Development Of Parthenogenetic Embryos Research Articles

158 EFFECTS OF AMINO ACIDS IN EMBRYO TRANSPORT MEDIA ON PORCINE EMBRYOS

Due to the distance from the laboratory to the recipient farm, several laboratories, including ours, carry somatic cell nuclear transfer (SCNT)-derived porcine embryos to the farm using a portable incubator for a few hours. If the embryos are nourished well during the transport, viability of embryos might be increased and cloning efficiency can be improved. TALP, which is widely used as a porcine embryo transport medium, lacks amino acids (AA). Proper supply of AA in the uterus is important for the development of pre-implantation embryos because AA have functions as osmolytes, metabolic regulators, or substrates and buffers of intracellular pH. Thus, supplementation of AA could affect the embryonic viability during the transport of SCNT-derived porcine embryos. The aim of this study is to determine whether the transport medium containing AAs affects the in vitro development of parthenogenetic embryos compared to TALP. Porcine zygote medium-5 (PZM-5) was chosen as transport medium containing AA due to its similarity in constituents with TALP except for the AA. Because PZM-5 contains sodium bicarbonate as a buffer system which can not cover wide variation of pH, 10 mM HEPES was added into PZM-5 (PZM+H) as it was normally done with TALP. Porcine cumulus–oocyte complexes (COC) were collected from ovaries of slaughtered pigs and cultured for 44 h using a two-step culture protocol. After denuded, matured oocytes were activated by thimerosal for 10 min followed by dithiothreitol for 30 min. The parthenogenetic embryos were cultured in PZM-5 for 2 days, monitored for cleavage, and loaded in a straw with TALP or PZM+H, respectively. Embryos were stored in a portable incubator (MTG, Bruckberg, Germany; no CO2) at 37°C for three hours and moved to PZM-5 drop for additional 5 days culture. The development was monitored on Day 7 after activation and blastocysts (BL) were collected for total cell number counts and RNA extraction. Ten BL from the TALP group and 11 BL from the PZM+H group were stained with 10 µg mL–1 bisbenzimide (Hoechst 33342) and were visualized for cell counting under fluorescence microscopy. Messenger RNA was extracted from 7 BL of the TALP and PZM+H groups and cDNA were synthesized. Quantitative real-time PCR were done to detect expression levels of apoptosis-related genes using the cDNA. The Bax/Bcl2 ratio was investigated as expression level of apoptosis-related genes and GAPDH was used as control. Each experiment was repeated at least 3 times. Data were analyzed by paired Student’s t-test using Graphpad Prism (version 5, Graphpad Software Inc., La Jolla, CA, USA). No difference was observed between the TALP and PZM+H groups with respect to blastocyst formation rate (22.46 ± 1.47% and 23.17 ± 2.13%, respectively) and total cell number (32.9 ± 2.22 and 37.09 ± 2.18, respectively). There was no significant difference between groups in the Bax/Bcl2 ratio. The use of PZM-5 media, which contains AA, did not affect the development and apoptosis of parthenogenetic embryos. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

Expression Pattern of Pluripotent Markers in Different Embryonic Developmental Stages of Buffalo (Bubalus bubalis) Embryos and Putative Embryonic Stem Cells Generated by Parthenogenetic Activation

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Altered Cell Cycle Gene Expression and Apoptosis in Post-Implantation Dog Parthenotes

Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.

Effect of Small Molecule Supplements during In Vitro Culture of Mouse Zygotes and Parthenogenetic Embryos on Hypoblast Formation and Stem Cell Derivation

Small molecule inhibitors are organic components that modulate signalling pathways and have the ability to change the differentiation state of cells. They have been used to increase the efficiency of induced pluripotent stem cell generation and to support stem cell derivation and culture. In this study, we aimed to evaluate the effects of small molecules on the development of mouse zygotes and parthenogenetic embryos. Three inhibitors (SC-1, PD0325901 and BIO) were added to the culture medium from the 2-cell stage onwards. We have observed that addition of an inhibitor of the fibroblast growth factor (FGF) pathway (SC-1 or PD0325901) compromises the segregation of hypoblast from the inner cell mass (ICM). Given no difference was observed in size of the ICM, but more epiblast cells were found in these embryos, we can conclude that this is caused by redirection of all ICM cells to the epiblast. We also determined the consequences of reduced hypoblast and increased epiblast formation on stem cell derivation efficiency. No significant difference was found between derivation rates from treated embryos as compared to controls. However, only under 2i + ROCKi conditions, stem cells could be derived with an efficiency of more than 90%. Addition of BIO, an activator of the WNT pathway, did not have any effects on hypoblast development or stem cell derivation. We have demonstrated that FGF signalling is crucial for hypoblast generation and small molecules can be efficiently used to inhibit this process both in zygotes and parthenogenetic embryos.

