H-Y typing in the ELISA

In a series of quantitative absorptions, biotin-conjugated monoclonal H-Y antibody was reacted with a plated source of H-Y antigen in the ELISA. H-Y was demonstrated in testis supernatant fluid of the mouse in this system, and in cells from males of the mouse, bovine and human and females of the chicken, thereby confirming its evolutionary persistence, and underscoring its presumptive role in the primary determination of vertebrate sex.     

Agonadism with positive H-Y antigen

A 30-year-old phenotypic female was investigated because of absence of the vagina. Her serum gonadotropins were elevated while testosterone was in the normal range for females. Stimulation with human chorionic gonadotropin (hCG) gave no response of testosterone. No uterus, Fallopian tubes, Wolffian derivatives, or the gonads were found at laparotomy. Chromosome analysis showed a 46 , XY karyotype. The expression of the H-Y antigen was positive. The results suggest that testicular degeneration in agonadism is not caused by a defective H-Y antigen.     

XY (H-Y+) gonadal dysgenesis

Summary The gonads of 4 phenotypically female individuals with XY chromosomal constitution and signs of virilisation were examined by light microscopy. Electron microscopic examination was also performed in two cases. Serological analysis of H-Y antigen titer yielded positive results. The matrix of the gonads is shown to be ovarian stroma, in which tubular and follicular structures are embedded. The epithelia of the follicles resemble granulosa cells of the ovary, the tubular epithelia are resemble Sertoli cells. Tubules and follicles both show extensive regressive changes. A varying number of Leyding cells/stroma lutein cells were found in each gonad. The different degree of development of testicular and ovarian structures in the dysgenetic gonads might be explained by a defect of the gonadal specific receptor for the H-Y antigen, this defect varying in time of occurrence, duration and severity.     

Correlation between testicular tissue and H-Y phenotype in intersex patients

H-Y antigen was tested in 13 humans with testicular tissue and disturbed primary and/or secondary sex differentiation. All cases typed H-Y positive. This finding is in agreement with the idea that testicular tissue only differentiates in the presence of H-Y antigen.     

H-Y antigen generating and receptor systems in abnormal sexual development

This paper reports data from one case of XY gonadal dysgenesis and one case of XY mixed gonadal dysgenesis. Their H-Y antigen expression was determined on peripheral blood leukocytes and on gonad-derived fibroblasts. The former case was found to be H-Y negative while the latter was H-Y positive. The origin of these disorders is discussed in terms of mutations affecting the H-Y antigen generating and receptor systems.     

H-Y antigen in 46,XY pure testicular dysgenesis

Clinical, cytogenetic, pathologic and histocompatibility-Y (H-Y) antigen studies were performed on a phenotypic female with primary amenorrhea and streak gonads. Pathological examination of tissues removed at total hysterectomy and bilateral salpingo-gonadectomy showed gonadoblastoma and dysgerminoma of left streak. A single F-body (Y chromosome) was found in buccal smears. Analysis of blood cells and tumor fibroblasts showed a 46, XY chromosome constitution (Q-banding). The data were consistent with a diagnosis of 46, XY pure testicular dysgenesis. Positive results for H-Y antigen were found in this case.     

Diagnostic applications of H-Y serology: H-Y negative phenotype in cells from 45, X/ 46, XY fetus with testes

Sexual dysmorphism should be considered likely in cases in which H-Y- phenotype and XY complement are found together. In the case described here, a pregnancy was terminated at nineteen weeks of gestation after 45,X and 46,XY cell lines were detected among cultured amniocytes. The fetus was a male with hypospadias and intraabdominal testes containing irregular tubules and hyperplastic interstitium. Cultured skin fibroblasts, containing 45,X and 46,XY lines in ratio of 18:2, were typed H-Y antigen negative. This underscores the danger of predicting gonadal type on the basis of somatic H-Y phenotype.     

