Exploring the Protective Effects and Mechanism of Huaji Jianpi Decoction against Nonalcoholic Fatty Liver Disease by Network Pharmacology and Experimental Validation.

This paper was designed to predict the mechanisms of the active components of Huaji Jianpi Decoction (HJJPD) against nonalcoholic fatty liver disease (NAFLD) based on network pharmacology-combined animal experiments. The candidate compounds of HJJPD and its relative targets were obtained from TCMSP and PharmMapper web server, and the intersection genes for NAFLD were discerned using OMIM, GeneCards, and DisGeNET. Then, the target protein-protein interaction (PPI) and component-target-pathway networks were constructed. Moreover, gene function annotation (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to study the potential signaling pathways associated with HJJPD's effect on NAFLD. Molecular docking simulation was preformed to validate the binding affinity between potential core components and key targets. Eventually, the candidate targets, the possible pathway, and the mechanism of HJJPD were predicted by the network pharmacology-based strategy, followed by experimental validation in the NAFLD mice model treated with HJJPD. A total of 55 candidate compounds and 36 corresponding genes were identified from HJJPD that are associated with activity against NAFLD, and then the network of them was constructed. Inflammatory response and lipid metabolism-related signaling pathways were identified as the critical signaling pathways mediating the therapeutic effect of the active bioactive ingredients on NAFLD. Compared with the model group, the liver wet weight, liver/body ratio, the levels of total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and high-density lipoprotein (HDL) in serum in the HJJPD low-dose (17.52 g/kg·d), medium-dose (35.04 g/kg·d), and high-dose (70.07 g/kg·d) groups significantly decreased (P < 0.05). Light microscope observation shows that HJJPD could control the degree of lipid denaturation of the mouse liver tissue to a great extent. RT-qPCR results show that the mRNA expression levels of peroxisome proliferative activated receptor gamma (PPARG), tumor necrosis factor-α (TNF-α), antiserine/threonine protein kinase 1 (AKT1), and prostaglandin-endoperoxide synthase (PTGS2) in the liver tissues of the three HJJPD groups (17.52 g/kg·d, 35.04 g/kg·d, and 70.07 g/kg·d) were significantly lower than those in the model group (P < 0.05). HJJPD can exert its effect by inhibiting hepatic steatosis and related mRNA expression and decreasing the levels of other liver-related indexes. This study suggested that HJJPD exerted its effect on NAFLD by modulating multitargets with multicompounds through multipathways. It also demonstrated that the network pharmacology-based approach might provide insights for understanding the interrelationship between complex diseases and interventions of HJJPD.

Recent evidence in support of traditional chinese medicine to restore normal leptin function in simple obesity.

Reducing the incidence of obesity is the focus of global attention, and traditional Chinese medicine (TCM) may play an important role in achieving this goal. Numerous studies have shown that most individuals with obesity have leptin resistance, exogenous leptin is ineffective in individuals with obesity, and the effect of leptin decreases with increased serum leptin levels in individuals with obesity. At present, there are many hypotheses regarding the mechanism of leptin resistance, but there is no definite conclusion. TCM has a long history of treating obesity, and single and compound TCM is an effective obesity treatment method. However, TCM's mechanism of action is complex and resists further weight loss drug development. In the last decade, network pharmacology has become an important tool for exploring the mechanism of compound TCMs. In this study, we reviewed the interrelation between TCM obesity treatment and leptin resistance, and network pharmacology studies of TCM intervention in simple obesity revealed that their targets overlap with the leptin pathway. We also summarized TCM pairs that effectively interfere with leptin resistance and their related intervention mechanisms, providing targets for anti-obesity drug development.

Study on the Effect and Mechanism of Huaji Jianpi Decoction on Simple Obesity.

