Identification and analysis of key circRNAs in the mouse embryonic ovary provides insight into primordial follicle development.

BACKGROUND: CircRNAs are a class of noncoding RNAs with tissue- and development-specific expression characteristics. In many mammals, primordial follicle development begins in the embryonic stage. However, the study of circRNAs in primordial follicle development in mice has not been reported. RESULTS: In this study, ovaries were collected from mouse foetuses at 15.5 days post coitus (dpc) and 17.5 dpc, which are two key stages of primordial follicle development. A total of 4785 circRNAs were obtained by using RNA-seq. Of these, 83 differentially expressed circRNAs were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these differential circRNAs were mainly involved in the regulation of reproductive development. Through qRT-PCR, back-splice sequence detection and enzyme digestion protection experiments, we found that circ-009346, circ-014674, circ-017054 and circ-008296 were indeed circular. Furthermore, circ-009346, circ-014674 and circ-017054 were identified as three key circRNAs by analysing their expression in the ovaries of mice at different developmental stages. The circRNA-miRNA-mRNA interaction network was constructed and validated for target miRNA and mRNA using qRT-PCR. The interacting genes circ-009346, circ-014674, and circ-017054 were subjected to KEGG enrichment analysis. We found that circ-014674 may participate in the assembly and reserve of primordial follicles through oestrogen and the Janus kinase (JAK) signal transducer and activator of transcription (STAT) signalling pathway (JAK-SATA). Circ-009346 and circ-017054 may have similar functions and are involved in the activation and growth of primordial follicles through the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling pathways. CONCLUSIONS: Based on our findings, three circRNAs associated with primordial follicle development were identified, and their potential mechanisms of regulating primordial follicle development were revealed. These findings will help us better understand the molecular mechanism of circRNAs in primordial follicles and provide important references and targets for the development of primordial follicles.

S100A4 Promotes BCG-Induced Pyroptosis of Macrophages by Activating the NF-κB/NLRP3 Inflammasome Signaling Pathway.

Pyroptosis is a host immune strategy to defend against Mycobacterium tuberculosis (Mtb) infection. S100A4, a calcium-binding protein that plays an important role in promoting cancer progression as well as the pathophysiological development of various non-tumor diseases, has not been explored in Mtb-infected hosts. In this study, transcriptome analysis of the peripheral blood of patients with pulmonary tuberculosis (PTB) revealed that S100A4 and GSDMD were significantly up-regulated in PTB patients' peripheral blood. Furthermore, there was a positive correlation between the expression of GSDMD and S100A4. KEGG pathway enrichment analysis showed that differentially expressed genes between PTB patients and healthy controls were significantly related to inflammation, such as the NOD-like receptor signaling pathway and NF-κB signaling pathway. To investigate the regulatory effects of S100A4 on macrophage pyroptosis, THP-1 macrophages infected with Bacillus Calmette-Guérin (BCG) were pre-treated with exogenous S100A4, S100A4 inhibitor or si-S100A4. This research study has shown that S100A4 promotes the pyroptosis of THP-1 macrophages caused by BCG infection and activates NLRP3 inflammasome and NF-κB signaling pathways, which can be inhibited by knockdown or inhibition of S100A4. In addition, inhibition of NF-κB or NLRP3 blocks the promotion effect of S100A4 on BCG-induced pyroptosis of THP-1 macrophages. In conclusion, S100A4 activates the NF-κB/NLRP3 inflammasome signaling pathway to promote macrophage pyroptosis induced by Mtb infection. These data provide new insights into how S100A4 affects Mtb-induced macrophage pyroptosis.

Human Pluripotent Stem Cell-Mesenchymal Stem Cell-Derived Exosomes Promote Ovarian Granulosa Cell Proliferation and Attenuate Cell Apoptosis Induced by Cyclophosphamide in a POI-like Mouse Model.

