Embryonic Developmental Competence Research Articles

Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility.

Fertility is crucial for enhancing the efficiency of livestock production, as it directly impacts the reproductive rates. A comprehensive understanding of the relationship between sperm quality and embryo development is key to optimizing reproductive outcomes and improving the quality of livestock. This study analyzed the developmental competence of in vitro embryos recovered from Bali cattle with normal or poor sperm motility. Nine bulls with normal fresh semen (NFS) or poor fresh semen (PFS) motility were ejaculated for semen. Semen ejaculates, including volume, motility, and sperm concentration, were evaluated immediately after collection to measure the quality of the fresh semen. Frozen semen was evaluated using computer-assisted semen analysis (CASA) for motility, progressive sperm motility, distance curve path, distance curve linear, distance straight line, average path velocity, curvilinear velocity, linear velocity, straightness (STR), linearity of forward progression (LIN), wobble, and average lateral head displacement (ALH). Bull groups were used to determine in vitro embryo cleavage ability after fertilization of Bali cattle. Ovaries of Bali cattle were collected by slicing, and only cytoplasmic oocytes with compact cumulus cells were used in this study. The oocytes were matured, and in vitro fertilization was performed using fertilization media with a final sperm concentration of 1.5 × 106 spermatozoa/mL. After 48 h, the embryo cleavage ability of the cultured oocytes was evaluated. There were significant differences in motility values between the NFS and PFS groups; however, there were no significant differences in the volume or sperm concentration. There was a significant difference in the LIN value between the groups but no significant differences in other CASA parameters. There were no significant differences in the cleavage rate and morula between the groups, but a positive correlation was observed between the cleavage rate and the morula and between the morula and ALH. A significant negative correlation was observed between the cleavage rate and STR and between the morula and STR; no significant differences were observed for other variables. Despite variations in sperm characteristics, both normal and poor sperm motility demonstrated comparable in vitro embryonic development competence. These findings provide important insights into the fertility potential of Bali bulls, providing valuable information that can enhance selection strategies to improve the quality of livestock production.

MiR-124-3p negatively impacts embryo implantation via suppressing uterine receptivity formation and embryo development

During embryo implantation, blastocyst interacts with the receptivity endometrium and the endometrial epithelium secretes nurturing fluid to support embryonic development. Interferon-λ (IFN-λ) is a novel, non-redundant regulator that participates in the fetal–maternal interaction; however, the precise molecular mechanism underlying its impact on uterine receptivity remains elusive. Here, microarray profiling revealed that 149 specific miRNAs were differentially expressed in the human endometrial cells following IFN-λ treatment. In particular, miR-124-3p expression was significantly reduced after IFN-λ treatment (p < 0.05). An in vivo mouse pregnancy model showed that miR-124-3p overexpression notably decreased embryo implantation rate and led to an aberrant epithelial phenotype. Furthermore, miR-124-3p negatively impacted the migration and proliferation of endometrial cells, and hindered embryonic developmental competence in terms of blastocyst formation and global DNA re-methylation. Downstream analysis showed that LIF, MUC1 and BCL2 are potential target genes for miR-124-3p, which was confirmed using western blotting and immunofluorescence assays. In conclusion, IFN-λ-driven downregulation of miR-124-3p during embryo implantation modulates uterine receptivity. The dual functional role of miR-124-3p suggests a cross-talk model wherein, maternal endometrial miRNA acts as a transcriptomic modifier of the peri-implantation endometrium and embryo development.

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

High Temperature-Induced m6A Epigenetic Changes Affect Early Porcine Embryonic Developmental Competence in Pigs.

N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10 h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.

