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Wheat (Triticum aestivum L.) is one of the world's most important staple crops, whose production is critical to feed the expanding population worldwide. The 90-kDa Heat Shock Protein 90 (HSP90) is a highly abundant chaperone protein involved in multiple cellular processes. It facilitates the folding of nascent preproteins for their maturation and functioning. This data described HSP90.2 clients identified from the whole genome of wheat. The HSP90.2 chaperome contains over 1500 proteins, most detected by the C terminus and full-length of HSP90.2. Over 60 % of the clients reside in the cytosol, nucleus, and chloroplasts. Cytoskeleton-related proteins are enriched in the chaperome of the N terminus of HSP90.2. The clients of the middle part of HSP90.2 contains several factors involved in ethylene biosynthesis and extracellular vesicle or organelle-related activities. Some clients related to plant hypersensitive response are induced by stripe rust. The presented dataset could isolate proteins regulated by HSP90.2 at the post-translational level.

Abstract Germline-somatic mutation interactions are universal and associated with tumorigenesis, but their role in breast cancer, especially in non-Caucasians, remains poorly characterized. We performed large-scale prospective targeted sequencing of matched tumor-blood samples from 4079 Chinese females, coupled with detailed clinical annotation, to map interactions between germline and somatic alterations. We discovered 368 pathogenic germline variants and identified 5 breast cancer DNA repair-associated genes (BCDGs; BRCA1/BRCA2/CHEK2/PALB2/TP53). BCDG mutation carriers, especially those with two-hit inactivation, demonstrated younger onset, higher tumor mutation burden, and greater clinical benefits from platinum drugs, PARP inhibitors, and immune checkpoint inhibitors. Furthermore, we leveraged a multiomics cohort to reveal that clinical benefits derived from two-hit events are associated with increased genome instability and an immune-activated tumor microenvironment. We also established an ethnicity-specific tool to predict BCDG mutation and two-hit status for genetic evaluation and therapeutic decisions. Overall, this study leveraged the large sequencing cohort of Chinese breast cancers, optimizing genomics-guided selection of DNA damaging-targeted therapy and immunotherapy within a broader population.

Introduction:Ganoderma lucidum (G. lucidum, Lingzhi) has long been listed as a premium tonic that can be used to improve restlessness, insomnia, and forgetfulness. We previously reported that a rat model of sporadic Alzheimer’s disease (sAD) that was induced by an intracerebroventricular injection of streptozotocin (ICV-STZ) showed significant learning and cognitive deficits and sleep disturbances. Treatment with a G. lucidum spore extract with the sporoderm removed (RGLS) prevented learning and memory impairments in sAD model rats.Method: The present study was conducted to further elucidate the preventive action of RGLS on sleep disturbances in sAD rats by EEG analysis, immunofluorescence staining, HPLC-MS/MS and Western blot.Results: Treatment with 720 mg/kg RGLS for 14 days significantly improved the reduction of total sleep time, rapid eye movement (REM) sleep time, and non-REM sleep time in sAD rats. The novelty recognition experiment further confirmed that RGLS prevented cognitive impairments in sAD rats. We also found that RGLS inhibited the nuclear factor-κB (NF-κB)/Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammatory pathway in the medial prefrontal cortex (mPFC) in sAD rats and ameliorated the lower activity of γ-aminobutyric acid (GABA)-ergic neurons in the parabrachial nucleus (PBN).Discussion: These results suggest that inhibiting the neuroinflammatory response in the mPFC may be a mechanism by which RGLS improves cognitive impairment. Additionally, improvements in PBN-GABAergic activity and the suppression of neuroinflammation in the mPFC in sAD rats might be a critical pathway to explain the preventive effects of RGLS on sleep disturbances in sAD.

As a crucial component of the male reproductive system, the epididymis plays multiple roles, including sperm storage and secretion of nutritive fluids for sperm development and maturation. The acquisition of fertilization capacity by sperm occurs during their transport through the epididymis. Compared with the testis, little has been realized about the importance of the epididymis. However, with the development of molecular biology and single-cell sequencing technology, the importance of the epididymis for male fertility should be reconsidered. Recent studies have revealed that different regions of the epididymis exhibit distinct functions and cell type compositions, which are likely determined by variations in gene expression patterns. In this research, we primarily focused on elucidating the cellular composition and region-specific gene expression patterns within different segments of the epididymis and provided detailed insights into epididymal function in male fertility.