Human Sperm Anatomy: Different Expression and Localization of Phosphatidylinositol 3-Kinase in Normal and Varicocele Human Spermatozoa

Abstract

Recent reports support the possible role of PI3K in sperm capacitation and acrosome reaction, although studies regarding PI3K identity in human sperm, under certain disease states such as varicocele, are still lacking. The authors, therefore, examined the expression profile and ultrastructural localization of PI3K in human semen samples, comparing healthy donors and patients with varicocele. The results obtained performing western blotting assay showed decreased PI3K expression in varicocele with respect to the “healthy” sperm. Immunogold labeling revealed human sperm cellular compartments containing PI3K, evidencing it in the head at both the membrane and nucleus and the entire tail, from the middle to the end piece of normal sperm. In varicocele PI3K label was confined to the head, with a strong reduction of specific reaction in the neck, middle piece, and tail. In conclusion, the data suggest that PI3K may play a role in the maintenance of male factor infertility associated with varicocele, and it may be further exploited as an additional molecular marker for the diagnosis of male infertility disorders.     

Positive H-Y antigen testing in a case of XY gonadal absence syndrome

H-Y antigen testing was positive in a case of pseudohermaphroditism due to XY gonadal absence syyndrome. In conclusion, H-Y antigen may be present even in the absence of testes. Also, the syndrome could not have originated from a defect in the H-Y antigen system.     

妊娠浓度雌、孕激素对小鼠H-Y皮肤移植物存活的影响及机制研究

目的:探讨妊娠浓度雌激素(E2)、孕激素(P4)对小鼠H-Y皮肤移植的影响及其机制。方法:建立小鼠去势模型,每天分别注射E2、P4及E2+P4联合注射,14 d后受鼠行H-Y皮肤移植并继续给药。检测移植前及移植后72 h外周血CD4+CD25+Foxp3+调节性T细胞(Treg)变化及外周血细胞因子水平;观察H-Y皮肤移植物的存活情况。结果:E2可提高外周血Treg的比例(P0.05),移植后Treg的比例进一步升高(P0.05);P4对Treg没有显著影响(P0.05),而E2+P4联合对Treg的影响与单独应用E2结果相似(P0.05)。移植后P4对Treg仍无明显影响(P0.05),而E2+P4联合亦不能进一步提高Treg的比例。细胞因子检测显示,E2、P4均能减少促炎因子分泌,并提高抗炎因子分泌(P0.05)。两者均能有效延长H-Y皮肤移植物存活(P0.05),且具有协同效应。结论:P4可通过诱导免疫偏移促进H-Y皮肤移植物的存活。而E2除诱导免疫偏移外,还通过提高Treg水平而延长移植物的存活时间。     

Presence of H-Y antigen in patients with ullrich-Turner syndrome and X-chromosome rearrangements

Cells from eight of ten patients with gonadal dysgenesis and an isochromosome for the long arm of X, (i(Xq)), have been found to be H-Y antigen-positive, using an assay that employs rat antiserum and Raji cells. In addition, two patients with del(Xq) were also found to be H-Y antigen-positive, whereas four patients in whom only a 45,X line was detected were H-Y antigen-negative. These findings suggest that the X chromosome plays a role in the expression of H-Y antigen in the absence of a Y chromosome. Since our patients with i(Xq) show no evidence of testicular differentiation, it is clear that there is not enough H-Y antigen on these patients' cells to direct the development of a testis. These findings are consistent with the view that the normal functioning of genes on the X and the Y chromosomes is necessary for testicular organogenesis to occur.     

45, X Constitution in a H-Y antigen positive boy with partial monosomy 5p

This is the second reported case of a boy with partial monosomy 5p and 45, X constitution. He has the typical clinical features of the Cri-du-chat syndrome and no Turner stigmata. The normal male development of external genitalia in the patient is related to the presence of the H-Y antigen.     