Ochratoxin A triggers endoplasmic reticulum stress through PERK/NRF2 signaling and DNA damage during early embryonic developmental competence in pigs

Ochratoxin A (OTA), a mycotoxin found in foods, has a deleterious effect on female reproduction owing to its endocrine-disrupting activity mediated through endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the mechanisms of OTA-induced ER stress in pig embryos during in vitro culture (IVC) are not yet fully understood. In the present study, porcine embryos were cultured for two days in an IVC medium supplemented with 0.5, 1.0, and 5.0 μM OTA, which led to an OTA-induced reduction in the developmental rate of blastocysts. The mRNA-seq transcriptome analysis revealed that the reduced blastocyst development ability of OTA-exposed porcine embryos was caused by ER stress, ultimately resulting in the accumulation of ROS and the occurrence of apoptosis. The expression levels of some UPR/PERK signaling-related genes (DDIT3, EIF2AK3, EIF2S1, NFE2L2, ATF4, EIF2A, and KEAP1) were found to differ in OTA-exposed pig embryos. OTA induces DNA damage by triggering an increase in RAD51/γ-H2AX levels and suppressing p-NRF2 activity. This effect is mediated through intracellular ROS and superoxide accumulation in the nuclei of porcine embryos. The cytotoxicity of OTA increased the activation of the PERK signal pathways (p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP) in porcine embryos, with abnormal distribution of the ER observed around the nucleus. Collectively, our findings indicate that ER stress is a major cause of decline in the development of porcine embryos exposed to OTA. Therefore, OTA exposure induces ER stress and DNA damage via oxidative stress by disrupting PERK/NRF2 signaling activity in the developmental competence of porcine embryos during IVC.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Cordycepin delays postovulatory aging of oocytes through inhibition of maternal mRNAs degradation via DCP1A polyadenylation suppression

Postovulatory aging leads to the decline in oocyte quality and subsequent impairment of embryonic development, thereby reducing the success rate of assisted reproductive technology (ART). Potential preventative strategies preventing oocytes from aging and the associated underlying mechanisms warrant investigation. In this study, we identified that cordycepin, a natural nucleoside analogue, promoted the quality of oocytes aging in vitro, as indicated by reduced oocyte fragmentation, improved spindle/chromosomes morphology and mitochondrial function, as well as increased embryonic developmental competence. Proteomic and RNA sequencing analyses revealed that cordycepin inhibited the degradation of several crucial maternal proteins and mRNAs caused by aging. Strikingly, cordycepin was found to suppress the elevation of DCP1A protein by inhibiting polyadenylation during postovulatory aging, consequently impeding the decapping of maternal mRNAs. In humans, the increased degradation of DCP1A and total mRNA during postovulatory aging was also inhibited by cordycepin. Collectively, our findings demonstrate that cordycepin prevents postovulatory aging of mammalian oocytes by inhibition of maternal mRNAs degradation via suppressing polyadenylation of DCP1A mRNA, thereby promoting oocyte developmental competence.

Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence.

Acquisition of germinal vesicle (GV) stage oocytes for fertility preservation (FP) offers several benefits over in vivo matured oocyte cryopreservation following ovarian stimulation, particularly for cancer patients necessitating immediate treatment. Two FP approaches for GV oocytes are available: vitrification before in vitro maturation (IVM) at the GV stage (GV-VI) or post-IVM at the metaphase II (MII) stage (MII-VI). The optimal method remains to be determined. In this study, mouse oocytes were collected without hormonal stimulation and vitrified either at the GV stage or the MII stage following IVM; non-vitrified in vitro matured MII oocytes served as the control (CON). The oocyte quality and developmental competence were assessed to obtain a better method for immediate FP. No significant differences in IVM and survival rates were observed among the three groups. Nevertheless, GV-VI oocytes exhibited inferior quality, including abnormal spindle arrangement, mitochondrial dysfunction, and early apoptosis, compared to MII-VI and CON oocytes. Oocyte vitrification at the GV stage impacted maternal mRNA degradation during IVM. In addition, the GV-VI group demonstrated significantly lower embryonic developmental competence relative to the MII-VI group. RNA sequencing of 2-cell stage embryos revealed abnormal minor zygotic genome activation in the GV-VI group. Vitrification at the GV stage compromised oocyte quality and reduced developmental competence. Consequently, compared to the GV stage, oocyte vitrification at the MII stage after IVM is more suitable for patients who require immediate FP.

