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. 2023 Oct 31;80(11):343. doi: 10.1007/s00018-023-04979-2

SRSF1 is essential for primary follicle development by regulating granulosa cell survival via mRNA alternative splicing

Xiaohong Yao 1,#, Chaofan Wang 1,#, Weiran Yu 1, Longjie Sun 1, Zheng Lv 1, Xiaomei Xie 1, Shuang Tian 1, Lu Yan 1, Hua Zhang 1, Jiali Liu 1,✉
PMCID: PMC11072053  PMID: 37907803

Abstract

Granulosa cell abnormalities are characteristics of premature ovarian insufficiency (POI). Abnormal expression of serine/arginine-rich splicing factor 1 (SRSF1) can cause various diseases, but the role of SRSF1 in mouse granulosa cells remains largely unclear. In this study, we found that SRSF1 was expressed in the nuclei of both mouse oocytes and granulosa cells. The specific knockout of Srsf1 in granulosa cells led to follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. Gene Ontology (GO) analysis of RNA-seq results revealed abnormal expression of genes involved in DNA repair, cell killing and other signalling pathways. Alternative splicing (AS) analysis showed that SRSF1 affected DNA damage in granulosa cells by regulating genes related to DNA repair. In summary, SRSF1 in granulosa cells controls follicular development by regulating AS of genes associated with DNA repair, thereby affecting female reproduction.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-023-04979-2.

Keywords: SRSF1, Granulosa cell, Alternative splicing, DNA damage

Introduction

The main function of the ovary is to produce germ cells (oocytes) [1]. The ovaries comprise oocytes, granulosa cells, and theca cells, among which oocytes provide genetic material. Oocytes and somatic cells exchange many regulatory signals that control oocyte metabolism, cytoskeletal remodelling, cell cycle progression, and fertilization, all of which are key events that initiate and maintain early embryogenesis [2–4]. Problems in any of the cell types in the ovary can lead to infertility in females.

POI affects female fertility. The histological characteristics of POI indicate that most follicles are histologically abnormal, and the follicles are atretic, characterized by partial to complete absence of granulosa cells [5, 6]. Ovarian granulosa cells in patients with POI exhibit significant changes in their proteomes, and a total of 2688 proteins have been identified, 70 of which are significantly differentially expressed [7]. Abnormal AS is one of the causes of POI. TP63 (human)/Trp63 (mouse) produces various AS isomer products at the C-terminus associated with female infertility, including POI [8]. A missense mutation in the HFM1 gene (c.3470G > A) alters the splicing of its mRNA in the ovary and may be one of the causes of POI [9].

mRNA splicing requires a set of fundamental factors called SR proteins. SRSF1 is the classic member of the SR protein family, and it is involved in the maturation of the spliceosome [10–12]. SRSF1 plays an integral role in maintaining cell homeostasis by regulating many complex biological pathways [13]. SRSF1 deficiency is associated with a variety of diseases; for example, SRSF1 regulates AS in breast cancer [14], promotes mammary epithelial cell transformation [15], regulates gene expression in the immune system [16], regulates T-cell homeostasis and function [17], promotes the proliferation, migration, invasion of hepatocellular carcinoma [18] and regulates primordial follicle formation and number determination during meiotic prophase I [19]. A previous study showed that SRSF1 inhibits porcine granulosa cell apoptosis and follicular atresia through a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis [20]. However, the role of SRSF1 in granulosa cell development in vivo has not been described.

To explore the function of SRSF1 in granulosa cells, we used Foxl2-CreERT2 mice to knock out Srsf1 specifically. The specific deletion of Srsf1 in granulosa cells resulted in follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. RNA-seq results showed that SRSF1 directly regulated the AS of genes related to DNA repair.

