Oocytes Research Articles

Pseudoinvasion and squamous metaplasia/morules in colorectal adenomatous polyp: a case report and literature review

BackgroundSubmucosal pseudoinvasion and squamous metaplasia (SM) are incidental and special morphological findings in colorectal adenomas, and both can mimic invasive carcinoma. The coexistence of these two findings further increases the risk of misdiagnosis, posing a great diagnostic challenge to pathologists. From 1979 to 2022, only 8 cases have been reported, which was extremely rare. In this report, we presented a case of sigmoid colon adenoma accompanied by pseudoinvasion and SM. Additionally, relevant literature was analyzed to summarize the clinical and pathological characteristics.Case presentationA 51-year-old Chinese male patient presented with fresh blood after defecation. Electronic colonoscopy revealed multiple polyps, which were removed using a snare and subjected to high-frequency electrocoagulation resection. The largest polyp, located in the sigmoid colon, was a thick pedunculated and lobulated polyp with a maximum diameter of 2.8 cm. The surface of the polyp showed slight ruggedness and redness, and it was sent for pathological examination. Grossly, the polyp had a lobulated and slightly rough surface. Microscopically, it showed a tubulovillous adenoma with focal high-grade dysplasia and mucosal muscle hyperplasia. Glandular elements were observed in the submucosal layer, forming a well-defined lobular structure. Some of the glands displayed cystic change, and focal SM could be seen within the adenoma. SM could manifest as discrete solid cell nests of varying sizes or cribriform-morular-like structures. Immunohistochemical staining showed that SM cells were diffusely positive for cytokeratin 5/6 (CK5/6); p40, p63, and cytokeratin 20 (CK20) were negative; while caudal type homeobox 2 (CDX2) was weakly positive. β-catenin showed abnormal nuclear expression, and an extremely low Ki67 proliferation index was observed.ConclusionsCoexistence of SM and pseudoinvasion in colorectal adenomas is highly rare. It is more commonly observed in males and tends to occur in the sigmoid colon. It primarily manifests in tubulovillous adenoma and tubular adenoma, with a majority of cases exhibiting a pedicle. Histologically, it is similar to invasive lesions. The cystic dilation of the submucosal glands, hemosiderin deposition, and the presence of a lamina propria around the submucosal glands without adjacent desmoplastic reaction, suggest pseudoinvasion rather than cancer. The bland cytological morphology and Immunohistochemical markers play a crucial role in distinguishing SM from true invasive lesions.

Primary Conjunctival Molluscum Contagiosum in a Patient With Ocular Graft-Versus-Host Disease.

Primary conjunctival molluscum contagiosum (MC) is rare and usually reported in patients with acquired immunodeficiency syndrome. In this study, we present a case of bilateral primary conjunctival MC in a patient with ocular graft-versus-host disease (oGVHD). This is a case report study. Clinical evaluation, in vivo confocal microscopy imaging, and histopathology were used to confirm the diagnosis. A 38-year-old woman with a history of allogeneic bone marrow transplant and secondary chronic oGVHD presented with ocular discomfort, redness, and dryness. On examination, clusters of white gelatinous nodular lesions, stained with fluorescein and Lissamine green, were observed on the bulbar conjunctiva, along with similar solitary nodular lesions in all quadrants of both eyes. In vivo confocal microscopy revealed nests of epithelial cells with bright inclusions measuring approximately 30-35 μm. Excisional biopsy confirmed the diagnosis of MC. A 6-month post-operative follow-up showed healed conjunctiva with no recurrence and improved ocular comfort. Molluscum contagiosum should be considered in the differential diagnosis of conjunctival lesions in patients with impaired immunity such as oGVHD. In diagnosing MC lesions, in vivo confocal microscopy proves to be valuable. In the absence of topical antiviral treatment, surgical excision is warranted.

Functional complementation of two splicing variants of Gustavus in Neocaridina denticulata sinensis during ovarian maturation

Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns—high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.

D4Z4 Hypomethylation in Human Germ Cells.

Expression of the double homeobox 4 (DUX4) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal (PEG3 and GTL2) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Improved The Aged Mouse IVM Oocytes Quality.