人干燥综合征B抗原重组体的构建及融合表达

目的克隆人干燥综合征B抗原基因(human sjogren’s syndrome antigen B,SSB)并进行原核表达,为使用重组抗原用于自身抗体的临床检测奠定基础。方法根据GenBank中检索到的人SSB cDNA序列,在5′非编码区和3′非编码区设计特异性引物,提取人源HeLa细胞总RNA作为模板,逆转录RT-PCR扩增人干燥综合征B抗原cDNA。PCR产物纯化后连接至载体PET-30a,导入大肠埃希菌DH5α,构建重组质粒PET-30a-SSB。对重组质粒进行酶切鉴定,选择阳性克隆测序。重组质粒导入大肠埃希菌BL21,阳性克隆经鉴定后在IPTG诱导下表达。结果RT-PCR扩增产物为1245bp。重组质粒PET-30a-SSB经EcoR Ⅰ和HindⅢ双酶切证实含目的基因片段。序列分析提示与GenBank中检索到的一致。SDS-PAGE和Western印迹结果显示融合蛋白相对分子量为73ku,具有天然SSB抗原活性。结论成功克隆SSB基因并表达融合蛋白。     

The H-Y Response in Mid-Gestation and Long After Delivery in Mice Primed Before Pregnancy

The mechanisms underlying maternal tolerance of the semi-allogeneic fetus are not completely understood. The maternal immune system's response to the male antigen, H-Y is an example of the conflicting evidence that both supports and refutes the idea that the immune system in pregnant females is fundamentally different from that in non-pregnant females. Although multiple pregnancies may inactivate H-Y specific T cells, the immune system of the pregnant female can also generate a cytotoxic response to this antigen. To help understand this apparent conflict, we immunized female mice against H-Y with male spleen cells before pregnancy and examined the subsequent anti H-Y response during mid-pregnancy. The pregnant mice studied were able to mount cytotoxic immune responses to H-Y that were equivalent to those generated in their non-pregnant counterparts. Moreover the experience of pregnancy did not impair the ability to maintain immunologic memory to H-Y. The data support the idea that pregnancy does not violate general rules of antigen specific immunity, even if the antigen is expressed on the fetus.     

人干燥综合征A抗原克隆表达与重组蛋白及其临床应用研究

克隆人干燥综合征A抗原基因(SSA-60kD),为SSA抗原的表达和使用重组抗原建立ELISA法用于自身抗体的临床检测.根据GenBank中检索到的人SSA-60kD cDNA序列,在5'和3'非编码区设计特异性引物,提取人源HeLa细胞总RNA作为模板,经RT-PCR扩增SSA-60kD抗原cDNA.PCR产物纯化后连接至载体PET-30a,导人大肠杆菌DH5,构建重组质粒PET-30a-sSA-60kD.对后者进行酶切鉴定、测序和分析.在大肠杆菌中表达,产生重组融合蛋白.RT-PCR扩增产物为1632bp.PET-30a-SSA-60kD经EcoR I和V双酶切证实含目的基因片段.序列分析提示与GenBank中一致.利用金属螯合亲和层析法和纯化,得到了高纯度的SSA-60kD重组蛋白.成功克隆人SSA-60kD基因,并构建PE-30a-ssA-60kD,建立了ELISA方法,检测了风湿病患者血清中抗SSA-60kD自身抗体.     

利用基因敲入技术在肝癌细胞系中稳定表达HBV的X抗原

目的利用基因诱捕载体整合到人类肝癌细胞系SMMC7721细胞的染色体基因中,建立稳定表达HBx蛋白的细胞系。方法通过电击转染将基因诱捕载体pU17导入人类肝癌细胞系SMMC7721细胞,经G418筛选,报告基因X-gal染色,PCR,Western印迹等方法检测HBxDNA的存在和蛋白质的表达。结果得到永久性高表达诱捕载体报告基因X-gal的阳性克隆;用Cre-LoxP置换系统,将构建好的HBx全长片段与诱捕载体的报告基因部分交换,HBx全长片段完整地整合在SMMC7721细胞的染色体基因中,并能从该细胞系中检测到HBx抗原。结论本实验提供了一种新的稳定表达蛋白的方法。该细胞系为制备、纯化X抗原和研究X基因调控提供了实验材料。     

Daudi supernatant, unlike other H-Y antigen sources, exerts a sex-reversing effect on embryonic chick gonad differentiation

