Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes.

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 µM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 µM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 µM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

In vitro maturation using an agarose matrix with incorporated extracellular matrix proteins improves porcine oocyte developmental competence by enhancing cytoplasmic maturation.

Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.

Alpha-lipoic acid improves the maturation and the developmental potential of goat oocytes in vitro.

Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25 µmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18-22 hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96 hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p < .05) and 5.41% (p < .05) compared with those in the control group, respectively. After 24 hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p < .01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p < .05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25 µmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.

Ginsenoside Rb1 protects porcine oocytes against methylglyoxal damage thus it improves the quality of parthenogenetic activation and in vitro fertilization embryos.

Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.

Melatonin protects against defects induced by malathion during porcine oocyte maturation.

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles.

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Melatonin Improves Parthenogenetic Development of Vitrified⁻Warmed Mouse Oocytes Potentially by Promoting G1/S Cell Cycle Progression.

This study aimed to investigate the effect of melatonin on the cell cycle of parthenogenetic embryos derived from vitrified mouse metaphase II (MII) oocytes. Fresh oocytes were randomly allocated into three groups: untreated (control), or vitrified by the open-pulled straw method without (Vitrification group) or with melatonin (MT) supplementation (Vitrification + MT group). After warming, oocytes were parthenogenetically activated and cultured in vitro, then the percentage of embryos in the G1/S phase, the levels of reactive oxygen species (ROS) and glutathione (GSH), and the mRNA expression of cell cycle-related genes (P53, P21 and E2F1) in zygotes and their subsequent developmental potential in vitro were evaluated. The results showed that the vitrification/warming procedures significantly decreased the frequency of the S phase, markedly increased ROS and GSH levels and the expression of P53 and P21 genes, and decreased E2F1 expression in zygotes at the G1 stage and their subsequent development into 2-cell and blastocyst stage embryos. However, when 10-9 mol/L MT was administered for the whole duration of the experiment, the frequency of the S phase in zygotes was significantly increased, while the other indicators were also significantly improved and almost recovered to the normal levels shown in the control. Thus, MT might promote G1-to-S progression via regulation of ROS, GSH and cell cycle-related genes, potentially increasing the parthenogenetic development ability of vitrified⁻warmed mouse 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.

Improved development by melatonin treatment after vitrification of mouse metaphase II oocytes.

The study was aimed to investigate the effect of melatonin on the development potential of mouse MII oocytes after cryopreservation. Mouse MII oocytes were subjected first to vitrification/warming and 2 h of in vitro culture (phase 1), then to parthenogenetic activation (PA) followed by in vitro culture of parthenogenetic embryos (phase 2). Different concentrations of melatonin (0, 10-9, 10-6 mol/L) were added to the medium during either phase 1, phase 2 or both phases. The fresh oocytes were used as control. When melatonin was used during both phases, 10-9 mol/L melatonin-treated group showed similar rates of cleavage and 4-cell embryo development compared with control, which were significantly higher than those of melatonin-free group, while the rates in either 10-6 mol/L melatonin-treated or melatonin-free groups were significantly lower than that in control. When 10-9 mol/L melatonin was added during either phase 1 or phase 2, both cleavage and 4-cell embryo development rates of either group were significantly lower than those of control. After oocyte vitrification/warming and PA, the ROS levels increased significantly and maternal-to-zygotic transition (MZT) related genes (Dcp1a, Dcp2, Hspa1a, Eif1ax, Pou5f1, Sox2) expression were disorganized. However, after 10-9 mol/L melatonin supplementation, the ROS levels decreased significantly compared with melatonin-free group, and the gene expressions were almost recovered to normal level of control group. These results demonstrated that 10-9 mol/L melatonin supplementation could increase the developmental potential of vitrified-warmed mouse MII oocytes, which may result from ROS scavenging activities and recovery of normal levels of the expressions of MZT-related genes.

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.

Pollen Morphology and Boron Concentration in Floral Tissues as Factors Triggering Natural and GA-Induced Parthenocarpic Fruit Development in Grapevine.

