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Granulosa cell-specific FOXJ2 overexpression induces premature ovarian insufficiency by triggering apoptosis via mitochondrial calcium overload

Journal of Ovarian Research volume 18, Article number: 75 (2025) Cite this article

Abstract

Background

Follicle development is a complicated biological process that produces mature oocytes, and requires nutrients, growth factors, and steroids produced by ovarian granulosa cells (GCs). High fork head box J2 (FOXJ2) expression might negatively regulate ovarian function; however, the mechanism is unclear. This study aimed to investigate the effect and mechanism of FOXJ2 overexpression in GCs on regulating follicle development and fertility.

Methods

A GC-specific conditional Foxj2 knock-in mouse model (Amh-cre; Foxj2tg/tg mouse) was generated. Reproductive phenotypes were compared between Amh-cre; Foxj2tg/tg and control mice using fertility evaluation, oocyte collection, estrus cycle analysis, hormone evaluation, and ovarian follicle assessment. Then, RNA sequencing and bioinformatic analyses were used to detect the altered transcriptome of GCs collected from the Amh-cre; Foxj2tg/tg and wild-type mice. Western blotting, transmission electron microscopy, immunofluorescence staining, and flow cytometry were used to explore apoptosis and mitochondrial calcium homeostasis. Furthermore, Chromatin immunoprecipitation-PCR and dual-luciferase reporter assays were used to detect the target gene of FOXJ2. Moreover, short hairpin RNA interference was performed on primary GCs and human ovarian granulosa-like tumor (KGN) cells to explore the relationship between FOXJ2 and its target gene in apoptosis and mitochondrial calcium overload.

Results

FOXJ2 overexpression in GCs led to reduced fertility, hormonal abnormalities, and follicle atresia, starting at the initiation of sexual maturity, resulting in a premature ovarian insufficiency (POI)-like phenotype. Increased apoptosis and mitochondrial calcium overload were detected in the GCs of Amh-cre; Foxj2tg/tg mice. Mcu (encoding a mitochondrial calcium uniporter) was observed to be upregulated in the GCs of the Amh-cre; Foxj2tg/tg mice and was a direct target of FOXJ2. Moreover, Mcu knockdown restored mitochondrial calcium homeostasis and reduced the apoptosis in the GCs of the Amh-cre; Foxj2tg/tg mice and in KGN cells transfected with FOXJ2-overexpression lentivirus.

Introduction

Follicles are the fundamental structural and functional units of the ovary, comprising oocytes and somatic cells. Granulosa cells (GCs), a type of somatic cell surrounding oocytes, play vital roles in follicle development by providing essential nutrients, growth factors, and hormones to the oocytes [1]. Their rapid growth, proliferation, and differentiation are distinctive signs of follicular development. Furthermore, GC apoptosis can induce atresia in non-dominant follicles, thereby concentrating nutrition resources to support the development of dominant follicles. However, excessive follicular atresia might lead to ovarian dysfunction, such as premature ovarian insufficiency (POI) and polycystic ovary syndrome [2, 3]. Therefore, the dynamics of GC proliferation, differentiation, metabolism, and apoptosis have a critical impact on oocyte quality and follicular development [4].

Transcription factors play pivotal roles in regulating ovarian GC functions, including follicular development, steroidogenesis, apoptosis, and response to hormone signals [5, 6]. Fork head box J2 (FOXJ2), a member of the FOX transcription factor family, is widely distributed in most tissues of mammals and other vertebrates [7, 8]. FOXJ2 plays significant roles in cell proliferation, differentiation, and migration by regulating downstream genes, and participating in embryonic development [9], spermatogenesis [10], tumorigenesis, and the progression of certain cancers [11, 12]. Follicular development involves highly complicated cell proliferation, differentiation and migration, but whether and how FOXJ2-regulated signal transduction pathways play a role in this process remain largely unknown. A preliminary study explored the biological function of FOXJ2 using Foxj2 transgenic mice [13] and found that the ovaries of the female Foxj2 transgenic mice showed a high percentage of anomalies and reabsorption sites. In addition, another study revealed that the activity of FOXJ2 was related to ovarian ageing [14]. These studies suggested that FOXJ2 overexpression might negatively regulate follicle development and ovarian function; however, the regulatory mechanism is not well characterized.

Considering the pivotal regulatory role of GCs in follicle development, we questioned whether overexpression of FOXJ2 in GCs would affect follicle development and female fertility.

Herein, using an ovarian GC-specific conditional Foxj2 knock-in mouse model (Amh-cre; Foxj2tg/tg), we showed that Foxj2 overexpression in mouse ovary GCs might affect mitochondrial calcium overload by upregulating a mitochondrial calcium uniporter (MCU). This might lead to apoptosis of GCs and atresia of follicles, ultimately resulting in the POI-like phenotype. Our data provides a new insight into the role of FOXJ2 in follicle development and the significance of mitochondrial calcium homeostasis in ovarian function.

Materials and methods

Animals

All mice were housed in a controlled barrier system with a 12-hour light/dark cycle and unlimited access to food and water. Amh-cre mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Foxj2 heterozygous knock-in mice (Foxj2tg/+) were generated by Beijing Biocytogen Co., Ltd. (Beijing, China). They were maintained in the C57BL/6J background. Amh-cre; Foxj2tg/tg (cre tgtg) female mice were final transgenic models with Foxj2 overexpression in the ovarian granulosa cells. Female littermates (Foxj2tg/tg or Foxj2tg/+) were served as controls. Genotypes of mice were identified using PCR. The primers are listed in Table S1. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine.

Fertility evaluation

Adult Amh-cre; Foxj2tg/tg females or their control female littermates were mated with fertile male mice according to the ratio of one male to one female for at least 3 months. The numbers of pups per litter and the date of delivery were recorded.

Collection of mouse ovarian granulosa cells (GCs)

The follicles in the ovary were punctured with 30-gauge needles, and the oocytes were removed from the cumulus oocyte complex under a stereomicroscope. The GCs were collected, stored in liquid nitrogen, and subsequently stored at -80 °C after removing the supernatant through centrifugation.

Cell culture of primary ovarian GCs and the human ovarian granulosa-like tumor (KGN) cell line

Female mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). After 44–48 h (hours), GCs were collected from follicles by puncturing with 30-gauge needles and then digested with hyaluronidase (H3506, Sigma-Aldrich, USA) for 5 min (minutes). After being terminated digestion and centrifugation, GCs were cultured in DMEM/F12 (c11330500bt, Gibco, USA) supplemented with 1% penicillin and streptomycin (15140122, Gibco, USA) and 10% fetal bovine serum (Gibco, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. The KGN cell line (Feiya Biotechnology Co., Ltd., China) was cultured under identical conditions to those of the GCs.

