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. 2026 Jan 17;17:83. doi: 10.1186/s13287-026-04908-7

CNOT3 resists ovarian aging by accelerating oocyte maturation and promoting ESCs differentiation

Nian Li 1, Enyuan Huang 1, Ruiqi Wang 1, Jiaqi Li 1,✉, Xiaolong Yuan 1,✉
PMCID: PMC12896153  PMID: 41547942

Abstract

Background

Ovarian aging (OA), which is characterized by a decline in the quality and quantity of oocytes, represents a major challenge in reproductive medicine. However, the therapeutic targets and therapeutic methods of OA remain poorly defined. Previous studies have suggested that the embryonic stem cells (ESCs) resist mammalian OA, yet the underlying molecular mechanisms are unclear.

Methods

To assess CNOT3’s role in OA and oocyte maturation, we employed RT-qPCR, Western blotting, micro-injection, and RNA seq. RNA-seq, RT-qPCR, and Western blot were used to prove the effects of Cnot3 on the differentiation of mouse ESCs into primordial germ cell-like cells (PGCLCs). Mouse ESCs were injected into mice to evaluate the therapeutic benefit of ESCs on OA.

Results

In this study, we demonstrated that the CCR4-NOT transcription complex subunit 3 (CNOT3) played a critical regulator of OA resistance. Our results revealed that the expression of CNOT3 significantly decreased in aging ovaries of pigs and mice, compared with young ovaries. Using RNA-seq and micro-injection, we proved that CNOT3 resisted porcine OA by accelerating oocyte maturation. Moreover, Cnot3 upregulated the expressions of pluripotent genes in mouse ESCs and promoted the differentiation of ESCs into PGCLCs in vitro. Importantly, we found that tail vein injection of ESCs resisted mouse OA, while the therapeutic effects of ESCs on OA were reversed by knockdown of Cnot3.

Conclusion

Overall, our results indicated that CNOT3 counteracted OA and enhanced the therapeutic benefit of ESCs on OA. These findings will provide useful information for the improvement of therapeutic methods of OA.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-04908-7.

Keywords: Ovarian aging, CNOT3, ESCs, Mammals

Introduction

As the reproductive organs of female mammals, the ovaries are composed of follicles, which serve as the basic functional units [1]. The normal growth, development, and ovulation of ovarian follicles are collectively regulated by oocytes [2], mesothelial cells [3], and granulosa cells [4] to maintain ovarian function and reproductive capacity of female mammals. The gradual reduction of follicles [5], the decline in the quantity and quality of oocytes [6], and the increased apoptosis in ovarian cells [7] during aging in female mammals contribute to the recession of ovarian function, ultimately leading to ovarian aging (OA). Previous studies have shown that the alleviation of OA decreases the prevalence of various ovarian dysfunctions and diseases [8]. In mice, Nur77 resists OA to prevent ovarian function decline by regulating the AKT pathway [9]. However, the key therapeutic targets of OA remain unclear, and the therapeutic methods of OA still require breakthroughs.

Embryonic stem cells (ESCs), isolated from the inner cell mass of mammalian blastocysts, exhibit pluripotency and self-renewal capacity [10]. Core transcription factors are highly expressed in ESCs to maintain pluripotency, including octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and nanog homeobox (NANOG) [11]. Furthermore, these pluripotent genes also regulate mammalian aging processes [12]. Oct4 upregulates intraserous Lmnb1 to counteract the somatic aging in mice [13], and NANOG enhances the regenerative potential of mesenchymal stem cells from aged humans to mitigate aging [14]. Moreover, CD47 molecule (CD47) promotes the survival and function of ESCs in vitro [15]. Activation of orthodenticle homeobox 2 (OTX2) induces the differentiation of ESCs into epiblast-like cells (EpiLCs) [16]. Notably, the EpiLCs derived from ESCs possess the potential to differentiate into primordial germ cell-like cells (PGCLCs) after initiating the expression of nanos C2HC-type zinc finger 3 (NANOS3) [17] and PR/SET domain 14 (PRDM14) [18]. Additionally, the apoptotic vesicles of mesenchymal stem cells restore the follicular development in aged mice by activating WNT/β-catenin pathway [19], and intravenous injection of ESCs inhibit ovarian fibrosis to alleviates mouse OA [20], suggesting the therapeutic value of stem cells in OA.

