Development Of Parthenogenetic Embryos Research Articles

In vitro maturation using porcine follicular fluid-derived exosomes as an alternative to the conventional method

Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 106 particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 107 particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 107 particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression.

Vitamin C Improves Oocyte In Vitro Maturation and Potentially Changes Embryo Quality in Cattle.

To obtain high-quality bovine oocytes, the effects of vitamin C (VC) on the IVM of bovine oocytes and early embryo development were investigated. The results showed the following. (1) The IVM medium containing 50 µg/mL VC improved the oocyte maturation rate but did not affect the parthenogenetic embryo development. (2) The IVC medium containing 20 µg/mL VC improved the cleavage rate of the IVF embryos and enhanced the mRNA transcriptions of pluripotency gene Oct4, Sox2, Cdx2, and Nanog in the blastocysts but had no effects on the blastocyst rate. (3) Combining supplementation of 50 µg/mL VC in IVM medium + 20 µg/mL VC in IVC medium (named as VC 50/20, similar hereinafter) elevated the cleavage rate of IVF embryos and enhanced the mRNA expressions of Oct4, Sox2, Cdx2, and Nanog in the blastocysts. (4) Combination of VC 0/20 and VC 50/20 enhanced the transcription of anti-apoptotic gene Bcl-2 and VC 50/0 weakened the transcription of pro-apoptotic gene Bax, while VC 0/40 and VC 0/60 increased Bax expression and diminished the Bcl-2/Bax ratio in blastocysts. Together, employing 50 µg/mL VC improves the IVM of bovine oocytes and combination of VC 50/20 potentially changes bovine embryo quality by enhancing the expressions of the pluripotency genes and regulating the expressions of apoptosis-related genes.

The walnut-derived peptide TW-7 improves mouse parthenogenetic embryo development of vitrified MII oocytes potentially by promoting histone lactylation

BackgroundPrevious studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative stress is the probable factor; however, the underlying mechanism remains unclear. The walnut-derived peptide TW-7 was initially isolated and purified from walnut protein hydrolysate. Accumulating evidences implied that TW-7 was a powerful antioxidant, while its prospective application in oocyte cryopreservation has not been reported.ResultHere, we found that parthenogenetic activation (PA) zygotes derived from vitrified MII oocytes showed elevated ROS level and delayed progression of pronucleus formation. Addition of 25 μmol/L TW-7 in warming, recovery, PA, and embryo culture medium could alleviate oxidative stress in PA zygotes from vitrified mouse MII oocytes, furtherly increase proteins related to histone lactylation such as LDHA, LDHB, and EP300 and finally improve histone lactylation in PA zygotes. The elevated histone lactylation facilitated the expression of minor zygotic genome activation (ZGA) genes and preimplantation embryo development.ConclusionsOur findings revealed the mechanism of oxidative stress inducing repressed development of PA embryos from vitrified mouse MII oocytes and found a potent and easy-obtained short peptide that could significantly rescue the decreased developmental potential of vitrified oocytes, which would potentially contribute to reproductive medicine, animal protection, and breeding.

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 μM CGA treatment during the invitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

Effects of Leonurine on oocyte maturation and parthenogenetic embryo development in sheep.

Leonurine (LEO), an alkaloid isolated from Leonurus spp., has anti-oxidant, anti-inflammatory and anti-apoptotic effects and can prevent damage caused by reactive oxygen species (ROS). These properties suggest that it can improve the maturation rate of oocytes and developmental ability of embryos, which are key parameters in animal breeding. In this study, the effects of LEO on invitro maturation and early embryonic development in sheep oocytes were evaluated. Among various doses examined (0, 10, 20 and 40 μM), a dose of 20 μM was optimal with respect to the oocyte maturation rate. Compared with estimates in the control group, GSH levels and mitochondrial membrane potential of sheep oocytes treated with 20 μM LEO were significantly higher, and 40 μM LEO would affect oocyte maturation. Additionally, ROS levels were significantly lower, expression levels of the antioxidant genes CAT and SOD1 were significantly higher, and there was no significant difference in GPX3 expression. The Bax/Bcl-2 ratio and Caspase-3 expression were significantly reduced in the 20 μM LEO group. During early embryonic development invitro, the cleavage rate and blastocyst rate were significantly higher in the 20 μM LEO treatment group compared to other groups. GSH levels and mitochondrial membrane potential were significantly higher, while ROS levels were significantly lower, and expression levels of the antioxidant genes CAT, GPX3 and SOD1 were significantly higher in eight-cell embryos treated with 20 μM LEO than in the control group. The Bax/Bcl-2 ratio and Caspase-3 levels were significantly decreased. In summary, LEO can reduce the effect of oxidative stress, improve the oocyte maturation rate and enhance embryonic development.

