CACNA2D1 promotes ovulation by regulating ovarian immune cell infiltration

In the previous single-cell transcriptome analysis of goat ovulation, we identified that CACNA2D1 may play a critical role in immune cell infiltration during the ovulation process. Therefore, this study aimed to investigate the function of CACNA2D1 in ovulation.

CACNA2D1-associated signaling was pharmacologically inhibited in a mouse ovulation model to evaluate ovarian immune cell infiltration, ovulatory output, and transcriptomic changes. In vitro, CACNA2D1 knockdown and overexpression were performed in ovarian granulosa cells to assess chemokine production, intracellular calcium signaling, CaMKII activation, and cell proliferation. Granulosa cell-mediated macrophage recruitment was functionally examined using a RAW264.7 transwell migration assay.

CACNA2D1 showed increased expression during the periovulatory period, and pharmacological inhibition of CACNA2D1-associated signaling in mice significantly reduced ovarian immune cell infiltration, particularly macrophages, accompanied by decreased ovulatory output. Ovarian transcriptomic analysis after inhibition revealed broad alterations in genes related to immune regulation, signal transduction, and cellular functional states. In granulosa cells, CACNA2D1 knockdown significantly reduced intracellular Ca²⁺ levels, decreased CaMKII phosphorylation, and suppressed the expression and secretion of immune recruitment-related factors, including IL6, CCL2, and CXCL12. Restoration of calcium signaling with A-23,187 rescued the reduced expression of these chemokines. Functionally, CACNA2D1 knockdown impaired granulosa cell-mediated RAW264.7 macrophage migration in a transwell co-culture system. CACNA2D1 also influenced granulosa cell proliferative activity, whereas its effect on macrophage-related responses appeared to be more closely associated with chemotactic signaling than with macrophage proliferation.

This study identifies CACNA2D1 as a periovulatory granulosa cell-associated regulator involved in calcium-dependent chemokine production and macrophage recruitment. These findings provide new insight into ovarian immune remodeling during ovulation and offer a potential molecular basis for future studies of follicular development, ovulation efficiency, and fertility regulation in livestock species.