Follicle isolation and GC collection
All experimental procedures were performed according to the guidelines for the care and use of laboratory animals and approved by the Animal Ethics Committee of Guangxi University. The pig ovaries were collected from a local slaughterhouse and returned to the laboratory in normal saline with high pressure treatment within 1 hour. The ovarian tissue was washed with 75% ethanol and cut with ophthalmic scissors. The single follicle with a diameter of 3-5 mm was separated and
Detection of autophagy in GCs from porcine follicles with different degrees of atresia
To examine the difference in GC autophagy of porcine follicles with different degrees of atresia, the GCs of the three groups were pooled. MDC staining, WB and TEM were implemented to detect autophagy. With increasing extent of atresia, the number of MDC-positive cells increased significantly and the LC3-II/LC3-I ratio showed an increasing trend. TEM showed that the GCs in the EAF and PAF groups had more autophagic vesicles than those in the HF group (Fig. 2).
Follicular atresia induced by GC autophagy
To check if GC can induce autophagy
Although the hypothalamic-pituitary-ovarian axis plays an important role in follicular atresia through endocrine regulation, more cellular processes such as autophagy are also thought to be involved in this regulation . Autophagy is an adaptive response to maintain cell homeostasis and normal physiological activity by removing intracellular waste through a lysosomal mechanism . However, excessive activation of autophagy can lead to programmed cell death . ovules in follicles
The present study found that the degree of follicular atresia was positively correlated with the autophagy level of GCs and negatively with the m6A modification level of GCs. In addition, autophagy can induce follicular atresia. The m6A modification may regulate GC autophagy by altering theULK1transcription levels. Our study reveals a possible mechanism by which the m6A modification regulates follicular atresia, providing new insight into the mechanism of follicular atresia
This work was jointly supported by theNatural Science Foundation of China(grant number32160788);Natural Science Foundation of Guangxi Province(grant number2021GXNSFDA075001,2020GXNSFBA297090);Project to improve basic skills for junior and junior high school teachers in Guangxi Province(2020KY07004);Self-funded scientific research project of the Health Commission of the Guangxi Zhuang Autonomous Region in China(Z-A20220028);Young Scientist Research Award Fund of Guangxi People's Hospital
CRediT Authorship Contribution Statement
Zhengda Li:Conceptualization, Resources, Formal Analysis, Writing - Original Draft.Ziyun Ruan:Resources, Research, Methodology, Writing - Original Draft.Yun Feng:resources, methodology.Yanxin Wang:Resources.Jun Zhang:Investigation.Canqiang Lu:Formal Analysis.Deshun Shi:Conceptualization, resources, fundraising.Fenghua Lu:Conception, acquisition of funding, writing – review & editing.
declarations of competing interests
The authors declare no conflict of interest.
Featured Articles (6)
Single-layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies the chromatin structure in boar sperm
Theriogenology, Band 201, 2023, S. 95-105
Storage of boar semen samples at 17°C for artificial insemination (AI) doses allows the bacteria to proliferate, necessitating antibiotics. This can contribute to the development of antimicrobial resistance (AMR). This study tested the presence of bacteria and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were divided into 500 ml tubes as control and low density colloid centrifugation (single layer centrifugation, SLC, 20% and 30% Porcicoll). Analyzes were performed on days 0, 3 and 7 (17°C) for microbial presence and sperm chromatin structure analysis: % DFI (DNA fragmentation) and % HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges) and chromomycin A3 (CMA3; chromatin condensation). In addition to comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these groups of variables were described by canonical correlation analysis (CCA). The results showed a significant decrease in some bacteria or complete clearance after SLC (particularly for P30). SLC also caused a decrease in %HDS and an increase in disulfide bonds and low- and medium-mBBr populations, indicating removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, particularly enterobacteria.P.aeuriginosa,AndK. varicella. In summary, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low density colloid not only removes bacteria from boar semen but also improves chromatin structure after selection.
Effect of crowding stress on growth performance, the antioxidant system, and humoral immunity in hybrid sturgeon
Aquaculture Reports, Volume 28, 2023, Item 101468
To investigate the effects of crowding stress on the physiological mechanisms of juvenile hybrid sturgeon (♀Acipenser the baths×♂Acipenser schrenckii, 23±1g body weight) an experiment was assigned to three initial stocking densities in running waters (LD = 100 and 150 fish/m3; MD = 200 and 250 fish/m3; and HD = 300 and 350 fish/m3) for 60 days. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to identify different metabolites between different stocking densities. Final weight, weight gain rate (WGR) and specific growth rate (SGR) decreased significantly with increasing stocking density (P<0.05). Gill filaments were injured in the MD and HD groups. Constriction stress significantly increased serum cortisol and glucose levels (P<0.05) and inhibited antioxidant capacity. However, stocking density did not affect innate immunity parameters in fish, including complement c3, immunoglobulin M (IgM), and lysozyme (LZM). These negative growth performances against overcrowding stress mainly affected pathways associated with ascorbate and aldarate metabolism, butanoate metabolism, and fatty acid catabolism. Gluconeogenesis and glycogenolysis were activated by the glucagon signaling pathway to accommodate the increased energy expenditure of fish under stress, which played a protective role against lipid peroxidation. Assessing these factors allows for a more complete understanding of the underlying physiological mechanisms associated with overcrowding stress in hybrid sturgeon.
Analyzes of broad metabolic profiles reveal mechanisms of metabolomic variations during testis development in Tibetan sheep (Ovis aries).