Methoxychlor induces oxidative stress and impairs early embryonic development in pigs.

Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs. Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development. Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

Effect of post-activation culture conditions on the development of parthenogenetic embryos in cattle

Purpose: to conduct a comparative analysis of the effect of commercial media BO-IVC and СR1aa at the stage of the activation and subsequent culture of artificially activated oocytes on the formation and quality of parthenogenetic bovine embryos.Materials and methods. 3 groups of disemeters of 50 goals in each were formed. In the first experimental group, the disemeted was in a meticulous manner with a ram-industrialist (artificial kriproporchid), in the second experimental-with a penEexctomed ram-industrialist. In the third (control) group, a producer ram was used. In the first experimental group of a ram-industrialist (artificial kriproporchid) with attached taps were released into a group of sheep twice a day for 1.5-2 hours. In the second experimental group of a penEctomed ram, it was placed in the corral to the disemetery in the morning for 3 hours. In the third group, the lamb producer was constantly with the disemets for two weeks, then he was changed on a new ram i.e. Used the methodology used in the farm. During the experiment, they observed the behavior of animals of all groups. In the experimental groups, after the detection of disemeters in the hunt, their natural insemination of the manufacturer was carried out. Based on the results of the subsequent oster, the effectiveness of the reproduction of sheep was evaluated.Results. The cleavage rate did not differ between the experimental groups, varying from 73,0 to 76,5%. Also, there was not found a significant effect of the conditions for post-activation culture of oocytes on their development before late morula and late blastocyst stage, which was for the CR1aa/CR1aa, CR1aa/BO-IVC and BOIVC/ BO-IVC groups 28,9±1,7, 40,4±7,5 and 36,0±6.4%, respectively. Meanwhile, we found out the effect of tested culture conditions on the ability of parthenogenetic embryos to overcome the 8-16 cell block and their quality on the late stages of embryo development. The rate of embryos with less than 16 nuclei was the highest in the CR1aa/CR1aa group (56,8±2,1 %). The replacement of CR1aa medium to BO-IVC medium (BO-IVC/BO-IVC group) significantly reduced this level (p&lt;0,05). The positive effect was enhanced when CR1aa medium was used at the stage of culture in the presence of 6-DMAP and cycloheximide, and subsequent embryo development was in BO-IVC medium (CR1aa/BO-IVC group) (p&lt;0.001). Furthermore, when we used the mixed variant of culture, the total cell number in parthenogenetic morula and blastocyst stage embryos increased (p&lt;0.05).Conclusion. Thus, the BO-IVC medium at the stages of post-activation and subsequent development of artificially activated bovine oocytes is comparable to the CR1aa medium in terms of the efficiency of obtaining parthenogenetic embryos at the blastocyst stage. Nevertheless, its replacement at the post-activation stage with CR1aa medium makes it possible to improve the quality of parthenogenetic embryos.

Interleukin-7 enhances in vitro development and blastocyst quality in porcine parthenogenetic embryos.

Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.

Miro1 depletion disrupts spatial distribution of mitochondria and leads to oocyte maturation defects.