During assisted reproductive technology (ART) treatment, the aged women, especially those over 35years old, have fewer mature oocytes and poorer quality of the oocytes comparing with the young women. In vitro maturation (IVM) technology facilitates the usage of immature oocytes, which is clinically important for the aged women. However, the maturation rate is low for the oocytes from the aged women. Human umbilical cord mesenchymal stem cells derived exosomes (HUCMSCs-exosomes), as important mediators of intercellular communication, have been widely used to restore ovarian function and improve female fertility. In this study, we isolated HUCMSCs-exosomes and collected the immature germinal vesicle oocytes from the naturally aged mouse model. And we added these HUCMSCs-exosomes to the conventional IVM culture system. The effects of HUCMSCs-exosomes on IVM oocytes were observed and analyzed from multiple aspects including maturation rate, spindle morphology, mitochondria function, and development potential. We found the quality of oocytes was improved by HUCMSCs-exosomes. Based on the results, we propose that HUCMSCs-exosomes may provide a novel and cell free strategy in the improvement of the IVM in elderly infertile women in the future.

CtIP regulates G2/M transition and bipolar spindle assembly during mouse oocyte meiosis

CtBP-interacting protein (CtIP) is known for its multifaceted roles in DNA repair and genomic stability, directing the homologous recombination-mediated DNA double-stranded break (DSBs) repair pathway via DNA end resection, an essential error-free repair process vital for genome stability. Mammalian oocytes are highly prone to DNA damage accumulation due to prolonged G2/prophase arrest. Here, we explore the functions of CtIP in meiotic cell cycle regulation via a mouse oocyte model. Depletion of CtIP by siRNA injection results in delayed germinal vesicle breakdown and failed polar body extrusion. Mechanistically, CtIP deficiency increases DNA damage and decreases the expression and nuclear entry of CCNB1, resulting in marked impairment of meiotic resumption, which can be rescued by exogenous CCNB1 overexpression. Furthermore, depletion of CtIP disrupts MTOCs coalescence at spindle poles as indicated by failed accumulation of γ-tubulin, p-Aurora kinase A, Kif2A, and TPX2, leading to abnormal spindle assembly and prometaphase arrest. These results provide valuable insights into the important roles of CtIP in the G2/M checkpoint and spindle assembly in mouse oocyte meiotic cell cycle regulation.

Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca2+, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca2+, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca2+ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

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.

Identification of novel cattle (Bos taurus) genes and biological insights of their function in pre-implantation embryo development

BackgroundAppropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes.ResultsWe identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10–8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10–7).ConclusionsOur results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclinB1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosisI. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphaseI (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclinB1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphaseII (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Wnt-5a/Ca2+ pathway modulates endogenous current and oocyte structure of Xenopus laevis

Wnt signaling plays an essential role in cellular processes like development, maturation, and function maintenance. Xenopus laevis oocytes are a suitable model to study not only the development but also the function of different receptors expressed in their membranes, like those receptors expressed in the central nervous system (CNS) including Frizzled 7. Here, using frog oocytes and recordings of endogenous membrane currents in a two-electrode path configuration along with morphological observations, we evaluated the role of the non-canonical Wnt-5a ligand in oocytes. We found that acute application of Wnt-5a generated changes in endogenous calcium-dependent currents, entry oscillatory current, the membrane's outward current, and induced membrane depolarization. The incubation of oocytes with Wnt-5a caused a reduction of the membrane potential, potassium outward current, and protected the ATP current in the epithelium/theca removed (ETR) model. The oocytes exposed to Wnt-5a showed increased viability and an increase in the percentage of the germinal vesicle breakdown (GVBD), at a higher level than the control with progesterone. Altogether, our results suggest that Wnt-5a modulates different aspects of oocyte structure and generates calcium-dependent endogenous current alteration and GVDB process with a change in membrane potential at different concentrations and times of the exposition. These results help to understand the cellular effect of Wnt-5a and present the use of Xenopus oocytes to explore the mechanism that could impact the activation of Wnt signaling.

Effects of orange peel extract (Citrus sinensis) treatment on zebrafish oocytes (Danio rerio) exposed to heat stress.