Premature ovarian insufficiency (POI) is a complex disease which causes amenorrhea, hypergonadotropism and infertility in patients no more than 40 years old. Recently, several studies have reported that exosomes have the potential to protect ovarian function using a POI-like mouse model induced by chemotherapy drugs. In this study, the therapeutic potential of exosomes derived from human pluripotent stem cell-mesenchymal stem cells (hiMSC exosomes) was evaluated through a cyclophosphamide (CTX)-induced POI-like mouse model. POI-like pathological changes in mice were determined by serum sex-hormones levels and the available number of ovarian follicles. The expression levels of cellular proliferation proteins and apoptosis-related proteins in mouse ovarian granulosa cells were measured using immunofluorescence, immunohistochemistry and Western blotting. Notably, a positive effect on the preservation of ovarian function was evidenced, since the loss of follicles in the POI-like mouse ovaries was slowed. Additionally, hiMSC exosomes not only restored the levels of serum sex hormones, but also significantly promoted the proliferation of granulosa cells and inhibited cell apoptosis. The current study suggests that the administration of hiMSC exosomes in the ovaries can preserve female-mouse fertility.

To explore the regulatory role of Wnt/P53/Caspase3 signal in mouse ovarian development based on LFQ proteomics.

Early ovarian follicular development is regulated by multiple proteins and signaling pathways, including the Wnt gene. To explore the regulatory mechanism of Wnt signaling on early ovarian follicular development, ovaries from 17.5 days post coitum (17.5 dpc) mice were collected and cultured in vitro for four days in the presence of IWP2 as a Wnt activity inhibitor and KN93 as a CaMKII inhibitor. LFQ proteomics technique was then used to analyze the significant differentially abundant (P-SDA) 93 and 262 proteins in the IWP2 and KN93 groups, respectively. Of these, 63 up-regulated proteins and 30 down-regulated proteins were identified for IWP2, along with 3 significant KEGG pathways (P < 0.05). For the KN93 group, 168 up-regulated proteins and 94 down-regulated ones were P-SDA, with 9 significant KEGG pathways also noted (P < 0.05). In both IWP2 and KN93 groups, key pathways (Wnt signaling pathway, Notch signaling pathway, P53 signaling pathway, TGF-ß signaling pathway, ovarian steroid production) and metabolic regulation (energy metabolism, metal ion metabolism) were found to be related to early ovarian follicular development. Finally, western blotting demonstrated the regulatory role of Wnt/P53/Caspase3 signaling pathway in mouse ovarian development. These results contribute new knowledge to the understanding of regulatory factors of early ovarian follicular development. SIGNIFICANCE: In this study, label-free quantification (LFQ) was used in combination with liquid chromatography-mass spectrometer (LC-MS/MS) to study potential changes in the proteomic profiles of embryonic mice subjected to Wnt inhibitor IWP2 and CaMKIIinhibitor KN93. In addition, bioinformatics and comparative analyses were performed using publicly available proteomics databases to further explore the underlying mechanisms associated with early mouse ovarian growth and development.

Cyproterone Acetate Mediates IRE1α Signaling Pathway to Alleviate Pyroptosis of Ovarian Granulosa Cells Induced by Hyperandrogen.

OBJECTIVE: Hyperandrogenemia (HA) is the main pathophysiological change that takes place in polycystic ovary syndrome (PCOS). Cyproterone acetate (CYA) is a drug commonly used to reduce androgen in patients with PCOS. Long-term and continuous exposure to HA can cause ovarian granulosa cells (GCs), pyroptotic death, and follicular dysfunction in PCOS mice. The aim of this study was to investigate whether CYA could ameliorate the hyperandrogenemia-induced pyroptosis of PCOS ovarian GCs by alleviating the activation of the IRE1α signaling pathway. METHODS: Firstly, thirty PCOS patients with HA as their main clinical manifestation were selected as the study group, and thirty non-PCOS patients were selected as the control group. The GCs and follicular fluid of the patients were collected, and the expression of pyroptosis-related proteins was detected. Secondly, a PCOS mouse model induced by dehydroepiandrosterone (DHEA) was constructed, and the treatment group model was constructed with the subcutaneous injection of cyproterone acetate in PCOS mice. The expression of pyroptosis-related protein in ovarian GCs was detected to explore the alleviating effect of CYA on the pyroptosis of ovarian GCs in PCOS mice. Thirdly, KGN cells-i.e., from the human GC line-were cultured with dihydrotestosterone, CYA, and ERN1 (IRE1α gene) small interfering RNA in vitro to explore whether CYA can alleviate the activation of the IRE1α signaling pathway and ameliorate the hyperandrogenemia-induced pyroptosis of PCOS ovarian GCs. RESULTS: The expression of pyroptosis-related proteins was significantly increased in ovarian GCs of PCOS patients with HA as the main clinical manifestation, and in the PCOS mouse model induced by DHEA. After treatment with CYA, the expression of pyroptosis-related proteins in the ovarian GCs of mice was significantly lower than that in PCOS mice. In vitro experiments showed that CYA could ameliorate KGN cells' pyroptosis by alleviating the activation of the IRE1α signaling pathway. CONCLUSION: This study showed that CYA could ameliorate the activation of the IRE1α signaling pathway in mouse GCs and KGN cells, and also alleviate pyroptosis in ovarian GCs. This study provides a new mechanism and evidential support for CYA in the treatment of PCOS patients.