Mitochondria are dynamic organelles that undergo regulated microtubule- and actin-mediated trafficking to meet local energy and metabolic needs. Mitochondrial trafficking may be particularly critical in large cells such as eggs and early embryos where spindle formation and polar body extrusion occur in specific regions of the cytoplasm. To investigate the role of mitochondrial distribution in oocytes we have targeted the mitochondrial membrane protein, MIRO1, which couples mitochondria to the motor protein-TRAK complex. Oocyte-specific deletion of MIRO1 leads to the formation of large aggregates of mitochondria in perinuclear and cortical compartments. Mitochondria remain capable of long-range trafficking during maturation, indicating redundancy in the mechanisms coupling mitochondria to motor proteins. Polar body extrusion in the absence of MIRO1 was reduced by approximately 20%. In MIRO1-deleted zygotes, mitochondria showed increased accumulation around the pronuclei but this did not affect mitochondrial distribution to daughter blastomeres. In vitro development of parthenogenetic embryos was also reduced, although no differences were found in the fertility of oocyte-specific Miro1 KO mice. These findings demonstrate MIRO1 acts as a mitochondrial adaptor, setting mitochondrial distribution in oocytes and early embryos, and disrupting this process compromises in vitro oocyte maturation and embryo development.

Mendelian segregation for parthenogenetic embryo development at the diploid level in the flowering plant Erigeron.

Parthenogenesis is the capacity of organisms to develop embryos from unfertilized eggs. When parthenogenesis is coupled with unreduced gamete formation (apomeiosis), genetically maternal progeny result. Genetic elucidation of this form of reproduction in plants, apomixis, has important agronomic implications. However, genetic characterization of apomeiosis and parthenogenesis has been problematic in part because the traits usually co-occur and are restricted to polyploids. In this work, the inheritance of parthenogenetic embryo development, by itself, was studied at the diploid level. Progeny resulting from a cross between a diploid (2n = 18), heterozygous, parthenogenetic pollen donor, and a diploid, wildtype, sexual seed parent were evaluated. Paternity was tested with conserved orthologous sequence (COS) markers, reproductive development of F1s was evaluated with microscopy of cleared ovules, and an amplified fragment length polymorphism (AFLP) marker (Eagc × Macg.615) co-segregating with parthenogenesis was characterized at the sequence level. Of 102 diploid biparental progeny, 47 exhibited parthenogenetic embryo and endosperm development, and 55 lacked development of the egg and central cell. This result is consistent with Mendelian inheritance for a single locus (P = 0.43). Isolation and sequencing of the AFLP marker indicates that it is likely a portion of a Ty-Gypsy retrotransposon. Attempts to develop a sequence-characterized amplified region marker from the AFLP were unsuccessful. This work shows that parthenogenesis can be transmitted simply at the diploid level. This advance is key in the development of a tractable system in Erigeron aimed at the identification of the parthenogenesis locus using genetic mapping strategies.

Phylogenetically Distant BABY BOOM Genes From Setaria italica Induce Parthenogenesis in Rice.

The combination of apomixis and hybrid production is hailed as the holy grail of agriculture for the ability of apomixis to fix heterosis of F1 hybrids in succeeding generations, thereby eliminating the need for repeated crosses to produce F1 hybrids. Apomixis, asexual reproduction through seed, achieves this feat by circumventing two processes that are fundamental to sexual reproduction (meiosis and fertilization) and replacing them with apomeiosis and parthenogenesis, resulting in seeds that are clonal to the maternal parent. Parthenogenesis, embryo development without fertilization, has been genetically engineered in rice, maize, and pearl millet using PsASGR-BABY BOOM-like (PsASGR-BBML) transgenes and in rice using the OsBABY BOOM1 (OsBBM1) cDNA sequence when expressed under the control of egg cell-specific promoters. A phylogenetic analysis revealed that BABY BOOM (BBM)/BBML genes from monocots cluster within three different clades. The BBM/BBML genes shown to induce parthenogenesis cluster within clade 1 (the ASGR-BBML clade) along with orthologs from other monocot species, such as Setaria italica. For this study, we tested the parthenogenetic potential of three BBM transgenes from S. italica, each a member of a different phylogenetic BBM clade. All transgenes were genomic constructs under the control of the AtDD45 egg cell-specific promoter. All SiBBM transgenes induced various levels of parthenogenetic embryo development, resulting in viable haploid T1 seedlings. Poor seed set and lower haploid seed production were characteristics of multiple transgenic lines. The results presented in this study illustrate that further functional characterization of BBMs in zygote/embryo development is warranted.