The CCR4-NOT transcription complex subunit 3 (CNOT3) has been proven to be involved in the transcription, translation and homeostasis of mRNAs [21], and is an important reproductive regulatory factor for mammals [22, 23]. Intriguingly, Cnot3-mediated mRNA decay and metabolism of Dand5 accelerates the mouse embryogenesis [24], while the absence of Cnot3 in mouse leads to the rapid loss of epiblast cells and the arrest of embryonic growth [25]. These results indicate that CNOT3 may regulates RNA metabolism and embryogenesis to postpone reproductive aging in mammals. Moreover, pluripotency is necessary during the process of ESCs differentiating into PGCLCs, and Cnot3 ensures the pluripotency of mouse ESCs [25]. The silence of Cnot3 induces the abnormal differentiation of mouse ESCs [26]. Therefore, we speculate that CNOT3 is a potential target for improving the therapeutic effects of ESCs on OA.

In our study, we found that CNOT3 maintained the expressions of pluripotent genes in mammalian ovaries. Using micro-injection, RNA-seq, and Co-IP, we identified that CNOT3 played a mediator in the remission of mammalian OA via promoting maturation of oocytes. Moreover, we demonstrated that Cnot3 possessed a positive regulation during the differentiation of mouse ESCs into PGCLCs, and Cnot3 enhanced the therapeutic benefit of ESCs on OA in vivo. In summary, our results provide new insights into the improvement of therapeutic methods of OA.

Results

CNOT3 possesses the potential to resist mammalian OA

We found that the expressions of pluripotent genes (e.g., OCT4, SOX2, and NANOG), the marker genes of EpiLCs (e.g., OTX2), PGCLCs (e.g., NANOS3), and oocytes (e.g., growth differentiation factor 9 [GDF9] and synaptonemal complex protein 3 [SYCP3]) were significantly downregulated in ovaries of aged pigs, compared to that of young pigs (Fig. 1A), and the protein levels of OCT4, SOX2, NANOG, OTX2, and NANOS3 in porcine ovaries were significantly decreased by OA (Fig. 1B). Similarly, OA significantly downregulated the mRNA levels of Oct4, Sox2, Nanog, Otx2, Gdf9, Sycp3, and Nanos3 in ovaries of mice (Fig. 1C). Moreover, OA reduced the expression of Oct4 protein in ovaries, and significantly decreased the protein levels of Sox2, Nanog, Otx2, and Nanos3 in ovaries of mice (Fig. 1D). Moreover, compared with young pigs (5-month-old), the mRNA (Fig. 1E) and protein (Fig. 1F) of CNOT3 showed markedly low expression in the ovarian tissue of aged pigs (36-month-old). The similar appearance was found in young (3-month-old) and aged mice (13-month-old) (Fig. 1G, H). Interestingly, CNOT3 in oocytes and various tissues in pigs and mice, e.g. heart, lung, spleen, muscle, and ovary. We found that CNOT3 showed specific expression in oocytes, compared that in the heart, lung, spleen, muscle, and ovary in pigs (Fig. 1I) and mice (Fig. 1J).

Fig. 1.

Fig. 1

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CNOT3 possesses the potential to resist mammalian OA. The mRNA levels of OCT4, SOX2, NANOG, OTX2, GDF9, SYCP3, and NANOS3 in the ovaries of young pigs and aged pigs (A). The protein levels of OCT4, SOX2, NANOG, OTX2, and NANOS3 in the ovaries of young pigs and aged pigs (B). The mRNA levels of Oct4, Sox2, Nanog, Otx2, Gdf9, Sycp3, and Nanos3 in the ovaries of young mice and aged mice (C). The protein levels of Oct4, Sox2, Nanog, Otx2, and Nanos3 in the ovaries of young mice and aged mice (D). The mRNA (E) and protein (F) of CNOT3 in the ovaries of young pigs and aged pigs. The mRNA (G) and protein (H) of Cnot3 in the ovaries of young mice and aged mice. The mRNA expression of CNOT3 in the heart, lung, spleen, muscle, ovary, and oocyte of pigs (I) and mice (J)

