A novel effect of sulforaphane on promoting mouse granulosa cells proliferation via the NRF2-TKT pathway

Abstract

Introduction: Granulosa cells (GCs) is essential for maintaining follicular development. Follicle-stimulating Hormone (FSH) has been demonstrated to effectively promote GCs proliferation, driving the establishment of various superovulation techniques for animal husbandry. However, these techniques face challenges, such as high costs, hormonal imbalances, and an increased risk of early ovarian dysfunction. Therefore, it is important to investigate new methods to improve GCs proliferation.

Objectives: This study aimed to investigate the effect of sulforaphane (SFN) on ovarian GCs proliferation and the underlying mechanisms.

Methods: A comparative transcriptomic analysis of ovaries from the control, SFN, and FSH groups was conducted to identify the primary factors contributing to high proliferative capacity. The role of SFN in the regulation of cell proliferation has been examined in mouse ovarian GCs. Gene interference, overexpression, CUT&TAG technology, and transcriptome analyses were performed to elucidate the underlying mechanisms of the nuclear factor E2-related factor 2 (NRF2)-transketolase (TKT) axis in mediating GCs proliferation.

Results: Our research revealed a previously unknown function of SFN, an isothiocyanate of plant origin that is prevalent in cruciferous vegetables, in facilitating the proliferation of mouse ovarian GCs. The efficacy of SFN in enhancing GCs proliferation is similar to that of FSH. At the mechanistic level, SFN promotes NRF2 to transport to the nucleus, which subsequently activates the key enzyme of the non-oxidative pentose phosphate pathway TKT. This activation is instrumental in generating ribose 5-phosphate, a critical precursor for amino acid and nucleotide biosynthesis that underpins the proliferation of GCs.

Conclusion: Collectively, our findings delineate a novel pathway by which SFN, through the NRF2-TKT axis, enhances the nucleotide pool and thereby supports the proliferation of mouse GCs, presenting novel avenues for exploration in reproductive biology and agricultural sciences.

Keywords: Cell proliferation; Granulosa cells; NRF2–TKT; Ribose 5-phosphate; Sulforaphane.

Graphical abstract

Fig. 1

SFN promotes the proliferation of follicular GCs both in vivo and in vitro. (A) The schematic diagram illustrates the experimental procedure of intraperitoneal injection in mice. (B) H&E staining of mouse ovarian tissue, depicting the maximum cross-section of the ovary. Scale bar: 500 μm. (C) Immunohistochemical staining for PCNA in mouse ovarian tissue, with corresponding statistical analysis of the proportion of PCNA-positive cells on the right. Scale bar: 100 μm. (D) PCNA protein levels of GCs in each groups were detected by western blot, followed by grayscale analysis. (E, F) EdU staining (red) shows GCs proliferation. (F) displays the analysis of EdU-positive cell proportion. Scale bar: 50 μm. (G) GCs were treated with SFN or DMSO for 24 h, and cell proliferation viability was detected using the CCK-8 assay. (H) After a 6-hour EdU labeling of GCs, fluorescent staining was conducted with red indicating EdU and blue representing DAPI, aiming to assess GCs proliferation. The right panel displays the proportion of EdU-positive cells. Scale bar: 50 μm. (I) Following a 12-hour treatment of GCs with SFN or DMSO, PCNA protein levels were assessed by western blot, followed by grayscale analysis. Data represent mean ± SD, and experiments were repeated a minimum of three times. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

SFN enhances glycolysis and pentose phosphate pathway by RNA-Seq. (A) Venn diagram showing the number of DEGs in the SFN vs Con and FSH vs Con. Validation of selected DEGs through qPCR is provided on the right. (B) Venn diagram showing the number of DEGs in the SFN vs Con and FSH vs Con. Validation of selected DEGs through qPCR is provided on the right. (C) Gene set enrichment analysis was performed using the whole gene list generated from RNA-seq. (D) Heat map of glycolysis and pentose phosphate pathway genes in each group. (E) The quantitative expression analysis of key DEGs was conducted using qPCR. (F–I) Glucose uptake capacity, lactate production, ATP and R5P levels were evaluated as indicators to demonstrate the activity of the glycolytic pathway. (J) The schematic diagram illustrates the enhancement of glycolysis and the PPP in GCs by SFN. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***P<0.001, by two-tailed Student’s t test.

Fig. 3

SFN facilitates GCs proliferation by increasing R5P levels. (A) Heat map depicting cell cycle-related gene expression in each group. (B, C) Flow cytometry was employed to assess alterations in the cell cycle of GCs treated with SFN for 24 h. Quantification and analysis of the data are presented in (C). (D) Cell proliferation of GCs was determined using the CCK-8 assay after 24-hour treatment with SFN or R5P. (E, F) Targeted metabolomics analysis was conducted in GCs to examine changes in amino acid and nucleotide levels after 24 h of SFN treatment. (G, H) Following a 6-hour EdU labeling of GCs, fluorescent staining was performed, with red indicating EdU and blue representing DAPI, to evaluate GCs proliferation. The proportion of EdU-positive cells is displayed in (H). Scale bar: 50 μm. (I) Western blot analysis was conducted to assess the protein levels of CDK2, cyclin E1, and PCNA in GCs following a 12-hour treatment with either SFN or R5P. Quantitative grayscale analysis is presented on the right. Data represent mean ± SD, and experiments were repeated a minimum of three times. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

