Parthenogenetic Activation Research Articles

Delayed Blastocyst Formation Reduces the Quality and Hatching Ability of Porcine Parthenogenetic Blastocysts by Increasing DNA Damage, Decreasing Cell Proliferation, and Altering Transcription Factor Expression Patterns.

The purpose of this study was to investigate the influence of blastocyst formation timing on the quality of porcine embryos derived from parthenogenetic activation. Newly formed blastocysts at days 6, 7, and 8 of culture [termed formation 6, 7, and 8 blastocysts (F6, F7, and F8 blastocysts)] were obtained, and a series of parameters related to the quality of blastocysts, including apoptosis incidents, DNA replication, pluripotent factors, and blastocyst hatching capacity, were assessed. Delayed blastocyst formation (F7 and/or F8 blastocysts) led to increased levels of ROS, DNA damage, and apoptosis while decreasing the mitochondrial membrane potential, DNA replication, Oct4 levels, and numbers of Sox2-positive cells. F7 blastocysts showed a significantly reduced hatching rate compared to F6 blastocysts; however, F8 blastocysts were unable to develop to the hatching stage. Collectively, our findings suggest a negative correlation between delayed blastocyst formation and blastocyst quality.

Precise Insertion of AttB Sequences in Goat Genome Using Enhanced Prime Editor.

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3' terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.

Exogenous expression of PGC-1α during in vitro maturation impairs the developmental competence of porcine oocytes

Objectives of the current study were to examine the effects of exogenous expression of PGC-1α, which is a transcription factor responsive for controlling mitochondrial DNA (mtDNA) replication, mitochondria quantity control, mitochondrial biogenesis, and reactive oxygen species (ROS) maintenance, in porcine oocytes during in-vitro maturation (IVM) on the developmental competence, as well as mitochondrial quantity and function. Exogenous over-expression of PGC-1α by injection of the mRNA construct into oocytes 20 h after the start of IVM culture significantly increased the copy number of mtDNA in the oocytes, but reduced the incidences of oocytes matured to the metaphase-II stage after the IVM culture for totally 44 h and completely suppressed the early development in vitro to the blastocyst stage following parthenogenetic activation. The exogenous expression of PGC-1α also significantly induced spindle defects and chromosome misalignments. Furthermore, markedly higher ROS levels were observed in the PGC-1α-overexpressed mature oocytes, whereas mRNA level of SOD1, encoded for a ROS scavenging enzyme, was decreased. These results conclude that forced expression of PGC-1α successfully increase mtDNA copy number but led to increased ROS production, evidently by downregulation of SOD1 gene expression, inducement of spindle aberration/chromosomal misalignment, and consequently reduction in the meiotic and developmental competences of porcine oocytes.

P-191 Pronuclear transfer overcomes embryonic developmental arrest in Padi6-deficient mice

