The Hippo Pathway Effectors YAP and TAZ Regulate LH Release by Pituitary Gonadotrope Cells in Mice

Abstract

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LβT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

Keywords: KO mice; TAZ; YAP; fertility; gonadotropins.

Figure 1.

Gonadotrope-specific conditional knockout validation. TAZ protein levels in gonadotrope cells from pituitary glands isolated from 10- to 12-week-old control (Yap^flox/flox;Taz^flox/flox) and cKO (Yap^flox/flox;Taz^flox/flox;Gnrhr^GRIC/+) male mice. Representative immunofluorescence images of staining for TAZ (red) in an adenohypophysis region with significant positive staining for the gonadotrope cell marker LHβ (green). Overlays (merge) of the 2 images are also shown with the nuclei labeled with DAPI (blue). Scale bars = 100 µm.

Figure 2.

Effects of gonadotrope-specific Yap/Taz knockout in male mice. For all analyses, samples were collected from 10- to 12-week-old cKO (n = 11) and control (n = 7-8) male mice. A, Blood samples were collected by cardiac puncture and serum obtained by centrifugation. LH and FSH levels were measured by RIA, and intratesticular testosterone was measured in testicular homogenates by ELISA. B, Assessment of testes and seminal vesical weight/corporal weight (gonadosomatic index). C, For sperm counting, cauda epididymides were collected, weighed and placed in prewarmed solution, and epididymal ducts were opened to release their contents. All data are expressed as means ± SEM. * P < 0.05, **P < 0.01.

Figure 3.

Effects of gonadotrope-specific Yap/Taz knockout in female mice. For hormonal assays and ovarian antral follicle counting, samples were collected from mature 42-day-old control (n = 7) and cKO (n = 7) female mice. A, Blood samples were collected by cardiac puncture and serum obtained by centrifugation. LH and FSH levels were measured by RIA. B, Both ovaries were weighed to determine mean ovarian weight, but only left ovaries were isolated and prepared for follicle counting (serially sectioned). All antral follicles were counted and scored as either healthy or atretic. Data (means ± SEM) represent raw follicle count numbers and were not adjusted to estimate the total ovarian follicle population. C, For the natural ovulation rate experiment, 8- to 10-week-old cKO (n = 7) and control (n = 7) female mice were housed with wild-type males and following mating, COCs were retrieved from the oviducts and counted. All data are expressed as means ± SEM. * P < 0.05, ***P < 0.001.

Figure 4.

Basal and GnRH-induced LH secretion in pituitary cells from cKO mice. Pituitary cells were isolated from 12- to 16-week-old (A) female and (B) male control and cKO mice and seeded into cell culture plates. After 24 hours, cells were treated or not with a single pulse of GnRH (1 μM) for 15 (left panels) or 30 minutes (right panels) and the cell culture medium was harvested for LH assessment by RIA. Different letters show statistically significant differences between groups. P < 0.05.

Figure 5.

Effects of the disruption of YAP/TAZ-TEAD interactions in an immortalized gonadotrope-like cell line. LβT2 gonadotrope-like cells were pretreated for 1 hour with 5 µM of verteporfin (VP; a small molecule inhibitor that interferes with YAP/TAZ binding to TEAD family of transcription factors) before challenge with a single pulse of GnRH (1 µM) for 15 minutes. A, Cell culture medium was harvested for LH assessment by RIA and B, cells were collected for RNA extraction. mRNA levels were assessed by RT-qPCR and normalized to the housekeeping gene Rpl19. Different letters show statistically significant differences between groups. P < 0.05.

Figure 6.

Expression pattern of gonadotropin synthesis-related genes in whole pituitary gland. For all analyses, pituitary glands were collected from 10- to 12-week-old mice. A, Data represent control (n = 5) and cKO (n = 7) female mice. B, Data represent control (n = 8) and cKO (n = 11) male mice. mRNA levels of the indicate genes were determined by RT-qPCR and normalized to the housekeeping gene Rpl19. Data are expressed as means ± SEM. P > 0.05 in all cases.