Effects of bisphenol A and tauroursodeoxycholic acid on maturation of porcine oocytes and parthenogenetic development of embryos.

Prolonged exposure of bisphenol A (BPA) has adverse effects on in vitro maturation (IVM) of oocytes, but treatment with tauroursodeoxycholic acid (TUDCA) can improve the IVM and development of embryos. The purpose of this study was to investigate the effects of BPA and both BPA and TUDCA on IVM and parthenogenetic development of embryos. The results showed that BPA treatment adverse effects on the cumulus expansion index, survival rate, polar body rate, mitochondrial distribution of the oocytes after maturation culture, and that it also decreased the cleavage rate and blastocyst rate of embryos after parthenogenetic develpoment. In addition, BPA treatment upregulated expression of genes related to endoplasmic reticulum stress and apoptosis and increased the intracellular reactive oxygen species (ROS) level, while it decreased expression of genes related to cumulus expansion. However, the supplementation of TUDCA relieved these adverse effects of BPA except polar body rate, blastocyst rate, and expression of BCL2 and PTGS1. In conclusion, the supplementation of TUDCA can partly attenuate the negative effects of BPA on IVM and parthenogenetic development of embryos, possibly by modification of the expression of genes related to endoplasmic reticulum stress, apoptosis and cumulus expansion, intracellular ROS level, and mitochondrial distribution.

The full model of micropipette aspiration of cells: A mesoscopic simulation.

Studies on the interaction between cells and micromanipulation tools are necessary to optimize the procedures and improve the developmental potential of cells. The molecular dynamics simulation is not possible for such a large-scale simulation, and the spring-damped viscoelastic models and the constitutive equations of the continuum are usually adopted to model the cells as a whole without consideration of the different properties presented by the heterogeneous subcellular components. In this study, we utilized coarse-grained modeling to develop a subcellular model of suspension cell dynamics and a model of a holding micropipette for the fixation of a suspension cell, and designed a large-scale, accurate mesoscopic simulation environment for specific cell micromanipulation. We established a triangular mesh cell membrane and a uniformly distributed, non-intersecting cytoskeleton network and added polymerization/depolymerization processes to connect the cytoskeleton chains with the membrane and cross-linking proteins. In the cell aspiration model, we adopted the profile of the reversed Poiseuille flow to calibrate the viscosity of the fluid and set the bounce-back condition and the appropriate solid-fluid force coefficient to realize non-slip flow at the boundary. The rheological properties of the cells during micropipette aspiration were further analyzed in the simulation by varying parameters such as the inner diameter of the micropipette, negative pressure, and maximum bond length. The model well reproduced the experimentally observed cell deformation phenomenon at low and high pressures. The dynamic response of the cell elongation observed from the simulation was consistent with that obtained from the analysis of the experimental data collected from a custom-designed micromanipulation system. STATEMENT OF SIGNIFICANCE: In this study, we extended the coarse-grained modeling of cells by developing a relatively large-scale micromanipulation environment consisting of a subcellular cell dynamics model and a fluid flow model for cell aspiration. We simulated cytoskeleton filaments that were uniformly distributed in space via applying Harmonic energy to model cytoskeleton with a high level of fidelity. The shortcoming of the soft repulsion in the solid-fluid interaction in the current simulation technique was solved by implementing the bounce-back boundary and the condition that the total force imposed by the wall particles on the fluid particles was equal to the pressure of the fluid. This work paved the way for understanding the mechanical properties of cells and improving the biological efficacy of micromanipulation.

Identification of significant imaging features for sensing oocyte viability.

The evaluation of oocyte viability in the laboratory is limited to the morphological assessment by naked eyes, but the realization that most normal-appearing oocytes may conceal abnormalities prompts the search for automated approaches that can detect the abnormalities imperceptible to naked eyes. In this study, we developed an image processing pipeline applicable to bright-field microscope images to quantify the causal relationship between the quantitative imaging features and the developmental potential of oocytes. We acquired 19 imaging features of approximately 700 oocytes and determined two imaging subtypes, namely viable and nonviable subtypes that correlated closely with a viability fluorescence indicator and cleavage rates. The causal relationship between these imaging features and oocyte viability was derived from a viability-oriented Bayesian network that was developed based on the Bayesian information criterion and Tabu search. Our experimental results revealed that entropy with mean Gray Level Co-Occurrence Matrix energy describing the uniformity and texture roughness of cytoplasm were salient features for the automated selection of promising oocytes that exhibited excellent developmental potential.