Abstract Study question Can pronuclear transfer (PNT) overcome embryonic developmental arrest (EDA) observed in the created Padi6-/- mouse model? Summary answer Applying PNT significantly improved the blastocyst rate of Padi6-/- zygotes, indicating its potential in overcoming female-related infertility. What is known already Various components of the subcortical maternal complex (SCMC), including peptidyl arginine deiminase 6 (PADI6), play a crucial role in early embryonic development and female fertility. Recent studies have revealed that mutations in PADI6 can result in EDA and thus female infertility, which is hard to overcome by current available treatment methods. The PNT technique, designed to replace compromised cytoplasm with a healthy counterpart, has been proposed to overcome specific female-related infertility indications. The Padi6-/- mouse displaying EDA, offers a valuable model to investigate the efficacy of PNT to overcome female-related infertility. Study design, size, duration From April 2022 to December 2023, an experimental study was conducted with CRISPR/Cas9 created Padi6 knockout mice. For fertility evaluation, Padi6-/- and Padi6+/- females were paired with wildtype males. Next, 30 wildtype and 25 Padi6-/- oocytes were collected for parthenogenetic activation. A total of 81, 54 and 145 zygotes were collected from wildtype, Padi6+/- and Padi6-/- females, respectively, for evaluating embryonic development and the effectiveness of PNT in overcoming EDA. Participants/materials, setting, methods The C57BL/6J Padi6-/- mouse model was established by introducing Padi6 c.1577_1578insT with CRISPR/Cas9. Female mice aged 6-12 weeks underwent ovarian hyperstimulation and mated with wildtype males. Oocytes were parthenogenetically activated with SrCl2 in presence of Cytochalasin D, and checked for 2-cell and blastocyst rates. PNT treatment was applied by transferring the 2PN from Padi6-/- to wildtype enucleated zygotes using electrofusion, and the efficiency was evaluated in terms of embryonic developmental potential. Main results and the role of chance After mating Padi6-/- female mice with wild-type males, no offspring could be obtained. In contrast, Padi6+/- females exhibited normal fertility, yielding an average of 7.3 offspring per litter. In addition, Padi6-/- males are also fertile. Twenty-eight wildtype oocytes (n = 30) survived parthenogenetic activation, with 85.71% (24/28) reaching the 2-cell stage, and 75% (18/24) subsequently forming blastocysts. In contrast, all Padi6-/- oocytes (n = 25) survived after activation, but only 20% (5/25) reached the 2-cell stage (P < 0.0001), with none progressing to blastocyst (P = 0.0039). Regarding zygote development, 82.14% (46/56) of wildtype zygotes and 98.14% (53/54) of Padi6+/-embryos reached the 2-cell stage, showing blastocyst rates of 95.65% (44/46) and 100% (53/53) respectively. For zygotes obtained from Padi6-/- females, 72.50% (87/120) of them progressed to the 2-cell stage (P = 0.1908, vs wildtype), but only 4.60% (4/87) reached blastocysts (P< 0.0001, vs wildtype), exhibiting the EDA phenotype. Following PNT to wildtype cytoplasm (n = 25), 23 reconstructed PNT zygotes were obtained. Of these, 91.30% (21/23) reached the 2-cell stage (P = 0.4924, vs wildtype), and 85,71% (18/21) developed to the blastocyst stage (P = 0.3151, vs wildtype), which is equivalent to the wildtype zygotes. Limitations, reasons for caution This study is limited by the small sample size of the PNT, which will still be extended. Next to the blastocyst rate, additional assessment methods such as multi-omics analyses will be employed to assess the efficiency and safety of PNT treatment. Wider implications of the findings This study provides further insight into the potential of the nuclear transfer technology for female-related infertility issues due to cytoplasmic incompetence. For couples experiencing recurrent EDA after ICSI due to inferior oocyte quality, PNT still offers them the possibility of having genetically related children. Trial registration number Not applicable

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

O-066 Parental inheritance of monopronuclear blastocysts from ICSI cycles and its association with specific morphokinetic traits: a retrospective observational study

Abstract Study question Are monopronuclear-originated blastocysts from ICSI prone to have uniparental genome and does it correlate with specific morphokinetic parameters? Summary answer Extended compaction was notably observed in parthenogenetic monopronuclear blastocysts, suggesting a potential correlation between morphokinetic traits and parental origin. What is known already Previous studies indicated monopronuclear embryos with larger pronucleus exhibit a propensity to develop into blastocysts as normal fertilized embryos, which was considered as evidence supporting the presence of a diploid genome enclosed within a singular pronucleus.However, diploid blastocysts may also arise from haploid spontaneous diploidization following parthenogenetic or androgenetic activation, particularly in ICSI-derived blastocysts. Although certain characteristics were discerned in prior morphokinetic studies on monopronuclear blastocysts, these embryos were not proceeded to comprehensive chromosomal analysis to authenticate the association with parental inheritance, which requires further clarification and investigation. Study design, size, duration This retrospective cohort study, conducted in a university-affiliated reproductive medical center, focused on ICSI-derived monopronuclear blastocysts (n = 151) obtained from 145 PGT cycles spanning January 2013 to December 2022. Among these, nineteen were subject to morphokinetic analysis within a time-lapse system, while 35 bipronuclear-originated sibling blastocysts from the same cycles comprised the control group. Notably, the morphokinetic analysis incorporated 10 uniparental monopronuclear blastocysts, with 7 originating from parthenogenesis and the remaining from androgenesis. Participants/materials, setting, methods The data was collected from patients who had at least one biopsied ICSI-derived monopronuclear blastocyst between 2013 and 2022 at the reproductive center. The genetic analysis of blastocysts were performed using SNP array or NGS. Morphokinetic recordings were obtained from blastocysts cultured in time-lapse system along with euploid bipronuclear sibling blastocysts in the same cycles. All annotations underwent manual scrutiny and standardization by the same embryologist to ensure consistency. Main results and the role of chance This study explored links between genetic origin and morphokinetics in ICSI-derived monopronuclear blastocyst through combine analysis. Among 151 blastocysts, 53 (35.1%) were biparental euploidies, while 20% demonstrated uniparental patterns, with 18 confirmed as parthenogenetic and 7 as androgenetic. The ratio of parthenogenesis to androgenesis was 2.6:1, aligning with reported figures. Pronuclear metrics revealed that pronuclei area of monopronuclear blastocysts were notably larger (91∼171 μm2) than that of maternal-origin pronuclei but equivalent to paternal-origin pronuclei from bipronuclear blastocysts.Kinetic analysis indicated that pronuclei of monopronuclear blastocysts vanished 1.9 hours earlier than those of bipronuclear blastocysts, and furtherly biparental monopronuclear blastocysts had a 2.74-hour shorter visible duration compared to bipronuclear blastocysts, whereas uniparental monopronulcei showed no significant difference. Following pronucleus disappearance, the compaction period of monopronuclear blastocysts lasted 2.85 hours longer than bipronuclear blastocysts. Notably, in parthenogenetic uniparental monopronuclear blastocysts, this gap expanded to 10.92 hours. Additionally, there was a significantly higher proportion of asymmetrical halo and dysmorphism in monopronuclear blastocysts (78.95%, 63.16%) compared to bipronuclear blastocysts (40.00%, 34.29%), but neither differences was related to parental origins. The study corroborated a high incidence of uniparental diploidy and identified the discrepancies in pronuclear parameters and compaction before blastulation of monopronuclear blastocysts with different parental origins. Limitations, reasons for caution The scarcity of ICSI-derived monopronuclear blastocysts undergoing PGT and time-lapse culture posed challenges in discerning additional morphokinetic distinctions related to parental inheritance and conducting in-depth analyses for androgenetic blastocysts. It is noted that parental ploidy results from NGS were deduced from targeted chromosome, which may not represent the whole genome. Wider implications of the findings Our results confirmed the high risk of uniparental diploidy in ICSI-derived monopronuclear blastocysts, suggesting the necessity of comprehensive screening. We pinpointed morphokinetic features in monopronuclear embryos associated with parental origin. While it remains unreliable to gauge parental compositions through morphokinetics, these distinctions could serve as supplementary indicators evaluating monopronuclear embryos. Trial registration number not applicable

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

P-260 In Vitro Fertilization (IVF) using magnetotactic sperm cells and their prospects for assisted in vivo reproduction

Abstract Study question Are bovine Magnetotactic Sperm Cells (MSC) able to fertilize an oocyte in-vitro and Which would be the main requirements for translating this technology to in-vivo? Summary answer IVF using highly purified MSC result in embryo development until blastocyst stage. We demonstrate different alternatives to control multiple MSC, individually and as a swarm. What is known already Medical microrobots have appeared as the new generation of drug delivery vehicles. They are tiny devices in the order of a few micrometers, able to move in biological environments by different means, physical, chemical, or biohybrid. They are functionalized to deliver targeted therapies for drugs, cells, or other payloads. In assisted reproduction, sperm-hybrid microcarriers have been pioneered by our group, and various in vitro demonstrations have been done to assist sperm cells with motion deficiencies or low count and to deliver anticancer drugs for treating, for example, cervical and ovarian cancer, but so far in-vitro fertilization has never been shown. Study design, size, duration Experimental quantitative study design. A total of 520 bovine cumulus-oocyte complexes were used for in vitro fertilization. The analyzed sample groups were using bovine frozen sperm cells, MSC, or by evaluating parthenogenetic activation. A power of 0.98 with an a-error of 0.05 was used for the IVF experiments. Sperm samples in the control as well as the micromotor groups have been analyzed for different sperm parameters. Participants/materials, setting, methods Bovine MSC are created by thawing frozen sperm cells and adding polystyrene microparticles. Different sperm parameters were evaluated to check the effects of the particles in the cells. After developing a purification method for the MSC, bovine ovaries were collected from a slaughterhouse nearby and, after in vitro maturation, were used for the IVF experiments. Ultrasound, and photoacoustic imaging setups with a set of electromagnets were employed for real-time manipulation and visualization of MSC. Main results and the role of chance Sperm viability was significantly higher in the MSC group compared to the control group 6h after formation (72.3 ± 5.0% vs 35.0 ± 4.1%), and after 24 hours, both viabilities decreased drastically to 16.6 ± 3.9% and 7.7 ± 1.7% respectively, probably a possible local antioxidant effect by the microparticles through the formation of a protein corona. In the case of motility, the MSC group presented significantly higher motility up to 6h (64.0 ± 1.5%), and in all the measured times, the motility of the MSC group was higher than the control group. Purification of the micromotor sample was performed, but an average of 63 free sperm was still on the MSC sample. IVF experiments were performed, and blastocyst development was observed in IVF control (2x106 sperm/ml), medium concentration (same concentration as MSC group), and the MSC groups (275.000 sperm/ml). No blastocyst formation was observed in the parthenogenetic, parthenogenetic + particles, and low-concentration groups (number of free sperm). The MSC group was significantly different in cleavage and blastocyst rates compared to the low concentration groups, with p-values of 0.04552 and 0.005865, respectively, which suggest that fertilization may have occurred by the micromotors instead by the free sperm in the sample. Limitations, reasons for caution More studies of these MSC in the different gynecological tissues and on embryo development are needed to ensure the safety of future human translation. In addition, developing a detection system for micromotors in deep tissue is needed to perform in vivo experiments. Wider implications of the findings MSC show an alternative to efficiently guide efficiently sperm cells in different biological environments and can be a promising alternative method for assisted fertilization, and the treatment of a variety of gynecological diseases. They can also be used as exploratory tools to learn more about sperm migration in-vivo, among others Trial registration number European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program No. 853609

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.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus–oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Acetic acid affects porcine oocyte metabolism and improves oocyte developmental ability

Improvement in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism.

Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

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.

Agarose-based 3D culture improved the developmental competence of oocyte-granulosa complex isolated from porcine preantral follicle

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established.The present study indicated that the use of a modified medium with Ascorbic Acid (50 μM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3–0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac–H3-K9) and methylation of histone H3 at lysine 4 (Me–H3–K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.

C-phycocyanin improves the developmental potential of cryopreserved human oocytes by minimizing ROS production and cell apoptosis.

The cryopreservation process damages oocytes and impairs development potential. As a potent antioxidant, C-phycocyanin (PC) regulates reproductive performance. However, its beneficial effects on vitrified human oocytes remain unknown. In this study, human GV-stage oocytes obtained from controlled ovarian hyperstimulation (COH) cycles were randomly allocated to three groups: fresh oocyte without freezing (F group), vitrification in medium supplemented with PC (P group), and vitrification in medium without PC as control group (C group). After warming, viable oocytes underwent in vitro maturation. Our results showed that 3 μg/mL PC treatment increased the oocyte maturation rate after cryopreservation. We also found that PC treatment maintains the regular morphological features of oocytes. After PC treatment, confocal fluorescence staining showed a significant increase in the mitochondrial membrane potential of the vitrified oocytes, along with a notable decrease in intracellular reactive oxygen species and the early apoptosis rate. Finally, after in vitro maturation and parthenogenetic activation, vitrified oocytes had a higher potential for cleavage and blastocyst formation after PC treatment. Our results suggest that PC improves the developmental potential of cryopreserved human GV-stage oocytes by attenuating oxidative stress and early apoptosis and increasing the mitochondrial membrane potential.

Elevated chorionic gonadotropic hormone in transgenic mice induces parthenogenetic activation and ovarian teratomas

Both male and female reproductive functions are impacted by altered gonadotrophin secretion and action, which may also influence the development of endocrine tumours. To ascertain if chronic hypersecretion of human chorionic gonadotropin (hCG) contributes to the development of gonadal tumours, double transgenic (TG) mice that overexpress hCGα- and β-subunits were analysed. By the age of two months, ovarian tumours with characteristics of teratomas developed with 100% penetrance. Teratomas were also seen in wild-type ovaries orthotopically transplanted into TG mice, demonstrating an endocrine/paracrine mechanism for the hCG-induced ovarian tumorigenesis. Both in vitro and in vivo experiments showed oocyte parthenogenetic activation in TG females. In addition, ovaries showed reduced ovulatory gene expression, inhibited ERK1/2 phosphorylation, and impaired cumulus cell expansion. Hence, persistently high endocrine hCG activity causes parthenogenetic activation and development of ovarian teratomas, along with altered follicle development and impaired ERK1/2 signalling, offering a novel mechanism associated with the molecular pathogenesis of ovarian teratomas.

A rare germline BMP15 missense mutation causes hereditary ovarian immature teratoma in human

Ovarian immature teratomas (OITs) are malignant tumors originating from the ovarian germ cells that mainly occur during the first 30 y of a female's life. Early age of onset strongly suggests the presence of susceptibility gene mutations for the disease yet to be discovered. Whole exon sequencing was used to screen pathogenic mutations from pedigrees with OITs. A rare missense germline mutation (C262T) in the first exon of the BMP15 gene was identified. In silico calculation suggested that the mutation could impair the formation of mature peptides. In vitro experiments on cell lines confirmed that the mutation caused an 84.7% reduction in the secretion of mature BMP15. Clinical samples from OIT patients also showed a similar pattern of decrease in the BMP15 expression. In the transgenic mouse model, the spontaneous parthenogenetic activation significantly increased in oocytes carrying the T allele. Remarkably, a mouse carrying the T allele developed the phenotype of OIT. Oocyte-specific RNA sequencing revealed that abnormal activation of the H-Ras/MAPK pathway might contribute to the development of OIT. BMP15 was identified as a pathogenic gene for OIT which improved our understanding of the etiology of OIT and provided a potential biomarker for genetic screening of this disorder.

Improving the meiotic competence of small antral follicle-derived porcine oocytes by using dibutyryl-cAMP and melatonin.

We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts. The study consisted of three experiments. The first one examined oocyte electroporation with and without subsequent IVF and found that electroporated oocytes had higher activation rates, increased occurrence of a single pronucleus, and no effect on sperm penetration. Cleavage rates improved in electroporated oocytes, but blastocyst rates remained constant. Genotype analysis revealed a significant increase in the proportion of parthenotes in the electroporated groups compared to the IVF control (30.2 % vs. 6.8 %). The second experiment compared two electroporation media, Opti-MEM and Nuclease-Free Duplex Buffer (DB). DB induced higher oocyte degeneration rates, and lower cleavage and blastocyst rates than Opti-MEM, while parthenogenetic formation remained consistent (60.0 and 48.5 %). In the third experiment, the timing of electroporation relative to IVF was evaluated (1 h before IVF, immediately before IVF and 7 h after IVF). Electroporation immediately before IVF resulted in higher activation rates and different pronuclear proportions compared to the other timing groups. The penetration rate was higher in the immediate electroporation group, and cleavage rate improved in all electroporated groups compared to the control. Blastocyst rates remained constant. Genotyping revealed no significant differences in parthenote proportions among the timing groups, but these were higher than the control (56.25 %, 63.89 %, 51.61 %, 2.44 %, respectively), and showed higher mutation rates when electroporation was performed 7 h after IVF. Overall, this comprehensive study sheds light on the potential of electroporation for creating genetically modified embryos and the importance of media selection and timing in the process, the best media being the Opti-MEM and the more efficient timing regarding mutation rate, 7 h post-IVF, even when the parthenote formation did not differ among electroporated groups. Further studies are needed to reduce the parthenogenetic activation while maintaining high mutation rates to optimize the use of this procedure for the generation of gene-edited pig embryos by oocyte/zygote electroporation.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.