CNOT3 promotes the maturation of porcine oocytes

To explore the specific functions of CNOT3 in the regulation of OA, the effects of CNOT3 on the oocyte maturation in pigs were verified. The expression of CNOT3 in the oocytes cultured in vitro at 24 h was significantly higher than that at 0 h and 48 h (Fig. 2A). The OE-CNOT3 significantly promoted the maturation of porcine oocytes, while the opposite results were observed in si-CNOT3 group (Fig. 2B). In addition, the porcine oocytes that had been microinjected with the mRNA (OE-CNOT3) and small interfering RNA (si-CNOT3) of CNOT3 were subjected to RNA-seq, respectively. We identified 7998 differential expressed genes (DEGs) between OE-NC and OE-CNOT3 (Fig. 2C), including eukaryotic translation initiation factor 4E (EIF4E) [27] and cytochrome P450 family 27 subfamily A member 1 (CYP27A1) [28], the key factors of follicular development, and kruppel-like factor 10 (KLF10) [29], the pluripotency-related gene. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these DEGs were markedly enriched in NF-kappa B, TGF-beta, and Ovarian steroidogenesis signaling pathways (Fig. 2D). We found that these DEGs were annotated to extracellular matrix by Gene Ontology (GO) analysis (Fig. 2E). Similarly, 4911 DEGs between si-NC and si-CNOT3 were identified (Fig. 2F), including EIF4E, CYP27A1, and KLF10. The KEGG (Fig. 2G) and GO (Fig. 2H) analyses indicated that these DEGs were enriched in Apoptosis pathway and extracellular exosome. Further, using Co-IP, we explored whether there were interactions between CNOT3 protein and key proteins in the signaling pathways of NF-kappa B (e.g., P105 and P65) [30], Apoptosis (e.g., BAX and Caspase-8) [31], Ovarian steroidogenesis (e.g., CYP11A1, CYP19A1, FSHR) [32], TGF-beta (e.g., mTOR) [33], and extracellular matrix (e.g., COL4A1 and FN1) [34], and we confirmed that mTOR and FN1 proteins are targets of CNOT3 protein (Fig. 2I).

Fig. 2.

Fig. 2

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CNOT3 promotes the maturation of porcine oocytes. A The expression level of CNOT3 in the porcine oocytes cultured in vitro for 0 h, 24 h, and 48 h. B The maturation rates of oocytes treated with OE-NC, OE-CNOT3, si-NC, and si-CNOT3. C The DEGs between the porcine oocytes treated with OE-NC and OE-CNOT3. The KEGG (D) and GO (E) analytical results of DEGs between the oocytes treated with OE-NC and OE-CNOT3. F The DEGs between the porcine oocytes treated with si-NC and si-CNOT3. The KEGG (G) and GO (H) analytical results of DEGs between the oocytes treated with si-NC and si-CNOT3. I The interaction between CNOT3 protein and key proteins in the signaling pathways of NF-kappa B, Apoptosis, Ovarian steroidogenesis, TGF-beta, and extracellular matrix

Cnot3 accelerates the differentiation of mouse ESCs into PGCLCs

The mouse ESCs were induced into PGCLCs in vitro, and the induced process was shown in Fig. 3A. The mRNA levels of Oct4, Sox2, and Nanog in ESCs significantly exceeded mouse embryonic fibroblasts (MEFs), the feeder cells for ECSs (Fig. 3B). Similarly, the protein levels of Oct4 and Sox2 in ESCs significantly exceeded MEFs (Fig. 3C), suggesting the successful cultivation of ESCs. The marked activation of Otx2’s mRNA (Fig. 3D) and protein (Fig. 3E) indicated the differentiation of ESCs into EpiLCs, while the expression of Cd47 was not significantly changed (Fig. 3D). Moreover, we found that the mRNAs (Fig. 3F) of Nanos3 and Prdm14, and Nanos3 protein (Fig. 3G) were significantly upregulated in the induced PGCLCs, compared with EpiLCs. Notably, the expressions of Cnot3’s mRNA (Fig. 3H) and protein (Fig. 3I) were increased gradually during the differentiation process of ESCs into PGCLCs. We further performed RNA-seq for ESCs and PGCLCs, and 1612 DEGs between ESCs and PGCLCs were identified (Fig. 3J). The KEGG analysis indicated that these DEGs were enriched in the signaling pathways of Calcium and Insulin secretion (Fig. 3K). Furthermore, these DEGs were annotated to transmembrane transporter complex after GO analysis (Fig. 3L).

Fig. 3.

