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Oocyte loss, a natural process that accelerates as women approach their mid-30s, poses a significant challenge to female reproduction. Recent studies have identified DNA damage as a primary contributor to oocyte loss, but the mechanisms underlying DNA damage accumulation remain unclear. Here, we show that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. Incomplete DNA repair in aged oocytes results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. We found that DNA repair proteins are arranged in spatially distinct DNA repair compartments that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. We demonstrate alterations in these compartments with age, including substantial changes in the levels of key DNA repair proteins and a shift toward error-prone DNA repair pathways. In addition, we show that reduced cohesin levels make aged oocytes more vulnerable to persistent DNA damage and cause changes in DNA repair compartments. Our study links DNA damage accumulation in aged oocytes, a leading cause of oocyte loss, to cohesin deterioration and changes in the organization, abundance, and response of DNA repair machinery. [Display omitted] • Aged oocytes accumulate DNA damage and have a reduced capacity for DNA repair • DNA repair proteins are organized into distinct nuclear DNA repair compartments • Aged oocytes show changes in DNA repair machinery, favoring error-prone repair • Age-related cohesin loss also contributes to reduced capacity for DNA repair Oocytes accumulate DNA damage with advancing maternal age, leading to their eventual death and loss of reproductive potential. Sharma et al. show that aged oocytes accumulate DNA damage due to reduced DNA repair capacity, changes in DNA repair compartments, loss of cohesin, and a shift toward error-prone repair pathways. [ABSTRACT FROM AUTHOR]

Background: Diminished ovarian reserve (DOR) is clinically characterized by a decrease in the number of available ovarian follicles and a decline in the quality of oocytes, accompanied by hormonal changes. Low quality of DOR oocyte leads to impaired embryo development, an increased risk of aneuploid pregnancies and miscarriages. However, the specific pathogenic mechanism remains unclear, posing a significant challenge for assisted reproductive technology. Methods: For the first time, our study employed single-cell RNA sequencing to reveal the altered transcriptomic landscape of DOR oocytes at GV stage after ovarian stimulation. Differentially expressed genes analysis (DEGs), functional enrichment analysis, weighted gene co-expression network analysis (WGCNA) and protein-protein interactions network analysis were performed. Results: We found 132 up-regulated genes and 466 down-regulated genes in DOR oocytes, with the down-regulated genes primarily enriched in mitochondrial function and translation. Hub genes, identified through integrated analysis of WGCNA and DEGs, were further validated in DOR and control oocytes using RT-qPCR. By utilizing hub genes and employing transcription factor enrichment tools, it had been predicted that pleomorphic adenoma gene 1 (PLAG1) played a crucial role as a transcriptional regulatory factor in DOR oocytes. Additionally, we conformed the PLAG1-IGF2 axis was dysregulated in DOR oocytes. Conclusions: Transcriptome analysis revealed that DOR oocytes exhibited mitochondrial dysfunction and translational defects, and the PLAG1-IGF2 axis might be a potential contributor for the low quality of DOR oocytes. [ABSTRACT FROM AUTHOR]

Asynchronous nuclear and cytoplasmic maturation in human oocytes is believed to cause morphological anomalies after controlled ovarian hyperstimulation. Vacuolar protein sorting 34 (VPS34) is renowned for its pivotal role in regulating autophagy and endocytic trafficking. To investigate its impact on oocyte development, oocyte‐specific knockout mice (ZcKO) are generated, and these mice are completely found infertile, with embryonic development halted at 2‐ to 4‐cell stage. This infertility is related with a disruption on autophagic/mitophagic flux in ZcKO oocytes, leading to subsequent failure of zygotic genome activation (ZGA) in derived 2‐cell embryos. The findings further elucidated the regulation of VPS34 on the activity and subcellular translocation of RAS‐related GTP‐binding protein 7 (RAB7), which is critical not only for the maturation of late endosomes and lysosomes, but also for initiating mitophagy via retrograde trafficking. VPS34 binds directly with RAB7 and facilitates its activity conversion through TBC1 domain family member 5 (TBC1D5). Consistent with the cytoplasmic vacuolation observed in ZcKO oocytes, defects in multiple vesicle trafficking systems are also identified in vacuolated human oocytes. Furthermore, activating VPS34 with corynoxin B (CB) treatment improved oocyte quality in aged mice. Hence, VPS34 activation may represent a novel approach to enhance oocyte quality in human artificial reproduction. [ABSTRACT FROM AUTHOR]

Production of oocytes from pluripotent cell cultures in a dish represents a new paradigm in stem cell and developmental biology and has implications for how we think about life. The spark of life for the next generation occurs at fertilization when sperm and oocyte fuse. In animals, gametes are the only cells that transmit their genomes to the next generation. Oocytes contain in addition a large cytoplasm with factors that direct embryonic development. Reconstitution of mouse oocyte and embryonic development in culture provides experimental opportunities and facilitates an unprecedented understanding of molecular mechanisms. However, the application of in vitro gametogenesis to reproductive medicine or infertility treatment remains challenging. One significant concern is the quality of in vitro‐derived oocytes. Here, we review the current understanding and identify limitations in generating oocytes in vitro. From this basis, we explore opportunities for future improvements of the in vitro approach to generating high‐quality oocytes. [ABSTRACT FROM AUTHOR]

Antioxidants have a well-established effect on general health and are essential in preventing oxidative damage to cells by scavenging free radicals. Free radicals are thought to be neutralized by these substances, which include polyphenols, β-carotene, and vitamins C and E, reducing cellular damage. On the other hand, recent data indicates that consuming excessive amounts of antioxidants may have side effects. Apoptosis and cell signaling are two beneficial physiological processes that are affected by excessive supplementation. Other negative effects include paradoxical enhancement of oxidative stress and unbalanced cellular redox potential. Overdosing on particular antioxidants has been associated with increased medication interactions, cancer progression, and fatality risks. Additionally, the complex impacts they may have on fertility might be both useful and adverse, depending on the quantity and duration of usage. This review delves into the dual role of antioxidants and emphasizes the importance of employing antioxidants in moderation. Antioxidant overconsumption may disrupt the oxidative balance necessary for normal sperm and oocyte function, which is one of the potential negative effects of antioxidants on fertility in both males and females that are also investigated. Although modest usage of antioxidants is generally safe and useful, high levels of antioxidants can upset hormonal balance, impair sperm motility, and negatively impact the outcomes of assisted reproductive technologies (ART). The findings emphasize the need to use antioxidant supplements in a balanced way, the importance of further research to optimize their use in fertility treatments, and the importance of supporting reproductive health to avoid adverse effects. [ABSTRACT FROM AUTHOR]