Materials and methods

Mice

C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Srsf1F/F mice and Foxl2-CreERT2 mice were generated as previously reported [21, 22]. Srsf1F/F mice were generated in the laboratory of Prof. Xiangdong Fu (University of California, San Diego, USA) and kindly provided by Prof. Yuanchao Xue (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). To knockout Srsf1 in mouse ovarian granulosa cells as early as possible, the mice were injected with 20 mg/kg tamoxifen (T5648; Sigma, St. Louis, MO, USA) at 1 day postpartum (dpp), 3 dpp, and 5 dpp to activate Cre recombinase. All the mice were housed in the pathogen-free Animal Care Facility of China Agricultural University under a 12-h light/12-h dark cycle at an ambient temperature of 21 ± 2 °C, and they were given free access to food and water.

Identification of mouse genotypes

DNA was extracted from mouse tails using the HotSHOT method, and PCR was used for genotyping. The PCR primers used are listed in Supplementary Table 1. The PCR conditions were as follows: 95 °C for 5 min; 36 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s; 72 °C for 5 min; and holding at 4 °C. The primers produced PCR products of 346 bp for the wild-type Srsf1 allele and 386 bp for the floxed allele.

Histological analysis of ovary and follicle counts

Ovaries were prepared for histological analysis by immediately placing them in cold 4% paraformaldehyde for fixation. Fixation was performed overnight at 4 °C, and ovaries were transferred to 70% ethanol, dehydrated, and embedded in paraffin. Tissues were sectioned at a thickness of 5 µm. Tissue sections were stained with hematoxylin.

The ovarian sections were analyzed with a slide scanner (VENTANA DP 200, Roche, Switzerland), and the follicles were counted in all sections with clear oocyte nuclei. Every five ovarian sections were analysed. There are five types of follicles: primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles, according to the morphology and number of granulosa cell layers [23].

Immunofluorescence (IF) staining

Ovarian sections were dewaxed and then microwaved in 0.01 M sodium citrate buffer (pH = 6.0) for antigen retrieval. The prepared sections were blocked with 10% normal goat serum (SP-9000; Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature (RT) and then incubated with the indicated primary antibodies (Table S2). After being washed three times with PBS, the samples were incubated with the indicated secondary antibodies (Table S2) at a 1:400 dilution for 2 h at RT and with 4′,6-diamidino-2-phenylindole (DAPI) (10236276001; Roche Applied Science, Basel, Switzerland) at RT for 10 min. The slides were washed three times in PBS and mounted with anti-fade mounting medium (P0128S; Beyotime Biotechnology, Shanghai, China). Finally, a Nikon A1 laser scanning confocal microscope (A1, Nikon, Japan) was used for image acquisition.

TUNEL

TUNEL assays were performed on 4% paraformaldehyde-fixed paraffin-embedded sections using the One Step TUNEL Apoptosis Assay Kit (C1088; Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions.

Cell proliferation assay

Mice were injected with 100 mg/kg 5-bromo-2´-deoxyuridine (BrdU) (B5002, Sigma, St. Louis, MO, USA) in a physiological saline solution by intraperitoneal injection. After 2 h, mouse ovaries were fixed and embedded in paraffin. Then, tissue sectioning, antigen retrieval, BrdU staining, and image acquisition were conducted as described in the IF methods.

Western blot

The ovaries were lysed in RIPA buffer (P0013B; Beyotime Biotechnology, Shanghai, China) and homogenized with a grinding rod. The protein extracts were subjected to electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (IPVH00010; Millipore, USA). The membranes were blocked with 5% nonfat milk (P0216-300 g; Beyotime Biotechnology, Shanghai, China) prepared in Tris-buffer saline plus 0.1% Tween-20 (TBST) at RT for 1 h and then incubated with primary antibodies overnight at 4 °C. Then, the membranes were incubated with secondary antibodies (Table S2) for 2 h. After washing with TBST three times, the membranes were visualized with BeyoECL Plus (P0018S; Beyotime Biotechnology, Shanghai, China) with the ECL system (5200, Tanon, China).

