Conditional Deletion of FOXL2 and SMAD4 in Gonadotropes of Adult Mice Causes Isolated FSH Deficiency

Abstract

The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common α gonadotropin subunit and the hormone-specific FSHβ subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.

Figure 1.

Validation of the efficiency and specificity of tamoxifen-induced recombination in gonadotropes. (A) Male iGRIC/+;Rosa26^YFP/+ mice were treated with oil (vehicle) or tamoxifen with one of five different treatment protocols [see schematic in panel (B)]. Cells in pituitary sections were labeled for YFP (green) and LHβ (red). Arrows indicate LHβ+/YFP− cells. (B) Top: quantification of LHβ-positive cells colabeled with YFP after different vehicle or tamoxifen treatments. Bottom: schematic representation of the different tamoxifen treatment protocols. (C) Immunofluorescence staining for FOXL2 (green) and LHβ (red) in pituitary sections from males in the four treatment groups. (D) RT-qPCR analyses of relative Foxl2 and Smad4 gene expression in YFP+ cells purified from control (black) and inducible KO (red) pituitaries 4 weeks after the last tamoxifen injection. Gene expression levels were normalized to the housekeeping gene Rpl19. Scale bars in panels (A) and (C), 50 µm. icKO, inducible conditional knockout; TAM, tamoxifen.

Figure 2.

Impaired fertility and ovarian follicular development in induced F2S4 cKO females. (A) Fertility assessment before and after oil or tamoxifen injections in control and induced F2S4 cKO females. The panels at the right indicate individual fertility data (litter size) for sterile (red dots) and subfertile females (cyan, royal blue, brown, and pink dots). The number of litters produced by subfertile mice is also shown. (B) Representative ovarian tissue sections in control and induced F2S4 cKO mice. Scale bar, 200 μm. (C) Ovarian and (D) uterine weights of control and induced F2S4 cKO females 1 month after oil or tamoxifen injections. Horizontal lines reflect group means (+SEM). Each data point represents one animal. Fertility of each group before injection was analyzed by one-way ANOVA. Fertility before and after treatment in iGRIC/+;TAM group was analyzed by Student t test. *P < 0.05. Avg., average; CL, corpus luteum; TAM, tamoxifen.

Figure 3.

Intact testicular weights and spermatogenesis in induced cKO males. (A) Testicular and (B) seminal vesicle weights from control and induced F2S4 cKO males 2 months after oil or tamoxifen injections. (C) Progressive sperm motility from caudal epididymides. (D) Sperm counts from snap frozen epididymides. (E) Representative testicular tissue sections from control and induced F2S4 cKO mice. Sertoli cells were identified with SOX9 using immunohistochemistry (brown stain). (F) Average Sertoli cell numbers per tubule per testis 2 months after oil or tamoxifen injection. Scale bar, 100 µm. There were no statistically significant differences between groups. TAM, tamoxifen.

Figure 4.

Reduced FSH production and secretion in induced F2S4 cKO mice. Serum FSH levels in (A) females and (B) males before (left) and after (right) tamoxifen or oil treatment. Individual serum FSH levels after injection are shown in the boxes at the right. Serum LH levels in (C) females and (D) males after treatment. (E and F) RT-qPCR analysis of relative mRNA levels of pituitary Fshb, Foxl2, Smad4, Cga, Lhb, and Gnrhr in females and males after treatment with oil or tamoxifen. All gene expression was normalized to the housekeeping gene Rpl19. Dashed lines represent mRNA levels in mice lacking the Foxl2 and Smad4 genes during development (20). Relative gene expression and serum FSH levels before and after treatment were analyzed separated by one-way ANOVAs followed by Dunn multiple comparisons test. Serum FSH levels before and after treatment in the same group were analyzed by Student t test. There were no significant differences between the control groups. *P < 0.05. TAM, tamoxifen.

Figure 5.

Reduced pituitary FSH protein content in induced F2S4 cKO mice. Pituitary FSH content in (A) females and (B) males after oil or tamoxifen treatment. Control female pituitaries were collected at random estrous cycle stages; cKO pituitaries were collected at the same time as their control littermates. (C and D) Pituitary LH content in (C) females and (D) males after treatment. (E) Immunofluorescence staining for LHβ (green) and FSHβ (red) in pituitaries of male control and induced F2S4 cKO mice. Data were analyzed by one-way ANOVA followed by Dunn multiple comparisons test. Scale bar, 50 µm. *P < 0.05. TAM, tamoxifen.

Figure 6.

Impaired basal and activin A–stimulated Fshb expression in cultured pituitary cells of induced F2S4 cKO mice. Primary pituitary cultures were prepared from control and induced F2S4 cKO (A) females and (B) males at least 1 month after oil or tamoxifen injection. Cells were treated with vehicle or 1 nM activin A. (C) Primary pituitary cultures were prepared from male and female (combined) iGRIC/+;Foxl2^fx/fx;Smad4^fx/fx mice or from (D) wild-type mice. Cells were then treated with 1.5 µM of 4-OHT or vehicle. Twenty-four hours later, cells were treated with vehicle or 1 nM activin A for 24 hours. In all panels, RNA was extracted and gene expression was assessed by RT-qPCR. Bars represent the means (+SEM) of three independent experiments. In (A) and (B), basal Fshb expression levels (black bars) were analyzed by one-way ANOVA followed by Dunn multiple comparisons test. Data in (C) and (D) were analyzed by two-way ANOVA followed by Sidak post hoc tests. Bars with different symbols (^, ^&, or *) differ significantly. ^#Significantly different from the other groups; activin A stimulation on Fshb expression in each group was analyzed by Student t test (red vs black bars). *P < 0.05. n.s., not significant; TAM, tamoxifen.