Background: As the major type of obesity in clinical, simple obesity has gained increasing attention in recent years. Depending on the etiology and pathogenesis of simple obesity and combined with clinical practice experience, Huaji Jianpi decoction (HJJPD) was established to invigorate the spleen and eliminate dampness; however, the underlying molecular mechanism is yet unclear. Materials and Methods: A simple obesity mouse model was established by feeding a high-fat diet to the animals, and the related indexes were analyzed. The mice were divided into the normal, positive control (orlistat), and HJJPD high-dose, medium-dose, and low-dose groups. After 6 weeks of administration, the curative effect of HJJPD was observed. Simple obesity is associated with leptin resistance. The leptin signal transduction pathways mainly include the JAK2-STAT3, AMPK-ACC, LepRb-IRS-PI3K-PDE3B-cAMP, and LepRb-SHP2-MAPKs (ERK1/2) pathways. Therefore, the networks of HJJPD acting on these four pathway-related targets were constructed using the network pharmacology method, and the key nodes were identified. Results: After 6 weeks of drug intervention, we found a good therapeutic effect of HJJPD on simple obesity in the mouse model. The biological network analysis showed that HJJPD plays a role in treating leptin resistance in simple obesity by acting on multiple targets in the JAK2-STAT3 pathway via various components. Also, HJJPD can improve leptin resistance in mice by enhancing the binding force of LEP and LEPRB and activating the LEP-mediated JAK2-STAT3 signaling pathway. Conclusion: In this study, animal experiments, network pharmacology, and molecular biology were combined to establish a mouse model of simple obesity, confirm the role of HJJPD in the treatment of simple obesity, and preliminarily reveal the related mechanism. Relevant research results will provide a basis for the treatment of simple obesity and the drug discovery.

Lipidomics Revealed Alteration of the Sphingolipid Metabolism in the Liver of Nonalcoholic Steatohepatitis Mice Treated with Scoparone.

Perturbation in sphingolipid metabolism has been regarded as a risk factor for nonalcoholic steatohepatitis (NASH) development, predisposing to inflammation, insulin resistance, and weight gain. Scoparone can regulate the level of ceramide in primary hepatocytes and effectively ameliorate hepatic inflammation, apoptosis, steatosis, and fibrogenesis in a mice model of NASH. Nevertheless, the potential effects of scoparone in sphingolipid metabolism, which is dysregulated in NASH, have not been explored so far. To uncover the impact of scoparone on sphingolipid metabolism in NASH and potential therapeutic targets for treating NASH, the liver tissue samples were collected and lipidomics analysis based on UPLC-QTRAP-MRM/MS was carried out. The collected raw data was handled with multivariate data treatment to discover the potential biomarkers in sphingolipid metabolism. Compared to the control group, 22 potential sphingolipid biomarkers were discovered in the NASH group, of which 10 were downregulated and 12 were upregulated. Orally administrated scoparone contributed to the reversal of the levels of these potential biomarkers. Ten differential metabolites showed a tendency of recovery compared to the control group and may be potential targets for scoparone to treat NASH. This study indicated that lipidomics can detect the perturbed sphingolipids to unravel the therapeutic effects of scoparone on NASH.

Separation of principal component dihydromyricetin from Ampelopsis grossedentata by high-speed counter-current chromatography and its interaction with corn starch.

Ampelopsis grossedentata (AG) is an industrial crop in the grape family, which has been used as a dual-purpose plant for medicine and tea with high medicinal values. However, little is reported on the separation technology of active components from AG and processing technology of AG products. High-speed counter-current chromatography (HSCCC) was applied to separate the principal component dihydromyricetin (DMY) from AG. DMY is added to starch-based products to improve food quality. The interaction between corn starch (CS) and DMY was investigated to predict and control the structure and function of starch-based foods. Results show that DMY with 97.13% purity was successfully obtained by HSCCC using a solvent system composed of light petroleum-ethyl acetate-methanol-water-trichloroacetic acid (1:3:1:3:0.01, v/v/v/v/v). Fourier-transform infrared spectroscopy (FT-IR) exhibits that the interactions between CS and DMY included hydrogen bond and noncovalent bond. X-ray diffraction (XRD) shows that DMY could increase the relative crystallinity of CS. Low-field nuclear magnetic resonance results (LF-NMR) imply that DMY decreased the spin relaxation time (T2 ) and inhibited the mobility of free water. Atomic force microscopy (AFM) results suggest that DMY changed the surface morphology of CS through hydrogen bond interaction. Moreover, the results of confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) indicate that DMY could enlarge the pores and change the microstructure of CS-DMY complexes. The findings promote the development of industrial CS-based products and utilization of corn crop.

Effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice.

Background: At present, the incidence of obesity is increasing. Several studies have shown that obesity can reduce male fertility by affecting spermatogenesis and semen quality. Traditional Chinese medicine (TCM) has fewer side effects and stable efficacy in the treatment of related diseases. This study aimed to investigate the effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice. Methods: The obese male mice model was constructed by using high-fat diet and the Huaji Jianpi Decoction was processed into an aqueous extract. Mice were allocated in the normal group (n=30) and five different treatment groups (n=50). Huaji Jianpi Decoction was applied in low-, medium- and high-dose [17.52 g/(kg·d), 35.04 g/(kg·d) and 70.07 g/(kg·d), respectively]. The body weight, body fat, testis wet weight, testis coefficient, and routine sperm parameters were detected and analyzed. Meanwhile, transmission electron microscope (TEM) was used to observe testis ultrastructure. reverse-transcription quantitative PCR (RT-qPCR) was used to measure the expression of tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). Results: Compared with normal mice (ND), the testis wet weight and testis coefficient of mice in the blank group were significantly decreased, while the number of mitochondria was observed to be decreased on testis ultrastructure examination, and apoptotic cells and germ cells in the spermatogenic tubules were shed. After Huaji Jianpi Decoction administration, the body fat and blood lipid levels of obese mice were decreased, the testis wet weight and testis coefficient were increased, and semen parameters were increased. Different doses of Huaji Jianpi Decoction could improve testicular weight, sperm density, sperm motility, forward motility, and total sperm motility. Huaji Jianpi Decoction could also downregulate TNF-α and MCP-1 expression and inhibit germ cell apoptosis to improve semen quality. Conclusions: Huaji Jianpi Decoction can improve the semen quality of high-fat diet-induced obese mice by reducing weight and lipid levels.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

Ultrasound assisted aqueous two-phase extraction of polysaccharides from Cornus officinalis fruit: Modeling, optimization, purification, and characterization.

Ultrasound assisted aqueous two-phase extraction of polysaccharides from Cornus officinalis fruit was modeled by response surface methodology (RSM) and artificial neural network (ANN), and optimized using genetic algorithm coupled with ANN (GA-ANN). Statistical analysis showed that the models obtained by RSM and ANN could accurately predict the Cornus officinalis polysaccharides (COPs) yield. However, ANN prediction was more accurate than RSM. The optimum extraction parameters to achieve the highest COPs yield (7.85 ± 0.09)% was obtained at the ultrasound power of 350 W, extraction temperature of 51 ℃, liquid-to-solid ratio of 17 mL/g, and extraction time of 38 min. Subsequently, the crude COPs were further purified via DEAE-52 and Sephadex G-100 chromatography to obtain a homogenous fraction (COPs-4-SG, 33.64 kDa) that contained galacturonic acid, arabinose, mannose, glucose, and galactose in a molar ratio of 34.82:14.19:6.75:13.48:12.26. The structure of COPs-4-SG was also characterized with UV-vis, fourier-transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), scanning electron microscopy (SEM), Congo-red test, and circular dichroism (CD). The findings provide a feasible way for the extraction, purification, and optimization of polysaccharides from plant resources.

Traditional Chinese medicine in COVID-19.

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across the globe, posing an enormous threat to public health and safety. Traditional Chinese medicine (TCM), in combination with Western medicine (WM), has made important and lasting contributions in the battle against COVID-19. In this review, updated clinical effects and potential mechanisms of TCM, presented in newly recognized three distinct phases of the disease, are summarized and discussed. By integrating the available clinical and preclinical evidence, the efficacies and underlying mechanisms of TCM on COVID-19, including the highly recommended three Chinese patent medicines and three Chinese medicine formulas, are described in a panorama. We hope that this comprehensive review not only provides a reference for health care professionals and the public to recognize the significant contributions of TCM for COVID-19, but also serves as an evidence-based in-depth summary and analysis to facilitate understanding the true scientific value of TCM.

Systematic analysis of the mechanism of hydroxysafflor yellow A for treating ischemic stroke based on network pharmacology technology.

