Concise Synthesis and Fertility-Promoting Activity of Thiamidol

In order to make the synthetic route of Thiamidol more efficient and easier to scale up,a new method was developed.The compound 2,4-dihydroxyacetophenone was dissolved in ethyl acetate and brominated with cupric bromide.After bromination,the mixture was filtered to remove cuprous bromide.The obtained ethyl acetate solution containing 2-bromo-1-(2,4-dihydroxyphenyl)ethanone was directly reacted with(2-methylpropanoyl)thiourea to obtain the precipitate of Thiamidol hydrobromide.The precipitate was neutralized by deacidification and recrystallized to afford Thiamidol with high purity.In addition,in the screening of fertility-promoting substances,Thiamidol was found to be effective in protecting human sperm from motility loss in vitro.This suggests that it may have potential application in fertility protection and promotion.

Comprehensive analysis of mitochondrial dynamic-related genes on their functions and prognostic values for glioblastoma multiforme

Glioblastoma multiforme(GBM)is the most malignant intracranial tumor in adults and its unique pathology leads to limited therapeutic benefits.1,2 Mitochondrial fusion and fission play an important role in carcinogenesis;fragmented mitochondria promote tumor cell proliferation and prolonged mitochondria lead to tumor cell apoptosis.3 Therefore,profiling the function and prognostic value of mitochondrial dynamics-related genes(MDRGs)is of great interest for GBM precision treatment.Here we focused on the expression,function,and genetic alterations of MDRGs and identified new DNA methylation sites being significantly associated with the survival of GBM patients using available data in public databases.

Elemental sulfur upregulated testicular testosterone biosynthesis by associating with altered gut microbiota in mice

Elemental sulfur has been used as a traditional Chinese medicine to treat the late-onset hypogonadism and impotence without a clarified mechanism for many hundreds of years.In the present study,mice were received sulfur or distilled water for 35 days by daily intragastric gavage at a dose of 250 mg/kg body weight.Then,the serum testosterone level and genes associated with testicular testosterone biosynthesis(TTB)were detected.The gut microbiota was also analyzed by 16S rRNA gene sequencing.Serum testosterone level was significantly increased by 291.1%in sulfur-treated mice.The H2S levels in serum and feces were significantly increased.The expression of genes associated with TTB including StAR,p450c17,3β-HSD,and P450scc in testes were significantly upregulated by Sulfur and NaHS,suggesting that sulfur promotes TTB depending on H2S.In addition,sulfur increased the diversity of gut microbiota and the abundance of several bacteria associated with sulfur metabolism,including genus Prevotella,which might be positively associated with serum level of testosterone in boys.Five pathways including bile secretion,carotenoid biosynthesis,lipid biosynthesis proteins,propanoate metabolism,and biosynthesis of type II polyketide products,were identified to associate with sulfur.Together,our results suggested that sulfur upregulated testicular testosterone biosynthesis via H2S,which was associated with alteration of gut microbiota in mice.Our study highlights a mechanism for the treatment of late-onset hypogonadism and impotence by sulfur.

Expansion of the mutant monkey through cloning

Non-human primates with gene-modifications have been considered as the ideal models for some complex human diseases that are extremely difficult to be recapitulated in other animal models, especially for neural disorders, due to the similarity between monkeys and human.

Untargeted metabolomic analysis of pregnant women exposure to perfluorooctanoic acid at different degrees

Perfluorooctanoic acid(PFOA)is a novel type of persistent synthetic organic pollutant,and its exposure on pregnant women can cause some adverse effects,such as pregnancy-induced hypertension,gestational diabetes mellitus,and preeclampsia.Therefore,understanding the metabolic changes caused by PFOA exposure is of great significance to protect pregnant women from its adverse effects.In this study,the metabolomes from the urine samples of pregnant women exposure to PFOA at different degrees were analyzed by GC-MS and LC-MS.The samples in different groups were distinguished and the differential metabolites were screened based on the VIP value,FC,and P-value of each comparison group through multivariate statistical analysis.The pathways related to differential metabolites were searched to reveal the effects of PFOA exposure on metabolic changes in pregnant women at different degrees.Finally,the ROC of differential metabolites was performed,and the differential metabolites with large area under the curve(AUC)values were selected and compared to identify the mutually differential metabolites.Meanwhile,these metabolites were fitted with a multivariable to explore if they could be used to distinguish different groups.The quantitative comparison of mutually differential metabolites revealed that the levels of L-cysteine,glycine,and 5-aminovaleric acid were positively correlated with the degree of PFOA exposure,indicating that different degrees of PFOA exposure could affect the synthesis or degradation of GSH and change the metabolism of oral or intestinal microbiota.Additionally,they may cause oxidative stress and abnormal fat metabolism in pregnant women.