Oocyte collection

Mice were injected intraperitoneally with 10 IU of PMSG for 48 h and followed by human chorionic gonadotropin (hCG) for 16 h. Oocytes from 3-week-old mice, in which endogenous hormonal interference was minimized, were collected from the ampullae and photographed for counting after the GCs were digested with hyaluronidase. Oocytes from 8-week-old mice were collected and stored at -80 °C for RT-PCR.

RNA extraction and RT-PCR

Total RNA was extracted using RNA simple Total RNA Kits (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Complementary DNA was prepared from each sample with an equal amount of RNA using PrimeScript RT Master Mix (Takara, Dalian, China). Real-time PCR was performed using TB Green Premix Ex Taq II (Takara), following the manufacturer’s protocol, on an Applied Biosystems 7500 instrument (ABI, Foster City, CA, USA). Relative quantification of the mRNA levels was calculated using the threshold cycle (CT) method 2−ΔΔCt with Actb (encoding β-actin) as the endogenous control. The primers are listed in Supplementary Table S2.

Western blotting analysis

Samples were extracted in cold radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, China) containing protease inhibitor cocktail (P1005, Beyotime, China). The proteins were collected in the supernatant after centrifugation at 13,400×g for 15 min at 4 °C. The protein lysates were resolved using 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated with antibodies overnight. After being washed three times, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The images were captured using a luminescent image analyzer (ImageQuant LAS 4000 mini, Chicago, IL, USA) with enhanced chemiluminescence kits (36208ES60, Yeasen). The antibodies employed were as follows: FOXJ2 (1:500, ab22857, Abcam), Cleaved caspase 3 (1:1000, cst9664, Cell Signaling Technology), Bcl-2 (1:1000, ab182858, Abcam), Bax (1:1000, 60267-1-Ig, Proteintech), γH2AX (1:1000, ab26350, Abcam), Mcu (1:2000, A22525, Abclonal) and β-actin (1:1000, ab8227, Abcam).

Enzyme-linked immunosorbent assay (ELISA) for hormones

Blood samples were collected from sacrificed mice. Serum was prepared and stored at − 80℃ until use. The levels of anti-Mullerian hormone (AMH), Estrogen (E2), and follicle-stimulating hormone (FSH) were measured with ELISA kits (Mlbio, China) according to the manufacturer’s instructions.

Vaginal smears of mice

Vaginal smears were taken at 10:00 am every morning for 17 days (about 3 cycles of estrus). The vagina was rinsed 3–5 times using a pipetting gun with 10ul of saline at the vaginal orifice. Subsequently, the saline containing vaginal fluid was smeared onto slides, stained with methylene blue after drying, and observed under a light microscope. Stages of the estrous cycle can be designated as proestrus (P), estrus (E), metestrus (M), and diestrus (D). The exact phase was determined by the presence, absence or proportional numbers of epithelial cells, keratinized cells, and leucocytes [15].

Assessment of ovarian follicle counts

Ovaries from mice were collected at postnatal day 1(PD1) and at 3, 4, 6, 8, 12 and 16 weeks after birth. The ovaries were fixed in 4% paraformaldehyde, dehydrated through a graded series of alcohol and xylene, embedded in paraffin, and then serially sectioned into 5-µm thick slices. These sections were stained with hematoxylin and eosin (HE). The numbers of atretic follicles and health follicles containing primordial, primary, secondary and antral follicles were counted every tenth section based on the well-accepted standards [16, 17].

RNA-seq and bioinformatic analysis

GCs from 6 weeks old mice were harvested for RNA extraction. After detection of RNA purity and integrity, complementary DNA library construction and quality inspection were performed using the above mRNA as the template. According to the effective concentration and data requirements, the library was pooled and then sequenced using the Illumina system (Illumina, San Diego, CA, USA). Then, differentially expressed genes (DEG) analysis and gene set enrichment analysis (GSEA) were carried out. The target genes of FOXJ2 were predicted by using the ARACNe algorithm [18, 19].

Transmission electron microscopy analysis

Ovaries were fixed in 2.5% glutaraldehyde phosphate buffer and trimmed to a size of 1 mm3. Images were randomly captured using a transmission electron microscope (Talos L120C TEM, Thermo Fisher Scientific, USA) at the Electron Microscopy Center of Shanghai Jiaotong University School of Medicine.

Immunohistochemistry (IHC) staining

The ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Five- micrometer-thick sections were rehydrated, antigen-retrieved, peroxidase-blocked, serum-sealed, and incubated with anti-Cleaved caspase 3 antibodies (1:1000, cst9664, Cell Signaling Technology). On the following day, the sections were incubated with goat anti-rabbit IgG antibody and followed by 3,3’-diaminobenzidine (DAB) chromogenic reaction and hematoxylin counterstaining. Images were captured by a microscope (Nikon, Tokyo, Japan). The integrated option density (IOD), area and mean density (IOD/area) value of the lHC sections were assessed using Fiji software (USA). The final mean density value of an IHC section was the average of five randomly selected fields [20].

Immunofluorescence staining

The GCs, maintained on cell slides, were fixed with 4% paraformaldehyde for 15 min at room temperature and subsequently perforated using 0.3% Triton X-100 buffer for 30 min. After being blocked in 5% BSA for 1 h, the GCs were incubated with antibodies against FOXJ2 (1:50, sc-514265, Santa Cruz Biotechnology), or MCU (1:50, A16281, ABclonal) at 4 °C overnight, followed by a 1-hour incubation at room temperature with Alexa fluor 488/594-labeled secondary antibody. After mounted on glass slides with antifade mounting medium with DAPI (36308ES20, Yeasen, China), the GCs were observed under a fluorescence microscope (Leica, Germany).

Apoptosis measurement

Cell apoptosis was measured with FITC Annexin V Apoptosis Detection Kits (556547, BD Pharmingen, USA) and Annexin V-APC/PI apoptosis kits (Dakewe Biotech Co., Ltd., China). Resuspended cells in 100 ul of binding buffer were added to a mixture containing 5 µL of FITC- or APC-conjugated Annexin V and 5 µL of propidium iodide (PI). The solutions were gently vortexed and then incubated in the dark at room temperature for 15 min. Subsequently, 400 ul of binding buffer was used to stop the reaction. The samples were analyzed using CytoFlex S (Beckman, Indianapolis, IN, USA) within 30 min.