Loss of WTAP Impairs Early Parthenogenetic Embryo Development

Simple SummaryWilms’ tumor 1-associating protein (WTAP) is a key subunit of the N6-methyl-adenosine (m6A) methyltransferase complex during porcine early embryo development. However, the role of WTAP in embryonic development is still unclear. In this study, we demonstrate that WTAP plays an indispensable role in embryonic development, and the loss of WTAP will promote the apoptosis of embryonic cells, and reduce the rate and quality of embryonic development.m6A is one of the most common and abundant modifications of RNA molecules present in eukaryotes. The methyltransferase complex, consisting of methyltransferase-like 3 (METTL3), METTL14, and WTAP, is responsible for the m6A modification of RNA. WTAP was identified as an mRNA splicing regulator. Its role as a regulatory subunit of the m6A methyltransferase complex in embryonic development remains largely unknown. To investigate the role of WTAP in porcine early embryonic development, si-WTAP was microinjected into porcine parthenogenetic zygotes. WTAP knockdown significantly reduced the blastocyst rate and global m6A levels, but did not affect the cleavage rate. Betaine was supplemented into the in vitro culture (IVC) to increase the m6A levels. Betaine significantly increased the global m6A levels but did not affect the blastocyst rate. Furthermore, the pluripotency genes, including OCT4, SOX2, and NANOG, were downregulated following WTAP knockdown. The apoptotic genes BAX and CASPASE 3 were upregulated, while the anti-apoptotic gene BCL2 was downregulated in WTAP knockdown blastocysts. TUNEL staining revealed that the number of apoptotic cells was significantly increased following WTAP knockdown. Our study indicated that WTAP has an indispensable role in porcine early embryonic development.

Mouse oocyte vitrification with and without dimethyl sulfoxide: influence on cryo-survival, development, and maternal imprinted gene expression.

Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr + DMSO), and vitrification without DMSO (Vitr - DMSO) on fresh/warmed oocyte survival, induced parthenogenetic activation, parthenogenetic embryo development, and embryonic maternal imprinted gene expression. In this prospective controlled laboratory study, mature B6C3F1 female mouse metaphase II oocytes were treated as: i) No-Vitr, ii) Vitr + DMSO/warmed, and iii) Vitr - DMSO/warmed with subsequent parthenogenetic activation and culture to the blastocyst stage. Oocyte cryo-survival, parthenogenetic activation and embryo development, parthenogenetic embryo maternal imprinted gene expression were outcome measures. Oocyte cryo-survival was significantly improved in Vitr + DMSO versus Vitr - DMSO at initial warming and 2h after warming. Induced parthenogenetic activation was similar between all three intervention groups. While early preimplantation parthenogenetic embryo development was similar between control, Vitr + DMSO, Vitr - DMSO oocytes, the development to blastocysts was significantly inferior in the Vitr - DMSO oocytes group compared to the control and Vitr + DMSO oocyte groups. Finally, maternal imprinted gene expression was similar between intervention groups at both the 2-cell and blastocyst parthenogenetic embryo stage. Inclusion of DMSO in oocyte vitrification solutions improved cryo-survival and developmental potential of parthenogenetic embryos to the blastocyst stage without significantly altering maternal imprinted gene expression.

In vitro maturation on ovarian granulosa cells encapsulated in agarose matrix improves developmental competence of porcine oocytes

In vivo, mammalian oocytes are surrounded by granulosa cells (GCs) that exist in a three-dimensional (3D) microenvironment with soft stiffness. The GCs play an important role for the in vivo growth and development of oocytes, through bidirectional communication between oocytes and GCs. To mimic the cellular microenvironment of a 3D organized follicle, this study designed a co-culture system using porcine ovarian GCs (pGCs) encapsulated in agarose matrix for in vitro maturation (IVM) of pig oocytes. We report the effects of our newly designed co-culture system on IVM and development of pig oocytes. Immature cumulus-oocyte-complexes (COCs) were matured on a 1% (w/v) agarose matrix encapsulated without or with pGCs. The number of pGCs within the agarose matrix was optimized by analyzing the in vitro development of parthenogenetic embryos. Moreover, the role of the ovarian stromal pGCs as feeder cells was assessed by analyzing the PA embryonic development. Subsequently, the effect of pGCs encapsulated in a 3D agarose matrix was evaluated for the developmental competence of pig oocytes by analyzing blastocyst formation after parthenogenetic activation (PA), intra-oocyte GSH and ROS contents, expression levels of BMP15 and BAX, TUNEL (terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling) assay, protein expression levels of BMP15, and intra-oocyte ATP levels. The optimized number of pGCs (5 × 104 cells/well) in a 3D agarose matrix led to a significantly higher blastocyst formation, increased BMP15 gene and protein expression, and intra-oocyte ATP levels; moreover, it induced significantly lower intra-oocyte ROS contents, pro-apoptotic BAX gene expression, and apoptotic index, compared to control. Our results demonstrate that application of pGCs as feeder cells encapsulated in the agarose matrix for IVM effectively increases the developmental competence of porcine oocytes.

