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Ligand of Numb-protein X 2 (LNX2) is an E3 ubiquitin ligase that is known to regulate Notch signaling by participating in NUMB protein degradation. Notch signaling is important for differentiation and proliferation in mammals, and plays a significant role in blastocyst formation during early embryonic development. In this study, we investigated Lnx2 in mouse preimplantation embryos. Expression analysis showed that Lnx2 is expressed in oocytes and preimplantation embryos. Lnx2-knockdown embryos normally progress to the morula stage, but the majority of them do not develop into normal blastocysts. Transcript analysis revealed that the expression levels of genes critical for cell lineage specification, including octamer-binding transcription factor 4 (Oct4), are increased in Lnx2 knockdown embryos. Furthermore, the expression levels of Notch and Hippo signaling-related genes are also increased by Lnx2 knockdown. Collectively, our results show that Lnx2 is important for blastocyst formation in mice, suggest that this may act via lineage specification of inner cell mass, and further show that Lnx2 may be involved in transcriptionally regulating various genes implicated in early embryonic development.

Oxidative stress, caused by the accumulation of reactive oxygen species (ROS) during acute myocardial infarction (AMI), is one of the main factors leading to myocardial cell damage and programmed cell death. Phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation, and apoptosis. Phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1) is an intrinsic inhibitor of PI3K in various tissues, but its functional role during AMI remains unknown. In this study, the anti-ischemic role of PIK3IP1 in an in vitro AMI setting was evaluated using H9c2 cells. The MTT assay demonstrated that cell viability decreased significantly via treatment with H2O2 (200–500 μM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 μM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase-3 were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced and PI3K-mediated apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. Our results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. Taken together, these findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interaction with the ETA-PI3Kr-AKT axis.

Male reproductive aging, or andropause, is associated with gradual age-related changes in testicular properties, sperm production, and erectile function. The testis, which is the primary male reproductive organ, produces sperm and androgens. To understand the transcriptional changes underlying male reproductive aging, we performed transcriptome analysis of aging testes in mice. A total of 31,386 mRNAs and 9387 long non-coding RNAs (lncRNAs) were identified in the mouse testes of diverse age groups (3, 6, 12, and 18 months old) by total RNA sequencing. Of them, 1571 mRNAs and 715 lncRNAs exhibited changes in their levels during testicular aging. Most of these aging-related transcripts exhibited slight and continuous expression changes during aging, whereas some (9.6%) showed larger expression changes. The aging-related transcripts could be classified into diverse expression patterns, in which the transcripts changed mainly at 3–6 months or at 12–18 months. Our subsequent in silico analysis provided insight into the potential features of testicular aging-related mRNAs and lncRNAs. We identified testis-specific aging-related transcripts (121 mRNAs and 25 lncRNAs) by comparison with a known testis-specific transcript profile, and then predicted the potential reproduction-related functions of the mRNAs. By selecting transcripts that are altered only between 3 and 18 months, we identified 46 mRNAs and 34 lncRNAs that are stringently related to the terminal stage of male reproductive aging. Some of these mRNAs were related to hormonal regulation. Finally, our in silico analysis of the 34 aging-related lncRNAs revealed that they co-localized with 19 testis-expressed protein-coding genes, 13 of which are considered to show testis-specific or -predominant expression. These nearby genes could be potential targets of cis-regulation by the aging-related lncRNAs. Collectively, our results identify a number of testicular aging-related mRNAs and lncRNAs in mice and provide a basis for the future investigation of these transcripts in the context of aging-associated testicular dysfunction.