[Wnt5a activates JNK to promote the proliferation and autophagy of KGN human granulosa cells].

Objective To investigate the effect of Wnt5a on autophagy in KGN human granulosa cells. Methods KGN human granulosa cells were treated with DMSO (control group), recombinant Wnt5a protein (rWnt5a), Wnt5a inhibitor IWP2 or BOX5, separately. The expression level of Wnt5a protein was detected by Western blot. The co-localization of the Wnt5a protein and the forkhead box L2 (FOXL2), a specific marker of granulosa cells, was observed by immunofluorescence cytochemical staining in rWnt5a group and IWP2 group. The proliferation of KGN cells was detected by CCK-8 assay. The effects of rWnt5a and IWP2 on autophagy of KGN cells and the expressions of c-Jun N-terminal kinase (JNK) and nuclear factor of activated T cells 1 (NFAT1) proteins were detected by Western blot. Results Compared with those in the control group, the expression of Wnt5a protein in the rWnt5a group was increased, cell proliferation was promoted, and the expressions of microtubule-associated proteins 1 light chain 3 (LC3), JNK, and NFAT1 were increased, while the expression of nucleoporin 62 (P62) protein was decreased. In contrast to the rWnt5a group, the expression of Wnt5a protein was decreased, cell proliferation was inhibited, and the expressions of LC3, JNK, and NFAT1 proteins were decreased, while the expression of P62 protein was increased in IWP2 group. Conclusion Wnt5a promotes the proliferation and autophagy of KGN human granulosa cells by activating JNK.

Effects and potential mechanism of Ca2+/calmodulin­dependent protein kinase II pathway inhibitor KN93 on the development of ovarian follicle.

Adequate regulation of the speed of follicular development has been reported to prolong the reproductive life of the ovary. The aim of the present study was to assess the potential effects and mechanism of the Ca2+/calmodulin­dependent protein kinase II (CaMKII) pathway on the development of ovarian follicle. In the present study, the expression of CaMKII was measured in the ovary of mice at different developmental stages by immunofluorescence, confirming that CaMKII has a role in follicular development. Subsequently, the 17.5 days post­coitus (dpc) embryonic ovaries were collected and cultured with KN93 for 4 days in vitro. It was revealed that KN93 inhibited the development of follicles, where it reduced the expression levels of oocyte and granulosa cell markers DEAD­box helicase 4 (DDX4) and forkhead box L2 (FOXL2). These results suggested that KN93 could delay follicular development. Proteomics technology was then used to find that 262 proteins of KN93 treated 17.5 dpc embryonic ovaries were significantly altered after in vitro culture. Bioinformatics analysis was used to analyze these altered proteins. In total, four important Kyoto Encyclopedia of Genes and Genome pathways, namely steroid biosynthesis, p53 signaling pathway and retinol metabolism and metabolic pathways, were particularly enriched. Further analysis revealed that the upregulated proteins NADP­dependent steroid dehydrogenase­like (Nsdhl), lanosterol synthase (Lss), farnesyl­diphosphate farnesyltransferase 1 (Fdft1), cytochrome P450 family 51 family A member 1 (Cyp51a1), hydroxymethylglutaryl­CoA synthase 1 (Hmgcs1), fatty acid synthase (Fasn) and dimethylallyltranstransferase (Fdps) were directly interacting with each other in the four enriched pathways. In summary, the potential mechanism of KN93 in slowing down follicular development most likely lies in its inhibitory effects on CaMKII, which upregulated the expression of Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps. This downregulated the expression of oocyte and granulosa cell markers DDX4 and FOXL2 in the follicles, thereby delaying follicular development. Overall, these results provide novel insight into the potential mechanism by which KN93 and CaMKII can delay follicular development.