The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry （FCM） and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies （MoAbs）, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase （cAMP-dependent protein kinase II, PRKAR2A）, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

CLONING AND IDENTIFICATION OF A RABBIT SPERM MEMBRANE PROTEIN GENE

Antibodies raised against rabbit sperm membrane protein(rSMP-B)interacted with a rattestis cytosolic protein with an estimated molecular weight of 70 kD.The clone expressingrSMP-B was isolated by epitope selection from a rat testis,λgt11 vector cDNA library,An Eco Rl-eut 2.0 kb cDNA fragment encoding rSMP-B was identified.The cDNA fragment(RSD-1)was isolated,subcloned and restriction mapped.Dot hybridization analysis of mRNAprepared from human,rabbit and rat testes showed that the gene encoding rSMP-B is ex-pressed in the testes of all three mammals.

Protein phosphatase PP1γ2 in sperm morphogenesis and epididymal initiation of sperm motility

The serine/threonine phosphatase （PP1） isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1γ2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1γ2 isoform is present in all mammalian spermatozoa studied： mouse, rat, hamster, bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1γ2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1γ2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppplcc gene, which encodes the PP1γ1 or PP1γ2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis. Our observations suggest that, in addition to motility, the protein phosphatase PP1γ2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa. （Asian J Androl 2007 July; 9： 445--452）

Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

Rat protein DE is an androgen-dependent cysteine-rich secretory protein （CRISP） synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 （S2）. The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 （CRISP-2） was capable of binding to rodent eggs, human epididymal AEG-related protein （ARP） and helothermine （from lizard saliva） were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. （Asian J Androl 2007 July; 9： 528-532）

Expression of Human Sperm Mem brane Protein in theRecom binantSalm onella Typhim urium Vaccine

A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.

Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

<abstract>Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensional electrophoresis （2-DE） analysis in the project of breeding a new variety of sheep. Differential expression proteins were detected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed through liquid chromatography tandem mass spectrometry （LC-MS/MS）. Among the 480 total protein spots displayed in 2-D gels, 6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center for Biotechnology Information databases （NCBI） indicated that differentially expressed proteins correspond to two proteins, identified to be enolase and transcription factor AP-2-alpha （TFAP-2c0. The two proteins were up-regulated in the irradiated sperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation in sheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheep reproduction and in clarifying the mechanisms involved in irradiation or space breeding.

Rat recombinant β-defensin 22 is a heparin-binding protein with antimicrobial activity

Approximately 40-50 β-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertility in the male reproductive tract. Rat β-defensin 22 is an epididymis-specific β-defensin expressed in segments 12-14 of the epididymis. This protein contains both β-defensin and lectin signature sequences, yet its antimicrobial activity and carbohydrate-binding ability have not been shown. We herein demonstrated the antimicrobial activity of recombinant rat β-defensin 22 against Escherichia coliand Candida albicans. Its lectinJike activity was also investigated by demonstrating its binding ability with heparin beads. This heparin-binding activity implies some potential roles for this defensin in determining the fertilisation capabilities of sperm.

Structure and function of epididymal protein cysteine-rich secretory protein-1

Cysteine-rich secretory protein-1 （CRISP-1） is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP- 1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract. （Asian J Androl 2007 July; 9： 508-514）

Protein profiles in various epididymal segments of normal and castrated rats

Aim: Epididymal proteins are known to play an important role in the maturation of spermatozoa, we ougnt to deter-mine if there are regional differences in androgen-dependent epididymal proteins. Methods: A group of adult rats wascastrated and epididymides were removed three days following castration. The epididymides were dissected into caput,corpus and cauda segments, homogenized, and proteins were fractionated by anion exchange HPLC. Proteins in select-ed fractions were resolved by SDS-PAGE and visualized by silver staining. Results: It was observed that the levels ofmultiple proteins drastically reduced in the various regions of epididymis of the orchiectomized rats. Conclusion: Theepididymal proteins appear to be useful markers to study androgenic action in the epididymis.

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

GOSSYPOL-INDUCED ALTERATIONS OF AMINOPHOSPHOLIPID COMPOSITION IN HUMAN SPERM

Gossypol-induced alterations of aminophospholipid composition in human sperm wereobserved and the effects of some factors on these alterations were investigated.The altera-tions of aminophospholipid composition of human sperm induced by gossypoI included aprogressive decrease in the levels of phosphatidylethanolamine（PE）and phosphatidylserine（PS）when gossypol concentrations ranged from 5-500μmol/L,and a progressive increase inthe level of（LPE）at lower gossypol concentrations（5-50μmol/L）.The conversion of PEinto lysophosphatidylethanolamine（LPE）was strongly enhanced by Ca2+and inhibitedby 0.5mmol/L EDTA,while PMSF,NEM,iodoacetamide and Zn2+had no effect.Thisconversion was weaker in sperm with stripped surface proteins than in normal sperm.In addition,three surface proteins（MW 80,60 and 40 kD）were fixed on the plasmamembrane by gossypol treatment.The significance of gossypol action and the possibilitythat sperm PLA2, PE and.some surface proteins might play an important role in the physio-logical acrosome reaetion of human sperm,as well as in oocyte penetration,are discussed.

