsequent use, for example in embryonic stem cell production., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

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., (© 2013 Wiley Periodicals, Inc.)

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed., (© 2013 Blackwell Verlag GmbH.)

Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes.

In livestock, parthenogenic embryos are simple to produce, but androgenetic embryos have been successfully produced only in sheep and cows. In the present study, matured porcine oocytes were enucleated by micromanipulation and then fertilized with sperm in vitro, thereby producing porcine androgenetic embryos. Porcine androgenetic embryos, which had only sperm genomes, were assessed for cleavage and for blastocyst formation 2 and 6 d after IVF, respectively. There was no difference in cleavage rate between androgenetic embryos and biparental IVF embryos (mean ± SD androgenetic: 65.5 ± 5.4%; biparental IVF: 63.2 ± 3.6%), but there was a difference in the rate of blastocyst formation (androgenetic: 4.5 ± 0.7%; biparental IVF: 30.2 ± 2.6%, P < 0.05). The average number of cells in Day 6 androgenetic blastocysts (34.3 ± 18.2) was lower (P < 0.05) than that in biparental IVF blastocysts (44.1 ± 19.5), but did not differ from that in parthenogenetic embryos (35.7 ± 16.7). The androgenetic embryos were transferred into recipient mothers to examine the competence of post-implantation development. Androgenetic fetuses were present on Days 21 and 25, but not on Days 28, 31, or 35. Of the six androgenetic fetuses recovered on Day 21, five had normal, translucent bodies, and two of these five had beating hearts. The four fetuses recovered on Day 25 were all non-viable. In conclusion, porcine androgenetic embryos initiated embryogenesis and had reached a viable fetal stage 21 days after IVF., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.

Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality., (Copyright © 2011 Wiley Periodicals, Inc.)

In our previous study (Tsuji et al., 2010 ), administration of hCG to recipients around the timing of implantation significantly increased the in vivo development of mouse embryos after somatic cell nuclear transfer (SCNT) until day 10.5, but did not increase the development to full term. The present study was undertaken to examine whether cotransfer of fertilized embryos or parthenogenetic embryos prevents the embryonic loss of SCNT embryos after day 10.5, allowing them to develop to full term. We found that compared with SCNT embryo transfer alone, full-term development of SCNT embryos slightly, but not significantly, increased by cotransfer of mouse hybrid blastocysts derived from BDF1 (C57BL/6×DBA) female×ICR male into the oviducts of recipients administered hCG (2.0% vs. 5.5%). This was not the case with the cotransfer of blastocysts from an ICR female×ICR male (2.5% vs. 2.2%) or parthenogenetic blastocysts from BDF1 female (3.0% vs. 2.0%). Furthernore, when SCNT blastocysts were transferred into the uteri of recipients, full-term development did not increase even with the cotransfer of hybrid blastocysts. The mechanisms of the effect of cotransfer of fertilized and parthenogenetic embryos on the full-term development of SCNT mouse embryos are discussed.

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The events resulting in compaction have an important influence on the processes related to blastocyst formation. To analyse the quality of the embryos obtained by somatic cell nuclear transfer (SCNT) in aspects different from previous studies, not only the number of blastomeres of cloned embryos during the initiation of compaction, but also the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos before and after compaction was preliminarily investigated in mouse. Our results showed that during compaction the number of blastomeres in SCNT embryos was fewer than that in intracytoplasmic sperm injection (ICSI) embryos and, before compaction, there was a uniform distribution of microvilli over the blastomere surface, but microvilli became restricted to an apical region after compaction in the four types of embryos. We also reported here that the time course of compaction in SCNT embryos was about 3 h delayed compared with that in ICSI embryos, while there was no significant difference between SCNT and ICSI embryos when developed to the 4-cell stage. We concluded that: (i) the cleavage of blastomeres in cloned embryos was slow at least before compaction; (ii) the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos was coherent before and after compaction; and (iii) the initiation of compaction in SCNT embryos was delayed compared with that of ICSI embryos.

Mammalian oocyte maturation and early embryo development processes are Ca(2+)-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca(2+) and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca(2+) was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca(2+) was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca(2+) was present throughout the blastomere. In PA embryos, Ca(2+) was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca(2+) in the SCNT embryos. However, Ca(2+) was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca(2+) showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca(2+) location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.

Parthenogenetic activation using zona-free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona-free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6-dimethylaminopurine (6-DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona-free oocytes when compared to zona-intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona-free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6-DMAP resulted in the highest blastocyst rate., (© 2010 Blackwell Verlag GmbH.)

Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Development of a porcine germinal vesicle oocyte (GVO) to a 4-cell stage embryo occurs during a transcriptionally silent period when the oocyte/embryo relies on maternally derived mRNA to encode proteins required for development. Regulation of translation and degradation of maternal mRNA is thought to be partially dependent on cytoplasmic polyadenylation elements (CPEs) within the 3' untranslated region of the mRNA. The goal of this study was to determine how CPE sites affect the abundance of mRNA during embryogenesis and parthenogenetic development, and how cordycepin, a 3'-deooxyadenosine (3'-dA) that inhibits poly-(A) tail formation, affects polyadenylation and transcript abundance. Expressed sequence tags (ESTs) from oocytes and 4-cell stage embryos were scanned for the presence of five consensus CPEs. Nineteen different transcripts containing one to three CPEs were selected, and transcript abundance was determined in GVO, metaphase II, 2-cell, and 4-cell stage embryos via real-time PCR while the length of the poly-(A) tail was determined by using a poly-(A) tail PCR (PAT) assays. Real-time PCR was performed on three biological and two technical replicates for each stage. There was no direct correlation between poly-(A) tail length, transcript abundance, and the CPE. In addition, the abundance of some messages was different if the embryo was the result of parthenogenetic activation. Cordycepin prevented polyadenylation of transcripts that normally undergo noticeable polyadenylation. Thus, CPEs may not be the only factors that regulate message stability, and parthenogenetic activation does not result in changes in transcript abundance that mimic in vitro fertilization., ((c) 2010 Wiley-Liss, Inc.)

Objective: To determine whether the preantral follicles in adult ovaries can generate developmentally competent oocytes after in vitro culture., Design: Prospective, animal-model study., Setting: Gamete and Stem Cell Biotechnology Laboratory, Seoul National University, Seoul, Korea., Animal(s): B6CBAF1 mice., Intervention(s): Preantral follicles collected from 8-week-old mice were cultured in vitro., Main Outcome Measure(s): Follicle development, embryogenesis, and embryonic stem cell characterization., Result(s): A mean of 50.3 preantral follicles were retrieved from one adult animal, which is significantly less than the number (88.7 follicles) retrieved from a prepubertal female. Extension of the culture period greatly improved oocyte maturation; increased follicular growth to the pseudo-antral (89%-91% vs. 32%) or mature oocyte stage (65%-77% vs. 13%) was observed after 12 or 13 days of culture compared with 9 days of culture. Blastocyst formation after parthenogenesis was detected in only one case; in comparison, the use of IVF yielded a large number of embryos that developed into blastocysts. A mean of 14.7 intrafollicular oocytes per animal were produced after 13 days of culture, and 41% of those developed into blastocysts after IVF. Embryonic stem cell-like colonies were established by subculturing the inner cell mass cells from the blastocysts., Conclusion(s): Developmentally competent oocytes can be generated by culturing adult preantral follicles. These results may help increase the feasibility of follicle culture systems.

Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus-oocytes-complexes (n=1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 microM of calcium ionophore (CaI) for 20 min then incubated for 4h with either 2mM 6-dimethylaminopurine (6-DMAP) (group 1, n=202) or with 10 microg/mL cycloheximide (CHX) (group 2, n=194). Group 3 (n=172) and group 4 (n=184), oocytes were pretreated with 5 microM ionomycin (Iono) for 5 min then incubated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 5 (n=161) and group 6 (n=155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 7 (n=196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 degrees C under 5% CO(2) in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater (P<0.05) than other treatments (10% for group 2, 47% for group 3, 27% for group 4 and 41% for group 6). The blastocyst developmental rate was better (P<0.05) in parthenotes following activation with Iono/6-DMAP (21%) compared to activation with Iono/CHX (12%). However, all were less than that achieved in the IVF group (35%). We conclude that parthenogenetic activation of camel oocytes with 6-DMAP is more effective than activation with CHX for all pre-treatments tested (CaI, Iono or ES). The viability of activated (n=15) or IVF (n=10) hatched-dromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of dromedary oocytes with CaI/6-DMAP or ES/6-DMAP is more effective than other treatments in terms of in vitro embryo development. This provides efficient activation conditions which may lead to the development of the somatic cell nuclear transfer procedure in dromedary.

Polarity formation in preimplantation embryos is controversial. To investigate the embryonic-abembryonic axis in the pig, porcine parthenotes were used to prevent the topological change caused by polyspermy as well as to avoid the influences of sperm entry position. For lineage tracing, DiI, a fluorescence dye, was injected into only a blastomere of the 2-cell stage embryos. If the first blastomere to divide was labeled, the embryo was included in the leading group, and while all others were included in the lagging group. In 60.5% of the blastocysts in the lagging group, the progeny of the labeled blastomeres formed the inner cell mass (ICM) and adjacent trophectoderm (TE) hemisphere; 62.1% of the blastocysts in the leading group had progeny of the labeled blastomeres distributed only to the TE (opposite of ICM). The rest of the lagging and leading groups showed random distributions. Unlike murine parthenotes, biased mitochondrial distribution was also found in porcine parthenotes (38.1%). Our findings indicate that the ;leading' blastomere of the 2-cell porcine parthenote forms the distal TE (abembryonic) and that the 'lagging' blastomere forms the remaining portion of the blastocyst, including the ICM (embryonic). Biased distribution of mitochondria in each 2-cell blastomere may contribute partly to this event.

