In mammals, spermatogenesis (the differentiation of male germ cells after meiosis) is a highly coordinated developmental process controlled by a set of genes collectively known as spermatogenesis genes.
Given that nuclear condensation during spermatogenesis gradually stops transcription, spermatogenic genes are transcribed early during the early stages of male reproductive development and stored as translationally inert messenger ribonucleoprotein (mRNP) in developing spermatozoa until they are needed for translation. These translationally inert mRNPs are typically put together to form germ granules, which are storage spaces for untranslated mRNAs in different kinds of germ cells. The process by which those mRNAs contained in translationally dormant mRNP granules are activated during late spermatogenesis is still poorly understood.
To understand how translationally inert mRNAs are activated during spermatogenesis, researchers from the University of Chinese Academy of Sciences, Shanghai University of Science and Technology, Shanghai Jiao Tong University, Wuhan University, Fudan University, and Nanjing Medical University screened for potential translation regulators in a new study by performing proteomic analysis of polysomes from mouse testis. FXR1, a member of the FXR (fragile X-related) protein family, stood out in this screen as a translational regulator of late-stage sperm. The results of the study were published in the August 12, 2022 issue of Science titled “LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs”.
By performing eCLIP and polyribosome analysis in combination with the construction of a germline-specific Fxr1 knockout (Fxr1cko) mouse model, they investigated whether FXR1 is required for translational activation in late spermatozoa.
To decipher the mechanism of FXR1-mediated translational regulation, they used immunoprecipitation and mass spectrometry to identify potential cofactors of FXR1 in mouse testis, observing FXR1 granule formation by liquid-liquid phase separation (LLPS) in late spermatocytes recruiting the translation factor and used the TRICK (translating RNA imaging by coat protein knock-off) reporter system to determine whether FXR1 LLPS is required for translation of target mRNAs in cells cultured in vitro.
To further investigate whether FXR1 LLPS is essential for translation of target mRNAs in mouse sperm, they used lentiviral testis transduction to ectopically express wild-type FXR1, the LLPS-deficient FXR1L351P mutant, and the LLPS-retained FXR1L351P-IDRFUS mutant in the testes of Fxr1cko mice. Finally, by generating germline-specific Fxr1L351P knock-in mice, they determined that FXR1 LLPS is indispensable for translation activation, spermatogenesis, and male fertility in mice with late spermatozoa.
These researchers discovered that testicular polyribosomes enriched for FXR1 at 35 days postnatal compared to 25 days postnatal, indicating a function for FXR1 in the translational activation of late-stage sperm. They determined that mouse germline-specific FXR1 deletion drastically decreased target mRNA translation in late spermatozoa and discovered 770 mRNAs that might be direct targets of FXR1 activation.
Mechanistic studies showed that FXR1 undergoes LLPS to form a lectin that assembles target mRNAs into mRNP particles and then recruits the translation complex to activate mRNAs stored in the mRNP particles.
These results demonstrate a critical role for FXR1 LLPS in the translational activation of mRNAs stored in mouse sperm and in male fertility in mice, indicating that FXR1 is an important translational activator controlling mouse spermatogenesis. This new study also emphasizes the significance of LLPS in in vivo developmental processes.
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