Mechanical Characterization and Modelling of Subcellular Components of Oocytes.

The early steps of embryogenesis are controlled exclusively by the quality of oocyte that linked closely to its mechanical properties. The mechanical properties of an oocyte were commonly characterized by assuming it was homogeneous such that the result deviated significantly from the true fact that it was composed of subcellular components. In this work, we accessed and characterized the subcellular components of the oocytes and developed a layered high-fidelity finite element model for describing the viscoelastic responses of an oocyte under loading. The zona pellucida (ZP) and cytoplasm were isolated from an oocyte using an in-house robotic micromanipulation platform and placed on AFM to separately characterizing their mechanical profiling by analyzing the creep behavior with the force clamping technique. The spring and damping parameters of a Kelvin-Voigt model were derived by fitting the creeping curve to the model, which were used to define the shear relaxation modulus and relaxation time of ZP or cytoplasm in the ZP and cytoplasm model. In the micropipette aspiration experiment, the model was accurate sufficiently to deliver the time-varying aspiration depth of the oocytes under the step negative pressure of a micropipette. In the micropipette microinjection experiment, the model accurately described the intracellular strain introduced by the penetration. The developed oocyte FEM model has implications for further investigating the viscoelastic responses of the oocytes under different loading settings.

Oocyte Penetration Speed Optimization Based on Intracellular Strain.

Oocyte penetration is an essential step for many biological technologies, such as animal cloning, embryo microinjection, and intracytoplasmic sperm injection (ICSI). Although the success rate of robotic cell penetration is very high now, the development potential of oocytes after penetration has not been significantly improved compared with manual operation. In this paper, we optimized the oocyte penetration speed based on the intracellular strain. We firstly analyzed the intracellular strain at different penetration speeds and performed the penetration experiments on porcine oocytes. Secondly, we studied the cell development potential after penetration at different penetration speeds. The statistical results showed that the percentage of large intracellular strain decreased by 80% and the maximum and average intracellular strain decreased by 25-38% at the penetration speed of 50 µm/s compared to at 10 µm/s. Experiment results showed that the cleavage rates of the oocytes after penetration increased from 65.56% to 86.36%, as the penetration speed increased from 10 to 50 µm/s. Finally, we verified the gene expression of oocytes after penetration at different speeds. The experimental results showed that the totipotency and antiapoptotic genes of oocytes were significantly higher after penetration at the speed of 50 µm/s, which verified the effectiveness of the optimization method at the gene level.

A Cell's Viscoelasticity Measurement Method Based on the Spheroidization Process of Non-Spherical Shaped Cell.

The mechanical properties of biological cells, especially the elastic modulus and viscosity of cells, have been identified to reflect cell viability and cell states. The existing measuring techniques need additional equipment or operation condition. This paper presents a cell's viscoelasticity measurement method based on the spheroidization process of non-spherical shaped cell. The viscoelasticity of porcine fetal fibroblast was measured. Firstly, we introduced the process of recording the spheroidization process of porcine fetal fibroblast. Secondly, we built the viscoelastic model for simulating a cell's spheroidization process. Then, we simulated the spheroidization process of porcine fetal fibroblast and got the simulated spheroidization process. By identifying the parameters in the viscoelastic model, we got the elasticity (500 Pa) and viscosity (10 Pa·s) of porcine fetal fibroblast. The results showed that the magnitude of the elasticity and viscosity were in agreement with those measured by traditional method. To verify the accuracy of the proposed method, we imitated the spheroidization process with silicone oil, a kind of viscous and uniform liquid with determined viscosity. We did the silicone oil's spheroidization experiment and simulated this process. The simulation results also fitted the experimental results well.