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.

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66(shc) and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66(shc) and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

Full mammalian development typically requires genomes from both the oocyte and spermatozoon. Biparental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis; that is, a maternal methylation imprint imposed during the oocyte growth period and a paternal methylation imprint imposed in pregonadal gonocytes. This leads to unequivalent expression of imprinted genes from the maternal and paternal alleles in embryos and individuals. It is possible to hypothesise that the maternal methylation imprint is necessary to prevent parthenogenesis, which extinguishes the opportunity for having descendents, whereas the paternal methylation imprint prevents parthenogenesis, ensuring that a paternal contribution is obligatory for any descendants. To date, there are several lines of direct evidence that the epigenetic modifications that occur during oocyte growth have a decisive effect on mammalian development. Using bimaternal embryos with two sets of maternal genomes, the present paper illustrates how parental methylation imprints are an obstacle to the progression of parthenogenesis.

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.

The purpose of this study was to investigate the effect of different activation treatments on the development of IVM-derived and cloned bovine embryos. The effect of oocyte age (20h versus 24h after IVM) on the blastocyst rate was also investigated. No differences in the percentage of blastocyst development were observed between the oocytes matured for 20 and 24h (15% versus 27%, p>0.05). Reconstructed oocytes activated 4h after fusion (fusion before activation, FBA) had a higher blastocyst rate than those oocytes activated immediately after electrofusion (fusion and activation simultaneously, FAS) (26% versus 5%, p<0.01). Blastocyst rates were significantly greater (p<0.01) for the reconstructed oocytes activated by calcium ionophore (A23187) combined with 6-dimethylaminopurine (6-DMAP) (51.6%) than for those activated with cycloheximide (CHX) plus cytochalasin B (CB) treatment (1h, 8.2%; 5h, 14.3%). However, the blastocyst rates were similar among reconstructed oocytes activated by electric pulses and A23187 (30.5% versus 42.2%) or by A23187 and ionomycin (36.7% versus 33.3%) combined with 6-DMAP, respectively. Blastocysts that developed from reconstructed oocytes activated by A23187 and 6-DMAP resulted in three pregnancies (3/9) and one live birth from 18 embryos transferred to recipient cows. Genotypic analysis of six bovine microsatellite markers by polymerase chain reaction confirmed that the cloned calf was genetically identical to the nuclear donor. In conclusion, reconstructed oocytes that derived from oocytes exposed to activation treatment 4h after electrofusion are more likely to develop to the blastocyst stage. The best treatment to activate reconstructed bovine oocytes in this study was A23187 combined with 6-DMAP.

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.

Oil is an indispensable material in micro-droplet culture; it prevents medium from evaporation, and its transparency facilitates monitoring. However, lipophilic factors in the medium can be absorbed into the oil overlay, and conversely, deleterious materials can diffuse into the medium. In the present study, we describe a novel oil-free microtube culture (MTC) system. Parthenogenetic mouse embryos were placed into 0.2-mL thin-wall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as a control. Embryos in MTC had a higher blastocyst formation rate (89.2%) and larger population of cells in the blastocysts (92.0+/-6.9; mean+/-S.E.M.) compared with drop culture (78.3% and 74.7+/-8.1; P<0.05 for each). The large blastocyst cell population in MTC was due to higher numbers of trophectoderm (TE) cells (70.5+/-5.9 versus 53.8+/-7.4; P<0.05) rather than inner cell mass cells. The presence of more TE cells was attributed to faster development in MTC. Embryos cultured in oil-covered MTC had fewer TE cells (61.5+/-5.6) than oil-free cultures (70.5+/-5.9; P<0.05). In conclusion, oil-free MTC was an alternative to conventional micro-drops, without the deleterious effects of oil.

