Oocyte Growth Research Articles

EB-SUN, a new microtubule plus-end tracking protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, whichencodes a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing MT plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in decreased MT density at the onset of mid-oogenesis (stage 7) and delays oocyte growth during late mid-oogenesis (stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduces MT density at stage 7. Hatching assays of single protein depletion reveal distinct roles for EB-SUN and EB1 in early embryogenesis, likely due to differences in theirexpression and binding partners. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, suggesting partial redundancy between these proteins.

A Significant Correlation of Glutamine and Interlukin-6 with Pregnancy Rate in Women Undergoing ICSI

Background: The composition of follicular fluid (FF) is significantly influenced by metabolic factors, and the analyses of metabolites in FF could reveal the correlation between metabolites and oocyte quality, embryo development, and pregnancy outcome. FF provides nutrients and growth factors that promote oocyte growth, development, and maturation. Objective: To investigate the relationship between serum and FF glutamine and FF IL-6 levels and their influence on the pregnancy rate in women who are undergoing an ICSI treatment cycle. Materials and Methods: A prospective cross-sectional study was performed on 90 women aged 20- 44 years who underwent ICSI treatment from July 2023 to February 2024. Serum and follicular fluid collected on the day of oocyte pick-up were used as biological material. The embryological laboratory examined the retrieved follicular fluid and collected oocytes using stereomicroscopy. ELISA analysed serum, FF glutamine and FF IL-6. The ICSI method was utilized for fertilization. The pregnancy test is done by measuring serum BHCG. Statistical data processing was conducted using the software package SPSS 23. Results: There was a highly significant correlation between FF glutamine and FF IL-6 (p < 0.001) and a positive correlation between FF glutamine and FF IL-6 with pregnancy rate ( p < 0.001) (p < 0.001 ). Conclusion The correlation of FF glutamine and FF IL-6 affects the pregnancy rate in women undergoing ICSI. FF glutamine and FF IL-6 can be considered predictors for pregnancy while serum glutamine has no significance.

Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid.

Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation-the peak breeding season (Clusters 4 and 6). The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

Integrative Omics Reveals the Metabolic Patterns During Oocyte Growth

Integrative Omics Reveals the Metabolic Patterns During Oocyte Growth

Changes in DNA repair compartments and cohesin loss promote DNA damage accumulation in aged oocytes.

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.

RNA surveillance by the RNA helicase MTR4 determines volume of mouse oocytes.

Oocytes are the largest cell type in multicellular animals. Here, we show that mRNA transporter 4 (MTR4) is indispensable for oocyte growth and functions as part of the RNA surveillance mechanism, which is responsible for nuclear waste RNA clearance. MTR4 ensures the normal post-transcriptional processing of maternal RNAs, their nuclear export to the cytoplasm, and the accumulation of properly processed transcripts. Oocytes with Mtr4 knockout fail to accumulate sufficient and normal transcripts in the cytoplasm and cannot grow to normal sizes. MTR4-dependent RNA surveillance has a previously unrecognized function in maintaining a stable nuclear environment for the establishment of non-canonical histone H3 lysine-4 trimethylation and chromatin reorganization, which is necessary to form a nucleolus-like structure in oocytes. In conclusion, MTR4-dependent RNA surveillance activity is a checkpoint that allows oocytes to grow to a normal size, undergo nuclear and cytoplasmic maturation, and acquire developmental competence.

Effects of Astaxanthin on the Physiological State of Porcine Ovarian Granulose Cells Cultured In Vitro.