Context. As a 'duty of care', it is important to test whether new forage plants for ruminants contain secondary compounds (PSCs) that affect reproductive performance. We have previously observed, a posteriori, that the presence of a methanolic extract of Biserrula pelecinus during maturation of sheep oocytes increased fertilisation rate and blastocyst development. This result needed to be verified a priori and, if the outcome was repeated, we needed to identify the plant secondary metabolite responsible. Aims. To test whether PSCs from B. pelecinus, when added to the oocyte maturation medium, improve fertilisation rate and blastocyst development; to test whether loliolide is the active molecule produced by B. pelecinus. Methods. Methanol-chloroform extracts of B. pelecinus were fractionated using rapid silica filtration and solvents of increasing polarity. Fractions at final concentrations of 0, 100 or 200 µg mL-1 were added to the medium used to mature sheep cumulus-oocyte complexes (COCs) and effects were determined for maturation, subsequent cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN). Results. Fraction BP-6 at 100 µgmL-1 reduced blastocyst rate (P < 0.05), but had no effect when the dose was doubled to 200 µg mL-1. Further fractionation using semi-preparative high-performance liquid chromatography showed loliolide as the most abundant compound in BP-6. Supplementation of the in vitro maturation medium with loliolide (0, 2.5, 5, 10 and 25 µgmL-1) did not affect any measure of embryo development. All COCs treated with B. pelecinus fractions reached the final stage of embryo development, blastocyst hatching. Total blastocyst cell number was not affected. Conclusion. The presence of fractions of B. pelecinus extract during in vitro oocyte maturation can reduce embryo development. Implications. In vitro techniques can detect potential effects of forages on reproduction. Some fractions from an extract of B. pelecinus when present during oocyte maturation can reduce embryo development. The abundant PSC, loliolide, was not responsible. There was no indication that a PSC in B. pelecinus improves outcomes. [ABSTRACT FROM AUTHOR]

To evaluate donor gamete utilization, patient satisfaction, and fertility treatment outcomes of patients pursuing treatment with donor gametes stratified by the desired race as well as ethnicity of the gamete donor. Survey study. Clinic. Patients planning to undergo treatment using donor sperm and/or donor oocytes at a single academic fertility clinic in the Southeastern United States between 2015 and 2020. None. Utilization rates of donor gametes, satisfaction with donor gamete selection, and fertility treatment outcomes stratified by race and ethnicity of patient, as well as that of their gamete donor. Four hundred fifty patients were eligible for inclusion and 170 (38%) responded to the survey. Among the respondents, 59% desired a non-Hispanic White gamete donor and 20% desired a non-Hispanic Black gamete donor. Patients seeking a non-Hispanic Black gamete donor had lower odds of using donor gametes (Odds ratio [OR], 0.13; 95% confidence interval [CI], 0.04–0.40) compared with individuals seeking a non-Hispanic White gamete donor. When evaluating satisfaction with donor gamete selection, patients seeking a non-Hispanic Black gamete donor reported lower satisfaction compared with individuals seeking a non-Hispanic White gamete donor (OR, 0.19; 95% CI, 0.09–0.43). When evaluating fertility outcomes, Non-Hispanic Black patients and those using non-Hispaninc Black gamete donors were found to have lower odds of successful conception compared with non-Hispanic White patients (OR, 0.18; 95% CI, 0.07–0.46) and individuals seeking non-Hispanic White gamete donors (OR, 0.26; 95% CI, 0.09–0.75), respectively. Patients seeking non-Hispanic Black donor gametes have lower utilization rates, less satisfaction with gamete donor selection, and lower odds of conception when compared with those seeking non-Hispanic White gamete donors. These findings highlight the need for more racial diversity within donor gamete banks, as well as within the donor pools available through agencies and fertility clinics. [ABSTRACT FROM AUTHOR]

This document provides the latest recommendations for the evaluation of potential sperm, oocyte, and embryo donors, as well as their recipients, incorporating recent information about optimal screening and testing for sexually transmitted infections, genetic diseases, and psychological assessments. This revised document incorporates recent information from the US Centers for Disease Control and Prevention, the US Food and Drug Administration, and the American Association of Tissue Banks, with which all programs offering gamete and embryo donation services must be thoroughly familiar, and replaces the document titled "Recommendations for gamete and embryo donation: a committee opinion," last published in 2013. [ABSTRACT FROM AUTHOR]

Flavopiridol (FP) is a plant-derived flavonoidis and used to treat cancers, fungal infections and inflammation-related diseases. However, it is not clear whether it has side effects on the female reproductive system. In this study, we aimed to investigate the toxic effects and potential underlying mechanisms of FP on oocyte maturation and cumulus cell expansion in mice. Cumulus-oocyte complexes (COCs) were cultured in vitro with FP of gradient concentration (50–1000 nM), according to the plasma concentration of FP in the clinical trial. The maturation rate and cumulus expansion index of oocytes were counted and studied by immunofluorescence staining, qRT-PCR, oocyte chromosome preparation and so on. The results showed that the FP-exposed COCs inhibited the oocyte maturation and cumulus cell expansion, leading to cell apoptosis in a dose dependent way. Oocytes exposed to 500 nM FP showed abnormalities in the spindle structure and chromosome arrangement, ultimately leading to the oocyte maturation arrest and aneuploidy. This may be due to the excessive oxidative stress caused by mitochondrial membrane potential damage and mislocalization. Therefore, when FP is used for cancer treatment, its side effects on the female reproductive system should be seriously considered. • Flavopiridol exposure leads to a decrease in the maturation rate of mouse oocytes. • Flavopiridol causes oxidative stress and mitochondrial dysfunction. • Flavopiridol causes abnormal spindle structure and chromosome arrangement. [ABSTRACT FROM AUTHOR]

Purpose: Recently, progesterone has been used to prevent LH surge instead of GnRH analogues during ART treatments, which is known as progesterone-primed ovarian stimulation (PPOS) protocol. During ART treatment, highly purified human menopausal gonadotropin (HP-hMG) and recombinant follicle stimulating hormone (rFSH) are two of the agents used for stimulation of antral follicles. The aim of this study is to compare the efficacy and success of HP-hMG and rFSH agents in the ovarian stimulation step of the PPOS protocol, which has not been previously reported in the literature. Methods: This retrospective study was conducted at a university hospital with patients who underwent IVF treatment using PPOS protocols in between January 2019 and July 2021. For ovarian stimulation, rFSH was used in group I and HP-hMG was used in group II. Mature oocyte ratio was the primary outcome, and live birth rate was the secondary outcome. Mann–Whitney and Chi-square tests were used for statistical analysis. All p values below 0.05 were considered significant. Results: Total numbers of follicles, oocytes, MII, and 2PN numbers obtained were similar between the two groups. The fertilization rates were 66.7% in the rFSH group and 64.3% in the HP-hMG group (p > 0.05). The pregnancy rates were 53.5% and 46.7% in the rFSH and HP-hMG groups, respectively. There was no statistically significant difference between pregnancy, abortus, and live birth rates. Conclusion: In this study, it is demonstrated that stimulation of oocytes with either rFSH or hMG in the PPOS protocol, which has been added to IVF treatment protocols in recent years, had no statistical difference regarding mature oocyte numbers and live birth rates between the two groups. These results are consistent with the previous literature which compared rFSH and hMG in GnRH agonist and antagonist protocols. [ABSTRACT FROM AUTHOR]

