FGA, a new target of histone acetylation, inhibits apoptosis of granulosa cells in follicles

Abstract

Granulosa cells (GCs) are the main supporting cells for follicles, and histone acetylation has been reported to regulate follicular development. However, the mechanism of histone acetylation regulating follicular development is still unclear in GCs. In this study, we found that FGA, fibrinogen alpha chain, mediated the survival and fate of GCs. Knockdowns of HDAC1 and HDAC3 significantly inhibited the mRNA level of FGA, while knockdown of HDAC2 notably decreased the protein level of FGA. Moreover, knockdown of HDAC2 repressed the chromatin accessibility and the enrichment level of H3K9ac at -1350/-1454 bp of FGA. In addition, FGA promoted GCs proliferation and cycle progression by up-regulating the expressions of PCNA and CCNE1, whereas it inhibited apoptosis by suppressing the expression of Caspase3. In vitro, FGA was likely to promote follicular development of pigs. In mice, FGA inhibited the apoptosis of GCs and increased the number of corpora lutea, as a result, elevating estradiol levels and advancing the day of pubertal initiation. Both in vitro and in vivo experiments, FGA promoted follicular development by up-regulating PCNA and CCNE1, while inhibited follicular apoptosis by down-regulating Caspase3 and Caspase9. Overall, knockdown of HDAC2 repressed transcription by reducing chromatin accessibility and decreasing H3K9ac binding at the FGA promoter. FGA inhibited apoptosis of GCs by suppressing the expression of Caspase3 and promoted follicular development. This study showed that FGA is a novel target for histone acetylation to regulate follicular development in mammals.

Keywords: Chromatin accessibility; FGA; Follicles; H3K9ac.

Fig. 1

Histone acetylation inhibits FGA to promote apoptosis in GCs. Changes in mRNA (a) and protein levels (b) in apoptosis signaling pathways after TSA treatment. Changes in mRNA (c) and protein (d) levels of FGA, HDACs, and H3K9ac after TSA treatment. The changes in mRNA and protein levels of apoptosis pathway genes and FGA after si-HDAC1 (e, f), si-HDAC2 (g, h) or si-HDAC3 (i, j) treatment. segmentation design of FGA promoter region (k). The level of accessibility modification (l) and enrichment (m) of H3K9ac on FGA promoter (P1, P2, P3, P4 and P5) in cells treated with si-HDAC2 was detected by chromosome accessibility and ChIP-qPCR. In all panels, the statistical significance of differences between means were assessed using Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significant difference)

Fig. 2

FGA promotes proliferation of ovarian GCs by accelerating cell cycle progression. The mRNA (a) and protein (b) levels of FGA in small (< 3 mm) and large (> 5 mm) follicles. The overexpression and interference efficiency of FGA (c). The effects of overexpression of FGA (d) and si-FGA (e) on the proliferation of cells. The effects of overexpression of FGA (f) and si-FGA (g) on the mRNA levels of genes related to cell proliferation. The effects of overexpression of FGA and si-FGA on the protein levels of genes related to cell proliferation (h). The effects of overexpression of FGA (d) and si-FGA (e) on the cell cycle. The effects of overexpression of FGA (k) and si-FGA (l) on the mRNA levels of genes related to cell cycle. The effects of overexpression of FGA and si-FGA on the protein levels of genes related to cell cycle (m). In all panels, the statistical significance of differences between means were assessed using Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significant difference)

Fig. 3

FGA interacts with Caspase3 to inhibit apoptosis. The effects of overexpression of FGA (a) and si-FGA (b) on the apoptosis of cells. The effects of overexpression of FGA (c) and si-FGA (d) on the mRNA levels of genes related to cell apoptosis. The effects of overexpression of FGA and si-FGA on the protein levels of genes related to cell apoptosis (e). CoIP detected that FGA binds to apoptosis-related proteins (f). CoIP reversed validation of apoptosis-related gene protein binding (g). Caspase3 in the nucleus and cytoplasm at the mRNA (h) and protein level (i). In all panels, the statistical significance of differences between means were assessed using Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significant difference)

Fig. 4

FGA promotes the growth of porcine follicles. Efficiency of lentivirus-mediated FGA overexpression (LV-FGA) (a) or FGA knockdown (sh-FGA) (b) on the mRNA level. LV-FGA and sh-FGA efficiency on the protein levels (c). The effects of LV-FGA and sh-FGA on days 1, 3, 5 of porcine follicles (d). TUNEL staining of LV-FGA and sh-FGA treated follicles was performed to assess the apoptotic rates of GCs (e). The nuclei stained by DAPI were blue and the positive apoptotic nuclei were green. The scale bar was 200 μm. The effects of LV-FGA (f) and sh-FGA (g) on the mRNA levels of genes of cell apoptosis. The effects of LV-FGA and sh-FGA on the protein (h) levels of genes of cell apoptosis. The effects of LV-FGA (i) and and sh-FGA (j) on the mRNA levels of genes of cell proliferation and cycle. The effects of LV-FGA and sh-FGA on the protein levels of genes of cell proliferation and cycle (k). In all panels, the statistical significance of differences between means was assessed using Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significant difference)

Fig. 5

FGA promotes the follicular growth in mice. Efficiency of lentivirus-mediated FGA overexpression (LV-FGA) (a) or FGA knockdown (sh-FGA) (b) on the mRNA level. LV-FGA and sh-FGA efficiency on the protein levels (c). The effects of LV-FGA and sh-FGA on the day of pubertal (d) and E2 (e) levels in mice. The HE (f) and TUNEL staining (g) of mouse ovary treated by LV-FGA and sh-FGA. CL represents corpus luteum, black arrows represent antral follicle and yellow arrows represent pre-antral follicles. The scale bar is 500 μm. The effects of LV-FGA (h) and sh-FGA (i) on the mRNA levels of cell apoptosis. The effects of LV-FGA and sh-FGA on the protein (j) levels of cell apoptosis. The effects of LV-FGA (k) and sh-FGA (l) on the mRNA levels of cell proliferation and cycle. The effects of LV-FGA and sh-FGA on the protein levels of cell proliferation and cycle (m). In all panels, the statistical significance of differences between means was assessed using Student’s t-test (* P < 0.05; ** P < 0.01; ns, no significant difference)