GV-stage Oocytes Research Articles

Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. N/A.

Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca2+, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca2+, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca2+ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

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

CXCR3 participates in asymmetric division of mouse oocytes by modulating actin dynamics

Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis.

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.

Focusing on the accumulation of chromatin/chromosomes around nucleoli and optimizing the timing of ICSI to facilitate the rescue in vitro maturation of denuded GV stage oocytes.

This study aims to achieve the methodological improvement of rescue IVM by predicting germinal vesicle breakdown (GVBD) and optimizing the timing of ICSI. Time lapse analysis was performed retrospectively to evaluated the relationship between the presence of AC around the nucleoli and GVBD. To find the optimal timing of ICSI, the time from the initiation of the first polar body extrusion to ICSI were measured, and the rates of fertilization at each point were calculated. The GVBD rate of GV stage oocytes with AC around the nucleoli was significantly higher than that of GV stage oocytes without AC. The GV stageoocytes required more time for nuclear maturation after polar body extrusion than MI oocytes, with GV stageoocytes taking 400-600min from polar body extrusion to the optimal timing of ICSI, while the MI stageoocytes took 200-400min. The GV stage oocytes resulted in the birth of healthy babies with the appropriate timing of ICSI. It was found that GV stageoocytes with AC around nucleoli can initiate GVBD and reach the MII stage with a high rate, and that GV stage oocytes required more time than MI stageoocytes to reach the optimal timing of ICSI. Considering these factors, ART laboratories may employ immature GV stageoocytes in routine ART procedures rather than discarding them.

The effects of pretreatment with Cyclosporin A and Docetaxel before vitrification of porcine immature oocytes on subsequent embryo development

In the present study, we attempted to improve the developmental competence of vitrified immature porcine oocytes by the preservation of mitochondrial properties using Cyclosporin A (CsA, inhibitor of mitochondrial membrane permeability transition) and Docetaxel (stabilizer of microtubules, hence mitochondrial distribution). In Experiment 1, Mitotracker red staining revealed reduced mitochondrial activity (MA) in vitrified/warmed oocytes at 0 and 22 h of in vitro maturation (IVM) compared with fresh ones. However, by at 46 h of IVM, MA levels in vitrified oocytes were similar to those in fresh control. Treatment of oocytes with CsA or Docetaxel improved MA at 0 h and 22 h of IVM compared with non-treated vitrified oocytes. However, there were no significant differences among groups in percentages of survival, maturation and embryo development after subsequent IVM and parthenogenetic activation. Nevertheless, a pretreatment with a combination of 10 µg/mL CsA and 0.05 µM Docetaxel improved the blastocyst formation of vitrified oocytes compared with non-treatment counterparts (11.2 ± 1.6% vs 5.9 ± 1.6%, P < 0.05). In conclusion, vitrification reduced mitochondrial activity in GV-stage oocytes during 0–22 h of IVM; however, it was normalized by 46 h IVM. Docetaxel or CsA pretreatment alone did not improve development competence of vitrified oocytes. However, pretreatment with a combination of CsA and Docetaxel could improve blastocyst formation rates.

LSM14B controls oocyte mRNA storage and stability to ensure female fertility

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

Xrcc5/Ku80 is required for the repair of DNA damage in fully grown meiotically arrested mammalian oocytes

Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging events. Recent studies have shown that DNA double-strand break repair occurs predominantly via the homologous recombination (HR) pathway in small non-growing meiotically arrested oocytes (primordial follicle stage). However, the DNA repair mechanisms employed by fully grown meiotically arrested oocytes (GV-stage) have not been studied in detail. Here we established a conditional knockout mouse model to explore the role of Ku80, a critical component of the nonhomologous end joining (NHEJ) pathway, in the repair of DNA damage in GV oocytes. GV oocytes lacking Ku80 failed to repair etoposide-induced DNA damage, even when only low levels of damage were sustained. This indicates Ku80 is needed to resolve DSBs and that HR cannot compensate for a compromised NHEJ pathway in fully-grown oocytes. When higher levels of DNA damage were induced, a severe delay in M-phase entry was observed in oocytes lacking XRCC5 compared to wild-type oocytes, suggesting that Ku80-dependent repair of DNA damage is important for the timely release of oocytes from prophase I and resumption of meiosis. Ku80 was also found to be critical for chromosome integrity during meiotic maturation following etoposide exposure. These data demonstrate that Ku80, and NHEJ, are vital for quality control in mammalian GV stage oocytes and reveal that DNA repair pathway choice differs in meiotically arrested oocytes according to growth status.