Measurement of the Ca2+ levels in mitochondria, Endoplasmic reticulum and cytoplasm

The fluorescent dye Rhod-2AM (40776ES50, Yeasen, China) was used to measure mitochondrial Ca2+ uptake in GCs and KGN cells according to the manufacturer’s instructions. Briefly, after being washed with Hank’s balanced salt solution without Ca2+, Mg2+ and phenol red (D-HBSS) (60148ES76, Yeasen, China), cells were incubated with 4 µM Rhod-2AM and 0.1% Pluronic F-127(60318ES60, Yeasen, China) for 30 min at 37 °C. They were then washed with D-HBSS. Images were immediately captured using a fluorescence microscope (Leica, Germany) at the wavelengths of 549 nm (excitation) and 578 nm (emission). The fluorescence intensity of Rhod-2AM were also measured using CytoFlex S (Beckman) at PE channel.

When measuring the mitochondrial Ca2+ uptake capacity, the GCs, which were maintained on a confocal focusing dish (D30-20-1.5P, Cellvis, USA), were incubated with Rhod-2AM containing 0.1% Pluronic F-127 for 30 min at 37 °C. Afterward, they were washed with D-HBSS and observed using a high-speed, high-resolution, multi-point scanning living cell confocal system (Nikon). The procedure was as follows: After a baseline recording of 1 min with a 15-s interval, CaCl2 solution was added to achieve a final concentration of 20 µM, and images were then recorded for 2 min with a 5-s interval.

The fluorescent dye Rhod-5 N AM (AAT-B21070, AAT Bioquest, USA) and Cal-590 AM (MX4535-50UG, Shanghai Maokang Biotechnology Co., Ltd. China) were used to measure Ca2+ levels in the endoplasmic reticulum and cytosol, respectively, in GCs, according to the manufacturer’s instructions. Briefly, after being digested and washed with D-HBSS, the GCs were incubated with 4 µM Rhod-5 N AM or Cal-590 AM containing 0.04% Pluronic F-127 for 60 min at 37 °C and then for 30 min at room time. Following another wash with D-HBSS, the Ca2+ levels in the endoplasmic reticulum and cytosol of the GCs were immediately measured using CytoFlex S (Beckman) at PE channel, respectively.

Cell transfection and lentivirus production

The plasmid, pLX304 containing human FOXJ2 exons, and pGIPZ-MCU shRNAs for KGN cells were purchased from the DNA Library Center of Shanghai Jiaotong University School of Medicine. The pCLenti-Mcu shRNAs for mouse GCs were prepared by OBiO Technology Corp., Ltd. (Shanghai). Detailed information about these shRNAs is listed in Supplementary Table S3. The target plasmids, along with the psPAX2 and pMD2.G packaging plasmids, were transfected into 293T cells using Lipofectamine™ 3000 (L3000015, ThermoFisher, USA) according to the manufacturer’s instructions. The viral supernatant was then collected and used to infect GCs and KGN cells.

ChIP-PCR

ChIP was performed using a ChIP Assay kit (17-10086, Millipore) according to the manufacturer’s protocol. Briefly, primary GCs from the Amh-cre; Foxj2tg/tg mice were cross-linked in 1% formaldehyde for 10 min at room temperature and then neutralized with glycine. Subsequently, the cells were lysed, and the nuclear lysates were sonicated. The solicated product was immunoprecipitated with primary antibodies (anti-HA-tag, 3724 S, Cell Signaling Technology) or control IgG at 4 °C overnight. The immunocomplexes were then captured using protein A/ G beads at 4 °C for 2 h. After washing with various washing buffers, the DNA components were extracted using a standard phenol-chloroform method. The purified chromatin-immunoprecipitated DNA was used to amplify the FOXJ2 binding sites within the Mcu promoter region using primers, which are listed in Supplementary Table S4.

Dual-luciferase reporter assay

The GCs were transfected with the plasmids pGL4-basic, pGL4-Mcu (constructed for the Mcu-3444 site “TTATTTAT”) or pGL4-Mcu-Mutant (containing mutated Mcu-3444 site “CGGCCGGG”) using Lipofectamine™ 3000 (L3000015, ThermoFisher, USA) according to the manufacturer’s instructions. The mutant plasmid was generated using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used to measure the activities of firefly luciferase and Renilla luciferase in the cell lysates collected 24 h post-transfection, according to the manufacturer’s instructions. The activity of firefly luciferase was normalized to that of Renilla luciferase.

Study population

The human granulosa cells were collected from 32 patients undergoing IVF-embryo transfer at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The control group consisted of 21 patients who met the following inclusion criteria: (1) age < 40 years; (2) infertility treatment due to tubal or male factors; (3) regular menstruation; (4) sufficient ovarian reserve, defined as a bilateral ovarian antral follicle count (AFC) ≥ 5 and anti-Müllerian hormone (AMH) levels ≥ 1.2 ng/mL; and (5) basal FSH levels between 5 IU/L and 10 IU/L. Eleven patients with biochemical primary ovarian insufficiency (bPOI) met the following criteria: (1) age < 40 years; (2) regular menstruation; (3) bilateral ovarian AFC < 5; and (4) basal FSH levels ≥ 10 IU/L. Patients with hyperprolactinemia (≥ 25 ng/mL), polycystic ovary syndrome, a history of radiotherapy or chemotherapy, ovarian surgery, malignant tumors, chromosomal abnormalities, severe heart diseases, or diabetes were excluded from the study.

Human granulosa cell collection

The follicular fluid was centrifuged for 3 min at 300 g and subsequently for 7 min at 500 g. The precipitates were then incubated with 0.01% hyaluronidase for 20 min at 37 °C. After incubation, the samples were transferred into lymphocyte separation medium (Servicebio, China) in an equal volume ratio and centrifuged at 800 g for 20 min. The granulosa cells were carefully collected from the interface layer and washed with PBS. Then, red blood cells mixed in the granulosa cells were discarded with Red Cell Lysis Buffer (Solarbio, China). The granulosa cells precipitates were stored at − 80 °C.

Statistical analysis

The data are presented as the mean ± SEM and were analysed using GraphPad Prism 9 (GraphPad Inc., La Jolla, CA, USA). Comparisons between two groups were analysed using Student’s t test. For comparisons among four groups, one-way ANOVA was employed, followed by the Bonferroni test for all pairwise multiple comparison. Statistical significance was set at P < 0.05.

Results

Overexpression of Foxj2 in mouse ovarian granulosa cells induces a POI-like phenotype

To explore the role of FOXJ2 in ovaries, we generated a GC-specific conditional Foxj2 knock-in mouse model (Amh-cre; Foxj2tg/tg) using the Cre-loxP recombination system. The breeding strategy is shown in Fig. 1A. The successful generation of Amh-cre; Foxj2tg/tg mice was determined using PCR genotyping (Fig. 1B, C). Quantitative real-time reverse transcription PCR (qRT-PCR) showed the ovary-specific overexpression of Foxj2 mRNA, while the expression levels did not change significantly in other tissues or in oocytes (Fig. 1D). Higher levels of FOXJ2 protein and Foxj2 mRNA were observed in the GCs of Amh-cre; Foxj2tg/tg mice compared with those in their control littermates (Fig. 1E–I), confirming the GC-specific overexpression of Foxj2.

