摘要: DNA double-strand breaks (DSBs) are highly toxic lesions that can cause genomic instability and can be repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. Despite extensive studies about DSB repair pathways, the roles of each pathway during meiotic maturation in oocytes are not well understood. Here we show that oocytes selectively utilize NHEJ and HR to repair DSBs during meiotic maturation. Inhibition of NHEJ impaired the meiotic maturation of oocytes with DNA damage by activating the spindle assembly checkpoint (SAC) with a concomitant increase in metaphase I (MI) arrest and DNA damage levels. In contrast, oocytes with DNA damage bypassed SAC-mediated MI arrest despite the presence of fragmented DNA when HR was inhibited. Notably, this bypass of SAC arrest by HR inhibition was associated with a loss of centromere integrity and subsequent impairment of chromosome architecture. Our results demonstrate that, while NHEJ is critical for the meiotic maturation of oocytes with DNA damage, HR is essential to maintain centromere integrity against DNA damage during meiotic maturation, revealing distinct roles of NHEJ and HR during meiotic maturation in mouse oocytes.

摘要: ObjectivesOogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. Materials and MethodsIn this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. ResultsDDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. ConclusionsThis study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.

摘要: The activation of hepatic stellate cells (HSCs) is the main cause of liver fibrogenesis in response to different etiologies of chronic liver injuries. HSCs are heterogeneous, but the lack of specific markers to distinguish different HSC subset hinders the development of targeted therapy for liver fibrosis. In this study, we aim to reveal new HSC subsets by cell fate tracking. We constructed a novel ReelinCreERT2 transgenic mouse model to track the fate of cells expressing Reelin and their progeny (Reelin(+) cells). And we investigated the property of Reelin(+) cells, such as differentiation and proliferation, in hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury models by immunohistochemistry. Our study revealed that Reelin(+) cells were a new HSC subset. In terms of activation, migration, and proliferation, Reelin(+) HSCs displayed different properties from Desmin(+) HSCs (total HSCs) in cholestatic liver injury model but shared similar properties to total HSCs in hepatotoxic liver injury model. Besides, we did not find evidence that Reelin(+) HSCs transdifferentiated into hepatocytes or cholangiocytes through mesenchymal-epithelial transition (MET). In this study, our genetic cell fate tracking data reveal that ReelinCreERT2-labelled cells are a new HSC subset, which provides new insights into targeted therapy for liver fibrosis.

摘要: Previous studies have revealed cellular heterogeneity in intervertebral discs (IVDs). However, the cellular and molecular alteration patterns of cell populations during degenerative progression remain to be fully elucidated. To illustrate the cellular and molecular alteration of cell populations in intervertebral disc degeneration (IDD), we perform single cell RNA sequencing on cells from four anatomic sites of healthy and degenerative goat IVDs. EGLN3(+) StressCs, TGFBR3(+) HomCs and GPRC5A(+) RegCs exhibit the characteristics associated with resistance to stress, maintaining homeostasis and repairing, respectively. The frequencies and signatures of these cell clusters fluctuate with IDD. Notably, the chondrogenic differentiation programme of PROCR+ progenitor cells is altered by IDD, while notochord cells turn to stemness exhaustion. In addition, we characterise CAV1(+) endothelial cells that communicate with chondrocytes through multiple signalling pathways in degenerative IVDs. Our comprehensive analysis identifies the variability of key cell clusters and critical regulatory networks responding to IDD, which will facilitate in-depth investigation of therapeutic strategies for IDD.

摘要: The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium-activated potassium (IKCa) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+](i)), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IKCa expression and an increase in [Ca2+](i). Serum-induced cell proliferation and increase in [Ca2+](i) were suppressed by IKCa inhibition with TRAM-34 or IKCa knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+](i) induced by serum or by IKCa activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.