Oocyte Development Research Articles

Toxic effects of microplastic and nanoplastic on the reproduction of teleost fish in aquatic environments.

Microplastics and nanoplastics are widely present in aquatic environments and attract significant scholarly attention due to their toxicity, persistence, and ability to cross biological barriers, which pose substantial risks to various fish species. Microplastics and nanoplastics can enter fish through their digestive tract, gills and skin, causing oxidative damage to the body and adversely affecting their reproductive system. Given that fish constitute a crucial source of high-quality protein for humans, it is necessary to study the impact of microplastics on fish reproduction in order to assess the impact of pollutants on ecology, biodiversity conservation, environmental sustainability, and endocrine disruption. This review explores the reproductive consequences of microplastics and nanoplastics in fish, examining aspects such as fecundity, abnormal offspring, circadian rhythm, gonad index, spermatocyte development, oocyte development, sperm quality, ovarian development, and changes at the molecular and cellular level. These investigations hold significant importance in environmental toxicology.

Variability in spruce beetle (Coleoptera: Curculionidae, Scolytinae) adult diapause and evidence for oocyte development prior to winter in a Colorado population.

Diapause regulates seasonal insect life cycles and may be highly variable within and among populations due to genetic and environmental variability. Both types of variation may influence how populations respond plastically or evolutionarily to changing climates. We assessed diapause variability in spruce beetle Dendroctonus rufipennis Kirby (Coleoptera: Curculionidae, Scolytinae), a major forest pest whose life cycle timing is regulated by both prepupal and adult diapauses. Using mating studies and ovary dissections, we tested for variability in adult diapause within and between collection sites in Colorado and Wyoming, USA. Ovary morphology suggested that most females from both sites enter diapause prior to egg formation (oogenesis) when reared at warm temperatures. Though previous studies suggested that adult diapause is obligate, we found that a small proportion of females from both populations terminated diapause without winter chilling in the lab. Moreover, we found that most female beetles sampled at the Colorado field site had mature ovaries relatively early in the fall, suggesting that transient exposure to low temperatures may potentiate pre-winter reproductive development. Adult diapause may act primarily as a block to prevent offspring production late in the season but not necessarily as an overwintering phenotype. Overall, our data do not suggest imminent life cycle shifts mediated by adult diapause, but if the observed variability is heritable, diapause regulation may evolve in response to changing environmental conditions.

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.

Changes in Inflammatory Cytokines, Vascular Markers, Cell Cycle Regulators, and Gonadotropin Receptors in Granulosa Cells of COVID-19 Infected Women

Background: COVID-19 infection can negatively affect multiple organ systems, including the reproductive system. Previous research has indicated altered levels of inflammatory markers in the reproductive tissues of women with chronic diseases. This study aimed to assess the expression of inflammatory, vascular, cell cycle, and gonadotropin receptor genes in the granulosa cells and oocytes of women with recent COVID-19 infection undergoing Assisted Reproductive Technology (ART), compared to healthy controls. Materials and Methods: The study involved 15 women who had tested positive for COVID-19 within three months of ART treatment and 15 age-matched healthy women as controls. Granulosa cells were collected during oocyte retrieval, and RNA was isolated to analyze gene expression using quantitative real-time PCR. The evaluated genes included inflammatory cytokines (IL-1B, TNF-α, IL-6, IL-8), vascular genes (VEGF, ANGPT1), cell cycle regulators (FOXL2, Cyclin D1, Cyclin D2, KLF4), and gonadotropin receptors (LHCGR, FSHR). Results: Results showed significantly higher expression of inflammatory cytokines in the granulosa cells of COVID-19 positive women, including IL-1B (4.2-fold), TNF-α (3.8-fold), IL-8 (2.5-fold), and IL-6 (3.2-fold). Vascular genes VEGF and ANGPT1 were also overexpressed, while FOXL2 was downregulated and Cyclin D1/D2 were upregulated in the study group. However, LH and FSH receptor expression remained similar between both groups. Conclusion: The present study demonstrates altered gene expression of inflammatory cytokines, vascular factors and cell cycle regulators in granulosa cells and oocytes of COVID-19 positive women undergoing ART. The dysregulated molecular pathways could potentially impair folliculogenesis and oocyte development in SARS-CoV-2 infected individuals.

