Comparative transcriptome profile analysis of granulosa cells from porcine ovarian follicles during early atresia.

At various stages of ovarian follicular development, more than 99% of follicles will be eliminated through a degenerative process called atresia. The regulatory mechanisms of atresia have been elucidated to some extent, involving hormones, growth factors, cytokines, and other factors. However, the stimuli initiating atresia in follicular granulosa cells remain unknown. In this study, we isolated the granulosa cells from porcine ovarian follicles (3-5 mm diameter) divided into healthy follicles (HFs) and early atretic follicles (EAFs). We applied high-throughput RNA sequencing to identify and compare differentially expressed genes (DEGs) between HFs and EAFs. A total of 31,694 genes were detected, of which 21,806 were co-expressed in six samples, and 243 genes (p < 0.05; FDR < 0.05) were differentially expressed (DEGs), including 123 downregulated and 120 upregulated in EAFs. GO analysis highlighted hormone metabolism, plasma membrane localization, and transporter activity. The pathway analysis indicated that 51 DEGs, involved in steroidogenesis, cell adhesion molecules, and TGF-beta signaling pathways, were highly related to atresia. Additionally, the interaction network of DEGs (p < 0.01; FDR < 0.05) using STRING highlighted LHR, ACACB, and CXCR4 as central nodes. In summary, this transcriptome analysis enriched our knowledge of the shifted mechanisms in granulosa cells during early atresia and provided novel perspectives into the atresia initiation.

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells.

The ubiquitin-specific peptidase 9 X-linked (USP9X) is one of the highly conserved members belonging to the ubiquitin-specific proteases (USPs) family, which has been reported to control substrates-mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF-ß) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin-proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR-143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR-143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3'-untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF-ß signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro-regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR-143) and the TGF-ß signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility.

SMARCA2 is regulated by NORFA-miR-29c, a novel pathway that controls granulosa cell apoptosis and is related to female fertility.

SMARCA2, an evolutionarily conserved catalytic ATPase subunit of SWI/SNF complexes, has been implicated in development and diseases; however, its role in mammalian ovarian function and female fertility is unknown. Here, we identified and characterized the 3'-UTR of the porcine SMARCA2 gene and identified a novel adenylate number variation. Notably, this mutation was significantly associated with sow litter size traits and SMARCA2 levels, due to its influence on the stability of SMARCA2 mRNA in ovarian granulosa cells (GCs). Immunohistochemistry and functional analysis showed that SMARCA2 is involved in the regulation of follicular atresia by inhibiting GC apoptosis. In addition, miR-29c, a pro-apoptotic factor, was identified as a functional miRNA that targets SMARCA2 in GCs and mediates regulation of SMARCA2 expression via the NORFA-SMAD4 axis. Although a potential miR-29c-responsive element was identified within NORFA, negative regulation of miR-29c expression by NORFA was not due to activity as a competing endogenous RNA. In conclusion, our findings demonstrate that SMARCA2 is a candidate gene for sow litter size traits, because it regulates follicular atresia and GC apoptosis. Additionally, we have defined a novel candidate pathway for sow fertility, the NORFA-TGFBR2-SMAD4-miR-29c-SMARCA2 pathway.This article has an associated First Person interview with the first author of the paper.

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging.

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

The Role of Circular RNAs in the Physiology and Pathology of the Mammalian Ovary.

Circular RNAs (circRNAs) are an abundant class of endogenous non-coding RNAs (ncRNAs) generated from exonic, intronic, or untranslated regions of protein-coding genes or intergenic regions. The diverse, stable, and specific expression patterns of circRNAs and their possible functions through cis/trans regulation and protein-coding mechanisms make circRNA a research hotspot in various biological and pathological processes. It also shows practical value as biomarkers, diagnostic indicators, and therapeutic targets. This review summarized the characteristics, classification, biogenesis and elimination, detection and confirmation, and functions of circRNAs. We focused on research advances circRNAs in the mammalian ovary under conditions including ovarian cancer, polycystic ovarian syndrome (PCOS), and maternal aging, as well as during reproductive status, including ovarian follicle development and atresia. The roles of circRNAs in high reproductive traits in domestic animals were also summarized. Finally, we outlined some obstructive factors and prospects to work with circRNA, aiming to provide insights into the functional research interests of circRNAs in the reproduction and gynecology areas.

