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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.


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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.
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f6: 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.

Mentions: PI3K/Akt is the major pathway that regulates the proliferation and survival of differentiating spermatogonia and spermatocytes3031, and Akt1 knockout males exhibit impaired reproductive ability due to increased germ cell apoptosis32. Deletion of PRL2 leads to compromised Akt activation due to elevated PTEN21. To evaluate whether deletion of PRL1 also affects the PTEN/PI3K/Akt pathway, we next investigated the signaling pathways in whole testis lysate by Western blot. Similar to what we observed in PRL2−/− mice, the testis from PRL1−/−/PRL2+/− mice also showed increased PTEN level and reduced Akt activation (Fig. 6A). More strikingly, deletion of one PRL1 allele in PRL2−/− mice further elevated the PTEN level and reduced pAkt, leading to more PARP cleavage (Fig. 6A). To further substantiate that the total PRL1 and PRL2 level negatively regulates PTEN expression in testis, we determined the total PRL1/2 and PTEN level as well as Akt activation in isolated germ cells by Western blot from all five genotypes (n = 6 for each genotype, Fig. 6B). Similar to the results from whole testis (Supplemental Table 1), the total PRL1/2 levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 65 ± 7%, 29 ± 6%, 32 ± 6%, and 13 ± 2% of the wild-type controls (100 ± 4%). As expected, relative PTEN/Actin levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 20 ± 23%, 39 ± 33%, 46 ± 34%, and 78 ± 26% higher than that of the wild-type (100 ± 19%). Consistently, the relative pAkt/Akt levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 102 ± 21%, 73 ± 16%, 70 ± 19%, and 45 ± 17% of the wild-type (100 ± 18%). Consistently, immunohistochemistry analysis also revealed elevated PTEN in PRL2−/−, PRL1−/−/PRL2+/− and, most strikingly, PRL1+/−/PRL2−/− testes (Fig. 6C,D). Moreover, re-expression of the PRL2 transgene in PRL1+/−/PRL2−/− testes successfully reduced PTEN level (Figure S5C,F, middle and bottom panels). Collectively, the data indicate that PRL1, like PRL2, is also involved in PTEN downregulation and there is a negative correlation between the total PRL1/2 level and the PTEN level.


Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis
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.
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f6: 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.
Mentions: PI3K/Akt is the major pathway that regulates the proliferation and survival of differentiating spermatogonia and spermatocytes3031, and Akt1 knockout males exhibit impaired reproductive ability due to increased germ cell apoptosis32. Deletion of PRL2 leads to compromised Akt activation due to elevated PTEN21. To evaluate whether deletion of PRL1 also affects the PTEN/PI3K/Akt pathway, we next investigated the signaling pathways in whole testis lysate by Western blot. Similar to what we observed in PRL2−/− mice, the testis from PRL1−/−/PRL2+/− mice also showed increased PTEN level and reduced Akt activation (Fig. 6A). More strikingly, deletion of one PRL1 allele in PRL2−/− mice further elevated the PTEN level and reduced pAkt, leading to more PARP cleavage (Fig. 6A). To further substantiate that the total PRL1 and PRL2 level negatively regulates PTEN expression in testis, we determined the total PRL1/2 and PTEN level as well as Akt activation in isolated germ cells by Western blot from all five genotypes (n = 6 for each genotype, Fig. 6B). Similar to the results from whole testis (Supplemental Table 1), the total PRL1/2 levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 65 ± 7%, 29 ± 6%, 32 ± 6%, and 13 ± 2% of the wild-type controls (100 ± 4%). As expected, relative PTEN/Actin levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 20 ± 23%, 39 ± 33%, 46 ± 34%, and 78 ± 26% higher than that of the wild-type (100 ± 19%). Consistently, the relative pAkt/Akt levels in germ cells isolated from PRL1−/−, PRL2−/−, PRL1−/−/PRL2+/− and PRL1+/−/PRL2−/− mice were 102 ± 21%, 73 ± 16%, 70 ± 19%, and 45 ± 17% of the wild-type (100 ± 18%). Consistently, immunohistochemistry analysis also revealed elevated PTEN in PRL2−/−, PRL1−/−/PRL2+/− and, most strikingly, PRL1+/−/PRL2−/− testes (Fig. 6C,D). Moreover, re-expression of the PRL2 transgene in PRL1+/−/PRL2−/− testes successfully reduced PTEN level (Figure S5C,F, middle and bottom panels). Collectively, the data indicate that PRL1, like PRL2, is also involved in PTEN downregulation and there is a negative correlation between the total PRL1/2 level and the PTEN level.

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.


Related in: MedlinePlus