Quantitative real-time PCR (qPCR) and reverse transcription PCR (RT‒PCR)

Total RNA was extracted by TRIzol Reagent (9109; TaKaRa, Dalian, China). One microgram of total RNA was used to synthesize first-strand cDNA (M170A; Promega, USA).

qPCR analysis was performed with Hieff UNICON® qPCR SYBR Green Master Mix (11198ES03; YEASEN, Shanghai, China). cDNA was diluted and used as the template for the real-time SYBR Green assay. Gapdh was used as the endogenous control. All gene expression was quantified relative to Gapdh expression (Table S3).

RT‒PCR analysis was performed with cDNA using 2 × Taq Master Mix (Dye Plus) for common PCR, and mature mRNA sequences were used to design primers for RT‒PCR (Table S4).

RNA immunoprecipitation (RIP)

Ovaries were lysed with cell lysis buffer (P0013, Beyotime Biotechnology, Shanghai, China). First, RNase inhibitor (1:20, R0102, Beyotime Biotechnology, Shanghai, China) was added. Two micrograms of IgG (A7016, Beyotime Biotechnology, Shanghai, China) and SRSF1 antibody (12929–2-AP, Proteintech, Chicago, USA) were bound to BeyoMag™ Protein A (P2102-1 ml, Beyotime Biotechnology, Shanghai, China). 10% of the ovary lysate supernatants were used as the input, and the remaining volume was incubated with BeyoMag™ Protein A overnight at 4 °C. Bound RNA was extracted using a Direct-zol RNA Microprep Kit (R2060, Zymo Research, Los Angeles, USA) and analysed by RT‒qPCR analysis.

RNA-seq

We called primordial follicular granulosa cells as pfGCs. For the convenience of description, Srsf1F/F mice are called PfGC- Srsf1+/+, Srsf1F/F; Foxl2-CreERT2 mice are called PfGC- Srsf1−/−. Ovarian samples were collected from PfGC- Srsf1+/+ and PfGC- Srsf1−/− mice at 8 dpp and sent to Novogene for RNA-seq analysis. Total RNA was isolated, and the quality of RNA samples was controlled by an Agilent 2100 bioanalyzer (2100, Agilent, USA). mRNA was randomly divided into fragments with fragmentation buffer, and the database was built in standard NEB mode. Sequencing was performed using an Illumina NovaSeq 6000 (Illumina, USA). Significant gene expression differences were analyzed, and GO enrichment analysis was conducted using clusterProfiler (version 3.8.1) software. AS was analyzed using rMATS (version 4.1.0) software.

Cell culture and cell transfection

The KK1 cell line was cultured in DMEM/F12 (C11330500BT, Gibco, USA) containing 10% fetal bovine serum (S500, Newzerum, Ltd., Christchurch, New Zealand) and 1% penicillin and streptomycin at 37 ℃ in a 5% CO2 incubator. The sequences were as follows: NC, sense (5´ -3´ ): UUCUCCGAACGUGUCACGUTT;

antisense (5´ -3´ ): ACGUGACACGUUCGGAGAATT;

siRNA-361, sense (5´–3´): GGGUUUGGAGGAUUUGGAATT; antisense (5´–3´): UUCCAAAUCCUCCAAACCCTT; siRNA-1204, sense (5´–3´): GCAGGUGUUGAUCCUAUUATT; antisense (5´–3´): UAAUAGGAUCAACACCUGCTT; siRNA-541, sense (5´–3´): GGACUUGGAUUUGGAGGAUTT; antisense (5´–3´): AUCCUCCAAAUCCAAGUCCTT; siRNA-1002, sense (5´–3´): GGAUGAUCAGACGGAAGUUTT; antisense (5´–3´): AACUUCCGUCUGAUCAUCCTT; siRNA was transfected into the KK1 cell line using Lipo8000™ (C0533; Beyotime Biotechnology, Shanghai, China) according to the manufacturer’ s instructions.

Statistical analysis

All the statistical analyses were performed with GraphPad Prism software (version 7.0) using Student’ s t-test. The data are presented as the means ± SEMs at least three independent experiments. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0. 01, ***P < 0.001, ****P < 0.0001. Mouse genome data (GRCm39) were used as the reference.