Fig. 3

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The expression of Cnot3 is gradually upregulated during the differentiation of mouse ESCs into PGCLCs. A The diagram of the method of mouse ESCs into PGCLCs. The mRNA (B) levels of Oct4, Sox2, and Nanog as well as the protein levels (C) of Oct4 and Sox2 in MEFs and ESCs. The mRNA (D) levels of Otx2 and Cd47, and the protein levels (E) of Otx2 in ESCs and EpiLCs. The mRNA (F) levels of Nanos3 and Prdm14, and the protein levels (G) of Nanos3 in EpiLCs and PGCLCs. The mRNA (H) and protein (I) levels of Cnot3 during the differential process of ESCs into PGCLCs. J The DEGs between ESCs and PGCLCs. The KEGG (K) and GO (L) analytical results of DEGs between ESCs and PGCLCs

We successfully transfected the overexpression vector (OE-Cnot3) and small interfering RNA (si-Cnot3) of Cnot3 into mouse ESCs, respectively (Fig. 4A). We found that OE-Cnot3 significantly weakened the mRNA expressions of Oct4, Sox2, and Nanog in ESCs, while si-Cnot3 significantly inhibited the expressions of Oct4 and Nanog (Fig. 4B). The OE-Cnot3 markedly increased the protein levels of Oct4 and Sox2, while si-Cnot3 markedly decreased the protein levels of Oct4, Sox2, and Nanog (Fig. 4C). Notably, the mRNA levels of Otx2, Cd47, Nanos3, and Prdm14 in ESCs were significantly upregulated by OE-Cnot3 (Fig. 4D). Similarly, the protein levels of Otx2 and Nanos3 were markedly increased after overexpression of Cnot3 in ESCs (Fig. 4E).

Fig. 4.

Fig. 4

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Cnot3 accelerates the differentiation of mouse ESCs into PGCLCs. A The efficiencies of OE-Cnot3 and si-Cnot3 in mouse ESCs. The effects of OE-Cnot3 and si-Cnot3 on the mRNA (B) and protein (C) levels in ESCs. D The effects of OE-Cnot3 on the mRNA expressions of Otx2, Cd47, Nanos3, and Prdm14 in ESCs. E The effects of OE-Cnot3 on the protein levels of Otx2 and Nanos3

Cnot3 improves the therapeutic effects of ESCs on OA in vivo

The PBS solution, ESCs, and ESCs treated with si-Cnot3 were injected into 13-month-old C57BL/6J female mice, the process was shown in Fig. 5A. We injected ESCs labeled with green fluorescent protein (GFP) into mice to investigate whether these ESCs could be delivered to the mouse ovaries. The results of RT-qPCR (Fig. 5B), Western blot (Fig. 5C), and fluorescence microscopy (Fig. 5D) proved the expressions of GFP’s mRNA and protein in the mouse ovaries of ESCs group, indicating that ESCs were integrated into mouse ovaries. The injection of ESCs significantly promoted the expressions of Oct4, Sox2, Nanog, anti-aging marker genes (e.g., sirtuin 1 [Sirt1] and sirtuin 6 [Sirt6]) [35], and significantly inhibited the expressions of aging marker genes (cyclin dependent kinase inhibitor 2 A [P16] and cyclin dependent kinase inhibitor 1 A [P21]) [36] and apoptotic marker genes (cysteine aspartic acid specific protease 3 [Caspase-3], cysteine aspartic acid specific protease 9 [Caspase-9], and BCL-2 interacting mediator of cell death [Bim]) [37] in the ovaries of aged mice (Fig. 5E). Interestingly, si-Cnot3 observably weakened the effects of ESCs on the expressions of above genes, excluding P16, Sirt1, and Sirt6 (Fig. 5E). Finally, we detected that ESCs promoted the follicular formation (Fig. 5F) and inhibited the cell apoptosis (Fig. 5G) in the ovaries of aged mice, while these effects of ESCs were reversed by si-Cnot3.

Fig. 5.

Fig. 5

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Cnot3 improves the therapeutic results of ESCs on OA in vivo. A The diagram of the method of tail vein injection. The expressions of GFP’s mRNA (B) and protein (C) in the ovaries of aging mice of Control and ESCs groups. (D) The location of GFP protein in the ovaries of 13-month-old mice of Control and ESCs groups. E The mRNA expressions of Oct4, Sox2, Nanog, P16, P21, Sirt1, Sirt6, Caspase-3, Caspase-9, and Bim in the ovaries of aging mice of Control, ESCs, and ESCs (si-Cnot3) groups. The follicular formation (F) and cell apoptosis (G) in the ovaries of aging mice of Control, ESCs, and ESCs (si-Cnot3) groups