Parthenocarpic fruit development (PFD) reduces fruit yield and quality in grapevine. Parthenocarpic seedless berries arise from fruit set without effective fertilization due to defective pollen germination. PFD has been associated to micronutrient deficiency but the relation of this phenomenon with pollen polymorphism has not been reported before. In this work, six grapevine cultivars with different tendency for PFD and grown under micronutrient-sufficient conditions were analyzed to determine pollen structure and germination capability as well as PFD rates. Wide variation in non-germinative abnormal pollen was detected either among cultivars as well as for the same cultivar in different growing seasons. A straight correlation with PFD rates was found (R2 = 0.9896), suggesting that natural parthenocarpy is related to defective pollen development. Such relation was not observed when PFD was analyzed in grapevine plants exposed to exogenous gibberellin (GA) or abscissic acid (ABA) applications at pre-anthesis. Increase (GA treatment) or reduction (ABA treatment) in PFD rates without significative changes in abnormal pollen was determined. Although these plants were maintained at sufficient boron (B) condition, a down-regulation of the floral genes VvBOR3 and VvBOR4 together with a reduction of floral B content in GA-treated plants was established. These results suggest that impairment in B mobility to reproductive tissues and restriction of pollen tube growth could be involved in the GA-induced parthenocarpy.

Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.

Effects of salinity and ultraviolet radiation on the bioaccumulation of mycosporine-like amino acids in Artemia from Lake Urmia (Iran).

We investigated the effects of salinity and artificial UV radiation on the accumulation of mycosporine-like amino acids (MAAs) in sexual and parthenogenetic Artemia from Lake Urmia. The nauplii hatched from the cysts were cultured until adulthood under two salinities (150 and 250 g L(-1) ) and two light treatments (PAR and PAR+UVR) in the laboratory. Finally, the Artemia were analyzed for their concentration of MAAs. In most of the cases, the higher salinity level applied was found to increase the MAA concentrations in both Artemia populations significantly. The acquisition efficiency of MAAs in both Artemia populations increased under exposure to UVR-supplemented photosynthetically active radiation (PAR) compared to those raised under PAR, except for Porphyra-334. It was observed that combination of UV radiation and elevated salinity significantly increased the bioaccumulation of MAAs. Thus, the presence of these compounds in these populations of Artemia may increase their adaptability for living in high-UV and high-salinity conditions prevailing in Lake Urmia. Higher concentrations of MAAs in the parthenogenetic population of Artemia could be probably attributed to its mono sex nature and higher adaptation capacities to extreme environmental conditions.

Effect of melatonin treatment on the developmental potential of parthenogenetic and somatic cell nuclear-transferred porcine oocytes in vitro.

Melatonin secreted from the mammalian pineal gland is a free-radical scavenger that protects tissues from cell damage. The present study examined the effects of addition of melatonin to the culture medium on the developmental potential of parthenogenetic and somatic cell nuclear-transferred (SCNT) porcine oocytes. Supplementation of the maturation medium with melatonin did not increase the maturation rate, the proportion of oocytes that cleaved and developed into blastocysts after parthenogenetic activation, or the blastocyst cell number compared to controls. When 10-7 M melatonin was added to the culture medium, the proportion of parthenogenetic oocytes that developed to the 2-cell and 4-cell stages was significantly higher than that of controls. The potential of melatonin-treated oocytes to develop into blastocysts was high but not significantly different from that of controls. The addition of 10-7 M melatonin to the culture medium did not increase the preimplantation development of SCNT oocytes. Melatonin treatment significantly reduced the levels of reactive oxygen species in 4-cell parthenogenetic and SCNT embryos, but did not reduce the proportion of apoptotic cells in parthenogenetic and SCNT blastocysts. Although the results indicated that parthenogenetic and SCNT melatonin -treated embryos had significantly lower levels of reactive oxygen species than controls, the potential of melatonin-treated embryos to develop into blastocysts was not significantly higher than that of controls, in contrast to previous reports. The beneficial effects of melatonin on the developmental potential of oocytes might depend on the culture conditions.

