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

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; Foxj2^tg/tg mouse) was generated. Reproductive phenotypes were compared between Amh-cre; Foxj2^tg/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; Foxj2^tg/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; Foxj2^tg/tg mice. Mcu (encoding a mitochondrial calcium uniporter) was observed to be upregulated in the GCs of the Amh-cre; Foxj2^tg/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; Foxj2^tg/tg mice and in KGN cells transfected with FOXJ2-overexpression lentivirus.