The wide application of ovine oocyte vitrification is limited by its relatively low efficiency. Nanoparticle is potentially to be used in cryopreservation technology for its unique characteristics with high biocompatibility, potent antioxidant property as well as superiority in membrane permeation and heat transduction. However, the effect of nanoparticle on ovine oocyte cryopreservation as well as the underlying mechanism has not been systematically evaluated. The objective of this study was to investigate the impact of nanoparticles on ovine oocytes cryopreservation and further identify the underlying mechanism. Firstly, the effects of Hydroxyapatite (HA) and Fe 3 O 4 nanoparticles on the developmental potential of vitrified ovine oocytes were determined, and the results showed that neither HA (VC = 85.95 ± 6.23 % vs. VH = 92.47 ± 8.11 %, P > 0.05) nor Fe 3 O 4 (VC = 85.95 ± 6.23 % vs. VF = 89.39 ± 6.32 %, P > 0.05) had adverse effect on the survival rate of vitrified-thawed oocytes. Notably, both HA (VC = 77.78 ± 0.09 % vs. VH = 44.00 ± 0.09 %, P ＜0.01) and Fe 3 O 4 (VC = 77.78 ± 0.09 % vs. VF = 51.67 ± 0.15 %, P ＜0.01) nanoparticles effectively reduced the level of oocyte apoptosis after freezing and thawing. What's more, HA could significantly improve the cleavage rate of frozen oocytes (VC = 33.79 ± 2.83 % vs. VH = 59.54 ± 4.13 %, P ＜0.05). Moreover, reduced reactive oxygen species (ROS) level (VC = 13.66 ± 0.47 vs. VH = 12.61 ± 0.53, P < 0.05), increased glutathione (GSH) content (VC = 60.69 ± 7.89 vs. VH = 87.92 ± 1.05, P < 0.05) and elevated mitochondrial membrane potential (MMP) level (VC = 1.43 ± 0.04 vs. VH = 1.63 ± 0.01， P ＜0.01) were observed in oocytes treated with HA nanoparticles when compared with that of the control group. Furthermore, Smart-RNA sequence technology was utilized to identify differentially expressed mRNAs (DEMs) induced by nanoparticles during cryopreservation. When compared with the control counterparts, a total of 721 DEMs (309 up-regulated and 412 down-regulated mRNAs) were identified in oocytes treated with HA, while 702 DEMs (480 up-regulated and 222 down-regulated mRNAs) were identified in oocytes treated with Fe 3 O 4. A comparison of DEMs showed that total 692 mRNAs were expressed in oocytes treated with HA and Fe 3 O 4. Notably, we discovered that 15 mRNAs were specially highly expressed in oocytes treated with HA, and Focal adhesion signaling pathway mainly contributed to the improved ovine oocyte quality after vitrification by alleviating oxidative stress. • HA nanoparticles improved the cleavage rate of frozen oocytes. • HA nanoparticles reduced reactive oxygen species level, increased glutathione and elevated mitochondrial membrane potential levels. • HA nanoparticles improved oocyte quality mainly by alleviating oxidative stress via Focal adhesion signaling pathway. [ABSTRACT FROM AUTHOR]

Acrylamide (AAM), a compound extensively utilized in various industrial applications, has been reported to induce toxic effects across multiple tissues in living organisms. Despite its widespread use, the impact of AAM on ovarian function and the mechanisms underlying these effects remain poorly understood. Here, we established an AAM‐exposed mouse toxicological model using 21 days of intragastric AAM administration. AAM exposure decreased ovarian coefficient and impaired follicle development. Further investigations revealed AAM would trigger apoptosis and disturb tricarboxylic acid cycle in ovarian tissue, thus affecting mitochondrial electron transport function. Moreover, AAM exposure decreased oocyte and embryo development potential, mechanically associated with pericentrin and phosphorylated Aurora A cluster failure, leading to meiotic spindle assembly defects. Collectively, these results suggest that AAM exposure may lead to apoptosis, glucose metabolic disorders, and mitochondrial dysfunction in ovary tissue, ultimately compromising oocyte quality. [ABSTRACT FROM AUTHOR]

The Aurora Kinases (AURKs) are a family of serine-threonine protein kinases critical for cell division. Somatic cells express only AURKA and AURKB. However, mammalian germ cells and some cancer cells express all three isoforms. A major question in the field has been determining the molecular and cellular changes when cells express three instead of two aurora kinases. Using a systematic genetic approach involving different Aurora kinase oocyte-specific knockout combinations, we completed an oocyte-AURK genetic interaction map and show that one genomic copy of Aurka is necessary and sufficient to support female fertility and oocyte meiosis. We further confirm that AURKB and AURKC alone cannot compensate for AURKA. These results highlight the importance of AURKA in mouse oocytes, demonstrating that it is required for spindle formation and proper chromosome segregation. Surprisingly, a percentage of oocytes that lack AURKB can complete meiosis I, but the quality of those eggs is compromised, suggesting a role in AURKB to regulate spindle assembly checkpoint or control the cell cycle. Together with our previous studies, we wholly define the genetic interplay among the Aurora kinases and reinforce the importance of AURKA expression in oocyte meiosis. [ABSTRACT FROM AUTHOR]

The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K 0.5 G A B A ~8 mM. l -Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2 -overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress. [ABSTRACT FROM AUTHOR]

Knowledge of the reproductive biology of fishes is essential for effective fisheries management. Information derived from an understanding of fish reproduction, such as size and age at maturity, is used in models to assess fish stocks and can affect estimates of important ecological processes such as recruitment, abundance, and trophic interactions. Common practices for determining the reproductive status of teleost fishes include macroscopic evaluation of gonads as well as histological analysis. However, macroscopic evaluation can be biased and histological analysis is time-consuming, resulting in limitations to spatial and temporal data availability. Here, we explore Raman spectroscopy of ovaries as a novel approach to rapidly determine the reproductive status of walleye pollock (Gadus chalcogrammus), a commercially and ecologically important species in theNorth Pacific. We used a twostage partial least-squares (PLS) regression analysis followed by a linear discriminant analysis (LDA) to classify walleye pollock ovary samples as physiologically mature or immature and to subsequently predict their histologically-determined reproductive stage based on the Raman spectra. Biologically mature samples with visible yolk differentiated from mature and immature samples (non-yolked; 99% accuracy). Nonyolked ovaries that were physiologically mature (either mature non-developing or previously spawned) were further differentiated from physiologically immature ovaries (93% accuracy). In addition, detailed, histologically-determined reproductive stages of yolked samples also differentiated via Raman spectroscopy, but with reduced accuracy (79% - 86% accuracy). Our results indicate that accurate identification of maturity status and the reproductive staging of oocytes of walleye pollock based on spectral data from ovaries is possible. This can provide a fast and efficient way to increase the availability of a key component of reproductive data to inform fisheries research and management. [ABSTRACT FROM AUTHOR]

