Research ArticleFertility

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

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Science Signaling  26 Mar 2019:
Vol. 12, Issue 574, eaao7232
DOI: 10.1126/scisignal.aao7232

Protamine dephosphorylation for fertility

During the final stage of spermatogenesis, protamines tightly package DNA in the mature sperm. Itoh et al. generated mice deficient in the heat shock protein and chaperone Hspa4l, which is implicated in spermatogenesis. The mice were infertile with malformed sperm heads, a phenotype similar to that of mice deficient in the phosphatase Ppp1cc2. The authors showed that Hspa4l was required to release Ppp1cc2 from a complex with other chaperones, enabling its translocation to chromatin. In vitro studies showed that Ppp1cc2 dephosphorylated protamine 2 at Ser56. Expression of the unphosphorylatable protamine 2 S56A mutant reversed the infertility of Hspa4l-deficient mice, suggesting that the dephosphorylation of protamine 2 at Ser56 is important for its role in sperm maturation.


The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l−/−; Prm2S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l−/− mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

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