Melatonin Restores the Developmental Competence of Heat Stressed Porcine Oocytes, and Alters the Expression of Genes Related to Oocyte Maturation.

Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10-9 M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

Robotic Label-Free Precise Oocyte Enucleation for Improving Developmental Competence of Cloned Embryos.

OBJECTIVE: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. METHODS: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. RESULTS: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. CONCLUSIONS: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. SIGNIFICANCE: Our method is expected to improve the extremely low success rate of animal cloning in the future.

Elevation of MPF and MAPK gene expression, GSH content and mitochondrial distribution quality induced by melatonin promotes porcine oocyte maturation and development in vitro.

The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10-6, 10-9, and 10-12 mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10-9mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10-6, and 10-12 mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10-6 mol/L melatonin was higher than with 10-12 and 0 mol/L melatonin, and the expression of the MAPK gene in 10-6 and 10-12 group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10-6 and 10-9 mol/L melatonin was significantly higher than the 10-12 mol/L melatonin and control. In conclusion, 10-9-10-6 mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.

Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability.

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus-oocyte complexes were cultured in TCM199 medium with non-treated (control), 10-5 M luzindole (melatonin receptor antagonist), 10-5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10-5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

Conserved microRNA mediates heating tolerance in germ cells versus surrounding somatic cells.

Mammalian fertility is reduced during heat exposure in the summer, but is regained as temperatures decrease in the autumn again. However, the mechanism underlying the phenomenon remains unknown. We investigated heat stress tolerance of germ cells and their surrounding somatic cells, and discovered that microRNA ssc-ca-1 was upregulated after heat stress in cultured porcine granulosa cells (GCs), but not in serum-starved GCs. Ssc-ca-1 inhibited heat shock protein 70 (Hsp70) expression through its 3'- and 5'-UTRs. Although Hsp70 mRNA transcription was induced in GCs by in vivo exposure to heat in the summer, ssc-ca-1 inhibited Hsp70 expression. In ovarian cultures, heat stress-induced Hsp70 expression in primordial but not in growing follicles; ssc-ca-1 expression did not change in primordial follicles, but increased in growing follicles. Consistently, ssc-ca-1 was present in testicular cells and exhibited the same function as in ovarian cells. It modulated the different Hsp70 expression between spermatogonial stem cells and Sertoli cells after scrotal heat stress. This mechanism is of relevance to mammalian fertility in parts of the world dominated by heat stress associated with global climate change.

Robotic Micropipette Aspiration for Multiple Cells.

As there are significant variations of cell elasticity among individual cells, measuring the elasticity of batch cells is required for obtaining statistical results of cell elasticity. At present, the micropipette aspiration (MA) technique is the most widely used cell elasticity measurement method. Due to a lack of effective cell storage and delivery methods, the existing manual and robotic MA methods are only capable of measuring a single cell at a time, making the MA of batch cells low efficiency. To address this problem, we developed a robotic MA system capable of storing multiple cells with a feeder micropipette (FM), picking up cells one-by-one to measure their elasticity with a measurement micropipette (MM). This system involved the following key techniques: Maximum permissible tilt angle of MM and FM determination, automated cell adhesion detection and cell adhesion break, and automated cell aspiration. The experimental results demonstrated that our system was able to continuously measure more than 20 cells with a manipulation speed quadrupled in comparison to existing methods. With the batch cell measurement ability, cell elasticity of pig ovum cultured in different environmental conditions was measured to find optimized culturing protocols for oocyte maturation.

Efficacy evaluation of two commercial modified-live virus vaccines against a novel recombinant type 2 porcine reproductive and respiratory syndrome virus.

