Oxidative stress controls lncRNA-mediated granulosa cell functions in a FoxO1-dependent manner

doi:10.21203/rs.3.rs-4703620/v1

Download PDF

Research Article

Oxidative stress controls lncRNA-mediated granulosa cell functions in a FoxO1-dependent manner

https://doi.org/10.21203/rs.3.rs-4703620/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Oxidative stress (OS) is intensely involved in female low fertility by altering the multi-omics such as transcriptome, miRome, and lncRNome in follicular cells and follicular fluid. However, the mechanism by which OS affects multi-omics dynamics is largely unknown. Here, we report that OS induces lncRNome dynamics in sow granulosa cells (sGCs) partially depending on the transcription factor activity of its effector FoxO1.

Results

2283 putative FoxO recognition elements (FREs) were identified in the promoters of 394 lncRNAs, accounting for 91.20% (394/432) of the lncRNAs stimulated by OS. ChIP and reporter assays showed that effector FoxO1 mediates OS regulation of the transcription activity of lncRNAs in a transcription factor activity-dependent manner. In sGCs, OS induces the transcription of NORSF, a nuclear lncRNA involved in sGCs functions and its mediated cell apoptosis via FoxO1. Furthermore, FoxO1 was identified as a transcription activator of NORSF in sGCs by interacting with the FRE motif of its promoter. Meanwhile, OS reduces the transcription of CYP19A1, which encodes an essential enzyme for estrogen synthesis, and 17β-estradiol (E2) release by sGCs via the FoxO1 and NORSF axis. Phenotypically, transcriptional dysregulation of NORSF transcription caused by two novel adjacent transitions in the promoter leads to decreased sow fertility.

Conclusion

These results suggest a model of OS-stimulated lncRNome dynamics of sGCs, and a new signaling pathway of OS influences sGC functions and sow fertility.

Oxidative stress

granulosa cell apoptosis

FoxO1

NORSF

sow fertility

The gene-environment interactions (GEI) determine all phenotypes of organisms, including sow reproductive traits, and their level determines the economic benefits of the swine industry. Oxidative stress (OS) is considered a common mechanism underlying various internal and external environmental factors (e.g., heat stress, density, social ranks, and feed mycotoxins) cause sow low fertility [1–3]. Some measures to reduce OS, such as adding antioxidant stress additives to feed or reducing density, can improve sow fertility [1, 3–5]. Similarly, in sow ovary, OS leads to various abnormalities (e.g., apoptosis, autophagy, meiotic arrest, and oxidative damage) of follicular cells, including granulosa cells (GCs), oocytes, and luteal cells, and multiple omics disorders, while antioxidants can reverse these processes [6–9]. A more recent reporter showed that OS affects sGC functions and sow fertility through miR-23a, the first causal microRNA (miRNA) for sow fertility traits [10].

Interestingly, OS has also been shown to control sow ovarian functions by interacting with non-coding RNAs [7, 11, 12]. For miRNAs, 55 miRNAs were discovered to participate in the stimulation of OS on sGC apoptosis [7]. OS induces sGC apoptosis and oxidative damage via the miR-192 and Acvr2a axis [11]. On the other hand, several miRNAs (e.g., miR-183 cluster and let-7a) rescue OS-inducing sGC apoptosis via interaction with FoxO1, a well-known sensor and effector of OS [13], and an apoptosis-inducing factor [14]. For long non-coding RNA (lncRNA), SDNOR restrained by OS, suppresses OS-induced sGC apoptosis and maintains the states and functions of GCs [12]. In addition, NORHA, a lncRNA up-regulated during sow follicular atresia, can synergistically induce FoxO1 expression with OS, thereby inducing an increase in the sGC apoptosis rate [13]. Overall, little is known about the interaction between OS and ncRNAs in regulating sow ovarian functions.

We previously demonstrated that OS can cause significant changes in the lncRNAome dynamics in sGCs and identified hundreds of differentially expressed lncRNAs (DELs) [12]. However, the mechanism by which OS influences the transcription of these DELs is still unclear. As is well known, the OS’s effector, FoxO1, is also a transcription factor (TF) that activates or inhibits target genes by interacting with the FoxO1 response elements (FREs) in their promoters [15, 16]. In this study, we sought to explore the OS regulating DELs transcription mechanism from the perspective of TF activity of its effector FoxO1.

Materials

The following reagents and kits were used: TRIzol (Angle Gene), H₂O₂ (Sigma, St. Louis, USA), transfection agent Lipofectamine 2000 (Thermofisher Scientific), trypsin (Thermofisher Scientific), HiScript II Q Select RT SuperMix (Vazyme) for reverse transcription, AceQ qPCR SYBR Green Master Mix (Vazyme), a DLR™ assay system for luciferase assay (Promega) and a Detection Kit for Estrodial (E2) (North Biotech). DMEM and RPMI 1640 medium were ordered from Gibco (Carlsbad, USA) and Hyclone (Logan, USA). For antibodies, anti-FoxO1 (1: 1000) and IgG (1:1000) were ordered from Sangon (Shanghai, China). KGN cells were ordered from GuanDao (Shanghai, China) for cell lines.