Abstract Background Breast fibroadenoma is the most common benign breast tumour. This study aimed to investigate the advantages and disadvantages of endoscopic-assisted resection via a gas-less transaxillary single-port approach for breast fibroadenoma in adolescent patients, compared with a traditional approach. Methods The clinical data of 83 patients with breast fibroadenoma treated in our hospital from October 2019 to October 2021 were collected for retrospective analysis. These patients were divided into an endoscopic-assisted surgery (ES) group (n = 39) and a traditional open surgery (OS) group (n = 44) according to the surgical approach. The operative time, intraoperative blood loss, incision length, postoperative complications, and patient satisfaction were compared between the two groups. Results The surgical cost was (5.1 ± 0.6) thousand Yuan [(0.7 ± 0.1) thousand US dollars] in the ES group and (3.5 ± 2.7) thousand Yuan [(0.5 ± 0.4) thousand US dollars] in the OS group, showing a statistically significant difference (p

(1) Background: Spermatozoa acquired motility and matured in epididymis after production in the testis. However, there is still limited understanding of the specific characteristics of sperm development across different species. In this study, we employed a comprehensive approach to analyze cell compositions in both testicular and epididymal tissues, providing valuable insights into the changes occurring during meiosis and spermiogenesis in mouse and pig models. Additionally, we identified distinct gene expression signatures associated with various spermatogenic cell types. (2) Methods: To investigate the differences in spermatogenesis between mice and pigs, we constructed a single-cell RNA dataset. (3) Results: Our findings revealed notable differences in testicular cell clusters between these two species. Furthermore, distinct gene expression patterns were observed among epithelial cells from different regions of the epididymis. Interestingly, regional gene expression patterns were also identified within principal cell clusters of the mouse epididymis. Moreover, through analysing differentially expressed genes related to the epididymis in both mouse and pig models, we successfully identified potential marker genes associated with sperm development and maturation for each species studied. (4) Conclusions: This research presented a comprehensive single-cell landscape analysis of both testicular and epididymal tissues, shedding light on the intricate processes involved in spermatogenesis and sperm maturation, specifically within mouse and pig models.

Introgression of the short arm of rye chromosome one into common wheat increases root biomass and drought tolerance, but the underlying genetic basis is unknown. Here, the authors report that dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate the differences of wheat root architecture.

Farmlands fertilized with livestock manure-derived amendments have become a hot topic in the dissemination of antibiotic resistance genes (ARGs). Field ponding water connects rice paddies with surrounding water bodies, such as reservoirs, rivers, and lakes. However, there is a knowledge gap in understanding whether and how manure-borne ARGs can be transferred from paddy soil into field ponding water. Our studies suggest that the manure-derived ARGs aadA1, bla1, catA1, cmlA1-01, cmx(A), ermB, mepA and tetPB-01 can easily be transferred into field ponding water from paddy soil. The bacterial phyla Crenarchaeota, Verrucomicrobia, Cyanobacteria, Choloroflexi, Acidobacteria, Firmicutes, Bacteroidetes, and Actinobacteria are potential hosts of ARGs. Opportunistic pathogens detected in both paddy soil and field ponding water showed robust correlations with ARGs. Network co-occurrence analysis showed that mobile genetic elements (MGEs) were strongly correlated with ARGs. Our findings highlight that manure-borne ARGs and antibiotic-resistant bacteria in paddy fields can conveniently disseminate to the surrounding waterbodies through field ponding water, posing a threat to public health. This study provides a new perspective for comprehensively assessing the risk posed by ARGs in paddy ecosystems.

Abstract. Background:. Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions. Methods:. Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730). Results:. Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ± 4.074, 72-h: 10.600 ± 3.517, 8-day: 84.770 ± 2.228), CD4+ SP cells (control: 15.770 ± 4.419, 72-h: 44.670 ± 3.089, 8-day: 6.367 ± 0.513), and CD8+ SP cells (control: 7.000 ± 1.916, 72-h: 34.030 ± 3.850, 8-day: 5.133 ± 0.647) were obviously different at different stages of ET. The polymorphism and length of TCR β chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly. Conclusion:. These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

It has been reported that several phytohormones are involved in the regulation of grain weight, but the role of jasmonic acids (JA) remains elusive. Here, via bulked segregant analysis (BSA)-based cloning, the authors show keto-acyl thiolase 2B involved in β-oxidation during JA synthesis is a positive regulator of wheat grain weight.