Phosphodiesterase 10A inhibitor PF-2545920 as a prospective agent for the clinical promotion of sperm motility

Phosphodiesterase(PDE)inhibitors can improve sperm motility in patients with asthenozoospermia.However,the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity.We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil.After seminal plasma was discarded,several semen samples were subjected to four treatments(control,PF-2545920,pentoxifylline,and sildenafil)to evaluate their ability to affect motility,viability,and spontaneous acrosome reactions.Intracellular calcium and adenosine triphosphate(ATP),mitochondrial membrane potential,and penetration through viscous medium were assessed by flow cytometry,luciferase,and hyaluronic acid after treatment with PF-2545920.Statistical analyses were performed using the analysis of variance statistical test.PF-2545920 elevated the percentage of motile spermatozoa compared to the control,pentoxifylline,and sildenafil groups at 10μmol l^(-1)(P<0.01).It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions(P<0.05).PF-2545920 also increased mitochondrial membrane potential(P<0.001)and altered intracellular calcium(P<0.05)in a dose-dependent manner,including increasing sperm hyaluronic acid penetrating ability(P<0.05).Therefore,PF-2545920 might be an excellent choiceforstimulatingthe spermmotility.

Downregulated nuclear lncRNA NRON inhibits SHP2/Wnt/β-catenin signaling and cardiomyocyte differentiation during the development of Tetralogy of Fallot

Tetralogy of Fallot(TOF)is the most common cyanotic congenital heart disease and the incidence of late cardiac death in long-term survivors continues to increase.1 So,there is an urgent need to explore the etiology and pathogenesis of TOF.The precise cause of TOF is currently unclear,and exploration of the pathogenesis has focused increasingly in recent years on the roles of noncoding gene products,especially long noncoding RNAs(lncRNAs).

Homozygous loss-of-function mutations in FSIP2 cause male infertility with asthenoteratospermia

Male infertility, as a major issue of human reproduction health, prevents successful natural conception. Asthenoteratospermia mainly presents one or multiple anomalies in head, neck and tail of spermatozoa, and impairs sperm function and motility (Coutton et al., 2015). Recurrent abnormalities of the fibrous sheath lead to multiple morphological abnormaliries of the sperm flagella (MMAF), which is a quite frequent type of asthenoteratospermia in male infertility (Chemes et al., 1987;Ben Khelifa et al, 2014).

LHCGR and ALMS1 defects likely cooperate in the development of polycystic ovary syndrome indicated by double-mutant mice

Polycystic ovary syndrome(PCOS)is a heterogeneous disorder with evidence of polygenetic components,and obesity may be a risk factor for hyperandrogen ism.Previous studies have shown that LHCGR is en riched in the ovary and LHCGR deficiency causes infertility without typical PCOS phenotypes.ALMS1 is implicated in obesity and hyperandrogenism,the common phenotypes among PCOS patients.Through whole-exome sequencing of 22 PCOS families and targeted candidate gene sequencing of additional 65 sporadic PCOS patients,we identified potential causative mutations in LHCGR and ALMS1 in a sibling-pair PCOS family and three sporadic PCOS patients.The expression of LHCGRL638 P in granulosa-like tumor cell line(KGN)cells promoted cyclic adenosine monophosphate production and granulosa cell proliferation,indicating that LHCGRL638 P is an activating mutation.Lhcgr~(L642 P/L642 P)mice showed an irregular estrous cycle,reduced follicles with dynamic folliculogenesis,and increased testosterone(T),estradiol(E2),and dehydroepiandrosterone.Lhcgr~(+/L642 P)AIms1~(+/PB)mice displayed increased T and E2 but decreased late secondary and preovulatory follicles.We showed that activating mutation of LHCGR likely plays important roles in the pathophysiology of PCOS involving abnormal reproductive physiology,whereas ALMS1 deficiency may promote anovulatory infertility via elevated androgens,suggesting that the disturbed LHCGR and ALMS1 cooperatively induce PCOS phenotypes,characterized as anovulation and hyperandrogenemia frequently observed in PCOS patients with obesity.

Master microRNA-222 regulates cardiac microRNA maturation and triggers Tetralogy of Fallot

Dear Editor,Tetralogy of Fallot(TOF)is the most common complex congenital heart disease.Besides gene mutations and copy number variants,altered protein function induced by posttranscriptional or translational regulation also contributes to the onset of TOF.1 MiRNAs are short noncoding RNAs that bind to the 3’-UTR of target mRNAs to repress protein production.However,the causal link between miRNAs and TOF and the underlying mechanism has not been established.