Effects of Different Vitrification Temperatures and Cryoprotective Agent Concentrations on Ultrastructure and Apoptosis Related Proteins in Bovine GV Stage Oocytes

The effects of different vitrification temperatures (VTs) and cryoprotective agent concentrations (CPAs) on the viability, ultrastructure, and apoptosis related proteins Bcl-2, Bax, and TP53 of bovine oocytes at the GV (germinal vesicle) stage were analyzed in this study. Cumulus oocyte complexes (COCs) were randomized into five groups: fresh (control), LHe 5.6 M (liquid helium, -269\dg, cryoprotectant concentration of 5.6 M), LHe 6.6 M (liquid helium, –269°C, cryoprotectant concentration of 6.6 M), LN 5.6 M (liquid nitrogen, –196°C, cryoprotectant concentration of 5.6 M), LN 6.6 M (liquid nitrogen, –196°C, cryoprotectant concentration of 6.6 M). Statistically, in the vitrification groups, LN 5.6 M group showed the lowest rate of morphologically normal, mature, and cleavage (58.1%; 32.9%; 12.9%). LHe 5.6 M showed the highest cleavage rate (39.6%), as well as effectively ameliorated ultrastructural damage, significantly reduced Bax protein expression, effectively increased Bcl-2 protein expression, reduced TP53 protein expression, in general, it increased the anti-apoptotic ability of the oocytes after vitrification. It was concluded that lower vitrification temperature and decreased cryoprotectant concentrations upregulate the expression of Bcl-2 protein and downregulate the expression of Bax and TP53 protein in oocytes, and enhance the anti-apoptotic capacity of oocytes, ameliorated the damage of the ultrastructure of the oocytes, perhaps improved the developmental capacity of bovine GV stage oocytes after vitrification can be partially attributed to the above changes.

Vitrification of porcine immature oocytes and zygotes results in different levels of DNA damage which reflects developmental competence to the blastocyst stage.

The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

A more accurate analysis of maternal effect genes by siRNA electroporation into mouse oocytes.

Maternal RNA and proteins accumulate in mouse oocytes and regulate initial developmental stages. Sperm DNA combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3; however, the effect of H3.3 on development post-fertilization remains unclear. Herein, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)-stage oocytes without removing cumulus cells. Oocyte-attached cumulus cells need to be removed during the traditional microinjection method; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. On introducing H3.3 siRNA at the GV stage, H3.3 was maintained in the maternal pronucleus and second polar body but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. These findings indicate that H3.3 protein was not incorporated into the paternal pronucleus, as it was repeatedly translated and degraded over a relatively short period. Conversely, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GV-stage oocytes without cumulus cell removal is not skill-intensive and is essential for the accurate analysis of maternal effect genes.

IGF2 reduces meiotic defects in oocytes from obese mice and improves embryonic developmental competency

BackgroundMaternal obesity is a global issue that has devastating effects across the reproductive spectrum such as meiotic defects in oocytes, consequently worsening pregnancy outcomes. Different studies have shown that such types of meiotic defects originated from the oocytes of obese mothers. Thus, there is an urgent need to develop strategies to reduce the incidence of obesity-related oocyte defects that adversely affect pregnancy outcomes. Multiple growth factors have been identified as directly associated with female reproduction; however, the impact of various growth factors on female fertility in response to obesity remains poorly understood.MethodsThe immature GV-stage oocytes from HFD female mice were collected and cultured in vitro in two different groups (HFD oocytes with and without 50 nM IGF2), however; the oocytes from ND mice were used as a positive control. HFD oocytes treated with or without IGF2 were further used to observe the meiotic structure using different analysis including, the spindle and chromosomal analysis, reactive oxygen species levels, mitochondrial functional activities, and early apoptotic index using immunofluorescence. Additionally, the embryonic developmental competency and embryos quality of IGF2-treated zygotes were also determined.ResultsIn our findings, we observed significantly reduced contents of insulin-like growth factor 2 (IGF2) in the serum and oocytes of obese mice. Our data indicated supplementation of IGF2 in a culture medium improves the blastocyst formation: from 46% in the HFD group to 61% in the HFD + IGF2-treatment group (50 nM IGF2). Moreover, adding IGF2 to the culture medium reduces the reactive oxygen species index and alleviates the frequency of spindle/chromosome defects. We found increased mitochondrial functional activity in oocytes from obese mice after treating the oocytes with IGF2: observed elevated level of adenosine triphosphate, increased mitochondrial distribution, higher mitochondrial membrane potentials, and reduced mitochondrial ultrastructure defects. Furthermore, IGF2 administration also increases the overall protein synthesis and decreases the apoptotic index in oocytes from obese mice.ConclusionsCollectively, our findings are strongly in favor of adding IGF2 in culture medium to overcome obesity-related meiotic structural-developmental defects by helping ameliorate the known sub-optimal culturing conditions that are currently standard with assisted reproduction technologies.