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) causing clinical disease outbreaks has been recently reported in China. The recombination occurring among PRRSV strains could lead to the emergence of novel and more virulent viruses. In our previous study, a novel recombinant type 2 PRRSV (TJnh1501) between NADC30-like and modified-live virus (MLV)-like derived from the Chinese highly pathogenic PRRSV was shown to have higher pathogenicity than NADC30-like PRRSV. It remains unknown whether the emergence of the novel recombinant PRRSV strain can lead to variable protection efficacy of the MLV vaccines. In this paper, two typical commercial MLV vaccines were used to evaluate their efficacy to block TJnh1501 infection and onset of clinical symptoms. Our results showed that both MLV vaccines could shorten the period of fever and reduce viral loads in sera, but were not able to reduce the clinical signs and lung lesions indicating that the two commercial MLV vaccines provide limited cross-protection efficacy against the novel recombinant type 2 PRRSV infection. This study gives valuable suggestions for the use of MLV vaccines to control PRRSV infection in the field.

A Simple Weighing Method for Spherical Cells.

This article presents a simple weighing method for spherical cells to avoid the high cost of correlated devices in traditional cell-weighing methods. In this method, the constant falling speeds of the spherical objects in liquid are derived to estimate their masses online. Using this method, the detected density of one type of microbead is highly in accordance with the known value. This method is proved to be capable of detecting tiny variations of the cell mass (at least within 1% of the cell mass). Finally, the proposed method is applied in nuclear transplantation operations, and, for the first time, the proper amount of the removed cytoplasm in porcine enucleation is estimated. The proposed method is able to weigh cells with a success rate of 92% at an average speed of 22 s/cell, and it can be performed on traditional microoperation systems, which makes it easily applicable in biological applications.

Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.

A novel pneumatic micropipette aspiration method using a balance pressure model.

This paper presents a novel micropipette aspiration (MA) method based on a common pneumatic micro-injection system. This method is the first to quantify the influence of capillary effect on aspiration pressure using a balance pressure model, and in return, uses the capillary effect to quantify the aspiration pressure. Subsequently, the seal between the cell and the micropipette is detected to judge and exclude the ineffective MA attempts. The rationality of the balance pressure model is validated by the designed micropipette-filling experiments. Through applied to elasticity-determination of the cells with different sizes, the feasibility and versatility of this MA method are proved. With abilities to quantify aspiration pressures and detect the seam between the cell and the micropipette, our method is expected to advance the application of the commercial pneumatic injector in the MA of cells. Moreover, with the quantified volume of the liquid entering into the micropipette during MA process, our method also has a potential applicability to the study of the permeability of the cell membrane in the future.

Effects of over-expression of TLR2 in transgenic goats on pathogen clearance and role of up-regulation of lysozyme secretion and infiltration of inflammatory cells.

BACKGROUND: Toll-like receptor 2 (TLR2) is important to host recognition of invading gram-positive microbes. In goats, these microbes can cause serious mastitis, anthrax, tetanus, and other problems. Transgenic goats constitutively over-expressing TLR2 in many tissues serve as a suitable model for the study of the role of TLR2 over-expression in bacterial clearance. RESULTS: Capra hircus TLR2 over-expression vector (p3S-LoxP-TLR2) was used to generate transgenic goats by egg microinjection. The integration efficiency was 8.57%. Real-time PCR and immunohistochemical results confirmed that the goats over-expressing the TLR2 gene (Tg) expressed more TLR2 than wild-type goats (WT). Monocyte-macrophages from the bloodstreams of transgenic goats were stimulated with synthetic bacterial lipoprotein (Pam3CSK4) and by the promotion of interleukin-6 (IL-6) and IL-10 expression in vitro. The oxidative damage was significantly reduced, and lysozyme (LZM) secretion was found to be up-regulated. Ear tissue samples from transgenic goats that had been stimulated with Pam3CSK4 via hypodermic injection showed that transgenic individuals can undergo the inflammation response very quickly. CONCLUSIONS: Over-expression of TLR2 was found to decrease radical damage to host cells through low-level production of NO and MDA and to promote the clearance of invasive bacteria by up-regulating lysozyme secretion and filtration of inflammatory cells to the infected site.

Mitochondrial distribution, ATP-GSH contents, calcium [Ca2+] oscillation during in vitro maturation of dromedary camel oocytes.