Sows

Fresh ovaries and ear tissue blocks were isolated from newly slaughtered commercial sows with sexually mature (Zhushun, Nanjing, China) and Yorkshire sows with fertility records (Kangle, Jintan, China), respectively.

Bioinformatics

Promoter sequences were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/), and the TF-binding sites (TFBSs) were predicted with the JASPAR database (http://jaspar.genereg.net/). DAVID bioinformatics (https://david.ncifcrf.gov/) was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Data for tissue expression and quantitative trait locus (QTLs) were obtained from the NCBI database and the Animal QTLdb database (https://www.animalgenome.org/), respectively. The DNA:dsRNA triplexes in the FoxO1 promoter were detected with Gaemons (http://www.gaemons.net/LongTarget).

Constructs and small interfering RNAs (siRNAs)

The overexpression plasmids of NORSF and FoxO1 were previously constructed [13, 17]. Promoters harboring the wild-type or mutated version of TFBSs were ligated into the pGL3-basic vector (Promega), and primers are given in Table S1. FoxO1- and NORSF-specific siRNAs (Table S2) were ordered from Generay (Shanghai, China).

Cells and treatment

sGCs were aspirated from antral follicles with 3–5 nm and cultured in DMEM with 15% FBS. KGN cells were cultured in RPMI 1640 with 15% FBS. Lipofectamine 2000 was used for cell transfection. After transfection for 12 h, sGCs were replaced into DMEM without FBS, and H₂O₂ was added at the final concentration of 150 µM and exposed for 2 h.

Reporter assays

KGN cells were co-treated with expression plasmids, reporter constructs, and pRL-TK for 24 h, and then collected for lysis and measurement. The luciferase reading of each sample was obtained from an enzyme-labeling instrument (Thermofisher Scientific, Rockford, USA) and normalized using the pRL-TK levels.

Chromatin immunoprecipitation (ChIP)

ChIP was conducted as described earlier [10]. After cross-linking in formaldehyde, precipitating chromatin by centrifugation, and treating with sonication, the samples were precleared with protein A/G agarose beads and incubated with anti-FoxO1 antibody. Primers (Table S3) and qPCR was applied to the input and bound DNA. Negative control was DNA enriched with IgG antibody; input control was unprocessed DNA.

Quantitative real-time PCR (qPCR)

GCs were collected at 24h post-transfection to extract total RNA with the TRIzol reagent. Reverse transcription and qPCR were conducted with corresponding kits and procedure [10]. The 2^−ΔΔCT method was employed to calculate the transcription level, with the GAPDH and RPLP0 serving as the reference controls. Primers are given in Table S4.

Fluorescence-activated cell sorting (FACS)

After washing twice with pre-cooled PBS, sGCs were dyed with 5 µL of each Annexin V and Propidium Iodide (PI) for 10 min. Then, sGCs were diluted with Binding Buffer and sorted with flow cytometry (Becton Dickinson, Franklin Lakes, USA). sGCs count in each quadrant is read with FlowJo v7.6 software (TreeStar, Nutley, USA).

SNV screening, genotyping, and association analysis

Identification of single nucleotide variations (SNVs) and genotyping were conducted with DNA sequencing. SAS v9.2 software (SAS Institute, Cary, USA) was employed for association analysis with linear models in literature [18]. Fertility traits include the total number of piglets born (TNB), the number of piglets born alive (NBA), the number of stillbirths (NSB), and the litter weight (LW). Primers are given in Table S5.

Statistics

Two-tailed T-test and one-way analysis of variance in Prism v9.0 software (GraphPad, USA) were used to evaluate data obtained from at least three biological replicates. The level of significance was accepted as P < 0.05.

OS stimulates the lncRNome of sGCs in a FoxO1-dependent manner.

To understand whether FoxO1 participates as a TF in OS-stimulated lncRNome dynamics of sGCs, we predicted the FRE motifs in the promoters of 432 OS-stimulated DELs [12]. A total of 2283 putative FRE motifs were identified, and 394 DELs were detected with at least one FRE motif, accounting for 91.20% (394/432) of the DELs stimulated by OS (Fig. 1A, and Table S6). GO and KEGG analysis showed that 909 potential cis-target mRNAs of 394 DELs are markedly enriched in pyruvate metabolism, fatty acid degradation, and regulation of actin cytoskeleton (Fig. 1B, S1, Table S7, and S8). Additionally, 101 potential cis-target mRNAs were differentially expressed in OS-stimulated sGCs, with 24.62% (97/394) of DELs having at least one cis-target mRNAs regulated by OS (Fig. 1C, Table S9). Together, these data suggest that OS stimulates lncRNome of sGCs, which may be mainly related to the TF activity of its effector FoxO1.