Molecular characterization and stage-dependent gene expression of gonadotropin receptors in Pacific bluefin tuna, Thunnus orientalis, ovarian follicles

To understand the physiological mechanisms by which pituitary-derived gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) regulate asynchronous oocyte development, we investigated the function and expression of Fsh and Lh receptors (Fshr and Lhr, respectively) in Pacific bluefin tuna (PBT, Thunnus orientalis). As a first, we cloned the full-length cDNAs encoding PBT Fshr and Lhr. Recombinant PBT Fsh and Lh single-chain proteins were produced in abundance using stable CHO-DG44 cell lines and were subsequently purified from the culture medium, culminating in their yields being 87.0 and 88.2%, respectively. An in vitro reporter assay using homologous recombinant Gths revealed that PBT Fshr and Lhr responded strongly to their corresponding ligands in a dose-dependent manner, with no cross-activation over a wide range of concentrations. Moreover, quantitative expression analysis of Fshr and Lhr at the follicle level showed that fshr gene expression was highly upregulated in the ovarian follicles through vitellogenesis, while lhr expression was significantly upregulated and peaked in fully vitellogenic ovarian follicles. These findings suggest that asynchronous-type oocyte development is primarily attributed to the differential function and expression of Gthrs, rather than the ligand, in PBT.

Which is the current knowledge on man-made endocrine- disrupting chemicals in follicular fluid? An overview of effects on ovarian function and reproductive health.

The increase in female reproductive disorders, such as polycystic ovary syndrome, endometriosis, and diminished ovarian reserve that lead to subfertility and infertility, has encouraged researchers to search and discover their underlying causes and risk factors. One of the crucial factors that may influence the increasing number of reproductive issues is environmental pollution, particularly exposure to man-made endocrine-disrupting chemicals (EDCs). EDCs can interfere with the ovarian microenvironment, impacting not only granulosa cell function but also other surrounding ovarian cells and follicular fluid (FF), which all play essential roles for oocyte development, maturation, and overall reproductive function. FF surrounds developing oocytes within an ovarian follicle and represents a dynamic milieu. EDCs are usually found in biological fluids, and FF is therefore of interest in this respect. This narrative review examines the current knowledge on specific classes of EDCs, including industrial chemicals, pesticides, and plasticizers, and their known effects on hormonal signaling pathways, gene expression, mitochondrial function, oxidative stress induction, and inflammation in FF. We describe the impact of EDCs on the development of reproductive disorders, oocyte quality, menstrual cycle regulation, and their effect on assisted reproductive technique outcomes. The potential transgenerational effects of EDCs on offspring through animal and first-human studies has been considered also. While significant progress has been made, the current understanding of EDCs' effects on ovarian function, particularly in humans, remains limited, underscoring the need for further research to clarify actions and effects of EDCs in the ovary.

High Carbohydrate, Fat, and Protein Diets Have a Critical Role in Folliculogenesis and Oocyte Development in Rats.

To date, there is no comparative data on the effects of carbohydrates, fat, and proteins, which are macronutrients, on female reproductive functions. Therefore, in this study, we investigated the effects of diets enriched with carbohydrates, fats, and proteins on folliculogenesis and oocyte development in female rats. 21-day-old rats that were just weaned were divided into 4 groups: control, carbohydrate, fat, and protein. The control group was fed with standard chow and the carbohydrate, fat, and protein groups were fed diets enriched with 75% carbohydrate, 60% fat, and 50% protein for 11 weeks, respectively. It was found that high-fat and high-protein diets caused an increase in the estrous cycle length compared to carbohydrate group (p < 0.05). Graafian follicle number decreased in the protein group compared to the control (p < 0.05). However, the atretic follicle number was higher in the fat group compared to the control group (p < 0.05). In the carbohydrate group, Zp1 was found to be lower than the control and protein groups, Zp2 was found to be lower than the control, and Zp3 was found to be lower than the fat group (p < 0.05). While BMP15 was similar between groups (p > 0.05), GDF9 was lower in all diet groups compared to the control (p < 0.05). Foxo3a was lower in the protein group compared to carbohydrate and control (p < 0.05). GAS2 was found to be higher in the control group than the fat group, and higher in the carbohydrate group than the fat and protein groups (p < 0.05). FSH, LH, Progesterone, and E2 levels were higher in all three diet groups than in the control (p < 0.05). Also, significant differences were observed between the groups regarding adiponectin, resistin, and leptin levels. Taken together, high carbohydrate, fat, and protein intake are associated with impairment of the menstrual cycle, depletion of the developing follicle types, and altered expression of folliculogenesis-specific genes and hormones. Therefore, long-term macronutrient diets may result in shortened reproductive periods and reduced fertilization potential in females in the long run.