The function of Nucleoporin 37 on mouse oocyte maturation and preimplantation embryo development.

Nucleoporin 37 (NUP37) has been reported to activate the YAP-TEAD signaling, which is crucial for early embryo development. However, whether NUP37 is involved in oocyte meiosis and embryo development remains largely unknown. The study aimed to clarify the function of Nup37 in oocyte maturation and early embryo development, and to explore the mechanism. The expression level and subcellular localization of NUP37 were explored. After knocking down of Nup37 by microinjecting interfering RNA (siRNA), the oocyte maturation rate, aberrant PB1 extrusion rate, and blastocyst formation rate were evaluated. In addition, the effect of the downregulation of Nup37 on YAP-TEAD signaling was confirmed by immunofluorescence staining and real-time quantitative PCR. NUP37 was highly expressed in oocytes and early embryos; it mainly localized to the nuclear periphery at mice GV stage oocytes and early embryos. Nup37 depletion led to aberrant PB1 extrusion at the MII stage oocyte and a decreased blastocyst formation rate. The reduction of NUP37 caused YAP1 mislocalization and decreased the expression of Tead1, Tead2, and Tead4 during mice embryo development, thus affecting the YAP-TEAD activity and embryo developmental competence. In summary, NUP37 played an important role in mice oocyte maturation and preimplantation embryo development.

Transcriptome analysis and potential mechanisms of bovine oocytes under seasonal heat stress

Heat stress is the major factor affecting cattle fertility but molecular mechanisms of deleterious impacts of elevated temperature on oocyte are still not well known. Therefore, the aim of this study is to gain a better understanding of the underlying molecular mechanism of how heat stress affects GV-stage and MII-stage oocytes and discover hub genes to heat resistance for cow oocytes. In this study, we used the bioinformatics approach to discover the differentially expressed genes between GV-stage and MII-stage oocytes, which were collected during spring and summer. When GV-stage oocytes were compared to MII-stage oocytes collected in July (Jul DEGs group) a total of 1068 genes were found as differentially expressed as a result of heat stress. Also, HSPA8, COPS5, POLR2L, PSMC6, and TPI1 were identified as the common top ranked genes for the Jul DEGs group. The highest connected hub gene for the Jul DEGs group was determined as HSPA8. Our results showed that different heat response mechanisms might be activated to protect oocytes from elevated temperatures in cattle. The identified genes and their associated pathways might play an important role in the response to heat stress that affects the oocytes in cattle.

Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

BackgroundDynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated.ResultsWe examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate.ConclusionsWe provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

Effects of prematuration culture with a phosphodiesterase-3 inhibitor on oocyte morphology and embryo quality in in vitro maturation.

ObjectiveThe study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. MethodsIn total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5.ResultsIn total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). ConclusionsCilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

Embryo culture media differentially alter DNA methylating enzymes and global DNA methylation in embryos and oocytes.

The effects of culture media on DNA methylation process, which is one of the epigenetic mechanisms, have not been clearly elucidated although it is known that in vitro culture conditions alter epigenetic mechanisms. This study was designed to address the question: does embryo culture media approach, sequential or single step, differentially affect DNA methylating enzymes and global DNA methylation. Mouse zygotes were cultured either in single step or sequential culture media until the blastocyst stage and in vivo developed blastocyst were utilized as control. Similarly, GV stage oocytes were in vitro matured either in single step or first step of sequential culture media. In vivo matured MII oocytes were used as control. The expression levels and cellular localization of Dnmt1 and 3a enzymes were analyzed by immunofluorescence and western blot analysis while global DNA methylation was evaluated by immunofluorescence. We found that signal intensities of Dnmt1 and Dnmt3a enzymes were significantly low in embryos or oocytes cultured in sequential media compared to single step media and control, which were comparable amongst themself. Similarly, global DNA methylation level in single step media and control groups was comparable but both was higher than the sequential media. This study demonstrated that composition of culture media may differentially affect DNA methylation levels in mouse embryos and oocytes. Since abnormal DNA methylation may cause aberrant oocyte or embryo development, we think that further studies are needed to test human embryos and oocyte, and to explain molecular mechanisms.

P–556 NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings: Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number Not applicable

P-556 Pre-selected for an award: NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number not applicable