OS and its effector FoxO1 have been shown to be related to the sow follicular atresia [10, 13]. Therefore, we conducted a joint analysis of the lncRNAome of sGCs stimulated by OS and the lncRNAome of sow follicular atresia [17], and found that out of 19 DELs during follicular atresia, two (i.e, LOC100512907 and LOC102164325) were stimulated by OS (Fig. 1D). Interestingly, the promoters of these two DELs all contain two FRE motifs, respectively (Fig. 1E). We next investigated whether FoxO1 mediates OS regulation of target lncRNAs in sGCs through direct interacting with their promoters. As expected, TF FoxO1 physically interacts with the promoters of these two DELs, and this interaction was stimulated by OS (Fig. 1F). Furthermore, reporter assays showed thatFoxO1 increases the promoter activities of these two DELs (Fig. 1G-H). Additionally, depleting FoxO1 can restrain the increase in the promoter activities of these four DELs caused by OS (Fig. 1I), indicating that FoxO1 can mediate OS regulation of the promoter activities of these two DELs. Taken together, our results support that OS controls the transcriptional activity of lncRNAs in sGCs in a FoxO1-dependent manner.

FoxO1 mediates OS induction of NORSF transcription and cell apoptosis.

We noticed that LOC102164325 (also known as non-coding RNA involved in sow fertility or NORSF) was intensely engaged in sow follicular atresia [17]. Thus, NORSF was selected as a target for FoxO1 mediated the role of OS for subsequent investigation. As expected, a remarkable rise in NORSF transcript was observed in sGCs under OS triggered by H₂O₂ exposure (Fig. 2A). Furthermore, depleting FoxO1 in sGCs resulted in a remarkable decrease in NORSF levels, while OS triggered by H₂O₂ exposure can reverse this process (Fig. 2B). The above results suggest that the effector FoxO1 mediates OS induction of lncRNA NORSF transcription in sGCs.

OS is well-known to be an essential factor in inducing cell apoptosis in sGCs [10, 13]. We herein first confirmed that OS triggered by H₂O₂ exposure could indeed induce sGC apoptosis (Fig. 2C). However, this process was reversed by depleting FoxO1 (Fig. 2C), confirming that OS induces cell apoptosis via its effector FoxO1 in sGCs. Similarly, depleting FoxO1 in sGCs resulted in a remarkable decrease in apoptosis rate. At the same time, overexpression of NORSF transcript can partially increase apoptosis rate (Fig. 2D). Furthermore, under OS triggered by H₂O₂ exposure, the same results were observed (Fig. 2C). Additionally, depleting NORSF transcript can restrain the decrease in apoptosis rate caused by overexpression of FoxO1 (Fig. 2E). These data support the conclusion that OS and its effector FoxO1 control sGC apoptosis through inducing NORSF transcription.

FoxO1 controls NORSF transcriptional activity via the FRE1 motif in its core promoter.

We first predicted the potential TFBSs in the core promoter of NORSF and identified 149 TFBSs of 79 TFs with a relative score ≥ 0.90 (Fig. 3A; Table S10). These TFs were significantly enriched in endometrial cancer, acute myeloid leukemia, and FoxO signaling pathway (Fig. S2). Electronic tissue expression profiling showed that 9 TFs, such as GATA4, FoxO1, and NFIX, have relatively high abundance in sow ovarian tissue (Fig. 3B; Table S11). Additionally, 4 TFs including FoxO1, NFIX, TCF7L1, and FoxO3 were observed to be stimulated by OS triggered by H₂O₂ exposure (Fig. 3C), using our previous transcriptome data from OS-stimulated sGCs [7]. Combined with previous ChIP assays, these data suggest that FoxO1 is a critical TF of NORSF in sGCs.

We have previously confirmed that TF FoxO1 directly interacts with the promoter of NORSF gene to enhance its transcriptional activity (Fig. 1). Furthermore, two FRE motifs are located at -121/-117 nt (FRE1 motif) and − 18/-11 nt (FRE2) (Fig. 3D). Therefore, we next sought to investigate that FoxO1 induces NORSF transcriptional activity via which motifs, and generated the reporter constructs of the NORSF core promoter harboring FRE motifs (Fig. 3D). Compared with empty vector, transfection of FoxO1 overexpression vector gave a remarkable induction in the activity of NORSF core promoter (Fig. 3E). Furthermore, FoxO1 induces the activity of NORSF promoter via the FRE1 motif in its core promoter (Fig. 3F). Together, combined with previous ChIP assay, our data suggest that FoxO1 is an inducer of NORSF in sGCs by physically interacting with the FRE1 motif of NORSF core promoter.

FoxO1 is a transcription activator of NORSF in sGCs.