Discussion

Many studies have shown that the decline in the quality and quantity of oocytes caused by OA heavily injures the reproductive capacity of mammals [6, 7]. However, the key factors that regulated OA remain to be further explored. CNOT3 has been proven to be the important gene during the growth of oocytes [23]. In this study, we found that the expression of CNOT3 was significantly downregulated in the ageing ovaries in pigs (Fig. 1E, F) and mice (Fig. 1G, H), indicating the potential functions to resist mammalian OA of CNOT3. The CNOT3 was detected the high expression in oocytes of pigs (Fig. 1I) and mice (Fig. 1J), compared with heart, lung, spleen, and ovary. Subsequently, using micro-injection, we proved that CNOT3 markedly promoted the maturation of porcine oocytes (Fig. 2B). We certified that the CNOT3-related genes participated in the regulation of the pathways related to oocyte growth, such as NF-kappa B [38], TGF-beta [39], Ovarian steroidogenesis [40], and Apoptosis [41] (Fig. 2D, G) using RNA-seq. Moreover, CNOT3-related genes were annotated in extracellular matrix [34] and extracellular exosome [42], key cell functions of oocyte growth (Fig. 2E, H). Notably, we detected the interaction of CNOT3 protein with mTOR and FN1 proteins in porcine oocytes, suggesting that CNOT3 regulated TGF-beta and extracellular matrix signaling pathways via targeting mTOR and FN1 proteins (Fig. 2I). These results indicated that CNOT3 resisted OA in mammals by facilitating oocyte maturation.

Previous studies have suggested that the high expression of pluripotent genes not only maintains self-renewal of ESCs [43], but also alleviate the aging process of mammalian bodies [12–14]. In our study, the expressions of pluripotent genes (e.g., OCT4, SOX2, NANOG), necessary genes for the differentiation of ESCs into PGCLCs (e.g., OTX2 and NANOS3) [16, 17], and the marker genes of oocytes (e.g., GDF9 and SYCP3) [44, 45] were significantly reduced in aging ovaries in pigs and mice, compared with young ovaries (Fig. 1A, C). The OA markedly downregulated the protein levels of OCT4, SOX2, NANOG, and OTX2 in pigs (Fig. 1B), and markedly downregulated the protein levels of Sox2, Nanog, Otx2, and Nanos3 in mice (Fig. 1D). Furthermore, ESCs are an important source for the generation of mammalian PGCLCs in vitro [46], and the PGCLCs derived from ESCs have been proven to be able to form oocytes in the ovaries of mice [47]. These results indicated that the integration of ESCs into ovaries might resist OA in mammals by expressing pluripotent genes and generating PGCLCs, while the therapeutic methods for treating OA with ESCs still need to be further explored.

Interestingly, we successfully induced mouse ESCs into PGCLCs in vitro (Fig. 3A-G) and found that the expression of Cnot3 gradually increased during the induction process (Fig. 3H, I). Moreover, RNA-seq analysis revealed that the DEGs between ESCs and PGCLCs were enriched in the signaling pathways and cell functions regulated by Cnot3, including the signaling pathways of Calcium [24] and Insulin secretion [48] as well as the cell function of transmembrane transporter complex [49] (Fig. 3J-L). Therefore, we further explored the specific functions of Cnot3 during the differentiation process of ESCs into PGCLCs in mice. We found that OE-Cnot3 significantly promoted the expressions of Oct4, Sox2, and Nanog in ESCs, while si-Cnot3 inhibited the expressions of Oct4 and Nanog (Fig. 4B). The OE-Cnot3 markedly increased the protein levels of Oct4 and Sox2 in ESCs, while si-Cnot3 markedly decreased the protein levels of Oct4, Sox2, and Nanog (Fig. 4C). Notably, the mRNA levels of Otx2, Cd47, Nanos3, and Prdm14 (Fig. 4D) as well as the Otx2 protein (Fig. 4E) were significantly upregulated in the ESCs treated with OE-Cnot3. The above results evidenced that Cnot3 maintained the pluripotency and survival of ESCs, and Cnot3 promoted the differentiation of ESCs into PGCLCs. Importantly, the tail vein injection of ESCs (Fig. 5A-D) observably resisted the OA in aging mice via promoting the follicular formation and inhibiting cell apoptosis, while knockdown of Cnot3 in ESCs reversed these effects (Fig. 5E-G), suggesting that CNOT3 might improve the therapy of ESCs on in mammalian OA.