In vitro cultures of intact chick gonads (organ cultures) and reaggregation cultures of dispersed gonad cells (roller cultures) were made. Gonads or gonad cells from 7-day-old chick embryos, at the stage when sex-specific differentiation begins, were cultured in the presence of presumed H-Y antigen-containing supernatants, or co-cultured in the presence of H-Y antigen-producing cell lines. The H-Y antigen-producing cells tested were of human, mouse, bovine and chicken origin. During organ culture, addition of supernatant of the human lymphoma cell line Daudi, or co-culture with Daudi cells, stimulated a clear proliferation of the germinal epithelium in male gonads, indicating feminization. A similar effect was obtained by treatment with estradiol. In reaggregation culture, the increase in nuclear size of germ cells was chosen as a parameter for feminization. A significant increase of germ cell nuclear size was observed in gonads cultured in the presence of Daudi supernatant. In both organ cultures and reaggregation cultures, other tested H-Y antigen sources and semi-purified H-Y antigen fractions did not exert significant effects on differentiation of the gonads or on the average area of the germ cell nuclei. These findings suggest that it is not H-Y antigen, but another protein produced by Daudi cells, that might be responsible for the sex-reversing effects.     

H-Y antigen expression in patients with X-autosomal translocations and gonadal dysgenesis

Cells from three patients with early gonadal failure and a balanced reciprocal translocation involving the long arm of the X chromosome and an autosome were studied. Fibroblasts from a patient with a similar balanced reciprocal translocation but normal reproductive capabilities were also studied. Two of the four patients were found to have serologically detectable H-Y antigen on their cells. Since H-Y antigen has been found on the cells of other patients with X chromosome abnormalities but without a Y chromosome, it is thought that the X chromosome plays a role in the regulation of H-Y antigen expression. This study suggests that the long arm of the X chromosome may be involved but the location of a regulatory gene cannot be identified in these studies. These cases do not permit us to implicate H-Y antigen as a cause of gonadal dysgenesis and early gonadal failure in females who have structurally abnormal X chromosomes.     

Immunohistochemical Localization of Concanavalin A and Wheat Germ Lectin Receptors in the Normal Human Spermatozoa*

ABSTRACT: A semiquantitative immunohistochemical technique for the detection of N-acetyl α-D-neuraminic acid, N-acetyl β-D-glucosamine and its β-(1 ? 4)-linked internal chains, α-D-glucopyranosyl and α-D-mannopyranosyl and its α-(1 ? 2)-linked internal chains and sterically related, nonreducing, end-chain residues of oligosaccharide chains of glycoproteins or glycolipids on the surface of membranes was developed using Con A and wheat germ lectins. When this method was applied to the localization of carbohydrate receptors on the membrane of the normal human spermatozoa, it was found that the Con A and wheat germ lectin receptors were mainly located in the equatorial and post nuclear cap with few receptors located in the acrosome and neck. None of them were found in the intermediate segment plus tail. Con A receptors were α-D-mannopyranosyl end-chain residues and wheat germ lectin receptors were N-acetyl β-D-glucosamine (1 ? 4)-linked internal chains. These groups occur together in the oligomannosidic type of N-glycosidic-linked oligosaccharide chains of glycoproteins and so the use of both lectins on desialycated membranes or on those which contain nonclustered N-acetyl neuraminic acid residues may be of help to localize this type of glycoprotein oligosaccharide chains. Con A receptors were not removed after proteases digestion, suggesting the possibility that they are part of intrinsic spermatozoal antigens.     

海藻糖和蛋黄在人类精子冻于保存中的保护作用

人类精子冻干保存中损伤除了来自环境溶液对精子生理活性的损伤外，还会来自于冷冻和干燥过程。目前精子冻干保存中保护剂选取基本上都从生理化学角度考虑，目的为抑制精子内酶活性，没有考虑对冷冻和干燥损伤的抑制。大量文献表明蛋黄能减小冷冻对精子的损伤，而二糖（特别是海藻糖）在细胞干燥中具有特殊保护作用。本研究在前人一般使用的精子冻干保护剂中加入海藻糖，或同时加入海藻糖和蛋黄，通过伊红Y染色、低渗膨胀和电镜显微观测实验，比较不同组成的保护剂对精子结构和膜的保护作用。结果表明，通过加入海藻糖修正的保护剂能提高对精子结构的保护效果，当同时加入海藻糖和蛋黄时效果更好。