SFN activates TKT, resulting in R5P levels increasement and causing GCs proliferation. (A) qPCR was used to detect the transcription levels of G6pdx and Tkt genes. (B) Relative protein levels in GCs were determined by western blot after 12-hour treatment with SFN or DMSO, quantitative grayscale analysis is presented on the right (C). (D) After knockdown or overexpression of Tkt in GCs, cells were treated with SFN or DMSO for 24 h, and cell proliferation viability was measured using the CCK-8 assay. (E) R5P levels were detected to evaluate the activity of the PPP. (F, G) Targeted metabolomics analysis was conducted in GCs to examine changes in amino acid and nucleotide levels after 24 h of treatment with either SFN or SFN+siTkt. (H) GCs were subjected to detect the expression levels of cell cycle-related genes in the Control, SFN and SFN+siTkt groups by qPCR. (I) After treating GCs for 12 h, western blot was conducted to evaluate the protein levels, quantitative grayscale analysis is presented on the right. (J, K) Flow cytometry was used to detect changes in the cell cycle of GCs after 24 h of treatment with SFN or SFN+siTkt. Quantification and analysis of the data are presented in (K). Data represent mean ± SD, and experiments were repeated a minimum of three times. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference.

Fig. 5

Nuclear accumulation of NRF2 is enhanced by SFN. (A) The DEGs identified in the RNA-seq data were subjected to analysis using Transcriptional Regulatory Relationships Unraveled by Sentence-based Text Mining (TRRUST). (B) Venn diagram showing the overlapping target genes identified from the analysis of NRF2 binding across three publicly available ChIP-seq datasets. (C, D) After treatment of GCs with SFN or DMSO for 12 h, total NRF2 and KEAP1 protein levels in GCs were detected by western blot, and the quantitative grayscale results are presented on the right. (E) The protein levels of NRF2 in both the cytoplasmic and nuclear fractions of GCs were individually assessed through western blot after a 12-hour treatment with SFN or DMSO, and quantitative grayscale values are provided below the blot. (F) Immunofluorescence staining of NRF2 was performed to examine its cellular localization in GCs. (G) Expression levels of Nrf2 and Nqo1 were detected by qPCR after a 12-hour treatment. Data represent mean ± SD, and experiments were repeated a minimum of three times. ***P<0.001, ns indicates no significant difference.

Fig. 6

SFN regulates TKT through NRF2-targeted modulation.(A) After 12 hourstreatment, GCs were subjected to assess the expression of Nrf2, Tkt and Nqo1 in the Control, SFN and SFN+siNrf2 groups by qPCR. (B) Conducting CUT&Tag sequencing, peak analysis was performed using IGV software, with the Control group in blue and the SFN group in red. (C, D) After Nrf2 knockdown or overexpression, western blot analysis was conducted to evaluate the protein levels of NRF2, NQO1, and TKT. ACTB was used for normalization, and the quantitative grayscale analysis is presented on the right. (E) R5P level detection was employed to evaluate the activity of PPP in all groups. (F, G) Targeted metabolomics analysis was conducted in GCs to examine changes in amino acid and nucleotide levels after 24 h of treatment with either SFN alone or SFN combined with siNrf2. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

SFN stimulates GCs proliferation via the NRF2-TKT pathway. (A, B) After a 24-hour treatment with either SFN or a combination of SFN and siNrf2, changes in the cell cycle of GCs were detected using flow cytometry. The data were quantified and analyzed in (B). (C, D) After treatment of GCs with SFN or DMSO for 12 h, CDK2, cyclin E1 and PCNA protein levels in GCs were detected by western blot, and the quantitative grayscale results are presented on the right. (D) Cell proliferation viability was assessed using the CCK-8 assay after knockdown or overexpression of Nrf2 in GCs, treated with SFN or DMSO for 24 h. (E, F) EdU labeling for 6 h in GCs, followed by fluorescent staining with red indicating EdU and blue representing DAPI, assessed GCs proliferation. (F) displays the proportion of EdU-positive cells. Scale bar: 50 μm. (G) Cell proliferation was detected using the CCK-8 assay in GCs treated with SFN or ML385 for 24 h. (C, D) Proliferation-related protein levels were detected by western blot after a 12-hour treatment of GCs with different treatments, and the quantitative grayscale analysis is presented on the right. (I) Western blot analysis assessed protein levels after a 12-hour treatment of GCs in the empty vector control, OE-Nrf2, and OE-Nrf2 + siTkt groups, quantitative grayscale analysis was performed to assess the relative protein levels. (J) The CCK-8 assay detected the cell proliferation activity of Control, OE-Nrf2, and OE-Nrf2 + siTkt groups after 24 h of GCs treatment. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

In vivo illustrating of the NRF2-TKT pathway in the regulation of GCs proliferation by SFN. (A) Relative protein levels of GCs in each groups were detected by Western blot, and quantitative grayscale values are provided below the blot. (B, C) Following EdU labeling of ovarian follicles, red fluorescence staining was used to evaluate GCs proliferation. (C) represents the analysis of the proportion of EdU-positive cells. Scale bar: 50 μm. (D, E) Immunohistochemical staining results for PCNA in mouse ovarian tissue, with corresponding statistical analysis of the proportion of PCNA-positive cells on the right (E). Scale bar: 100 μm. (F, G) Immunohistochemical staining outcomes for PCNA in mouse ovarian tissue, accompanied by statistical analysis depicting the proportion of PCNA-positive cells (G). Scale bar: 100 μm. (H) Relative protein levels of GCs in each groups were detected by western blot, and the quantitative grayscale results are presented on the right. Data show mean ± S.D, and each experiment was conducted with a minimum of three replicates. *p value < 0.05, **p value < 0.01, ***p value < 0.001 by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)