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SummaryThis 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.

Vitrification and warming can trigger premature meiosis in immature porcine oocytes. Our aim was to compare the efficacies of two meiotic inhibitors, dibutyryl-cAMP and roscovitine for the meiosis synchronization during in vitro maturation (IVM) of porcine oocytes vitrified at the germinal vesicle (GV) stage. We first compared the efficacy of 1 mM dibutyryl-cAMP and 25 μM roscovitine on meiotic arrest during the first 22 h of IVM. Dibutyryl-cAMP could maintain the GV stage in 83.5% of oocytes; however, roscovitine was even more effective (96.6%), whereas only 17.4% of the oocytes remained at the GV stage without these additives. Temporal meiotic arrest for 22 h by roscovitine did not reduce the percentage of oocytes reaching the Metaphase II stage during subsequent IVM. However, after parthenogenetic stimulation or in vitro fertilization, subsequent embryo development to the blastocyst stage was compromised after roscovitine treatment, whereas dibutyryl-cAMP improved the percentage of blastocyst development. In conclusion, dibutyryl-cAMP could derogate but not completely prevent premature meiosis in vitrified oocytes, whereas roscovitine could more efficiently prevent it. However, for embryo production, the use of roscovitine was disadvantageous, whereas the use of dibutyryl-cAMP was beneficial.

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4-8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

Premature meiotic arrest during in vitro maturation (IVM) of porcine oocytes after germinal vesicle breakdown is associated with microfilament degradation. We aimed to clarify (1) if such arrest occurs at the metaphase-I (MI) stage or the oocyte progresses to a so-called diploid metaphase-II (MII) stage and (2) if microfilament degradation is the cause or result of the meiotic arrest. The number and morphology of chromosomes in oocytes showing premature meiotic arrest at 44 h IVM (38 monovalents) was similar to those cultured in the presence of the actin polymerization-inhibitor cytochalasin-B, but different from those of MI-stage (19 bivalents), and MII-stage oocytes (19 monovalents) at 33 and 44 h of IVM, respectively. Immunostaining revealed similar frequencies of microfilament degradation in prematurely arrested and cytochalasin-B-treated oocytes (58.7% and 57.2%, respectively), which were higher (P 0.05) than those in MI- and MII-stage oocytes (10.6% and 6.8%, respectively). Induction of MI-arrest by nocodazole did not affect microfilament morphology. ATP and mRNA levels of microfilament-related genes in oocytes were similar among all groups. These results suggest that altered microfilament dynamics contribute to the formation of diploid metaphase spindles in oocytes, which fail to reach the MII stage. However, the cause of microfilament degeneration remains unclear.

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 (P0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P0.05) 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 (P0.05) 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 (P0.05) 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.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

The purpose of this study was to examine whether freeze-dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze-drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze-dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase-II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze-dried GVs with intact nuclear membrane and DNA stored at -20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze-dried GV stored for 15 or 30 days at -20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze-dried GVs.

We investigated whether high-quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis-shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p

We aimed to clarify the genomic characteristics of porcine endogenous retroviruses (PERVs) in Vietnamese native pig (VnP) breeds. First, we investigated genetic polymorphisms in β- and γ-like PERVs, and we then measured the copy numbers of infectious γ-like PERVs (PERV-A, B, and C). We purified genomic DNA from 15 VnP breeds from 12 regions all over the country and three Western pig breeds as controls, and investigated genetic polymorphisms in all known PERVs, including the beta (β)1-4 and gamma (γ)1-5 groups. PERVs of β1, β2, β3, and γ4 were highly polymorphic with VnP-specific haplotypes. We did not identify genetic polymorphisms in β4, γ1, or γ2 PERVs. We then applied a real-time polymerase chain reaction-based method to estimate copy numbers of the gag, pol, and env genes of γ1 PERVs (defined as A, B, and C). VnP breeds showed significantly lower copy number of the PERV genes compared with the Western pig breeds (on average, 16.2 and 35.7 copies, respectively, p < .05). Two VnP breeds showed significantly higher copy number compared with the other VnPs (p < .05). Our results elucidated that VnPs have specific haplotypes and a low copy number of PERV genes.