Theriogenology, Band 197, 2023, S. 116-126
In mammals, the testis is the organ with the highest transcriptional activity. After gene transcription, translation and post-translational protein modification, the transcriptional results are finally presented at the metabolic level. Metabolites, which are not only essential for cell signaling and energy transfer, but are also directly affected by the physiological and pathological changes in tissues and accurately reflect the physiological changes. The fact that the testes are oxygen-poor organs may explain why Sertoli cells and germ cells may use different metabolic pathways to obtain energy at their different developmental stages. Therefore, the study of metabolic changes during testicular development can better elucidate the metabolic profile of the testis, which is essential for uncovering characteristic metabolic pathways. The present study applied a large-scale UPLC-MS/MS-based metabolomics approach with large-scale detection, identification, and quantification to investigate the widespread metabolic changes during testicular development of Tibetan sheep. First, a total of 847 metabolites were detected in the sheep testicles and their changes as well as the three testicular development stages were further investigated. The results showed that these metabolites were grouped into amino acids and their derivatives, carbohydrates and their derivatives, organic acids and their derivatives, benzene and substituted derivatives, alcohols and amines, lipids, nucleotides and their derivatives, bile acids, coenzymes and vitamins, and hormones hormone-related compounds, etc. Among them, the most abundant metabolites in the testes were amino acids and lipid metabolites. The results showed that most lipids, carbohydrates with their derivatives, and alcohol and amine metabolites were high in sexually immature sheep, while organic acids, amino acids, and nucleotides showed a continuously increasing trend along with testicular developmental stages. Among them, levels of metabolites with antioxidant activity increased along with testicular development, while those associated with energy synthesis were downregulated with age. Further correlation analysis of each metabolite-metabolite pair emphasized the mutual influence between different metabolites throughout testicular development, suggesting a significant correlation between lipids and other metabolites. Finally, based on KEGG pathway analysis, we found that the metabolic pathways in Tibetan sheep testicular development were mainly clustered into energy metabolism, gonadal development, and antioxidant stress. Reactive oxygen species (ROS) are by-products of normal cellular metabolism and are unavoidable during testicular energy metabolism. Thus, the antioxidant stress function is a key process in maintaining the normal physiological function of the testicles. These results contributed to a broader view of the testis metabolome and to a comprehensive analysis of the metabolomic variation in different developmental stages of the testis and provided us with a theoretical basis to understand the metabolic mechanism of the ovine testis.
mRNA 3'-UTR-mediated translational control by PAS and CPE in sheep oocyte
Theriogenology, Band 201, 2023, S. 30-40
In oocytes, cytoplasmic polyadenylation and translation of maternal mRNAs is regulatedcis-Elements including polyadenylation signal (PAS) and cytoplasmic polyadenylation element (CPE) in 3'-UTR. Recent studies elucidate non-canonical polyadenylation mechanisms of translational regulation in mouse oocytes, which differs from that in Xenopus oocytes. However, it is still unclear whether this regulation works in rodent oocytes in the pet oocyte. Here, using sheep as an animal model, we cloned the 3' UTRs ofCpeb1orBtg4and ligated it into the pRK5 flagGfpVector. Variant numbers and positions of PASs and CPEs within the 3' UTRs were constructed to demonstrate their effects on translational control. Afterin vitro-Transcription and microinjection into oocytes in the fully grown germinal vesicle stage of sheep, the expression efficiency of mRNAs was demonstrated by the GFP and Flag expression. Our results indicate that: (i) PAS located at the proximal end of the 3'-UTR can mediate translation of the maternal mRNAs as long as they are distant from CPEs; (ii) the proximal PAS has a higher efficiency in regulating transcription than the distal; (iii) an increase in PAS number can promote translational activity more efficiently; (iv) a single CPE located near PAS (<50 bp) in 3' UTRs ofCpeb1orBtg4could partially suppress the translation. In 3ʹ UTRs fromBtg4two CPEs have a higher inhibitory effect, and three CPEs can completely inhibit mRNA translation. These results confirm the existence of the non-canonical mechanism in pet oocytes.
BDE-209 disrupted the proliferation and differentiation of spermatogonia during the mitotic process through the estrogen receptor α
Reproductive Biology, Volume 23, Issue 2, 2023, Item 100737
Exposure to decabromodiphenyl ether (BDE-209) caused spermatogenesis disorder resulting in poor sperm quality and has become a public concern in recent years. Spermatogenesis refers to the process by which the division of spermatogonia stem cells (SSCs) produces haploid spermatozoa, including mitosis, meiosis, and spermiogenesis. However, the mechanism of mitosis, including the proliferation and differentiation of BDE-209-induced spermatogonia dysfunction, remains largely unclear. Here, our data showed that BDE-209 exposure caused deterioration in sperm quality with disruption of seminiferous tubule structure in rats. Furthermore, BDE-209 exposure damages spermatogonia proliferation and differentiation with decreasing PLZF and cKit levels in the testis. Furthermore, rats exposed to BDE-209 decreased expression of ERα, while increased expression of Wnt3a and Wnt5a occurred. Mechanistically, propipyrazoletriol (PPT, a selective ERα pathway agonist) supplementation rescued sperm quality and attenuated the impairment of spermatogonia proliferation and differentiation in BDE-209-induced rats. Therefore, ERα plays a crucial role in the proliferation and differentiation of spermatogonia during the mitotic process. In conclusion, our study clarified the role of ERα in BDE-209-induced sperm proliferation and differentiation in rats and offers potential therapeutic application in poor sperm quality induced by BDE-209 exposure.
Current management of stage IIIA (N2) non-small cell lung cancer: role of perioperative immunotherapy and tyrosine kinase inhibitors
Thoracic Surgery Clinics, 2023
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