Long-distance chromatin interaction of IGF1 during embryonic and postnatal development in the liver of Sus scrofa.

The dynamics of chromatin have been the focus of studies aimed at characterizing gene regulation. Among various chromosome conformation capture methods, 4C-seq is a powerful technique to identify genome-wide interactions with a single locus of interest. Insulin-like growth factor 1 (IGF1) is a member of the somatotropin axis that plays a significant role in cell proliferation and growth. Determining the IGF1-involved genome-wide chromatin interaction profile at different growth stages not only is important for understanding IGF1 transcriptional regulation but also provides a representation of genome-wide chromatin transformation during development. Using the IGF1 promoter as a "bait", we identified genome-wide interactomes of embryonic (E70) and postnatal (P1 and P70) pig liver cells by 4C-seq. The IGF1 promoter interactomes varied significantly among the three developmental stages. The most active chromatin interaction was observed in the P1 stage, while the highest interaction variability was observed in the P70 stage. The identified 4C sites were enriched around transcription start sites, CpG sites and functional pig QTLs. In addition, the genes located in the interacting regions and the involved pathways were also analysed. Overall, our work reveals a distinct long-distance regulatory pattern in pig liver during development for the first time, and the identified interacting sites and genes may serve as candidate targets in further transcriptional mechanism studies and effective molecular markers for functional traits.

circRNA-Mediated Inhibin-Activin Balance Regulation in Ovarian Granulosa Cell Apoptosis and Follicular Atresia.

Ovarian granulosa cells (GC) play an essential role in the development and atresia of follicles. Emerging studies suggest that non-coding RNAs are involved in the regulation of GC apoptosis. Here, we aimed to analyze the function of ssc-circINHA-001, coded by the first exon of the inhibin subunit α gene (INHA), in resisting GC apoptosis and follicular atresia by enhancing the expression of the inhibin subunit ß A (INHBA) through a cluster of miRNAs. A higher expression of ssc-circINHA-001 in healthy follicles compared to early atretic follicles was detected by qRT-PCR. Its circular structure was confirmed by RNase R treatment and reversed PCR. The function of ssc-circINHA-001 in GC resistance to apoptosis was detected by in vitro transfection of its si-RNA. Furthermore, the dual-luciferase reporter assay suggested that ssc-circINHA-001 adsorbed three miRNAs, termed miR-214-5p, miR-7144-3p, and miR-9830-5p, which share the common target INHBA. A low expression of ssc-circINHA-001 increased the levels of the free miRNAs, inhibited INHBA expression, and thus raised GCs apoptosis through a shift from the secretion of activin to that of inhibin. Our study demonstrated the existence of a circRNA-microRNAs-INHBA regulatory axis in follicular GC apoptosis and provides insight into the relationship between circRNA function and its coding gene in inhibin/activin balance and ovarian physiological functions.

TGF-ß/SMAD4 signaling pathway activates the HAS2-HA system to regulate granulosa cell state.

Both TGF-ß/SMAD4 signaling pathway and HAS2-HA system have been shown to control granulosa cell (GC) state in mammalian ovary. However, the regulatory relationship between TGF-ß/SMAD4 signaling pathway and HA system in GCs is not well known. Here, we report that the TGF-ß/SMAD4 signaling pathway activates the HAS2-HA system by binding directly to the HAS2 promoter, ultimately controlling the GC state via the CD44-Caspase3 axis. SMAD4-induced HAS2 expression, HAS2-driven HA secretion, and HAS2-mediated GC state (proliferation and apoptosis) by interacting directly with the promoter region of the HAS2 gene. The CD44-Caspase3 axis, located downstream of the HAS2-HA system, was also activated by SMAD4 and the TGF-ß/SMAD4 signaling pathway. However, there was no feedback regulation of the TGF-ß/SMAD4 signaling pathway by the HAS2-HA system in GCs. In addition, we found that miRNA-26b attenuated HAS2 expression via SMAD4-dependent and -independent mechanisms. Our findings provide compelling evidence that HAS2 is a direct transcriptional target of SMAD4. They also reveal a novel mechanism by which the TGF-ß/SMAD4 signaling pathway controls the GC state and alters the structural components of GCs in porcine ovaries.

