Oocyte Maturation Research Articles

Sterigmatocystin declines mouse oocyte quality by inducing ferroptosis and asymmetric division defects

BackgroundSterigmatocystin (STE) is a mycotoxin widely found in contaminated food and foodstuffs, and excessive long-term exposure to STE is associated with several health issues, including infertility. However, there is little information available regarding the effects of STE toxin on the female reproductive system, particularly concerning oocyte maturation.MethodsIn the present study, we investigated the toxic effects of STE on mouse oocyte maturation. We also used Western blot, immunofluorescence, and image quantification analyses to assess the impact of STE exposure on the oocyte maturation progression, mitochondrial distribution, oxidative stress, DNA damages, oocyte ferroptosis and asymmetric division defects.ResultsOur results revealed that STE exposure disrupted mouse oocyte maturation progression. When we examined the cellular changes following 100 µM STE treatment, we found that STE adversely affected polar body extrusion and induced asymmetric division defects in oocytes. RNA-sequencing data showed that STE exposure affects the expression of several pathway-correlated genes during oocyte meiosis in mice, suggesting its toxicity to oocytes. Based on the RNA-seq data, we showed that STE exposure induced oxidative stress and caused DNA damage in oocytes. Besides, ferroptosis and α-tubulin acetylation were also found in STE-exposed oocytes. Moreover, we determined that STE exposure resulted in reduced RAF1 protein expression in mouse oocytes, and inhibition of RAF1 activity also causes defects in asymmetric division of mouse oocytes.ConclusionsCollectively, our research provides novel insights into the molecular mechanisms whereby STE contributes to abnormal meiosis.

Effects of Exogenous Regulation of PPARγ on Ovine Oocyte Maturation and Embryonic Development In Vitro.

Lactating oocytes consume a lot of energy during maturation, a large part of which comes from lipid metabolism. PPARγ is a key regulator of lipid metabolism. In this study, rosiglitazone (RSG), an activator of PPARγ, was added to a mature medium to investigate its effects on the levels of spindle and the chromosome arrangement, lipid deposition, reactive oxygen species (ROS), and glutathione (GSH) levels, oocyte secretion factors, apoptosis and lipid metabolism-related gene expression, and subsequent embryonic development during the maturation of sheep oocytes. The oocyte secretion factor affects gene expression related to apoptosis and lipid metabolism and subsequent embryonic development. The results showed that the proportion of spindle and normal chromosome arrangements increased in the 5 μM RSG treatment group, the lipid content increased after cell maturation, the ROS level decreased, and the GSH level increased. The expressions of oocyte secretion factor (GDF9 and BMP15), anti-apoptosis gene (BCL2), and lipid metabolism-related genes (ACAA1, CPT1A, PLIN2) were increased in the 5 μM treatment group. Finally, the development of blastocysts was examined. After the oocytes were treated with 5 μM RSG, the blastocyst rate and the gene expression of the totipotency gene (OCT4) were increased. It was concluded that increasing PPARγ activity during ovine oocyte maturation could promote lipid metabolism, reduce oxidative stress, and improve the ovine oocyte maturation rate and subsequent embryo development.

Ovarian Hyperstimulation Syndrome (OHSS): A Narrative Review and Legal Implications.

Infertility is a highly meaningful issue with potentially life-changing consequences, and its incidence has been growing worldwide. Assisted reproductive technology (ART) has made giant strides in terms of treating many infertility conditions, despite the risk of developing ovarian hyperstimulation syndrome (OHSS), a potentially life-threatening complication. This narrative review draws upon scientific articles found in the PubMed database. The search spanned the 1990-2024 period. Search strings used included "OHSS" or "ovarian hyperstimulation" and "IVF" and "GnRH" and "hCG"; 1098 results were retrieved and were ultimately narrowed down to 111 suitable sources, i.e., relevant articles dealing with the condition's underlying dynamics, management pathways, and evidence-based criteria and guidelines, crucial both from a clinical perspective and from the standpoint of medicolegal tenability. The following features constitute OHSS risk factors: young age, low body weight, and polycystic ovarian syndrome (PCOS), among others. GnRH antagonist can substantially lower the risk of severe OHSS, compared to the long protocol with a gonadotropin-releasing hormone (GnRH) agonist. However, a mild or moderate form of OHSS is also possible if the antagonist protocol is used, especially when hCG is used for the final maturation of oocytes. For women at risk of OHSS, GnRH agonist trigger and the freeze-all strategy is advisable. OHSS is one of the most frequent complications, with a 30% rate in IVF cycles. Providing effective care for OHSS patients begins with early diagnosis, while also evaluating for comorbidities and complications. In addition to that, we should pay more attention to the psychological component of this complication and of infertility as a whole. Compliance with guidelines and evidence-based best practices is essential for medicolegal tenability.

