Estrogen Receptor Beta Isoforms Interplay Integrates Nuclear and Extra‐Nuclear Signaling to Modulate Human Granulosa Cell Growth

ABSTRACT Estradiol (E2) regulates ovarian follicular development and granulosa cell (GC) function primarily through estrogen receptor β (ERβ). Human GCs express several ERβ isoforms, including ERβ1, ERβ2, ERβ4, and ERβ5, but how their relative abundance modulates estrogen signaling remains unclear. Here, we investigated the contribution of nuclear and extra‐nuclear ERβ signaling to human GC growth and function. HGrC1 granulosa cells lacking endogenous estrogen receptors were engineered to stably express individual or combined ERβ isoforms. Functional analyses showed that ERβ1 and ERβ4 inhibited GC proliferation, whereas ERβ2 and ERβ5 promoted cell growth. Co‐expression of all isoforms resulted in an overall inhibitory effect, indicating that GC responses depend on ERβ isoform balance rather than a single receptor. Reporter assays demonstrated that only ERβ1 activated estrogen response element‐dependent transcription, while the other isoforms suppressed E2‐dependent ERα transactivation. In contrast, ERβ2, ERβ4, and ERβ5 activated AP‐1 signaling in a ligand‐independent manner and attenuated ERβ1‐mediated repression, identifying AP‐1 as a central node of ERβ isoform crosstalk. RNA sequencing following selective ERβ1 depletion in primary human granulosa lutein cells revealed that ERβ1 is required for E2 responsiveness and regulates genes involved in membrane potential, ion transport, and cytoskeletal organization. Proteomic and biochemical analyses further identified isoform‐specific signaling networks involving ERK1/2, Akt, and CaMKII pathways. Together, these findings demonstrate that ERβ isoform balance orchestrates estrogen signaling through coordinated nuclear and extra‐nuclear mechanisms controlling GC growth and function.