Parthenogenetic Embryos Research Articles

The Influence of Oviductal and Uterine Fluid Supplementation on the In Vitro Development and Quality of Cloned Sheep Embryos.

Somatic cell nuclear transfer (SCNT) has great potential for the replication of high-commercial-value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate due to intrinsic factors of the technique itself, which leads to low rates of embryonic development and epigenetic alterations in cloned embryos. The objective of this study was to evaluate the effect of OF-UF on the intracellular concentrations of ROS and GSH and the development of cloned and parthenogenetic Ovis aries embryos. The results do not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF-UF. Furthermore, at 1% OF-UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos during the first divisions. Decreases in ROS and GSH levels were observed in the parthenogenetic blastocysts treated with 1% OF-UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS were observed. No effect of OF-UF was observed on embryonic development and redox balance in sheep embryos cloned via handmade cloning.

Developmental Competence of Somatic Cell Nuclear Transfer Embryos and Interspecies ICSI Zygotes From Bovine Small Antral Follicles.

Assisted reproductive technologies (ART) play a crucial role in conserving threatened wildlife species such as Bos gaurus. ART requires a large number of mature oocytes, and small antral follicles (SAFs) in the ovary are often used to obtain abundant sources of bovine oocytes. However, oocytes from SAFs often experience difficulty completing maturation and obtaining high quality and quantity of blastocyst formation compared to fully grown oocytes. This study aimed to increase the number of high-quality mature oocytes and improve their potential for ART applications in cloned and interspecies intracytoplasmic sperm injection (ICSI) embryos by utilising L-ascorbic acid (LAA) in pre invitro maturation (pre-IVM) culture. First, oocytes isolated from SAFs were cultured with the duration of pre-IVM 0, 6, 8, 10 h and different concentrations of LAA to determine good conditions for oocyte maturation. Then, mature oocytes were assessed for their developmental competence through parthenogenesis, cloned and interspecies ICSI embryos. The results showed that 8-h pre-IVM with 50 μg/mL LAA improved the maturation rate and developmental competence of parthenogenetic and clone embryos, especially, improving the high blastocyst quality by increasing cell number and expression of histone acetylation at lysine 9 (H3K9ac). In addition, the culture process improved the nuclear reprogramming of somatic cells after nuclear transfer into mature oocytes, resulting in an increased hatching rate of cloned embryos. It also enhanced the activation and the pronuclear formation rate of Gaurus-Taurus zygotes. Overall, the established pre-IVM culture method enhanced the meiotic and developmental competence of embryos. This procedure opened hope for the preservation of endangered species and other applications.

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.

Editorial Expression of Concern: Cell Lines Derived from Human Parthenogenetic Embryos Can Display Aberrant Centriole Distribution and Altered Expression Levels of Mitotic Spindle Check-point Transcripts.

Editorial Expression of Concern: Cell Lines Derived from Human Parthenogenetic Embryos Can Display Aberrant Centriole Distribution and Altered Expression Levels of Mitotic Spindle Check-point Transcripts.

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.

Behavior of Assembled Promyelocytic Leukemia Nuclear Bodies upon Asymmetric Division in Mouse Oocytes.

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are core-shell-type membrane-less organelles typically found in the nucleus of mammalian somatic cells but are absent in mouse oocytes. Here, we deliberately induced the assembly of PML-NBs by injecting mRNA encoding human PML protein (hPML VI -sfGFP) into oocytes and investigated their impact on fertilization in which oocyte/embryos undergo multiple types of stresses. Following nuclear membrane breakdown, preassembled hPML VI -sfGFP mRNA-derived PML-NBs (hmdPML-NBs) persisted in the cytoplasm of oocytes, forming less-soluble debris, particularly under stress. Parthenogenetic embryos that successfully formed pronuclei were capable of removing preassembled hmdPML-NBs from the cytoplasm while forming new hmdPML-NBs in the pronucleus. These observations highlight the beneficial aspect of the PML-NB-free nucleoplasmic environment and suggest that the ability to eliminate unnecessary materials in the cytoplasm of metaphase oocytes serves as a potential indicator of the oocyte quality.

Establishment of trophoblast cell line derived from buffalo (Bubalus bubalis) parthenogenetic embryo.

We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12-15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

P-587 Salidroside improves the reproductive capacity of aged mice via improving oocyte mitochondrial function