Female-specific mechanisms of meiotic initiation and progression in mammalian oocyte development.

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

The effect of cortisone on female zebrafish (Dania rerio): reducing reproductive capacity and offspring survival rate

Cortisone is a naturally occurring corticosteroid hormone known for its wide range of anti-inflammatory and immunosuppressive effects, and it is commonly found in various aquatic environments. Previous reports have shown that cortisone can have significant negative impacts on fish; however, its specific effects on fish reproduction have not been thoroughly investigated. In this study, female adult zebrafish were exposed to 0.0 (control), 3.9, 40.2, and 377.9 ng/L of cortisone for 60 days, and multiple endpoints were evaluated. The results showed that as the concentration of cortisone increased, there was an increase in the percentage of perinuclear oocytes and a decrease in the proportion of late-stage oocytes, indicating a stagnation in oocyte development. Additionally, female zebrafish exposed to cortisone exhibited decreased attraction to males and reduced mating intimacy. Furthermore, exposure to cortisone resulted in changes in the development and behavior of zebrafish embryos. At cortisone concentrations of 3.9 and 40.2 ng/L, fewer eggs were laid and the survival rate of fertilized eggs decreased. These observed effects are associated with abnormal transcription levels of genes (Star, Cyp11a1, Cyp17, Cyp19a, Cyp11b, Hsd11β2, Hsd17β3) related to the HPG axis. These findings provided new insights into understanding potential environmental risks associated with corticosteroids.

Identification, expression, and function analysis of Rbpms2 splicing variants in Japanese flounder gonad

Rbpms2, an RNA-binding protein with multiple splicing (Rbpms), can interact with RNAs to involve oocyte development, thereby influencing female sex differentiation in vertebrates. Here, two splicing variants of the Rbpms2 gene from Japanese flounder (Paralichthys olivaceus) were identified, namely Rbpms2.1 and Rbpms2.2. The two variants exhibited 98.22 % amino acid homology, both featuring an RNA recognition motif (RRM) domain spanning positions 98–170 amino acids. They were relatively conserved throughout phylogenetic evolution. Differently, the C-terminal region of the Rbpms2.1 contains five additional sequential amino acids (–VRDQP–) compared to Rbpms2.2. The real-time qPCR results demonstrated that Rbpms2.1 and Rbpms2.2 had relatively abundant expression in the gonads of adult Japanese flounder, with higher expression levels in the ovary compared to the testis (P < 0.05). In situ hybridization results showed strong positive expression of Rbpms2 mRNA in oocytes at stages I-III during the V stage of ovarian development. In the testis atstage IV, the expression of Rbpms2 mRNA was mainly concentrated on primary spermatocytes. Importantly, Rbpms2 binding sites were found in the 3′UTR, 5′UTR, and ORF regions of the sex-related genes including dmrt1, sox9, amh, foxl2, and wnt4. siRNA interference and overexpression analysis of Rbpms2.1 and Rbpms2.2 in primary cells of the ovary and testis showed that Rbpms2 can repress the expression of male-related genes (dmrt1, sox9, and amh) and significantly promote the expression of female-related genes (foxl2 and wnt4). Our results revealed that Rbpms2 may play a critical role by targeting the sex-related genes in the gonad development of Japanese flounder.

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.

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

Elucidating the Role of Sirtuin 3 in Mammalian Oocyte Aging.

