The Hippo pathway effectors YAP and TAZ interact with EGF-like signaling to regulate expansion-related events in bovine cumulus cells in vitro

Abstract

Purpose: To determine if the inhibition of the interaction between the Hippo effector YAP or its transcriptional co-activator TAZ with the TEAD family of transcription factors is critical for the cumulus expansion-related events induced by the EGF network in cumulus-oocyte complexes (COCs).

Methods: We performed a series of experiments using immature bovine COCs subjected to an IVM protocol for up 24 h in which cumulus expansion was stimulated with EGF recombinant protein or FSH.

Results: The main results indicated that EGFR activity stimulation in bovine cumulus cells (CC) increases mRNA levels encoding the classic YAP/TAZ-TEAD target gene CTGF. To determine if important genes for cumulus expansion are transcriptional targets of YAP/TAZ-TEAD interaction in CC, COCs were then subjected to IVM in the presence of FSH with or without distinct concentrations of Verteporfin (VP; a small molecule inhibitor that interferes with YAP/TAZ binding to TEADs). COCs were then collected at 6, 12, 18, and 24 h for total RNA extraction and RT-qPCR analyses. This experiment indicated that VP inhibits in a time- and concentration-dependent manner distinct cumulus expansion and oocyte maturation-related genes, by regulating EGFR and CTGF expression in CC.

Conclusions: Taken together, the results presented herein represent considerable insight into the functional relevance of a completely novel signaling pathway underlying cumulus expansion and oocyte maturation in monovulatory species. YAP/TAZ or CTGF may represent potential targets to improve the efficiency of IVM systems, not only for monovulatory species of agricultural importance as the cow, but for human embryo production.

Keywords: CTGF; Cow; EGF receptor; Oocyte in vitro maturation; Ovary; Verteporfin.

Fig. 1

Localization of YAP and TAZ in cumulus cells of immature bovine COCs. Immunohistochemistry (IHC) analysis was used to determine the cellular and subcellular localization of total YAP and total TAZ protein levels in cumulus-oocyte complexes (COCs) located in 3–8 mm bovine ovarian follicles (CC, cumulus cell; MGC, mural granulosa cells; TC, theca cells; SC, stromal cells). Representative IHC images of staining for YAP and TAZ (10 and 20 × , respectively) and their antibody control (NEG) showing negative staining of total YAP and total TAZ in MGC and TC

Fig. 2

Validation of the EGF signaling–related induced cumulus expansion models. COCs were randomly divided into groups (n = 5 COCs/group per replicate) and were pretreated without (Control and EGF groups) or with 6 μM of AG 1478 (a selective inhibitor of EGFR) for 1 h before adding 10 ng/mL of EGF (A) or pretreated without (Control and FSH groups) or with 6 μM of AG 1478 for 1 h before adding 500 ng/mL of FSH (B). Cumulus expansion was assessed by measuring COC area (µm²) in each COC at 0 and 24 h post-EGF (A) or post-FSH (B). Data represent the mean ± SEM of five independent replicates and asterisk (*) represents statistical difference in comparison to respective Control (P < 0.05)

Fig. 3

Effect of the selective EGFR inhibitor AG 1478 on mRNA levels encoding the classic YAP/TAZ-TEAD target gene CTGF in cumulus cells post-EGF or FSH. COCs were randomly divided into groups (n = 5 COCs/group per replicate) and were pretreated without (Control and EGF groups) or with 6 μM of AG 1478 (a selective inhibitor of EGFR) for 1 h before adding 10 ng/mL of EGF (A) or pretreated without (Control and FSH groups) or with 6 μM of AG 1478 for 1 h before adding 500 ng/mL of FSH (B) for 6, 12, 18, and 24 h. Messenger RNA abundance was measured in cumulus cells by real-time qPCR and normalized to the housekeeping genes H2AF2 and RPS18. Data represent the mean ± SEM of five independent replicates. Different letters show statistically significant differences between treatments in the same time point (P ˂0.05)

Fig. 4

Inhibition of YAP/TAZ-TEAD interaction affects the expression of critical FSH-induced cumulus expansion–related genes. COCs were randomly divided into groups (n = 5 COCs/group per replicate) and were pretreated or not for 1 h with different doses of Verteporfin (VP; 0.1, 0.3, and 0.5 μM), a small molecule inhibitor that interferes with YAP/TAZ binding to TEAD proteins, followed with FSH (500 ng/mL) treatment for 6, 12, 18, and 24 h. Messenger RNA abundance was measured in cumulus cells by real-time qPCR and normalized to the housekeeping genes H2AF2 and RPS18. Data represent the mean ± SEM of five independent replicates. Different letters show statistically significant differences between treatments in the same time point (P ˂0.05)

Fig. 5

Effect of Verteporfin (VP) on FSH-induced cumulus expansion. COCs were randomly divided into groups (n = 5 COCs/group per replicate) and were pretreated or not for 1 h with different doses of Verteporfin (VP; 0.1, 0.3, and 0.5 μM) followed with FSH (500 ng/mL) for 24 h. Representative images of the effect of VP treatments on FSH-induced COCs expansion at 0 and 24 h of culture