Next, we investigate the effect of TF FoxO1 on endogenous NORSF transcription in sGCs. Compared with empty vector, transfection of FoxO1 expression construct gave a remarkable induction in the levels NORSF transcript in sGCs (Fig. 4A). By contrast, silencing of FoxO1 gave a noteworthy reduction in the levels of NORSF transcript in sGCs (Fig. 4B). Combined with reporter assay and ChIP experiment, these data support that FoxO1 is a vital transcription activator of lncRNA NORSF in sGCs.

Our previous report showed that NORSF is a nuclear lncRNA in sGCs [17]. Nuclear lncRNAs usually control the transcription of targets by forming a DNA:dsRNA triplex with their promoters [19, 20]. Interestingly, five putative triplex target sites (TTSs) of the NORSF transcript were detected in the porcine FoxO1 promoter, and five putative triplex-forming oligonucleotides (TFOs) were detected in the NORSF transcript (Fig. 4C), suggesting that nuclear NORSF may potentially bind to the FoxO1 promoter. However, we did not observe significant changes in FoxO1 mRNA levels in sGCs overexpressing NORSF transcript (Fig. 4D-E), indicating that NORSF has no feedback regulatory effect on its transcription activator FoxO1 transcription in sGCs.

The FoxO1 and NORSF axis mediates OS reduction of E2 release.

NORSF is a vital modulator of E2 release by sGCs [17]. We next investigated whether its transcription activator FoxO1 regulates E2 release by sGCs. Compared with empty vector, transfection of FoxO1 expression construct gave a remarkable reduction in E2 concentration in the culture medium of sGCs (Fig. 5A). In contrast, silencing of FoxO1 gave a remarkable induction in E2 concentration (Fig. 5B), indicating that FoxO1 restrains E2 release by sGCs. Furthermore, depleting NORSF transcript can restrain the decrease in E2 release caused by FoxO1 (Fig. 5A), while overexpressing NORSF transcript can restrain the increase in E2 release caused by depleting FoxO1 (Fig. 5B). These data suggest that NORSF mediates FoxO1 reduction of E2 release in sGCs.

NORSF has been shown to suppress E2 release in sGCs by inhibiting the transcription of CYP19A1, a gene that encodes an essential enzyme for estrogen synthesis [17]. We therefore investigated whether FoxO1 regulates CYP19A1 transcription in sGCs. Compared with empty vector, transfection of FoxO1 expression construct gave a remarkable reduction in CYP19A1 transcription in sGCs (Fig. 5C), whereas silencing of FoxO1 gave a remarkable induction in CYP19A1 transcription (Fig. 5D), indicating that FoxO1 reduces CYP19A1 transcription in sGCs. Furthermore, depleting NORSF transcript can restrain the decrease in CYP19A1 transcription caused by FoxO1 (Fig. 5C), while overexpressing NORSF transcript can restrain the increase in CYP19A1 transcription caused by depleting FoxO1 (Fig. 5D). These data suggest that NORSF and CYP19A1 axis mediates FoxO1 reduction of E2 release in sGCs.

Similarly, we also showed that, like its effector FoxO1, OS induces the decrease in CYP19A1 transcription, while inactivation of FoxO1 can reverse this process (Fig. 5E), indicating that OS restrains CYP19A1 transcription via FoxO1. Meanwhile, under OS triggered by H₂O₂ exposure, overexpressing NORSF can restrain the increase in CYP19A1 transcription caused by depleting FoxO1 (Fig. 5E). These data suggest that OS restrains CYP19A1 transcription via the FoxO1 and NORSF axis.

NORSF is a candidate gene for sow fertility.

Notably, two adjacent SNVs (C to T and G to A) were observed at -360/-359 nt in the promoter, not the core promoter of Yorkshire sow NORSF gene (Fig. 6A). In this Yorkshire sow population (n = 310), three genotypes were detected for both SNVs, with the genotype AA at SNV g.-360C > T being a rare genotype with a genotype frequency of less than 1% (2/310) (Fig. 6B-G). Furthermore, these two SNVs are low to moderate polymorphic loci, with PICs of 0.108 and 0.271, respectively (Table S12). However, neither of these two SNVs was remarkably associated with sow fertility traits such as TNB, NBA, and LW (Fig. S3, and S4).

Interestingly, two adjacent SNVs g.-360C > T and g.-359G > A exhibit a linkage disequilibrium, and form four haplotypes H1 (C-G), H2 (C-A), H3 (T-G), and H4 (T-A) in the Yorkshire sow population. Haplotypes H1 and H2 are major haplotypes with 74.84% and 19.03% frequency, while H4 is a rare genotype with a frequency of 1.29% (Fig. 6F-G). Furthermore, compared with sows with the haplotype combination H1H3, a remarkable increase in the average litter size of TNB, NBA, and NSP traits was found in sows with H1H2. The average litter size of the NSP trait was observed in sows with H1H1 (Fig. 6J-N), suggesting that NORSF is a candidate gene for sow fertility traits, and H3 is an unfavorable haplotype of sow fertility.