However, there may be some limitations in our study. For example, although we investigated the regulation of CNOT3 on mammalian OA, the upstream mechanisms of the expression of CNOT3 remain to be explored. Previous study has indicated that high DNA methylation of Cyp11a1 promoter accelerates mouse OA by disturbing steroidogenesis in ovary [50]. Apoptosis of ovarian granulosa cells activated by oxidative stress promotes the OA of humans [51] and pigs [38]. Therefore, oxidative stress and DNA methylation may be important directions for elucidating the mechanisms of low CNOT3 expression in the aging ovaries of mammals.

Conclusions

Overall, CNOT3 showed low expression in the aging ovaries of mice and pigs, and it resisted OA via promoting the maturation of oocytes. Meanwhile, CNOT3 maintained the expressions of pluripotent genes in mouse ESCs and accelerated the differentiation of ESCs into PGCLCs, thereby improving the therapeutic effect of ESCs on OA. These works will provide the useful information and insights for the therapy of OA.

Materials and methods

Animals

Three young (2-month-old) and three aged (13-month-old) female C57BL/6J mice were obtained from GemPharmatech Co., Ltd (Jiangsu, China). After adaptive feeding, all mice were fed in the environment with a room temperature of 20–25 ℃, humidity of 55% ± 10%, and under a 12 h light/dark cycle. The spinal dislocation method was used to euthanize the young and aged female mice, and their ovaries were collected. The ovaries of three young (5-month-old) and three aged (36-month-old) sows were collected from a local livestock farm, and the porcine ovarian samples were collected after slaughtering. All the ovarian samples were washed twice with pre-cooling PBS and then stored at −80℃ for subsequent experiments. The authors state that this work has been reported in line with the ARRIVE guidelines 2.0.

Treatment in vivo of ESCs

The mouse ESCs were obtained from Cyagen-Bio Co., Ltd (Product code: MUAES-01101, Guangzhou, China) and Talent-Bio Co., Ltd (Product code: CC-0014, Guangzhou, China). The method for in vivo ESCs treatment was shown in Fig. 5A (Source: SciDraw, License agreement: CC-BY, Authors: Yunan Chen, Nicolás De Francesco, and Diogo Losch De Oliveira). Nine 13-month-old female mice were assigned into Control (n = 3), ESCs (n = 3), and ESCs (si-Cnot3) (n = 3) groups, randomly. The tail vein injection was performed on the mice after adaptive feeding. Control group mice were injected with 100 µL of PBS solution, the ESCs group mice were in injected with 100 µL of cell suspension containing 4 × 106 ESCs, and the ESCs (si-Cnot3) group were injected with 100 µL of cell suspension containing 4 × 106 ESCs treated with knockdown of Cnot3. The tail vein injection was given once 5 days, for a total of 3 times, and the mice were fed normally for 5 days. The mice were euthanized using spinal dislocation method, and the ovarian samples were collected.

The induction of ESCs into PGCLCs

The ESCs were seeded in the 6-wells plate containing MEFs feeder layer cells. 2i/L medium, the DMEM/F12 medium containing 1000 U/mL LIF (MCE, USA), 5% FBS, and 5% KSR was used for the culture of ESCs, and ESCs were incubated at 37℃ under 5% CO2. The mouse ESCs were induced into PGCLCs based on the method described [52]. The 1 × N2 (MCE, USA), 1 × B27 (MCE, USA), 1% KSR, 20 ng/mL Activin A (MCE, USA), and 12 ng/mL bFGF (MCE, USA) were added into DMEM/F12 medium to prepare the EpiLCs differential medium. The ESCs were induced to EpiLCs after culturing in the EpiLCs differential medium for 2 days. The 15% KSR, 500 ng/mL BMP4 (MCE, USA), 500 ng/mL BMP8a (MCE, USA), 100 ng/mL SCF (MCE, USA), 50 ng/mL EGF (MCE, USA), and 1000 U/mL LIF were added into GMEM medium to prepare PGCLCs differential medium. The EpiLCs were induced to PGCLCs after culturing in the PGCLCs differential medium for 4 days. The transfections of plasmid and siRNA were performed using Lipofectamine™ 3000 (Thermo Scientific, USA). The siRNA of CNOT3 were purchased from Dongze Biotechnology Co., Ltd (Guangzhou, China), and the sequence of siRNA is 5’-GGACAAGCGCAAACUCCAATT-3’.