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

Potential mechanism prediction of indole-3-propionic acid against diminished ovarian reserve via network pharmacology, molecular docking and experimental verification

BackgroundOxidative stress (OS) is one of the major causes of ovarian aging and dysfunction. Indole-3-propionic acid (IPA) is an indole compound derived from tryptophan with free radical scavenging and antioxidant properties, and thus may have potential applications in protecting ovarian function, although the exact mechanisms are unknown. This study aims to preliminarily elucidate the potential mechanisms of IPA that benefit ovarian reserve function through network pharmacology, molecular docking, and experimental verification.MethodsThe related protein targets of IPA were searched on SwissTargetPrediction, TargetNet, BATMAN-TCM, and PharmMapper databases. The potential targets of diminished ovarian reserve (DOR) were identified from OMIM, GeneCards, DrugBank, and DisGeNET databases. The common targets were uploaded directly to the STRING database to construct PPI networks. We then performed GO and KEGG enrichment analysis on the targets. Subsequently, molecular docking and molecular dynamics simulation were used to validate the binding conformation of IPA to candidate targets. Furthermore, we carried out in vitro experiments to validate the prediction results of network pharmacology.ResultsWe identified a total of 61 potential targets for the interaction of IPA with DOR. The PPI network topological parameter analysis yielded 13 hub genes for DOR treatment. The GO biological process enrichment analysis identified 293 entries, mainly enriched in aging, signal transduction, response to hypoxia, negative regulation of apoptotic process, and positive regulation of cell proliferation. The KEGG enrichment analysis mainly included lipid and atherosclerosis, progesterone-mediated oocyte maturation, AGE-RAGE, relaxin, estrogen, and other signaling pathways. The molecular docking further revealed the direct binding of IPA with six hub proteins including NOS3, AKT1, EGFR, PPARA, SRC, and TNF. In vitro experiments showed that IPA pretreatment attenuated H2O2-induced cellular oxidative stress damage, while IPA exerted cytoprotective and antioxidant damage effects by regulating the six hub genes and antioxidant proteins.ConclusionWe systematically illustrated the potential protective effects of IPA against DOR through multiple targets and pathways using network pharmacology, and further verified the cytoprotective effect and antioxidant properties of IPA through in vitro experiments. These findings provide new insights into the targets and molecular mechanisms whereby IPA improves DOR.

The impact of low oocyte maturity ratio on blastocyst euploidy rate: a matched retrospective cohort study

ObjectiveTo investigate the association between a low oocyte maturity ratio from in vitro fertilization cycle and blastocyst euploidy.MethodsA total of 563 preimplantation genetic testing (PGT) cycles (PGT cycles with chromosomal structural rearrangements were excluded) were performed between January 2021 and November 2022 at our center (average oocyte maturity rate: 86.4% ± 14.6%). Among them, 93 PGT cycles were classified into the low oocyte maturity rate group (group A, < mean − 1 standard deviation [SD]), and 186 PGT cycles were grouped into the average oocyte maturity rate group (group B, mean ± 1 SD). Group B was 2:1 matched with group A. Embryological, blastocyst ploidy, and clinical outcomes were compared between the two groups.ResultsThe oocyte maturity (metaphase II [MII oocytes]), MII oocyte rate, and two pronuclei (2PN) rates were significantly lower in group A than in group B (5.2 ± 3.0 vs. 8.9 ± 5.0, P = 0.000; 61.6% vs. 93.0%, P = 0.000; 78.7% vs. 84.8%, P = 0.002, respectively). In group A, 106 of 236 blastocysts (44.9%) that underwent PGT for aneuploidy were euploid, which was not significantly different from the rate in group B (336/729, 46.1%, P = 0.753). However, euploid blastocysts were obtained only in 55 cycles in group A (55/93, 59.1%), which was lower than the rate in group B (145/186, 78.0%, P = 0.001). The clinical pregnancy rate in group B (73.9%) was higher than that in group A (58.0%) (P = 0.040).ConclusionOur results suggest that a low oocyte maturity ratio is not associated with blastocyst euploidy but is associated with fewer cycles with euploid blastocysts for transfer, lower 2PN rates, and lower clinical pregnancy rates.

