Mutation of S461, in the GOLGA3 phosphorylation site, does not affect mouse spermatogenesis.

Background: Golgin subfamily A member 3 (Golga3), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites, is involved in protein transport, cell apoptosis, Golgi localization, and spermatogenesis. Although it has been previously reported that nonsense mutations in Golga3 cause multiple defects in spermatogenesis, the role of Golga3 in the testis is yet to be clarified. Methods: Immunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. Golga3 S461L/S461Lmice were generated using cytosine base editors. Fertility tests as well as computer-assisted sperm analysis (CASA) were then performed to investigate sperm motility within caudal epididymis. Histological and immunofluorescence staining were used to analyze testis and epididymis phenotypes and TUNEL assays were used to measure germ cell apoptosis in spermatogenic tubules. Results: Immunofluorescence co-localization showed reduced Golgi localization of GOLGA3S465L with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3S465L, confirming S461 as the phosphorylation site. Golga3 is an evolutionarily conserved gene and Golga3 S461L/S461Lmice were successfully generated using cytosine base editors. These mice had normal fertility and spermatozoa, and did not differ significantly from wild-type mice in terms of spermatogenesis and apoptotic cells in tubules. Conclusions: Golga3 was found to be highly conserved in the testis, and GOLGA3 was shown to be involved in spermatogenesis, especially in apoptosis and Golgi complex-mediated effects. Infertility was also observed in Golga3 KO male mice. Although GOLGA3S465Lshowed reduced localization in the Golgi with some expression in the cytoplasm, this abnormal localization did not adversely affect fertility or spermatogenesis in male C57BL/6 mice. Therefore, mutation of the S461 GOLGA3 phosphorylation site did not affect mouse spermatogenesis.

PABPN1L assemble into "ring-like" aggregates in the cytoplasm of MII oocytes and is associated with female 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.

Stable DNA Aptamer-Metal-Organic Framework as Horseradish Peroxidase Mimic for Ultra-Sensitive Detection of Carcinoembryonic Antigen in Serum.

Carcinoembryonic antigen (CEA) is an important broad-spectrum tumor marker. For CEA detection, a novel type of metal-organic framework (MOF) was prepared by grafting CEA aptamer-incorporated DNA tetrahedral (TDN) nanostructures into PCN-222 (Fe)-based MOF (referred as CEAapt-TDN-MOF colloid nanorods). The synthesized CEAapt-TDN-MOF is a very stable detection system due to the vertex phosphorylated TDN structure at the interface, possessing a one-year shelf-life. Moreover, it exhibits a significant horseradish peroxidase mimicking activity due to the iron porphyrin ring, which leads to a colorimetric reaction upon binding toward antibody-captured CEA. Using this method, we successfully achieved the highly specific and ultra-sensitive detection of CEA with a limit of detection as low as 3.3 pg/mL. In addition, this method can detect and analyze the target proteins in clinical serum samples, effectively identify the difference between normal individuals and patients with colon cancer, and provide a new method for the clinical diagnosis of tumors, demonstrating a great application potential.