The physiological state of Granulosa cells (GCs) is intricately linked to the growth and development of oocytes. Oxidative stress has been found to cause damage to GCs in vitro. Astaxanthin (AST), a well-known natural ketone-type carotenoid, has demonstrated strong antioxidant properties. This study investigates the impact of astaxanthin supplementation on the physiological state of porcine ovarian granulosa cells cultured in vitro. Variations in morphology, apoptosis, reactive oxygen species (ROS) levels, and the expression of apoptosis and anti-oxidation-related genes in porcine GCs from different passages were observed. Significant morphological changes, increases in apoptosis, and decreases in antioxidant capacity resulting from passage were observed. Subsequently, treatment with 5 μmol/L astaxanthin significantly enhanced cell viability, proliferation, antioxidant capacity and mitochondrial function while also regulating the estradiol (E2) and progesterone (P4) levels. Additionally, the gene expression of antioxidation, E2, and P4 synthesis markers was assessed, revealing reduced apoptosis and ROS levels in porcine GCs. In conclusion, supplementation with 5 μmol/L astaxanthin in vitro effectively enhances the physiological condition of porcine GCs and optimizes the culture system for these cells in vitro. Optimizing the culture system of porcine GCs in vitro can simulate the function of granulosa cells in vivo and provide a theoretical reference for further promoting follicular development, which is beneficial to improving sow fertility in actual production.

Melatonin facilitates oocyte growth in goats and mice through increased nutrient reserves and enhanced mitochondrial function.

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 invivo 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.

Rejuvenation of aged oocyte through exposure to young follicular microenvironment.

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.

Node Date Estimation of Diversified Species of Ray-Finned Fish Based on Morphological and Molecular (Vitellogenin 3) Characters Using Bayesian Analysis

Fish and other oviparous and ovoviviparous animals contain vitellogenin (Vtg), an egg yolk precursor phospholipoglycoprotein, which is the prerequisite for oocyte growth during oogenesis. Vtg is also employed as a biomarker to assess the reproductive health of female fish. In this study, we select 34 ray-finned fish under the class Actinopterygii to investigate the evolutionary time scale of their diversification age based on the molecular (vtg3 gene) as well as the combined (morphological and vtg3 gene) characters using different evolutionary models in Bayesian analysis. The analysis indicates that the species of different orders evolved in different epochs. In the case of molecular-based analysis, the orders Cyprinodontiformes and Perciformes are diversified in early Eocene to Oligocene, Anabantiformes in late Eocene, Cypriniformes in Oligocene & Salmoniformes Miocene. In the case of morpho-molecular based analysis the order Cyprinodontiformes is diversified in early Eocene to Oligocene, Anabantiformes in late Eocene, Perciformes in late Eocene to Oligocene, Cypriniformes in Oligocene to Miocene & Salmoniformes Oligocene to Pliocene. However, both analyses suggest that most of the selected species are diversified in Miocene, Oligocene, Eocene, and Paleocene Epoch between 5 – 65 MYA.

From follicle to blastocyst: microRNA-34c from follicular fluid-derived extracellular vesicles modulates blastocyst quality

BackgroundWithin the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent).MethodsBovine antral follicles were dissected, categorized as small (2–4 mm) or large (5–8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM).ResultsWe identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect.ConclusionThis study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

Oocyte-specific deletion of eukaryotic translation initiation factor 5 causes apoptosis of mouse oocytes within the early-growing follicles by mitochondrial fission defect-reactive oxygen species-DNA damage.