Fig. 1
figure 1

Conditional knock-in of Foxj2 in mouse ovary granulosa cells. A Breeding strategy for Amh-cre; Foxj2tg/tg (Cre tgtg) mice. B, C Genotypes of the mice: 1: Amh-cre; Foxj2tg/tg; 2: Foxj2tg/tg; 3, 5: Amh-cre; Foxj2tg/+; 4: Foxj2tg/+ mice. D Relative mRNA expression levels of Foxj2 in different mouse tissues between the Amh-cre; Foxj2tg/tg and the control mice quantified by qRT-PCR. Data are presented as the mean ± SEM (n = 3), normalized to Actb (β-Actin) transcript levels. *P < 0.05. E, F Representative immunohistochemical staining images of FOXJ2 in the ovaries of Amh-cre; Foxj2tg/tg and control mice (E), along with an analysis of the average optical density (F). Scale bar: 50 μm. Data are presented as the mean ± SEM (n = 4). **P < 0.01. G qRT-PCR analysis of Foxj2 mRNA levels in the isolated ovarian granulosa cells between Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 12), normalized to Actb (β-Actin) transcript levels. **P < 0.01. H, I Representative western blot images (H) with analysis of grey values (I) of FOXJ2 protein levels in the isolated ovarian granulosa cells between Amh-cre; Foxj2tg/tg and control mice. β-Actin was used as a loading control. Data are presented as the mean ± SEM (n = 4). **P < 0.01

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The ovary size and relative weight (ovary weight/body weight) of the Amh-cre; Foxj2tg/tg mice decreased significantly compared with those of their control littermates at 16 weeks after birth (Fig. 2A, B). To test whether Foxj2 overexpression affected female fertility, 2-month-old adult Amh-cre; Foxj2tg/tg females, as well as control female littermates, were mated with fertility-proven adult males (one male to one female). The result showed that the Amh-cre; Foxj2tg/tg females were almost sterile, and the interval between the first and second litters was significantly longer than that of the control litters, demonstrating that overexpression of Foxj2 in mouse ovarian GCs has a severe negative effect on female fertility (Fig. 2C, D). Next, we examined the estrous cycle, which is analogous to the human menstrual cycle. Regular estrous cycles were observed in the control female littermates, lasting for 4–6 days, whereas irregular estrous cycles with prolonged diestrus were observed in Amh-cre; Foxj2tg/tg females (Fig. 1E–H), indicating that Foxj2 overexpression in GCs resulted in a reduced frequency of ovulation and fewer pregnancies. Actually, after intraperitoneal injection of pregnant mare’s serum gonadotropin for 48 h, followed by human chorionic gonadotropin for 16 h in 3-week-old mice, the number of oocytes in the Amh-cre; Foxj2tg/tg females was significantly reduced compared with that in the control female littermates (Fig. 2I, J). In addition, increased follicle stimulating hormone levels, and decreased anti-Mullerian hormone and estrogen levels were observed in the Amh-cre; Foxj2tg/tg female’s serum compared with those in their control female littermates (Fig. 2K–M), which were similar to the hormone profile of patients with POI.

Fig. 2
figure 2

The premature ovarian insufficiency (POI)-like phenotype of Amh-cre; Foxj2tg/tg female mice. A Gross morphology of the ovaries from control and Amh-cre; Foxj2tg/tg females (Cre-tgtg). B Ratio of the ovary to body weight from control and Cre-tgtg females. Data are presented as the mean ± SEM (n = 3 or 4). *P < 0.05. C Pups per litter sired by control and Cre-tgtg females. Data are presented as the mean ± SEM (n = 6 or 10). ****P < 0.0001. D Interval days between the first and second litter from control and Cre-tgtg females. Data are presented as the mean ± SEM (n = 3 or 4). **P < 0.01. E Representative images of vaginal smears showing different stages of the estrous cycle of mice. Scale bar: 50 μm. F Typical changes observed in an estrous cycle during a 17-day period in control and Cre-tgtg females. Abbreviations: P - Proestrus, E - Estrus, M - Postestrus, D - Diestrus. G Proportion of each stage of the estrous cycle over a 17-day period in control and Cre-tgtg females. Data are presented as the mean ± SEM (n = 5). ****P < 0.0001. H The average length of the estrous cycle. Data are presented as the mean ± SEM (n = 5). ****P < 0.0001. I Representative images of MII oocytes collected from one ovary of 3-week-old control and Amh-cre; Foxj2tg/tg females. Scale bar: 100 μm. J The number of MII oocytes from each pair of ovaries of 3-week-old control and Amh-cre; Foxj2tg/tg females. Data are presented as the mean ± SEM (n = 7 or 3). ***P < 0.001. K, L and M Levels of follicle stimulating hormone (FSH), estrogen(E2), and anti-mullerian hormone (AMH) in serum, measured by ELISA, in control and Cre-tgtg females. Data are presented as the mean ± SEM (n = 15). ***P < 0.001. ****P < 0.0001

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Overexpression of Foxj2 in granulosa cells induces follicular atresia

To determine whether and when follicular development is impacted by Foxj2 overexpression, we compared follicle numbers in developing ovaries in the Amh-cre; Foxj2tg/tg mice and their control littermates. By 1 day after birth, there were only primordial follicles in the ovary, and the numbers of primordial follicles in the Amh-cre; Foxj2tg/tg mice were comparable to those in their control littermates (Fig. 3A, B), indicating the same ovarian reserve between the two kinds of mice. At 3–4 weeks old, there were no significant differences in the numbers of all levels of ovarian follicles in the Amh-cre; Foxj2tg/tg mice compared with those in their control littermates (Fig. 3C, D). At 6 weeks old, compared with those in their control littermates, the Amh-cre; Foxj2tg/tg mice showed decreased numbers of healthy follicles (i.e., the sum of primordial, primary, second, antral follicles) and increased numbers of atretic follicles (Fig. 3E, F). The phenotypes of 8-week-old and 12 to 16-week-old Amh-cre; Foxj2tg/tg mice were similar to those of the 6-weekold mice, except for decreased numbers of primordial, second, and antral follicles (Fig. 3G–J). Overall, these data showed that the numbers of atretic follicles increased in the Amh-cre; Foxj2tg/tg mice from 6 weeks after birth, suggesting that overexpression of Foxj2 in GCs might negatively affect follicular development and induce follicular atresia, possibly representing the cause of the POI-like phenotype observed in the mice.