Caffeine improves the developmental competence of parthenogenetic embryos derived from aging porcine oocytes

Oocytes are committed to deterioration in quality as they aged due to a long duration manipulation which leads to the reduced success rate of somatic cell nuclear transfer (SCNT). Caffeine with an effect to maintain the maturation-promoting factor (MPF) from the metaphase of oocytes is expected to enhance the quality of the aging oocytes. To investigate the timely treatment of caffeine to rescue aging oocytes, caffeine was supplemented after in vitro maturation (IVM) or during metaphase I – metaphase II (MI – MII) transition. First, the effect of caffeine after IVM of oocytes was examined. After IVM for 42 h, oocytes were left for aging within 6 or 8 hours in supplement with various concentrations of caffeine (0, 5 and 10 mM), and then, examined the quality of embryo from aged oocyte through parthenogenesis activation. We found that 5 mM caffeine for the first 6 hours of aging process was suggested to improve the early development of parthenogenetic diploid embryos. However, the cytoplasmic homogeneity is significantly reduced in aging oocyte compared to fresh oocyte and it could not be improved by caffeine treatment. Next, the effect of caffeine during MI – MII transition of oocyte was examined. Caffeine was supplemented during MI – MII transition (27 – 42 h) of IVM. Then mature oocytes were left for aging within 6 h to examine on aging porcine oocyte quality via parthenogenesis embryos. The results indicated that 5 mM caffeine during MI-MII transition could efficiently rescue aged oocytes and improve the development of embryos derived from aging oocytes to four-cell, eight-cell and blastocyst stage as compared to fresh oocytes. Especially, these aged oocytes treated by caffeine could improve the cytoplasmic homogeneity in embryos and the quality of blastocysts by increasing cell number similar to fresh oocytes.

CHIR99021 and rpIL6 promote porcine parthenogenetic embryo development and blastocyst quality

Signaling pathways and transcription factors are involved in porcine embryonic development. Here, we demonstrate that glycogen synthase kinase-3 (GSK3) inhibitor, CHIR99021 and recombinant porcine interleukin-6 (rpIL6) significantly promote porcine parthenogenetic blastocyst formation (49.23 ± 8.40% vs 32.34 ± 4.15%), with increased inner cell mass (ICM) cell numbers (7.72 ± 2.30 vs 4.28 ± 1.60) and higher expression of pluripotent genes, such as OCT4, SOX2 and NANOG. Furthermore, CHIR99021 and rpIL6 improve blastocyst quality with increased blastocyst hatching percentage (16.19 ± 1.96% vs 10.25 ± 1.12%) and subsequently porcine pluripotent stem cells (pPSCs) derivation efficiency. These results advance the understanding of porcine pre-implantation development and provide evidences in improving the blastocyst quality.

Fatty acid induced lipolysis influences embryo development, gene expression and lipid droplet formation in the porcine cumulus cells†.

