Activation Of Porcine Oocytes Research Articles

Chrysoeriol Improves the Early Development Potential of Porcine Oocytes by Maintaining Lipid Homeostasis and Improving Mitochondrial Function.

Our previous study established that chrysoeriol (CHE) can reduce reactive oxygen species (ROS) accumulation, apoptosis, and autophagy in vitro culture (IVC) of porcine embryos. However, the role of CHE in oocyte maturation and lipid homeostasis is unclear. Herein, we aimed to elucidate the effect of CHE on porcine oocyte competence in vitro maturation (IVM) and subsequent embryo development. The study chooses parthenogenetic activated porcine oocytes as the research model. The study revealed that the cumulus expansion index and related gene expressions are significantly elevated after supplementing 1 μM CHE. Although there were no significant differences in nuclear maturation and cleavage rates, the blastocyst formation rate and total cell numbers were significantly increased in the 1 μM CHE group. In addition, CHE improved the expression of genes related to oocyte and embryo development. ROS was significantly downregulated in all CHE treatment groups, and intracellular GSH (glutathione) was significantly upregulated in 0.01, 0.1, and 1 μM CHE groups. The immunofluorescence results indicated that mitochondrial membrane potential (MMP) and lipid droplet (LD), fatty acid (FA), ATP, and functional mitochondria contents significantly increased with 1 μM CHE compared to the control. Furthermore, CHE increased the expression of genes related to lipid metabolism, mitochondrial biogenesis, and β-oxidation.

Alpha-lipoic acid attenuates heat stress-induced apoptosis via upregulating the heat shock response in porcine parthenotes

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes.

Porcine oocytes matured in a chemically defined medium are transcriptionally active

The statement that fully-grown porcine oocytes (oocytes from follicles with diameter from 3 to 6 mm) are transcriptionally quiescent is not as strongly supported as it was before. Currently, we know that there is a difference between the transcription profile of germinal vesicle (GV) and metaphase II (MII) oocytes. The goal of our study was to compare the transcription profile of GV, germinal vesicle breakdown (GVBD), metaphase I (MI), and MII oocytes matured in the chemically defined medium FLI. Oocytes were sequenced, and the results were subsequently validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We detected multiple differentially transcribed mRNAs, of which many were upregulated. Among them we found mRNAs necessary for protein production, mitochondrial functions and cytoplasmic maturation. Collectively, these data support the hypothesis that transcription activity in fully-grown porcine oocytes is necessary for key processes during their successful maturation in vitro in a chemically defined maturation medium.

Evaluation of developmental competence of Sus scrofa domesticus (L.) oocyte-cumulus complexes after intra- and extraovarian vitrif ication.

The aim of the present study was to identify the inf luence of extra- (EOV) and intraovarian vitrif ication (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO + 1.8 M EG; CPS-3 – 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG + 7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrif ication, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % f luid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Signif icant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrif ied groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells f inished the maturation ( p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4 ± 7.5 μA and 149.2 ± 11.3 μA, respectively, p ≤ 0.05). For the f irst time, pre-implantation embryos were obtained from oocytes treated by IOV.

Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

A fast and reliable protocol for activation of porcine oocytes

Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca2+ and Zn2+ levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different time intervals. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with PKCa + CDK1i for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different times to a Zn2+ chelator (TPEN) following Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximise oocyte activation and embryo development. Next, we observed that treatment with PKCa + CDK1i for 4 h after TPEN for 15 min decreased embryo development compared to TPEN alone. Lastly, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs 49.2%) and blastocyst from cleaved embryos (65.5% vs 46.2%) compared to CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol compared with CT-245 and the non-activated (N-A) group. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a fast and effective protocol for activation of porcine oocytes.

Effect of Kisspeptin on the Developmental Competence and Early Transcript Expression in Porcine Oocytes Parthenogenetically Activated with Different Methods.

