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.

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.

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.

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.

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.

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.

Comparative miRNA expression profile analysis of porcine ovarian follicles: new insights into the initiation mechanism of follicular atresia.

Follicular atresia occurs in every stage of ovarian development, which is relevant to female fertility. In the past decade, increasing studies have confirmed that miRNAs, a class of short non-coding RNAs, play an important role in follicular atresia by post-transcription regulation of their target genes. However, the function of miRNAs on follicular atresia initiation is unknown. In the present study, high-throughput small RNA sequencing was performed to analyze differential miRNA expression profiles between healthy (HF) follicles and early atretic (EAF) follicles. A total of 237 conserved miRNA were detected, and the miR-143 is the highest expressed in follicles. Meanwhile, we also found wide sequence variations (isomiRs) in porcine ovarian miRNA, including in 5'un-translation region, core seed sequences and 3'untranslation region. Furthermore, we identified 22 differentially expressed miRNAs in EAF groups compared to HF group, of which 3 miRNAs were upregulated, as well as 19 miRNAs were downregulated, and then the RT-PCR was performed to validate these profiles. The target genes of these differentially expressed miRNAs were predicted by using miRwalk, miRDB, and Targetscan database, respectively. Moreover, the gene ontology and KEGG pathway enrichment established that the regulating functions and signaling pathways of these miRNAs contribute to follicular atresia initiation and cell fate. In conclusion, this study provides new insights into the changes of miRNAs in early atretic follicles to demonstrate their molecular regulation in ovarian follicular atretic initiation.

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.

Effective Quality Breeding Directions-Comparison and Conservative Analysis of Hepatic Super-Enhancers between Chinese and Western Pig Breeds.

The transcriptional initiation of genes is closely bound to the functions of cis-regulatory elements, including promoters, typical enhancers (TEs), and recently-identified super-enhancers (SEs). In this study, we identified these cis-regulatory elements in the livers of two Chinese (Meishan and Enshi Black) and two Western (Duroc and Large White) pig breeds using ChIP-seq data, then explored their similarities and differences. In addition, we analyzed the conservation of SEs among different tissues and species (pig, human, and mouse). We observed that SEs were more significantly enriched by transcriptional initiation regions, TF binding sites, and SNPs than other cis-elements. Western breeds included fewer SEs in number, while more growth-related QTLs were associated with these SEs. Additionally, the SEs were highly tissue-specific, and were conserved in the liver among humans, pigs, and mice. We concluded that intense selection could concentrate functional SEs; thus, SEs could be applied as effective detection regions in genomic selection breeding.

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.

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.

SMAD4 Inhibits Granulosa Cell Apoptosis via the miR-183-96-182 Cluster and FoxO1 Axis.

The miR-183-96-182 cluster is a polycistronic miRNA cluster necessary for ovarian functions in mammals. However, its transcriptional regulation in the ovary is largely unclear. In this study, we characterized the promoter region of the porcine miR-183-96-182 cluster, and showed that SMAD4 may function as a transcriptional activator of the miR-183-96-182 cluster in GCs through direct binding to SBE motifs in its promoter. SMAD4 may inhibit GC apoptosis via suppression of FoxO1, an effector of GC apoptosis and a direct target of the miR-183-96-182 cluster, by inducing the miR-183-96-182 cluster, and this process may be regulated by the TGF-ß/SMAD signaling pathway. Our findings uncovered the regulatory mechanism of miR-183-96-182 cluster expression in GCs and demonstrated that TGF-ß1/SMAD4/miR-183-96-182 cluster/FoxO1 may be a potential pathway for regulating follicular atresia and female reproduction.

NORHA, a novel follicular atresia-related lncRNA, promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis.

BACKGROUND: Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility, which are regulated by many factors such as microRNAs (miRNAs), which constitute a class of noncoding RNAs (ncRNAs). However, little is known about long noncoding RNAs (lncRNAs), which constitute another ncRNA family that regulate follicular atresia. RESULTS: A total of 77 differentially expressed lncRNAs, including 67 upregulated and 10 downregulated lncRNAs, were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing. We characterized a noncoding RNA that was highly expressed in atretic follicles (NORHA). As an intergenic lncRNA, NORHA was one of the upregulated lncRNAs identified in the atretic follicles. To determine NORHA function, RT-PCR, flow cytometry and western blotting were performed, and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress. To determine the mechanism of action, bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay were performed, and the results showed that NORHA acted as a 'sponge', that directly bound to the miR-183-96-182 cluster, and thus prevented its targeted inhibition of FoxO1, a major sensor and effector of oxidative stress. CONCLUSIONS: We provide a comprehensive perspective of lncRNA regulation of follicular atresia, and demonstrate that NORHA, a novel lncRNA related to follicular atresia, induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.

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.

miR-2337 induces TGF-ß1 production in granulosa cells by acting as an endogenous small activating RNA.