Results

Expression pattern of SRSF1 in mouse ovaries

To explore the function of SRSF1 in mouse ovarian granulosa cells, we first characterized the SRSF1 expression pattern during follicle development. qPCR analyses of ovaries on different days showed that Srsf1 mRNA expression increased from 1 dpp to 21 dpp (Fig. 1A). Western blot was used to measure the protein expression level of SRSF1. The results were consistent with the qPCR results (Fig. 1B). To further determine the localization of SRSF1 in mouse ovaries, we conducted IF, and SRSF1 was highly expressed in the nuclei of oocytes and granulosa cells (Fig. 1C). These data suggest that SRSF1 in granulosa cells may play essential roles in ovary development.

Fig. 1.

Fig. 1

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Expression pattern of SRSF1 during follicular development. A Relative Srsf1 mRNA expression levels in ovaries on different days. Student’ s t-test was used for statistical analysis.The results are presented as the means ± SEMs. Three independent experiments were performed for each data. B The SRSF1 protein levels were measured by Western blot on different days. Student’ s t-test was used for statistical analysis.The results are presented as the means ± SEMs. n = 4. C The localization of SRSF1 in 1 dpp, 7 dpp, 14 dpp, and 21 dpp ovaries was determined by IF. Scale bars, 100 μm

SRSF1 was absent in granulosa cells of primary follicles in Srsf1−/− ovaries

To study the role of SRSF1 in granulosa cells, mice with the Srsf1 conditional floxed allele (Srsf1Fl/Fl), in which exons 2, 3, and 4 of Srsf1 were flanked by two loxP sites, were crossed with Foxl2-CreERT2 mice (Fig. 2A, S1A). To activate the Cre enzyme, mice were injected with 20 mg/kg of tamoxifen every another day, starting on 1 dpp and ending on 5 dpp (Fig. 2A). Mouse genotypes were determined by PCR (Fig. 2B). IF showed a specific loss of SRSF1 in primary follicular granulosa cells at 7 dpp (Fig. 2C). However, SRSF1 was still present in the pfGCs (Fig. S1B), which may be due to the accumulation of undegraded SRSF1 protein in PfGCs.

Fig. 2.

Fig. 2

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SRSF1 was successfully knocked out in granulosa cells at 7 dpp. A Injection of tamoxifen to induced knock out exon. B WT; Srsf1F/+; Foxl2-CreERT2, and Srsf1F/F; Foxl2-CreERT2 genotype determination. C Localization of SRSF1 in PfGC-Srsf1+/+ and PfGC-Srsf1−/−ovary. The area indicated by the dashed white circle represents granulosa cells. Scale bars, 50 μm

Knockout of Srsf1 in ovarian granulosa cells resulted in follicular development arrest

To further identify the function of SRSF1 in granulosa cells, we used haematoxylin staining to distinguish the differences in ovarian histology on different days in PfGC-Srsf1+/+ and PfGC-Srsf1−/−. The histological analyses at 7 dpp revealed that PfGC-Srsf1+/+ and PfGC-Srsf1−/− contained primordial follicles and primary follicles (Fig. 3A-a–d). We counted whole ovarian follicles and found that PfGC-Srsf1+/+ and PfGC-Srsf1−/− did not differ significantly in the numbers of primordial follicles and primary follicles, while the number of secondary follicles was reduced in PfGC-Srsf1−/− compared with PfGC-Srsf1+/+ (Fig. 3B). These results suggest that the loss of Srsf1 in granulosa cells leads to the inhibition of follicular development. At 14 dpp, there was a difference in ovarian sizes between PfGC-Srsf1+/+ and PfGC-Srsf1−/− (Fig. 3A-e, g). When the total number of follicles remained unchanged, more follicles in PfGC-Srsf1+/+ developed into secondary follicles, but few primary follicles developed into secondary follicles in PfGC-Srsf1−/−, and the number of atretic follicles in PfGC-Srsf1−/− significantly increased (Fig. 3B). At 21 dpp, the difference in ovarian morphology was more pronounced between PfGC-Srsf1+/+ and PfGC-Srsf1−/− (Fig. 3A-i, k). The follicle counts at 21 dpp showed the same trend as those at 14 dpp. There was no significant difference in the total number of follicles between PfGC-Srsf1+/+ and PfGC-Srsf1−/−, but compared with the PfGC-Srsf1+/+group, the PfGC-Srsf1−/− group had fewer secondary follicles, and antral follicles, while the number of atretic follicles was increased (Fig. 3B). These data revealed that specific knockout of Srsf1 in ovarian granulosa cells could lead to follicular development arrest, and many follicles of the PfGC-Srsf1−/− group were blocked in the primary follicle stage.