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells. Synopsis: Oocytes plays a central role in the success of reproduction, yet the phosphorylation dynamics during mammalian oocyte development remain poorly understood. This resource article describes the comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes, and reveals novel molecular mechanisms controlling meiotic maturation. The phosphorylation landscape of oocytes during in vivo maturation is delineated. Novel phosphosites in oocytes and conserved phosphosites across multiple species are identified. Key kinases orchestrating the regulation of oocyte meiosis are pinpointed Phosphatase dynamics during meiotic maturation are deciphered. This resource article provides the most comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes to date, revealing novel molecular control mechanisms. [ABSTRACT FROM AUTHOR]

Background: Oocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes. Methods: This study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles. Results: At the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes. Conclusions: Our findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation. [ABSTRACT FROM AUTHOR]

As interest in deep space travel grows exponentially, understanding human adaptation in becoming an interplanetary species is crucial. This includes the prospect of reproduction. This review summarizes recent updates and innovations in assisted reproductive technologies (ART) on Earth, while also discussing current challenges and areas for improvement in adapting ART studies to the space environment. We discuss the critical components of ART - gamete handling and preparation, fertilization, embryo culture, and cryopreservation - from the daily practice perspective of clinical embryologists and reproductive endocrinologists and lay out the complicated path ahead. In vitro embryo development in low Earth orbit and beyond remains questionable due to synergetic effects of microgravity and radiation-induced damage observed in simulated and actual in-space mammalian studies. Cryopreservation and long-term storage of frozen samples face substantial obstacles - temperature limitations, lack of trained personnel, and absence of adapted cosmic engineering options. We touch on recent innovations, which may offer potential solutions, such as microfluidic devices and automated systems. Lastly, we stress the necessity for intensive studies and the importance of an interdisciplinary approach to address numerous practical challenges in advancing reproductive medicine in space, with possible implications for both space exploration and terrestrial fertility treatments. [ABSTRACT FROM AUTHOR]

Super‐resolution (SR) microscopy is a cutting‐edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures.Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step.We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two‐channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high‐resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes.Lay Abstract: Super‐resolution (SR) microscopy is a cutting‐edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high‐resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures. [ABSTRACT FROM AUTHOR]

Mitochondrial plasticity supports oogenesis in vertebrates. Dormant oocytes have mitochondrial morphology suggestive of low mitochondrial activity. Dormant human and Xenopus oocytes have their mitochondria clustered in the Balbiani body and suppress Complex I to avoid generation of damaging reactive oxygen species. Mouse oocytes fine-tune mitochondrial localisation and membrane potential to increase ATP production at energy-demanding sites during maturation. Major differences exist in mitochondrial distribution and physiology among vertebrates, including between evolutionary closer species, such as human and mouse. Mitochondria have a crucial role in cellular function and exhibit remarkable plasticity, adjusting both their structure and activity to meet the changing energy demands of a cell. Oocytes, female germ cells that become eggs, undergo unique transformations: the extended dormancy period, followed by substantial increase in cell size and subsequent maturation involving the segregation of genetic material for the next generation, present distinct metabolic challenges necessitating varied mitochondrial adaptations. Recent findings in dormant oocytes challenged the established respiratory complex hierarchies and underscored the extent of mitochondrial plasticity in long-lived oocytes. In this review, we discuss mitochondrial adaptations observed during oocyte development across three vertebrate species (Xenopus , mouse, and human), emphasising current knowledge, acknowledging limitations, and outlining future research directions. [ABSTRACT FROM AUTHOR]

Rabbits have played a significant role in both livestock production and the advancement of reproductive scientific research. Their unique biological traits, including induced ovulation and a reproductive process that closely mirrors that of humans, have been pivotal in their use as a model. Moreover, their body size is perfectly aligned with the 3Rs principles: Replacement, Reduction, and Refinement. Consequently, techniques for gamete collection and embryo recovery, followed by their use in artificial insemination or embryo transfer, are characterized by being minimally invasive. However, refining in vitro fertilization and embryo culture techniques continues to present challenges. The incorporation of cutting‐edge genomic editing tools, such as CRISPR/Cas9, has reestablished rabbits as essential models in genetic and biomedical research, driving scientific progress. This review aims to describe the most effective reproductive biotechnologies for both male and female rabbits and how these methodologies are in line with the 3Rs principles—Replacement, Reduction, and Refinement—highlighting their significance in conducting ethical research. [ABSTRACT FROM AUTHOR]

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68 , the Drosophila homolog of Spo11 , to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region. [ABSTRACT FROM AUTHOR]

Meiosis is regulated in sexually dimorphic manners in mammals. In females, the commitment to and entry into meiosis are coordinated with the developmental program of oocytes. Female germ cells initiate meiosis within a short time window during the fetal period and then undergo meiotic arrest until puberty. However, the genetic mechanisms underlying the orchestration of oocyte development and meiosis to maximize the reproductive lifespan of mammalian females remain largely elusive. While meiotic initiation is regulated by a sexually common mechanism, where meiosis initiator and Stimulated by Retinoic Acid Gene 8 (STRA8) activate the meiotic genes, the female‐specific mode of meiotic initiation is mediated by the interaction between retinoblastoma (RB) and STRA8. This review highlights the female‐specific mechanisms of meiotic initiation and meiotic prophase progression in the context of oocyte development. Furthermore, the downstream pathway of the RB‐STRA8 interaction that may regulate meiotic arrest will be discussed in the context of oocyte development, highlighting a potential genetic link between the female‐specific mode of meiotic entry and meiotic arrest. [ABSTRACT FROM AUTHOR]

Mammalian oocytes undergo a long‐term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication‐dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication‐dependent H3.1/H3.2 and replication‐independent H3.3, decrease with age. Aging‐associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging‐associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging‐associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice. [ABSTRACT FROM AUTHOR]

Mitochondria are essential organelles with specialized functions, which play crucial roles in energy production, calcium homeostasis, and programmed cell death. In oocytes, mitochondrial populations are inherited maternally and are vital for developmental competence. Dysfunction in mitochondrial quality control mechanisms can lead to reproductive failure. Due to their central role in oocyte and embryo development, mitochondria have been investigated as potential diagnostic and therapeutic targets in assisted reproduction. Pharmacological agents that target mitochondrial function and show promise in improving assisted reproduction outcomes include antioxidant coenzyme Q10 and mitoquinone, mammalian target of rapamycin signaling pathway inhibitor rapamycin, and nicotinamide mononucleotide. Mitochondrial replacement therapies (MRTs) offer solutions for infertility and mitochondrial disorders. Autologous germline mitochondrial energy transfer initially showed promise but failed to demonstrate significant benefits in clinical trials. Maternal spindle transfer (MST) and pronuclear transfer hold potential for preventing mitochondrial disease transmission and improving oocyte quality. Clinical trials of MST have shown promising outcomes, but larger studies are needed to confirm safety and efficacy. However, ethical and legislative challenges complicate the widespread implementation of MRTs. [ABSTRACT FROM AUTHOR]

To find a useful tool for estimating the minimum number of metaphase II (MII) oocytes needed to obtain at least one euploid blastocyst according to female age. Retrospective analysis of in vitro fertilization (IVF) treatment cycles with preimplantational genetic testing for aneuploidies (PGT-A) performed over 5 years in IVIRMA Valencia (Spain), January 2017–March 2022. Approval from the Institutional Review Board of IVI Valencia (2204-VLC-040-CR). Private infertility clinic in Spain. Eligible patients were undergoing their first IVF-PGT-A treatment cycle, in which at least one MII oocyte was obtained, regardless of oocyte and semen origin. Oocyte donation cycles were included in the donor group (≤34 years old). Treatment cycles from women with their own oocytes were selected only when the oocytes were aged ≥35 years (patient group). Only trophoectoderm biopsies performed on days 5 or 6 of development and analyzed using next-generation sequencing were included. Preimplantational genetic testing for aneuploidy cycles because of a known abnormal karyotype were excluded. Not applicable. Number of MII oocytes needed to obtain one euploid blastocyst according to female age. A total of 2,660 IVF-PGT-A treatment cycles were performed in the study period in the eligible population (patients group = 2,462; donors group =198). The mean number of MII oocytes needed to obtain one euploid blastocyst increased with age, as did the number of treatment cycles that did not get at least one euploid blastocyst. An adjusted multivariate binary regression model was designed using 80% of the patient group sample (n = 2,462; training set). A calculator for the probability of obtaining at least one euploid blastocyst was created using this model. The validation of this model in the remaining 20% of the patient group sample (n = 493; validation set) showed that it could estimate the event of having at least one euploid blastocyst with an accuracy of 72.0%. Our results show a preliminary model capable of predicting the number of MII oocytes needed to obtain at least one euploid blastocyst according to female age, calculated with the largest database of IVF-PGT-A treatment cycles ever used for this purpose, including only treatment cycles using next-generation sequencing on trophoectoderm biopsies. Once this model has been properly validated, it could help with decision-making for both clinicians and patients coming to an infertility clinic. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to evaluate the effectiveness of oral L-carnitine administration in patients after treatment failure to lay the groundwork for targeted in vivo use. Methods and materials: A total of 515 In Vitro Fertilization (IVF) patients undergoing subsequent cycles were included after applying exclusion criteria. They were divided into a control group of 362 patients and a study group of 153 patients who received oral L-carnitine until oocyte retrieval.140 patients were matched according to maternal age, infertility duration, body mass index (BMI), day three top-quality embryos rate, by propensity score matching (PSM). The study investigated the relationship between L-carnitine treatment and in vivo oocyte maturation, normal fertilization, and subsequent embryo development. Results: Following PSM, initial differences in BMI and Day3 top-quality embryo rate between groups were nullified, we created two comparable cohorts with highly similar characteristics. In the subsequent cycles, the study group showed significant improvements in in vivo oocyte maturation rate at retrieval (p=0.002), normal in vitro fertilization rate (p=0.003), blastocyst formation rate (p=0.003), and usable blastocyst rate compared to controls. Although there was no significant difference in the top-quality embryo rate on Day 3, the study group showed a 10% increase in the upper quartile (55.35% vs. 66.67%). The cumulative clinical pregnancy and live birth rates showed a significant improvement (59.82% vs. 68.42%, p=0.004, 47.41% vs. 59.80%, p=0.002). Furthermore, self-control analysis revealed substantial enhancements (p<0.001) in all outcome measures following L-carnitine administration, resulting in the birth of 74 healthy neonates without congenital anomalies. Conclusion: We theorized that daily oral intake of L-carnitine before oocyte retrieval could boost oocyte quality and embryonic development, thus improving IVF outcomes. Ongoing investigations hold the potential to offer valuable insights into the applications and mechanisms underlying the therapeutic effectiveness of L-carnitine. [ABSTRACT FROM AUTHOR]

The noninvasive and label‐free electrical characterization of single cells is increasingly gaining interest over the last years. Besides cancer cells, oocytes are in the focus of those investigations. The study of oocytes is relevant for two areas: on the one hand, the quality of mammalian oocytes is important in artificial reproduction technology for a high fertilization yield. On the other hand, amphibian oocytes are well established to be used as host cells for the characterization of ion‐channel kinetics for drug screening. For both areas, easy‐to‐perform and reliable characterization techniques are needed. This mini‐review summarizes important developments of characterizing oocytes with microfluidic systems in combination with observer‐independent noninvasive electrical sensing. Such miniaturized microfluidic systems can be improved to facilitate multiple oocyte characterizations simultaneously for higher throughput screening. [ABSTRACT FROM AUTHOR]

Correct chromosome segregation is essential to preserve genetic integrity. The two protein kinases, Aurora B and its meiotic homolog Aurora C, regulate attachments between chromosomal kinetochores and microtubules, thereby contributing to the accuracy of the chromosome segregation process. Here we performed a detailed examination of the localization and activity of Aurora B/C kinases, their partner Incenp and the kinetochore target Hec1, during the second meiotic division in mouse oocytes. We found that a majority of Aurora B and C changed their localization from the outer kinetochore region of chromosomes at prometaphase II to an inner central region localized between sister centromeres at metaphase II. Depletion of the Aurora B/C pool at the inner central region using the haspin kinase inhibitor 5-iodotubercidin resulted in chromosome misalignments at the metaphase II stage. To further understand the role of the Aurora B/C pool at the central region, we examined the behaviour of single chromatids, that lack a central Aurora B/C pool but retain Aurora B/C at the outer kinetochores. We found that kinetochore-microtubule attachments at single chromatids were corrected at both prometaphase II and metaphase II stages, but that single chromatids compared to paired chromatids were more prone to misalignments following treatment of oocytes with the Aurora B/C inhibitory drugs AZD1152 and GSK1070916. We conclude that the Aurora B/C pool at the inner central region stabilizes chromosome alignment during metaphase II arrest, while Aurora B/C localized at the kinetochore assist in re-establishing chromosome positioning at the metaphase plate if alignment is lost. Collaboratively these two pools prevent missegregation and aneuploidy at the second meiotic division in mammalian oocytes. [ABSTRACT FROM AUTHOR]

Embryo development is stimulated by calcium (Ca2+) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a "toolkit", a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca2+ from the egg's intracellular stores which in mammals is followed by repetitive Ca2+ spikes known as Ca2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca2+ signal and describe the steps of the signal transduction mechanism that activates the egg's developmental program and turns it into an embryo. [ABSTRACT FROM AUTHOR]

Oogenesis involves two phases: initial volumetric growth driven by nutrient accumulation and subsequent nuclear maturation. While melatonin (MLT) has been employed as a supplement to enhance the quality of fully grown oocytes during nuclear maturation phase, its impact on oocyte growth remains poorly studied. Here, we provide in vivo evidence demonstrating that follicle‐stimulating hormone increases MLT content in ovary. Administration of MLT improves oocyte growth and quality in mice and goats by enhancing nutrient reserves and mitochondrial function. Conversely, MLT‐deficient mice have smaller oocytes and dysfunctional mitochondria. Exploring the clinical implications of MLT in promoting oocyte growth, we observe that a brief 2‐day MLT treatment enhances oocyte quality and reproductive performance in older mice. These findings highlight the role of MLT in regulating oocyte growth and provide a specific treatment window for optimizing oocyte quality and reproductive performance in female animals. [ABSTRACT FROM AUTHOR]

Background: Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring. Results: Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed. Conclusions: These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health. [ABSTRACT FROM AUTHOR]

Methoxychlor (MXC) is a widely used organochlorine pesticide primarily targeting pests. However, MXC has been found to negatively impact the reproductive system of both humans and animals, triggering oxidative stress and apoptosis. Melatonin (MLT), an endogenous hormone, possesses various benefits, including circadian rhythm regulation and anti-inflammatory and antioxidative stress effects. Moreover, MLT plays a crucial role in the development of animal germ cells and embryos. This study aimed to investigate the impact of MLT on porcine oocytes exposed to MXC. The experimental findings revealed that 200 μM MXC had detrimental effects on the maturation of porcine oocytes. However, the addition of 10−8 M MLT mitigated the toxic effects of MXC. MXC induced oxidative stress in porcine oocytes, leading to an increase in reactive oxygen species and impairing mitochondrial function. Consequently, oocyte quality was affected, resulting in elevated levels of early apoptosis and DNA damage, ultimately negatively impacting subsequent embryonic development. However, the addition of MLT showed the potential to ameliorate the damage caused by MXC. In conclusion, our results suggest that MLT exhibits a protective effect against MXC-induced damage to porcine oocyte maturation. [ABSTRACT FROM AUTHOR]

The aim of this study was to prevent initial changes in Drosophila melanogaster oocytes under simulated weightlessness and hypergravity at the 2 g level. Phospholipids with polyunsaturated fatty acids in the tail groups (essential phospholipids) at a concentration of 500 mg/kg of nutrient medium were used as a protective agent. Cell stiffness was determined using atomic force microscopy, the change in the oocytes' area was assessed as a mark of deformation, and the contents of cholesterol and neutral lipids were determined using fluorescence microscopy. The results indicate that the administration of essential phospholipids leads to a decrease in the cholesterol content in the oocytes' membranes by 13% (p < 0.05). The stiffness of oocytes from flies that received essential phospholipids was 14% higher (p < 0.05) and did not change during 6 h of simulated weightlessness or hypergravity, and neither did the area, which indicates their resistance to deformation. Moreover, the exposure to simulated weightlessness and hypergravity of oocytes from flies that received a standard nutrient medium led to a more intense loss of cholesterol from cell membranes after 30 min by 13% and 18% (p < 0.05), respectively, compared to the control, but essential phospholipids prevented this effect. [ABSTRACT FROM AUTHOR]

The field of reproductive biology has made significant progress in recent years, identifying specific molecular players that influence oocyte development and function. Among them, sirtuin 3 (SIRT3) has attracted particular attention for its central role in mediating mitochondrial function and cellular stress responses in oocytes. So far, studies have demonstrated that the knockdown of SIRT3 leads to a decrease in blastocyst formation and an increase in oxidative stress within an embryo, underscoring the importance of SIRT3 in maintaining the cellular redox balance critical for embryonic survival and growth. Furthermore, the literature reveals specific signaling pathways, such as the SIRT3- Glycogen synthase kinase-3 beta (GSK3β) deacetylation pathway, crucial for mitigating oxidative stress-related anomalies in oocyte meiosis, particularly under conditions like maternal diabetes. Overall, the emerging role of SIRT3 in regulating oocyte mitochondrial function and development highlights the critical importance of understanding the intricate connections between cellular metabolism, stress response pathways, and overall reproductive health and function. This knowledge could lead to the development of novel strategies to support oocyte quality and fertility, with far-reaching implications for assisted reproductive technologies and women's healthcare. This commentary aims to provide an overview of the importance of SIRT3 in oocytes by synthesizing results from a multitude of studies. The aim is to elucidate the role of SIRT3 in oocyte development, maturation, and aging and to identify areas where further research is needed. [ABSTRACT FROM AUTHOR]

Simple Summary: Sperm cells are specialized cells that are highly susceptible to different kinds of damage, impairing the success of in vitro fertilization and, consequently, of assisted reproductive techniques (ART). Oxidative stress plays a significant role in this context causing DNA injury, reduced motility, and compromising the fertilization ability of sperm. The use of antioxidants aims to mitigate the oxidative stress, protect sperm cells from damage, and enhance their fertilization potential. Urolithin A (UA) is a natural compound with proven anti-aging and antioxidant effects in various cells, but its effect on bovine sperm has not been tested yet. The objective of this study was to investigate the impact of UA on bovine sperm function and fertilization rate, thus contributing to animal reproductive development. The results showed that UA improved sperm motility quality and reduced oxidative stress levels in a dose-dependent manner. The use of UA as an antioxidant therapy may have a significant impact on the advancement of reproductive biotechnologies. Reactive oxygen species (ROS) play a critical role in the functional competence of sperm cells. Conversely, excessive generation of ROS can impair sperm function, including their fertilization ability. Urolithin A (UA), a gut bacteria-derived metabolite produced from the transformation of ellagitannins, with anti-aging and antioxidant properties, was investigated for the first time in bovine sperm cells in the present study. Firstly, different doses of UA (0, 1, and 10 μM; 8–16 sessions) were used during the capacitation process of frozen-thawed bovine sperm. Sperm motility was assessed using optical microscopy and CASA. Sperm vitality (eosin-nigrosin), ROS, and ATP levels, as well as mitochondrial membrane potential (JC1) and oxygen consumption were evaluated. A second experiment to test the effect of different doses of UA (0, 1, and 10 μM; 9 sessions) in both the capacitation medium, as above, and the fertilization medium, was also implemented. The embryonic development and quality were evaluated. UA, at a concentration of 1 μM, significantly improved sperm movement quality (p < 0.03). There was a trend towards an increase in the oxygen consumption rate (OCR) of capacitated sperm with 1 μM and 10 μM UA supplementation. Moreover, an increase in ATP levels (p < 0.01) was observed, accompanied by a reduction in ROS levels at the higher UA concentration. These results suggest that UA may enhance spermatozoa mitochondrial function, modifying their metabolic activity while reducing the oxidative stress. Also, the number of produced embryos appears to be positively affected by UA supplementation, although differences between the bulls may have mitigated this effect. In conclusion, presented results further support previous findings indicating the potential therapeutic value of UA for addressing reproductive sub/infertility problems and improving ART outcomes. In addition, our results also reinforce the important bull effect on ART and that male sperm bioenergetic parameters should be used to predict spermatozoa functionality and developmental potential. [ABSTRACT FROM AUTHOR]

HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea. Recently, emerging evidences indicate that HT-2 also disturbs the reproductive system. In this study, we investigated the impact of HT-2 toxin exposure on the organelles of porcine oocytes. Our results found that the abnormal distribution of endoplasmic reticulum increased after HT-2 treatment, with the perturbation of ribosome protein RPS3 and GRP78 expression; Golgi apparatus showed diffused localization pattern and GM130 localization was also impaired, thereby affecting the Rab10-based vesicular transport; Due to the impairment of ribosomes, ER, and Golgi apparatus, the protein supply to lysosomes was hindered, resulting in lysosomal damage, which further disrupted the LC3-based autophagy. Moreover, the results indicated that the function and distribution of mitochondria were also affected by HT-2 toxin, showing with fragments of mitochondria, decreased TMRE and ATP level. Taken together, our study suggested that HT-2 toxin exposure induces damage to the organelles for endomembrane system, which further inhibited the meiotic maturation of porcine oocytes. • HT-2 caused abnormal distribution of ER and GRP78-based ER stress in oocytes. • HT-2 disrupted GM130-based Golgi apparatus and Rab10-based vesicular transport. • HT-2 induced lysosome damage and disrupted LC3-based autophagy in oocytes. • HT-2 disturbed mitochondria distribution, MMP and ATP production in oocytes. [ABSTRACT FROM AUTHOR]

During aging, oocytes display cytoskeleton dynamics defects and aneuploidy, leading to embryonic aneuploidy, which in turn causes miscarriages, implantation failures, and birth defects. KIF15 (also known as Hklp2), a member of the kinesin-12 superfamily, is a cytoplasmic motor protein reported to be involved in Golgi and vesicle-related transport during mitosis in somatic cells. However, the regulatory mechanisms of KIF15 during meiosis in porcine oocytes and the connection with postovulatory aging remain unclear. In present study, we found that KIF15 is expressed during porcine oocyte maturation, and its localization is dependent on microtubule dynamics. Furthermore, the level of KIF15 expression decreased in postovulatory aged oocytes. The decrease in KIF15 blocked polar body extrusion, thereby hindering oocyte maturation. We demonstrated that KIF15 defects contributed to abnormal spindle morphologies and chromosome misalignment, possibly due to microtubule instability, as evidenced by microtubule depolymerization after cold treatment. Additionally, our data indicated that KIF15 modulates HDAC6 to affect tubulin acetylation in oocytes. Taken together, these results suggest that KIF15 regulates HDAC6-related microtubule stability for spindle organization in porcine oocytes during meiosis, which may contribute to the decline in maturation competence in aged porcine oocytes. • KIF15 associates with microtubule dynamics and decreases in aged porcine oocytes. • KIF15 is essential for polar body extrusion in porcine. • KIF15 regulates spindle formation through microtubule instability in porcine oocytes. • KIF15 modulated HDAC6 to affect tubulin acetylation in porcine oocytes. [ABSTRACT FROM AUTHOR]

Caenorhabditis elegans hermaphrodite presents a unique model to study the formation of oocytes. However, the size of the model animal and difficulties in retrieval of specific stages of the germline have obviated closer systematic studies of this process throughout the years. Here, we present a transcriptomic level analysis into the oogenesis of C. elegans hermaphrodites. We dissected a hermaphrodite gonad into seven sections corresponding to the mitotic distal region, the pachytene region, the diplotene region, the early diakinesis region and the 3 most proximal oocytes, and deeply sequenced the transcriptome of each of them along with that of the fertilized egg using a single-cell RNA-seq (scRNA-seq) protocol. We identified specific gene expression events as well as gene splicing events in finer detail along the gonad and provided novel insights into underlying mechanisms of the oogenesis process. Furthermore, through careful review of relevant research literature coupled with patterns observed in our analysis, we delineate transcripts that may serve functions in the interactions between the germline and cells of the somatic gonad. These results expand our knowledge of the transcriptomic space of the C. elegans germline and lay a foundation on which future studies of the germline can be based upon. [ABSTRACT FROM AUTHOR]

Introduction: Co-administration of clomiphene citrate during the early phase in in vitro fertilisation (IVF) cycles has been used by clinicians for several years to improve outcomes. Materials and methods: This is a single centre retrospective study involving 222 patients with 81 patients underwent IVF using co-administration of Clomiphene Citrate (CC) with gonadotropins (study group - group A) and 141 patients used only gonadotropins (control group - group B) were compared. The outcome data of the IVF cycles were focused on four major aspects of which are the total gonadotropin usage, number of oocytes obtained, fertilisation rate and top-quality embryos produced. Results: The Mean ± Standard Deviation (SD) of patients age is (34.72 ± 3.25) and (34.39 ± 3.39) years old from group A and B respectively. Mean ± (SD) of total usage of gonadotropins (1834.57 ± 477.34iu) vs (2645.04 ± 669.66iu), (p< 0.0001), mean number of oocytes ± (SD) retrieved (8.54 ± 5.95) vs (10.32 ± 6.67), (p = 0.048), mean ± (SD) fertilisation rate (39.82 ± 30.46) vs (49.04 ± 27.62), (p=0.027) and mean percentage ± (SD) of the top-quality embryos are (17.96 ± 25.68) vs (28.02 ± 32.19), (p=0.02) from group A and B respectively. Conclusion: Co-administration of CC with gonadotropins in IVF reduces the total amount of gonadotropins required in the IVF cycle however it also reduces the number of oocytes retrieved, fertilization rate and top quality embryos. [ABSTRACT FROM AUTHOR]

The present experiment was conducted during January, 2018 to March, 2019 in the laboratory, Dept. of Veterinary Physiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam (781022), India to investigate the effect of epidermal growth factor on in vitro maturation, fertilization and early embryonic development of cattle oocytes. A total of 318 nos. cattle ovaries were collected from slaughter houses for one year and 1089 nos. culturable oocytes were collected. 381 out of 1089 nos. culturable oocytes were subjected to EGF supplemented serum and serum free basic maturation media. The mean percentage of in-vitro maturation of cattle oocytes based on polar body extrusion was found to be significantly higher in EGF supplemented serum free basic maturation media (70.00±14.49) than the serum basic maturation media without EGF (54.17±7.19) respectively. But the mean percentages of in vitro maturation of cattle oocytes based on the cumulus expansion and polar body extrusion were found to be significantly higher in EGF supplemented serum free basic maturation media (77.59±5.48 and 70.00±14.49) than the value recorded in serum free basic maturation media without EGF (64.20±3.77 and 45.95±8.19). The mean cleavage percentages recorded at 4 cell, 8 cell, 16 cell and morula stages of embryos in EGF supplemented serum free culture media were found to be significantly higher than the value recorded in serum culture media without EGF. From the present experiment, it can be inferred that addition of EGF in serum free basic maturation media had little beneficial effect than serum basic maturation media. [ABSTRACT FROM AUTHOR]