Fig. 3
figure 3

The number of atretic follicles increases from postnatal week 6 in the Amh-cre; Foxj2tg/tg mice. A Micrographs of H&E-stained ovarian sections from control mice and Amh-cre; Foxj2tg/tg mice (Cre tgtg) at postnatal day 1 (PD1). Scale bar: 50 μm–20 μm. B Comparison of the number of primordial follicles in mice at PD1. Data are presented as the mean ± SEM (n = 4). C, E, G, I Micrographs of H&E-stained ovarian sections from control and Cre tgtg mice at postnatal weeks 3 to 4 w (3–4 w) (C), 6 w (E), 8 w (G), and 12 to16 w (12–16 w) (I). Scale bar: 100 μm–50 μm. Black pentagrams indicate the atretic follicles. D, F, H, J Comparison of the number of primordial follicles, primary follicles, secondary follicles, antral follicles, atretic follicles, and health follicles between control and Cre tgtg mice at 3–4 w (n = 3) (D), 6 w (n = 3) (F), 8 w (n = 4) (H), 12–16 w (n = 6) (J). Data are presented as the mean ± SEM. *P < 0.05. **P < 0.01. ***P < 0.001. The red, yellow, blue and green arrows indicate primordial follicles, primary follicles, secondary follicles and antral follicles, respectively. The black pentagonal stars represent atretic follicles. Health follicles are defined as the sum of primordial, primary, secondary, and antral follicles

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Increased apoptosis and mitochondrial calcium overload in the granulosa cells of the Amh-cre; Foxj2 tg/tg mice

To uncover the molecular mechanism by which Foxj2 overexpression led to increased atretic follicles, RNA sequencing (RNA-seq) of the GCs from the 6-weekold Amh-cre; Foxj2tg/tg and wild-type (WT) mice was conducted. Six-week-old mice were selected for RNA-seq due to the initial observation of an increase in the number of atretic follicles in the Amh-cre; Foxj2tg/tg mice at this age. There were 4303 differentially expressed genes (DEGs) in the GCs between the Amh-cre; Foxj2tg/tg and WT mice, including 2161 upregulated genes and 2142 downregulated genes (Fig. 4A). Gene set enrichment analysis revealed that the apoptotic signaling pathway and mitochondria calcium homeostasis pathway were hyperactive in the GCs of Amh-cre; Foxj2tg/tg mice (Fig. 4B, C). We then verified whether increased apoptosis and abnormal mitochondria calcium levels were present in the Amh-cre; Foxj2tg/tg mouse GCs.

Fig. 4
figure 4

Increased apoptosis and mitochondrial calcium overload in the GCs of 6-week-old Amh-cre; Foxj2tg/tg mice. A A Volcano diagram depicting differentially expressed genes. Red dots represent 2162 upregulated genes; green dots represent 2142 downregulated genes; blue dots represent 22,761 genes with no significant differences in expression. B, C Gene set enrichment analysis (GSEA) -based GO enrichment plots for representative gene sets: intrinsic apoptotic signaling pathway (B) and mitochondrial calcium ion homeostasis (C). D, E Transmission electron microscopy (TEM) images of ovaries from 6-week-old control mice and Amh-cre; Foxj2tg/tg mice. Abbreviations: O - oocyte; ZP - zona pellucida; M - mitochondria. Red dashed lines delineate the borders of the ZP. Red arrows indicate transport vesicles within the ZP. Yellow arrows point to the microvilli of granulosa cells (GCs) in the ZP. Scale bar: 5 μm (top panel of D), 2 μm (bottom panel of D and top panel of E), 500 nm (bottom panel of E). F, G Representative immunohistochemical staining (IHC) images (F) and statistical results (G) of Cl-caspase3 protein levels in ovaries of Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 6). **P < 0.01. H, I Representative western blot images (H) and statistical results (I) of Cl-caspase3, bcl-2, bax, γH2AX protein levels in isolated ovarian granulosa cells from Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 4). *P < 0.05, **P < 0.01. J, K, L Quantitative analysis of mean fluorescence intensity of Rhod-2AM (representing mitochondrial Ca2+ levels) (J), Rhod-5 N AM (representing of endoplasmic reticulum Ca2+ levels) (K), Cal-590 AM (representing cytoplasmic Ca2+ levels) (L) in GCs from Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 3). *P < 0.05, ****P < 0.0001

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Firstly, transmission electron microscopy was performed to observe the ultrastructure of the ovary. Loosely arranged GCs with swollen mitochondria and even apoptotic GCs, characterized by chromatin condensation and nuclear fragmentation by cleavage, were observed in the ovaries of Amh-cre; Foxj2tg/tg mice. Moreover, we found a thinner zona pellucida, fewer transportation vesicles, and decreased microvilli of GCs in the zona pellucida of the Amh-cre; Foxj2tg/tg mice, which indicated that apoptosis of GCs could impair nutrient exchange between GCs and oocytes, leading to increased follicle atresia (Fig. 4D, E). Meanwhile, compared with that in their control littermates, a stronger signal of cleaved-caspase3-positive GCs was observed in the Amh-cre; Foxj2tg/tg mice (Fig. 4F, G). Next, we detect certain specific apoptotic markers using GCs collected from ovaries. Western blotting analysis showed that the levels of cleavedcaspase3 and γH2AX (H2A.X variant histone) were increased, and the ratio of Bcl-2 (B-Cell CLL/lymphoma 2) to Bax (BCL2 associated X protein) was decreased in the GCs of the Amh-cre; Foxj2tg/tg mice compared with those of their control littermates (Fig. 4H, I). Taken together, these results showed that overexpression of Foxj2 in GCs triggered apoptosis in the GCs of Amh-cre; Foxj2tg/tg mice, which might induce follicular atresia in the mice (Fig. 3).

Then, we detected mitochondria calcium levels (Ca2+[mt]) using Rhod-2 Acetoxymethyl ester (Rhod 2AM) staining in primary cultured GCs. The results showed that the levels of Ca2+[mt] in the GCs of Amh-cre; Foxj2tg/tg mice were upregulated to a higher level than those in the control GCs (Fig. 4J). In addition, to explore where the overload of Ca2+ in the mitochondria came from, we examined the endoplasmic reticulum (ER) Ca2+ levels using Rhod-5 N AM and the cytosolic Ca2+ levels using Cal-590 AM. As shown in Fig. 4K and L, the mean fluorescence intensity of Rhod-5 N AM was remarkably reduced and the mean fluorescence intensity of Cal-590 AM did not change in the Amh-cre; Foxj2tg/tg GCs compared with those in the control GCs, indicating the overload of Ca2+[mt] might have derived from the ER.