Fig. 3.

Fig. 3

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Loss of SRSF1 in granulosa cells led to follicular development arrest. A Follicular morphology of PfGC-Srsf1+/+ and PfGC-Srsf1−/− at 7 dpp, 14 dpp and 21 dpp ovary. The red boxes represent atretic follicles in figure A-h,l. Black scale bars, 100 μm; white scale bars, 1 mm. B The numbers of ovarian follicles of PfGC- Srsf1+/+ and PfGC- Srsf1−/− at 7 dpp, 14 dpp, and 21 dpp were counted. PrF, primordial follicles; PF, primary follicles; SF, secondary follicles; AF, antral follicles; ATF, atretic follicles. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. *P < 0.05, **P < 0. 01, ***P < 0.001, ****P < 0.0001

Loss of SRSF1 led to abnormal proliferation and apoptosis in granulosa cells

The absence of secondary follicles in mouse PfGC-Srsf1−/− suggests that granulosa cell proliferation and apoptosis may be abnormal. To test our hypothesis, we examined granulosa cell proliferation by BrdU incorporation at 7 dpp (Fig. 4A). Cell counting results showed that the BrdU immunoreactive (BrdU+) cell ratio in PfGC-Srsf1−/− was significantly lower than that in PfGC-Srsf1+/+ (Fig. 4B), indicating that the proliferation rate of PfGC-Srsf1−/− was decreased. Furthermore, we examined the apoptosis of granulosa cells. The TUNEL results showed no positive cells in PfGC-Srsf1+/+ at 7 dpp, 10 dpp, and 14 dpp. However, the apoptosis of PfGC-Srsf1−/− occurred at 10 dpp and increased at 14 dpp (Fig. 4C, D), and granulosa cell apoptosis occurs in follicles that are transformed from primary follicles to secondary follicles. These results suggest that knockout Srsf1 in granulosa cell can slow granulosa cell proliferation and increase apoptosis, ultimately leading to follicular atresia.

Fig. 4.

Fig. 4

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Loss of SRSF1 affected granulosa cell proliferation and apoptosis. A BrdU signal in 7 dpp ovaries were detected by IF. Scale bars, 50 μm. B The percentage of BrdU+ cells among granulosa cells. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. **P < 0. 01. C Images of PfGC- Srsf1+/+ and PfGC- Srsf1−/− ovaries were obtained after TUNEL staining at 7 dpp, 10 dpp and 14 dpp. Scale bars, 100 μm. D The TUNEL-positive cell proportion was determined at different times. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. **P < 0.01