BRAFV600E is the most common mutated gene in thyroid cancer and is most closely related to papillary thyroid carcinoma(PTC). We investigated the value of elasticity and grayscale ultrasonography for predicting BRAFV600E mutations in PTC.Methods138 patients with PTC who underwent preoperative ultrasound between January 2014 and 2021 were retrospectively examined. Patients were divided into BRAFV600E mutation-free group (n=75) and BRAFV600E mutation group (n=63). Patients were randomly divided into training (n=96) and test (n=42) groups. A total of 479 radiomic features were extracted from the grayscale and elasticity ultra-sonograms. Regression analysis was done to select the features that provided the most information. Then, 10-fold cross-validation was used to compare the performance of different classification algorithms. Logistic regression was used to predict BRAFV600E mutations.ResultsEight radiomics features were extracted from the grayscale ultrasonogram, and five radiomics features were extracted from the elasticity ultrasonogram. Three models were developed using these radiomic features. The models were derived from elasticity ultrasound, grayscale ultrasound, and a combination of grayscale and elasticity ultrasound, with areas under the curve (AUC) 0.952 [95% confidence interval (CI), 0.914−0.990], AUC 0.792 [95% CI, 0.703−0.882], and AUC 0.985 [95% CI, 0.965−1.000] in the training dataset, AUC 0.931 [95% CI, 0.841−1.000], AUC 0. 725 [95% CI, 0.569−0.880], and AUC 0.938 [95% CI, 0.851−1.000] in the test dataset, respectively.ConclusionThe radiomic model based on grayscale and elasticity ultrasound had a good predictive value for BRAFV600E gene mutations in patients with PTC.

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

Loss of cardiomyocytes is a vital manifestation and predisposing factor of many cardiovascular diseases and will eventually lead to heart failure (HF). On the other hand, adult mammalian cardiomyocytes have a very limited regenerative capacity and cannot achieve self-repair of the myocardium after injury. Therefore, it is necessary to promote regeneration and repair of the myocardium through effective intervention means. Exercise plays an important role in the prevention and rehabilitation of cardiovascular diseases. Exercise can improve ischemia-reperfusion injury, reduce the size of the infarcted area, and improve the quality of life of patients. In addition, exercise has also been shown to be able to elevate the proliferative potential of adult cardiomyocytes and promote myocardial regeneration. Studies have shown that newly formed cardiomyocytes in adult mammalian hearts are mainly derived from pre-existing cardiomyocytes. By regulating various cytokines, transcription factors, and microRNAs (miRNAs), exercise can promote the dedifferentiation and proliferation of pre-existing cardiomyocytes to form new cardiomyocytes. Therefore, this paper focuses on the recent research progress of exercise-induced adult cardiomyocyte proliferation and explores its potential molecular mechanism.

Abstract Background Rib fractures account for a fairly high proportion of chest injuries, ranging from 55 to 80%. The most common mechanisms of injury include: traffic accident, extrusion and falls from significant heights. Besides, the surgical treatment of multiple rib fractures has been accepted by more and more medical professionals. We reported 5 clinical cases of patients with multiple rib fractures undergoing open reduction and internal fixation using 3D printing technology. Case presentation Retrospective analysis of 5 clinical cases of multiple rib fractures from January 2017 to August 2018 in our hospital. A preoperative CT thin slice scan was used to reconstruct the 3D model according to the scanning results, and 3D printing technology was adopted to prepare the rib model. Preoperative reconstruction of the rib’s normal shape and lock plate for the shaped ribs was created according to reconstructed model. For multiple fractures especially patients with severely deformed rib shape, it is suggested to intraoperative shape directly to the metal bone plate fixed on the ribs on both ends of the fracture line, in order to establish a basic support frame. The other various fracture section can be fixed on the lock plate respectively. Postoperative chest radiographs of the 5 patients showed that the internal fixations were in good and natural shape. The thoracic contour was well formed and symmetrically with the contralateral side. Conclusion Making the rib model and the pre-shaped titanium alloy rib locking plate using 3D printing technology, provided a more minimally invasive and precisely individualized treatment for some rib fracture operations.

This paper investigates the event-triggered H∞ filtering for networked control systems (NCSs) with quantization and network-induced delays. With consideration of the limited capacity of the communication channels in NCSs, a quantizer is proposed to quantize control signals before being transmitted into the next node. First, an event-triggered scheme is addressed between the quantizer and the fuzzy filter, which aims to mitigate transmission rate and improve the usage of network resource. Based on the event-triggered scheme, the fuzzy filtering error system is established with quantization and delays. Second, a novel Lyapunov-Krasovskii functional is constructed, and the Writinger inequality is used to deal with the integral item of the derivative of the Lyapunov-Krasovskii functional, which can obtain more useful items. Then, a new stability criterion is addressed to ensure that the filtering error system is asymptotically stable with a prescribe H∞ performance level. By introducing matrix decoupling technique, the fuzzy filter is designed without the coupling matrices. Finally, numerical simulations are given to show the effectiveness of the proposed method.