Mcu is upregulated in the GCs of the Amh-cre; Foxj2 tg/tg mice and is a target of FOXJ2

FOXJ2 is a transcription factor; therefore, we explored the potential target genes regulated by FOXJ2 in GCs. Firstly, 1744 predicted target genes of FOXJ2 were obtained through bioinformatic analyses, and 740 of the 1744 predicted target genes overlapped with the DEGs between the Amh-cre; Foxj2tg/tg and WT mouse GCs (Fig. 5A). Three out of the 740 overlapped genes were associated with mitochondria calcium homeostasis: Mcu (encoding mitochondrial calcium uniporter), Bcap31 (encoding B cell receptor associated protein 31), and Pdzd8 (encoding PDZ domain containing 8). We then detected the expression levels of the subunits of the MCU complex using qRTPCR. Compared with those in the GCs of the control littermates, the mRNA expression levels of Mcu and Smdt1, which correlate positively with Ca2+[mt] uptake, were significantly upregulated, while the mRNA expression levels of Mcur1, Micu1, Micu2, Micu3, and Mcub did not change significantly in the GCs of the Amh-cre; Foxj2tg/tg mice (Fig. 5B), which was consistent with the results of the RNA-seq data. Considering that Mcu is one of the predicted target genes of FOXJ2, we hypothesized that FOXJ2 overexpression might induce mitochondria calcium overload by targeting Mcu, which required further testing.

Fig. 5
figure 5

MCU is upregulated in GCs of Amh-cre; Foxj2tg/tg mice and is a target of FOXJ2. A A Venn diagram illustrating differentially expressed genes (DEGs) (purple) and the predicted target genes of FOXJ2 (yellow) using the ARACNe algorithm. B Validation of DEGs related to the mitochondrial calcium uniporter complex using qRT-PCR assay. Data are presented as the mean ± SEM (n = 4). *P < 0.05; ***P < 0.001. C Expression and co-localization of MCU and FOXJ2 in GCs using immunofluorescent staining (IF). Scale bar: 50 μm. D, E The binding sites of FOXJ2 to Mcu promoter region in GCs from Amh-cre; Foxj2tg/tg mice analyzed by ChIP-PCR using an anti-HA-tag antibody. Data are presented as the mean ± SEM (n = 3). **P < 0.01. F Verification of FOXJ2 binding to the Mcu promoter using a Dual-luciferase reporter assay. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01

Full size image

Firstly, we showed that MCU and FOXJ2 were both located in GCs using immunofluorescent staining (Fig. 5C). In addition, the expression levels of Mcu mRNA and MCU protein were upregulated in the Amh-cre; Foxj2tg/tg mouse GCs (Supplementary Fig. S1 B, C). To further investigate whether Mcu is a target gene of FOXJ2 in vivo, we examined the binding of FOXJ2 to the upstream regulatory region of Mcu (transcription start site to approximately − 5000 bp) in GCs using chromatin immunoprecipitation-PCR (ChIP-PCR). The results showed that in the Amh-cre; Foxj2tg/tg mouse GCs, the fold enrichment of FOXJ2 binding to the Mcu upstream regulatory region, amplified using the Mcu − 3444 primers, was significantly higher than that of the negative control (Fig. 5D, E), suggesting that FOXJ2 might bind to the Mcu − 3444 site. Next, we identified that FOXJ2 directly binds to the Mcu − 3444 site using a dual-luciferase reporter assay (Fig. 5F).

Overexpression of Foxj2 induces mitochondrial calcium overload and apoptosis by targeting MCU in the Amh-cre; Foxj2 tg/tg mouse GCs

Previously, we confirmed that Foxj2 overexpression triggered GC apoptosis and mitochondrial calcium overload, and Mcu is a target of FOXJ2. Next, we wanted to determine whether mitochondrial calcium overload and apoptosis induced by Foxj2 overexpression could be rescued if the expression of Mcu was knocked down.

Firstly, we established short hairpin RNAs (shRNAs) targeting Mcu (pCLentiMcu shRNAs) in mouse GCs, and their interference efficiency was confirmed (Supplementary Fig. S1 A). The expression levels of Mcu mRNA and MCU protein in the GCs of the Amh-cre; Foxj2tg/tg mice were both reduced by the pCLentiMcu shRNAs, indicating their successful construction (Supplementary Fig. S1 B, C). Then, we examined Ca2+[mt] signals by staining with Rhod-2AM in GCs. The results showed that the fluorescent intensity in GCs of the Amh-cre; Foxj2tg/tg mice was significantly increased compared with that of the control GCs, indicating Ca2+[mt] overload in the GCs of the Amh-cre; Foxj2tg/tg mice, which was consistent with our previous result (Fig. 4M). After treatment of Amh-cre; Foxj2tg/tg mouse GCs with pCLenti-Mcu shRNAs, the levels of Ca2+[mt] decreased, reaching a level similar to that of the control GCs (Fig. 6A, B). To further measure the Ca2+[mt] uptake capacity, Rhod-2AM fluorescence intensity in GCs was captured before and after adding CaCl2 to the culture medium. The result showed that after the addition of CaCl2, the Ca2+[mt] levels were obviously elevated compared with the baseline in the GCs of the Amh-cre; Foxj2tg/tg mice, while slight changes in the magnitude of the rise were observed in the Amh-cre; Foxj2tg/tg mouse GCs treated with pCLenti-Mcu shRNAs, which was comparable to those in the control GCs (Fig. 6D). Representative images captured during the imaging recording process are shown in Fig. 6C. The above results demonstrated that the mitochondrial calcium overload in GCs induced by Foxj2 overexpression could be rescued by knocking down Mcu.

Fig. 6
figure 6

Targeting MCU rescues FOXJ2 overexpressioninduced mitochondrial calcium overload and apoptosis in GCs. A A representative image of Rhod-2AM loaded GCs in each group. Scale bar: 50 μm. B Quantitative analysis of the mean fluorescence intensity of Rhod-2AM (indicating mitochondrial Ca2+ levels) in GCs from each group. Data are presented as the mean ± SEM (n = 44–55). ****P < 0.0001. C Representative images of Rhod-2AM loaded GCs in each group at the initial recording time, 5 s, and 2 min after exogenous Ca2+ loading. Scale bar: 5 μm. D Time-course records of Rhod-2AM fluorescence intensity in GCs from each group. Data are presented as the mean ± SEM (n = 4). E, F Representative flow cytometry images (E) and quantitative analysis (F) of apoptosis levels in GCs from each group. Data are presented as the mean ± SEM (n = 4). * P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001

Full size image

Furthermore, we investigated apoptosis in GCs using flow cytometry. The result showed that the early apoptosis rate (Annexin V positive and propidium iodide (PI) negative), the late apoptosis rate (Annexin V positive and PI positive), and the total apoptosis rate (the sum of the early and late apoptosis rates) were increased in the GCs of Amh-cre; Foxj2tg/tg mice compared with those in their control littermates. After treatment of Amh-cre; Foxj2tg/tg mouse GCs with pCLenti-Mcu shRNAs, the elevated apoptosis rates were decreased, reaching those of the control GCs (Fig. 6E, F). In addition, treatment of Amh-cre; Foxj2tg/tg mouse GC cells with an inhibitor of Ca2+[mt] uptake, Ru360, also decreased the apoptosis rates in the GCs of the Amh-cre; Foxj2tg/tg mice (Fig. 6E, F).