Functional SNVs in the promoter mainly control the transcriptional activity of targets [10, 21]. To understand the impact of the g.-360C > T and g.-359G > A on transcriptional activity, the reporters of the NORSF promoter with four haplotypes were generated, respectively (Fig. 6O). Reporter assay showed that these two SNVs significantly alter the activity of the NORSF promoter, with lower haplotype H2 activity and higher haplotype H3 activity (Fig. 6P). This is consistent with the function of NORSF in sGCs and the fertility phenotype of haplotype combination. These data support that NORSF is a functional candidate gene for sow fertility traits, as its two SNVs synergistically influence transcriptional activity and sow fertility.

FoxO1 is a component of the Forkhead box O (FOXO) family of TFs that are known to respond to many biological stressors, including OS, which plays a crucial role in both physiological and disease processes [22–24]. On the one hand, FoxO1 serves as an effector in response to OS, with the rise in OS-stimulated cells such as sGCs [7], MCF7 cells [25], and KGN cells [26]. On the other hand, FoxO1 also acts as a TF to transmit OS signals to the nucleus, activate or inhibit the transcription of downstream genes, and mediate OS's regulation of cellular functions [27, 28]. However, the extent to which the TF activity of effector FoxO1 plays a role in the OS-stimulated transcription is mainly unknown. In this study, taking lncRNAs in sGCs as an example, we showed that more than 90% of OS-stimulated lncRNAs may be transcriptionally regulated by its effector and TF FoxO1, and demonstrated for the first time that FoxO1 mediates OS stimulation of the transcription of lncRNAs (e.g., LOC100512907, LOC102164325, L0C102167708, and LOC102162300). In addition, we also noticed that multiple cis-target mRNAs of OS-stimulated lncRNAs have been proven to be modulated by OS [28, 29]. Interestingly, FoxO1 is the cis-target mRNA of OS-stimulated LOC102160118 in Sus scrofa Chr.3. In contrast, SLC25A15, another cis-target mRNA of LOC102160118, is oxidative stress and the metabolism-related gene has been shown to be sever as an biomarker for predicting the adverse events of patients with stomach adenocarcinoma [30]. 6-pyruvoyltetrahydropterin synthase (PTS) and beta-carotene oxygenase 2 (BCO2), two cis-target mRNAs of NORSF, have all been shown to be involved in OS [29, 31]. Taken together, our findings argue that OS-stimulates lncRNome dynamics in its effector FoxO1 transcription activity-dependent manner.

OS and its effector FoxO1 are crucial endogenous environmental and TFs that regulate cell states, functions, metabolisms, and structures [15, 32, 33]. In the ovary, OS and its effector FoxO1 have regulatory effects on multiple cell types, such as GC autophagic injuries, mitochondrial impairments and apoptosis in humans, and impaired viability, reduced proliferation, and cell cycle arrest in mice [33, 34], oocyte maturation and quality in mice and meiotic arrest in sows [35, 36], and luteal cell apoptosis and luteolysis in rat [37], and are involved in ovarian aging and diseases [35, 38]. In sGCs, OS and its effector FoxO1 are also modulators of cell viability, apoptosis, and autophagy [11, 12, 39]. OS induced by various concentrations (e.g., 100, 150, 200, 300, 500, and 1000 µM) of H₂O₂ and exposure times (e.g., 1.5, 2.0, 6.0, and 12.0 h) can induce sGC apoptosis and oxidative damage, whereas antioxidant factors can rescue sGC apoptosis [10–13, 40]. Furthermore, FoxO1 levels were increased in OS-stimulated sGCs, and dysfunction of FoxO1 rescues OS-induced sGC apoptosis, indicating that FoxO1 mediated OS-induced sGC apoptosis [13, 40]. In this study, we also demonstrated that FoxO1 mediated OS-induced sGC apoptosis. More importantly, we also showed that OS restrains E2 release, a primary function of GCs, and FoxO1 mediated this process via NORSF and the estrogen synthesis pathway. Thus, our findings not only define a new function of OS and its effector FoxO1 in sGCs but also reveal a new mechanism by which OS controls sGC functions and provides a new pathway for regulating sGC functions, the FoxO1/NORSF/CYP19A1 pathway.