Effect of Endoplasmic Reticulum Stress on Porcine Oocyte Maturation and Parthenogenetic Embryonic Development In Vitro1

X-box-binding protein 1 (XBP1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP1 was weak in mature oocytes and at the 1-, 2-, and 8-cell stages of embryos but abundant at the germinal vesicle (GV), 4-cell, morula, and blastocyst stages. In addition, RT-PCR revealed that both spliced XBP1 (XBP1-s) and unspliced XBP1 (XBP1-u) were expressed at the GV, 4-cell, morula, and blastocyst stages. Tunicamycin, an ER stress inducer, induced active XBP1 protein in nuclei of 4-cell embryos. Next, porcine embryos cultured in the presence of tauroursodeoxycholate, an ER stress inhibitor, were studied. Total cell numbers and the extent of the inner cell mass increased (P < 0.05), whereas the rate of nuclear apoptosis decreased (P < 0.05). Moreover, expression of the antiapoptotic gene BCL2 increased, whereas expression of the proapoptotic genes BCL2L1 (Bcl-xl) and TP53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress-mediated apoptosis in vitro.

The effect of horse serum on in vitro development of porcine parthenogenetic embryos

The objective of this study was to examine the effect of different sera and serum-like substances on the preimplantation development of porcine parthenogenetic embryos. Chemically activated (calcium ionophore A23187+cytochalasin B) pig oocytes were pre-cultured for five days. On day 5, the parthenogenetic embryos were treated with porcine follicular fluid (PFF), fetal bovine serum (FBS), horse serum (HS) or porcine serum albumin (PSA), and were cultured two more days. Horse serum was found to be the most effective protein source in enhancing parthenote development judging by blastocyst formation and hatching. Next, three different concentrations of HS (10, 20 and 30%) were used to determine the optima HS concentration needed to improve the development of porcine parthenogenetic embryos. All HS concentrations increased the blastocyst cell number and decreased the incidence of blastocyst apoptotic cells with 20% being the most effective. In conclusion, horse serum enhanced parthenogenetic embryo development and the quality of porcine parthenogenetic embryos.

CDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos

The retarded development of parthenote embryo could be due to aberrant epigenetic imprinting, which may disrupt many aspects and lead to conceptus demise. The present work was conducted to: 1) compare the development of in vitro produced (IVP) and parthenogenetically developed (P) buffalo embryos from the 2-cell to blastocyst stage; 2) investigate the global gene expression profile and search for new candidate transcripts differing between IVP and P buffalo blastocyst using cDNA microarray analysis (validated by Real Time PCR); 3) follow the particular expression patterns of PLAC8 and OCT4 genes at five different stages of preimplantation development by Real Time PCR; and 4) study the expression of CDX2 at the blastcocyst stage. Cleavage rate was higher (P < 0.05) in P than IVP buffalo embryos, while, progression to blastocyst and number of cells per blastocyst were lower (P < 0.05) in P than IVP blastocysts. Microarray analysis indicate that 56 differentially expressed genes between the two groups, of which 51 genes (91.07%) were up-regulated, and five genes were downregulated in IVP blastocyst, using 1.4 fold-changes as a cutoff. Differentially expressed genes are related to translation, nucleic acid synthesis, cell adhesion and placentation. Validation of candidate genes revealed that the transcript abundance of PTGS2, RPS27A, TM2D3, PPA1, AlOX15, RPLO and PLAC8 were downregulated (7/8) in parthenote blastocyst compared to the IVP blastocyst. PLAC8 gene expression was higher (P < 0.05) at 2-cell, morula and blastocyst stages in IVP embryos compared with parthenote embryos. The OCT4 gene expression was higher (P < 0.05) in 2-cell, 4-cell, 8-cell and blastocysts produced in vitro. In conclusion, the retarded development of parthenogenetic buffalo embryos could be due to downregulation of genes related to translation, nucleic acid synthesis, cell adhesion, and placental development. The low expression of PLAC8 and OCT4 during the different stages of development may be responsible, in part, to the failure of development of parthenote buffalo embryos.

Nlrp2, a Maternal Effect Gene Required for Early Embryonic Development in the Mouse

Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (∼118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse.