SRSF1 deficiency in granulosa cells resulted in abnormal ovarian transcriptome

To investigate changes in gene expression after Srsf1 deletion in granulosa cells, we performed RNA-seq sequencing on ovaries from PfGC- Srsf1+/+ and PfGC-Srsf1−/− mice at 8 dpp. The correlation coefficients within and between the groups were calculated according to the FPKM values of all the genes in each sample. The squared Pearson correlation coefficients (R2) of the gene expression levels between samples were more significant than 0.92, indicating that the experiment was reliable and that the sample selection was reasonable (Fig. S2A). We found that 1543 genes were differentially expressed, with 881 upregulated and 662 downregulated genes in PfGC-Srsf1−/− ovaries (Fig. 5A). Cluster analysis was performed on the differential gene sets, and genes with similar expression patterns were grouped. The differentially expressed genes in the control and knockout groups are presented as a clustered heatmap (Fig. 5B). We then performed GO analysis to identify the enriched biological processes for each group of differentially expressed genes (DEGs). The upregulated genes in PfGC-Srsf1−/− were significantly involved in autophagy, reproductive system development, regulation of apoptotic signalling pathway, signal transduction by p53 class mediator, ERK1 and ERK2 cascade, and cell killing (Fig. 5C). The downregulated genes in PfGC-Srsf1−/− were significantly involved in the regulation of DNA repair, cAMP biosynthetic process and so on (Fig. 5D). Cluster analysis of the upregulated and downregulated signalling pathways of cell killing, regulation of the apoptotic pathway, signal transduction by p53 class mediators and regulation of DNA repair indicated that the knockout of SRSF1 in ovarian granulosa cells affected multiple biological processes that lead to cell apoptosis (Fig. S2B). Moreover, qPCR was used to verify the expression of related genes. The qPCR results were consistent with the RNA-seq results (Fig. 5E).

Fig. 5.

Fig. 5

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SRSF1 deficiency in granulosa cells altered gene expression patterns. A Volcano plot showing the distribution of differentially expressed genes identified by RNA-seq. (pvalue <  = 0.05, |log2FoldChange|> = 0). B Heatmap of the DEGs between PfGC-Srsf1+/+ and PfGC-Srsf1−/− at 8 dpp. C GO analysis of upregulated genes in PfGC-Srsf1−/−. The overrepresented terms, gene counts, and qValue are shown. D GO analysis of downregulated genes in PfGC-Srsf1−/−. The overrepresented terms, gene counts, and qValue are shown. E The mRNA expression of 8 dpp ovaries. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. *P < 0.05, **P < 0. 01, ***P < 0.001, ****P < 0.0001

SRSF1 participated in AS affecting DNA repair

AS is essential for regulating gene expression and generating proteome diversity. Data analysis on cKO vs. WT showed that 249 genes underwent AS, of which 29 splicing events were classified as alternative 3´ splice sites (A3SS), 34 splicing events were classified as alternative 5´ splice sites (A5SS), 142 splicing events were classified as skipped exons (SE), 28 splicing events were classified as retained introns (RI), and 16 splicing events were classified as mutually exclusive exons (MXE) (Fig. 6A). SE accounted for the highest proportion (Fig. 6B). The GO enrichment analysis of AS revealed that lysosome organization, regulation of apoptotic signalling pathway, DNA repair, negative regulation of translation, mitotic spindle organization, negative regulation of growth, and single-stranded RNA binding-related genes were associated alterations in AS form (Fig. 6C). We used Integrative Genomics Viewer (IGV, version 2.10.2) software to visualize AS, and RT‒PCR was used to analyze AS patterns (Fig. S3A). Therefore, we successfully validated the AS of four DNA repair-related genes. The results showed that Chd1l has an A5SS splicing form, and Herc2, Usp3, and Nup54 have SE AS forms. Using RT‒PCR verification, the results showed that SRSF1 regulates the AS of Chd1l, Herc2, Usp3, and Nup54 pre-mRNA in the ovary, thus affecting DNA repair (Fig. 6D). Western blot results showed that NUP54 and USP3 protein levels were significantly decreased (Fig. 6E). In addition, the results revealed that PPT1 and NDUFS3 protein levels were reduced (Fig. S3B). RIP was used to verify whether SRSF1 directly binds to RNA, and the verification results showed that genes were significantly enriched in the anti-SRSF1 group, indicating that SRSF1 directly binds to pre-mRNA and participates in AS (Fig. 6F) (Fig. S3C). To further verify whether Srsf1 deletion affects DNA repair in ovarian granulosa cells, granulosa cells were stained with γ-H2AX at 7 dpp, 10 dpp, and 14 dpp. The results showed no positive cells among PfGC-Srsf1+/+ at 7 dpp, 10 dpp, and 14 dpp, whereas DNA damage increased in granulosa cells from 10 to 14 dpp in the PfGC-Srsf1−/− group (Fig. 6G, H), suggesting that Srsf1 deletion leads to irreparable DNA damage through abnormal AS.