OS is well known to be intensely involved in sow fertility by interacting with transcripts related to sow fertility [10, 41, 42]. Firstly, multiple OS-stimulated transcripts, including mRNAs (e.g., FZD4 and Acvr2a), miRNAs (e.g., miR-192 and miR-370), and lncRNAs (e.g., lnc2300 and SDNOR), have been proved to be closely related to follicular atresia, a disadvantageous factor for sow fertility [7, 11, 12, 18, 42, 43]. Secondly, multiple OS-stimulated transcripts were discovered to be localized within QTLs for sow fertility traits, such as VEGFA (QTL ID: 584), miR-182 (QTL ID: 24290), and lncRNA LOC100512907 (QTL ID: 7536), which are potential candidate genes for sow fertility traits. Of them, some transcripts such as TGF-β1, PRLR, and miR-27a have been demonstrated to be candidate genes for sow fertility traits [44, 45]. Several transcripts have been confirmed to be causal genes for sow fertility traits. For example, miR-23a, a miRNA located within a QTL (ID: 909) for sow fertility traits, is identified as a causal gene for the TNB trait, as a point mutation in its promoter reduced GCs response to OS [10]. NORSF, a lncRNA activated by OS, not only rose during sow follicular atresia, inducing sGC apoptosis and restraining E2 release but also located in two QTLs (ID: 517 and 7462) for sow fertility traits [13, 17]. In this study, we further demonstrated that NORSF induced by OS is a cause gene for Yorkshire sow fertility traits (e.g., TNB, NBA, and NSP), as two novel SNVs in its promoter lead to its transcription dysregulation and a decrease in sow fertility. However, further investigation is needed on the mechanism underlying NORSF transcriptional dysregulation caused by variants. Together, our findings support the conclusion that OS is intensely involved in such fertility but also provides a target for anti-OS in sow reproductive regulation and a genetic marker (two causal variants) for molecular breeding in swine.

In summary, we provided evidence that OS stimulates lncRNome dynamics of sGCs in an effector FoxO1-dependent manner; that is, FoxO1 acts as a TF to interact with the FRE motifs of target promoters. Furthermore, FoxO1 mediated OS-stimulated sGC apoptosis, and E2 release via a new pathway, FoxO1/NORSF/CYP19A1 pathway (Fig. S5). Additionally, NORSF induced by OS is a cause gene for Yorkshire sow fertility traits. Overall, our findings expand the new functions of OS in female reproduction and the mode underlying its regulation of downstream target transcription, providing a new target for improving sow fertility through an anti-OS strategy.

CYP19A1

cytochrome P450 family 19 subfamily A member 1

DELs

differentially expressed lncRNAs

17β-estradiol

TNB

total number of piglets born

FoxO1

forkhead box O1

FRE

FoxO recognition element

LncRNA

long non-coding RNA

litter weight

miRNA

microRNA

NBA

number of piglets born alive

NORSF

non-coding RNA involved in sow fertility

NSB

number of stillbirths

Oxidative stress

sGC

sow granulosa cell

SNV

single nucleotide variation

TFO

triplex-forming oligonucleotide

TTS

triplex target site

Author Contributions

Conception and design: QL. Collection and assembly of data: WS and MW. Data analysis and interpretation: WS, MW, YW, YL, and XD. Manuscript writing: WS and QL. Final approval of the manuscript: all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2022YFD1600903) and the National Natural Science Foundation of China (32272842).

