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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Generation of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ 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 PRL2βˆ’/βˆ’, PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males and females were significantly less than those of wild-type and PRL1βˆ’/βˆ’ males and females. Body weights of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males were also significantly less than those of PRL2βˆ’/βˆ’ males. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5035919&req=5

f1: Generation of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ 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 PRL2βˆ’/βˆ’, PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males and females were significantly less than those of wild-type and PRL1βˆ’/βˆ’ males and females. Body weights of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males were also significantly less than those of PRL2βˆ’/βˆ’ males. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).

Mentions: Given the lower expression of PRL1 relative to PRL2, loss of PRL1 may be compensated by the remaining PRL2, whereas loss of PRL2 cannot be fully rescued by PRL1202122. However, since mice deficient in both PRL1 and PRL2 were embryonic lethal, we hypothesized that PRL1 and PRL2 have similar functions during development and that the combined total level of PRL1 and PRL2 dictates phenotype severity. To test this hypothesis, we then generated PRL1+/βˆ’/PRL2βˆ’/βˆ’ mice and PRL1βˆ’/βˆ’/PRL2+/βˆ’ mice. These mice also displayed a survival disadvantage as evidenced by the reduced birth rate (Table 2). PCR analysis was used to determine the deletion of PRL1 and PRL2 alleles (Fig. 1A). Deletion of the PRL protein was confirmed by Western blot on lung lysates from mice with different genotypes using PRL1/2 antibody (Fig. 1B). To examine the expression pattern of all PRLs, protein lysates from different organs were subjected to Western blot analysis using PRL1/2 and PRL3 antibodies. As shown in Figure S4A, both PRL1 and PRL2 are broadly expressed in all the organs we assayed. However, endogenous PRL3 is only moderately detectable in brain, lung, colon, small intestine, spleen, thymus, muscle and heart, which is consistent with the most recent report9. Given the same affinity and specificity of the PRL1/2 antibody towards PRL1 and PRL2, the intensity of the PRL1 band (upper band) and PRL2 band (lower band) reflects the relative level of endogenous PRL1 and PRL2. Overall, the level of PRL1 protein is lower than PRL2 in the organs we measured, from ~40% of PRL2 in testis to ~5% of PRL2 in muscle (Figure S4B). Supplemental Table 1 lists the combined PRL1 and PRL2 level relative to that of the wild-type in different organs of mice with different genotypes.


Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis
Generation of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ 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 PRL2βˆ’/βˆ’, PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males and females were significantly less than those of wild-type and PRL1βˆ’/βˆ’ males and females. Body weights of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males were also significantly less than those of PRL2βˆ’/βˆ’ males. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035919&req=5

f1: Generation of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ 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 PRL2βˆ’/βˆ’, PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males and females were significantly less than those of wild-type and PRL1βˆ’/βˆ’ males and females. Body weights of PRL1βˆ’/βˆ’/PRL2+/βˆ’ and PRL1+/βˆ’/PRL2βˆ’/βˆ’ males were also significantly less than those of PRL2βˆ’/βˆ’ males. **p < 0.01, ***p < 0.001 (Student’s t test). Data are representative of at least three independent experiments (mean and SD).
Mentions: Given the lower expression of PRL1 relative to PRL2, loss of PRL1 may be compensated by the remaining PRL2, whereas loss of PRL2 cannot be fully rescued by PRL1202122. However, since mice deficient in both PRL1 and PRL2 were embryonic lethal, we hypothesized that PRL1 and PRL2 have similar functions during development and that the combined total level of PRL1 and PRL2 dictates phenotype severity. To test this hypothesis, we then generated PRL1+/βˆ’/PRL2βˆ’/βˆ’ mice and PRL1βˆ’/βˆ’/PRL2+/βˆ’ mice. These mice also displayed a survival disadvantage as evidenced by the reduced birth rate (Table 2). PCR analysis was used to determine the deletion of PRL1 and PRL2 alleles (Fig. 1A). Deletion of the PRL protein was confirmed by Western blot on lung lysates from mice with different genotypes using PRL1/2 antibody (Fig. 1B). To examine the expression pattern of all PRLs, protein lysates from different organs were subjected to Western blot analysis using PRL1/2 and PRL3 antibodies. As shown in Figure S4A, both PRL1 and PRL2 are broadly expressed in all the organs we assayed. However, endogenous PRL3 is only moderately detectable in brain, lung, colon, small intestine, spleen, thymus, muscle and heart, which is consistent with the most recent report9. Given the same affinity and specificity of the PRL1/2 antibody towards PRL1 and PRL2, the intensity of the PRL1 band (upper band) and PRL2 band (lower band) reflects the relative level of endogenous PRL1 and PRL2. Overall, the level of PRL1 protein is lower than PRL2 in the organs we measured, from ~40% of PRL2 in testis to ~5% of PRL2 in muscle (Figure S4B). Supplemental Table 1 lists the combined PRL1 and PRL2 level relative to that of the wild-type in different organs of mice with different genotypes.

View Article: PubMed Central - PubMed

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&minus;/&minus;/PRL2+/&minus; male mice show testicular atrophy phenotype similar to PRL2&minus;/&minus; mice. More strikingly, deletion of one PRL1 allele in PRL2&minus;/&minus; 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+/&minus;/PRL2&minus;/&minus; 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.

No MeSH data available.