Fig. 6.

Fig. 6

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SRSF1 participation in AS affected DNA repair. A Different types of AS are caused by SRSF1 deficiency in granulosa cells. B The proportions of different types of AS are shown in pie charts. C GO enrichment analysis of AS events. D Splicing pattern and RT-PCR validation of Chd1l, Herc2, Usp3, and Nup54. Integrative Genomics Viewer (IGV, version 2.10.2) was used to visualize and confirm AS events in RNA-seq data. E Western blot results revealed the NUP54 and USP3 protein levels in PfGC- Srsf1+/+ and PfGC- Srsf1−/− ovaries. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. *P < 0.05. F The results of RIP enrichment. Student’ s t-test was used for statistical analysis. The data are presented as the means ± SEMs. n = 3. *P < 0.05, ***P < 0.001, ****P < 0.0001. G Images of PfGC- Srsf1+/+ and PfGC- Srsf1−/− ovaries were obtained at 7 dpp, 10 dpp, and 14 dpp after staining for γ-H2AX. Scale bars, 100 μm. H The proportion of γ-H2AX-positive cells was counted at different times. Student’ s t-test was used for statistical analysis. n = 3. ***P < 0.001

To detect whether deletion of Nup54 in KK1 cell lines leads to DNA damage, we successfully knocked down Nup54 in KK1 cell lines using siRNA, siRNA-1204 has the highest interference efficiency (Fig. S4A). Immunofluorescence results showed that the percent of the γ-H2AX positive cell in knockdown group was large than in control group, indicating that DNA damage occurred in KK1 cell line after knocking down Nup54 (Fig. S4B).

Discussion

SR proteins are a series of highly conserved serine/arginine-rich RNA binding proteins and are important splicing factors [24, 25]. SRSF1 is a member of the SR protein family [13]. Srsf1 deletion in different tissues can lead to a variety of diseases, and Srsf1 KO results in embryonic lethality in mice [21]. However, the role of SRSF1 in granulosa cell function is poorly understood. Our study showed that SRSF1 was expressed in both oocytes and granulosa cells during follicular development. To explore the role of SRSF1 in granulosa cells, we successfully knocked out SRSF1 in the granulosa cells of the primary follicles by Foxl2-CreERT2 mice. Morphological analysis of the ovaries showed that follicles of PfGC-Srsf−/− ovaries were stunted, primary follicles failed to convert to secondary follicles, and a large number of atretic follicles were observed at 21 dpp. This study demonstrated that SRSF1 was required for follicle development in granulosa cells.

To further explore the cause of follicular atresia, we examined whether there were problems in the proliferation and apoptosis of granulosa cells. BrdU is an analogue of nucleotides and thymidine that is used to detect changes in proliferating cells [26]. BrdU staining of the ovary of 7dpp mice showed that the proliferation of PfGC-Srsf1−/− granulosa cells was significantly slowed down. To further investigate the physiological status of reduced granulosa cell proliferation, TUNEL staining was performed on ovarian tissues at different developmental stages. The results showed that the number of apoptotic granulosa cells was increased in PfGC-Srsf1−/−. This is consistent with previous reports, in which knockdown of Srsf1 induces cell cycle arrest in mASMCs and inhibits mASMC proliferation [27], and SRSF1 regulates the apoptosis and proliferation and promotes the transformation of mammary epithelial cells [15]. These results indicate that SRSF1 regulates cell proliferation and apoptosis in different cell types.