The collected and culture of porcine oocytes

The porcine ovarian were purchased from a large slaughterhouse and cleaned twice with PBS. The follicular fluid was separated from 3 to 5 mm antral follicles for the collection of porcine oocytes. Transferred the follicular fluid was into 15 mL centrifuge tube, and removed the liquid supernatant after standing for 10 min. Resuspend the cell precipitate with DPBS, and aspirated the cumulus-oocyte complex using glass needle under the microscope. Using 0.1% Hyaluronidase treated cumulus-oocyte complex to dispel cumulus cells. The oocytes were placed in 4-wells plate and cultured with M199 medium containing 0.05 IU/mL FSH (MCE, USA), 1 mM Sodium Pyruvate (Thermo Scientific, USA), and 1% Penicillin-Streptomycin (Hyclone, USA).

The micro-injection of porcine oocytes

The oocytes were assigned into OE-NC (n = 60), OE-CNOT3 (n = 60), si-NC (n = 60), and si-CNOT3 (n = 60) groups, and 3 biological replicates were set for each group. For the OE-CNOT3 group, each oocyte was micro-injected with 10 pL of CNOT3 overexpression vector solution at a concentration of 200 ng/µL, and the oocytes of si-CNOT3 group were micro-injected with 10 pL of CNOT3 siRNA solution at a concentration of 5 nmol. The oocytes of OE-NC and si-NC were micro-injected with 10 pL of ddH2O and siRNA-NC, respectively. The oocytes were regarded as maturation by the extrusion of first polar body after culturing for 24 h, and the maturation rate of oocytes in each group was calculated.

Real-time quantitative PCR

The samples of tissue and cell used for extracting RNA need to be stored at −80℃. All RNA samples were extracted using RNAfast200 Kit (FastGene, Shanghai). The cDNA was obtained using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The cDNA samples for RT-qPCR need to be diluted by 5 to 10 times. The Hieff® qPCR SYBR Green Master Mix (YEASEN, Shanghai) and CFX96 Touch Real-Time PCR system (Bio-Rad, USA) were used to quantitate the mRNA levels of genes in different cDNA samples. Using glyceraldehyde phosphate dehydrogenase (GAPDH) as reference gene. The relative expressions of genes were calculated using 2−ΔΔCT method. The primers for RT-qPCR were synthesized from Sangon Biotech Co., Ltd (Shanghai, China), and the primer sequences were provided in Supplementary Material 1.

Co-immunoprecipitation

The BersinBio™ Co-Immunoprecipitation (Co-IP) Kit (Guangzhou, China) was used to verified the interacting proteins of CNOT3. Firstly, the porcine follicular fluid was aspirated and at least 500 oocytes were isolated. We lysed oocytes using Cell lysis buffer. Subsequently, the lysate was divided into two equal parts. We added 3 ug of CNOT3 antibody (11135-1-AP, Proteintech) into one part as IP group, and we added 3 ug of IgG antibody (BersinBio, Guangzhou) to the other part as negative control. The antibody-protein complexes were recovered using Protein A/G-MagBeads after overnight incubation. Finally, the Co-IP productions of IP and IgG groups were eluted by Elution buffer for subsequent western blot experiments. The total protein sample of oocytes (Input group) was used as positive control.

Western blot

The total protein samples were extracted using RIPA lysis buffer (Beyotime, Shanghai). The proteins were transferred to PVDF membranes after SDS-PAGE gels. Using 5% skimmed milk blocked PVDF membranes at room temperature. Blocked PVDF membranes were washed twice with TBST solution, and then incubated with anti-CNOT3 (DF12583, Affinity, 1:1000), anti-OCT4 (60242-1-Ig, Proteintech, 1:5000), anti-SOX2 (11064-1-AP, Proteintech, 1:1000), anti-NANOG (14295-1-AP, Proteintech, 1:5000), anti-OTX2 (CY9339, Abways, 1:1000), anti-NANOS3 (21679-1-AP, Proteintech, 1:1000), anti-Caspase-8 (AF6442, Affinity, 1:1000), anti-mTOR (AF6308, Affinity, 1:1000), anti-FSHR (AF5242, Affinity, 1:1000), anti-COL4A1 (AF0510, Affinity, 1:1000), anti-FN1 (AF5335, Affinity, 1:1000), anti-CYP19A1 (40809, SAB, 1:1000), anti-P105 (21017, SAB, 1:1000), anti-CYP11A1 (13363-1-AP, Proteintech, 1:2000), anti-P65 (10745-1-AP, Proteintech, 1:2000), anti-BAX (50599-2-Ig, Proteintech, 1:20000), anti-GFP (380634, ZenBio, 1:1500), anti-GAPDH (10494-1-AP, Proteintech, 1:5000), anti-Tubulin (AF7010, Affinity, 1:5000) at 4℃ overnight. The membranes were incubated with Goat Anti-Rabbit IgG Secondary Antibody HRP Conjugated (L3012, SAB, 1:10000) and Goat Anti-Mouse IgG Secondary Antibody HRP Conjugated (L3032, SAB, 1:20000) at room temperature for 2 h after cleaning. The images were obtained by Clinx ChemiScope System (Clinx, Shanghai), and the protein bands were quantitated by the Clinx Gel Analysis software. All unedited blot images are provided in Supplementary Material 2.