Recent studies showed the modulatory effect of kisspeptin (KP) on calcium waves through the cell membrane and inside the cell. Spermatozoon can induce similar ooplasmic calcium oscillations at fertilization to trigger meiosis II. Here, we evaluated the effect of KP supplementation with 6-dimethylaminopurine (6-DMAP) for 4 h on embryonic development after oocyte activation with single electric pulse, 5 µM ionomycin, or 8% ethanol. Compared to control nonsupplemented groups, KP significantly improved embryo developmental competence electric- and ethanol-activated oocytes in terms of cleavage (75.3% and 58.6% versus 64% and 48%, respectively, p < 0.05) and blastocyst development (31.3% and 10% versus 19.3% and 4%, respectively, p < 0.05). MOS expression was increased in electrically activated oocytes in presence of KP while it significantly reduced CCNB1 expression. In ionomycin treated group, both MOS and CCNB1 showed significant increase with no difference between KP and control groups. In ethanol-treated group, KP significantly reduced CCNB1 but no effect was observed on MOS expression. The early alterations in MOS and CCNB1 mRNA transcripts caused by KP may explain the significant differences in the developmental competence between the experimental groups. Kisspeptin supplementation may be adopted in protocols for porcine oocyte activation through electric current and ethanol to improve embryonic developmental competence.

Localization of Autophagosome in Porcine Follicular Cumulus-oocyte Complex

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during In Vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

C-Phycocyanin supplementation during in vitro maturation enhances pre-implantation developmental competence of parthenogenetic and cloned embryos in pigs

C-Phycocyanin (C-PC), a protein from green microalgae, has been suggested to possess various biological activities, including antioxidant and free radical scavenging properties. The aim of the current study was to explore the effects of C-PC on the maturation of porcine oocytes and subsequent developmental competence after parthenogenetic activation and somatic cell nuclear transfer (SCNT) as well as the underlying mechanisms. There was no significant improvement in nuclear maturation rates between the control and C-PC supplementation groups (1, 3, 5, 10 μg/mL). However, supplementation of 5 μg/mL C-PC in the maturation medium significantly increased blastocyst formation and hatching rates after parthenogenetic activation (59.6 ± 3.6% and 33.0 ± 2.6% vs. 49.8 ± 3.5% and 27.4 ± 2.4%, respectively). In addition, the presence of C-PC during the maturation period significantly improved blastocyst formation rates and total cell numbers after SCNT (24.8 ± 1.9% and 42.2 ± 3.3 vs. 21.6 ± 2.2% and 39.5 ± 3.4, respectively) compared to the control group. Furthermore, cellular proliferation and the expression of pluripotency-related genes (SOX2 and NANOG) were increased in cloned blastocysts derived from the C-PC supplemented group. Importantly, C-PC supplementation during maturation not only improved cumulus expansion and increased the expression of cumulus expansion-related genes (HAS2, PTX3, and PTGS2), but also enhanced antioxidant capacity, improved mitochondria function, and decreased cathepsin B activity in porcine oocytes. These results demonstrate that C-PC may be useful for improving porcine oocyte quality and subsequent developmental competence in embryos.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment.

The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2mM 6-dimethylaminopurine for 4h and those activated by an EP and treated with 0.01µg/mL anisomycin for 4h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture.

