Synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells

Figures & data

Table 1. Primer sequences.

Primer name	Primer sequence (5’–3’)	Melting temperature (°C)	Product length (bp)	GenBank number
CTNNB1	F:TCATTGGCAGCAGCAGTCATAT	56.6	253	NM_205081.3
	R: CATTCCTTCATCCAGTGTTTCG	53.7
SF1	F: GCGGGAGGAATAAGTT	47.9	169	NM_205077.2
	R: ATGGCGTGGATGCTGT	54.5
StAR	F:GACAACATGGAGCAGATGGGCGACTGG	66.3	160	NM_204686.3
	R: GCCGGGAGCACCGAACACTCACAA	67.1
CYP11A1	F: ACCGCAACAAGCCCTACG	58.9	238	NM_001001756.2
	R: CACACAGACTCCAAGGCAAAG	56.5
β-actin	F: CACGATGGAGGGGCCGGACTCATC	67.1	241	NM_001101.5
	R: TAAAGACCTCTATGCCAACACAGT	55.4

CTNNB1, catenin beta-1; SF1, nuclear receptor subfamily 5 group A member 1; StAR, steroidogenic acute regulatory; CYP11A1, cytochrome P450 family 11 subfamily A member 1; β-actin, actin-beta.

Table 2. siRNA sequences.

Name	Sequence (5’–3’)
siRNA-CTNNB1	5′-CCCUAUGAUGGAACAUGAATT-3′
	5′-UUCAUGUUCCAUCAUAGGGTT-3′
siRNA-SF1	5′-GCUCCACUUUCAUAGCACUTT-3′
	5′-AGUGCUAUGAAAGUGGAGCTT-3′
siRNA-CYP11A1	5′-GGCUGACAAAUGUAUCCAATT-3′
	5′-UUGGAUACAUUUGUCAGCCTT-3′
siRNA-NC	5′-UUCUCCGAACGUGUCACGUTT-3′
	5′-ACGUGACACGUUCGGAGAATT-3′

Figure 1. β-catenin promotes the secretion of progesterone in GCs. (a) Relative level of CTNNB1 after transfection with pcDNA3.1-CTNNB1. (b) Relative expression of CTNNB1 mRNA after transfection with siRNA-CTNNB1. (c) Relative expression of StAR after transfection with pcDNA3.1-CTNNB1. (d) Relative expression of StAR after transfection with siRNA-CTNNB1. (e) Relative expression of CYP11A1 after transfection with pcDNA3.1-CTNNB1. (f) Relative expression of CYP11A1 after transfection with siRNA-CTNNB1. (g) Levels of P4 after transfection with pcDNA3.1-CTNNB1. (h) Levels of P4 after transfection with siRNA-CTNNB1. Results are presented as means ± SE; n = 3; * P < 0.05; ** P < 0.01.

Figure 2. β-catenin and SF1 co-promote the secretion of progesterone in GCs. (a) Relative expression of SF1 after transfection with pcDNA3.1-SF1 cells. (b) Relative expression of SF1 after transfection of with three different siRNAs-SF1. (c) Relative expression of CYP11A1 after cotransfection with pcDNA3.1-CTNNB1 and pcDNA3.1-SF1. (d) Levels of secreted P4, measured by ELISA, after cotransfection with pcDNA3.1-CTNNB1 and pcDNA3.1-SF1. (e) Relative expression of CTNNB1 and SF1 after cotransfection with pcDNA3.1-CTNNB1 and siRNA3-SF1. (f) Relative expression of CYP11A1 after cotransfection with pcDNA3.1-CTNNB1 and siRNA3-SF1. (g) Level of secreted P4 after cotransfection with pcDNA3.1-CTNNB1 and siRNA3-SF1. Results are presented as means ± SE; n = 3; * P < 0.05; ** P < 0.01; a, b, and c indicate significant different values (P < 0.05).

Figure 3. SF1 and CYP11A1 co-promote the secretion of progesterone in GCs. (a) Relative expression of CYP11A1 after transfection of with three different siRNAs-CYP11A1. (b) Relative expression of SF1 and CYP11A1 after cotransfection with pcDNA3.1-SF1 and siRNA2-CYP11A1. (c) Relative expression of CYP11A1 after transfection with pcDNA3.1-SF1. (d) Relative expression of CYP11A1 after transfection with siRNA3-SF1. (e) Level of secreted P4 in cells cotransfected with pcDNA3.1-SF1 and siRNA2-CYP11A1. Results are presented as means ± SE; n = 3; * P < 0.05; ** P < 0.01; a, b, and c indicate significant different values (P < 0.05).