Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis

Abstract

The PRL phosphatases are oncogenic when overexpressed but their in vivo biological function is less well understood. Previous gene deletion study revealed a role for PRL2 in spermatogenesis. We report here the first knockout mice lacking PRL1, the most related homolog of PRL2. We found that loss of PRL1 does not affect spermatogenesis and reproductive ability of male mice, likely due to functional compensation by the relatively higher expression of PRL2 in the testes. However, PRL1^-/-/PRL2^+/- male mice show testicular atrophy phenotype similar to PRL2^-/- mice. More strikingly, deletion of one PRL1 allele in PRL2^-/- male mice causes complete infertility. Mechanistically, the total level of PRL1 and PRL2 is negatively correlated with the PTEN protein level in the testis and PRL1^+/-/PRL2^-/- mice have the highest level of PTEN, leading to attenuated Akt activation and increased germ cell apoptosis, effectively halting spermatozoa production. These results provide the first evidence that in addition to PRL2, PRL1 is also required for spermatogenesis by downregulating PTEN and promoting Akt signaling. The ability of the PRLs to suppress PTEN expression underscores the biochemical basis for their oncogenic potential.

Figure 1. Generation of PRL1^−/−/PRL2^+/− and PRL1^+/−/PRL2^−/− mice.

(A) PCR strategy for genotyping using mouse tail DNA. (B) Endogenous PRL1 and PRL2 protein products were determined by Western blot on lung lysates from different genotypes. (C,D) Body weights of 4-weeks-old male mice (C) or female mice (D) with different genotypes were determined. Body weights of PRL2^−/−, PRL1^−/−/PRL2^+/− and PRL1^+/−/PRL2^−/− males and females were significantly less than those of wild-type and PRL1^−/− males and females. Body weights of PRL1^−/−/PRL2^+/− and PRL1^+/−/PRL2^−/− males were also significantly less than those of PRL2^−/− males. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).

Figure 2. PRL1 also contributes to the normal testis development.

(A) Representative testis picture from five different genotypes. (B,C) Testis weights (B) or testis weight/body weight ratios (C) from five different genotypes were determined. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).

Figure 3. Deletion one PRL1 allele in PRL2^−/− results in male infertility due to no sperm production.

(A) Sexual desire was measured by plug rate. (B,C) Reproductive performance was measured by pregnancy rate (B) and little size (C). (D) The sperm number was determined by calculating the sperms from epididymis. (E) Caudal epididymis sections from 3 months old male mice were histologically examined by H&E staining. Scale bar = 50 μm. (F) Electron microscopic analysis of epididymal sperm from the indicated genotypes. The typical “9 + 2” microtubule structure indicates the transverse section of the sperm. Scale bar = 500 nm. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).

Figure 4. PRL1 also plays a role in maintaining spermatogenesis.

(A) Testis sections from 3 months or 2 weeks old male mice were histologically examined by H&E staining and immunohistochemical staining for Vimentin, PLZF and Kit. (B–E) Relative seminiferous tubule diameters (B), number of Vimentin positive cell per seminiferous tubule (C), number of c-Kit positive cell per tubule (D) and number of PLZF positive cell per tubule (E) in testis sections from all five genotypes were quantificated. For each mouse, at least 20 tubules were counted. *p < 0.05, **p < 0.01 (Student’s t test). Data are representative of at least three independent experiments (mean and SD). Scale bar = 50 μm.

Figure 5. PRL1 is also required to prevent germ cells from apoptosis.

(A) Testis sections from 3 month old male mice were histologically examined by immunohistochemical staining for Cleaved PARP and PCNA. (B,C) Number of cleaved-PARP positive cell per field (B) and percentage of PCNA positive cells per tubule (C) in testis sections from five genotypes were determined. For each mouse, at least 10 fields or 20 tubules were counted. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD). Scale bar = 50 μm.

Figure 6. Total PRL1 and PRL2 negatively correlated with PTEN.

(A) Alterations of key signaling pathway molecules in testis from different genotypes by Western blot. (B) Upper panel: germ cells isolated from testis of male mice with different genotypes were analyzed for PTEN, pAkt, Akt, Actin, PRL1 and PRL2 by Western blot. Lower panel: relative level of either PTEN/Actin, pAkt/Akt or total PRL1 and PRL2/Actin in isolated germ cells from different genotypes. (C) PTEN immunohistochemistry analysis in seminiferous tubules of testis with different genotypes. (D) Quantification of PTEN IHC staining by ImageJ IHC Image Analysis Toolbox. *p < 0.05 (Student’s t test) compared to wild-type; #p < 0.05 (Student’s t test) compared to PRL2^−/− and PRL1^−/−/PRL2^+/−. Scale bar = 50 μm.