An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility

Emergy-Based Sustainability Evaluation of the Mulberry-Dyke and Fish-Pond System on the South Bank of Taihu Lake, China

The Taihu Lake drainage basin is the birthplace of the Mulberry-dyke and Fish-pond System (MFS), a traditional eco-agricultural system. In 2017, the largest and best-preserved “Zhejiang Huzhou Mulberry-dyke and Fish-pond System” located by the South Bank of Taihu Lake, China was recognized as Globally Important Agricultural Heritage Systems (GIAHS) by the Food and Agriculture Organization of the United Nations (FAO), and its value has been appreciated. As a dynamic heritage, the sustainable development of MFS is a fundamental requirement of the conservation of GIAHS. In this regard, it is necessary to figure out an approach to evaluating the status of its sustainable development. This paper analyzes and contrasts the emergy embodied in the three patterns of MFS over different periods, then constructs an index system of sustainability evaluation involving the production and consumption processes based on that. Finally, it provides the evaluation and analysis. The three patterns of MFS differ in the system structure. In the Ming and Qing Dynasties (abbreviated as Ming-Qing pattern), MFS was an integrated system compromised of mulberry cultivation, silkworm breeding, fish breeding, and sheep breeding, while other patterns exclude sheep breeding, but increase the input of fertilizer, and add the production of mulberry-leaf tea and other local specialties. The results show that the MFS in the Ming-Qing pattern has the highest integrated evaluation index of sustainable development, followed by the traditional MFS pattern and the new MFS pattern employed nowadays. This indicates that the current capability of sustainable development has decreased compared to that in the Ming and Qing Dynasties. The integrated evaluation index regarding the consumption process of the new MFS pattern is higher than the traditional one, suggesting that it needs to promote sustainability in the production process, especially via the utilization rates of renewable resources and wastes

Heat shock protein 90 has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-responsive sperm function in human sperm.

Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function

Addition of Organic Acids and Lactobacillus acidophilus to the Leguminous Forage Chamaecrista rotundifolia Improved the Quality and Decreased Harmful Bacteria of the Silage

This study aimed to investigate the effects of citric acid, malic acid, and Lactobacillus acidophilus (L) on fermentation parameters and the microbial community of leguminous Chamaecrista rotundifolia silage. Fresh C. rotundifolia was treated without any additive (CK), or with L (106 CFU/g fresh weight), different levels (0.1, 0.3, 0.5, and 1% fresh weight) of organic acid (malic or citric acid), and the combinations of L and the different levels of organic acids for 30, 45, and 60 days of ensiling. The effects of malic acid and citric acid were similar during the ensiling process. Treatment with either citric or malic acid and also when combined with L inhibited crude protein degradation, lowered pH and ammonia nitrogen, and increased lactic acid concentration and dry matter content (p < 0.05). The neutral detergent fiber and acid detergent fiber increased initially and then decreased with fermentation time in all treatments (p < 0.05). Increasing the level of organic acid positively affected the chemical composition of C. rotundifolia silage. In addition, the addition of 1% organic acid increased the relative abundance of Lactobacillus, while the relative abundances of Clostridium and Enterobacter decreased at 60 days (p < 0.05). Moreover, both organic acids and combined additives increased (p < 0.05) the relative abundance of Cyanobacteria at 60 days of fermentation. We concluded that adding malic acid, citric acid, and L combined with an organic acid could improve the quality of C. rotundifolia silage and increase the relative abundance of beneficial bacteria. The addition of organic acid at a level of 1% was the most effective

Mechanism of Cell Wall Polysaccharides Modification in Harvested ‘Shatangju’ Mandarin (<i>Citrus reticulate</i> Blanco) Fruit Caused by <i>Penicillium italicum</i>