(1) Background: Deoxynivalenol (DON) and zearalenone (ZEA) are type B trichothecene mycotoxins that exert serious toxic effects on the reproduction of domestic animals. However, there is little information about the toxicity of mycotoxins on testis development in Equus asinus. This study investigated the biological effects of DON and ZEA exposure on Sertoli cells (SCs) of Equus asinus; (2) Methods: We administered 10 μM and 30 μM DON and ZEA to cells cultured in vitro; (3) Results: The results showed that 10 μM DON exposure remarkably changed pyroptosis-associated genes and that 30 μM ZEA exposure changed inflammation-associated genes in SCs. The mRNA expression of cancer-promoting genes was remarkably upregulated in the cells exposed to DON or 30 μM ZEA; in particular, DON and ZEA remarkably disturbed the expression of androgen and oestrogen secretion-related genes. Furthermore, quantitative RT-PCR, Western blot, and immunofluorescence analyses verified the different expression patterns of related genes in DON- and ZEA-exposed SCs; (4) Conclusions: Collectively, these results illustrated the impact of exposure to different toxins and concrete toxicity on the mRNA expression of SCs from Equus asinus in vitro.

Sternal fracture is a common complication of chest trauma but has a low incidence. Various treatments have been developed to reconstruct sternal fractures. Among these approaches, analgesia, corset fixation, and open reduction with plate internal fixation have been suggested. The use of newly developed minimally invasive plate osteosynthesis is a feasible method. In this study, we report a case involving a 54-year-old man with a sternal fracture accompanied by bilateral pleural effusion and a small amount of right-sided pneumothorax. The patient was treated with minimally invasive plate osteosynthesis. The operation was successful and the postoperative recovery was good. No pneumothorax or complications such as chest pain, paresthesia, or wound infection were observed at the 1-year follow-up visit. Additionally, the bilateral pleural effusion had been completely absorbed. The incision in the lower chest was aesthetic and minimally traumatic. This case describes a novel method for internal fixation of sternal fractures.

Zearalenone (ZEA), one of the most prevalent estrogenic mycotoxins, is mainly produced by Fusarium fungi and has been proven to affect the reproductive capacity of animals. Exposure of farm animals to ZEA is a global public health concern because of its toxicity and wide distribution in animal feeds. In vitro and in vivo experiments indicate that ZEA possesses estrogenic activity in mice, swine, Equus asinus and cattle. The precise mechanism of the reproductive toxicity of ZEA has not been established yet. This article reviews evidence on the deleterious effects of ZEA on mammalian folliculogenesis from early to final oogenesis stages. Such effects include impaired granulosa cell (GC) development and follicle steroidogenesis, reduced oocyte nest breakdown, damaged meiotic progression, poor fetal oocyte survival, accelerated primordial follicle activation and enhanced follicle atresia. These phenomena may result in reproductive and non-reproductive problems in domestic animals. In addition, emerging data indicates that ZEA may cause mRNA expression changes in the GCs. In general, E. asinus is more sensitive than swine to ZEA exposure. Finally, results of in vivo animal studies and in vitro tests are reported and discussed.

Zearalenone (ZEA) is a natural contaminant existing in food and feed products that exhibits a negative effect on domestic animals’ reproduction. Donkeys possess high economic value in China and are at risk of exposure to ZEA. However, few information is available on ZEA-induced toxicity and no report on toxicity in donkeys can be found in scientific literature. We investigated the biological effects of ZEA exposure on donkey granulosa cells (dGCs) by using RNA-seq analysis. ZEA at 10 and 30 μM were administered to GCs within 72 h of in vitro culture. ZEA at 10 μM significantly altered the tumorigenesis associated genes in dGCs. Exposure to 10 and 30 μM ZEA treatment significantly reduced mRNA expression of PTEN, TGFβ, ATM, and CDK2 genes, particularly, the ZEA treatment significantly increased the expression of PI3K and AKT genes. Furthermore, immunofluorescence, RT-qPCR, and Western blot analysis verified the gene expression of ZEA-exposed GCs. Collectively, these results demonstrated the deleterious effect of ZEA exposure on the induction of ovarian cancer related genes via the PTEN/PI3K/AKT signaling pathway in dGCs in vitro.