The distribution and expression of vascular endothelial growth factor A (VEGFA) during follicular development and atresia in the pig.

The involvement of vascular endothelial growth factor A (VEGFA) in ovarian physiological processes has been widely reported, but the location and role of VEGFA during follicular atresia remain unknown. This study investigated the distribution and expression of VEGFA during porcine follicular development and atresia. Pig ovaries were obtained, individual medium-sized (3-5mm in diameter) antral follicles were separated and classified into healthy, early atretic or progressively atretic groups. Immunobiology and quantitative techniques were used to investigate the varied follicular distribution of VEGFA at both the morphological and molecular level. The results indicated that VEGFA protein expression peaked in tertiary follicles, mostly distributed in the thecal and inner granulosa layers, during follicular development while VEGFA mRNA was mainly expressed in the inner granulosa layers. Additionally, healthy antral follicles showed a significantly higher expression of VEGFA than atretic follicles in both theca and granulosa cells. Knockdown of VEGFA using siRNA revealed an antiapoptosis effect of VEGFA in cultured pig granulosa cells. Our results increase the knowledge of VEGFA functions in follicles.

Detection of the effects and potential interactions of FSH, VEGFA, and 2-methoxyestradiol in follicular angiogenesis, growth, and atresia in mouse ovaries.

Ovarian follicular development is a complex process that requires codevelopment of the perifollicular vascular network, which is closely regulated by angiogenic factors, gonadotropins, sex steroids, and their metabolites. To detect the effects of vascular endothelial growth factor 120 (VEGF120), follicle-stimulating hormone (FSH), and 2-methoxyestradiol (2ME2) on follicular angiogenesis during development and atresia, we treated sexually immature and mature female mice with VEGF120, FSH, 2ME2, and FSH receptor (FSHR) antagonist singly or in combination via intraperitoneal injection. The number of follicles and their perifollicular angiogenesis and atresia rates at different developmental stages were examined in paraffin sections after hematoxylin and eosin staining. The results showed that the exogenous factors have specific and precise effects on developmental, angiogenesis, and atresia processes in follicles of different sizes in mature and immature mice. Perifollicular angiogenesis was regulated by VEGFA and closely related to follicular development and atresia. 2ME2 affected angiogenesis through VEGFA and might regulate atresia directly. FSH might control VEGFA function via both transcriptional and posttranscriptional mechanisms because FSHR was required for achieving VEGFA functions at all the follicular development stages. The present study presents insights into the mechanisms of FSH, 2ME2, and VEGFA in follicular development and disorders and provides a foundation for the development of new therapeutic strategies.

MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis.

MicroRNAs (miRNAs) are short, noncoding RNAs that posttranscriptionally regulate gene expression. In the past decade, studies on miRNAs in ovaries have revealed the key roles of miRNAs in ovarian development and function. In this review, we first introduce the development of follicular atresia research and then summarize genome-wide studies on the ovarian miRNA profiles of different mammalian species. Differentially expressed miRNA profiles during atresia and other biological processes are herein compared. In addition, current knowledge on confirmed functional miRNAs during the follicular atresia process, which is mostly indicated by granulosa cell (GC) apoptosis, is presented. The main miRNA families and clusters, including the let-7 family, miR-23-27-24 cluster, miR-183-96-182 cluster and miR-17-92 cluster, and related pathways that are involved in follicular atresia are thoroughly summarized. A deep understanding of the roles of miRNA networks will not only help elucidate the mechanisms of GC apoptosis, follicular development, atresia and their disorders but also offer new diagnostic and treatment strategies for infertility and other ovarian dysfunctions.

miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-ß signaling by interacting with the TGFBR1 promoter.