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

Calcineurin role in porcine oocyte activation

Calcineurin is required for oocyte exit from meiotic block in metaphase II (MII) stage in invertebrates and also in lower vertebrates. However, the role of calcineurin in mammalian oocyte activation is still unclear. The aim of this study was to determine whether calcineurin is involved in the processes regulating porcine oocyte activation. Indirect immunofluorescence demonstrated localization of both calcineurin subunits, CnA and CnB, especially in the cortex area of MII oocytes, in vitro fertilized and also parthenogenetically activated oocytes. After activation, the fluorescence intensity of the protein in the cortex area of oocytes remains unchanged; the protein calcineurin in the cytoplasm was recorded mainly around the pronuclei. Treatment of matured oocytes with calcineurin inhibitors, cyclosporin A (CsA) and hymenistatin I (HS-I), followed by activation with calcium ionophore A23187, significantly decreased the rate of activated oocytes compared to oocytes that were treated only with calcium ionophore (Ca-Io), (CsA+Ca-Io 25.0% v. Ca-Io 83.3%; HS-I+Ca-Io 32.5% v. Ca-Io 85.0%). Compared to the control, CsA treatment of matured oocytes followed by activation with Ca-Io did not affect the activity level of metaphase-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in activated oocytes evaluated by kinase activity assay. Simultaneous staining of calcineurin and cortical granule content in matured oocytes showed that calcineurin distributed in the cortical area of the oocyte has not been colocalized with cortical granules content. On the other hand, the calcineurin inhibition before parthenogenetic activation leads to a reduction of the cortical reaction level compared to oocytes that were not treated with CsA (complete exocytosis: CsA+Ca-Io 2.6% v. Ca-Io 83.9%; sum of cortical granule brightness: CsA + Ca-Io 0.69 v. Ca-Io 0.15). Our results showed that calcineurin is involved in the process of pig oocyte activation and cortical granule exocytosis; however this regulation seems to be MPF and MAPK independent.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities.

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

Transgenesis through Blastomeres Transfection

Parthenogenetically activated porcine oocytes were subjected to pronase 0.3% (in PBS w/v) for 90 seconds to digest the zona pellucida. Zonafree blastomres were transfected with piggybac transposon DNA using Fugene HD transfection reagent (Promega Corp., Madison, WI, USA).

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre- and Post-Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre- and post-activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre- or post-electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1min pre- and 3min post-activation showed significantly higher blastocyst development than 0min pre- and 0min post-activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0min pre- and 0min post-activation. In conclusion, exposure of oocytes to mannitol for 1min pre- and 3min post-activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1min pre- and 3min post-activation.

Involvement of Dynamin 2 in actin-based polar-body extrusion during porcine oocyte maturation.

Mammalian oocyte meiotic maturation involves a unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic GTPases involved in membrane trafficking and actin dynamics, whose roles in mammalian oocyte maturation have not been determined. In this study, we used porcine oocytes to show that Dynamin 2 accumulated at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. Inhibiting Dynamin 2 activity in porcine oocytes with the specific inhibitor dynasore resulted in failed polar-body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment disrupted the formation of the actin cap and cortical granule-free domain, which negatively impacted spindle positioning. Moreover, the distribution of ARP2, a key actin-nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin 2 possibly regulates porcine oocyte maturation through its effects on actin-mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization.

Gynogenetic Activation of Porcine Oocytes

The possibility of fertilization without male contribution to the embryonic genome was investigated in pig oocytes. Mature oocytes were co-incubated with sperm, and in an attempt to prevent the incorporation of the sperm head into the ooplasm, the actin polymerization inhibitor cytochalasin B was added to the fertilization medium. We found that perturbing actin filament integrity did not affect the pattern of the sperm-induced Ca(2+) signal or the process of cortical granule exocytosis, and it did not alter the percentage of activated oocytes compared to the control (oocytes fertilized in the absence of the inhibitor). However, over 20% of the cytochalasin B-treated oocytes formed only a single pronucleus after fertilization, indicating that the inhibitor blocked sperm head incorporation at least in some oocytes. In most cases, cytochalasin B also prevented the integration of the male chromosomes into the embryonic genome as determined by the absence of the SRY gene in the embryonic blastomeres or by the frequency of embryos showing green fluorescence after sperm from a GFP-transgenic boar was used for fertilization. Finally, the percentage of embryos that developed beyond the four-cell stage and the total number of nuclei in the resultant blastocysts were higher when oocytes reconstructed by nuclear transfer were activated by fertilization in the presence of cytochalasin B compared to the control group, where activation was induced by electroporation. These results suggest that fertilization in the presence of cytochalasin B can induce oocyte activation while it also prevents integration of the male genome into the embryo. This method has the potential to be used as an alternative to inducing embryonic development after nuclear transfer.