Zona-free and with-zona parthenogenetic embryo production in goat ( Capra hircus) — effect of activation methods, culture systems and culture media

Studies on parthenogenetic activation of oocytes are important to improve the efficiency of nuclear transfer as artificial activation of oocytes is an essential component of nuclear transfer protocol. The present study was carried out to investigate the effects of different activation methods, culture systems and culture media on in vitro development of zona-free and with-zona parthenogenetic embryos in goat. In case of zona-free parthenogenesis, there was a significant (p < 0.05) increase in cleavage rate and blastocyst yield when oocytes were activated by electrical pulse (76.29 ± 0.52% and 19.07 ± 0.39% respectively) than when Ca-ionophore was used for activation (63.45 ± 0.73% and 14.09 ± 0.65% respectively). The quality of blastocysts was evaluated by determination of cell number and by expression profile of pluripotent related gene Oct-4. No significant (p < 0.05) difference was found in quality of blastocysts produced by different activation methods. In culturing of zona-free parthenogenetic embryos, flat surface (FS) was proved to be the best system. A significant (p < 0.05) decrease in cleavage rate and blastocyst yield was found in Microdrop culture of zona-free embryos (43.67 ± 2.08% and 0.72 ± 0.72% respectively) in comparison to WOW of zona-free embryos (73.88 ± 1.70% and 15.51 ± 1.34% respectively) and FS of zona-free (75.14 ± 0.81% and 23.93 ± 2.71% respectively) as well as with-zona (72.16 ± 1.55% and 18.16 ± 0.68%) embryos. Zona-free flat culture system yielded significantly (p < 0.05) higher blastocyst rate than zona-free WOW system as well as with-zona flat culture system. The zona-free and with-zona parthenogenetic embryos were cultured in three different media — Research Vitro Cleave media from Cook® Australia (RVCL), Embryo Development Medium (EDM) and Modified Synthetic Oviductal Fluid (mSOF). In case of zona-free parthenogenesis, significant (p < 0.05) increase was found in blastocyst development rate in RVCL medium (18.61 ± 1.52%) than EDM (11.29 ± 0.77%) or mSOF (11.53 ± 1.86%). In case of with-zona parthenogenesis, RVCL medium and EDM were found superior to mSOF. The results of the study will be helpful to improve the efficiency of nuclear transfer in goat.

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.

Effects of Activation Methods on DNA Synthesis and Development of Parthenogenetic Porcine Embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.

Efficient Transgene Expression in IVF and Parthenogenetic Bovine Embryos by Intracytoplasmic Injection of DNA–Liposome Complexes

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA-liposome complexes (0.5, 5, 50, 500 ng pCX-EGFP/μl). The highest EGFP-embryos rates were obtained using 500 ng pCX-EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA-liposome complexes into pre-fertilized oocytes and presumptive zygotes, 16 and 24 h post-fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp-liposome was injected 16 h post-fertilization. The percentages of positive embryos for the 24-h post-fertilization and pre-fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp-liposome complexes into pre-activated oocytes, and 3 and 11 h post-activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post-activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.

105 IN VIVO DEVELOPMENT OF CANINE PARTHENOTES AND THE EXPRESSION PATTERN OF Igf2/Igf2r GENES

Parthenogenesis is the process by which an oocyte develops without fertilization, resulting in parthenogenetic embryos carrying only maternal chromosomes. Until now, little information has been available on the post-activation development of parthenogenetic embryos, and there are no reports about canine post-implantation development of parthenogenetically activated oocytes. Thus, the objective of this study was to investigate the development of parthenogenetic canine embryos when implanted in vivo, and the subsequent post-implantation development of such canine parthenogenetic fetuses. Also, we examined expression patterns of Igf2 and its receptor (Igf2r), which are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in other mammalians, to gain insight into the role of genomic imprinting during uniparental development. In vivo matured dog oocytes were obtained by flushing oviducts of mixed breed bitches ∼72 h after ovulation. The denuded oocytes (n = 48; 5 replicates) were subjected to chemical activation by incubation in a culture medium containing 10 μM calcium ionophore (Sigma, St. Louis, MO, USA) for 4 min and then in a culture medium supplemented with 1.9 mM of 6- dimethylaminopurine (Sigma) for 4 h at 39°C. Parthenogenetic embryos were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic embryos was compared with that of artificially inseminated controls. Normal and parthenogenetic fetuses, obtained from recipients on Day 28, 30, and 32 of pregnancy, were analysed for gross external morphology and Igf2/Igf2r gene expression examined. Data were analysed using SAS and means compared by Student’s t-test. The in vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rate of parthenotes was 56.3%, which was significantly lower than those of the control (79.5%; P &lt; 0.05). The weight of parthenogenetic fetuses and placentae recovered from uteri at 28, 30, and 32 day of pregnancy were significantly lighter than those of the control (P &lt; 0.05), whereas the appearance of recovered parthenogenetic fetuses were comparable to those of in vivo fertilized fetuses. We found that both Igf2 and Igf2r were expressed in canine parthenotes but the expression level of Igf2 in the parthenotes was significantly lower than the control (P &lt; 0.05). The expression level of Igf2r in the parthenotes was comparable with the control. These results confirmed that the protocols used in our present study were suitable for activating the canine oocyte artificially and to support the viability and developmental potential of canine embryos up to the mid-gestation stage. It will provide an opportunity to determine the reason for developmental differences between parthenogenetic and fertilized embryos, and will be a useful model system for elucidating the roles of parental genomes in mammalian postimplantation development. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), Institute for Veterinary Science, and Nature Balance Korea.

Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation, and Subsequent Parthenogenetic Embryo Development

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.

Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos

This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 μg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 μg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 μg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.

Parthenogenetic Activation of Pig Oocytes Using Pulsatile Treatment with a Nitric Oxide Donor

The nitric oxide donor (+)-S-nitroso-N-acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short-term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2-h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short-term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4-cell stage. Thirty-minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.

A-type lamin dynamics in bovine somatic cell nuclear transfer embryos

The persistence of A-type nuclear lamin in somatic cell nuclear transfer (SCNT) embryos has been proposed as a marker for incomplete nuclear reprogramming. Using monoclonal antibodies to A/C- (A/C-346 and A/C-131C3) and B-type lamin, we compared distribution during early development of bovine IVF, parthenogenetic and SCNT embryos. A/C-346 staining was observed in the pronuclei of IVF embryos and in nuclei at the two-cell stage, but was not detected in subsequent cleavage stages up to and including hatched blastocysts. In contrast, A/C-131C3 and anti-lamin B2 stained all preimplantation stage embryos. Parthenogenetic and SCNT embryos had similar staining patterns to IVF embryos for all three antibodies, demonstrating correct nuclear architecture reprogramming. Inhibiting protein synthesis with cycloheximide (CHX) in parthenogenetic and SCNT embryos did not affect lamin A/C localisation, suggesting that lamin A/C is maternal in origin. However, activation with CHX delayed lamin A/C incorporation compared with 6-dimethylaminopurine activation. In SCNT embryos, staining for both A/C- and B-type lamin was delayed compared with parthenotes, although lamin B2 incorporation preceded lamin A/C in both. In conclusion, the lamin A/C distribution in SCNT bovine embryos paralleled that of IVF and parthenogenetic controls and therefore is not a marker of incomplete reprogramming.

342 EFFECTS OF MELATONIN ON PREIMPLANTATION DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS

In mammalian species, melatonin is a multi-functional pineal gland hormone that regulates several circadian and seasonal rhythms including reproduction. However, the melatonin study was not common as to the oocytes in the pig. Recently, we reported that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine oocyte IVM and we also reported an existence of melatonin receptor on the cumulus cells and granulose cells (Kang JT et al. 2009 J. Pineal Res. 46(1), 22-28). In this study, as adding further experiments rather than our previous study, we investigated effect of exogeneous melatonin (10 ng mL-1) on the porcine oocytes and analyzed possible factors which can be responsible for that results. Oocytes were recovered by aspiration of slaughterhouse ovaries, and then matured in TCM-199 supplemented with EGF, insulin, pyruvate, cystine, and gonadotropin. Expression of apoptosis-related genes mRNA in oocytes cultured with melatonin were evaluated by real-time PCR (Exp 1), cumulus cell expansion on COC was assessed on the microscopes during in vitro maturation (Exp 2), and developmental effects between melatonin treatement group and non-treatment group on the in vitro culture of parthenogenetically activated oocytes was investigated (Exp 3). In results, oocytes matured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl (anti-apoptotic gene) and Bax (proapoptotic gene) by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher compared to the control while the expression of Bax was decreased relative to the control (P &lt; 0.05). Cumulus cell expansion was evaluated under a stereomicroscope at 22 h, 44 h during IVM. Representative photomicrographs of porcine COC at the start of the IVM, after 22 h and 44 h treatment with melatonin, are shown in Figure. After 22 h of melatonin treatment, cumulus cells were visually expanded compared with non-treatment group. We analyzed significantly greater proportion of parthenogenetically activated oocytes developed to blastocyst when the IVM medium was supplemented with melatonin. Melatonin treatment in the IVM has consequently beneficial effect on the blastocyst formation rates on the development of porcine parthenogenetic embryos (15.4%) compared to non-treatment group (10.7%, P &lt; 0.05). However, cleavage frequency was not affectedby the treatment. In conclusion, the present study demonstrated that melatonin had a beneficial effect on the development of parthenogenetically activated porcine embryos, probably through decreased apoptosis rate and increased cumulus cell expansion. This study was supported by Korean MKE, MEST (BK21 program), and Hanhwa L&amp;C

150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P &lt; 0.05) higher in RVCL and EDM media.