Porcine granulosa cell transcriptomic analyses reveal the differential regulation of lncRNAs and mRNAs in response to all-trans retinoic acid in vitro.

The active metabolite of vitamin A, all-trans retinoic acid (ATRA), is involved in the proliferation and differentiation of granulosa cells, and promotes the follicular development, oocyte maturation, and ovulation in mammals. This study aims to investigate the ATRA induced potential long noncoding RNAs (lncRNAs) that regulate the expression of genes associated with granulosa cell proliferation and follicular development. The lncRNA and mRNA profiles of porcine granulosa cells from ATRA treatment and control group in vitro were constructed through RNA sequencing. Meanwhile, the sequencing data were verified using quantitative polymerase chain reaction (qPCR). A total of 86 differentially expressed lncRNAs and 128 differentially expressed genes (DEGs) were detected in granulosa cells after ATRA treatment. The qRT-PCR results were consistent with the RNA-seq data. Functional annotation analysis revealed that the DEGs were remarkably enriched in ovary function and reproduction which contained FoxO, Hippo, Oocyte meiosis, mTOR signaling pathway, as well as several pathways associated with hormone regulation like Oxytocin signaling pathway and Steroid hormone biosynthesis. Moreover, an interaction network of lncRNAs and their cis-target DEGs was constructed, and 7 differentially expressed lncRNAs and 6 cis-target DEGs were enriched in ovarian steroidogenesis and reproduction. These findings expand the lncRNA catalogue and provide a basis for further studies on the mechanism of ATRA-mediated lncRNA regulation of follicular development in pigs.

Proteome and metabolomic profile of Mongolian horse follicular fluid during follicle development

During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid were analyzed by mass spectrometry. The results showed that: in LFF vs MFF, 20 differential abundant protein (DAP) and 88 differential abundant metabolites (DAM) were screened out; In SFF vs MFF, 3 DAPs and 65 DAMs were screened out; In MFF vs SFF, 24 DAPs and 35 DAMs were screened out. The analysis of differential proteins and metabolites showed that glycerophosphate hydrolysis decreased during follicular development, and proteins played a major role in metabolism and binding. In addition, DAMs and DAPs are co-enriched in the “linoleic acid metabolism” pathway. Combinatorial analysis reveals the dynamic profile of follicular fluid during follicular development and provides fundation for further exploring the function of follicular fluid in Mongolian horse.

Genome-wide association study of growth and reproductive traits based on low-coverage whole-genome sequencing in a Chubao black-head goat population

The Chubao black-head goat is a novel hybrid breed that combines the advantages of Macheng black goats, such as good reproductive performance, strong adaptability, and resistance to rough feeding, with the superior growth and meat characteristics of Boer goats. Given the substantial economic importance of growth (such as birth weight, body height, body length, and chest circumference across different growth stages) and reproductive traits (particularly average litter size after first parity), the aim of this study was to identify significant SNPs and candidate genes associated with these traits in Chubao black-head goats. Through whole-genome sequencing (with 34 goats at approximately 15× coverage and 466 goats at approximately 1× coverage), genotype imputation, and quality control, 22,665,331 SNPs were identified and subsequently used for genetic analyses. Heritability estimates indicated that growth traits exhibit moderate to high heritability (ranging from 0.297 ± 0.071 to 0.535 ± 0.118), while reproductive traits demonstrated low to moderate heritability (with a value of 0.220 ± 0.108). By performing FarmCPU-based genome-wide association studies, we identified 48 potentially significant SNPs associated with growth traits and 7 with reproductive traits. Additionally, 85 candidate genes (such as COL14A1, ZNF148, and TTC39C) linked to growth traits were identified and enriched in pathways associated with fundamental molecular biological activities such as protein deubiquitination, regulation of mRNA stability, and the MAPK signaling pathway. Furthermore, 10 candidate genes (such as SOHLH2, CCNA2, and SOX7) associated with reproductive traits were identified and enriched in pathways related to specific reproductive processes such as oocyte differentiation, endoderm formation, and progesterone-mediated oocyte maturation. Overall, these findings provide valuable preliminary insights into the molecular mechanisms underlying growth and reproductive traits in Chubao black-head goats. However, further functional validation is needed to effectively use these potential SNPs and candidate genes in improving the breeding of these traits in this breed.