SMAD7 disrupts the TGF-ß signaling pathway by influencing TGFBR1 stability and by blocking the binding of TGFBR1 to SMAD2/3. In this study, we showed that SMAD7 attenuated the TGF-ß signaling pathway in ovarian granulosa cells (GCs) by regulating TGFBR1 transcriptional activity. To function as a transcription factor, SMAD7 downregulated the mRNA levels of TGFBR1 via direct binding to the SMAD-binding elements (SBEs) within the promoter region of pig TGFBR1. We also showed that SMAD7 enhanced porcine GC apoptosis by interrupting TGFBR1 and the TGF-ß signaling pathway. Interestingly, miR-181b, a microRNA that is downregulated during porcine follicular atresia, was identified to be directly targeting SMAD7 at its 3'-UTR. By inhibiting SMAD7, miR-181b could inhibit GC apoptosis by activating the TGF-ß signaling pathway. Our findings provide new insights into the mechanisms underlying the regulation of the TGF-ß signaling pathway by SMAD7 and miR-181b.

Initiation of follicular atresia: gene networks during early atresia in pig ovaries.

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involved in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (Sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly upregulated in early atretic with respect to healthy group and 308 were downregulated. Similar expression trends were observed between microarray data and quantitative RT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with (1) a crosstalk of cell apoptosis, autophagy and ferroptosis rather than change of typical apoptosis markers, (2) dramatic shift of steroidogenic enzymes, (3) deficient glutathione metabolism and (4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.

Androgen receptor and miRNA-126* axis controls follicle-stimulating hormone receptor expression in porcine ovarian granulosa cells.

Androgen, which acts via the androgen receptor (AR), plays crucial roles in mammalian ovarian function. Recent studies showed that androgen/AR signaling regulates follicle-stimulating hormone receptor (FSHR) expression in follicles; however, the detailed mechanism underlying this regulation remained unknown. Here, we demonstrate that AR and miR-126* cooperate to inhibit FSHR expression and function in pig follicular granulosa cells (pGCs). In pGCs, overexpression of AR decreased, whereas knockdown increased, FSHR mRNA and protein expression; however, neither manipulation affected FSHR promoter activity. Using a dual-luciferase reporter assay, we found that the FSHR gene is a direct target of miR-126*, which inhibits FSHR expression and increases the rate of AR-induced apoptosis in pGCs. Collectively, our data show for the first time that the AR/miR-126* axis exerts synergetic effects in the regulation of FSHR expression and apoptosis in pGCs. Our findings thus define a novel pathway, AR/miR-126*/FSHR, that regulates mammalian GC functions.

MicroRNA-26b functions as a proapoptotic factor in porcine follicular Granulosa cells by targeting Sma-and Mad-related protein 4.

Sma- and Mad-related protein 4 (SMAD4) is the central mediator of the transforming growth factor beta signaling pathway and is closely related to mammalian reproductive ability and the development of ovarian follicles. However, little is currently known about the role of SMAD4 in mammalian follicular granulosa cell (GC) apoptosis or its regulation by miRNAs. Here, we found that the porcine SMAD4 protein was expressed at high levels in GCs and oocytes from primary, preantral, and antral follicles, and only slightly expressed in theca cells; its expression level was down-regulated in apoptotic ovarian GCs, suggesting that SMAD4 may be involved in ovary development and selection. Overexpression and knockdown of SMAD4 increased the proliferation and apoptosis of cultured porcine GCs, respectively. In addition, the use of miRNA mimics and luciferase reporter assays revealed that miRNA-26b (miR-26b) functions as a proapoptotic factor in porcine follicular GCs by targeting the 3'-untranslated region of the SMAD4 gene. Overexpression of miR-26b in follicular GCs suppressed SMAD4 mRNA and protein levels, resulting in down-regulation of the antiapoptotic BCL-2 gene and the promotion of GC apoptosis. Furthermore, transforming growth factor beta 1 (TGF-beta1) down-regulates miR-26b expression in porcine GCs. Taken together, these data suggest that SMAD4 plays a critical role in porcine follicular GC apoptosis and follicular atresia and that miR-26b may have a proapoptotic role in GCs by regulating the expression of SMAD4 in the transforming growth factor beta signaling pathway.