P-191 The impact of resveratrol supplementation as antioxidant on embryonic development in older women (over 40years) undergoing IVF

Abstract Study question Does the resveratrol supplementation in human embryo culture medium improve embryo quality and pregnancy outcomes in older women? Summary answer Resveratrol significantly improved good quality embryo rates. These results are thought to be due to the biological activities of resveratrol as an antioxidant. What is known already Reproductive aging involves age-related declines in ovarian function and development capacity. After initiating meiosis, germ cells arrest at the dictyate stage of prophase I in the ovary for 10-50 years in women. Accumulated reactive oxygen species (ROS) affect oocytes adversely. Antioxidants reduce ROS-induced damage to oocytes and resveratrol is an important antioxidant polyphenolic compound. Resveratrol has positive effects on fertility, it improves the development of blastocysts in aged animal and it could enhance the maturation and quality of oocytes from aged women. However, there are just a few studies there are clarified relationship between resveratrol and embryos in older women. Study design, size, duration The study retrospectively included 966 patients with aged over 40 years to evaluate the impact of resveratrol on embryo quality and IVF pregnancy outcomes between January 2019 and December 2021. Participants/materials, setting, methods The oocytes from aged female (over 40years) were divided into 2 groups, fertilized and cultured in MRC media (MRC#D13 (day 1 ∼ 3) and MRC#D46 (day 4 ∼ 6 culture medium); Maria Medical Foundation, South Korea) in the presence or absence of resveratrol (0.5 μM; Sigma-Aldrich, USA). Fertilization, good quality embryo, clinical pregnancy, and implantation rate after embryo transfer were compared among between the two groups. Data analyzed using the SPSS and T-test p value &amp;lt;0.05 considered statistically significant. Main results and the role of chance There was no significant difference in female age (42.5 ± 2.2 vs. 42.3 ± 2.0, P = 0.358), No. of previous ART cycles (3.6 ± 2.3 vs. 3.7 ± 2.5, P = 0.680), thickness of endometrium (9.2 ± 1.5 vs. 9.4 ± 1.4, P = 0.312), maturation rate per oocytes (74.6% vs. 75.8%, P = 0.290), fertilization rate (81.5% vs. 80.0%, P = 0.165), and No. of transferred embryos (2.4 ± 0.6 vs. 2.4 ± 0.6, P = 0.818). However, good quality embryo rate (44.0% vs. 36.4%, p &amp;lt; 0.05) in the resveratrol group was 7.6% higher than the control group. Pregnancy rate (26.8% vs. 22.7%, P = 0.167) and implantation rate (11.9% vs. 9.9%, P = 0.131) were slightly higher than control group but this was not statistically significant. Limitations, reasons for caution This is a retrospective study that generates strong conclusions, but it could be interesting to test it in a prospective study. The study was conducted at a single IVF center. Embryo transfer result is based on implantation rate, while the live birth data for all pregnancies are not yet available. Wider implications of the findings This is the first study to investigate the effects of resveratrol on the embryonic developmental competence and pregnancy potential of older women (over 40years). Trial registration number not applicable

P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes

Abstract Study question Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice? Summary answer Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes. What is known already In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts. Study design, size, duration The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer. Participants/materials, setting, methods Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls. Main results and the role of chance In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p &amp;lt; 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p &amp;lt; 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p &amp;lt; 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p &amp;lt; 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p &amp;lt; 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p &amp;lt; 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p &amp;lt; 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing. Limitations, reasons for caution Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique. Wider implications of the findings Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human. Trial registration number Not applicable

Testis-specific knockout of Kdm2a reveals nonessential roles in male fertility but partially compromises spermatogenesis

