Search

Currently, incubators with a time-lapse system are widely used for in vitro embryo production in several species, however, their effect on alpaca embryo development compared to conventional incubators remains unknown. The aim of this study was to compare early in vitro embryo development in alpacas using a time-lapse incubator system versus a conventional incubator. Ovaries were obtained from a slaughterhouse and 1048 cumulus-oocyte complexes (COCs) were collected and in vitro matured for 26 h in either a time-lapse system (n = 542) or a conventional incubator (n = 542). All the matured COCs were fertilised for 24 h with fresh spermatozoa recovered from epididymis obtained from the slaughterhouse. The presumptive zygotes in both systems were cultured in a commercial one-step culture medium, and the embryo development was evaluated at 48, 120 and 168 h after fertilisation. Our results showed no differences in maturation rates between the time-lapse system and the conventional incubator (79.9% vs. 55.0%, p = 0.080). Cleavage rates at 48 h were also similar between the time-lapse and conventional systems: 20.5% vs. 20.8% for embryos at the two- to four-cell stage, and 6.0% vs. 7.0% for embryos above the four-cell stage (p > 0.05). At 120 h after fertilisation, the percentage of embryos were more evenly distributed across all stages (below eight-cell, eight-cell and above eight-cell stages) in both systems (p > 0.05). At 168 h, no differences in the blastocyst rates and blastocyst quality were observed between the time-lapse and conventional systems. In conclusion, in vitro embryo production in alpacas can be successfully carried out using the time-lapse incubation system or a conventional incubator with comparable results. Further study would focus on the correlation between the developmental patterns and the quality of alpaca embryos, as well as the comparison of in vivo embryo development between the two incubation systems., (© 2025 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in three different time windows using quartiles, with 465 oocytes (22.0 %) between 28 and 30 h (first quartile), 1078 oocytes (51.0 %) >30 and 31.7 h (second and third quartiles), and 571 oocytes (27.0 %) >31.7 and 34 h (fourth quartile). Using logistic regression models, the effect of duration of IVM with and without holding was tested on nuclear maturation, cleavage, blastocyst, and pregnancy rates. The three IVM intervals did not show differences in nuclear maturation (respectively 64.5 ± 0.48 %, 65.7 ± 0.47 %, and 67.3 ± 0.47 %), cleavage (respectively 59.7 ± 0.49 %, 58.5 ± 0.49 %, and 64.8 ± 0.48 %), blastocyst (respectively 17.5 ± 0.38 %, 19.0 ± 0.39 %, and 20.8 ± 0.41 %) nor pregnancy rates (respectively 65.4 ± 0.49 %, 70.3 ± 0.46 %, and 74.2 % ± 0.44) (P ≥ 0.38). Oocyte holding prior to IVM did not affect the results either (P ≥ 0.15). In conclusion, oocyte holding and IVM duration between 28 and 34h do not significantly affect outcomes, allowing flexibility in the planning of clinical OPU-ICSI in horses., Competing Interests: Declaration of interest We have nothing to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During fetal development, primordial oocytes maintain their developmental potential through a ROS-minimizing metabolic mechanism. Maturation increases ROS levels, causing stress and damage, which are countered by in vivo antioxidants. In vitro maturation (IVM) worsens this due to fewer antioxidant presence and medium factors. To address this, we evaluated the effects of incorporating various natural antioxidants in the porcine oocyte IVM media. Our findings revealed that 10 μM Dendrobine (DEN), 1 μM Polydatin (PD), 20 μM Limonin (LIM), and 25 μM Nobiletin (NOB) significantly improved the first polar body extrusion rates (p < 0.05), reduced ROS, and increased GSH levels. Individual addition of 100 μg/mL Lycium barbarum polysaccharides (LBP), 0.1 μM Kaempferol (KAE), 250 μM Salidroside (SAL), 10 μM Curcumin (CUR), DEN, PD, LIM, and NOB to the porcine IVM system showed that KAE, LIM, NOB, and LBP treatments yielded the most favorable results. At the gene level, LIM, LBP, and NOB were found to upregulate the expression levels of GPX1, SIRT1, and TFAM, while downregulating Caspase3 and increasing the BCL2/BAX ratio. The inclusion of LIM, NOB, and LBP, either alone or in combination, into the IVM media effectively alleviated oxidative stress in porcine oocytes, decreased cell apoptosis, preserved mitochondrial membrane potential, and enhanced the blastocyst rate. These results offer valuable insights for optimizing the porcine oocyte IVM culture system., Competing Interests: Declaration of competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zheng Feng reports administrative support was provided by Science and Technology Department of Guangdong Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Assisted reproductive technologies (ARTs) involve the laboratory manipulation of gametes and embryos to help couples with fertility problems become pregnant. One of these procedures, controlled ovarian stimulation, uses pharmacological agents to induce ovarian and follicular maturation in vivo. Despite the effectiveness in achieving pregnancy and live births, some patients may have complications due to over-response to gonadotropins and develop ovarian hyperstimulation syndrome. In vitro maturation (IVM) of oocytes has emerged as a technique to reduce the risk of ovarian hyperstimulation syndrome, particularly in women with polycystic ovary syndrome, and for fertility preservation in women undergoing oncological treatment. Although there are some limitations, primarily due to oocyte quality, recent advances have improved pregnancy success rates and neonatal and infant outcomes. Different terms have been coined to describe variations of IVM, and the technique has evolved with the introduction of hormones to optimize results. In this review, we provide a comprehensive overview of IVM relating hormonal priming, culture system and media, and clinical indications for IVM with its reproductive outcomes during ARTs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ovarian stimulation (OS) is a crucial component of clinical IVF treatment that strongly influences outcomes. As such, it is useful to understand the indicators for successful OS during IVF. As OS leads to multiple follicular recruitment, it can be quantified as number of oocytes retrieved. Optimal OS should help to maximize the number of oocytes, thus improving preclinical laboratory outcomes. Optimal preclinical outcomes should ultimately lead to clinical outcomes with maximal efficacy, safety, and cost-effectiveness. To help guide successful OS, this review details prognostic factors and appropriate endpoints for an optimal OS at each stage of the IVF cycle., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: None to declare., (© 2025. The Author(s).)