To further investigate the effect of Srsf1 deletion on granulosa cell development, 8 dpp ovaries were harvested for RNA-seq. The results showed that 881 genes were upregulated and 662 genes were downregulated at the transcriptome level. GO analysis of upregulated and downregulated genes revealed some associations with regulating the apoptotic signalling pathway, signal transduction by p53 class mediators, cell killing, and regulation of DNA repair-related signalling pathways. The results of qPCR verification were consistent with those of RNA-seq. These results suggest that SRSF1 may affect granulosa cell survival through multiple pathways.

SRSF1 may need to bind to a specific target pre-mRNA to perform its splicing function. We found that 249 genes underwent AS, and SEs accounted for the highest proportion. GO analysis was performed on the genes that were regulated by AS, and multiple phenotype-related signalling pathways were enriched. We successfully verified the AS of genes that are associated with signalling pathways such as DNA repair using RT‒PCR. It has been documented that HERC2, NUP54, USP3, and CHD1L are involved in DNA repair in different cell types [28–35], and the present study showed that the expression of the DNA damage-related proteins USP3 and NUP54 was decreased in PfGC-Srsf1−/−, which was closely related to POI [36, 37]. And knocking out Nup54 in KK1 cell lines also causes DNA damage. Furthermore, the RIP results showed that SRSF1 directly binds to RNAs to regulate AS. However, SRSF1 regulates AS events related to the maintenance of survival and proliferation in dental progenitors by targeting Cdc45, Incenp, Rif1, Ncor1, Cttn, Gak, Picalm, Hnrnpd, Srrm2, and Lsm6 pre-mRNAs [38]. Additionally, SRSF1 can regulate oocyte meiosis by binding to different target Msh5 and Six6os1 pre-mRNAs [19]. Thus, SRSF1 regulates the survival of different cells and development through AS.

In conclusion, our study showed that Srsf1 deletion leads to abnormalities in multiple signalling pathways, such as DNA repair and cell killing. SRSF1 participation in AS affects DNA damage in granulosa cells, which in turn leads to follicular development arrest (Fig. 7). Abnormal AS can lead to granulosa cell apoptosis, which provides new information about clinical POI.

Fig. 7.

Fig. 7

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Schematic diagram of the mechanism by which SRSF1 regulates granulosa cell survival

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1815 KB) (1.8MB, docx)

Acknowledgements

We thank Prof. Yuanchao Xue (Institute of Biophysics, Chinese Academy of Sciences, Beijing) for sharing Srsf1Fl/Fl mice, Prof. Chao Wang (China Agricultural University, Beijing) and Shuyang Yu for thoughtful discussions and suggestions, and all the members of Prof. Hua Zhang and Chao Wang laboratory for helpful discussions and comments. We thank Novogene for their assistance with the RNA-seq experiments.

Author contributions

XY, CW, HZ, and JL conceived and designed the entire project. XY, CW, and WY performed the experiments. XY, CW, WY, LS, ZL, XX, ST, and LY contributed to breeding mice. XY, CW, JL, and WY analyzed the data. XY, CW, and JL wrote the manuscript. All authors discussed the results and commented on the manuscript.

Funding

This work was supported by the National Key Research & Developmental Program of China [2021YFF1000603; 2018YFC1003701]. Funding for open access charge: China Agricultural University.

Availability of data and materials

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. The RNA-seq data be deposited in GEO(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE228975)under accession number GSE228975.

Declarations

Conflict of interest

Conflict of interest statement. None declared.

Ethics approval and consent to participate

This study was approved by China Agricultural University laboratory animal welfare and animal experimental ethical inspection (Issue No. AW11402202-3–6).

Consent for publication

Not applicable.

Footnotes

Publisher's Note

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

Xiaohong Yao, Chaofan Wang have contributed equally.

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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 file1 (DOCX 1815 KB) (1.8MB, docx)

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. The RNA-seq data be deposited in GEO(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE228975)under accession number GSE228975.

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