Changes in Specific Granulosa Cell mRNA Abundance During Early Avian Follicular Development.

Abstract

Daily ovulations in poultry require nearly continuous follicular development, selection and maturation. Granulosa cell differentiation involves dramatic upregulation of transcripts involved in progesterone production, including STAR, HSD3B and CYP11A1. Other transcripts that are critical regulators of mammalian preantral granulosa cells are also expressed in avian granulosa cells. These include members of the TGF-beta super-family (BMP2, BMP4, BMP6 and AMH), several transcription factors (FOXO3, FOXL2, GATA4, SMAD9 and WT1), and other secreted products (PCSK6 and WNT4). However, the regulation of these transcripts during early avian follicular development is unknown. Based on this information, we hypothesize that levels of transcripts involved in steroidogenesis will increase during development of small avian follicles (0.5-4 mm in diameter) and that other transcripts will be useful markers to track differentiation of avian granulosa cells in vivo and in vitro. In our first study, we collected 0.5, 1, 2, and 4 mm follicles from the ovaries of white leghorn hens (n=6) in active egg production. The oocyte cytoplasm was removed by bisecting the follicles with 30 ga needles and washing the granulosa cell "shells" through 3 changes of media before freezing in liquid nitrogen. Total RNA was isolated from the granulosa cells and levels of STAR, HSD3B, CYP11A1, BMP2, BMP4, BMP6, AMH, FOXO3, FOXL2, GATA4, WT1, PCSK6, WNT4, and SMAD9 mRNA were determined by qPCR using the 2-ddct method and ACTB (beta-Actin) mRNA as the normalizer. Based on our findings, the growth of follicles from 0.5 to 1 mm is an important transition during avian follicular development. The mRNA for STAR, HSD3B and CYP11A1 increased 15-20 fold in GC from 1 mm compared to 0.5 mm follicles and remained elevated in 2 and 4 mm follicles (P<0.05). BMP2, BMP4 and BMP6 mRNA decreased 50-95% in GC from 1-4 mm follicles compared to 0.5 mm follicles, while AMH increased ~2 fold in GC from 1-2 mm follicles and then decreased in 4 mm follicles below levels observed in 0.5 mm follicles (P<0.05). FOXL2, GATA4, WT1 and WNT4 mRNA decreased 40-95%, while FOXO3, PCSK6 and SMAD9 mRNA increased 2, 3 and 20 fold, respectively, as follicles grew from 0.5 to 4 mm (P<0.05). In a second study, we cultured small 0.5 mm follicles from white leghorn hens (n=6) on collagen coated plates for 7 days to determine if the follicles would differentiate in vitro. Total RNA was isolated from fresh and cultured follicles and the same transcripts as in experiment 1 were measured by qPCR. Interestingly, STAR, HSD3B and CYP11A1 mRNA were downregulated during culture to almost undetectable levels compared to fresh follicles (P<0.05). However, the pattern of expression for the other transcripts measured was similar to the in vivo experiment. BMP2, BMP4, BMP6, AMH, FOXL2, GATA4, WT1 and WNT4 mRNA decreased 70-95% in cultured follicles, while FOXO3 and SMAD9 mRNA increased 4 and 7 fold, respectively, during culture (P<0.05). PCSK6 mRNA increased during in vivo follicular development, but did not differ between fresh and cultured follicles (P<0.05). We conclude that the growth of avian follicles from 0.5 to 1 mm is an important transition during avian follicular development and some, but not all, of the observed in vivo changes are recapitulated during culture. (poster)