Loss of growth differentiation factor 9 causes an arrest of early folliculogenesis in zebrafish – a novel insight into its action mechanism

Abstract

Growth differentiation factor 9 (GDF9) is the best characterized growth factor released by the oocyte; however, most information about GDF9 functions comes from studies in the mouse model. In this study, we created a mutant for Gdf9 gene (gdf9-/-) in zebrafish using TALEN approach. The loss of Gdf9 caused a complete arrest of follicle development at primary growth (PG) stage. These follicles eventually degenerated, and all mutant females gradually changed to males through sex reversal, which could be prevented by mutation of the male-promoting gene dmrt1. Interestingly, the phenotypes of gdf9-/- could be rescued by mutation of inhibin a (inha-/-) but not estradiol, suggesting a potential role for the activin-inhibin system in Gdf9 actions. In gdf9 null follicles, activin bAa (inhbaa) expression decreased dramatically; however, its expression rebounded in the double mutant (gdf9-/-;inha-/-). These results clearly indicate that although endocrine hormones such as follicle-stimulating hormone (FSH) are important for folliculogenesis, the activation of PG follicles requires intrinsic factors from the oocyte, such as Gdf9, which in turn works on the neighboring follicle cells to trigger follicle activation, probably via activins.

Phenotypical Rescue of Bmp15 Deficiency by Mutation of Inhibinα(inha) Provides Novel Clues to How Bmp15 Controls Zebrafish Folliculogenesis

“…Our recent study showed that the loss of gdf9 in zebrafish resulted in a complete arrest of follicles at PG stage without accumulation of cortical alveoli. Interestingly, this phenotype could be rescued by simultaneous mutation of inhibin α subunit ( inha -/-) (Chen et al, 2022). This raised an interesting question about the relationship between Bmp15 and inhibin.…”

“…Interestingly, the loss of bmp15 and gdf9 resulted in follicle blockade at different developmental stages. The mutation of gdf9 caused a complete arrest of follicle development at PG stage without formation of cortical alveoli (PG-PV transition) (Chen et al, 2022) whereas bmp15 deficiency arrested follicles at PV stage (PV-EV transition) without any signs of vitellogenesis characterized by yolk granule accumulation. To study potential interactions between bmp15 and gdf9 , we generated a double mutant ( bmp15-/-;gdf9-/- ) using a gdf9 mutant we recently reported (ZFIN line number: umo18) (Chen et al, 2022).…”

“…The mutation of gdf9 caused a complete arrest of follicle development at PG stage without formation of cortical alveoli (PG-PV transition) (Chen et al, 2022) whereas bmp15 deficiency arrested follicles at PV stage (PV-EV transition) without any signs of vitellogenesis characterized by yolk granule accumulation. To study potential interactions between bmp15 and gdf9 , we generated a double mutant ( bmp15-/-;gdf9-/- ) using a gdf9 mutant we recently reported (ZFIN line number: umo18) (Chen et al, 2022). Although the double mutant showed similar phenotype to that of gdf9-/- mutant at 90 dpf with most follicles arrested at PG stage, some follicles in most females (9/13) could break the blockade at PG stage to enter early PV stage (PV-I), but not further to PV-II and III as seen in bmp15-/- (Fig.…”

“…Since inha deficiency could partially rescue the follicle blockade in both gdf9 (Chen et al, 2022) and bmp15 mutants as described above, we also created a triple mutant ( bmp15-/-;gdf9-/-;inha-/- ) to see if inha mutation had any impact on the double mutant of bmp15 and gdf9 . The results showed that inha mutation could also help the double mutant ( bmp15-/-;gdf9 ) to overcome the follicle blockade to enter vitellogenic growth with formation of both cortical alveoli and yolk granules (Fig.…”

“…We first created a bmp15 mutant by CRISPR/Cas9. In contrast to the gdf9 mutant ( gdf9-/- ) whose follicles were arrested at primary growth (PG) stage or PG-PV transition (Chen et al, 2022), the follicles in female bmp15 mutant ( bmp15-/- ) were also completely arrested, but at later stage, i.e. , previtellogenic (PV) stage or PV-EV (early vitellogenic) transition, leading to female infertility.…”