Insulin-like growth factor 2 promotes osteogenic cell differentiation in the parthenogenetic murine embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent and can differentiate into all somatic cell types. ESCs are an alternative solution to hard tissue regeneration and skeletal tissue repair to treat bone diseases and defects using regenerative strategies. Parthenogenetic ESCs (PESCs) may be a useful alternative stem cell source for tissue repair and regeneration. The defects in full-term development of this cell type enable researchers to avoid the ethical concerns related to ESC research. Moreover, in female patients, if the PESCs are derived from oocytes, then they will have that patient's genetic information. Here, we present data demonstrating that osteogenic differentiation of PESCs can be promoted by insulin-like growth factor 2 (IGF2). PESCs were plated onto Petri dishes with ESC culture medium supplemented with or without IGF2, followed by culturing of the cells for 1 week. PESCs formed floating aggregates called embryoid bodies (EBs). An osteogenic lineage was induced from the EBs by incubating them in medium containing serum, ascorbic acid, ß-glycerophosphate, and retionic acid, with or without IGF2, for 20 days. Gene expression of specific osteoblastic markers such as osteocalcin, osteopontin, osteonectin, bone sialoprotein, collagen type-I, alkaline phosphatase, and Runx2 (Cbfa-I) was analyzed by real-time polymerase chain reaction. The expression level of osteocalcin, osteopontin, osteonectin, and alkaline phosphatase was twofold higher in IGF2-treated PESC derivatives than IGF2-naive PESC derivatives. In vivo experiments were also performed using a critical-sized calvarial defect mouse model. Ten weeks after cell transplantation, more bone tissue regeneration was observed in the IGF2-treated PESC transplantation group than in IGF2-naive PESC transplantation group. Both our in vitro and in vivo data indicate that IGF2 induces osteogenic differentiation of PESCs. Addition of IGF2 may reactivate imprinting genes in PESCs that are only expressed in the paternal genome and are normally silent in PESCs. Our findings provide insights into the mechanisms of skeletal tissue repair and the imprinting mechanisms active in stem cells.

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

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

Zinc availability regulates exit from meiosis in maturing mammalian oocytes.

Cellular metal ion fluxes are known in alkali and alkaline earth metals but are not well documented in transition metals. Here we describe major changes in the zinc physiology of the mammalian oocyte as it matures and initiates embryonic development. Single-cell elemental analysis of mouse oocytes by synchrotron-based X-ray fluorescence microscopy (XFM) revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. Perturbation of zinc homeostasis with a cell-permeable small-molecule chelator blocked meiotic progression past telophase I. Zinc supplementation rescued this phenotype when administered before this meiotic block. However, after telophase arrest, zinc triggered parthenogenesis, suggesting that exit from this meiotic step is tightly regulated by the availability of a zinc-dependent signal. These results implicate the zinc bolus acquired during meiotic maturation as an important part of the maternal legacy to the embryo.

A fertilization-independent developmental program triggers partial fruit development and senescence processes in pistils of Arabidopsis.

The pistil is the specialized plant organ that enables appropriate pollination and ovule fertilization, after which it undergoes growth and differentiation to become a fruit. However, in most species, if ovules are not fertilized around anthesis the pistil irreversibly loses its growth capacity. We used physiological, molecular, and transcriptomic tools to characterize the post-anthesis development of the unfertilized Arabidopsis (Arabidopsis thaliana) pistil. Surprisingly, developmental processes that have been previously described in developing Arabidopsis fruits, such as the collapse of the adaxial epidermis, differentiation of a sclerenchyma layer in the adaxial subepidermis and the dehiscence zone, and valve dehiscence, were also observed in the unfertilized pistil. We determined that senescence is first established in the transmitting tract, stigma, and ovules immediately after anthesis, and that the timing of senescence in the stigma and ovules correlates with the loss of fruit-set responsiveness of the pistil to pollen and the hormone gibberellin (GA), respectively. Moreover, we showed that mutants with altered ovule development have impaired fruit-set response to the GA gibberellic acid, which further indicates that the presence of viable ovules is required for fruit-set responsiveness to GAs in the unfertilized pistil. Our data suggest that a fertilization-independent developmental program controls many of the processes during post-anthesis development, both in unfertilized pistils and seeded fruits, and point to a key role of the ovule in the capacity of pistils to undergo fruit set in response to GA.

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos.

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

Activation and parthenogenetic development of pig oocytes exposed to ultrasound in media containing different concentrations of Ca2+.

The present study was carried out to examine the activation and parthenogenetic development of pig oocytes after exposure to ultrasound in sorbitol media supplemented with different concentrations of Ca2+. The activation rates (68.8-75.6%) of oocytes exposed to ultrasound in media containing 0.1-1.0 mM Ca2+ were significantly (P<0.05) higher than those (54.3-58.3%) of oocytes exposed to ultrasound in media containing 0-0.05 mM Ca2+. When oocytes were exposed to ultrasound in media containing 0.1-0.5 mM Ca2+, the blastocyst formation rates (20.5-21.3%) were significantly (P<0.05) higher than those (3.3-6.0%) of oocytes exposed to ultrasound in media containing 0, 0.05 or 1.0 mM Ca2+. The results of the present study showed that the concentration of Ca2+ in the medium used for exposure to ultrasound affects the activation and parthenogenetic development of pig oocytes and showed that the optimal Ca2+ concentration is 0.1-0.5 mM.