Chunling Shen

Prss37 Is Required for Male Fertility in the Mouse

ABSTRACT In order to understand the mechanisms of mammalian fertilization, studies using genetically manipulated animals have provided us with plenty of interesting and valuable information on the genetic factors affecting male fertility. In the present work, we demonstrate for the first time that Prss37, a previously uncharacterized putative trypsin-like serine protease, is required for male fertility. Prss37 is highly and exclusively expressed in the testis of adult mice, especially in the elongating spermatids during spermiogenesis, and almost vanishes in the mature sperm of mice. Mice deficient for Prss37 show male infertility, but their mating activity, spermatogenesis, sperm morphology, and motility remain unaffected. In vivo fertilization assays revealed that Prss37−/− mice exhibited a markedly decreased fertilization rate (2.3% vs. 70% of that in control mice) accompanied by the defect in sperm migration from uterus into oviduct. In vitro study further showed sperm were incapable of sperm-egg recognition/binding when zona-intact eggs were exposed to Prss37−/− sperm, in which mature Adam3 was completely undetectable. Interestingly, however, Prss37−/− sperm were able to fertilize cumulus-intact oocytes in vitro. These data clearly indicate that Prss37 deficiency causes the absence of mature Adam3 in sperm and a defect in sperm migration from uterus into oviduct, which mainly accounts for male infertility of Prss37-null mice, while the defect in sperm-zona binding seems irrelevant to the fertilizing ability of Prss37−/− sperm.

A point mutation in Fgf9 impedes joint interzone formation leading to multiple synostoses syndrome

Human multiple synostoses syndrome (SYNS) is an autosomal dominant disorder characterized by multiple joint fusions. We previously identified a point mutation (S99N) in FGF9 that causes human SYNS3. However, the physiological function of FGF9 during joint development and comprehensive molecular portraits of SYNS3 remain elusive. Here, we report that mice harboring the S99N mutation in Fgf9 develop the curly tail phenotype and partially or fully fused caudal vertebrae and limb joints, which mimic the major phenotypes of SYNS3 patients. Further study reveals that the S99N mutation in Fgf9 disrupts joint interzone formation by affecting the chondrogenic differentiation of mesenchymal cells at the early stage of joint development. Consistently, the limb bud micromass culture (LBMMC) assay shows that Fgf9 inhibits mesenchymal cell differentiation into chondrocytes by downregulating the expression of Sox6 and Sox9. However, the mutant protein does not exhibit the same inhibitory effect. We also show that Fgf9 is required for normal expression of Gdf5 in the prospective elbow and knee joints through its activation of Gdf5 promoter activity. Signal transduction assays indicate that the S99N mutation diminishes FGF signaling in developmental limb joints. Finally, we demonstrate that the conformational change in FGF9 resulting from the S99N mutation disrupts FGF9/FGFR/heparin interaction, which impedes FGF signaling in developmental joints. Taken together, we conclude that the S99N mutation in Fgf9 causes SYNS3 via the disturbance of joint interzone formation. These results further implicate the crucial role of Fgf9 during embryonic joint development.

Nhe5 deficiency enhances learning and memory via upregulating Bdnf/TrkB signaling in mice

Nhe5, a Na+/H+ exchanger, is predominantly expressed in brain tissue and is proposed to act as a negative regulator of dendritic spine growth. Up to now, its physiological function in vivo remains unclear. Here we show that Nhe5‐deficient mice exhibit markedly enhanced learning and memory in Morris water maze, novel object recognition, and passive avoidance task. Meanwhile, the pre‐ and post‐synaptic components, synaptophysin (Syn) and post‐synaptic density 95 (PSD95) expression levels were found increased in hippocampal regions lacking of Nhe5, suggesting a possible alterations in neuronal synaptic structure and function in Nhe5−/− mice. Further study reveals that Nhe5 deficiency leads to higher Bdnf expression levels, followed by increased phosphorylated TrkB and PLCγ levels, indicating that Bdnf/TrkB signaling is activated due to Nhe5 deficiency. Moreover, the corresponding brain regions of Nhe5−/− mice display elevated ERK/CaMKII/CREB phosphorylation levels. Taken together, these findings uncover a novel physiological function of Nhe5 in regulating learning and memory, further implying Nhe5 as a potential therapeutic target for improving cognition.

Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice.

Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compounds. Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell (HSC) activation, attenuates collagen I (II), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (Tgf-β1) expression. The results indicate an important role of Rdh13 and suggest RDH13 as a possible new therapeutic target for CCl4-induced fibrosis.

Establishment, characterization, and application of pAcr-SP-NTP-EGFP transgenic mice in visualizing the oviduct-migrating ability of sperm from Prss37-null mice

Transgenic mouse model with fluorescently labeled sperm has extensive application value. It is an auxiliary tool for investigating the mechanism of fertilization, especially for visualizing the oviduct-migrating ability of sperm in vivo. Here, we produced transgenic mouse lines whose sperm were tagged with enhanced green fluorescent protein (EGFP) according to the previously described method. Polymerase chain reaction analysis of tail-tip genomic DNA identified 13 founders, of which 5 male founders produced offspring to form transgenic lines. We showed that EGFP was testis-specifically expressed, sharing similar expression pattern with endogenous acrosin. It has luminal side restricted distribution in seminiferous tubules and acrosomal aggregation in mature sperm. In addition, interstrain hybridization obtained Prss37(-/-)EGFP(tg/+) males produced sperm with impaired oviduct-migrating ability as visualized under fluorescence microscope, compared with Prss37(+/+)EGFP(tg/+) counterparts. These results indicate that a transgenic mouse model with fluorescently labeled sperm has been successfully established and it is a useful tool for evaluating the oviduct-migrating ability of sperm.