Mutations in several translation initiation factors are closely associated with premature ovarian insufficiency (POI), but the underlying pathogenesis remains largely unknown. We generated eukaryotic translation initiation factor 5 (Eif5) conditional knockout mice aiming to investigate the function of eIF5 during oocyte growth and follicle development. Here, we demonstrated that Eif5 deletion in mouse primordial and growing oocytes both resulted in the apoptosis of oocytes within the early-growing follicles. Further studies revealed that Eif5 deletion in oocytes downregulated the levels of mitochondrial fission-related proteins (p-DRP1, FIS1, MFF and MTFR) and upregulated the levels of the integrated stress response-related proteins (AARS1, SHMT2 and SLC7A1) and genes (Atf4, Ddit3 and Fgf21). Consistent with this, Eif5 deletion in oocytes resulted in mitochondrial dysfunction characterized by elongated form, aggregated distribution beneath the oocyte membrane, decreased adenosine triphosphate content and mtDNA copy numbers, and excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Meanwhile, Eif5 deletion in oocytes led to a significant increase in the levels of DNAdamageresponse proteins (γH2AX, p-CHK2 and p-p53) and proapoptotic proteins (PUMA and BAX), as well as a significant decrease in the levels of anti-apoptotic protein BCL-xL. These findings indicate that Eif5 deletion in mouse oocytes results in the apoptosis of oocytes within the early-growing follicles via mitochondrial fission defects, excessive ROS accumulation and DNA damage. This study provides new insights into pathogenesis, genetic diagnosis and potential therapeutic targets for POI. Eif5 deletion in oocytes leads to arrest in oocyte growth and follicle development. Eif5 deletion in oocytes impairs the translation of mitochondrial fission-related proteins, followed by mitochondrial dysfunction. Depletion of Eif5 causes oocyte apoptosis via ROS accumulation and DNA damage response pathway.

Attenuation of endoplasmic reticulum stress improves invitro growth and subsequent maturation of bovine oocytes

Endoplasmic reticulum (ER) stress interferes with developmental processes in oocyte maturation and embryo development. Invitro growth (IVG) is associated with low developmental competence, and ER stress during IVG culture may play a role. Therefore, this study aimed to examine the effect of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, on the IVG of bovine oocytes to understand the role of ER stress. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles (1.5–1.8 mm) and allowed to grow in vitro for 5 days at 38.5 °C in a humidified atmosphere containing 5 % CO2. Basic growth culture medium was supplemented with TUDCA at various concentrations (0, 50, 100, 250, and 500 μM). After IVG, oocyte diameters were similar among groups, but the antrum formation rate tended to be higher in the TUDCA 100 μM group. The mRNA expression levels of ER stress-associated genes (PERK, ATF6, ATF4, CHOP, BAX, IRE1, and XBP1) in OGCs were downregulated in the TUDCA 100 μM group than those in the control group. Moreover, the TUDCA 100 μM group exhibited reduced ROS production with higher GSH levels and improved in vitro-grown oocyte maturation compared with those in the control group. In contrast, no difference in the developmental competence of embryos following invitro fertilization was observed between the control and TUDCA 100 μM groups. These results indicate that ER stress could impair IVG and subsequent maturation rate of bovine oocytes, and TUDCA could alleviate these detrimental effects. These outcomes might improve the quality of oocytes in IVG culture in assisted reproductive technology.

Temporal metabolomics analysis reveals the metabolic patterns in goat cumulus cells during oocyte maturation

Cumulus cells play a crucial role in the oocyte growth and maturation processes through providing necessary nutrients and growth signals by gap junction communication. However, a global overview of metabolic events in goat cumulus cells is still lacking. In the present study, we collected cumulus cells from goat cumulus-oocyte complexes (COCs) at different developmental stages. Metabolomics analysis was performed to investigate the global metabolic patterns in cumulus cells during oocyte in vitro maturation. In particular, we revealed the several significantly altered metabolic pathways and metaboliccharacteristics in goat cumulus cells, including the accumulation of fatty acids, steroid hormones metabolism, active catabolism of arginine during meiotic resumption, and a progressive decline in nucleotide metabolism. In conclusion, the dataset generated by our metabolomic profiling will provide valuable information to understand the key metabolic pathways and metabolites involved in COCs development.