Abstract Study question The potential application value of salidroside as a reproductive anti-aging drug. Summary answer After old mice were intraperitoneally injected with salidroside, the reproductive function was significantly improved. This improvement is due to salidroside’s enhancement of mitochondrial function. What is known already The main cause of fecundity decline with maternal age is ovarian aging, which is characterized by decreases in follicle number and oocyte quality, However, there are currently no approved drugs for the treatment of ovarian aging. Salidroside has been regarded as an effective medicine to prevent and treat high-altitude illnesses. Emerging studies found that it also has a variety of pharmacological properties, such as anti-aging, antioxidation, anti-inflammatory and antivirus. In terms of anti-aging, salidroside supplements can extend the lifespan of fish Nothobranchius guentheri, reverse the premature senescence of human skin fibroblasts and ameliorate age-related disease. Study design, size, duration In this study, salidroside was used in two ways: First, we injected salidroside into naturally aging mice via intraperitoneal injection for 14 consecutive days and observed the fertility and ovarian reserve function of mice. Subsequently, we added salidroside to the oocyte IVM culture medium to observe changes in oocyte and mitochondrial function. Participants/materials, setting, methods Experiments were conducted in vivo and in vitro. In vivo, naturally, 10-month-old C57/B6 mice were used as treatment models. The mice were injected with 50 mg/kg salidroside every day for a total of 14 days. Then, the corresponding reproductive function. In vitro, We added 25uM salidroside to the IVM solution and conducted IVM experiments on 10-month-old GV oocytes. CGS and spindle staining observed the oocyte maturation. Main results and the role of chance We found that salidroside can increase the number of MII oocytes excreted in 10-month-old female mice, and at the same time, increase the number of pups per litter and the total number of pups. Follicle statistics showed that the addition of salidroside could increase the follicle reserve of 10-month-old female mice. Collection of GV stage oocytes. In the in vitro IVM treatment group, salidroside can delay oocyte GVBD. Improve the GVBD condition and polar body excretion of aged oocytes, At the same time, parthenogenetic activation showed that the salidroside treatment group could significantly improve the developmental ability of old parthenogenetic embryos. After 14 days of in vivo treatment, GV oocytes were collected to detect mitochondrial function. It was found that salidroside could increase the mitochondria distribution, enhance changes in mitochondrial membrane potential, and improve ROS scavenging ability. The same results were also obtained in the in vitro IVM treatment group. Limitations, reasons for caution This study found that salidroside treatment can improve ovarian aging, but the specific molecular mechanism is not clear, which is not conducive to the use of salidroside as a clinical medication. Wider implications of the findings In this study, we discovered the potential therapeutic effect of salidroside in ovarian aging and made a preliminary exploration of the therapeutic mechanism, which provides important insights into salidroside as a drug for ovarian aging. Trial registration number not applicable

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.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

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.

DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro.

Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.

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.

Allelic reprogramming of chromatin states in human early embryos.

The reprogramming of parental epigenomes in human early embryos remains elusive. To what extent the characteristics of parental epigenomes are conserved between humans and mice is currently unknown. Here, we mapped parental haploid epigenomes using human parthenogenetic and androgenetic embryos. Human embryos have a larger portion of genome with parentally specific epigenetic states than mouse embryos. The allelic patterns of epigenetic states for orthologous regions are not conserved between humans and mice. Nevertheless, it is conserved that maternal DNA methylation and paternal H3K27me3 are associated with the repression of two alleles in humans and mice. In addition, for DNA-methylation-dependent imprinting, we report 19 novel imprinted genes and their associated germline differentially methylated regions. Unlike in mice, H3K27me3-dependent imprinting is not observed in human early embryos. Collectively, allele-specific epigenomic reprogramming is different in humans and mice.

Oocyte activation is a cytoplasm-confined event so far: what about the nucleus?

The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.

127 Pregnancy improvement following transfer of parthenogenetic embryos after artificial insemination in dairy cows is dependent on interval from calving

127 Pregnancy improvement following transfer of parthenogenetic embryos after artificial insemination in dairy cows is dependent on interval from calving

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 Vibration and Tilting Mechanical Loading Device on Parthenogenetic Embryos Development of Mice Immature Oocytes.

The growth and development of early mammalian embryos mainly take place in the fallopian tube, which not only provides nutrients for embryonic growth and development but also offers suitable mechanical conditions. The embryo culture system established in assisted reproductive technology mainly simulates the environment in which oocytes and embryos grow and develop in vivo. However, current in vitro embryo culture is mainly static and cannot completely mimic the mechanical environment in which embryos grow and develop in vivo. Therefore, to more accurately simulate the mechanical environment of embryos in the fallopian tube, we have developed a dynamic culture device to investigate the effects of mechanical stimulation on the in vitro maturation of immature oocytes and their parthenogenetic developmental potential. Immature mice oocytes were subjected to in vitro maturation by static culture and vibration (3 Hz, 6 Hz) with tilting for 15∼16 hours. The maturation of oocytes was observed after the culture period. The mature oocytes were activated by parthenogenesis and the rate of embryo compaction and formation of parthenogenetic blastocysts was analyzed. The results showed that using 3 Hz vibration and tilting can significantly improve the parthenogenetic development potential of immature mice oocytes.

Two-headed parthenogenetic lizard embryo from southern Georgia

Abstract Visual inspection of several embryos of the parthenogenetic lizard Darevskia armeniaca revealed the presence of an embryo with axial bifurcation (“two-headed”) on the stage of pigmented eyes and plate-like limb buds with a distinct border at the edges. This is the third recorded case of axial bifurcation in D. armeniaca, although two previous cases were mentioned without further discussion. Here the bicephalic embryo is described in detail and the potential reasons are discussed. The analysis of the literature on axial bifurcation in lacertids suggests that this type of developmental disorder may be particularly common in this parthenogenetic form. Hybridization may cause multiple developmental disorders, including incomplete separation of twin embryos; all parthenogenetic Darevskia have a hybrid origin, and this may cause the disorders rather than parthenogenetic reproduction by itself. The hypothesis needs further study with more representative samples of parthenogenetic and sexually reproducing Darevskia.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.