Ovulation, fertilization, and embryo development are orchestrated and synchronized processes essential for the optimal health of offspring. Postovulatory aging disrupts this synchronization and impairs oocyte quality. In addition, oocyte aging causes fertilization loss and poor embryo development. This investigation aimed to unravel the endpoint of in vitro oocyte aging in common carp (Cyprinus carpio) to understand the involvement of apoptosis in postovulatory oocyte death. It was observed that the fertilization ability significantly declined (P < 0.001) at 8-h poststripping (HPS), subsequently triggering apoptosis in the advanced stage of oocyte aging, i.e., 48 HPS. This process included an increase in proapoptotic transcripts (fas, bax, cathepsin D, caspase 8, caspase 9, and caspase 3a) (P < 0.05), elevated levels of caspase 3 protein (P < 0.05), and activation of caspase 3 enzyme (P < 0.001), a key player in apoptosis, in aging oocytes. Furthermore, the effects of oocyte aging on the embryonic apoptosis machinery were examined in embryos at 5-h postfertilization (HPF) and 24 HPF derived from fresh and aged oocytes. Expression of apoptotic genes and caspase enzyme activity remained at the basal level in 5 HPF (early blastula embryos) from both fresh and aged oocytes. In contrast, the zymogenic and active forms of caspase 3 increased in 24 HPF embryos from 8-h-aged oocytes (P < 0.01) compared with those from fresh oocytes. Thus, apoptosis intensified in 24 HPF embryos from aged oocytes without affecting the apoptotic machinery of early blastula embryos. Our findings demonstrate that apoptosis initiated by the Fas/FasL system is an important physiological process accompanying oocyte aging in common carp., (© The Author(s) 2025. Published by Oxford University Press on behalf of the American Society of Animal Science.)

In Brief: Ovulation, the female reproductive process whereby fertilizable oocytes are released from a mature ovarian follicle, is widely seen in all animals that sexually reproduce. This paper reveals that, despite the remarkably diverse styles of ovulation among animal taxa, most ovulate via follicle rupture and suggests that the rupture process may operate via similar cellular mechanisms across animals., Abstract: Ovulation is the process by which a fertilizable oocyte is extruded from the interior of the follicle. Herein, we conducted a literature survey to explore the ovulation patterns of 11 sexually reproducing species belonging to 10 animal phyla. These results indicate a large variety of ovulation patterns. Further comparative biological and evolutionary considerations of these results led us to conclude that most female animals ovulate via follicle rupture. We propose that in all animals that ovulate by follicle rupture, two cellular events may be critically involved in the process: i) the disintegration of cell junctional systems leading to intracellular cytoskeleton rearrangement in the follicle cells and ii) the degradation of extracellular matrix (ECM) proteins filled between follicle cells. These events may result in follicular cell deformation and increased motility, both of which are necessary for the formation of a path through which an oocyte escapes from the follicle. In addition to the requirement of ECM degradation for disintegrating cell junctions, intensive ECM protein degradation at the apical region of the follicle probably became increasingly important in late-evolving animals, such as vertebrates, in which a thick follicle wall containing a large abundance of ECM proteins is formed. We also considered hypothetical scenarios for the evolution of ovulation in these animals. Furthermore, this article discusses future problems that need to be solved for a more comprehensive understanding of ovulation in the animal kingdom.

The generation of mammalian embryos by in vitro culture is hampered by the failure of many of the embryos to develop to the blastocyst stage. This problem occurs even when cumulus-oocyte complexes (COCs) with good morphology are visually selected and used for culture. Because cumulus cells are important for oocyte maturation and subsequent embryo development, here we compared gene expression patterns in cumulus cells of COCs that developed in vitro to the blastocyst stage with those of COCs that failed to develop. Cumulus cells were aspirated from bovine COCs selected for in vitro culture. Oocyte developmental competence was evaluated by screening for cleavage and development to the blastocyst stage. The collected cumulus cells were used to quantify mRNA levels of FSH receptor (FSHR), insulin-like growth factor-1 receptor (IGF-1R), anti-Müllerian hormone (AMH), AMH receptor II (AMHRII), epidermal growth factor receptor (EGFR), estrogen receptor β (ERβ), B cell lymphoma/leukemia-2 associated X (Bax), and cysteine-aspartic acid protease-3 (Caspase-3). We found that the expression levels of FSHR, IGF-1R, AMH, and EGFR were higher in cumulus cells from COCs that developed to blastocysts as compared with those that failed to develop, whereas expression levels of Bax and Caspase-3 were lower in cumulus cells of COCs that matured to the blastocyst stage. Positive correlations were found between FSHR and IGF-1R expression (r = 0.59) and between ERβ and EGFR expression (r = 0.43) in cumulus cells from COCs that developed to the blastocyst stage. Our findings indicate that gene expression levels in cumulus cells are correlated with the developmental competence of bovine oocytes. Measurement of gene expression in cumulus cells therefore offers a non-invasive means of predicting oocyte developmental competence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yoshikazu Nagao reports financial support was provided by Utsunomiya University. Yoshikazu Nagao reports a relationship with Utsunomiya University that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper presents a microneedle thermocouple probe designed for temperature measurements in biological samples, addressing a critical need in the field of biology. Fabricated on a Silicon-On-Insulator (SOI) wafer, the probe features a doped silicon (Si)/chrome (Cr)/gold (Au) junction, providing a high Seebeck coefficient, rapid response times, and excellent temperature resolution. The microfabrication process produces a microneedle with a triangular sensing junction. Finite Element Analysis (FEA) was employed to evaluate the thermal time constant and structural integrity in tissue, supporting the probe's suitability for biological applications. Experimental validation included temperature measurements in ex-vivo tissue and live Xenopus laevis oocytes. Notably, intracellular thermogenesis was detected by increasing extracellular potassium concentration to depolarize the oocyte membrane, resulting in a measurable temperature rise. These findings highlight the probe's potential as a robust tool for monitoring temperature variations in biological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: The process of maturing ovine oocyte in vitro has not yet been raised with acceptable results., Objective: This study was designed to evaluate the γ-oryzanol effect as a supplement of maturation media on the development of ovine oocytes to blastocyst., Methods: Aspirated from ovine ovaries, morphologically normal cumulus-oocyte complexes (COCs) were matured in media supplemented with or without 5 µM γ-oryzanol. Matured oocytes were divided into two parts: one evaluated for their nuclear maturation, the level of GSH and ROS, mitochondrial membrane potential (MMP) and the pattern of transcription in oocytes and respective cumulus cells (CCs), and another subjected to fertilisation and culture to assess the development of oocytes to the blastocyst., Results: γ-Oryzanol improved the proportion of cleaved embryos and total blastocysts in the treated group, which was linked to improved MMP, higher levels of intracellular GSH and lower levels of ROS. A lower proportion of MI and GVBD was recorded for treated oocytes in comparison with control, although the proportion of MII oocytes was not different between groups. The treated oocytes and CCs showed downregulation of genes related to apoptosis (BAX and CASP-9) and upregulation of genes related to antioxidative status (NRF2, CAT and SOD). In conclusion, our results demonstrated the improved developmental outcome of supplemented oocytes so that the antioxidant response and higher enzymatic activity were maintained, and the generation of ROS was turned off; therefore, a novel alternative for counteracting oxidative stress in ovine oocytes undergoing maturation was offered by γ-oryzanol through an antioxidative pathway., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Aim: In developed nations, women have increasingly deferred childbearing, leading to a rise in demand for infertility treatments and the widespread use of assisted reproductive technologies. However, despite advancements in in vitro fertilization (IVF), live birth rates among women over 40 remain suboptimal. Mitochondrial dysfunction is widely recognized as a key factor in the processes driving the age-related deterioration in both the quantity and quality of oocytes. We aim to summarize current insights into ovarian aging, with a particular focus on pathways that impair mitochondrial function, and explore directions for future research., Methods: Electronic databases were searched for articles published up to June 30, 2024., Results: Ongoing ovulation, luteolysis, and menstruation trigger exogenous reactive oxygen species (ROS)-mediated oxidative stress that damages mitochondrial DNA. This, in turn, reduces nuclear gene expression, compromises mitochondrial oxidative phosphorylation, and diminishes adenosine 5' triphosphate production. Persistent endogenous ROS further exacerbate mitochondrial DNA damage and aneuploidy, ultimately causing irreversible chromosomal abnormalities, leading to oocyte aging., Conclusions: We have delineated the pathway from oxidative stress to ovarian aging. Early detection and management of ovarian aging present challenges and opportunities to enhance IVF treatment strategies., (© 2024 Japan Society of Obstetrics and Gynecology.)