Taken together, the above results demonstrated that overexpression of Foxj2 induced mitochondrial calcium overload and apoptosis by targeting MCU in mouse GCs.

Inhibiting mitochondrial calcium overload by targeting MCU rescues FOXJ2 overexpression-induced apoptosis in human KGN cells

To verify whether the above results observed in mouse GCs could be replicated in human GCs, we examined the levels of Ca2+[mt] and apoptosis in the human ovarian granulosa-like tumor (KGN) cell line. Firstly, we generated a stable KGN cell line overexpressing FOXJ2 (Lentivirus-FOXJ2) (Supplementary Fig. S1 D, E). Next, we transfected KGN cells with pGIPZ-MCU shRNA plasmids. GFP fluorescence and qRT-PCR assays demonstrated highly efficient transfection and obvious MCU knockdown by pGIPZ-MCU shRNAs (Supplementary Fig. S1 F, G). Then, we demonstrated that the MCU mRNA and protein levels in the KGN cell line stably overexpressing FOXJ2 were decreased after the addition of pGIPZ-MCU shRNAs, indicating successful construction of the pGIPZ-MCU shRNAs (Supplementary Fig. S1 H, I).

We then detected Ca2+[mt] levels and apoptosis in the KGN cells stably overexpressing FOXJ2 (Lentivirus-FOXJ2-KGN). Compared with the control KGN cells, increased mean fluorescent intensity of Rhod-2AM in the Lentivirus-FOXJ2-KGN cells was observed, suggesting that overexpression of FOXJ2 induced a higher Ca2+[mt] level in the KGN cells. Following treatment with pGIPZ-MCU shRNAs in the Lentivirus-FOXJ2-KGN cells, the levels of Ca2+[mt] were decreased to a degree comparable to those of control KGN cells (Fig. 7A).

Fig. 7
figure 7

Targeting MCU rescues FOXJ2 overexpression-induced mitochondrial calcium overload and apoptosis in KGN cells. A Quantitative analysis of the mean fluorescence intensity of Rhod-2AM (indicating mitochondria Ca2+ levels) in KGN cells of each group using flow cytometry. Data are presented as the mean ± SEM (n = 3). ***P < 0.001. ****P < 0.0001. B, C Representative flow cytometry images (B) and corresponding quantitative analysis (C) of apoptosis levels in KGN cells from each group. Data are presented as the mean ± SEM (n = 3). ***P < 0.001. ****P < 0.0001. D, E Representative flow cytometry images (D) and quantitative analysis (E) of apoptosis levels in KGN cells of each group. Data are presented as the mean ± SEM (n = 3). * P < 0.05. ***P < 0.001. ****P < 0.0001

Full size image

Using flow cytometry, we found that the early, late, and total apoptosis rates were markedly enhanced in the Lentivirus-FOXJ2-KGN cells compared with those in the control KGN cells, and the elevated apoptosis rates could be alleviated by transfection of pGIPZ-MCU plasmids (Fig. 7B, C). In addition, application of Ru360 also reduced the apoptosis rates in the Lentivirus-FOXJ2-KGN cells (Fig. 7D, E).

Consistently, these results demonstrated that overexpression of FOXJ2 induced Ca2+[mt] overload and apoptosis by targeting MCU in human KGN cells.

To verify whether the above conclusion holds for patients with POI, we collected primary human GCs from patients with biochemical POI (bPOI) to measure the expression levels of FOXJ2 and MCU mRNA. The results showed that the levels of FOXJ2 and MCU mRNA in the GCs of patients with bPOI were upregulated compared with those in GCs collected from the control patients (Supplementary Fig. S2 A, B).

Discussion

In the present study, we showed that overexpression of Foxj2 in ovarian GCs triggered apoptosis and mitochondrial calcium overload, leading to follicle atresia, and finally to a POI-like phenotype in the Amh-cre; Foxj2tg/tg mice, characterized by severely decreased fertility, disordered estrous cycle, reduced ovulation, and hormonal abnormalities. Knocking down Mcu (a verified target of FOXJ2), or treatment with an inhibitor of Ca2+[mt] uptake (Ru360), restored mitochondrial calcium homeostasis and reduced apoptosis in the GCs of Amh-cre; Foxj2tg/tg mice and in human KGN cells stably overexpressing FOXJ2.

Apoptosis promotes proper development and eliminates damaged cells in healthy condition [21], whereas uncontrolled and excessive apoptosis in ovarian GCs could induce atresia of follicles, contributing to POI and ovarian aging [22,23,24]. Apoptosis of GCs functions as an initiator of follicle atresia, at an earlier stage than oocyte apoptosis [25]. In this study, we found that overexpression of Foxj2 in GCs induced their apoptosis, leading to increased follicle atresia and finally, a POI-like phenotype. Previous studies have also reported that upregulation of FOXJ2 induced apoptosis of lung cancer cells [26, 27]. In addition, Li et al. reported that the circ_0006459/miR-940/FOXJ2 axis was responsible for the cell inflammation and apoptosis after ischemic stroke [28]. The above studies indicated that overexpression of FOXJ2 can lead to apoptosis of different cells via different mechanisms.

The mechanisms of GC apoptosis leading to POI involve reactive oxygen species overload and oxidative stress [29], mitochondrial dysfunction [30, 31], ER Ca2+ overloaded, and ER stress [23], as well as intracellular Ca2+ disturbance [22]. In this study, we showed that Ca2+[mt] uptake was elevated in the GCs of the Amh-cre; Foxj2tg/tg mice. Moreover, inhibiting Ca2+[mt] overload using shRNAs against Mcu or Ru360 could rescue FOXJ2 overexpression-induced apoptosis in mouse GCs and human KGN cells. These results demonstrated that FOXJ2-induced Ca2+[mt] overload resulted in apoptosis. In addition, Ru360 alleviates apoptosis by inhibiting Ca2+[mt] overload in vitro, indicating that it might be a potential treatment for POI, which should be further confirmed using an in vivo study.