Transforming growth factor-ß1 (TGF-ß1) is essential for ovarian function and female fertility in mammals. Herein, we identified three completely linked variants, including two known variants referred to as c.1583A > G and c.1587A > G and the novel variant c.2074A > C in the porcine TGF-ß1 3'-UTR. An important role of these variants in Yorkshire sow fertility was revealed. Variants c.1583A > G and c.1587A > G were located at the miRNA response element (MRE) of miR-2337 and affected miR-2337 regulation of TGF-ß1 3'-UTR activity. Interestingly, miR-2337 induces, not reduces the transcription and production of TGF-ß1 in granulosa cells (GCs). Mechanistically, miR-2337 enhances TGF-ß1 promoter activity via the MRE motif in the core promoter region and alters histone modifications, including H3K4me2, H3K4me3, H3K9me2, and H3K9ac. In addition, miR-2337 controls TGF-ß1-mediated activity of the TGF-ß signaling pathway and GC apoptosis. Taken together, our findings identify miR-2337 as an endogenous small activating RNA (saRNA) of TGF-ß1 in GCs, while miR-2337 is identified as a small activator of the TGF-ß signaling pathway which is expected to be a new target for rescuing GC apoptosis and treating low fertility.

SMAD4-induced knockdown of the antisense long noncoding RNA BRE-AS contributes to granulosa cell apoptosis.

Antisense long noncoding RNAs (AS-lncRNAs), a sub-class of lncRNAs, are transcribed in the opposite direction from their overlapping protein-coding genes and are implicated in various physiological and pathological processes. However, their role in female reproduction remains largely unknown. Here, we report that BRE-AS, an AS-lncRNA transcript from intron 10 of the protein-coding gene BRE, is involved in granulosa cell (GC) apoptosis. Based on our previous RNA sequencing data, we identified 28 AS-lncRNAs as important in the initiation of porcine follicular atresia, with BRE-AS showing the most significant upregulation in early atretic follicles. In this study, gain- and loss-of-function assays demonstrated that BRE-AS induces early apoptosis in GCs. Mechanistically, BRE-AS acts in cis to suppress the expression of BRE, an anti-apoptotic factor, via direct interaction with the pre-mRNA transcript of the latter, inducing increased GC apoptosis. Notably, we also found that BRE-AS was upregulated in SMAD4-silenced GCs. SMAD4 was identified as a transcriptional repressor of BRE-AS because it inhibits BRE-AS expression and BRE-AS-mediated GC apoptosis. In conclusion, we not only identified a novel AS-lncRNA related to the early apoptosis of GCs and initiation of follicular atresia but also described a novel regulatory pathway, SMAD4/BRE-AS/BRE, coordinating GC function and female fertility.

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.

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.

miR-130a/TGF-ß1 axis is involved in sow fertility by controlling granulosa cell apoptosis.

TGF-ß1 is a ligand of the TGF-ß superfamily and an important cytokine that regulates ovarian functions including follicular development, steroid production, ovulation, luteinization, and female fertility. However, little is known about the regulation of TGF-ß1 expression in ovary. Here, we identified that TGF-ß1 is a functional target of miR-130a in porcine ovarian granulosa cells (GCs). The 3'-UTR sequence of TGF-ß1 gene (1137 bp in length) in Large White (LW) pig was isolated, and multiple RNA regulatory elements (RREs), including several binding motifs of different miRNAs, were identified in this region. Luciferase activity assay showed that miR-130a dramatically suppresses the 3'-UTR luciferase activity of TGF-ß1 gene, and further inhibits the expression of TGF-ß1 in porcine GCs. FACS revealed that miR-130a acts as a pro-apoptotic factor and promotes GC apoptosis by inhibiting TGF-ß1. Two novel linked mutations (-573G > A and -540T  >  C) were identified in the promoter region of ssc-miR-130a, but their polymorphisms are not associated with sow reproductive traits. Importantly, combined genotype analysis with a known mutation (c.1583 A  >  G) in the 3'-UTR of porcine TGF-ß1 gene showed a significant association with reproductive performance in LW sow population. Overall, our findings defined a novel regulatory axis, miR-130a/TGF-ß1 axis, which is involved in regulating sow fertility.

miR-361-5p Mediates SMAD4 to Promote Porcine Granulosa Cell Apoptosis through VEGFA.

Follicular atresia is an inevitable degenerative process that occurs in mammalian ovarian follicles. The molecular events involved in atresia, particularly granulosa cell apoptosis, have long attracted researchers' attention. Vascular endothelial growth factor A (VEGFA) is downregulated during follicular atresia in porcine ovaries and serves as an inhibitor of apoptosis in granulosa cells. In addition, transforming growth factor (TGF)-ßsignaling has been considered a central trigger in granulosa cell apoptosis. However, the link between TGF-ß signaling and VEGFA is unknown. We proved that miR-361-5p is significantly upregulated during the atresia process and that it promotes GC apoptosis by directly targeting the VEGFA 3'UTR. In addition, we revealed that the miR-361-5p coding gene MIR361 was significantly downregulated by SMAD4, the central intracellular mediator of TGF-ß signaling, that bound to the MIR361 promoter. In conclusion, our findings expanded what is known about VEGFA posttranscriptional regulation and revealed a complete SMAD4/miR-361-5p/VEGFA regulatory network in ovarian granulosa cell apoptosis. These data provide useful references for follicular atresia and ovarian physiological function studies.