The pig oocyte maturation protocol differs from other mammalian species due to dependence on follicular fluid (FF) supplementation. One of the most abundant components of the porcine follicular fluid are fatty acids (FAs). Although evidence from other mammalian models revealed a negative impact of saturated fatty acids (SFA) on developmental competence of oocytes, pig has not yet been widely analyzed. Therefore, we aimed to investigate whether supplementation of IVM medium with 150 μM of stearic acid (SA) and oleic acid (OA) affects lipid content and expression of genes related to fatty acid metabolism in porcine cumulus–oocyte complexes and parthenogenetic embryo development. We found significant influence of fatty acids on lipid metabolism in cumulus cells without affecting the oocyte proper. The expression of ACACA, SCD, PLIN2, FADS1, and FADS2 genes was upregulated (P < 0.01) in cumulus cells, while their expression in oocytes did not change. The increase in gene expression was more pronounced in the case of OA (e.g., up to 30-fold increase in PLIN2 transcript level compared to the control). The number of lipid droplets and occupied area increased significantly in the cumulus cells and did not change in oocytes after SA treatment. Oleic acid improved the blastocyst rate (48 vs 32% in control), whereas stearic acid did not affect this parameter (27%). Additionally, we have discovered a phenotypic diversity of LD in cumulus cells in response to FA supplementation, suggesting extensive lipolysis in response to SA. Stearic acid excess in maturation media led to the formation of multiple micro lipid droplets in cumulus cells.

Production and Characterization of Mouse Diploid Parthenogenetic Blastocyst Developed in Phosphate-Free Medium

Parthenogenesis is an artificial oocytes activation process without paternal contribution. Blastocyst, derived from parthenogenesis, is one of potential source for pluripotent stem cell propagation. Unfortunately, previous studies reported that parthenogenetic embryo did not achieve exhilarating blastocyst rate. One of the component that predicted inhibit parthenogenetic embryo development is phosphate. Therefore, we try to modify culture medium in order to overcome that problem. The aim of this research was to produce and analyze the characteristics of parthenogenetic blastocyst developed in phosphate-free medium. Mouse oocytes obtained from adult female DDY by superovulation. The activator was strontium chloride 10 mM and diploidization with cytochalasin B 5 μg/ml. Medium for activation and culture medium were modified rat 1 cell embryo medium (MR1ECM) which is phosphate free. The results showed that parthenotes that were cultured in phosphate free medium reached higher blastocyst rate compared to the other groups. The increase of phosphate in culture medium lead to impaired parthenogenetic embryos development. Further experiment was made to analyze the differences between fertilized and parthenogenetic embryo in this medium. The experiment showed that diploid parthenogenetic could achieve high blastocyst rate (30.9±1.3%). The quality of diploid parthenogenetic blastocyst, based on cells number, viability, and ICM ratio, was lower than fertilized blastocyst.

Effects of PXD101 and Embryo Aggregation on the In Vitro Development of Mouse Parthenogenetic Embryos.

To improve the isolation efficiency of parthenogenetic embryonic stem cells (pESCs) in mice, it is necessary to optimize the method to increase in vitro developmental competence of mice parthenogenetic blastocysts. Therefore, this study aims to investigate an optimal method for the production of mouse parthenogenetic blastocysts and isolation of pESC colonies by comparing the effects of two methods: (1) the treatment of histone deacetylase inhibitor PXD101 before, during, or after parthenogenetic activation; (2) parthenogenetic embryo aggregation; and (3) their combination treatment. The results suggest that application of PXD101 treatment and embryo aggregation could both improve the development of mouse parthenogenetic blastocysts (50 nM PXD101 treated 4 hours during activation and further 4 hours after activation: 40.0% vs. 20.0%; p < 0.05; two-cell embryo aggregation: 38.3% vs. 20.0%; p < 0.05) and also enhance the isolation rate of pESC colonies (PXD101: 33.3% vs. 11.8%; p < 0.05; two-cell embryo aggregation: 36.4% vs. 11.8%; p < 0.05). The combination of their treatments had the higher rate of parthenogenetic blastocyst development (41.7%) and significantly higher rate of pESC colony isolation from parthenogenetic blastocysts (45.0%); therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 8 hours and then aggregated at two-cell stage with 0.25% pronase for 10 minutes in our self-made concave) is considered the optimal way for the in vitro development of parthenogenetic blastocysts and subsequent pESC colony isolation in mice, opening new opportunities for application of this combination method to improve the parthenogenetic embryo development in other species.

Induction of autophagy promotes porcine parthenogenetic embryo development under low oxygen conditions.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.