The regulatory mechanism of circadian clock gene <i>Cry</i> on estrogen secretion in Bactrian camels

The Bactrian camel is an essential livestock species in arid desert regions, but its unique reproductive pattern results in low fertility. Its estrus period primarily occurs during winter to adapt to harsh climatic conditions; however, the specific mechanisms underlying its seasonal reproduction remain unclear. In this study, the expression and distribution of circadian clock protein cryptochrome 1 (Cry1), melatonin receptors (MT1/MT2) and estrogen receptor alpha (ERα) in the ovary and uterus of the Bactrian camel were investigated. Using cultured ovarian granulosa cells, the cells treated with melatonin (Mel) or overexpressed Cry to assess the expression of estradiol (E2) and genes related to the MEK/ERK-ERα signaling pathway, aiming to elucidate the regulatory roles of Cry and Mel in reproduction. The results showed that these proteins were widely expressed in the corpus luteum, granulosa cell layer, follicular fluid, and uterine endometrial epithelial and stromal cells, indicating their involvement in estrogen secretion and uterine function. In vitro experiments demonstrated that Mel treatment or Cry overexpression enhanced E2 secretion, reduced cAMP levels, upregulated Raf, and activated the MEK/ERK-ERα pathway. Moreover, Mel and MT increased Cry expression, while Cry overexpression also upregulated MT. Cry1-induced activation of the MEK/ERK-ERα pathway was dependent on MT1/MT2, whereas Cry2-mediated activation was independent of MT1/MT2. Dual-luciferase reporter assays revealed that Cry regulates MT1 transcriptionally. This study provides foundational insight into the regulation of seasonal reproduction in Bactrian camels.