Ovarian stimulation (OS) truly is an art. There exists a myriad of protocols used to achieve the same goal: stimulating the ovaries to produce more than one mature oocyte to improve the chance of a live birth. However, considerable debate remains as to whether OS impacts oocyte and endometrial quality to affect in vitro fertilization outcomes. Although "more is better" has long been considered the best approach for oocyte retrieval, this review challenges that notion by examining the influence of stimulation on oocyte quality. Likewise, improved outcomes after frozen blastocyst transfer suggest that OS perturbs endometrial preparation and/or receptivity, although correlating changes with implantation success remains a challenge. Therefore, the focus of this review is to summarize our current understanding of perturbations in human oocyte quality and endometrial function induced by exogenous hormone administration. We highlight the need for further research to identify more appropriate markers of oocyte developmental competence as well as those that define the roles of the endometrium in the success of assisted reproductive technology., Competing Interests: Declaration of Interests A.J.H. has nothing to disclose. B.E.W. has nothing to disclose. E.S.S. has nothing to disclose. D.K.G has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In mammalian reproduction, testis-specific protein IZUMO1 and its receptor JUNO on the oocyte surface are essential for sperm-oocyte recognition, binding, and membrane fusion. However, these factors alone are insufficient to accomplish cytoplasmic membrane fusion. It is believed that other gametic proteins interact with them to facilitate sperm-oocyte interaction on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. In this study, Copper Transport Protein 2 (CTR2) has been identified on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. CTR2 directly interacts with both IZUMO1 and JUNO, colocalizing with IZUMO1 on the sperm head and with JUNO on the oocyte membrane. Treatment of the capacitated sperm and zona pellucida-free oocytes with anti-CTR2 antibody resulted in a significant decrease in fertilization rates in IVF experiments. These findings suggest that CTR2 plays an important role in mammalian fertilization by interacting with IZUMO1 and JUNO, providing new insights into the molecular mechanisms of mammalian sperm-oocyte adhesion and fusion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Ovum Pick-Up (OPU) technique offers a rapid path to genetic improvement. Embryonic genome activation (EGA) is crucial for successful embryonic development and occurs in two phases: minor EGA (MN EGA ) from 2-cell to 8-cell stages, and major EGA (MJ EGA ) from 8-cell to 16-cell stages. Supplementation with alpha-lipoic acid (ALA) during MN EGA using abattoir derived-oocytes has been shown to enhance in vitro embryo production. Two experiments were carried out in the study at hand. Experiment I aimed to evaluate ALA effects on embryo development and quality during MJ EGA using abattoir-derived oocytes. Oocytes were treated with either a Control in vitro culture (IVC) medium or 2.5 µM ALA during MJ EGA . The ALA presented a trend to increase the blastocyst cell number on day 7 (P = 0.06), along with reducing ROS levels and increasing GSH levels (P ≤ 0.05). Experiment II aimed to verify whether the results from abattoir- derived oocytes could be extrapolated to OPU-derived oocytes. The treatments included a Control, 2.5 µM ALA during MN EGA , and 2.5 µM ALA during MJ EGA . The ALA supplementation during MJ EGA improved the total blastocyst rate on day 8 (P = 0.04), with a trend towards increased blastocyst cell number on day 7 (P = 0.09). No improvements were observed in MN EGA . Overall, ALA supplementation during MJ EGA is more effective than during MN EGA for improving the embryonic quality of OPU-derived oocytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

It has long been speculated that the mechanical properties of the human oocyte can be an indicator for oocyte viability. Recent studies have demonstrated that embryo implantation rates, following Intra-Cytoplasmic Sperm Injection (ICSI) procedures, may be increased if the shear modulus value of the oocyte Zona Pellucida (ZP) is taken into consideration during embryo transfer. The shear modulus was determined by an iterative oocyte specific finite element (FE) analysis based on the clinical ICSI data. Nevertheless, the results obtained from the computational analysis may depend on the choice of the constitutive model used for the ZP. In the current study, three different hyper-elastic strain energy density functions (SEDF's) are considered. The Neo-Hookean (NH), Mooney-Rivlin (MR) and Ogden models (OG) were used to determine the ZP shear modulus from ICSI clinical data. The sensitivity of each SEDF in extracting the oocyte specific shear modulus is examined, for the first time. It is demonstrated that the NH and MR models are more sensitive than the OG model for determining the ZP shear modulus from clinical ICSI data. It is also demonstrated that the accuracy of ZP shear modulus identification process greatly depends on the value of suction pressure applied during the ICSI procedure., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: The clinical part of this study was conducted in a single Fertility and IVF Unit in a tertiary medical center during the years (2020–2023). The procedure was approved by Soroka university medical center review board (IRB reference 316-23-SOR). All procedures performed in the study were in accordance with the ethical standards of the institution research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Patients were recruited to the study after obtaining their written informed consent., (© 2024. The Author(s).)

Gametes in many species develop in cysts-clusters of germ cells formed by incomplete cytokinesis-that remain connected through intercellular bridges (ICBs). These connections enable sharing of cytoplasmic components between germ cells and, in the female germ line, enrich select cells in the cyst to become the oocyte(s). In mice, germline cysts of variable sizes are generated during embryonic development, thought to result from cyst fractures. Studies of fixed samples failed to capture fracture events, and thus, the mechanism remained elusive. Here, we use high-resolution live imaging of germ cells within their native tissue environment to visualize germline cyst dynamics. With this novel approach, we reveal a striking motile phenotype of gonad-resident germ cells and show that this randomly oriented cell-autonomous motile behavior during cyst formation underlies fracture events. Conversely, we show that stabilized ICBs help resist excessive fracturing. Additionally, we find that motility and thus fracture rates gradually decrease during development in a sex-dependent manner, completely ceasing by the end of cyst-forming divisions. These results lead to a model where the opposing activities of developmentally regulated cell motility and stable ICBs give rise to cysts of variable sizes. We corroborate these results by developing a model that uses experimentally measured fracture rates to simulate cyst formation and fracture and show that it can reproduce experimentally measured cyst sizes in both male and female. Understanding how variable cysts form will enable further studies of mammalian oocyte selection and establishment of the ovarian reserve., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cut-open oocyte Vaseline gap technique is a powerful electrophysiological method for the characterization of ion channels. However, traditional amplifiers for cut-open oocyte Vaseline gap are labor intensive and require significant user expertise. We introduce an innovative, open-source digital amplifier system with high-speed digitization and software-controlled electronics for computer-driven automation. This system compares well to existing commercial systems in terms of conventional specifications of step response (current peak at 25μs and decay of 36μs time constant), current noise (1.0 nA at 3-kHz bandwidth), and dynamic range (96.9 dB). Additionally, it unlocks new methods through close integration of the amplifier and software, including machine-learning techniques for tuning capacitive compensation waveforms, achieving a 100-fold suppression of mean-squared transient current, and impedance measurement methods to identify system components such as membrane capacitance and electrode resistances. For future extensions, the design has unique attributes such as real-time digital signal processing for feedback, multiple input and multiple output, and allows for user customization. By providing open-source access to the circuit board designs, control software, and field-programmable gate array code on GitHub, this approach aims to foster cross-disciplinary collaboration and facilitate instrument customization enabling previously inaccessible electrophysiology experiments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epigenetic mechanisms, including DNA methylation and histone modifications, govern chromatin arrangement in sperm, enhancing motility and safeguarding DNA integrity for accurate epigenetic inheritance. Abnormal methylation is linked to poor sperm quality and fertility issues, underscoring the need to study sperm DNA methylation and its impact on sperm function and embryo development in assisted reproductive technology. In this study, processed spermatozoa from 75 normozoospermic and 15 abnormal ejaculates were examined for sperm global DNA methylation levels using a colourimetric absorbance method. Although semen characteristics were poor in abnormal ejaculates, no significant correlation was found between sperm global DNA methylation levels and sperm characteristics in either normozoospermic or abnormal cohorts. However, mean global DNA methylation levels were significantly lowered in abnormal sperm samples compared to normozoospermic samples ( p  < 0.05). Furthermore, injecting spermatozoa from these patients (N = 50) into donor oocytes did not show a significant relationship between sperm global DNA methylation and embryo developmental competence. These findings highlight the limitation of sperm global DNA methylation as a biomarker for embryo development and quality.

Traditionally, sperm and embryos were studied using microscopy to assess morphology and motility. However, OMICS technologies, especially transcriptomic analysis, are now being used to screen the molecular dynamics of fertility markers at cellular and molecular levels, with high sensitivity. Transcriptomics is the study of the transcriptome - RNA transcripts produced by the genome - using high-throughput methods to understand how the RNAs are expressed. In this review, we have discussed gene contributions to sperm structure and function and their role in fertilization and early embryo development. Further, we identified miRNAs shared by sperm, oocytes, and early embryos and their roles in fertilization and early embryo development., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Published by Elsevier B.V.)

The objective of this study was to investigate the early follicular apoptosis in canine ovarian follicles by examining the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX proteins throughout the estrous cycle associated with oocyte maturation. Follicular cells from preantral and antral follicles of varying sizes were isolated and grouped based on follicle type and estrous phase. Antral follicles underwent flow cytometry analysis, whereas preantral follicles were subjected to Western blotting. The meiotic capacity of oocytes from these different follicle types was evaluated through in vitro maturation. Statistical analysis included ANOVA and Duncan's test. Results showed fluctuations in BCL-2 and BAX levels across different follicular stages and estrous phases. BCL-2 levels increased (P<0.05) with follicular development in antral follicles, particularly during estrus, while BAX exhibited variations peaking (P<0.05) during estrus. The BCL-2/BAX ratio in antral follicles was higher (P<0.05) in estrus and diestrus compared to anestrus and proestrus. Additionally, BCL-2 and BAX proteins were detected in preantral follicles, with varying expression levels (P<0.05) across estrous phases. The BCL-2/BAX ratio in preantral follicles was highest (P<0.05) during anestrus and estrus and decreased (P<0.05) in proestrus and diestrus. Oocytes from preantral follicles did not reach the MII stage, regardless of the levels of BCL-2/BAX. Conversely, oocytes obtained from large follicles during estrus showed the highest (P<0.05) maturation percentages, which were associated with the highest BCL-2/BAX ratio. These findings provide insights into the dynamic patterns of BCL-2 /BAX in canine follicles across the estrous cycle, shedding light on their potential roles in oocyte development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Research Question: Can spermatozoa penetrate maturing metaphase I (MI) oocytes, and render subsequent development following conventional IVF in a mouse model?, Design: ICR mice were used in this study. Metaphase II (MII) cumulus-oocyte complexes (COC) harvested 15 h after injection of human chorionic gonadotrophin (HCG) were used for IVF as the control group (Group 1). In the treatment group (Group 2), maturing MI COC harvested 7 h after HCG injection were used for IVF. Fertilization, pronuclear formation, cleavage, blastocyst formation, DNA methylation status, chromosome number and live birth rates were used to evaluate the developmental dynamics and competency of maturing MI oocytes following conventional IVF., Results: Maturing MI COC were fertilized using conventional IVF, and sperm penetration at MI-telophase I triggered oocyte activation. Most embryos resulting from fertilized MI oocytes developed to blastocyst stage during preimplantation development, albeit a substantial proportion of them were triploids due to the absence of the second meiotic division. Some of the embryos derived from fertilization of maturing oocytes were able to implant and gave rise to full-term development., Conclusion: Maturing MI COC from follicles before ovulation could be used for mouse IVF, and fertilized MI oocytes had high potential for development. Healthy offspring can be generated from maturing MI COC following conventional IVF. MI COC may represent a valuable source of 'usable' biomaterial in assisted reproduction. However, many embryos derived from MI COC via IVF have abnormal chromosome numbers in the mouse model. The implications of these findings for human IVF remain to be investigated., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Growth differentiation factor 9 ( GDF9) is an oocyte-specific paracrine factor involved in bidirectional communication, which plays an important role in oocyte developmental competence. In spite of its vital role in reproduction, there is insufficient information about exact transcriptional control mechanism of GDF9. Hence, present study was undertaken with the aim to study the expression of basic helix-loop-helix (bHLH) transcription factors (TFs) such as the factor in the germline alpha (FIGLA), twist-related protein 1 (TWIST1) and upstream stimulating factor 1 and 2 (USF1 and USF2), and nuclear receptor (NR) superfamily TFs like germ cell nuclear factor (GCNF) and oestrogen receptor 2 (ESR2) under three different in vitro maturation (IVM) groups [follicle-stimulating hormone (FSH), insulin-like growth factor-1 (IGF1) and oestradiol)] along with all supplementation group as positive control, to understand their role in regulation of GDF9 expression. Buffalo cumulus-oocyte complexes were aspirated from abattoir-derived ovaries and matured in different IVM groups. Following maturation, TFs expression was studied at 8 h of maturation in all four different IVM groups and correlated with GDF9 expression. USF1 displayed positive whereas GCNF, TWIST1 and ESR2 revealed negative correlation with GDF9 expression. TWIST1 & ESR2 revealing negative correlation with GDF9 expression were found to be positively correlated amongst themselves also. GCNF & USF1 revealing highly significant correlation with GDF9 expression in an opposite manner were found to be negatively correlated. The present study concludes that the expression of GDF9 in buffalo oocytes remains under control through the involvement of NR and bHLH TFs.

We aimed to establish efficient donor cells to produce piglets by somatic cell nuclear transfer (SCNT) of the endangered Vietnamese Ỉ pig. In Experiment 1, we assessed the effects of cell passages on the in vitro development of SCNT embryos. Cells with five and six passages showed significantly cleaved and blastocyst formation rates (86.72 and 86.64; 35.68 and 35.51, respectively, P < 0.05). The highest average total cell number per blastocyst was observed in groups of cells with five and six passages (50.45 and 50.18, respectively). Experiment 2 was performed to assess the sex of donor cells on the subsequent development of SCNT embryos. There was no significant difference in the cleaved and blastocyst formation rates, and the average total cell between female and male groups (86.51 % vs 86.94 % and 35.31 % vs 35.08 %, 50.29 % vs 50.67 %, respectively, P > 0.05). Experiment 3 was performed to assess the effect of cell lines on the development of SCNT embryos. Our results showed no significant difference in the success rate of fibroblast nuclear transfer into recipient oocytes, the cleaved and blastocyst formation rates, and the average total cell number per blastocyst among the cell lines 6004, 9154, 9155, 9156 and 9157 (P > 0.05). Experiment 4 was performed to assess the ability of SCNT embryos to induce pregnancy and to develop term. SCNT embryos were produced from Ỉ fibroblast cells established based on the results of Experiments 1, 2 and 3. Transfer of blastocyst stage embryos into 19 recipients (100-120 embryos in each) resulted in 14 pregnancies, in which 8 pregnant females terminated on Day 22-42 and 6 others produced 20 cloned piglets from donor cells of a female pig but 5 piglets died before birth and 15 healthy cloned piglets. However, 3 out of 15 healthy piglets died of unknown causes within 24h of birth and 3 out of 15 healthy piglets died at 3-5 days of age due to diarrhoea, 9 out of 15 healthy piglets are now 3 months of age. Finally, we established a protocol for the donor cell production which enabled the production of the endangered Ỉ pig embryos by SCNT and maximized blastocyst production rate by more than 35 % and pregnant rate after the transfer of cloned Ỉ pig embryos to recipients at 73.68 % for the first time in Vietnam., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The efficacy of in vitro embryo production (IVEP) in equines is relatively limited compared to other species due to the lack of a reliable superovulation technique, limited availability of cumulus oocyte complexes (COCs), low in vitro oocyte maturation (IVM) and fertilization rates. Extracellular vesicles (EVs), which are nanoparticles involved in intercellular signaling in the ovarian environment, have shown potential as supplements to improve oocyte development during IVM. This study tested the hypothesis that EVs from small (< 20 mm) ovarian follicles could enhance fertilization rates in mares. Follicular fluid was collected postmortem, and EVs were isolated and characterized. The IVM process was conducted with or without EVs (200 µg EV protein/ml). EV internalization during IVM was examined using fluorescent labeling and confocal microscopy. Following intracytoplasmic sperm injection (ICSI), presumptive zygotes were cultured in a time-lapse system. Confocal microscopy confirmed EV internalization by COCs. Nanoparticle tracking analysis showed that obtained EVs were submicron-sized, and flow cytometry identified surface markers CD81 and CD63 on a subpopulation of EVs. Transmission electron microscopy revealed the characteristic disk shape of EV isolates. After culture, 196 oocytes (36.84 %) exhibited a first polar body and were subjected to ICSI. The EV-treated group showed a significantly higher fertilization rate (34.7 % vs. 20.2 %; P < 0.05), reduced degeneration, and increased cleavage efficiency (P < 0.1). Despite early embryonic arrest in both groups, these results suggest that follicular fluid-derived EVs could play a supportive role in equine IVF procedures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 10 6 particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 10 7 particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 10 7 particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human oocyte maturation is a lengthy process that takes place over the course of which oocytes gain the inherent ability to support the next developmental stages in a progressive manner. This process includes intricate and distinct events related to nuclear and cytoplasmic maturation. Nuclear maturation includes mostly chromosome segregation, whereas rearrangement of organelles, storage of mRNAs and transcription factors occur during cytoplasmic maturation.Human oocyte maturation, both in vivo and in vitro, occurs through a process that is not yet fully understood. However, it is believed that the second messenger, cyclic adenosine monophosphate (cAMP), plays a pivotal role in the upkeep of the meiotic blocking of the human oocyte. Relatively high levels of cAMP in the human oocyte are required to maintain meiosis blocked, whereas lower levels of cAMP in the oocyte enable meiosis to resume. Oocyte cAMP concentration is controlled by a balance between adenylate cyclase and phosphodiesterases, the enzymes responsible for cAMP generation and breakdown.In addition to nuclear maturation, the female gamete requires a number of complicated structural and biochemical modifications in the cytoplasmic compartment to be able to fertilize normally. According to ultrastructural studies, during the transition from the germinal vesicle stage to metaphase II (MII), several organelles reorganize their positions. The cytoskeletal microfilaments and microtubules found in the cytoplasm facilitate these movements and regulate chromosomal segregation.The aim of this review is to focus on the nuclear and cytoplasmic maturation by investigating the changes that take place in the process of oocytes being competent for development.

In vitro production of porcine embryos is a complicated process that includes in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Insufficient cytoplasmic maturation, slow zona reaction and improper embryo culture conditions will compromise the efficiency of porcine embryo production in vitro . Previous studies have shown that insulin-transferrin-selenium (ITS) in IVM or IVC medium could improve porcine oocyte maturation, decrease polyspermy fertilization and promote subsequent embryonic development in vitro . However, the effect of ITS both in IVM and IVC media on porcine embryo production in vitro hasn't been elucidated. In this study, we found that 1.0% ITS supplementation in IVM/IVC media promoted the expansion of cumulus cells, raised mitochondrial membrane potential, increased ATP content and reduced ROS level in matured oocytes, improved blastocyst rate and the cell number of blastocyst, simultaneously. In conclusion, the IVM/IVC media supplemented with 1.0% ITS can improve the efficiency of porcine embryo production in vitro .

The objective of this research was to assess the viability and developmental potential of feline oocytes following in vitro maturation (IVM), vitrification, and post-warming incubation with resveratrol. In the first experiment, warmed oocytes were incubated with 0.2 μM, 2 μM, or 20 μM resveratrol for 2 h. Oocytes treated with 0.2 μM resveratrol had the highest viability (68.89 %), as assessed by fluorescein diacetate and ethidium bromide staining, while higher concentrations were associated with diminished oocyte viability. In the second experiment, the warmed oocytes were inseminated following the 2-h incubation with the three concentrations of resveratrol. The presumptive zygotes were then maintained in culture and their development evaluated. The highest cleavage rate was observed when the oocytes had been incubated with 0.2 μM resveratrol (88.34 %), which was higher than for the control group (without resveratrol (75 %)). Moreover, this concentration of resveratrol also augmented the blastocyst formation rate. While the vitrification of oocytes often results in diminished developmental potential in the ensuing embryos, attributed to cryopreservation-induced injury, the utilization of low concentrations of resveratrol enhances the procedure's efficacy., (Copyright © 2024. Published by Elsevier Inc.)

Dengue fever poses a global public health threat, with 2.5 billion people at risk of infection each year. Because the Aedes albopictus is the primary vector of dengue, it is closely monitored and handled. The efficiency of Dengue eradication is strongly dependent on understanding a female mosquito's physiological age. This study addresses key entomological issues about the impact of previtellogenic nutrition on egg production mechanisms. Ovarian development included two distinct periods: previtellogenesis and vitellogenesis. Sugar intake during previtellogenesis influences the size of the blood meal. The major parameter influencing the vitellogenesis process is the presence of a hematophagous feeding event following sugar concentration. Upon subjecting female mosquitoes to sucrose, the ovarian follicles entered the third phase of previtellogenesis. Once females feed on blood following sucrose, ovarian development enters the vitellogenesis, and the oocyte cytoplasm reveals that the yolk granules are organized in one or two rows like a crown, increasing oocyte size. Females fed 15% sucrose before a blood meal, have the largest vitellogenic growth, and follicular size, which is seven times greater than those fed water only. Fecundity increased by 78.7% by adding 7% sucrose to the diet. Mitochondria within oocytes increase, most likely due to their transportation from the nurse cells, where the yolk is synthesized. This study describes in detail the histological alterations detected in the ovaries during the previtellogenesis as well as those associated with yolk formation, suggesting that yolk protein deposition in the oocyte is associated with blood meal, independent of sucrose feeding. RESEARCH HIGHLIGHTS: Adult nutrition during previtellogenesis significantly impacts various biological parameters and the physiological age of adults of Aedes albopictus. Female mosquitoes experienced significant growth in vitellogenic development, vectorial capacity, and follicular size after consuming a diet with 15% sucrose before a blood meal., (© 2024 Wiley Periodicals LLC.)

Female fertility preservation via complete in vitro folliculogenesis is still chimerical. Due to many factors affecting the efficiency of isolation and culture of preantral follicles, the improvement of techniques geared to fertility preservation in higher mammals seems to be at an impasse. We need an objective view of the current stand to understand how to progress further. As such, a survey was conducted to analyze the relative distribution of studies performed in ten mammalian species on preantral follicle culture available on PubMed. Using the bovine as a reference model, we explore some factors influencing data variation that contribute to the difficulty in reproducing studies. While years of research have enabled the recapitulation of folliculogenesis from as modest as the early antral follicle stage ex vivo, in vitro preantral folliculogenesis remains elusive. Herein, we revisit the classical evidence that laid the foundations for understanding preantral folliculogenesis and review the length, breadth, and depth of information that the era of big data has currently levied. Moving forward, we recognize the urgency of synthesizing the multi-disciplinary approaches to mimic folliculogenesis in vitro to achieve a translational landscape of infertility at individual and large-scale conservation levels., Competing Interests: Declarations. Conflicts of interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The current narrative review aims to summarize the relation of mitochondrial content (MC) and mitochondrial DNA copy number (MDCN) in spermatozoa with sperm penetrability, and to discuss the various determining factors during the process of spermatogenesis in mammals. There are many potential factors associated with the quantitative alteration of MC and MDCN in male gametes from spermatogenesis to ejaculation. Particularly, spermatogenesis may be the first step to jointly contribute to an incomplete reduction of MC and MDCN in spermatozoon. It appears to be now quite clear that some abnormalities during spermatogenesis and oxidative stress are the main factors highly associated with the quantitative change of MC and MDCN in spermatozoa, consequently affecting sperm quality and their penetrability into oocytes. Currently, a series of proteins contributing to form sperm midpiece during spermatogenesis and cytoplasmic elimination during spermiation have been currently identified. The present review provides insight into how these factors interact with sperm MC and MDCN, and handholds to gain a better understanding of their roles. This review also highlights the uniqueness of normal fertile spermatozoa which have relatively lower MC and MDCN, but have mitochondria that function completely in multiple pivotal physiological pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In vitro embryo production (IVP) technology has been increasingly applied to beef cattle breeding. In vitro maturation (IVM) technology is the basis of IVP. However, the quality of in vitro-generated mature oocytes is still poor. Mitochondria are the energy factories of oocytes, so they are crucial for oocyte quality. OPA1 is a protein located on the mitochondrial inner membrane, and its main function is to mediate mitochondrial inner membrane fusion. This work demonstrated that OPA1 is expressed at different stages of meiosis in bovine oocytes. The inhibition of OPA1 activity resulted in a reduced rate of first polar body excretion from bovine oocytes and disruption of the spindle structure. OPA1 deficiency impacted mitochondria by leading to mitochondrial dysfunction, promoting mitochondrial fission, and inducing mitophagy through the PINK1/Parkin pathway. Taken together, our findings suggest that OPA1 is essential for bovine oocyte maturation and that OPA1 deficiency leads to mitochondrial dysfunction and promotes mitochondrial fission as well as mitophagy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous research has highlighted the significant role of progestins and glucocorticoids in fish oocyte maturation and ovulation. To clarify the molecular mechanisms underlying these processes, comprehensive investigations were conducted using a cyp17a2 mutant Nile tilapia ( Oreochromis niloticus ) model. Analysis revealed pronounced Cyp17a2 expression in ovarian somatic cells of the tilapia. Female cyp17a2 -deficient mutants exhibited markedly reduced levels of 17,20β-dihydroxy-4-pregnen-3-one (DHP) and cortisol/cortisone, leading to delayed meiotic initiation and impaired oocyte maturation and spawning. Notably, supplementation with human chorionic gonadotrophin (hCG), DHP, and cortisol effectively induced germinal vesicle breakdown (GVBD) and facilitated oocyte release with follicular cell layers in cyp17a2 -/- females. Additionally, cyp17a2 -/- and rescued cyp17a2 -/- females showed elevated transcription of steroidogenic enzymes involved in 17β-estradiol (E2) production compared to spawning wild-type females. Moreover, the reduction in Akt phosphorylation observed in cyp17a2 -deficient females and upon inhibitor treatment impaired hCG-induced oocyte maturation. Conversely, activation of the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) signaling pathway partially rescued the oocyte maturation impairment caused by cyp17a2 mutation. Overall, these findings provide functional evidence supporting the critical role of Cyp17a2 in DHP and cortisol biosynthesis, which, in turn, facilitates oocyte maturation and ovulation through activation of the PI3K-Akt signaling pathway in fish.

In Brief: O-GlcNAc plays an important role in many age-related diseases. This study shows that O-GlcNAc participates in oocyte aging and that reducing O-GlcNAc levels in aging oocytes improves oocyte quality., Abstract: With an increase in the mean age at parturition worldwide, female reproductive aging has become a key health problem. Advanced maternal age is reflected by decreased oocyte quality; however, the molecular mechanisms of oocyte aging are uncharacterized. O-linked N-acetylglucosamine (O-GlcNAc), a dynamic posttranslational modification, plays a critical role in the development of many age-related diseases; yet, it remains unclear whether and how O-GlcNAc participates in oocyte aging. Here, we found that global O-GlcNAc was elevated in normal biological aging mice oocytes (9 months), which were characterized by meiotic maturation failure and impaired mitochondrial function. Specifically, O-GlcNAc targeted the mitochondrial fission protein dynamic-related protein 1 to mediate mitochondrial distribution in the process of aging. Using the O-GlcNAcase (OGA) pharmacological inhibitor Thiamet-G and Oga knockdown (Oga-KD) to mimic the age-related high O-GlcNAc in young oocytes from 6-8 week-old mice mimicked the phenotype of oocyte aging. Moreover, reducing O-GlcNAc levels in aging oocytes restored spindle organization to improve oocyte quality. Our results demonstrate that O-GlcNAc is a key regulator of meiotic maturation that participates in the progression of oocyte aging.

In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C. elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28 ELYS . Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28 ELYS . Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2 M18BP1 (or MEL-28 ELYS ) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2 M18BP1 and MEL-28 ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2 M18BP1 recruits MEL-28 ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C. elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2 M18BP1 and MEL-28 ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024.)

The levels of nicotinamide adenine dinucleotide (NADH) dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2, a subunit of NADH dehydrogenase) decrease in aged tissues, and these reductions may be partly associated with age-related conditions such as Parkinson's disease. Aging leads to many mitochondrial defects, such as biogenesis disruption, dysfunction, defects in the mitochondrial membrane potential, and production of reactive oxygen species, that may be highly related to NDUFS2 expression. The relationship between NDUFS2 and postovulatory oocyte aging in pigs remains unknown. In this study, we investigated changes in NDUFS2 expression during postovulatory aging (POA). Furthermore, NDUFS2 was knocked down via dsRNA microinjection at the MII stage to evaluate the effects on mitochondrial-related processes during POA. The mRNA expression of NDUFS2 decreased significantly after 48-h aging compared with that in fresh oocytes. NDUFS2 knockdown (KD) significantly impaired the maintenance of oocyte morphology and blastocyst development of embryos after POA. The levels of PGC1α (mitochondrial biogenesis-related proteins) decreased significantly after NDUFS2 KD, while the level of GSNOR, a protein denitrosylase, was reduced by NDUFS2 KD after 48 h of aging. These data suggest that NDUFS2 is vital for maintaining the oocyte quality during POA in pigs., Competing Interests: Conflict of Interest: The authors declare that they have no competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