Dromedary camel oocytes have the ability to spontaneous parthenogenetic activation and development in vivo and in vitro. The present study was conducted to investigate changes in mitochondrial distribution, adenosine triphosphate (ATP), and glutathione (GSH) contents and [Ca(2+)] oscillation during in vitro maturation and spontaneous parthenogentic activation of dromedary camel oocytes. Dromedary camel cumulus-oocyte complexes (COCs) were matured in TCM199 medium supplemented with 10% FCS + 10 µg/mL FSH + 10 IU hCG + 10 IU eCG + 10 ng/mL EGF and 50 µg/mL gentamycine. Maturation was performed at 38.5 °C under 5% CO(2) in humidified air for 40 h. After maturation and removal of cumulus cells, oocytes were classified into: immature cultured (Group 1); metaphase II (M II, Group 2); and spontaneously parthenogenetically activated (with 2 polar bodies, Group 3); cleaved embryos (Group 4); and immature oocytes served as a control (Group 5). Cytoplasmic mitochondrial distribution, ATP-GSH contents, calcium [Ca(2+)] oscillation were determined. Results indicated that M II and spontaneously parthenogenetically activated oocytes represent 37.53% and 32.67% of the cultured oocytes, respectively, and 3.3% cleaved and developed to 2-16-cell stage embryos. Mitochondrial distribution, ATP-GSH contents and [Ca(2+)] oscillation were significantly (P < 0.01) differ between immature and matured dromedary camel oocytes. Mitochondrial distribution showed clustering form in matured oocytes without polar body. High polarized mitochondrial distribution (HPM) was detected in M II and spontaneously parthenogenetically activated oocytes, and the intensity of MitoTracker Red was higher in spontaneously parthenogenetically activated than M II. ATP-GSH contents and the duration of [Ca(2+)] oscillation were significantly (P < 0.01) higher in spontaneously parthenogenetically activated than M II oocytes or that matured without polar body. In conclusion, the higher incidence of spontaneously parthenogenetically activated in vitro matured dromedary camel oocytes could be attributed to the high polarized mitochondrial distribution associated with significantly higher ATP-GSH contents and duration of [Ca(2+)] oscillation.

Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species.

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called "oocyte aging". Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H(2)O(2)), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H(2)O(2) treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H(2)O(2) treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H(2)O(2) groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.

Abnormal changes in mitochondria, lipid droplets, ATP and glutathione content, and Ca(2+) release after electro-activation contribute to poor developmental competence of porcine oocyte during in vitro ageing.

The present study aims to investigate major changes in porcine oocytes during ageing in vitro. After the oocytes were cultured for 44, 56, 68 and 80 h, changes to porcine oocytes in ultrastructure, mitochondrial distribution, glutathione (GSH) and ATP content, Ca(2+) release patterns and developmental competence after electro-activation were observed. Mitochondria were evenly distributed in oocytes at 44 h, aggregated in clusters or in peripheral cytoplasm at 68 h and dimly dispersed throughout ooplasm at 80 h. Mitochondrial shape during ageing was also observed by transmission electron microscopy (TEM) at the same time intervals. Most mitochondria were spherical at 44 h, and became elongated when the culture time was extended to 68 h and 80 h. Moreover, mitochondrial clustering became increasingly loose from 56 h. Lipid droplets in oocytes appeared prominent and electron-dense at 44 h, but electron density was lost at 56 h. Lipid droplets were solidified as of 68 h. There was an age-dependent decrease in ATP content per oocyte. Glutathione content per oocyte decreased significantly and remained lower after 56 h. Amplitudes of [Ca(2+)] rise decreased dramatically following 56 h, and the time required for [Ca(2+)] to plateau became shorter after electro-activation with prolonged culture time. Cleavage and blastocyst rates of aged oocytes progressively decreased, while the fragmentation rate gradually increased after electro-activation. It is concluded that abnormal changes in mitochondria, lipid droplets, Ca(2+) release after electro-activation, and ATP and GSH content in oocytes during ageing may result in poor developmental competence of parthenotes.