Ca2+[mt] plays a variety of roles in physiological and pathological processes. On the one hand, it can promote the efficiency of the tricarboxylic acid cycle and electron transfer chain [32]. It also can regulate metabolism by adjusting the activity of glucose transporters [33]. On the other hand, excessive Ca2+ in the mitochondrial matrix is termed Ca2+[mt] overload, which can induce the excessive opening of the mitochondrial permeability pore and the overproduction of reactive oxygen species, resulting in cell death [34, 35]. Ca2+[mt] overload has been demonstrated to be associated with cell apoptosis in many diseases [36], including Alzheimer’s disease, Parkinson’s disease, stroke, and myocardial infarction; however, its association with female reproductive diseases has rarely been reported. Only one study declared that pauerarin, a Chinese traditional medicine, ameliorated the secretory function and apoptosis of ovarian GCs in polycystic ovarian syndrome mice by preventing cytosolic Ca2+ influx into mitochondria [37]. In this study, we showed that Ca2+[mt] uptake was elevated in the GCs of the Amh-cre; Foxj2tg/tg mice by targeting MCU. The MCU complex is the most important channel for Ca2+ intake by mitochondria, and is composed of MCU, EMRE (essential MCU regulator, also known as single-pass membrane protein with aspartate rich tail 1, encoded by Smdt1), MCUR1 (mitochondrial calcium uniporter regulator 1), MICU1 (mitochondrial calcium uptake 1), MICU2 (mitochondrial calcium uptake 2), MICU3 (mitochondrial calcium uptake 3), and MCUb (mitochondrial calcium uniporter dominant negative subunit beta) [38]. In this study, we found that only Mcu and Smdt1, which correlate positively with Ca2+[mt] uptake, were significantly upregulated in the GCs of the Amh-cre; Foxj2tg/tg mice. In addition, we found that FOXJ2 directly bound to the upstream regulatory region of Mcu at the −3444 bp site. How can such a distant region regulate Mcu transcription? We speculate that the region at −3444 bp likely functions as a FOXJ2−dependant enhancer. When FOXJ2 binds to this site, it might loop the chromatin to contact with the promoter of Mcu to active its expression, or it might recruit coactivators to active Mcu expression, which requires further investigation.

The ER is the most important storage organelle for intracellular Ca2+ [39]. Ca2+ is released from ER into the cytosol via the ryanodine receptor (RYR) and inositol 1,4,5-trisphosphate receptor (IP3R) channels, then into mitochondria via the MCU complex [40]. Ca2+ from the ER can also be directly transferred into mitochondria via mitochondria-associated ER membranes (MAMs), which is a dynamic structure containing thousands of proteins, playing a significant role in the regulation of Ca2+ homeostasis and the signal transduction between the ER and the mitochondria [39]. In this study, we preliminary explored the source of Ca2+[mt]. The flow cytometry results, showing decreased ER Ca2+ levels with unchanged cytosolic Ca2+ levels in the GCs of Amh-cre; Foxj2tg/tg mice, suggested that Ca2+[mt] might come straight from the ER rather than the cytosol, which suggested a possible relationship between MAMs and follicle development. Further analysis targeting MAMs in the GCs of Amh-cre; Foxj2tg/tg mice is required to provide innovative ideas for the treatment of POI.

Currently, POI is categorized into three clinical phases - occult, biochemical, and overt (previously termed premature ovarian failure) - based on disease progression [41, 42]. Patients with occult POI have regular menstruation and normal levels of FSH, although they manifest as reduced fertility. Biochemical POI (bPOI) is characterized by fluctuating basal FSH (10–40 IU/L) while retaining spontaneous menstruation. Overt POI presents amenorrhea and is accompanied by persistently elevated FSH levels exceeding 40 IU/L. In this study, we found that FOXJ2 and MCU mRNA levels were upregulated in GCs collected from patients with bPOI. The results indicated that FOXJ2 overexpression-induced Ca2+[mt] overload by targeting MCU might be associated with bPOI, which requires further investigation.

Conclusions

In summary, overexpression of Foxj2 in GCs aggravated apoptosis via MCU-mediated mitochondrial calcium overload, leading to follicle atresia, ultimately resulting in a POI-like phenotype. Our findings provide a new insight into the role of FOXJ2 in follicle development and the significance of mitochondrial calcium homeostasis in ovarian function.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

GCs:

Granulosa cells

FOXJ2:

Fork head box J2

POI:

Premature ovarian insufficiency

MCU:

Mitochondrial calcium uniporter

KGN:

Human ovarian granulosa-like tumor

RNA-seq:

RNA sequencing

WT:

Wild−type

DEGs:

Differentially expressed genes

ER:

Endoplasmic reticulum

ChIP-PCR:

Chromatin immunoprecipitation-PCR

shRNAs:

Short hairpin RNAs

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Acknowledgements

Thanks to the Core Facility of Basic Medical Sciences at Shanghai Jiao Tong University School of Medicine for processing the samples and analysing the TEM images.

Funding

This work was supported by Shanghai Municipal Health Commission (grant No. 202340286 to J.W.), by National Natural Science Foundation of China (grant No. 82293661, 82473455 to Y.T) and National Science Foundation of Shanghai (grant No. 24ZR1441700 to Y.T).

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Author notes

  1. Yunxia Zhang and Qiqian Wu contributed equally to this work.

Authors and Affiliations

  1. Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Building 5, Room 506 280 South Chongqing Road, Huangpu District, Shanghai, 200025, China

    Yunxia Zhang, Qiqian Wu, Yanqin Hu, Yujie Tang & Jingwen Wu

  2. Shanghai Key Laboratory of Reproductive Medicine, Shanghai, 200025, China

    Furong Bai

  3. Department of Andrology, Center for Men’s Health, Department of ART, Institute of Urology, Shanghai General Hospital, Urologic Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China

    Bufang Xu

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  1. Yunxia Zhang
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Contributions

J.W., Y.T. and B.X. conceived and designed the research. Y.Z., Q.W., F.B. and Y.H. performed the bench experiments. Y.Z., Q.W and J.W. analyzed the data. Y.Z. and J.W. wrote the manuscript. All authors reviewed and agreed with the contents of the manuscript.

Corresponding authors

Correspondence to Bufang Xu, Yujie Tang or Jingwen Wu.

Ethics declarations

Ethics approval and consent to participate

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine (ethics approval number: A-2022-076). The procedure of human primary granulosa cell collection was approved by the ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (ethics approval number: 2020104a) and written informed consent was obtained from all participants.

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Not applicable.

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The authors declare no competing interests.

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Zhang, Y., Wu, Q., Bai, F. et al. Granulosa cell-specific FOXJ2 overexpression induces premature ovarian insufficiency by triggering apoptosis via mitochondrial calcium overload. J Ovarian Res 18, 75 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13048-025-01651-0

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Journal of Ovarian Research

ISSN: 1757-2215

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