Smarca 2 (Brahma) and Smarca 4 (Brahma related gene 1, BRG1) alternatively occupy the catalytic site of SWI/SNF chromatin remodeling complexes. Mammalian embryos undergo a dramatic amount of epigenetic remodeling during cleavage development, which plays key roles in regulating both gene transcription and the developmental potential of the embryo. In order to understand how the epigenetic state of cleavage stage embryos is regulated, it is important to identify the factors that mediate epigenetic changes during cleavage development. In this study we hypothesized that altered expression of Smarca 2 would have profound effects on embryo development. The objectives of this study were to determine the expression pattern of Smarca 2 and determine the effects of Smarca 2 overexpression in cleavage stage parthenogenetic porcine embryos. Smarca 2 transcripts are most abundant in germinal vesicle (GV) stage oocytes and decline progressively during cleavage development. At the blastocyst stage, Smarca 2 transcripts are reduced by 18-fold (GV stage oocyte vs. blastocyst stage embryo, P < 0.05). Parthenogenetic porcine embryos injected with mRNA encoding wild type human Smarca 2 exhibited a dramatic developmental arrest as compared to noninjected embryos, embryos injected with GFP mRNA, or mRNA encoding a dominant negative version of human Smarca 2 (P < 0.01). This work demonstrates the importance of Smarca 2 containing SWI/SNF chromatin remodeling complexes in preimplantation porcine embryos and how perturbing the amount of Smarca 2 in porcine embryos disrupts cleavage development., ((c) 2007 Wiley-Liss, Inc.)

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.

We have successfully produced healthy piglets following cryopreservation of embryos derived from oocytes matured and fertilized in vitro. The appropriate timing of cryopreservation pretreatment (removal of cytoplasmic lipid droplets [delipation] and vitrification) was initially determined using parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. Viable embryos were obtained at the highest rate when embryos were delipated at the four- to eight-cell stages (Day 2 of embryo culture) and were vitrified approximately 15 h later (Day 3) by means of the minimum volume cooling method. After cryopreservation of embryos derived from oocytes matured and fertilized in vitro under the most appropriate conditions, 401 embryos were transferred to five recipient gilts, and the recipients all became pregnant. At autopsy of one of the recipients, which had received 47 embryos, eight fetuses (17.0%) were found. Three recipients each gave birth to two to four piglets (1.4%-6.0%). These results demonstrate that normal offspring can be produced from vitrified porcine embryos derived from IVM oocytes by a strategic combination of delipation and vitrification at the early cleavage stages. This approach has great potential in the reproduction of micromanipulated porcine embryos, such as cloned and sperm-injected embryos, produced from IVM oocytes.

Objective: To compare directly in vitro developmental competence between parthenogenetically activated and intracytoplasmic sperm injection (ICSI)-fertilized oocytes., Design: For each patient, three metaphase II oocytes were randomized to the ICSI procedure, while n-3 were allocated to parthenogenetic activation., Setting: University hospital infertility unit., Patients: Thirty-eight patients, aged 35.2 +/- 3.3 years (mean +/- SD) selected for ICSI., Interventions: After 1 hour from denudation, oocytes were either fertilized by ICSI (n = 114) or chemically activated (n = 104). Fertilized and activated oocytes were cultured for up to 3 and 5 days, respectively., Main Outcome Measures: Development rate, cell number, and morphological grade during culture., Results: The two groups showed no significant differences between rates of fertilization and parthenogenetic activation, development, and blastomere number on days 2 and 3 of culture. However, parthenotes showed a lower morphological grade, and a significantly lower proportion went on cleaving to day 3, when only activated rather than total numbers of oocytes were considered. On day 5 after activation, nine oocytes (8.6%) reached the blastocyst stage, representing 12.9% of parthenotes., Conclusions: Since most parameters examined in this study were similar between activated and fertilized oocytes, parthenogenetic activation may be a useful tool for the preclinical evaluation of experimental procedures.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells., (Copyright 2006 Wiley-Liss, Inc.)

Mouse parthenogenetic embryos (PEs) are developmentally arrested until embryo day (E) 9.5 because of genomic imprinting. However, we have shown that embryos containing genomes from non-growing (ng) and fully grown (fg) oocytes, i.e. ng(wt)/fg(wt) PE (wt, wild type), developed to E13.5. Moreover, parthenogenetic development could be extended to term by further regulation of Igf2 and H19 expression using mice with deletion of the H19 transcription unit (H19Delta13) together with its differentially unit (DMR). To gain an insight into the extended development of the parthenotes to term, we have here investigated the expression levels of paternally imprinted genes in ng(H19Delta13)/fg(wt) PE throughout their development. In ng(H19Delta13)/fg(wt) Pes that died soon after recovery, the expression of Igf2 and H19 was restored to the appropriate levels except for low Igf2 expression in the liver after E15.5. Further, the paternally expressed Dlk1 and Dio3 were repressed, while the expression levels of the maternal Gtl2 and Mirg were twice those of the controls. However, the above-mentioned four genes showed almost normal expression in the surviving ng(H19Delta13)/fg(wt) PEs. The methylation analysis revealed that the intragenic DMR of the Dlk1-Gtl2 domain was hypermethylated in the ng(H19Delta13)/fg(wt) PEs that survived, but not in the PEs that died soon after recovery. The present study suggests that two sets of co-ordinately regulated but oppositely expressed genes, Igf2-H19 and Dlk1-Gtl2, act as a critical barrier to parthenogenetic development in order to render a paternal contribution obligatory for descendants in mammals.

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.