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

Antidepressants as new endocrine disruptors? – transcriptomic profiling in gonads of Mytilus trossulus exposed to norfluoxetine

In this study an impact of norfluoxetine (NFLU) on Mytilus trossulus gonads was investigated focusing on sex-related differences in hormonal changes, gene expression, and transcriptomic profiling. Sex-specific differences in gonadal serotonin levels were found. NFLU stimulates serotonin synthesis and/or transport in female gonads, potentially accelerating oocyte maturation and gamete release. In males, NFLU decreases serotonin level what likely leads to impeding sperm maturation and thus spawning delay. Transcriptomic analyses highlighted the presence of NFLU-induced changes in gene expression related to gametogenesis and neurotransmission. In females, NFLU upregulated genes associated with oocyte development and downregulated those involved in sperm maturation. NFLU-treated males exhibited mixed effects in their genes in relation to spermatogenesis. Additionally, sex-related differences in the expression of the CYP450 genes responsible for detoxification were found. Overall, norfluoxetine acts as an endocrine-disrupting chemical and impacts gonadal serotonin levels and gene expression, potentially disrupting reproductive success of M. trossulus.

Lactation affects postweaning metabolic profiles, but not follicle size in multiparous sows

Young sows mobilise body reserves to support milk production during lactation, resulting in a negative energy balance (NEB). This NEB affects the development of follicles and oocytes that give rise to the next litter. Decreased IGF1 levels due to a NEB are thought to play a role in this process. As this has hardly been studied in multiparous sows, the current study focused on relations between lactation BW loss (%), metabolic hormones, and follicle development in multiparous sows at Day 0 and Day 4 after weaning. A total of 31 sows of parity 4.7 ± 2.5 were killed at either Day 0 or Day 4 after weaning. Average BW loss during lactation was 3.3 ± 4.5%, while average backfat loss was 4.1 ± 0.3 mm. The metabolic profile confirmed the metabolic impact of lactation as both non-esterified fatty acid (NEFA), and creatinine levels were higher at Day 0 than that at Day 4. Conversely, serum levels of IGF1 and growth differentiation factor 15 levels were lower on Day 0 than on Day 4. A higher BW loss (%) was related to higher NEFA levels on Day 0, but not on Day 4. IGF1 concentrations in serum and follicle fluid were similar at Day 0 and Day 4 and were not related to follicle size on these days. In conclusion, although lactation affected postweaning metabolic profiles in these multiparous sows, follicle size was not related to these profiles, probably due to the relatively mild BW loss of these sows. IGF1 concentrations were less affected by lactation and did not seem to limit follicle development, as it does in sows experiencing high weight loss.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

VPS34 Governs Oocyte Developmental Competence by Regulating Mito/Autophagy: A Novel Insight into the Significance of RAB7 Activity and Its Subcellular Location.

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

Comparative transcriptome analysis reveals molecular mechanisms of the effects of light intensity and photoperiod on ovarian development in Procambarus clarkii (Girard, 1852)

Procambarus clarkii (Girard, 1852) has important economic value in China and internationally. In this research, the comparative transcriptome analysis was used to reveal molecular mechanisms of influences of photoperiod and light intensity on ovarian development in P. clarkii for the first time. Some genes (such as laminin, collagen, integrin beta, catenin) and pathways (including TGF-beta signaling pathway, focal adhesion, ECM–receptor interaction) associated with ovarian development and oocyte maturation were significantly upregulated. Some genes related to circadian clock (such as CLK, PER) were identified in this research. The results indicated that when light intensity or photoperiod increased, P. clarkii could up-regulate the expression levels of the laminin and collagen, thereby synthesizing related proteins, promoting meiosis of the oocytes, thus increasing the number of oocytes in the ovary. At the same time, P. clarkii could up-regulate the expression levels of integrin beta, integrin alpha 6, and diacylglycerol to synthesize related proteins, thereby promoting the formation of proteins and fats such as triglycerides, these proteins and fats can provide material basis for maturation and development of oocytes, resulting in oocyte maturation and ovarian development. P. clarkii could synthesize related proteins by upregulating expression levels of genes (such as catenin), these proteins or hormones can adhere to other actins (such as integrins), thereby stabilizing the morphology of the oocytes and ensuring normal development. Meantime, the increase in light intensity or photoperiod could cause release GSH and VTG, resulting in oocytes development and maturation. The data in this research can reveal molecular mechanisms of impacts of photoperiod and light intensity on oocyte maturation and ovarian development in P. clarkii, can offer crucial genomic data for studying developmental mechanisms of ovary and oocyte in crustacean.

Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy

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.