Alternanthera philoxeroides is a notoriously invasive weed that can readily adapt to different environmental conditions. Control of this weed is difficult, and it spreads easily and causes damage to native habitats and agriculture. In this study, our goal was to investigate the molecular mechanisms that lead to the ability of A. philoxeroides to invade new habitats, to adapt to environmental stresses, and to cause damage. We developed a simple and highly effective potato virus X-based virus-induced gene silencing (VIGS) approach. The VIGS approach was first used to silence the phytoene desaturase gene, which resulted in the expected photo-bleaching phenotype. Next, the VIGS approach was used to silence two additional genes, drought-induced protein gene 15 (ApDRI15) and salinity-induced protein gene 1 (ApSI1). When ApDRI15 was knocked down, the plants were more sensitive to drought stress than the control plants, with smaller leaves, shorter internodes, and lower biomass. The ApDRI15-silenced plants had lower relative water content, lower free proline levels, and higher water loss rates than the control. Silencing of ApSI1 significantly decreased tolerance to salinity, and the ApSI1-silenced plants were withered and smaller. These results indicate that the pgR107 VIGS approach is a simple and highly effective tool for dissecting gene function in A. philoxeroides. Further experiments with the VIGS approach will enhance our understanding of the molecular mechanisms of the adaptability and plasticity of A. philoxeroides and improve our ability to combat the damage caused by this weed.

Methyl 3-amino-6-methoxythieno [2,3-b] quinoline-2-carboxylate (PU-48) is a novel diuretic urea transporter inhibitor. The aim of this study is to investigate the profile of plasma pharmacokinetics, tissue distribution, and excretion by oral dosing of PU-48 in rats. Concentrations of PU-48 within biological samples are determined using a validated high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. After oral administration of PU-48 (3, 6, and 12 mg/kg, respectively) in self-nanomicroemulsifying drug delivery system (SNEDDS) formulation, the peak plasma concentrations (Cmax), and the area under the curve (AUC0–∞) were increased by the dose-dependent and linear manner, but the marked different of plasma half-life (t1/2) were not observed. This suggests that the pharmacokinetic profile of PU-48 prototype was first-order elimination kinetic characteristics within the oral three doses range in rat plasma. Moreover, the prototype of PU-48 was rapidly and extensively distributed into thirteen tissues, especially higher concentrations were detected in stomach, intestine, liver, kidney, and bladder. The total accumulative excretion of PU-48 in the urine, feces, and bile was less than 2%. This research is the first report on disposition via oral administration of PU-48 in rats, and it provides important information for further development of PU-48 as a diuretic drug candidate.

BACKGROUND: No specific antiviral agent against hand foot and mouth disease (HFMD) is available for clinical practice today. OBJECTIVE: To evaluate the efficacy and safety of Jinzhen oral solution in treating uncomplicated HFMD. METHODS: In this randomized, double-blind, placebo-controlled trial, 399 children aged 1 to 7 years with laboratory confirmed HFMD were randomized to receive Jinzhen oral liquid or placebo 3 times daily for 7 days with a 3-day follow-up. The primary outcomes were time to the first disappearance of oral ulcers and vesicles on hand or foot and time to the first normalization of temperature (fever clearance). RESULTS: There were 199 children enrolling into the Jinzhen group including 79 with fever and 200 into the placebo group including 93 with fever. Jinzhen reduced the time to the first disappearance of oral ulcers and vesicles on hand or foot to 4.9 days (95% CI, 4.6 to 5.2 days), compared with 5.7 days (95% CI, 5.4 to 6.0 days) in the placebo group (P = 0.0036). The median time of fever clearance was shorter in the 79 children who received Jinzhen (43.41 hrs, 95% CI, 37.05 to 49.76) than that in the 93 children who received placebo (54.92 hrs, 95% CI, 48.16 to 61.68) (P = 0.0161). Moreover, Jinzhen reduced the risk of symptoms by 28.5% compared with placebo (HR, 0.7150, 95% CI, 0.5719 to 0.8940, P = 0.0032). More importantly, treatment failure rate was significantly lower in the Jinzhen group (8.04%) compared with that in the placebo group (15.00%) (P = 0.0434). The incidence of serious adverse events did not differ significantly between the two groups (9 in Jinzhen group vs. 18 in placebo, P = 0.075). CONCLUSIONS: Children with HFMD may benefit from Jinzhen oral liquid treatment as compared with placebo. TRIAL REGISTRATION: Chinese Clinical Trial Registry (http://www.chictr.org/en/) ChiCTR-TRC-10000937.

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day-old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.