P-661 Comparative and descriptive molecular analysis of cumulus and luteinized mural granulosa cells for steroidogenic function in IVF patients

Abstract Study question Do cumulus granulosa cells (CCs) have steroidogenic function and gonadotropin response like luteinized mural granulosa cells (LMGCs)? Summary answer Yes. Both types of granulosa cells have similar expression levels for the steroidogenic enzymes and hCG/LH receptors. What is known already CCs and LMGCs have distinct roles in follicular development, oocyte growth and maturation, cumulus expansion, ovulation, and luteinization. CCs support oocytes, especially during meiotic resumption, ovulation, and fertilization. They exhibit specialized functions in response to hormones like LH and participate in bidirectional communication with the oocyte. Although LMGCs are primarily responsible for steroidogenic function and progesterone (P4) production, very limited data is available as to whether CC themselves express the steroidogenic enzymes and gonadotropin receptors and produce sex steroids at basal or after gonadotropin stimulation in humans. We aimed to address this issue in the current study. Study design, size, duration An in-vitro basic science study was designed to compare CCs and LGMCs in the context of vitality, steroidogenic capabilities (both in baseline and response to rec-hCG (10 IU/ml), and EGF pathway responses. The assessment was planned to compare hormonal assay in spent culture and transcriptional levels. The study’s duration encompassed the period of ovarian stimulation, oocyte retrieval, and subsequent cellular analysis. Participants/materials, setting, methods 24 IVF patients undergoing ovarian stimulation with progestin primed (PPOS) and GnRH antagonist protocols for different infertility etiologies were included. Ovarian stimulation and ovulation trigger were done using rec-FSH and rec-hCG, respectively. CCs were collected from the denudation plate. LMGCs were obtained from follicular aspirates during the OPU and cultured individually for each patient without pooling in culture plates. After overnight incubation, the cells were treated with rec-hCG overnight. RNA isolation was performed. Main results and the role of chance The expression of the steroidogenic enzymes (StAR, SCC, 17B-HSD, 3B-HSD, Aromatase), LH receptor and EGF-like amphiregulin (AREG) in CCs were comparable to LMGCs on quantitative RT-PCR analysis. In line with this finding, we found they produced similar amounts of estradiol (E2) and progesterone (P4) in culture. Furthermore, similar to LMGCs, hCG treatment of the CCs resulted in a significant increase in P4 output, indicative of the presence of functional LH receptors. When the culture period was extended to six days, CCs retained their vitality and steroidogenic function like LMGCs and produced detectable E2 and P4. Limitations, reasons for caution Caution should be exercised when interpreting our data as it is unclear if the parameters analyzed here vary according to the infertility etiology, stimulation protocol, mode of trigger and any underlying disease that concomitantly present. Wider implications of the findings Prominent steroidogenic activity of CCs might have other important implications for a successful pregnancy. Moreover, similar expression profiles of steroidogenic pathways and hCG/LH receptors between CCs and LMGCs indicate that CCs can also be used to understand better the luteal function and intricate crosstalk between in oocyte microenvironment. Trial registration number N/A

P-223 Follicular fluid from women stimulated with hMG protocol displays altered energy metabolism and this impacts embryo morphokinetics