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4 M sucrose. The oocytes were then cultured for 22 h in a chemically defined porcine oocyte medium (POM) supplemented with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 equine chorionic gonadotrophin, 10 IU mL−1 human chorionic gonadotrophin, and 1 mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.

Boar sperm freeze-dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra-cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze-dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up-regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10(6) /mL) was lower (P 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone-stimulated Ban and Landrace. The percentages of two-cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non-stimulated Ban (4.0 ± 3.8%) was lower (P

Contents We tested the effects of resveratrol both as a pre-treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre-treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non-vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non-vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification-related damages.

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P

We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22 hr, last 22 hr, or entire 44 hr duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P < 0.05) and obvious induction of cumulus expansion were observed over the whole time span (44 hr) in the IVM group. When cumulus cells were removed at 22 hr and denuded oocytes were further cultured, pLIF showed no effect on maturation rate. Oocytes matured in pLIF-supplemented medium showed a tendency for more rapid blastocyst development (21.1% vs. 16.2%, P = 0.0715). Examination of transcripts and proteins of the LIF signaling pathway in COCs revealed that LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) are present in both cumulus cells and oocytes. The amount of phosphorylated STAT3 (p-STAT3) markedly increased in both cumulus cells and oocytes cultured in pLIF-supplemented media, although oocyte p-STAT3 disappeared after 44 hr of IVM. These results suggest that the LIF/STAT3 pathway is functional during IVM of porcine oocytes, and supplementing pLIF in the IVM medium can improve oocyte maturation by activating this pathway.

The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.

Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been studied in mammals other than the human and mouse. In the present study, the effect of knockdown of SUV39H1 and SUV39H2 genes on telomere length was examined in porcine embryonic stem-like cells (pESLCs) and porcine embryonic fibroblasts (PEFs). The telomeres in SUV39H1 and SUV39H2 knockdown (SUV39KD) pESLCs (37.1 ± 0.9 kb) were longer (P

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Although telomeres are elongated during morula-to-blastocyst transition in cloned embryos, it is still unknown whether donor cell types have any effect on this elongation. In the present study, we examined the changes of telomere length during morula-to-blastocyst transition in cloned porcine embryos using different types of donor cells. Porcine embryonic stem-like cells (pESLCs), porcine cumulus cells (PCs), and porcine embryonic fibroblasts at passages 7 and 10 (PEF7s and PEF10s, respectively) were used as donor cells. Telomere lengths of pESLCs (35.8±1.5 kb), PCs (24.4±0.5 kb), PEF7s (18.7±0.6 kb), and PEF10s (17.2±0.1 kb) were significantly different. In contrast, telomere length in morulae derived from pESLCs (18.2±0.3 kb), PC (17.8±0.7 kb), PEF7 (18.5±0.3 kb), and PEF10 (18.4±0.4 kb) did not differ significantly. Likewise, telomeres in blastocysts derived from pESLCs (22.3±1.5 kb), PCs (23.5±2.6 kb), PEF7s (20.2±1.0 kb), and PEF10s (20.9±1.0 kb) had similar lengths. However, telomeres in blastocysts were significant longer (p0.05) compared with morulae in each group. Relative telomerase activities of morulae derived from pESLCs (4.2±0.4), PCs (4.0±0.5), PEF7s (5.1±0.4), and PEF10s (4.9±0.4) were significantly lower (p0.01) than those of blastocysts derived from pESLCs (8.2±1.1), PCs (8.6±0.6), PEF7s (12.5±2.9), and PEF10s (8.3±1.1). In conclusion, the telomere elongation in cloned pig embryos that occurred during morula-to-blastocyst transition may be related to the rise of telomerase activity. The telomere elongation may also be independent of the type and telomere length of the donor cell.

