Disruption of Epidermal Growth Factor Receptor but Not EGF Blocks Follicle Activation in Zebrafish Ovary

Abstract

Folliculogenesis is controlled by intimate communications between oocytes and surrounding follicle cells. Epidermal growth factor (EGF/Egf) is an important paracrine/autocrine factor in vertebrate ovary, and it is well known for its stimulation of oocyte maturation. However, the role of EGF signaling through its receptor (EGFR/Egfr) in ovarian folliculogenesis is poorly understood, especially at early stages of follicle development. In this study, we created zebrafish mutants for Egf (egf−/−) and Egfr (egfra−/− and egfrb−/−) by CRISPR/Cas9 technique. Surprisingly, these mutants all survived well with little abnormality in growth and development. Spermatogenesis and folliculogenesis were both normal in egf−/− males and females. Their fecundity was comparable to that of the wildtype fish at 4 months post-fertilization (mpf); however, the fertilization rate of mutant eggs (egf−/−) decreased significantly at 7 mpf. Interestingly, disruption of egfra (egfra−/−) led to failed follicle activation with folliculogenesis being blocked at primary–secondary growth transition (PG-SG transition), leading to female infertility, whereas the mutant males remained fertile. The mutant ovary (egfra−/−) showed abnormal expression of a substantial number of genes involved in oxidative metabolism, gene transcription, cytomembrane transport, steroid hormone biosynthesis, and immune response. The stunted PG oocytes in egfra−/− ovary eventually underwent degeneration after 6 months followed by sex reversal to males with functional testes. No abnormal phenotypes were found in the mutant of truncated form of EGFR (egfrb). In summary, our data revealed critical roles for EGFR signaling in early folliculogenesis, especially at the PG-SG transition or follicle activation.

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

“…Like Gdf9, EGF ligands and EGFR may represent another paracrine pathway within the follicle that mediates signaling from oocyte to follicle cells. Our recent study showed that disruption of egfra in zebrafish also blocked follicle development at PG-PV transition followed by sex reversal to males [59], which is similar to the phenotypes of gdf9 mutant observed in the present study. These results indicate that follicle development in zebrafish ovary is finely controlled by multiple factors from the oocyte and that the regulation of folliculogenesis involves many checkpoints for regulation by both internal and external signals.…”

“…We isolated the PG and early PV follicles from the control fish (gdf9+/-) and PG follicles from gdf9-/-fish (no PV follicles in the mutant) to analyze expression of genes by real-time qPCR, including gonadotropin receptors (fshr and lhcgr), ovarian aromatase (cyp19a1a), epidermal growth factor receptor (egfra) and members of the activin-inhibin family (inhibin a subunit inha, activin b subunits inhbaa, inhbab and inhbb). Most of these genes showed increased expression during the PG-PV transition in the control fish (fshr, cyp19a1a, egfra, inha, inhbaa and inhbab) except lhcgr and inhbb, which agreed well with our previous studies [58][59][60][61][62]. However, their expression showed little change in mutant PG follicles compared to stage-matched PG follicles from the control except activin bAa (inhbaa).…”

“…Interestingly, although EGF also stimulated expression of all activin subunits in zebrafish follicle cells [87] and the loss of egfra gene led to similar phenotypes to those of gdf9 mutant, viz. blockade at PG-PV transition followed by sex reversal to males, inha mutation could not rescue the phenotypes of egfra mutant [59], suggesting differential mechanisms underlying actions of Gdf9 and EGF family ligands.…”

Role of EGFR expressed on the granulosa cells in the pathogenesis of polycystic ovarian syndrome

“…Additionally, by a discovery-validation GWAS on PCOS patients and controls identifified from electronic health record using an algorithm based on Rotterdam criteria, Zhang et al confirmed that the ERBB4-YAP1-WWTR1 network played a potential role of EGFR and Hippo signaling in the pathogenesis of PCOS (29). Nevertheless, the relationship between EGFR and PCOS has not yet been fully investigated (15). Bidirectional communication between somatic granulosa cells and the oocyte is an essential factor for folliculogenesis, which is orchestrated by endocrine and paracrine mechanisms (30).…”

“…One previous study found that the blockage of EGFR induced apoptosis in granulosa cells (14). A recent study reported that folliculogenesis was blocked at the primary-secondary growth transition in zebrafish mutants upon EGFR knockout by CRISPR/Cas9 technique, leading to female infertility (15). In cattle ovary, studies identified that the protein expression of EGFR was higher in the antral follicles than that in other follicular growth stages, which indicates that EGFR may promote follicular development during follicles growth waves (15).…”