Heat stress (HS) can negatively impact oocytes by disrupting mitochondrial activity, increasing the production of reactive oxygen species, and decreasing antioxidant levels. This study investigated the impact of orange peel (OP) exposure on zebrafish oocytes (ZOs) diameter, survival rate, and germinal vesicle breakdown (GVBD) during HS. We investigated the antioxidant effect of flavonoids (concentration = 328.58 ppm) derived from OP (Citrus sinensis) added to in vitro maturation (IVM) media of ZOs (Danio rerio) under non-heat stress (NHS) and HS conditions to mimic in vivo HS conditions due to the global warming phenomenon on females. ZO in stage 3 (n = 1080) was treated with 4 μL of OP extract (not treated/control) under HS: 32°C (Heat stress 32°C solution/Heat stress 32°C orange peel [HS32S/HS32O]) and 34°C (Heat stress 34°C solution/Heat stress 34°C orange peel [HS34S/HS34O]); and NHS: 28°C (Non-heat stress solution/Non-heat stress orange peel [NHSS/NHSO]), during maturation. After 24 h of maturation, we observed the oocyte diameter, survival rate, and GVBD rate. The data were analyzed with IBM Statistics 23 software using two-way analysis of variance and Kruskal-Wallis (p < 0.05). The highest oocyte diameter data were in NHS treated with OP extract (NHSO) group (0.759 ± 0.01; mean ± standard error) compared with HS group using and without OP extract (HS32S [0.583 ± 0.02]; HS32O [0.689 ± 0.02]; HS34S [0.554 ± 0.02]; and HS34O [0.604 ± 0.02]). The survival rate of OP treated group, namely, NHSO (93% ± 3%), HS32O (85% ± 2%), and HS34O (80% ± 2%) was higher than that of the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). ZO treated with OP extract (NHSO [93% ± 3%], HS32O [85% ± 2%], and HS34O [80% ± 2%]) showed a higher GVBD rate than the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). It revealed that OP can enhance the oocyte diameter, survival rate, and GVBD rate of ZO under NHS and HS. Further investigation should be conducted to determine the effect of OP extract (C. sinensis) on in vivo conditions in females as an alternative treatment to face global warming.

Mature oocyte found during ovarian tissue cryopreservation in an early adolescent female.

A 15-year-old female with Hodgkin's lymphoma underwent ovarian tissue cryopreservation for preserving fertility in Reproductive Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medical after receiving one course of chemotherapy. During the ovarian tissue cryopreservation, one MⅡmature oocyte and three germinal vesicle oocytes were found. The three immature oocytes underwent in vitro maturation but failed. Ultimately, one mature oocyte and 12 ovarian cortex slices were cryopreserved using vitrification. This case indicates that for patients with established gonadal axis feedback, ovarian tissue cryopreservation may not be the only method for fertility preservation. It is advisable to consider ovarian stimulation and oocyte retrieval for oocyte cryopreservation. Alternatively, for individuals in the ovulation phase of their menstrual cycle, attempting oocyte retrieval before ovarian tissue cryopreservation to obtain mature oocytes from the natural cycle, followed by oocyte cryopreservation, may enhance the likelihood of successful fertility preservation.

The regulatory roles of Smad2/3 protein and SMURF2 gene expression in granulosa cells of germinal vesicle and metaphase II oocytes in polycystic ovarian syndrome: A case-control study.

The impaired functions of granulosa cells (GCs) in the delayed development and immaturity of oocytes have been reported in polycystic ovary syndrome (PCOs). Even with ovarian stimulation, a large number of oocytes in these patients are still in the stage germinal vesicle (GV). The levels of Smad2/3, phosphorylated Smad2/3 (P-Smad2/3), the expression of SARA, Smad4, and SMURF2 genes in the GCs surrounding metaphase II (MII) or GV oocytes in PCOs women were investigated. GCs of MII and GV oocytes were isolated from 38 women with PCOs and the expression levels of SARA, Smad4, and SMURF2 in surrounding GCs of MII and GV oocytes were determined using reverse-transcription polymerase chain reaction. Also, Smad2/3 and P-Smad2/3 proteins were determined using western blotting. The expression level of SMURF2 was significantly higher in GCs surrounding GV oocytes compared with that of GCs encompassing MII oocytes (p 0.001). At the same time, no significant differences were observed in SARA and Smad4 expression levels in GCs surrounding GV and MII oocytes. A lower level of P-Smad2/3 was also found in GCs GV oocytes compared with GCs of MII oocytes (p 0.001). It seems that P-Smad2/3 plays a role in oocyte development, and the downregulation of this protein is associated with a defect in the maturation of GV oocytes. On the other hand, the upregulation of the SMURF2 gene also affects the growth process of GCs and the maturation of GV oocytes.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 μM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during development†.