In the present study, we propose an alternative technique called cytoplast fusion to improve the maturation rate and developmental competence of growing oocytes collected from early antral follicles in pigs. We examined whether the fusion of a growing oocyte with the cytoplast from a fully-grown oocyte (CFR group) could better promote maturation and developmental competence of the growing oocyte compared to germinal vesicle (GV) transfer (GVTR group). After 44 h of in vitro maturation (IVM), most growing oocytes (GR group) were still arrested at the GV stage (64.0 ± 5.1%); this number was significantly higher (P < 0.01) than that of the other groups. No matured oocyte was observed in the GR group. The maturation rate of GVTR oocytes was significantly improved (18.8 ± 3.5%) compared with that of growing oocytes. The proportion of oocytes that reached the metaphase-II (M-II) stage in the CFR group (37.8 ± 2.0%) was significantly higher (P < 0.05) than that in the GVTR group, although still lower than that in the control group (75.2 ± 4.4%). No blastocyst was derived from growing oocytes. Among in vitro fertilized GVTR oocytes, 3.0 ± 1.9% developed into blastocysts; however, this percentage showed an insignificant increase compared with the GR group. On the other hand, the percentage of CFR embryos that developed into blastocysts (12.0 ± 4.3%) was significantly higher than that of GR embryos (0.0%), although still lower than that of control embryos (27.0 ± 5.5%). Total cell number in blastocysts in the GVTR group (23.3 ± 6.9) was significantly lower (P < 0.05) than that in the control group (50.4 ± 5.0). Meanwhile, the total cell number in blastocysts derived from CFR oocytes (36.3 ± 4.8) was comparable to that of the control group. In summary, cytoplast fusion significantly improves maturation rate and developmental competence of growing oocytes compared with GV transfer.

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.

With 4 figures Summary We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

The pig is considered to be a suitable source of cells and organs for xenotransplants, as well as a transgenic animal to produce specific proteins, given the biological similarities it shares with human beings. However, the in vitro embryo production system in pigs is inefficient compared with those in other mammals, such as cattle or mice. Although numerous modifications have been applied to improve the efficiency of in vitro embryo production systems in pigs, not much progress has been made to overcome the problem of polyspermy, and low developmental ability due to insufficient cytoplasmic abilities of in vitro matured oocytes and improper culture conditions for the in vitro produced embryos. Recent achievements, such as the establishment of chemically defined medium and utilization of 'zona hardening' technique, have gained some success. However, further research for the reduction of polyspermy and detrimental effects of the culture systems in pigs is still needed.

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.

Boar sperm freeze‐dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra‐cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze‐dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up‐regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus–oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P

Totipotent and pluripotent cells display different degrees of cellular plasticity. After fertilization, embryonic cells transit naturally from a totipotent to a pluripotent state. Major changes in nuclear architecture, chromatin mobility and gene expression occur during this transition. In particular, nuclear architecture has recently emerged as a potential regulator of heterochromatin formation in the early embryo. Future research should address whether a causal, functional link between nuclear organization and gene regulation is a general theme during reprogramming and the formation of pluripotent cells.

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10(6) /mL) was lower (P 0.01) compared with Landrace (4160 ± 42 × 10(6) /mL). However, sperm characteristics before and after freezing in Ban and Landrace were similar. The numbers of follicles with diameter larger than 2 mm per ovary in Ban females treated with equine chorionic gonadotropin and human chorionic gonadotropin (27.1 ± 1.3) were higher (P 0.05) than those in Landrace (12.9 ± 2.0) and in non-hormone stimulated Ban (no 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone-stimulated Ban and Landrace. The percentages of two-cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non-stimulated Ban (4.0 ± 3.8%) was lower (P 0.05) than that in Landrace (15.3 ± 1.8%). In conclusion, an effective IVP system for good quality embryos in Ban, that is essential for genetic conservation of this breed, was established.