Establishment of an in vitro culture model of theca cells from hierarchical follicles in ducks

Abstract

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro Theca interna cells experienced the logarithmic phase on d1-d2, the plateau phase on d2-d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1-d3, the plateau phase on d3-d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Keywords: characteristics; culture model; duck; theca cell.

Figure 1. Morphological characteristics of theca cells

Morphological characteristics of duck theca cells in vitro as seen under a microscope (×200). (A) Theca cells in vitro cultured at d2. (B) Theca cells in vitro cultured at d4. The theca interna cells are marked by white arrows, and the theca externa cells are marked by black arrows.

Figure 2. Morphological characteristics and growth curve of theca cells

Morphological characteristics and growth curve of duck theca cells in vitro as seen under a microscope (×100). (A–G) Morphology of duck theca cells on d1-d7 in vitro. (H) Growth curve of duck theca cells that were cultured in vitro (P<0.05).

Figure 3. Expression profiles of CYP17A1/19A1 mRNA during theca cell culture

Relative mRNA expression of the CYP17A1 and CYP19A1 genes in duck theca cells on d1–d7. The letters at the top of each bar represents the significant differences between the gene expression in various stages (P<0.05).

Figure 4. immunofluorescence identification by CYP17A1/19A1 and FSHR antibodies

Fluorescent image of theca cells and granulosa cells marked by their specific protein antibodies. (A and B) Control and treatment groups stained with CYP17A1 antibody to mark theca cells. (C and D) Control and treatment groups stained with CYP19A1 antibody to mark theca cells. (E) Treatment group stained with FSHR antibody to mark theca cells. (F and G) Control and treatment group stained with FSHR antibody to mark granulosa cells.