Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis

Abstract

Male patients with Peutz-Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 (LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene (Lkb1(cko)) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1(cko) testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1(cko) testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1(cko) phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1(cko) testicular phenotype. Pten(cko) mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients.

Figure 1.

Conditional deletion of LKB1 in mouse testes. (A) Colocalization of mTOR with β-catenin (a; a marker for adherens junctions; arrowheads), vimentin (b; a marker for Sertoli cells apical extensions; arrowheads) and tyrosinated α-tubulin (c; a marker for Sertoli cell microtubules; arrowheads) by IF. (B) (a and b) Localization of LKB1 in control and Lkb1^cko mutant testes (Bc) by IF. Arrowheads indicate Sertoli cell nuclei and asterisks indicate interstitial Leydig cells. Western blot and semiquantitative analyses of LKB1 in control and mutant testes (Bc). Gross testes from Lkb1 control and mutant mice (Bd). Weight of Lkb1^cko mutant and control testes (Be) and seminal vesicles (Bf). Columns represent the mean in n= 3 testes, bars equal the SEM and asterisks indicate P< 0.05. Bars: 50 μm. Nuclei are stained with DAPI.

Figure 2.

Premature germ cell loss and Sertoli cell only tubules of Lkb1^cko, Tsc1^cko and Tsc2^cko but not in Pten^cko mice testes. (A–F) Histology of control and Lkb1^cko mutant testes by H&E. (G and H) Epididymides from control and mutant mice. Histology of control and Tsc1^cko (I–N) testes. (O) Gross picture of control and Tsc1^cko mutant testes. Arrows in (B and J) indicate vacuolated seminiferous epithelium. Arrows in (E), (F), (M), and (N) are pointing towards Sertoli cells. (P) Weight of adult Tsc1^cko, Tsc2^cko and control testes. Columns represent the mean in n= 3 testes, bars equal the SEM and asterisks indicate P< 0.05. Histology of control, Tsc2^cko (Q–T), and Pten^cko (U–X) mutant testes. (M), (N), (S), (T), (W) and (X) are higher magnification images of the boxed areas in K, L, Q, R, U and V, respectively. Arrowheads in (S), (T), (W) and (X) indicate Sertoli cells. Insets in (Q and U) show adult testes from control and mutant mice. Bars: 50 μm.

Figure 3.

Overactivation of mTOR signaling in Lkb1^cko, Tsc1^cko and Tsc2^cko testes. IHC for mTOR (A–E), pmTOR (F–J), S6 (K–O) and pS6 (P–T) in Lkb1^fl/fl, Lkb1^cko, Tsc1^cko, Tsc2^cko and Pten^cko testes. Black arrowheads in (A), (F), (K), (P) are pointing towards Sertoli cells. (U and X) Vacuolation (black arrowheads) of seminiferous epithelium next to Sertoli cell positive (white arrowhead) for pS6 immunostaining. (X), (Y), and (Z) are higher magnification images of the boxed areas in U, V and W, respectively. Seminiferous tubules in (U–Z) with elongated spermatids (black arrow) are marked by asterisks. Bars: 50 μm.

Figure 4.

Disruption of the Sertoli cell polarity and testicular junctions in Lkb1^cko, Tsc1^cko and Tsc2^cko mice. Expression of pAMPKα (A), TEX14 (B; a marker of testicular intercellular junctions), β-catenin (C), N-cadherin (D; markers of adherens junctions), ZO-1 (E; a marker for tight junctions), tyrosinated α-tubulin (F; a marker for Sertoli cell microtubules), SOX9 (a Sertoli cell nuclear marker)/vimentin (G; a marker for Sertoli apical extensions) and Phalloidin (H) in Lkb1^fl/fl, Lkb1^cko, Tsc1^cko, Tsc2^cko and Pten^cko testes. Arrowheads in (C), (D), (E) indicate positive staining for β-catenin, N-Cadherin and ZO-1 at the site of the blood–testis barrier. Arrowheads in (F) indicate Sertoli cell microtubules. White-dotted lines mark the basement membrane of the seminiferous tubules. Arrowheads in (G) indicate the Sertoli cells apical extensions. Bars: 50 μm. Nuclei are stained with DAPI.

Figure 5.

Proliferation of Sertoli cells in Lkb1^cko, Tsc1^cko and Tsc2^cko adult murine testes. Colocalization of PCNA (a marker for proliferating cells) and SOX9 (a nuclear marker for Sertoli cells) in Lkb1^fl/fl (A) Lkb1^cko, (B) Tsc1^cko, (C) Tsc2^cko, (D) and Pten^cko (E) testes. Arrowheads in (A–E) represent Sertoli cells. White-dotted lines indicate the basement membranes of the seminiferous tubules. Bars: 50 μm.