Basic Phenotyping of Male Fertility from 2019 to 2020 at the Human Sperm Bank of Fudan University

The aim of this cross-sectional survey was to analyze the semen parameters of volunteers from the Human Sperm Bank of Fudan University,as well as the related factors influencing these parameters.From January 2019 to December 2020,semen parameters from a total of 5214 men were included in this survey.The Kruskal-Wallis test was used to detect differences associated with several independent variables.A total of 5214 volunteers were included.The volunteers were registered in 33 provinces,autonomous regions,municipalities(including Macao and Taiwan)and 294 prefecture-level cities.The average age of volunteers was 27.40 years.Overall,76.50%of the volunteers had a college education or higher.Volunteers with BMI values of 18.5-23.9 kg/m2 accounted for 60.70%of participants.Semen parameters were significantly different according to season,education level,duration of abstinence,age group and BMI.The basic male fertility phenotypes(semen parameters)showed new trends in the study period,and accurate long-term tracking of male semen parameters will help researchers to better understand the changes in male fertility phenotypes(semen).

Whole exome sequencing and trio analysis to broaden the variant spectrum of genes in idiopathic hypogonadotropic hypogonadism

Dozens of genes are associated with idiopathic hypogonadotropic hypogonadism(IHH)and an oligogenic etiology has been suggested.However,the associated genes may account for only approximately 50%cases.In addition,a genomic systematic pedigree analysis is still lacking.Here,we conducted whole exome sequencing(WES)on 18 unrelated men affected by IHH and their corresponding parents.Notably,one reported and 10 novel variants in eight known IHH causative genes(AXL,CCDC141,CHD7,DMXL2,FGFR1,PNPLA6,POLR3A,and PR0KR2),nine variants in nine recently reported candidate genes(DCAF17,DCC,EGF,IGSF10,NOTCH1,PDE3A,RELN,SLIT2,and TRAPPC9),and four variants in four novel candidate genes for IHH(CCDC88C,CDON,GADL1,and SPRED3)were identified in 77.8%(14/18)of IHH cases.Among them,eight(8/18,44.4%)cases carried more than one variant in IHH-related genes,supporting the oligogenic model.Interestingly,we found that those variants tended to be maternally inherited(maternal with n=17 vs paternal with n=7;P=0.028).Our further retrospective investigation of published reports replicated the maternal bias(maternal with n=46 i^s paternal with n=28;P=0.024).Our study extended a variant spectrum for IHH and provided the first evidence that women are probably more tolerant to variants of IHH-related genes than men.

Carrier Rate Analysis of Single-Gene Disorders Based on 1000 Genome Project and Exome Aggregation Consortium Data

Objective:Screening variants underlying the single-gene disorder in the general population can help reduce the incidences of birth defects.To determine the most prevalent pathogenic variants causing autosomal recessive diseases,we investigated the frequencies of these variants in six major geographic ancestry groups from Exome Aggregation Consortium(ExAC)database and 26 populations from the 1,000 Genome Project,including three Chinese ethnic groups.Methods:We selected 64 autosomal recessive diseases and collected corresponding causal genes and variants from ClinVar for the analysis.The RS(reference single-nucleotide polymorphism)IDs of these variants were used to search the corresponding VCF file from the 1,000 Genomes Project and ExAC databases.We calculated the frequencies of heterozygotes of each disease variants in the 1,000 Genomes Project and ExAC samples and compared the distribution of disease alleles among different populations.Results:Our analysis revealed that 1,151/212 variants were carried by 60,706/2,504 individuals sequenced in the ExAC/1,000 Genomes Project.The average number of autosomal recessive disease alleles carried by samples from ExAC and 1,000 Genomes Project were 0.53 and 0.68,respectively.These disease alleles showed differential distribution among populations,and some disease alleles were significantly enriched in certain ethnic groups.In addition,1-2 main pathogenic variants were identified in each disease.Meanwhile,several ClinVar variants with relatively high frequency(>1%)in the samples were found to be benign instead of“conflicting evaluations of pathogenicity.”Conclusions:Our observations revealed that main pathogenic variants existed in certain autosomal recessive disease,suggesting that screening of disease hypermutations in different populations is valuable in reducing the occurrence of birth defects.