From stability to reliability: Unveiling the un-biased reference genes in porcine ovarian granulosa cells under different conditions.

Selection of optimal reference genes (RGs) is fundamental for functional genomics studies and gene expression analysis, which are two main approaches to identify functional genes and their expression patterns. However, no systematic study has identified the suitable RGs in porcine ovarian granulosa cells (GCs) which are essential for follicle fate and sow fertility. In this study, the expression profiles of 12 widely-used RGs (GAPDH, RPLP0, ACTB, TUBA1B, EIF3K, PPIA, ATP5F1, B2M, HPRT1, UBC, RPS3, and EEF1A1) in porcine GCs during follicular development and under different abiotic stresses were systematically investigated. Expression stability of the candidate RGs were comprehensively accessed by five statistical algorithms including ΔCt, NormFinder, BestKeeper, geNorm, and RefFinder, indicating that RPS3 and PPIA are the optimal RGs during follicular development, EEF1A1 and RPLP0 are most stable under oxidative stress and inflammation, while ATP5F1, B2M, and RPS3 have higher stability under starvation and heat stress. Notably, the most commonly used RGs (ACTB, GAPDH, and TUBA1B) exhibited low stability in GCs. Reliability of stable RGs was verified by RT-qPCR and showed that selection of the stable RGs significantly improved the detection accuracy of qPCR, which confirms once again that the stability of RGs should not be taken for granted. Our findings identified optimal RG sets in porcine GCs under different conditions, which is helpful in future studies to accurately identify the key regulators and their expression patterns during follicular development in sows.

The super-enhancer repertoire in porcine liver.

The transcriptional initiation of genes is inextricably bound with the functions of cis-regulatory sequences. The pig is one of the most important livestock species and an ideal animal model for biomedical studies. At the same time, the liver is a critical organ with diverse and complex metabolic functions. Here, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with high-throughput sequencing to profile the chromatin landscape of histone H3 lysine 27 acetylation (H3K27ac), histone H3 lysine 4 monomethylation (H3K4me1), and CCAAT enhancer-binding protein ß (C-EBPß) in the 70-d-old porcine liver, compared the different profiles among the three markers and their associated stitched-enhancers by stitching and sorting the peaks within 12.5 kb (Pott and Lieb, 2015) and generated the porcine liver-specific super-enhancers (SEs) by the combination of three markers. Compared to typical enhancers (TEs) and other stitched-enhancers, liver-specific SEs showed a higher density of cis-motifs and SNPs, which may recruit more tissue-specific vital TFs. The expression profiles in fetal and 70-d-old pigs proved that a large proportion of SE-associated genes were up-regulated and were more related to hepatic metabolisms and detoxification pathways. Our results illustrated the difference and connection among promoter and enhancer markers, identified the features of liver SEs and their associated genes, and provided novel insight into cis-element identification, function, and liver transcriptional regulation.

The cis-regulatory elements including promoters, enhancers, and newly identified super-enhancers (SEs), which were reported to function both promoter and enhancer capabilities, play critical roles in selective gene expression during development and disease. To reveal and compare the characteristics of these cis-elements in liver, we first performed a genome-wide profile of H3K27ac, H3K4me1, and C-EBPß, then constructed their associated stitched-enhancers respectively. Porcine liver-specific SEs were generated by overlapping the three stitched-enhancers. The genomic and genic location, TF binding sites and SNP distribution patterns were compared among these cis-elements. We found that stitched-enhancers gather in regions with higher gene densities and locate closer to the transcription starting sites. Additionally, SEs showed higher density of TF binding sites and SNPs. To access the transcriptional consequences of liver SEs, we first analyzed the genes locationally associated with SEs. The KEGG results suggested that these genes are significantly involved in metabolisms, detoxification, and autophagy pathways. We also detected the liver gene expression profiles using RNA-seq and noticed that SE-associated genes are more likely to be up-regulated. Our results provided novel information on the identification, function, and transcriptional regulation of cis-elements in the liver.