Variants in NLRP2 and ZFP36L2, non-core components of the human subcortical maternal complex, cause female infertility with embryonic development arrest.

The subcortical maternal complex (SCMC), which is vital in oocyte maturation and embryogenesis, consists of core proteins (NLRP5, TLE6, OOEP), non-core proteins (PADI6, KHDC3L, NLRP2, NLRP7), and other unknown proteins that are encoded by maternal effect genes. Some variants of SCMC genes have been linked to female infertility characterized by embryonic development arrest. However, so far, the candidate non-core SCMC components associated with embryonic development need further exploration and the pathogenic variants that have been identified are still limited. In this study, we discovered two novel variants [p.(Ala131Val) and p.(Met326Val)] of NLRP2 in patients with primary infertility displaying embryonic development arrest from large families. In vitro studies using 293T cells and mouse oocytes, respectively, showed that these variants significantly decreased protein expression and caused the phenotype of embryonic development arrest. Additionally, we combined the 'DevOmics' database with the whole exome sequence data of our cohort and screened out a new candidate non-core SCMC gene ZFP36L2. Its variants [p.(Ala241Pro) and p.(Pro291dup)] were found to be responsible for embryonic development arrest. Co-immunoprecipitation experiments in 293T cells, used to demonstrate the interaction between proteins, verified that ZFP36L2 is one of the human SCMC components, and microinjection of ZFP36L2 complementary RNA variants into mouse oocytes affected embryonic development. Furthermore, the ZFP36L2 variants were associated with disrupted stability of its target mRNAs, which resulted in aberrant H3K4me3 and H3K9me3 levels. These disruptions decreased oocyte quality and further developmental potential. Overall, this is the first report of ZFP36L2 as a non-core component of the human SCMC and we found four novel pathogenic variants in the NLRP2 and ZFP36L2 genes in 4 of 161 patients that caused human embryonic development arrest. These findings contribute to the genetic diagnosis of female infertility and provide new insights into the physiological function of SCMC in female reproduction.

A transcriptomics-based analysis of mechanisms involved in the sex-dependent effects of diazepam on zebrafish

Diazepam (DZP) is a universally detected emerging pollutant in aquatic ecosystems. Although the sex-dependent effects of DZP on fish have been properly established, the underlying mechanisms remain unclear. In this study, zebrafish of both sexes were separately exposed to DZP (8 μg/L) for 21 days, and the alteration of the behaviors, brain amino acid neurotransmitter contents, and transcriptomic profiles were investigated. Although DZP exposure showed a sedative effect on both sexes, significantly reduced cumulative duration of high mobility and willingness to encounter the opposite sex were only observed in females. However, DZP significantly enhanced the brain levels of glutamate and glutamine in males but not in females. Transcriptome analysis identified more different expression genes (DEGs) in females (322 up-regulated and 311 down-regulated) than in males (138 up-regulated genes and 38 down-regulated). The DEGs in both sexes were significantly enriched in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of the synaptic vesicle cycle, indicating a possible pathway for the sedative effects of DZP on zebrafish. DZP exhibited different or even opposing regulatory patterns on gene expression in the brains of females and males, providing some insights into its sex-dependent impacts on the behaviors and brain neurotransmitter contents in zebrafish. Moreover, enrichment analysis also suggested that DZP exposure may affect the oocyte maturation in female zebrafish, which highlights the need to study its reproductive and transgenerational toxicity to fish species.

Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.

A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development

The chromosome segregation and cell division programs associated with somatic mitosis and germline meiosis display dramatic differences such as kinetochore orientation, cohesin removal, or the presence of a gap phase.1,2,3,4,5,6 These changes in chromosome segregation require alterations to the established cell division machinery.5,6 It remains unclear what aspects of kinetochore function and its regulatory control differ between the mitotic and meiotic cell divisions to rewire these core processes. Alternative RNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternative mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell-type-specific expression. Reciprocally, precisely eliminating expression of the germline-specific DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.

Acrolein exposure affects ovarian function by interfering with glycolysis and mitochondrial energy metabolism in mouse

Acrolein is a widespread contaminant found in both diet and environment, entering the human body through food, alcohol, smoking, and exposure to fuel combustion fumes. While prior studies have highlighted acrolein's harmful impact on oocyte quality and early embryonic development in vitro, the specific mechanisms by which acrolein affects the female reproductive system in vivo remain poorly understood. This study first confirmed that in vitro acrolein exposure disrupts spindle morphology and chromosome alignment during the mid-MI stage of oocyte development, thus hindering oocyte maturation. Besides, exposure to acrolein not only stunts growth in mice but also impairs ovarian development, decreases the ovarian coefficient, disrupts follicular development, and increases the count of atretic follicles in vivo. Additional research has shown that acrolein exposure reduces the activity of key enzymes in glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle within the ovaries. It also suppresses mitochondrial complex expression and disturbs the balance between mitochondrial fission and fusion, as confirmed by metabolomic analyses. Moreover, acrolein exposure in vivo induced granulosa cell apoptosis and reduced oocyte number. In summary, acrolein exposure impairs glucose metabolism and induces mitochondrial dysfunction in the ovaries.

Granulosa cells provide transcriptomic information on ovarian follicle dynamics in southern white rhinoceros

Much remains unknown about the reproductive physiology of southern white rhinoceros (SWR) and the effect of ovarian stimulation prior to ovum pickup (OPU) have not been fully elucidated. Granulosa cells (GC) provide valuable insight into follicle growth and oocyte maturation status. The goals of this study were to evaluate transcriptomic changes in GC from three stages of follicle development and to identify biomarkers possibly associated with follicular growth and maturation as a result of ovarian stimulation. GC collected from SWRs following OPU were assigned stages based upon follicle size. Total RNA was isolated, and cDNA libraries were prepared and sequenced on a NovaSeq 6000. All bioinformatics analyses were performed utilizing the Galaxy web platform. Reads were aligned to CerSimCot1.0, and the manual curation was performed with EquCab3.0. Overall, 39,455 transcripts (21,612 genes) were identified across follicle stages, and manual curation yielded a 61% increase in gene identification from the original annotation. Granulosa cells from preovulatory follicles expressed the highest number of unique transcripts. The following seven biomarkers were determined based upon cluster analysis and patterns of expression: COL1A1, JMY, FBXW11, NRG1, TMPO, MACIR and COL4A1. These data can be used to potentially evaluate the effects of different ovarian stimulation protocols on follicle dynamics, improve OPU results, and support conservation efforts in this species.

IP3R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage

Calcium ions (Ca2+) regulate cell proliferation and differentiation and participate in various physiological activities of cells. The calcium transfer protein inositol 1,4,5-triphosphate receptor (IP3R), located between the endoplasmic reticulum (ER) and mitochondria, plays an important role in regulating Ca2+ levels. However, the mechanism by which IP3R1 affects porcine meiotic progression and embryonic development remains unclear. We established a model in porcine oocytes using siRNA-mediated knockdown of IP3R1 to investigate the effects of IP3R1 on porcine oocyte meiotic progression and embryonic development. The results indicated that a decrease in IP3R1 expression significantly enhanced the interaction between the ER and mitochondria. Additionally, the interaction between the ER and the mitochondrial Ca2+ ([Ca2+]m) transport network protein IP3R1-GRP75-VDAC1 was disrupted. The results of the Duolink II in situ proximity ligation assay (PLA) revealed a weakened pairwise interaction between IP3R1-GRP75 and VDAC1 and a significantly increased interaction between GRP75 and VDAC1 after IP3R1 interference, resulting in the accumulation of large amounts of [Ca2+]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 [Ca2+]m chelating agent ruthenium red (RR) or ROS scavenger N-acetylcysteine (NAC), the oocytes developmental abnormalities, oxidative stress and apoptosis caused by Ca2+ overload were improved. In conclusion, our results indicated IP3R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage.

Canine oocyte nuclear maturation with Nano-ozone (NZS) supplementation: The alterations of antioxidant, and oxidant status and CDK1, cyclin B1 expressions

This study aims to evaluate the effects of nano-ozone solution (NZS) on canine oocyte nuclear maturation, associated with the alterations of antioxidant and oxidant status and cyclin-dependent kinase 1 (CDK1), cyclin B1 gene expressions. Oocytes were cultured in four distinct concentrations of NZS (0.5, 1, 2, and 5 µg/mL) and parthenogenetically activated. The rates of oocytes arrested at the Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (MI), and Metaphase II (MII) stages were statistically different among groups (P < 0.05). The oocytes cultured in 1 µg/mL NZS yielded the best oocyte maturation rate at the MI and MII stages; however, the lowest maturation and high degeneration rates were observed in Group E. The measurements of Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Ferric Reducing/Antioxidant Power assay (FRAP) were performed from IVM culture media. No statistical difference was observed in SOD and MDA results (P > 0.05). GSH levels were statistically significant between Group A-Group E (p = 0.003), Group B-Group E (p = 0.045), and Group E-Group D (p = 0.021). The culture media in Group D and Group E had high FRAP concentrations and significantly differed between groups (P < 0.05). CDK1, and cyclin B1 genes, which are subunits of maturation-promoting factor (MPF), are upregulated in Group B and Group C, while are downregulated in oocytes of Group E.This study showed that low, controlled doses of NZS (1 µg/mL) supplementation could improve the meiotic competence of canine oocytes and lead to positive response in expressions of CDK1 and cyclin B1 on the gene level.

Luteinizing Hormone Receptor Mutation (LHRN316S) Causes Abnormal Follicular Development Revealed by Follicle Single-Cell Analysis and CRISPR/Cas9

Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHRN316S mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHRN316S mice exhibited reduced numbers of corpora lutea and ovulation. The LHRN316S mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHRN316S female mice with ICR wild type male mice revealed that the infertility rate of LHRN316S mice was 21.4% (3/14). Litter sizes from LHRN316S mice were smaller than those from control wild type female mice. The oocytes from LHRN316S mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHRN316S mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.Graphical

Autophagy and Female Fertility: Mechanisms, Clinical Implications, and Emerging Therapies.

Autophagy, an evolutionarily conserved cellular mechanism essential for maintaining internal stability, plays a crucial function in female reproductive ability. In this review, we discuss the complex interplay between autophagy and several facets of female reproductive health, encompassing pregnancy, ovarian functions, gynecologic malignancies, endometriosis, and infertility. Existing research emphasizes the crucial significance of autophagy in embryo implantation, specifically in the endometrium, highlighting its necessity in ensuring proper fetal development. Although some knowledge has been gained, there is still a lack of research on the specific molecular impacts of autophagy on the quality of oocytes, the growth of follicles, and general reproductive health. Autophagy plays a role in the maturation, quality, and development of oocytes. It is also involved in reproductive aging, contributing to reductions in reproductive function that occur with age. This review explores the physiological functions of autophagy in the female reproductive system, its participation in reproductive toxicity, and its important connections with the endometrium and embryo. In addition, this study investigates the possibility of emerging treatment approaches that aim to modify autophagy, using both natural substances and synthetic molecules, to improve female fertility and reproductive outcomes. Additionally, this review intends to inspire future exploration into the intricate role of autophagy in female reproductive health by reviewing recent studies and pinpointing areas where current knowledge is lacking. Subsequent investigations should prioritize the conversion of these discoveries into practical uses in the medical field, which could potentially result in groundbreaking therapies for infertility and other difficulties related to reproduction. Therefore, gaining a comprehensive understanding of the many effects of autophagy on female fertility would not only further the field of reproductive biology but also open new possibilities for diagnostic and treatment methods.

Smart RNA Sequencing Reveals the Toxicological Effects of Diisobutyl Phthalate (DiBP) in Porcine Oocytes.

Diisobutyl phthalate (DiBP) is commonly used in the plastics industry, and recent studies have shown that environmental exposure and accumulation in the food chain caused inflammation in some organs. However, the underlying mechanisms by which DiBP affects oocyte quality have not yet been fully defined. We used immunostaining and fluorescence to evaluate the effects of DiBP exposure and demonstrated that it impaired the morphology of matured porcine oocytes through generation of cytoplasmic fragmentation, accompanied by the perturbed dynamics of the spindle and actin cytoskeleton, misdistributed endoplasmic reticulum, as well as partial exocytosis of cortical granules and ovastacin. Moreover, analysis of Smart RNA-seq found that DiBP-induced aberrant oocyte maturation could be induced by abnormal mitochondrial function and apoptosis. Importantly, we discovered that supplementation with pyrroloquinoline quinone (PQQ) significantly attenuated the meiotic abnormalities induced by DiBP exposure through the modulation of reactive oxygen species levels. Our findings demonstrated that DiBP exposure adversely affects oocyte meiotic maturation and that PQQ supplementation was an effective strategy to protect oocyte quality against DiBP exposure.