One of the major causes of oocyte quality deterioration, chromosome segregation abnormalities manifest mainly during meiosis I, which occurs before and during ovulation. However, currently, there is a technical limitation in the introduction of mRNA into premature oocytes without impairing embryonic developmental ability. In this study, we established a low-invasive electroporation (EP) method to introduce mRNA into pre-ovulatory, germinal vesicle (GV) mouse oocytes in an easier manner than the traditional microinjection method. The EP method with an optimized impedance value resulted in the efficient introduction of mRNAs encoding enhanced green fluorescent protein (EGFP) into the GV oocytes surrounded by cumulus cells at a survival rate of 95.0%. Furthermore, the introduction of histone H2B-EGFP mRNA into the GV oocytes labeled most of the oocytes without affecting the blastocyst development rate, indicating the feasibility of the visualization of oocyte chromosomal dynamics that enable us to assay chromosomal integrity in oocyte maturation and cell count in embryonic development. The establishment of this EP method offers extensive assays to select pre-implantation embryos and enables the surveying of essential factors for mammalian oocyte quality determination.

KIT in oocytes: a key factor for oocyte survival and reproductive lifespan

The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).

Bisphenol M inhibits mouse oocyte maturation in vitro by disrupting cytoskeleton architecture and cell cycle processes

Bisphenol M (BPM), an alternative to bisphenol A (BPA), is commonly utilized in various industrial applications. However, BPM does not represent a safe substitute for BPA due to its detrimental effects on living beings. This research aimed to assess the influence of BPM exposure on the in vitro maturation of mouse oocytes. The findings revealed that BPM exposure had a notable impact on the germinal vesicle breakdown (GVBD) rate and polar body extrusion (PBE) rate throughout the meiotic progression of mouse oocytes, ultimately resulting in meiotic arrest. Investigations demonstrated that oocytes exposure to BPM led to continued activation of spindle assembly checkpoint. Further studies revealed that securin and cyclin B1 could not be degraded in BPM-exposed oocytes, and meiosis could not realize the transition from the MI to the AI stage. Mechanistically, BPM exposure resulted in abnormal spindle assembly and disrupted chromosome alignment of oocytes. Additionally, abnormal positioning of microtubule organizing center-associated proteins implied that MTOC may be dysfunctional. Furthermore, an elevation in the acetylation level of α-tubulin in oocytes was observed after BPM treatment, leading to decreased microtubule stability. In addition to its impact on microtubules, BPM exposure led to a reduction in the expression of the actin, signifying the disruption of actin assembly. Further research indicated a heightened incidence of DNA damage in oocytes following BPM exposure. Besides, BPM exposure induced alterations in histone modifications. The outcomes of this experiment demonstrate that BPM exposure impairs oocyte quality and inhibits meiotic maturation of mouse oocytes.

The inhibition roles of RAB23 gene in granulosa cell proliferation and progesterone synthesis of hen ovarian prehierarchical follicles

ABSTRACT 1. The proliferation of granulosa cells is vital for the development and recruitment of hen ovarian prehierarchical follicles (PF). The RAB23 protein is a member of the Rab family, belonging to the GTPase family. This study studied the regulatory roles of the RAB23 gene in PF. 2. The expression of RAB23 was significantly increased in granulosa cells (GC) during PF growth and was highest in GC at 6–8 mm diameter (p < 0.05). The RAB23 protein was mainly expressed in the GC, oocytes (OC) as well as somatic cells (SC) of the PF. 3. The mRNA expression of FSHR, CCND1,CYP11A1, StAR and HSD3B1 was significantly increased in the siRNA RAB23 group (p < 0.05). Additionally, protein expression of FSHR, CCND1, CYP11A1, HSD3B1 was significantly increased (p < 0.05) after GC were transfected with RAB23-specific siRNA. Protein expression of StAR in the siRNA RAB23 group was numerically higher than that in the positive control (PC) and negative control (NC) groups. The GC proliferation rate and progesterone synthesis of the prehierarchical follicles in hen ovaries were markedly increased in vitro (p < 0.05). 4.This study revealed that RAB23 might play an inhibitory role in GC proliferation and progesterone synthesis during the prehierarchical follicles development in vitro.

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.