Lacking of palladin leads to multiple cellular events changes which contribute to NTD

BackgroundThe actin cytoskeleton-associated protein palladin plays an important role in cell motility, morphogenesis and adhesion. In mice, Palladin deficient embryos are lethal before embryonic day (E) 15.5, and exhibit severe cranial neural tube and body wall closure defects. However, the mechanism how palladin regulates the process of cranial neural tube closure (NTC) remains unknown.MethodsIn this paper, we use gene knockout mouse to elucidate the function of palladin in the regulation of NTC process.ResultsWe initially focuse on the expression pattern of palladin and found that in embryonic brain, palladin is predominantly expressed in the neural folds at E9.5. We further check the major cellular events in the neural epithelium that may contribute to NTC during the early embryogenesis. Palladin deficiency leads to a disturbance of cytoskeleton in the neural tube and the cultured neural progenitors. Furthermore, increased cell proliferation, decreased cell differentiation and diminished apical cell apoptosis of neural epithelium are found in palladin deficient embryos. Cell cycle of neural progenitors in Palladin-/- embryos is much shorter than that in wt ones. Cell adhesion shows a reduction in Palladin-/- neural tubes.ConclusionsPalladin is expressed with proper spatio-temporal pattern in the neural folds. It plays a crucial role in regulating mouse cranial NTC by modulating cytoskeleton, proliferation, differentiation, apoptosis, and adhesion of neural epithelium. Our findings facilitate further study of the function of palladin and the underlying molecular mechanism involved in NTC.

Rdh13 deficiency weakens carbon tetrachloride-induced liver injury by regulating Spot14 and Cyp2e1 expression levels

Mitochondrion-localized retinol dehydrogenase 13 (Rdh13) is a short-chain dehydrogenase/reductase involved in vitamin A metabolism in both humans and mice. We previously generated Rdh13 knockout mice and showed that Rdh13 deficiency causes severe acute retinal light damage. In this study, considering that Rdh13 is highly expressed in mouse liver, we further evaluated the potential effect of Rdh13 on liver injury induced by carbon tetrachloride (CCl4). Although Rdh13 deficiency showed no significant effect on liver histology and physiological functions under regular culture, the Rdh13–/– mice displayed an attenuated response to CCl4-induced liver injury. Their livers also exhibited less histological changes and contained lower levels of liver-related metabolism enzymes compared with the livers of wild-type (WT) mice. Furthermore, the Rdh13–/– mice had Rdh13 deficiency and thus their liver cells were protected from apoptosis, and the quantity of their proliferative cells became lower than that in WTafter CCl4 exposure. The ablation of Rdh13 gene decreased the expression levels of thyroid hormone-inducible nuclear protein 14 (Spot14) and cytochrome P450 (Cyp2e1) in the liver, especially after CCl4 treatment for 48 h. These data suggested that the alleviated liver damage induced by CCl4 in Rdh13–/– mice was caused by Cyp2e1 enzymes, which promoted reductive CCl4 metabolism by altering the status of thyroxine metabolism. This result further implicated Rdh13 as a potential drug target in preventing chemically induced liver injury.

Tafa-2 plays an essential role in neuronal survival and neurobiological function in mice

Abstract Tafa is a family of small secreted proteins with conserved cysteine residues and restricted expression in the brain. It is composed of five highly homologous genes referred to as Tafa-1 to -5. Among them, Tafa-2 is identified as one of the potential genes responsible for intellectual deficiency in a patient with mild mental retardation. To investigate the biological function of Tafa-2 in vivo, Tafa-2 knockout mice were generated. The mutant mice grew and developed normally but exhibited impairments in spatial learning and memory in Morris water maze test and impairments in short- and long-term memory in novel object recognition test, accompanied with increased level of anxiety-like behaviors in open-field test and elevated plus maze test, and decreased level of depression-like behaviors in forced-swim test and tail-suspension test. Further examinations revealed that Tafa-2 deficiency causes severe neuronal reduction and increased apoptosis in the brain of Tafa-2−/− mice via downregulation of PI3K/Akt and MAPK/Erk pathways. Conformably, the expression levels of CREB target genes including BDNF, c-fos and NF1, and CBP were found to be reduced in the brain of Tafa-2−/− mice. Taken together, our data indicate that Tafa-2 may function as a neurotrophic factor essential for neuronal survival and neurobiological functions.

Ablation of Tacr2 in mice leads to gastric emptying disturbance

Tacr2 is one of the G protein‐coupled receptors(GPCRs) that mediate the biological actions of tachykinins. It is abundantly expressed in the gastrointestinal (GI) system and is thought to play an important role in GI motility, secretion, and visceral sensitivity. Previously, the physiological and pathophysiological functions of Tacr2 were mainly studied using Tacr2 selective agonists or antagonists. Here, we seek to investigate the effect of Tacr2 disruption in mice to provide further insights.