In this study, we analyzed the mechanism of hydroxysafflor yellow A (HSYA) for treating ischemic stroke (IS) based on network pharmacology tools, and verified the kernel targets via animal experiments. The targets of HSYA were collected via PharmMapper server and the IS-related targets were searched using Genecards, Online Mendelian Inheritance in Man, Therapeutic Target, and Disgenet databases. The targets identified from the above two steps were overlapped to acquire candidate targets involved in the effects of HSYA for treating IS. Subsequently, the Database for Annotation, Visualization, and Integrated Discovery was used for gene ontology analysis and the Kyoto encyclopedia of genes and genomes pathway analysis. Cytoscape 3.7.1 was applied to establish the component-target-pathway network. Potential core targets were obtained by protein-protein interaction analysis. Furthermore, Autodock Vina was used to identify core genes, and animal experiments was used to verify the expression level of core genes. On the basis of the modified neurologic severity score and the results of 2,3,5-Triphenyltetrazolium chloride and Hematoxylin-eosin staining, we confirmed that HSYA reduced the infarct volume in rats and protected neuronal cells in the hippocampal region after IS. Western blot and immunohistochemical staining showed that HSYA increased the expression of epidermal growth factor receptor, hypoxia inducible factor 1 alpha, and endothelial nitric oxide synthase (P < 0.05). The effects of HSYA on IS are mediated through several targets and pathways related to the regulation of oxidative stress and the renewal of cell and blood vessels while improving post-ischemic brain impairment.

A network pharmacology approach to investigate the mechanism of Shuxuening injection in the treatment of ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuening injection (SXNI), a popular herbal medicine, is an extract of Ginkgo biloba leaves (GBE), and is used to treat ischemic stroke (IS) in China. However, its specific active ingredients and molecular mechanisms in IS remain unclear. AIM OF THE STUDY: The purpose of the research is to identify the main active ingredients in GBE and explore its molecular mechanisms in the treatment of IS. MATERIALS AND METHODS: The main active components of GBE were discerned through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) database, and absorption, distribution, metabolism and excretion (ADME) analysis. The targets related to IS were obtained using Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and Disgenet. We discovered an intersection of genes. Subsequently, protein-protein interaction (PPI) networks were constructed with Cytoscape 3.7.1 and the String database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to analyze the intersection of targets via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8. Built on the above analysis, we made a Compound-Target-Pathway (C-T-P) network. Autodock Vina was used for molecular docking analysis. Maestro 11.9 was used to calculate the root-mean-square deviation (RMSD). Animal experiments were performed to verify the core targets. Triphenyl tetrazolium chloride (TTC) staining was used to calculate the infarct volume in rats. Hematoxylin-eosin (HE) staining was employed to observe the morphology of hippocampal neuron cells. RT-qPCR was applied to detect relative mRNA levels, and protein expression was determined using Western blotting. RESULTS: Molecular docking showed that PTGS2, NOS3 and CASP3 docked with small molecule compounds. According to RT-qPCR and Western blotting, mRNA and protein expression of PTGS2 and CASP3 were up-regulated (P < 0.05), and mRNA and protein levels of NOS3 were down-regulated (P < 0.05). CONCLUSIONS: SXNI can treat IS through multiple targets and routes, and reduce the apoptosis of neuron cells in brain tissue by inhibiting inflammation and regulating the level of oxidative stress, thereby protecting rats brain tissue.

Preliminary investigation of the effects of an FCF inhibitor interference with Septin in the early stage embryos in mice.