In-vivo Mutagenicity of Protein-modified Enterococcus faecalis

The mutagenicity of the protein-modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test.The test substance was designed with three dose groups (1,2 and 5 g/kg·bw) and intragastrically administrated,with cyclophosphamide as a positive control and normal saline as a normal control.The micronucleus rate and sperm abnormality rate were measured.The results showed that the micronucleus rates and sperm abnormality rates in the different dose groups were not significantly different from those in normal control group ( P >0.05),while the positive control group was significantly higher than the normal control group ( P <0.01).The conclusion is that the protein-modified E.faecalis was tested to be negative in both the mouse bone marrow micronucleus test and mouse sperm abnormality test,suggesting that it has no mutagenicity in vivo.

血清TNF-α、FS、Sp17Ab对子宫内膜异位症合并不孕症患者腹腔镜术后IVF-ET助孕妊娠结局的预测价值

目的探讨血清肿瘤坏死因子-α(TNF-α)、卵泡抑素(FS)、抗精子蛋白17抗体(Sp17Ab)对子宫内膜异位症(EMT)合并不孕症患者腹腔镜术后体外受精-胚胎移植(IVF-ET)助孕妊娠结局的预测价值。方法选择2020年1月至2022年2月在空军军医大学第二附属医院接受腹腔镜手术后IVF-ET助孕的EMT合并不孕症患者157例为研究对象,根据临床妊娠结果分为妊娠组和未妊娠组。比较两组IVF-ET助孕前血清TNF-α、FS和Sp17Ab水平,采用多因素Logistic回归分析IVF-ET助孕妊娠结局的影响因素,采用受试者工作特征(ROC)曲线分析血清TNF-α、FS、Sp17Ab对IVF-ET助孕失败的预测价值。结果妊娠组IVF-ET助孕前血清TNF-α、FS、Sp17Ab水平均低于未妊娠组(P<0.05)。妊娠组美国生殖医学学会(ASRM)分期Ⅲ~Ⅳ期患者占比低于未妊娠组,EMT生育指数(EFI)、基础促黄体生成素(LH)、基础促卵泡激素(FSH)高于未妊娠组,而获卵数、可移植胚胎数多于未妊娠组,差异均有统计学意义(P<0.05)。多因素Logistic回归分析结果显示,ASRM分期为Ⅲ~Ⅳ期及TNF-α、FS、Sp17Ab水平升高是IVF-ET助孕的独立危险因素(P<0.05),EFI升高、获卵数增多、可移植胚胎数增多是IVF-ET助孕的保护因素(P<0.05)。血清TNF-α、FS、Sp17Ab单项及联合检测预测IVF-ET助孕失败的曲线下面积(AUC)分别为0.691、0.775、0.688和0.862,联合检测的预测效能优于各指标单独检测。结论血清TNF-α、FS、Sp17Ab与EMT合并不孕症患者腹腔镜术后IVF-ET助孕妊娠结局具有密切的关系,联合检测TNF-α、FS、Sp17Ab对EMT合并不孕症患者腹腔镜术后IVF-ET助孕失败具有较高的预测价值。

精浆蛋白质对家畜精子功能的研究进展

精浆主要由睾丸、附睾、输精管以及副性腺的分泌物混合而成,它被认为与精子存活和保护精子在雌性生殖道中抵抗免疫系统的攻击有关。蛋白质作为精浆中含量最丰富的成分之一,其与精浆的功能息息相关,已有许多研究关注精浆蛋白对精子功能的影响,并试图鉴定其作为雄性生育力标志物的潜力。本文综述了精浆的主要组成成分及不同家畜的精浆蛋白质组成,总结了不同家畜精浆蛋白质对精子功能的影响,为进一步了解家畜精浆的生理功能及其影响雄性繁殖力的作用机制提供更多参考。

癌-睾丸抗原SPANXB在肝癌中的表达及其影响肝癌进展的机制研究

目的·分析癌-睾丸抗原(cancer-testis antigen,CTA)家族成员SPANXB(sperm protein associated with the nucleus on the X chromosome B)在肝癌中的表达及其与肝癌患者预后之间的相关性,并探究SPANXB对肝癌细胞增殖的影响及其潜在机制。方法·利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中的肝癌样本数据,分析SPANXB在肝癌组织中的表达及其与患者生存期的相关性。构建稳定敲低SPANXB与稳定过表达SPANXB的肝癌细胞系,利用活细胞成像实验、EdU细胞增殖实验和平板克隆形成实验评估SPANXB对肝癌细胞增殖的影响。通过RNA测序(RNA-sequence,RNA-seq)探究SPANXB调控肝癌细胞增殖的相关通路,并利用细胞周期实验验证SPANXB对肝癌细胞周期的影响。采用免疫沉淀-质谱联用技术(immunoprecipitation-mass spectrometry,IP-MS)探索与SPANXB相互作用的蛋白,并使用免疫共沉淀(co-immunoprecipitation,Co-IP)进行验证。结果·SPANXB mRNA在肝癌组织中的表达高于正常组织(P=0.003),且与肝癌患者的生存期呈负相关。稳定敲低SPANXB可降低肝癌细胞的增殖能力、克隆形成能力,而稳定过表达SPANXB则可促进这些过程。RNA-seq的结果显示,SPANXB的敲低可下调DNA复制与G1/S细胞周期转换相关通路,细胞周期实验的结果显示SPANXB的敲低可导致肝癌细胞周期发生改变。IP-MS和Co-IP结果显示,SPAXNB与有丝分裂停滞缺陷2样蛋白1(mitotic arrest deficient 2-like protein 1,MAD2L1)、WD重复域蛋白5(WD repeat domain 5,WDR5)等细胞周期相关蛋白存在相互作用。结论·SPANXB的高表达与肝癌的预后呈负相关,其可能通过与MAD2L1、WDR5相互作用调控细胞周期并增强肝癌细胞的增殖活性。