Is the Hippo Pathway Effector Yes-Associated Protein a Potential Key Player of Dairy Cattle Cystic Ovarian Disease Pathogenesis?

Abstract

Cystic ovarian disease (COD) in dairy cattle is characterized by preovulatory follicles that become cysts, fail to ovulate and persist in the ovary; consequently, interfering with normal ovarian cyclicity. The intraovarian key players that orchestrate the alterations occurring in the preovulatory follicle and that culminate with cyst formation and persistence, however, remain uncertain. Interestingly, the Hippo pathway effector yes-associated protein (YAP) has been described in humans and mice as a key player of anovulatory cystic disorders. To start elucidating if YAP deregulation in ovarian follicle cells can be also involved in the pathogenesis of COD, we have generated a series of novel results using spontaneously occurring cystic follicles in cattle. We found that mRNA and protein levels of YAP are significantly higher in granulosa (GCs) and theca cells (TCs) isolated from cystic follicles (follicular structures of at least 20 mm in diameter) in comparison to respective cell types isolated from non-cystic large follicles (≥12 mm). In addition, immunohistochemistry and Western blot analyses used to determine YAP phosphorylation pattern suggest that YAP transcriptional activity is augmented is cystic GCs. These results were confirmed by a significant increase in the mRNA levels encoding for the classic YAP-TEAD transcriptional target genes CTGF, BIRC5 and ANKRD1 in GCs from follicle cysts in comparison to non-cystic large follicles. Taken together, these results provide considerable insight of a completely novel signaling pathway that seems to play an important role in ovarian cystic disease pathogenesis in dairy cattle.

Keywords: ANKRD1; COD; CTGF; CYR61; Hippo; TEAD; anovulation; cow; cyst; granulosa cells; ovary; theca cells.

Figure 1

Molecular validation of spontaneous cystic follicle samples. Relative messenger RNA abundance was measured by real-time qPCR and normalized to the geometric mean of three housekeeping genes H2AFZ, GAPDH and RLP19. Granulosa (A) and theca cells (B) were isolated from ovarian follicular cysts (structures of at least 20 mm in diameter; Cyst group) and from non-cystic large follicles (≥12 mm; Control group). Data represent the mean ± SEM from granulosa (n = 10) and theca cells (n = 4) independent follicle samples per group. An asterisk (*) indicates significant difference between groups (p < 0.05).

Figure 2

Steroids concentration in follicular fluid of control and cyst groups. Follicular fluid steroid concentrations for estradiol (A), progesterone (B) and testosterone (C) were measured by liquid chromatography–tandem triple quadrupole mass spectrometry. Data represent the mean ± SEM of independent follicle samples (n = 10) per group. An asterisk (*) indicates significant difference between groups (p < 0.05).

Figure 3

Expression pattern of total and phosphorylated YAP proteins in spontaneous cystic follicles in cows. Immunohistochemistry (IHC) analysis was used to compare the cellular and subcellular localization of total and phospho-YAP (P-YAP; Ser 127) proteins in control non-cystic large follicles (A) and in spontaneous cystic follicles (B). The red lines differentiate the distinct cell layers observed in the images (G: granulosa cells; T: theca cells; S: stromal cells). Representative IHC images of staining for total and phospho-YAP (objective 63×) and their antibody control (NEG) showing negative staining of total YAP and phospho-YAP (objective 63×). Structure and cellular architecture of both groups can be observed in the H&E representative images.

Figure 4

Expression levels for YAP (total and phospho forms), TEADs and classic YAP-TEAD target genes in cystic granulosa cells. Granulosa cells were isolated from ovarian follicular cysts and from non-cystic large control follicles. Total and phospho-YAP (pYAP; Ser 127) and Pan-TEAD protein levels (A) were measured by Western blot (WB) and normalized to β-actin (n = 5 for each group). Relative messenger RNA abundance (B) was measured by real-time qPCR and normalized to the geometric mean of three housekeeping genes H2AFZ, GAPDH and RLP19 (n = 10 for each group). Data represent the group mean ± SEM. An asterisk (*) indicates difference between groups (p < 0.05).

Figure 5

Expression levels for YAP (total and phospho forms), TEADs and classic YAP-TEAD target genes in cystic theca cells. Theca cells were isolated from ovarian follicular cysts and from non-cystic large control follicles. Total and phospho-YAP (pYAP; Ser 127) and Pan-TEAD protein levels (A) were measured by Western blot (WB) and normalized to β-actin (n = 4 for each group). Relative messenger RNA abundance (B) was measured by real-time qPCR and normalized to the geometric mean of three housekeeping genes H2AFZ, GAPDH and RLP19 (n = 4 for each group). Data represent the group mean ± SEM. An asterisk (*) indicates difference between groups (p < 0.05).