Expression and function analysis of Rspo1 in oocyte primary growth of Qi River crucian carp (Carassius auratus)

Abstract

RSPO1, also referred to R-spondin1, plays a crucial role in female sex determination and differentiation in both mammals and teleosts. Nevertheless, limited documentation exists regarding its contribution to the subsequent ovarian development in teleosts. In this research, Qi River crucian carp (Carassius auratus) was employed as a model organism to investigate the function of Rspo1 during the initial stages of ovarian growth. To clarify the expression patterns, a polyclonal antibody targeting Rspo1 was generated. According to Western blot analysis, Rspo1 was able to identify a band that matched the size of Rspo1 in adult Qi River crucian carp. Furthermore, during the development of the ovary, the expression of Rspo1 peaked at 90 days after hatching (dah), a critical period for the ovary development in Qi River crucian carp, then decreased. Immunohistochemical (IHC) analysis revealed the widespread presence of Rspo1 in oogonia, stage I and II oocytes, as well as certain stage III oocytes, stromal cells, and granulosa cells throughout the ovary. To further confirm the essential roles of Rspo1 in early ovary development, we used dsRNA to suppress its expression for a duration of 30 days starting at 90 dah. Following this, there was a significant reduction in the number of oocytes. Additionally, the expression of genes associated with female development, such as β-catenin, foxl2, cyp19a1a, and figlα, exhibited a noticeable decline. Conversely, the expression of apoptosis-promoting factors caspase3 and p53 increased significantly. The gene ki-67, serving as a marker for cell proliferation, experienced a decrease. These findings suggest that Rspo1 may exert influence on ovarian development through the regulation of germ cell proliferation. The inhibition of Rspo1 expression during the crucial phase of ovarian growth in Qi River crucian carp is anticipated to induce somatic cell apoptosis and perturb the structural integrity of the ovary. Our investigation offers significant elucidation on the molecular mechanisms governing the involvement of Rspo1 in the initial stages of ovarian development in teleosts.