Simple Summary: Lipid metabolism plays an important role in the development of oocytes and early embryos. Peroxisome proliferators-activated receptor γ is one of the key nuclear transcription factors that regulate lipid metabolism. Their main function is to regulate the expression of downstream genes and participate in the deposition and decomposition of intracellular lipids, thus regulating the level of intracellular lipid metabolism. Studies have shown that PPARγ can also be used as an antioxidant to reduce cell oxidative stress. Energy metabolism and oxidative stress are important factors affecting the development of oocytes and early embryos. In this study, the PPARγ agonist rosiglitazone was used to study the effects of PPARγ on oxidative stress and lipid metabolism in sheep oocytes and early embryos. The results showed that PPARγ could alleviate oxidative stress, improve the ability of oocytes to develop in vitro, and then improve the reproductive performance of sheep. Lactating oocytes consume a lot of energy during maturation, a large part of which comes from lipid metabolism. PPARγ is a key regulator of lipid metabolism. In this study, rosiglitazone (RSG), an activator of PPARγ, was added to a mature medium to investigate its effects on the levels of spindle and the chromosome arrangement, lipid deposition, reactive oxygen species (ROS), and glutathione (GSH) levels, oocyte secretion factors, apoptosis and lipid metabolism-related gene expression, and subsequent embryonic development during the maturation of sheep oocytes. The oocyte secretion factor affects gene expression related to apoptosis and lipid metabolism and subsequent embryonic development. The results showed that the proportion of spindle and normal chromosome arrangements increased in the 5 μM RSG treatment group, the lipid content increased after cell maturation, the ROS level decreased, and the GSH level increased. The expressions of oocyte secretion factor (GDF9 and BMP15), anti-apoptosis gene (BCL2), and lipid metabolism-related genes (ACAA1, CPT1A, PLIN2) were increased in the 5 μM treatment group. Finally, the development of blastocysts was examined. After the oocytes were treated with 5 μM RSG, the blastocyst rate and the gene expression of the totipotency gene (OCT4) were increased. It was concluded that increasing PPARγ activity during ovine oocyte maturation could promote lipid metabolism, reduce oxidative stress, and improve the ovine oocyte maturation rate and subsequent embryo development. [ABSTRACT FROM AUTHOR]

Abstract Background Diminished ovarian reserve (DOR) is clinically characterized by a decrease in the number of available ovarian follicles and a decline in the quality of oocytes, accompanied by hormonal changes. Low quality of DOR oocyte leads to impaired embryo development, an increased risk of aneuploid pregnancies and miscarriages. However, the specific pathogenic mechanism remains unclear, posing a significant challenge for assisted reproductive technology. Methods For the first time, our study employed single-cell RNA sequencing to reveal the altered transcriptomic landscape of DOR oocytes at GV stage after ovarian stimulation. Differentially expressed genes analysis (DEGs), functional enrichment analysis, weighted gene co-expression network analysis (WGCNA) and protein-protein interactions network analysis were performed. Results We found 132 up-regulated genes and 466 down-regulated genes in DOR oocytes, with the down-regulated genes primarily enriched in mitochondrial function and translation. Hub genes, identified through integrated analysis of WGCNA and DEGs, were further validated in DOR and control oocytes using RT-qPCR. By utilizing hub genes and employing transcription factor enrichment tools, it had been predicted that pleomorphic adenoma gene 1 (PLAG1) played a crucial role as a transcriptional regulatory factor in DOR oocytes. Additionally, we conformed the PLAG1-IGF2 axis was dysregulated in DOR oocytes. Conclusions Transcriptome analysis revealed that DOR oocytes exhibited mitochondrial dysfunction and translational defects, and the PLAG1-IGF2 axis might be a potential contributor for the low quality of DOR oocytes.

Abstract Background Oocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes. Methods This study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles. Results At the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes. Conclusions Our findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation.

Abstract As interest in deep space travel grows exponentially, understanding human adaptation in becoming an interplanetary species is crucial. This includes the prospect of reproduction. This review summarizes recent updates and innovations in assisted reproductive technologies (ART) on Earth, while also discussing current challenges and areas for improvement in adapting ART studies to the space environment. We discuss the critical components of ART - gamete handling and preparation, fertilization, embryo culture, and cryopreservation - from the daily practice perspective of clinical embryologists and reproductive endocrinologists and lay out the complicated path ahead. In vitro embryo development in low Earth orbit and beyond remains questionable due to synergetic effects of microgravity and radiation-induced damage observed in simulated and actual in-space mammalian studies. Cryopreservation and long-term storage of frozen samples face substantial obstacles - temperature limitations, lack of trained personnel, and absence of adapted cosmic engineering options. We touch on recent innovations, which may offer potential solutions, such as microfluidic devices and automated systems. Lastly, we stress the necessity for intensive studies and the importance of an interdisciplinary approach to address numerous practical challenges in advancing reproductive medicine in space, with possible implications for both space exploration and terrestrial fertility treatments.

Abstract Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P

Objective: To investigate whether the presence of hepatitis B virus (HBV) in oocytes and embryos affects pregnancy outcomes for in vitro fertilization and embryo transfer (ET) as well as is related to the vertical transmission of HBV to children. Design: Retrospective cohort study. Setting: A university-affiliated fertility center. Patient(s): This study included 167 couples with at least 1 hepatitis B surface antigen–seropositive partner. These couples underwent in vitro fertilization–ET, and the discarded oocytes and embryos had been tested for HBV. Couples with HBV-positive oocytes or embryos were categorized as the positive group, whereas those couples with HBV-negative oocytes and embryos served as the negative group. Intervention(s): None. Main Outcome Measure(s): Pregnancy outcomes and the rate of children’s HBV infection. Result(s): The pregnancy outcomes of fresh and frozen ETs were not associated with the presence of HBV in the oocytes and embryos. Of the 106 infants born, 1 child whose mother tested positive for hepatitis B surface antigen but had negative oocytes and embryos was infected with HBV. Additionally, 26.09% of children who had been administered passive immunization and active vaccinations did not reach protective levels of anti-HBV antibodies (hepatitis B surface antibodies) and became nonresponders. The negative rate of children’s hepatitis B surface antibody was associated with the presence of HBV in oocytes and embryos (odds ratio, 3.01; 95% confidence interval, 1.04–9.25). Conclusion(s): The presence of HBV in oocytes and embryos did not affect pregnancy outcomes or result in the vertical transmission of HBV to the offspring of HBV carriers. Follow-up is needed for HBV-vaccinated children with an HBV-infected parent. Booster vaccinations are necessary for continued protection.

Abstract Background Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring. Results Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed. Conclusions These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health.

The purpose of this research was to determine the correlation between the weight of the ovary, the number of follicles, and the quality of the oocytes from the culling female bovine obtained from the slaughterhouse. The variation may affect the potential of the ovary to produce follicles. Paired ovaries were obtained from female bovines. Follicle numbers were recorded and divided into three size categories (small: 3 mm, medium: < 3 mm-8 mm, and large: > 8 mm). Oocytes were aspirated and the number of oocytes was recorded and graded into four categories (grades A, B, C, and D). There was a positive correlation between the weight of the ovary and the number of follicles, which is 0.560 with the regression equation y = 3.52 + 0.501 x. There was a positive correlation between the number of follicles and the number of oocytes, which is 0.546 with the regression equation y =2.48 + 1.204 x. There was a positive correlation between the number of oocytes and the grade of oocytes, which is 0.520, with the regression equation y = 0.93 + 0.800 x. There was no correlation between the weight of the ovary and oocyte grade A, which is 0.013.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).