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Zhao Y, Flowers WL, Saraiva A, Yeum KJ, Kim SW. Effect of social ranks and gestation housing systems on oxidative stress status, reproductive performance, and immune status of sows. J Anim Sci. 2013;91(12):5848–58.
Papatsiros VG, Katsogiannou EG, Papakonstantinou GI, Michel A, Petrotos K, Athanasiou LV. Effects of phenolic phytogenic feed additives on certain oxidative damage biomarkers and the performance of primiparous sows exposed to heat stress under field conditions. Antioxid (Basel). 2022;11(3):593.
Huang S, Zhang L, Luo J, Wu D, Ma K, Chen Y, et al. Cysteamine and n-acetyl-cysteine alleviate placental oxidative stress and barrier function damage induced by deoxynivalenol. J Agric Food Chem. 2023;71(18):6846–58.
Huang S, Wu Z, Huang Z, Hao X, Zhang L, Hu C, et al. Maternal supply of cysteamine alleviates oxidative stress and enhances angiogenesis in porcine placenta. J Anim Sci Biotechnol. 2021;12(1):91.
Zhang P, Jiang G, Wang Y, Yan E, He L, Guo J, et al. Maternal consumption of l-malic acid enriched diets improves antioxidant capacity and glucose metabolism in offspring by regulating the gut microbiota. Redox Biol. 2023;67:102889.
Tatemoto H, Muto N, Sunagawa I, Shinjo A, Nakada T. Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid. Biol Reprod. 2004;71(4):1150–7.
Du X, Li Q, Cao Q, Wang S, Liu H, Li Q. Integrated analysis of miRNA-mRNA interaction network in porcine granulosa cells undergoing oxidative stress. Oxid Med Cell Longev. 2019;2019:1041583.
Zhang X, Chen Y, Li H, Chen B, Liu Z, Wu G, et al. Sulforaphane acts through NFE212 to prevent hypoxia-induced apoptosis in porcine granulosa cells via activating antioxidant defenses and mitophagy. J Agric Food Chem. 2022;70(26):8097–110.
Mierzejewski K, Kurzynska A, Gerwel Z, Golubska M, Stryinski R, Bogacka I. Pparbeta/delta ligands regulate oxidative status and inflammatory response in inflamed corpus luteum-an in vitro study. Int J Mol Sci. 2023;24(5):4993.
Wang S, Li Y, Zeng Q, Yang L, Du X, Li Q. A mutation in endogenous saRNA miR-23a influences granulosa cells response to oxidative stress. Antioxid (Basel). 2022;11(6):1174.
Zhang J, Ren Q, Chen J, Lv L, Wang J, Shen M, et al. Downregulation of miR-192 alleviates oxidative stress-induced porcine granulosa cell injury by directly targeting ACVR2A. Cells. 2022;11(15):2362.
Huo Y, Li Q, Yang L, Li X, Sun C, Liu Y, et al. SDNOR, a novel antioxidative lncRNA, is essential for maintaining the normal state and function of porcine follicular granulosa cells. Antioxid (Basel). 2023;12(4):799.
Yao W, Pan Z, Du X, Zhang J, Liu H, Li Q. NORHA, a novel follicular atresia-related lncRNA, promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis. J Anim Sci Biotechnol. 2021;12(1):103.
Zhang JQ, Wang XW, Chen JF, Ren QL, Wang J, Gao BW, et al. Grape seed procyanidin B2 protects porcine ovarian granulosa cells against oxidative stress-induced apoptosis by upregulating let-7a expression. Oxid Med Cell Longev. 2019;2019:1076512.
Rodriguez-Colman MJ, Dansen TB, Burgering B. FOXO transcription factors as mediators of stress adaptation. Nat Rev Mol Cell Biol. 2024;25(1):46–64.
Zhang X, Evans TD, Chen S, Sergin I, Stitham J, Jeong SJ, et al. Loss of macrophage MTORC2 drives atherosclerosis via foxo1 and il-1beta signaling. Circ Res. 2023;133(3):200–19.
Du X, Liu L, Wu W, Li P, Pan Z, Zhang L, et al. Smarca2 is regulated by NORFA-miR-29c, a novel pathway that controls granulosa cell apoptosis and is related to female fertility. J Cell Sci. 2020;133(23):jcs249961.
Wang M, Wang Y, Yang L, Du X, Li Q. Nuclear lncRNA NORSF reduces E2 release in granulosa cells by sponging the endogenous small activating RNA miR-339. Bmc Biol. 2023;21(1):221.
Zhang X, Jiang Q, Li J, Zhang S, Cao Y, Xia X, et al. KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA-DNA triplexes and hp1 binding. Nat Cell Biol. 2022;24(11):1617–29.
Ali T, Rogala S, Krause NM, Bains JK, Melissari MT, Wahrisch S, et al. Fendrr synergizes with wnt signalling to regulate fibrosis related genes during lung development via its RNA:dsDNA triplex element. Nucleic Acids Res. 2023;51(12):6227–37.
Miao Y, Zhao Y, Wan S, Mei Q, Wang H, Fu C, et al. Integrated analysis of genome-wide association studies and 3D epigenomic characteristics reveal the BMP2 gene regulating loin muscle depth in yorkshire pigs. Plos Genet. 2023;19(6):e1010820.
Chan JD, Scheffler CM, Munoz I, Sek K, Lee JN, Huang YK, et al. Foxo1 enhances car t cell stemness, metabolic fitness and efficacy. Nature. 2024;629(8010):201–10.
Doan AE, Mueller KP, Chen AY, Rouin GT, Chen Y, Daniel B, et al. Foxo1 is a master regulator of memory programming in car t cells. Nature. 2024;629(8010):211–8.
Zhang X, Ge L, Jin G, Liu Y, Yu Q, Chen W, et al. Cold-induced FoxO1 nuclear transport aids cold survival and tissue storage. Nat Commun. 2024;15(1):2859.