Lysine-specific histone demethylase 2 (Kdm2a) is a regulatory factor of histone modifications that participates in gametogenesis and embryonic development. The mis-regulation of Kdm2a can lead to aberrant gene expression, thereby contributing to abnormal cell proliferation, differentiation, apoptosis, and tumorigenesis. However, due to the potential confounding effects that are secondary to the loss of Kdm2a function from the soma in existing whole-animal mutants, the in vivo function of Kdm2a in spermatogenesis for male fertility remains unknown. Herein, we focus on exploring the spatiotemporal expression profile and biological functions of Kdm2a in the spermatogenesis and fertility of male mice. A testis-specific knockout Kdm2a model (Kdm2a cKO) was established by using the Stra8-Cre/loxP recombinase system to explore the roles of Kdm2a in male fertility. Our results showed that Kdm2a was ubiquitously expressed and dynamically distributed in multiple tissues and cell types in the testis of mice. Surprisingly, Kdm2a-deficient adult males were completely fertile and comparable with their control (Kdm2aflox/flox) counterparts. Despite the significantly reduced total number of sperm and density of seminiferous tubules in Kdm2a cKO testis accompanied by the degeneration of spermatogenesis, the fertilization ability and embryonic developmental competence of the Kdm2a cKO were comparable with those of their control littermates, suggesting that Kdm2a disruption did not markedly affect male fertility, at least during younger ages. Furthermore, Kdm2a homozygous mutants exhibited a lower total number and motility of sperm than the control group and showed notably affected serum 17β-estradiol concentration. Interestingly, the transcriptome sequencing revealed that the loss of Kdm2a remarkably upregulated the expression level of Kdm2b. This effect, in turn, may induce compensative effects in the case of Kdm2a deficiency to maintain normal male reproduction. Together, our results reveal that Kdm2a shows spatiotemporal expression during testicular development and that its loss is insufficient to compromise the production of spermatozoa completely. The homologous Kdm2b gene might compensate for the loss of Kdm2a. Our work provides a novel Kdm2a cKO mouse allowing for the efficient deletion of Kdm2a in a testis-specific manner, and further investigated the biological function of Kdm2a and the compensatory effects of Kdm2b. Our study will advance our understanding of underlying mechanisms in spermatogenesis and male fertility.

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

Near-infrared fluorophore IR-61 improves the quality of oocytes in aged mice via mitochondrial protection

Maternal aging is associated with a decline in oocyte quality, which leads to the decreased fertility. Therefore, developing approaches to reduce aging-induced deterioration of oocyte quality in older women is important. Near-infrared cell protector-61 (IR-61), a novel heptamethine cyanine dye, has the potential for antioxidant effects. In this study, we found that IR-61 can accumulate in the ovaries and improved ovarian function of naturally aged mice; it also increased the oocyte maturation rate and quality by maintaining the integrity of the spindle/chromosomal structure and reducing the aneuploidy rate. In addition, the embryonic developmental competence of aged oocytes was improved. Finally, RNA-sequencing analysis indicated that IR-61 might perform the beneficial effects on aged oocytes by regulating mitochondrial function, this was confirmed by immunofluorescence analysis of mitochondrial distribution and reactive oxygen species. Taken together, our findings demonstrate that IR-61 supplementation in vivo can increase oocyte quality and protect oocytes from aging-induced mitochondrial dysfunction, and thus could improve the fertility of older women and efficiency of assisted reproductive technology.

Rapamycin encourages the maintenance of mitochondrial dynamic balance and mitophagy activity for improving developmental competence of blastocysts in porcine embryos in vitro.

Rapamycin induces autophagosome formation and activity during oocyte maturation, improved fertilization ability of matured oocytes, and early embryonic developmental competence. However, potential changes in mitochondrial fission and mitophagy via regulation of autophagy in early porcine embryonic development have not been previously studied. Here, we investigated embryonic developmental ability and quality of porcine embryos 2 days after in vitro fertilization and following treatment with 1 and 10 nM rapamycin. As a results, 1 nM rapamycin exposure significantly improved (p < 0.05) blastocyst developmental competence compared to that in nontreated embryos (nontreated: 26.2 ± 5.7% vs. 1 nM rapamycin: 35.3 ± 5.1%). We observed autophagic (LC3B) and mitochondrial fission protein expression (dynamin-related protein-1 [DRP1] and pDRP1-Ser616) at the cleavage stage of 1 and 10 nM rapamycin-treated porcine embryos, using Western blot and immunofluorescence analyses. Interestingly, 1 nM rapamycin treatment significantly improved autophagy formation, mitochondrial activation, and mitochondrial fission protein levels (p < 0.05; p-DRP1 [Ser616]) at the cleavage stage of porcine embryos. Additionally, mitophagy was significantly increased in blastocysts treated with 1 nM rapamycin. In conclusion, our results suggest that rapamycin promotes blastocyst development ability in porcine embryos through mitochondrial fission, activation, and mitophagy in in vitro culture.

Selective Translation of Maternal mRNA by eIF4E1B Controls Oocyte to Embryo Transition.

Maternal messenger ribonucleic acids (mRNAs) are driven by a highly orchestrated scheme of recruitment to polysomes and translational activation. However, selecting and regulating individual mRNAs for the translation from a competitive pool of mRNAs are little-known processes. This research shows that the maternal eukaryotic translation initiation factor 4e1b (Eif4e1b) expresses during the oocyte-to-embryo transition (OET), and maternal deletion of Eif4e1b leads to multiple defects concerning oogenesis and embryonic developmental competence during OET. The linear amplification of complementary deoxyribonucleic acid (cDNA) ends, and sequencing (LACE-seq) is used to identify the distinct subset of mRNA and its CG-rich binding sites within the 5' untranslated region (UTR) targeted by eIF4E1B. The proteomics analyses indicate that eIF4E1B-specific bound genes show stronger downregulation at the protein level, which further verify a group of proteins that plays a crucial role in oocyte maturation and embryonic developmental competence is insufficiently synthesized in Eif4e1b-cKO oocytes during OET. Moreover, the biochemical results in vitro are combined to further confirm the maternal-specific translation activation model assembled by eIF4E1B and 3'UTR-associated mRNA binding proteins. The findings demonstrate the indispensability of eIF4E1B for selective translation activation in mammalian oocytes and provide a potential network regulated by eIF4E1B in OET.

Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes.

Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.

Glycine regulates lipid peroxidation promoting porcine oocyte maturation and early embryonic development.

In vitro-cultured oocytes are separated from the follicular micro-environment in vivo and are more vulnerable than in vivo oocytes to changes in the external environment. This vulnerability disrupts the homeostasis of the intracellular environment, affecting oocyte meiotic completion, and subsequent embryonic developmental competence in vitro. Glycine, one of the main components of glutathione (GSH), plays an important role in the protection of porcine oocytes in vitro. However, the protective mechanism of glycine needs to be further clarified. Our results showed that glycine supplementation promoted cumulus cell expansion and oocyte maturation. Detection of oocyte development ability showed that glycine significantly increased the cleavage rate and blastocyst rate during in vitro fertilization (IVF). SMART-seq revealed that this effect was related to glycine-mediated regulation of cell membrane structure and function. Exogenous addition of glycine significantly increased the levels of the anti-oxidant GSH and the expression of anti-oxidant-related genes (glutathione peroxidase 4 [GPX4], catalase [CAT], superoxide dismutase 1 [SOD1], superoxide dismutase 2 [SOD2], and mitochondrial solute carrier family 25, member 39 [SLC25A39]), decreased the lipid peroxidation caused by reactive oxygen species (ROS) and reduced the level of malondialdehyde (MDA) by enhancing the functions of mitochondria, peroxisomes and lipid droplets (LDs) and the levels of lipid metabolism-related factors (peroxisome proliferator activated receptor coactivator 1 alpha [PGC-1α], peroxisome proliferator-activated receptor γ [PPARγ], sterol regulatory element binding factor 1 [SREBF1], autocrine motility factor receptor [AMFR], and ATP). These effects further reduced ferroptosis and maintained the normal structure and function of the cell membrane. Our results suggest that glycine plays an important role in oocyte maturation and later development by regulating ROS-induced lipid metabolism, thereby protecting against biomembrane damage.

Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development

To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. Laboratory study. Private fertility clinic. Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. Transcriptomic profiles comparison between different stages of early development in AGs and PGs. The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.