HE staining and TUNEL assay

The ovulation and cell apoptosis of ovarian tissues were detected by (Hematoxylin-eosin) HE staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. For the HE staining, the mouse ovaries were sliced into maximum transverse sections, and then fixed with 4% paraformaldehyde. Subsequently, the fixed sections were stained by HE. For the TUNEL assay, the ovarian sections were washed with PBS. The cleaned sections were repaired with proteinase K after dehydrating, and then incubated with TUNEL solution for 1 h in darkness. Finally, the images were obtained by the Nikon ECLIPSE Ti2 fluorescence microscope (Japan).

RNA sequencing and data analysis

The total RNA of tissue and cell was extracted by TRIzol reagent (TaKaRa, Tokyo, Japan). The quality control of RNA samples and the removal of ribosomal RNA were performed by NanoPhotometer spectrophotometer and the RiboMinuTM Eukaryote Kit (Thermo Scientific, USA), respectively. The RNA samples were sequencing with the Illumina NovaSeq X Plus or DNBSEQ-T7, and the low-quality reads was eliminated. The cleaned reads data were mapped to the GRCm39 reference genome using HISAT2 software [53], and the Stringtie software [54] was used to recombine transcripts. The trend of gene expression between different experimental groups were characterized based on FPKM values obtained from RSEM software [55]. Using the DESeq2 [56] of R software to perform differential analysis, and |log2FC| > 1 and FDR < 0.05 as the cut-off.

Statistical analysis

All experimental data are shown as mean ± standard deviation (SD) from at least 3 independent replicate experiments. The t-test was used to detect significant differences, and P value < 0.05 was judged as the statistical significance. The GraphPad Prism software was used to draw graphs.

Supplementary Information

Supplementary Material 1 (112.7KB, pdf)
Supplementary Material 2 (660.2KB, pdf)

Acknowledgements

Not applicable.

Artificial intelligence (AI)

The authors declare that they have not use AI-generated work in this manuscript.

Author contributions

N.L., X.Y., and J.L. designed experiments. E.H. performed the experiments in vivo. N.L. performed the experiments in vitro. R⋅W conducted the RNA-seq data analysis. N.L. and X.Y. evaluated results and wrote the manuscript with revising by X.Y. and J.L.

Funding

This study was supported by grants from the Science and Technology Program of Guangzhou (2024B03J1305), the Guangdong Basic and Applied Basic Research Foundation (2024B1515020112, 2024A1515012999, 2023A1515030054 and 2023A1515010364), Modern Agricultural Industrial Technology System lnnovation Team of Guangdong Province (2024CXTD22), and the National Center of Technology Innovation for Pigs (NCTIP-XDB14).

Data availability

All the RNA-seq data have been deposited the Sequence Read Archive (SRA) database under accession number PRJNA1302674.

Declarations

Ethics approval and consent to participate

All the animal experiments were conducted with “The Instructive Notions with Respect to Caring for Laboratory Animals”, and approved by Animal Care and Use Committee of Sun Yat-sen University Cancer Center (Approval ID: L025504202308010) on 27th August 2023. The approved title is: The effects of small molecule compounds (digitoxin, thapsigargin, and cinobufagin) on the development of ovary and oocyte in mice.

Consent for publication

All authors agree to publish this version of the article.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jiaqi Li, Email: jqli@scau.edu.cn.

Xiaolong Yuan, Email: yxl@scau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (112.7KB, pdf)
Supplementary Material 2 (660.2KB, pdf)

Data Availability Statement

All the RNA-seq data have been deposited the Sequence Read Archive (SRA) database under accession number PRJNA1302674.

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