The objective of the present study was to investigate the effects of for chlorfenuron (FCF) interference with the septin protein on early stage embryos in mice. The 1­cell embryos were collected and divided into an FCF interference group and a control group. The FCF interference group was cultured in FCF media and the control group was cultured in dimethyl sulphoxide media at 37˚C with 5% CO2 until the desired phase was achieved. Septin2 protein expression was detected using immunofluorescence and western blot analysis. Blastocyst α­tubulin was stained by immunofluorescence to observe the alterations in spindles and microtubules. The rate of early embryo development into blastocysts was significantly reduced following FCF treatment (P<0.05). In the control group, septin2 was observed with a confocal microscope; septin2 was expressed in embryos at all stages and mainly in the blastomeres from the 2­cell stage onwards, with the expression concentrated in the nuclei of the blastomeres as identified by strong fluorescence. In the FCF interference group, septin2 was weakly expressed in the nuclei of blastomeres at the 2­ and 4­cell stages, and in the granulated blastomeres at the 4­ and 8­cell stages. Expression was barely observed in and following the morula. Granulation was observed starting from the 4­ and 8­cell stages. Compared with the control group, the FCF interference group exhibited irregular microtubules, abnormal spindle morphology and disordered chromosome arrangement in the blastocysts. The septin2 protein was expressed throughout the early stage embryo from the 2­cell stage to the blastocyst and localized in the nuclei of blastomeres. When the septin protein experienced interference by the FCF inhibitor, septin2 protein expression was reduced, which simultaneously resulted in abnormal embryonic development, uneven cytoplasmic division, various sizes and a reduced number of blastomeres, granulation in the blastomeres, disordered blastocyst microtubule distribution, spindle shape alterations and an abnormality of chromosome arrangement.

Necessity to Evaluate Epigenetic Quality of the Sperm for Assisted Reproductive Technology.

Spermatogenesis is a highly complex process involving dynamic erasure and establishment of epigenetic modifications. Abnormal epigenetic marks have been shown to result in altered spermatogenesis and even cause male infertility. Assisted reproductive technology, with directly dealing and selecting the morphology qualified sperm, can be applied to treat male infertility, which may carry significant risks of passing on epigenetic defects to next generations. Selecting epigenetic qualified sperm is an effective way to reduce the risk of inheritance between generations. In this review, we mainly assess the risks of sperm with epigenetic defects and their effects on offspring health, to provide some basic information for the quality control of sperm via detecting their epigenetic biomarkers, which is essential for successfully treating male infertility via assisted reproductive technology.

[Research progress in obesity-induced low fertility in males].

The prevalence of obesity in men of reproductive age is globally increasing. Obesity alters the ratio of testosterone to estradiol and the homeostasis of leptin and other hormone levels by interfering with the hypothalamus-pituitary-gonadal axis. In addition, it may change epigenetic modifications and intergenerational transmission, which would affect the health of the offspring. Both of the pathways reduce male fertility, which may be associated with the obesity-induced change in the levels of some hormones and consequently the alteration of epigenetic modifications. This review focuses on the adverse effects of obesity on male fertility by influencing endocrine hormones and epigenetic modifications, and further discusses the effects of endocrine hormones on male fertility by epigenetic modification, aiming to provide some basic data for the prevention and treatment of obesity-related male fertility in clinical practice.

Epigenetic dynamics and interplay during spermatogenesis and embryogenesis: implications for male fertility and offspring health.

Mapping epigenetic modifications and identifying their roles in the regulation of spermatogenesis and embryogenesis are essential for gaining fundamental medical understandings and for clinical applications. More and more evidence has shown that specific epigenetic modifications are established during spermatogenesis, which will be transferred into oocyte via fertilisation, and play an important role in the early embryo development. Defects in epigenetic patterns may increase the risk of abnormal spermatogenesis, fertilisation failure, early embryogenesis abnormality and several other complications during pregnancy. This review mainly discusses the relationship between altered epigenetic profiles and reproductive diseases, highlighting how epigenetic defects affect the quality of sperm and embryo.

[Effects of obesity on global genome DNA methylation and gene imprinting in mouse spermatozoa].

OBJECTIVE: To investigate the influence of high fat diet-induced obesity (HFDIO) on the differentially methylated region (DMR) of the imprinted gene and global genome methylation of sperm DNA. METHODS: We performed bisulfite sequencing on the DMR of the imprinted gene and global genome methylation of sperm DNA in the mouse model of HFDIO. RESULTS: No statistically significant differences were found between the HFDIO model and normal control mice in MEG3-IG (93.73 vs 97.26%, P = 0.252), H19 (98.00 vs 97.83%, P = 0.920), IGF2 (97.34 vs 96.25%, P =0.166), IGF2R (1.43 vs 1.11%, P = 0.695), PEG3 (0.19 vs 0.38%, P = 0.537), MEST (0.23 vs 0.68%, P = 0.315), NNAT (0.31 vs 0.00%, P = 0.134), or SNRPN (1.88 vs 3.13%, P = 0.628). A total of 8 942 DMRs were detected across the sperm genome (P <0.05). Gene functional enrichment analysis indicated that the enriched terms with the largest numbers of genes were the metabolic process (n = 1 482), RNA synthesis (n = 779), and transcription (n = 767). CONCLUSIONS: The methylation level underwent no significant change in the DMRs of the imprinted genes from the mice with HFDIO, but the CG methylation of the genes involved in the metabolic process, RNA synthesis and transcription were significantly altered.