Monoclonal Antibodies Reveal Novel Localization of SPAG11E in Spermatozoa and the Antifertility Potential of SPAG11E Motifs

Objective: SPAG11E is the first β-defensin that has been reported to activate Ca;uptake and sperm motility. However, the exact subcellular localization and interaction of SPAG11E with sperm remain controversial because of the lack of qualified antibody tools. SPAG11E is also a potential male antifertility target because SPAG11E fragment conjugated with a carrier protein exhibits male contraceptive vaccine potential.However, the fine B-cell epitope motifs of SPAG11E have not been analyzed, which hampered further exploration of the potential target.Methods: Polyclonal and monoclonal antibodies（mcAbs） of mature SPAG11E were raised and qualified with Western blotting. Subcellular localization of SPAG11E was revealed by Western blotting, immunohistochemistry staining, and electron microscopy. B-cell epitopes of rat SPAG11E were mapped by Western blotting using polyclonal and mcAbs. Based on the conservation of the identified epitope motifs between rat and mouse SPAG11E, antifertility potential of the epitope motifs was evaluated by the offspring of the males compromised with specific mcAbs.Results: SPAG11E antibodies of high quality were obtained and all B-cell epitope motifs of rat SPAG 11 E were mapped, in which conserved epitope motifs of SPAG11E in various species were discovered. The epitope motifs recognized by mcAbs were identified respectively. With mcAbs, rat SPAG11E was proved to be expressed in the caput region of epididymis. A novel finding was that SPAG 11 E was located in the flagella and nuclei of sperm as revealed by immunoelectron microscopy. In addition, the males treated with mcAbs（3#-1 and 10#B4） showed apparently fewer offspring.Conclusions: SPAG11E revealed a β-defensin with novel localization in sperm flagellum and nucleus with qualified antibodies. All B-cell epitope motifs of rat SPAG11E were determined, and the antifertility potential was proved by corresponding mcAbs.

Novel compound heterozygous variants in dynein axonemal heavy chain 17 cause asthenoteratospermia with sperm flagellar defects

The axoneme of the human sperm has a very similar ultrastructure to motile cilia,the"9+2"structure,comprising nine peripheral double microtubules plus two central pairs(Nicastro et al.,2006;Ishikawa,2017).A proteomics analysis identified more than700 proteins exclusively in the sperm tail,and abnormal expression of related genes can cause male infertility with multiple morphological abnormalities of flagella(MMAF),including absent,coiled,short,and irregular-caliber flagella(Baker et al.,2013;Coutton et al.,2015).

Pan-Specific Antibodies as Novel Tools to Detect Valyllysine

Objective:Amino acyl modification of lysine residues is an essential mechanism of nutrient sensing that regulates various biological functions including reproduction.At present,the lack of pan-specific antibodies for a recently identified lysine valylation hinders the characterization and detection of this modification.The objective of this study is to raise pan-specific antibodies that may facilitate the identification of novel expression patterns of lysine valylation.Methods:Chicken ovalbumin was valylated as an immunogen to raise polyclonal antibodies(PcAbs)in rabbits.The population of the pan-specific antibodies recognizing valylated lysine was purified using the chemically synthesized valylated peptides consisting of random amino acids.The specificity of the antibodies was evaluated using ELISA,dot blots,Western blots,and immunohistochemistry(IHC)staining in human epididymis as well.Results:A preliminary and simple strategy to make an anti-valylated lysine PcAb was developed.The recognition of the antibodies to valyllysine was evaluated as pan specific.This was useful for the detection of the newly identified valyl modification using ELISA,dot blots,and Western blots.The antibodies were also successfully utilized in IHC assays,which revealed novel valyllysine modification patterns in epididymis tissues of human.Conclusions:A new antibody tool was provided for the study of lysine valylation.The novel expression patterns of valyllysine in the epididymis suggest that this modification may be involved in sperm maturation.

Cyclooxygenase-2 and Decidual Immune Cells

Cyclooxygenase-2(COX-2)is a rate-limiting enzyme in arachidonic acid(AA)metabolism.COX-2 and its products(prostanoids)serve versatile biological functions during pregnancy.Numerous evidences demonstrate special reprogramming of COX-2-catalyzing AA metabolism in decidual immune cells(DICs),particularly in decidual macrophages,corresponding to special gestational phases.This review summarizes the reprogramming of COX-2-catalyzing AA metabolism in DICs as well as the immunoregulation of diverse COX-2-generating prostanoids in DICs during the different phases of gestation.

Rare variants in TULP3 abolish the suppressive effect on sonic hedgehog signaling and contribute to human neural tube defects

To the Editor:Neural tube defects(NTDs)are among the most common human birth defects affecting about 1/1000 live births worldwide.1 The etiology of NTDs is attributed to complex genetic and environmental risk factors.Over 200 genes have been identified to cause NTDs in animal models,suggesting the involvement of distinct molecular basis in NTD etiology,including the Sonic hedgehog(Shh)signaling.