(304) CCAAT/enhancer-binding proteins alpha and beta downstream of follicle-stimulating hormone mediate ovarian folliculogenesis and vascularization

CCAAT/enhancer-binding proteins alpha and beta downstream of follicle-stimulating hormone mediate ovarian folliculogenesis and vascularization
Hanxue Hannah Zhang¹; Yi Athena Ren¹
1. Department of Animal Science, Cornell University, Ithaca, NY, 14853, USA.



Abstract Text:

CCAAT-enhancer binding proteins alpha (C/EBPa) and beta (C/EBPb) are transcription factors that play essential roles in regulating ovarian function. Previous studies have shown that the preovulatory luteinizing hormone (LH) surge induces the expression of Cebpa and Cebpb in ovarian granulosa cells via the cAMP/protein kinase A (PKA)/ERK1/2 signaling cascade. A conditional knockout mouse model (Cebpa/b gc-/-) in which the expression of Cre recombinase is driven by the Cyp19a1 promoter to induce gene deletion specifically in ovarian granulosa cells from the early antral follicle stage exhibited blocked ovulation, impaired granulosa cell luteinization, and abnormal corpus luteum vascularization, highlighting the importance of Cebpa and Cebpb in the preovulatory ovary. However, whether C/EBPα and C/EBPβ regulate earlier stages of folliculogenesis remains unclear.

Follicle-stimulating hormone (FSH) is crucial for follicle development, particularly in the transition from preantral to antral follicle stages. Multiple lines of evidence support that FSH regulates Cebpa and Cebpb in other tissues and Cebpb in cultured granulosa cells and follicles. However, the precise mechanism by which FSH regulates Cebpa and Cebpb in the ovary remains unclear, and the biological processes controlled by these transcription factors downstream of FSH have not been defined. To investigate whether and through which signaling pathways FSH regulates Cebpa and Cebpb during folliculogenesis, we performed RT-qPCR on granulosa cells collected from pregnant mare serum gonadotropin (PMSG)-stimulated mice (PMSG was used to mimic the effects of FSH in vivo) and from in vitro culture with FSH treatment. PMSG stimulation in vivo had minimal effect on Cebpa whereas Cebpb was induced in granulosa cells within 1 h post-PMSG injection. Treatment of FSH in vitro transiently (in 0.5-1 h) induced Cebpa and Cebpbexpression in a dose- and time-dependent manner. Furthermore, treatment of in vitro cultured granulosa cells with inhibitors targeting intracellular signaling pathways downstream of FSH revealed that FSH-induced Cebpa expression is mediated via PKA and ERK1/2 signaling, whereas Cebpb expression is regulated specifically through ERK1/2 but not PKA.

To determine the biological processes regulated by C/EBPa and C/EBPb downstream of FSH during different stages of follicle development, we conducted bulk RNA sequencing of granulosa cells from Cebpa/b gc+/+ and Cebpa/b gc-/- mice at the immature stage (day 21; no PMSG) and 48 h post-PMSG injection. Deletion of Cebpa and Cebpbresulted in profound transcriptional dysregulation at both time points, predominantly affecting genes involved in steroidogenesis and vasculature function - key processes regulated by FSH during folliculogenesis.

As a functional validation of the RNA sequencing data, we compared ovarian vasculature and follicle growth between controls and mutants. To this end, we collected ovaries from Cebpa/b gc+/+ and Cebpa/b gc-/- mice at 0 h post-PMSG with vascular structures labeled by Alexa 649-conjugated lectin, and performed whole-mount imaging and quantification. Our analyses revealed that Cebpa/b gc-/- mutants exhibited reduced ovarian vascular volume. Additionally, these mutants displayed a reduced number of antral follicles and a reduction in the number of small antral follicles (less than 200mm). These observations suggest defective preantral to early antral transition and potentially delayed follicular growth in the absence of C/EBPa and C/EBPb.

In conclusion, our study provides evidence that C/EBPα and C/EBPβ mediate the effects of FSH on folliculogenesis, in particular in the preantral to antral follicle transition and ovarian vascularization. These insights could contribute to the development of targeted therapies for infertility related to follicular dysfunction.