Jose E, March-Steinman W, Wilson BA, Shanks L, Parkinson C, Alvarado-Cruz I, et al. Temporal coordination of the transcription factor response to H₂O₂ stress. Nat Commun. 2024;15(1):3440.
Zhang M, Zhang Q, Hu Y, Xu L, Jiang Y, Zhang C, et al. MiR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation. Cell Death Dis. 2017;8(10):e3088.
Li N, Liu B, Xiong R, Li G, Wang B, Geng Q. HDAC3 deficiency protects against acute lung injury by maintaining epithelial barrier integrity through preserving mitochondrial quality control. Redox Biol. 2023;63:102746.
Chen Z, Wang R, Zhu Y, Huang Z, Yang X, Li Q, et al. A novel circular RNA, circsqstm1, protects the endothelial function in atherosclerosis. Free Radic Biol Med. 2023;209(Pt 2):301–19.
Wu L, Lu P, Guo X, Song K, Lyu Y, Bothwell J, et al. β-carotene oxygenase 2 deficiency-triggered mitochondrial oxidative stress promotes low-grade inflammation and metabolic dysfunction. Free Radic Biol Med. 2021;164:271–84.
Dong Y, Yuan Q, Ren J, Li H, Guo H, Guan H, et al. Identification and characterization of a novel molecular classification incorporating oxidative stress and metabolism-related genes for stomach adenocarcinoma in the framework of predictive, preventive, and personalized medicine. Front Endocrinol (Lausanne). 2023;14:1090906.
Hara S, Fukumura S, Ichinose H. Reversible S-glutathionylation of human 6-pyruvoyl tetrahydropterin synthase protects its enzymatic activity. J Biol Chem. 2019;294(4):1420–7.
Howes AC, Perisic O, Williams RL. Structural insights into the activation of ataxia-telangiectasia mutated by oxidative stress. Sci Adv. 2023;9(39):eadi8291.
Zhang L, Liu K, Liu Z, Tao H, Fu X, Hou J et al. Fetal hypoxia expose caused autophagy in ovary granulosa cells via PI3K/Akt/FoxO1 pathway and mitigated by melatonin subheading. J Adv Res. 2023:11: S2090-1232(23)00345-4.
Mu H, Cai S, Wang X, Li H, Zhang L, Li H, et al. RNA binding protein IGF2BP1 meditates oxidative stress-induced granulosa cell dysfunction by regulating MDM2 mRNA stability in an m6A-dependent manner. Redox Biol. 2022;57:102492.
Zhang Y, Bai J, Cui Z, Li Y, Gao Q, Miao Y, et al. Polyamine metabolite spermidine rejuvenates oocyte quality by enhancing mitophagy during female reproductive aging. Nat Aging. 2023;3(11):1372–86.
Li X, Duan J, Wang S, Cheng J, Chen H, Zhang Z, et al. Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway. J Anim Sci Biotechnol. 2023;14(1):22.
Tang Z, Chen J, Zhang Z, Bi J, Xu R, Lin Q, et al. Hif-1alpha activation promotes luteolysis by enhancing ROS levels in the corpus luteum of pseudopregnant rats. Oxid Med Cell Longev. 2021;2021:1764929.
Srnovrsnik T, Virant-Klun I, Pinter B. Heavy metals and essential elements in association with oxidative stress in women with polycystic ovary syndrome-a systematic review. Antioxid (Basel). 2023;12(7):1398.
Zhang JQ, Ren QL, Chen JF, Gao BW, Wang XW, Zhang ZJ, et al. Autophagy contributes to oxidative stress-induced apoptosis in porcine granulosa cells. Reprod Sci. 2021;28(8):2147–60.
Kong C, Su J, Wang Q, Liu K, Fu R, Sui S. Signaling pathways of Periplaneta americana peptide resist H₂O₂ induced apoptosis in pig-ovary granulosa cells through FoxO1. Theriogenology. 2022;183:108–19.
Du X, Li Q, Yang L, Liu L, Cao Q, Li Q. Smad4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis. Cell Death Dis. 2020;11(5):373.
Zhou X, He Y, Pan X, Quan H, He B, Li Y, et al. Dnmt1-mediated lncRNA IFFD controls the follicular development via targeting gli1 by sponging miR-370. Cell Death Differ. 2023;30(2):576–88.
Wang M, Wang Y, Yao W, Du X, Li Q. Lnc2300 is a cis-acting long noncoding RNA of cyp11a1 in ovarian granulosa cells. J Cell Physiol. 2022;237(11):4238–50.
Pang P, Li Z, Hu H, Wang L, Sun H, Mei S, et al. Genetic effect and combined genotype effect of ESR, FSHβ, ctnnal1 and miR-27a loci on litter size in a large white population. Anim Biotechnol. 2019;30(4):287–92.
Wang L, Du X, Li Q, Wu W, Pan Z, Li Q. MiR-2337 induces tgf-beta1 production in granulosa cells by acting as an endogenous small activating RNA. Cell Death Discov. 2021;7(1):253.

Download PDF

Reviewers agreed at journal
15 Aug, 2024
Reviewers invited by journal
16 Jul, 2024
Editor assigned by journal
09 Jul, 2024
First submitted to journal
07 Jul, 2024

You are reading this latest preprint version

Oxidative stress controls lncRNA-mediated granulosa cell functions in a FoxO1-dependent manner

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Introduction

Materials and Methods

Materials

Sows

Bioinformatics

Constructs and small interfering RNAs (siRNAs)

Cells and treatment

Reporter assays

Chromatin immunoprecipitation (ChIP)

Quantitative real-time PCR (qPCR)

Fluorescence-activated cell sorting (FACS)

SNV screening, genotyping, and association analysis

Statistics

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1