Abstract Study question Does different type of ovarian stimulation affect follicle metabolism? Summary answer Patients stimulated hMG-only show increased levels of glucose and MDA in follicular fluid and longer Cc3 embryo time compared to patients stimulated rFSH and hMG. What is known already Oocyte growth and maturation strongly rely on energy metabolism. However, there is a notable lack of studies examining the influence of ovarian stimulation protocols on individual follicular fluid. While certain studies indicate changes in genes associated with glucose metabolism in patients subjected to different protocols, there remains a need for prospective studies in this field. Study design, size, duration This is a single-center prospective analysis from March 2022 to August 2022 including 20 patients. From the total, 14 were stimulated with rFSH + hMG and 6 with hMG only. A total of 81 embryos were submitted to morphokinetics analysis. Patients with Polycystic Ovary Syndrome (PCOS), Ovarian insufficiency, Endometriosis in stages III and IV, and patients who have already undergone other attempts at the clinic were excluded. Participants/materials, setting, methods Patients were firstly assessed by age, body mass index (BMI), and dosage of stimulation hormones. Puncture of follicles was performed individually after system wash and only follicular fluid (FF) with no visible blood contamination were submitted for analysis. FFs were analyzed for glucose, lactate, insulin (specific fluorometric assays), and MDA (important oxidative stress marker). Morphokinetics analyses were performed for all available embryos from day 0 until day 6. Main results and the role of chance Body Mass Index (BMI) and ages were similar among the categories (p = 0.388 and p = 0.373, respectively). FSH dosage used in both groups was not different (p = 0.85) but LH used was higher in patients treated with human menopausal gonadotropin (hMG) (p = 0.002). Women whose treatment was hMG presented more glucose in FF (p = 0.02) and MDA (p = 0.02) while no differences were observed for insulin and lactate content (p = 0.57 and p = 0.72 respectively). There was a significant increase of 3º Cell Cicle (Cc3) time of embryos from women treated with hMG only. Taken together, these data suggest that hMG is modifying the glucose metabolism by interrupting glycolysis in the oocyte, resulting in a higher amount in FF, which increases Malondialdehyde (MDA) levels. Considering that oocytes from women treated with hMG only had an altered metabolic profile, this could impact the further embryo development, with consequences specifically at the time of the embryonic metabolic shift. Limitations, reasons for caution The limited number of patients is a limitation of the study and the lack of standardization in the use of trigger, despite of not having significant difference. Wider implications of the findings Understanding the implication of hormones in follicular microambient might help individualization of hormones usage and improve the outcomes of IVF treatment. Trial registration number 44895621.1.0000.5594

O-038 Impact of maternal aging on the DNA repair landscape in mouse oocytes

Abstract Study question How does maternal aging impact DNA damage accumulation and DNA repair capacity in mammalian oocytes? Summary answer Age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in DNA repair capacity. What is known already Oocyte loss occurs progressively from birth and accelerates as women approach their mid-30s, posing a significant challenge to female reproductive health. Recent studies have highlighted the role of the DNA repair response in driving ovarian ageing and oocyte loss. Aged oocytes accumulate elevated levels of DNA damage. Unless repaired, persistent DNA damage activates DNA repair checkpoints, causing the elimination of defective oocytes. Although we know DNA double-strand breaks (DSBs) in oocytes increase with maternal age, surprisingly, little is known about why this damage is not repaired efficiently. Study design, size, duration Oocytes were isolated from ovaries of 8 weeks or 68-86-week-old CD1 mice, which correspond to reproductively young and old females respectively. Over 600 and 1000 oocytes from aged any young females were used to analyse DNA damage levels, DNA repair effciency, DNA repair compartments and for other live assays. Participants/materials, setting, methods DNA damage was induced in CD1 oocytes from young and aged mice using neocarzinostatin. Levels of DNA damage and DNA repair proteins were quantified using immunofluoresnce confocal/STED microscopy. High-resolution light sheet microscopy was used to perform live imaging of dynamics of DNA damage foci and chromosome segregation during meiosis. Main results and the role of chance Our work demonstrates that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. We further show that incomplete DNA repair results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. To investigate the causes of the age-related decline in DNA repair, we systematically studied the localization and abundance of several DNA repair proteins in oocytes from young and aged mice. We found that DNA repair proteins localized in spatially distinct DNA repair compartments in oocyte nuclei, that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. Several DNA repair proteins, their corresponding compartments, and their response to DNA damage were altered in aged oocytes. Lastly, we demonstrate that age-related cohesin loss increases DNA damage levels and reduces DNA repair capacity. We hypothesize that cohesin dissociation with maternal ageing may make chromosomes prone to accumulate DNA damage without proper DNA repair. Thus, age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in reproductive potential. Limitations, reasons for caution Neocarzinostatin, a radiomimetic drug used to induce DNA DSBs in mouse oocytes in vitro, may not fully represent sources of DNA damage during aging. The CD1 mouse model and ages were selected to recapitulate equivalent advanced maternal age in women. Further investigation is needed to confirm these findings in humans. Wider implications of the findings Overall, we propose that altered DNA repair machinery and cohesin loss contribute to persistent DNA damage and reduced DNA repair capacity in aged oocytes, which can cause chromosome defects and fragmentation, and eventually trigger oocyte death, thereby accelerating the loss of female fertility. Trial registration number Not Applicable