Ovum Pick Up (OPU) is a minimally invasive technique widely used in cattle and mares for oocyte retrieval, involving ultrasound-guided puncture of ovarian follicles. It has been demonstrated that this technique is safe for its repeated use in the same female without affecting her reproductive health, allowing for the retrieval of oocytes in individuals regardless of their reproductive status. The oocytes obtained through OPU can subsequently be used for in vitro embryo production (IVP) using assisted reproductive techniques (ARTs) or be cryopreserved in biobanks for their future use. Traditionally, the minimally invasive technique of choice performed in vivo in domestic and wild felines was LOPU (laparoscopic-guided ovum pick up). The present study was designed to explore if ultrasound-guided OPU in the domestic cat is safe and effective. In an initial series of ex vivo experiments (n = 92 ovaries, n = 434 oocytes), the effect of different aspiration pressures for oocyte collection was explored. These experiments identified 43 mmHg as the optimal aspiration pressure, resulting in the highest recovery rate and a favorable maturation and blastocyst rate. Subsequently, 16 grade I and II oocytes were retrieved by OPU and 101 oocytes were retrieved following ovariectomy and slicing. Sixteen oocytes obtained with each technique were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). A total of 14 presumptive zygotes were selected for in vitro culture (IVC) from each group (OPU and slicing), obtaining a cleavage rate of 57.1 % and 64.2 %, a morula rate of 28.5 % in both groups, and a blastocyst rate of 7.14 % and 14.2 % respectively. The hormonal stimulation protocol was well-tolerated, with no adverse effects observed. Moreover, no complications arose during the ovariectomy performed post-OPU. The use of this technique in domestic cats represents a significant step forward in terms of safety, replicability, and invasiveness, serving as a valuable model for its application in wild felids species. Additional research involving a greater number of animals is required to validate these encouraging findings., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fertilization capacity and embryo survival rate are decreased in postovulatory aging oocytes, which results in a reduced reproductive rate in female animals. However, the key regulatory genes and related regulatory mechanisms involved in the process of postovulatory aging in oocytes remain unclear. In this study, RNA-Seq revealed that 3237 genes were differentially expressed in porcine oocytes between the MII and aging stages (MII + 24 h). The expression level of FOXM1 was increased at the aging stage, and FOXM1 was also observed to be enriched in many key biological processes, such as cell senescence, response to oxidative stress, and transcription, during porcine oocyte aging. Previous studies have shown that FOXM1 is involved in the regulation of various biological processes, such as oxidative stress, DNA damage repair, mitochondrial function, and cellular senescence, which suggests that FOXM1 may play a crucial role in the process of postovulatory aging. Therefore, in this study, we investigated the effects and mechanisms of FOXM1 on oxidative stress, mitochondrial function, DNA damage, and apoptosis during oocyte aging. Our study revealed that aging oocytes exhibited significantly increased ROS levels and significantly decreased GSH, SOD, T-AOC, and CAT levels than did oocytes at the MII stage and that FOXM1 inhibition exacerbated the changes in these levels in aging oocytes. In addition, FOXM1 inhibition increased the levels of DNA damage, apoptosis, and cell senescence in aging oocytes. A p21 inhibitor alleviated the effects of FOXM1 inhibition on oxidative stress, mitochondrial function, and DNA damage and thus alleviated the degree of senescence in aging oocytes. These results indicate that FOXM1 plays a crucial role in porcine oocyte aging. This study contributes to the understanding of the function and mechanism of FOXM1 during porcine oocyte aging and provides a theoretical basis for preventing oocyte aging and optimizing conditions for the in vitro culture of oocytes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Cumulus-oocyte complex (COC) expansion and oocyte maturation are crucial processes for embryo development and fertility across species. Although miR-29b has been detected in porcine ovarian granulosa cells, its specific role in regulating oocyte maturation remains largely unknown. In this study, using the pig as a model, we report that over-expression of miR-29b lead to a decrease of COC expansion area and inhibits oocyte maturation (P<0.05). This suppression correlated with a decrease expression of COC-expansion-associated genes, including SHAS2, ADAMTS1, ADAMTS2, ADAMTS17 and PTX 3 in both mural granulosa cells (mGCs) and cumulus granulosa cells (cGCs). Further investigation revealed that miR-29b over-expression induces reactive oxygen species (ROS) accumulation in both mGCs and cGCs, conversely, knock-down of miR-29b reverses all these effects. Treatment with the antioxidant β-mercaptoethanol alleviates ROS accumulation, rescues COC expansion and restores oocyte polar body formation impaired by miR-29b mimics. Computational analysis predicted CYCS, the gene encoding cytochrome C, as a potential target of miR-29b. Subsequent examination demonstrated that miR-29b downregulates CYCS at both mRNA and protein levels. Dual-luciferase reporter assays further confirmed that miR-29b interacts with the 3'-untranslated region (3'UTR) of CYCS. Over-expression of CYCS decreases ROS accumulation and promotes COC expansion (P<0.05). These results indicate that miR-29b regulates COC expansion and oocyte maturation in vitro by inducing ROS, likely through targeting of CYCS. This study sheds light on the role of miR-29b in oocyte maturation and provides insight into the regulatory function of miRNAs in ovarian physiology., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall in vitro maturation efficiency and early embryonic stages., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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) Ca 2+ , and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca 2+ , 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, Ca 2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare that they have no interest conflict., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Calcium ions (Ca 2+ ) regulate cell proliferation and differentiation and participate in various physiological activities of cells. The calcium transfer protein inositol 1,4,5-triphosphate receptor (IP 3 R), located between the endoplasmic reticulum (ER) and mitochondria, plays an important role in regulating Ca 2+ levels. However, the mechanism by which IP 3 R1 affects porcine meiotic progression and embryonic development remains unclear. We established a model in porcine oocytes using siRNA-mediated knockdown of IP 3 R1 to investigate the effects of IP 3 R1 on porcine oocyte meiotic progression and embryonic development. The results indicated that a decrease in IP 3 R1 expression significantly enhanced the interaction between the ER and mitochondria. Additionally, the interaction between the ER and the mitochondrial Ca 2+ ([Ca 2+ ] m ) transport network protein IP 3 R1-GRP75-VDAC1 was disrupted. The results of the Duolink II in situ proximity ligation assay (PLA) revealed a weakened pairwise interaction between IP 3 R1-GRP75 and VDAC1 and a significantly increased interaction between GRP75 and VDAC1 after IP 3 R1 interference, resulting in the accumulation of large amounts of [Ca 2+ ] m . These changes led to mitochondrial oxidative stress, increased the levels of reactive oxygen species (ROS) and reduced ATP production, which hindered the maturation and late development of porcine oocytes and induced apoptosis. Nevertheless, after treat with [Ca 2+ ] m chelating agent ruthenium red (RR) or ROS scavenger N-acetylcysteine (NAC), the oocytes developmental abnormalities, oxidative stress and apoptosis caused by Ca 2+ overload were improved. In conclusion, our results indicated IP 3 R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Context The number of developmentally competent cumulus-oocyte complexes (COCs) retrieved during Ovum Pick-Up (OPU) determines success in both bovine and human assisted reproduction. Follicular flushing for COC retrieval is practicsed widely in humans but not in cattle. Aims To determine the benefits of follicular flushing in cattle and assess the merits of a novel 16G double-lumen needle ('OxIVF') that flushes laterally to the needle shaft. Methods Experiment 1 flushed 655 antral follicles (≥7mm) from 255 abattoir-derived cattle ovaries. In Experiment 2, 12 Holstein heifers underwent two cycles of OPU in a cross-over design comparing both needle types. In Experiment 3, 11 Holstein heifers underwent two cycles of OPU using the OxIVF needle in a cross-over design: flushing (≥7mm follicles) vs a 'Hybrid' approach of flushing (≥7mm follicles) and aspiration (5-7mm follicles); followed by two cycles of standard follicle aspiration (>5mm follicles). Key results In Experiment 1, COC recovery was greater (P =0.034) for the OxIVF vs Standard needle (mean±s.e.; 74.1±2.10% vs 67.0±2.23%); yield of Grade 1 COCs was also greater (20.1±1.97% vs 8.2±1.38%; P P =0.045) for the OxIVF vs Standard needle (89.1±2.98% vs 79.6±3.47%). Day 6 embryo yield was also greater (P =0.017) for the OxIVF vs Standard needle (87.2±4.38% vs 67.6±6.73%). In Experiment 3, recovery of COCs was greater (P =0.033) for 'Flush' vs 'Aspirate' groups (82.1±5.06% vs 66.2±3.48%). However, number of Day 8 blastocysts for the 'Hybrid' vs 'Flush' approach (9.2±1.39vs 6.5±1.05 per cycle) did not reach statistical significance. Conclusions Follicular flushing using the OxIVF needle, embracing the 'Hybrid' approach, has the potential to increase oocyte retrieval and blastocyst number per donor cycle in cattle but requires further validation. Implications Larger scale studies will seek to confirm benefits of follicular flushing using the OxIVF needle in cattle. Future studies should consider applications in both equine and human assisted reproduction.

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM + ) or in α-MEM + plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM + presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM + (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type-specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues., Competing Interests: EB, KV, LZ, CG, SE, MS, JS, FD No competing interests declared, (© 2024, Bomba-Warczak, Velez et al.)