Background: Eukaryotic elongation factor-2 kinase (Eef2k) is a protein kinase associated with the calmodulin-induced signaling pathway and an atypical alpha-kinase family member. Eef2kmediated phosphorylation of eukaryotic translation elongation factor 2 (Eef2) can inhibit the functionality of this protein, altering protein translation. Prior work suggests Eef2k to be overexpressed in breast, pancreatic, brain, and lung cancers wherein it may control key processes associated with apoptosis, autophagy, and cell cycle progression. The functional importance of Eef2k in the testes of male mice, however, has yet to be clarified. Methods: A CRISPR/Cas9 approach was used to generate male Eef2k-knockout mice, which were evaluated for phenotypic changes in epididymal or testicular tissues through histological and immunofluorescent staining assays. In addition, TUNEL staining was conducted to assess the apoptotic death of cells in the testis. Fertility, sperm counts, and sperm motility were further assessed.Results: Male Eef2k-knockout mice were successfully generated, and exhibited normal fertility and development. No apparent differences were observed with respect to spermatogenesis, sperm counts, or germ cell apoptosis when comparing male Eef2k −/− and Eef2k +/+ mice. Conclusions: Male Eef2k-knockout mice remained fertile and were free of any evident developmental or spermatogenic abnormalities, suggesting Eef2k to be dispensable in the context of male fertility.

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Infertilityaffects 10% -15% of familiesworldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. We constructeda mouse model (Pabpn1l-/-) simulatingthe splicing abnormality of human PABPN1Landfound that the female was sterile and the male was fertile. The Pabpn1l-/-oocytes can be produced, ovulated and fertilized normally, but cannot develop beyond the 2-cell stage. UsingRNA-Seq, we found a large-scale upregulation of RNA in Pabpn1l-/-MII oocytes. Of the 2401 transcripts upregulatedin Pabpn1l-/-MII oocytes, 1523transcripts (63.4%) were also upregulatedin Btg4-/-MII oocytes, while only 53transcripts (2.2%) were upregulatedin Ythdf2-/-MII oocytes. We documentedthat transcripts in zygotes derived from Pabpn1l -/-oocytes have a longer poly(A) tail than the control group, aphenomenon similar to that in Btg4-/-mice. Surprisingly,the poly(A) tail of these mRNAswas significantly shorter in the Pabpn1l -/-MII oocytes than in the Pabpn1l +/+. Theseresults suggest that PABPN1L is involved in BTG4-mediated maternal mRNA degradation, and may antagonizepoly(A) tail shortening in oocytesindependentlyof its involvement in maternalmRNA degradation. Thus,PABPN1L variants could be a genetic marker offemale infertility.

Infertility affects 10–15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A recent study showed that poly(A)binding protein nuclear 1-like (PABPN1L) recruited BTG anti-proliferation factor 4 (BTG4) to mRNA 3′-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated a PABPN1L-antibody and found “ring-like” PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L–EGFP proteins spontaneously formed “ring-like” aggregates in vitro. This phenomenon is similar with CCR4–NOT catalytic subunit, CCR4-NOT transcription complex subunit 7 (CNOT7), when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l−/− and Pabpn1l  tm1a/tm1a) simulating the intron 1–exon 2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l−/− MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l−♀/+♂zygote. Both the Btg4 and CCR4-NOT transcription complex subunit 6 like (Cnot6l) genes are necessary for the deadenylation process and Pabpn1l−/− resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4−♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l−♀/+♂zygote were also stabilized in Pabpn1l−♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4–NOT-mediated maternal mRNA degradation and PABPN1L variants on intron 1–exon 2 could be a genetic marker of female infertility.