An Insight into the Roles of Zona Pellucida in Growth and Development of Mammalian Ocytes and Embryos: Changes of Age-related and Cryopreservation: A Review

The zona pellucida (ZP) is a extracellular transparent coat surrounding the oocyte and preimplantation embryo in mammals. The ZP separates between the surrounding cumulus cells and oocyte. It plays a pivotal role in oocyte growth and fertilization and early embryo development. Zona pellucida formation and glycoproteins, roles of ZP on female fertility and infertility, ZP micromanipulation on embryo development and changes of ZP upon vitrification of oocytes is discussed. Zona pellucida is formed of glycoproteins during ovarian follicle growth and development. It protects the oocyte from physical or chemical damage, initiates fertilization, prevents polyspermy and supports early embryonic development. The ZP is responsible for recognition between oocytes and sperm. Mutations in glycoproteins cause female infertility characterized by empty follicle syndrome and oocyte degeneration. The ZP removal is detrimental for embryo implantation. Furthermore, there is a biophysical role of ZP during vitrification of oocytes. Hence, the current review article was designed to collect, discuss and highlight the characters of zona pellucida, roles in female fertility and infertility, ZP micromanipulation on embryo development and changes of ZP upon vitrification.

Analysis of molecular identity and function of putative serotonin receptors in the Giant clam (Tridacna crocea) and the potential role of 5-HT1D-like receptor in reproduction

5-hydroxytryptamine (5-HT) is a secondary metabolite that interacts with a corresponding receptor, mediating various processes in organisms. However, the molecular function and characteristics of 5-HT receptor (5-HTR) in giant clam gonads remain unknown. In this study, we cloned full-length complementary DNA (cDNA) of four putative 5-HTRs from Tridacna crocea, designated Tc5-HTR1A-like, Tc5-HTR1D-like, Tc5-HTR2-like and Tc5-HTR4-like genes. SMART analysis revealed that all four Tc5-HTR-like proteins contained a seven-transmembrane structural domain, spanning from GN and ending at Y. Phylogenetic analysis showed that the four Tc5-HTR-like proteins were clustered into four distinct groups. The results of RT-qPCR showed that Tc5-HTR-like genes were widely expressed in all tested tissues, including gonads, suggesting that the function of Tc5-HTR-like genes involves the regulation of reproduction, including gametogenesis and embryonic development. Notably, the Tc5-HTR1D-like gene is highly expressed in oocytes of hermaphrodite and female stages. Estradiol-17β (E2) induced the upregulation of Tc5-HTR1A-like, Tc5-HTR1D-like and Tc5-HTR2-like genes, as well as downregulation of Tc5-HTR4-like gene in the gonad within 72 h. In situ hybridization showed that the Tc5-HTR1D-like gene had a positive signal in oocytes, indicating its role in oogenesis. The knockdown of the Tc5-HTR1D-like gene resulted in a significant reduction in oocyte growth and the number of oocytes. Whole-mount in situ hybridization and RT-qPCR analyses suggested that the spatiotemporal expression pattern of the Tc5-HTR1D-like gene is related to the physiological activities of embryonic development. Our data demonstrate that Tc5-HTR-like genes, especially Tc5-HTR1D-like gene, are essential for regulating gamete growth in T. crocea.

Stem cell-conditioned medium improves methylation patterns and quality of caprine preantral follicles.

Conditioned medium from Wharton's jelly mesenchymal stem cells improved tissue and preantral follicle outcomes, preventing adverse effects of oxidative stress, apoptosis, and epigenetic changes. This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton's jelly mesenchymal stem cells (WJ-MSCs). In the invivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.