Modification of cell wall polysaccharide in the plant plays an important role in response to fungi infection. However, the mechanism of fungi infection on cell wall modification need further clarification. In this study, the effects of Penicillium italicum inoculation on &#8216;shatangju&#8217; mandarin disease development and the potential mechanism of cell wall polysaccharides modification caused by P. italicum were investigated. Compared to the control fruit, P. italicum infection modified the cell wall polysaccharides, indicated by water-soluble pectin (WSP), acid-soluble pectin (ASP), hemicellulose and lignin contents change. P. italicum infection enhanced the activities of polygalacturonase (PG), pectin methylesterase (PME), and the expression levels of xyloglucanendotransglucosylase/hydrolase (XTH) and expansin, which might contribute to cell wall disassembly and cellular integrity damage. Additionally, higher accumulation of reactive oxygen species (ROS) via decreasing antioxidant metabolites and the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) also contributed to the cell wall polysaccharides modification. Meanwhile, the gene expression levels of hydroxyproline-rich glycoprotein (HRGP) and germin-like protein (GLP) were inhibited by pathogen infection. Altogether, these findings suggested that cell wall degradation/modification caused by non-enzymatic and enzymatic factors was an important strategy for P. italicum to infect &#8216;shatangju&#8217; mandarin

Proteomic profiling of 24-epibrassinolide-induced chilling tolerance in harvested banana fruit

The mechanism of 24-epibrassinolide (EBR)-induced chilling tolerance in harvested banana fruit was investigated. Results showed that EBR pretreatment remarkably suppressed the development of chilling injury (CI) in harvested banana fruit during 12 days of cold storage at 8 degrees C, as indicated by lower CI index in treated fruit. Physiological measurements exhibited that EBR treatment reduced the relative electrolyte leakage and malondialdehyde (MDA) content while increased the chlorophyll fluorescence (Fv/Fm), total soluble solids (TSS) and ratio of TSS and titratable acidity. Furthermore, the differentially accumulated proteins of banana fruit in response to EBR and cold treatment were investigated by employing gel-based proteomic in combination with MALDI-TOF-TOF MS and LC-ESI-MS/MS analyses. There were fifty five protein spots to be successfully identified. Notably, most of up-regulated proteins by EBR treatment were related to energy biosynthesis, stress response and cell wall modification. In contrast, proteins involved in protein degradation and energy consumption were down-regulated by EBR treatment. These results suggest that EBR treatment could enhance the defense ability, promote the synthesis and utilization of energy, as well as maintain the protein function via enhancing protein biosynthesis and inhibiting protein degradation, consequently contributing to improvement of cold tolerance in harvested banana fruit. Significance: To extend our understanding of chilling injury (CI) of harvested banana fruit, we reported the effect of 24-epibrassinolide (EBR) on CI of banana fruit when stored at 8 degrees C. It was the first report on the comprehensive proteomic analysis of banana fruit in response to EBR treatment at low temperature. EBR pretreatment significantly reduced CI in harvested banana fruit. Fifty five protein spots were successfully identified. Notably, the most of up-regulated proteins by EBR treatment were related to energy biosynthesis, stress response and cell wall modification. In contrast, proteins involved in protein degradation and energy consumption were down regulated. These results suggest that exogenous EBR treatment could enhance the defense ability and maintain high energy status. Meanwhile, EBR treatment maintained protein function via enhancing protein biosynthesis and inhibiting protein degradation. These results may help us to understand the molecular mechanism of the chilling tolerance induced by EBR treatment and broaden the current knowledge of the mechanism of CI of harvested banana fruit

Increased co-expression of PD1 and TIM3 is associated with poor prognosis and immune microenvironment heterogeneity in gallbladder cancer

Abstract Background The effectiveness of immune checkpoint inhibitors in treating gallbladder cancer (GBC) remains unsatisfactory. Recently, several new immune checkpoints have been identified. However, investigations exploring these immune checkpoints in GBC are limited. In this study, we aim to investigate the expression patterns and clinical implications of various immune checkpoints, and further characterize the spatial and quantitative heterogeneity of immune components in GBC. Methods We employed single and multiplex immunohistochemistry to evaluate the expression of five immune checkpoint markers and four immune cell markers in the primary tumor core, hepatic invasion margin, and liver metastasis. Subsequently, we analyzed their interrelationships and their prognostic significance. Results We observed a robust positive correlation between PD1/TIM3 expression in GBC (R = 0.614, P < 0.001). The co-expression of PD1/TIM3 exhibited a synergistic effect in predicting poor prognosis among postoperative GBC patients. Further analysis revealed that the prognostic significance of PD1/TIM3 was prominent in the subgroup with high infiltration of CD8 + T cells (P < 0.001). Multiplex immunohistochemistry reveals that PD1 + TIM3 + FOXP3 + cells constitute a significant proportion of FOXP3 + TILs in GBC tissue. Moreover, the co-high expression of PD1 and TIM3 is positively correlated with the accumulation of CD8 + TILs at the hepatic invasion margin. Lastly, our findings indicated reduced expression levels of immune checkpoints and diminished immune cell infiltration in liver metastases compared to primary tumors. Conclusions Increased co-expression of PD1/TIM3 is associated with poor prognosis in GBC patients and is related to the heterogeneity of immune microenvironment between GBC primary tumor and its hepatic invasion margin or liver metastases, which may be a potential target for future immunotherapy of GBC

Androgen regulation and developmental change of the HongrES1 expression.

<p>(A) Northern blot analysis of adult rat epididymal RNAs from precastration (0 d) and bilateral castration for 1, 3, 5, and 7 days (1 d, 3 d, 5 d, and 7 d) as well as for 1, 3, 5, and 7 days after the initial injection of testosterone propionate applied to the 7 d-castrated rats (7+1 d, 7+3 d, 7+5 d, and 7+7 d). The total RNAs were pooled from four to seven animals at each time-point. (B) The relative expression levels of HongrES1 mRNA (hybridization density of HongrES1 mRNA/18S ribosomal RNA) in the rat cauda epididymis and the serum testosterone level (expressed in nanomoles per liter) during androgen manipulation (n = 4–7). (C) Northern analysis of HongrES1 mRNA and 18S rRNA during development. The samples were collected at 15 d, 30 d, 45 d, 60 d, 90 d, 120 d, 270 d, 360 d, 450 d and 720 d respectively. RNAs were pooled from three animals per group. (D) Western blot analysis of HongrES1 and β–actin proteins during development. The samples were collected at 15 d, 30 d, 45 d, 60 d, 90 d, 120 d, 270 d, 450 d and 720 d respectively. The total proteins were pooled from three animals each group. (E) The relative amounts of HongrES1 mRNA (hybridization density of HongrES1 mRNA/18S ribosomal RNA) and protein (immunoreactivity density of HongrES1 protein/β–actin protein) in the rat epididymis at different developmental stages (n = 3).</p

The localization of HongrES1 protein in the rat epididymis.

<p>(A) Rabbit polyclonal antisera were raised against HongrES1 recombinant protein (antigen) and the specificity of the antibody towards HongrES1 was verified by Western blotting. (B) Western analysis of HongrES1 protein in total tissue protein extracts from caput (Cap), corpus (Cor) and cauda (Cau) of the epididymis and testis (Te). Blot was probed with monoclonal antibody against GAPDH to assess protein loading. (C) Western analysis of HongrES1 protein in total protein extracts of sperm from caput (Cap), corpus (Cor) and cauda (Cau) regions of the epididymis. β–actin was used for loading control. (D) The change of molecular masses of HongrES1 protein in total tissue protein of cauda epididymis before (−) and after (+) deglycosylation by Peptide N-Glycosidase F (PNGase-F). (E–G) The immunohistochemical staining showed the expression pattern of HongrES1 protein in the whole epididymis (E) and the epithelial cells of the cauda epididymis (F), meanwhile, the immunoreactivity are also detected in the lumen (E and F). Preimmune serum at the same condition showed background level of immunoreactivity (G). (H) The subcellular localization of HongrES1 protein is determined by using cell-specific antibodies. The immunofluorenscence of HongrES1 (FITC-labeled, green) and ATP6E (Rhodamine labeled for clear cells, red) are shown by confocal microscopy. (I) The localization of HongrES1 protein on the spermatozoa by indirect influorescence assays. The positive HongrES1 immunoreactivity is localized the head of sperm from cauda (Cau) region, but not from caput (Cap) and corpus (Cor) regions of epididymis. (J) The HongrES1 binding pattern on the whole head of the cauda sperm. Sperm DNA was stained with propidium iodide (PI) in red color.</p