[Association of single nucleotide polymorphisms(SNPs) in Pygo2 coding gene with idiopathic oligospermia and azoospermia].

Male infertility is often associated with a decreased sperm count. Pygo2 gene is expressed in the elongating spermatid when chromatin remodeling occurs, thus it is possible that impairment of Pygo2 function could lead to spermatogenic arrest, reduction of sperm count and subsequent infertility. The aim of this study was to detect mutations in Pygo2 that lead to idiopathic oligospermia and azoospermia in human. DNA was isolated from venous blood from 77 fertile and 195 idiopathic oligospermic or azoospermic men. PCR-sequencing analysis was performed for the 3 coding regions of Pygo2. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2 and Mutation Taster software to determine possible changes in protein structure that could affect phenotype. Of the 195 patients analyzed, sufficient gene sequencing was accomplished for 178 men (30 mild or moderate oligospermic, 57 severe oligospermic and 91 azoospermic men). Three previously reported non-synonymous SNPs were identified in azoospermic and severe oligospermic patients and not in mild and moderate oligozoopermic or normozoospermic men. SNP rs61758740 (M141I) causes the replacement of a hydrophobic amino acid with another hydrophobic amino acid, rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic amino acid and rs141722381 (N261I) causes the replacement of a hydrophilic amino acid with another hydrophobic amino acid. The data predicted by three different software programs showed that SNP rs141722381 results in the damage of tertiary protein structure and thus could be involved in relevant diseases. The study demonstrates that SNPs in the coding region of Pygo2 gene may be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

Sperm selection based on electrostatic charge.

Charge is a fundamental property of all forms of matter that exhibit attraction or repulsion in the presence of another charged particle. This electrokinetic property occurs when the particles exhibiting a net negative or positive charge are subjected to an external electric field that exerts an electrostatic force between them. Sperm surface membranes exhibit varying levels of electrostatic potential that are proportional to the levels of sialic acid residue acquired on the cell surface during maturation. Electrostatic charge-based sperm separation is a recently developed technique that uses an electric field to isolate mature sperm with reduced levels of DNA fragmentation. Two methods for the separation of sperm based on electrostatic charge, the Zeta method and a commercially available electrophoretic method using the SpermSep Cell Sorter 10, are discussed in this chapter including a detailed protocol for sperm separation based on the Zeta method.

Transition of basic protein during spermatogenesis of Fenneropenaeus chinensis (Osbeck, 1765).

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.

[Methylation and acetylation of histones during spermatogenesis].

Spermatogenesis is a highly complex and unique differentiation process. This process involves development of spermatogonia into spermatocytes, formation of haploid spermatids, and maturation of spermatozoa. It features stage- and testis-specific gene expression, mitotic and meiotic divisions, and the histone-protamine transition. The epigenetic modification plays an important role in meiotic recombination, formation of the synaptonemal complex, sister chromatid cohesion, spermiogenesis during postmeiotic stages, gene expression repression, and heterochromatin formation. The mark of the repressive and/or activating histone methylation and acetylation has a defined composition. It not only ensures proper chromosome pairing and successful bivalent segregation but also mediates highly orchestrated expression of meiosis-specific genes. The incorrect histone methylation and/or acetylation during spermatogenesis will directly affect the establishment and maintenance of epigenetic patterns, resulting in abnormal spermatogenic cells and even male infertility. This article is an effort to review the dynamic changes of methylation and acetylation of histones during spermatogenesis, as well as the regulatory mechanism of the enzymes involved in these processes, which provides some basic information for further study of the epigenetic events during spermatogenesis and the prevention of male infertility.