Deep learning based de-overlapping correction of projections from a flat-panel micro array X-ray source: Simulation study.

PURPOSE: Flat-panel X-ray source is an experimental X-ray emitter with target application of static computer tomography (CT), which can save imaging space and time. However, the X-ray cone beams emitted by the densely arranged micro-ray sources are overlapped, causing serious structural overlapping and visual blur in the projection results. Traditional deoverlapping methods can hardly solve this problem well. METHOD: We converted the overlapping cone beam projections to parallel beam projections through a U-like neural network and selected structural similarity (SSIM) loss as the loss function. In this study, we converted three kinds of overlapping cone beam projections of the Shepp-Logan, line-pairs, and abdominal data with two overlapping levels to corresponding parallel beam projections. Training completed, we tested the model using the test set data that was not used at the training phase, and evaluated the difference between the test set conversion results and their corresponding parallel beams through three indicators: mean squared error (MSE), peak signal-to-noise ratio (PSNR) and SSIM. In addition, projections from head phantoms were applied for generalization test. RESULT: In the Shepp-Logan low-overlapping task, we obtained a MSE of 1.624×10-5, a PSNR of 47.892 dB, and a SSIM of 0.998 which are the best results of the six experiments. For the most challenging abdominal task, the MSE, PSNR, and SSIM are 1.563×10-3, 28.0586 dB, and 0.983, respectively. In more generalized data, the model also achieved good results. CONCLUSION: This study proves the feasibility of utilizing the end-to-end U-net for deblurring and deoverlapping in the flat-panel X-ray source domain.

A transmission measurement-based spectrum estimation method incorporating X-ray tube voltage fluctuation.

Background: The energy spectrum is the property of the X-ray tube that describes the energy fluence per unit interval of photon energy. The existing indirect methods for estimating the spectrum ignore the influence caused by the voltage fluctuation of the X-ray tube. Methods: In this work, we propose a method for estimating the X-ray energy spectrum more accurately by including the voltage fluctuation of the X-ray tube. It expresses the spectrum as the weighted summation of a set of model spectra within a certain voltage fluctuation range. The difference between the raw projection and the estimated projection is considered as the objective function for obtaining the corresponding weight of each model spectrum. The equilibrium optimizer (EO) algorithm is used to find the weight combination that minimizes the objective function. Finally, the estimated spectrum is obtained. We refer to the proposed method as the poly-voltage method. The method is mainly aimed at the cone-beam computed tomography (CBCT) system. Results: The model spectra mixture evaluation and projection evaluation showed that the reference spectrum can be combined by multiple model spectra. They also showed that it is appropriate to choose about 10% of the preset voltage as the voltage range of the model spectra, which can match the reference spectrum and projection quite well. The phantom evaluation showed that the beam-hardening artifact can be corrected using the estimated spectrum via the poly-voltage method, and the poly-voltage method provides not only the accurate reprojection but also an accurate spectrum. The normalized root mean square error (NRMSE) index between the spectrum generated via the poly-voltage method and the reference spectrum could be kept within 3% according to above evaluations. There existed a 1.77% percentage error between the estimated scatter of polymethyl methacrylate (PMMA) phantom using the two spectra generated via the poly-voltage method and the single-voltage method, and it could be considered for scatter simulation. Conclusions: Our proposed poly-voltage method could estimate the spectrum more accurately for both ideal and more realistic voltage spectra, and it is robust to the different modes of voltage pulse.

SDNOR, a Novel Antioxidative lncRNA, Is Essential for Maintaining the Normal State and Function of Porcine Follicular Granulosa Cells.

Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR's regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs.