乙型肝炎病毒DNA在肝炎患者精子染色体上的整合

乙型肝炎（ｈｅｐａｔｉｔｉｓＢ，ＨＢ）患者的体液和组织中广泛存在乙型肝炎病毒（ＨＢＶ），因而认为ＨＢＶ具有泛嗜性［１］。分子生物学研究表明，ＨＢＶＤＮＡ能在乙肝患者精子染色体上整合［２，３］。但ＨＢＶ在人精子染色体上的整合特点及其可能机理尚不清楚。我...     

抗精子抗体测定的临床应用

早在１８９９年Ｍｅｅｃｎｉｋｏｆｆ及Ｌａｎｄｓｅｅｉｎｅｒ发现，将异种动物精子注入机体后可产生抗体。一年后Ｍｅｅｃｎｉｋｏｆｆ证实豚鼠在注入自体精子后，血液中有抗精子抗体存在。后来用精子免疫雌性动物，结果出现暂时性不育，同时血清中产生了高滴度的抗精子抗体（ＡＳＡＢ）。大量研究发现在不育人群中抗精子抗体存在于血清中有较高的发生率。不论是男性还是女性不育患者血清中都可能出现抗精子抗体。检测这种自身抗体是除细胞遗传学分析以外，检测男性不育较为理想的方法。可以对男性不育患者进行综合分析和辅助诊断。是对细…     

牛精子头后部质膜分区的研究

检测牛精子头 存在的不同质膜区域。方法先用钙离子载体Ａ２３１８７诱导精子发生顶体反应,然后用水浴超声波和低速离心法分离得到纯牛精子头。直接用顶体反应后精子和低渗处理的顶体反应后精子头免疫Ｂａｌｂ／ｃ母鼠和新西兰公兔,制备抗牛精子抗体,     

人类精子载体介导的γ－氨基丁酸的摄取，反映高亲合性的γ－氨基丁酸转运体存在于人类精子

人类精子载体介导的γ－氨基丁酸的摄取，反映高亲合性的γ－氨基丁酸转运体存在于人类精子ＡＡａｎｅｓｅｎ，ｅｔａｌＢｉｏｌｏｇｙｏｆＲｅｐｒｏｄｕｃｔｉｏｎ．１９９６１５４（４）８４１～８４６人类精子有载体介导摄取一ｔａ基丁酸（Ｇ＾Ｂ＾）的能力，胞外基质...     

微波局部照射小鼠睾丸对睾丸、附睾和精子凝集素受体含量和分布的影响

本实验应用２４５０ＭＨｚ微波，局部照射ＢＡＬＢ／ｃ小鼠睾丸，在肛温达４１±０．５℃后维持１５分钟，分别照射１或３次，以凝集素（ＷＧＡ、ＳＢＡ、Ｃｏｎ－Ａ）作为分子探针，于动物受照后１周、２和５个月观察微波辐射对各级生精细胞、附睾及精子糖脂和糖蛋白含量及分布的影响。结果表明，生精细胞中ＷＧＡ、Ｃｏｎ－Ａ受体的含量均显著下降（Ｐ＜０．０１），形态异常的精子中三种凝集素受体的含量均显著增加，且分布特性有改变。受照睾丸内呈增生的血管内皮ＳＢＡ反应为阳性。本研究结果证明，微波确可引起生殖细胞的损伤。     

微波局部照射小鼠睾丸对睾丸,附睾和精子凝集素受体含量和分…

本实验应用２４５０ＭＨｚ微波，局部照射ＢＡＬＢ／ｃ小鼠睾丸，在肛温达４１±０．５℃后维持１５分钟，分别照射１或３次，以凝集素（ＷＧＡ、ＳＢＡ、Ｃｏｎ－Ａ）作为分子探针，于动物受照后１周、２和５个月观察微波辐射对各级生精细胞、附睾及精子糖脂和糖蛋白含量及分布的影响。结果表明，生精细胞中ＷＧＡ，Ｃｏｎ－Ａ受体的含量均显著下降（Ｐ＜０．０１），形态异常的精子中三种凝集素受体的含量均显著增加，且分布特性有     

微波局部照射小鼠睾丸对精子染色体的影响

精细胞是男性遗传物质代代相传的唯一联系，了解微波辐射对生精细胞遗传物质的影响，将有益于微波的应用和防护。本实验应用２４５０ＭＨ：微波局部照射ＢＡＬＢ／ｃ小鼠睾丸，在睾九内温度达４１±０．５℃后维持１５分钟。对动物分别照射１或３次，于东次照射后二个月观察精子单倍染色体。结果显示，精子染色体的改变主要为非整倍体的增加，但和对照间无显著性差异（Ｐ＞０．０５）．该结果说明，一定剂量的微波照射，对生殖细胞的遗传物质无显著的远后效应。     

慢性乙型肝炎患者精子中乙型肝炎病毒DNA定位

国内外学者曾用斑点杂交和Ｓｏｕｔｈｅｒｎ印迹杂交法检测精液中乙型肝炎病毒 (ＨＢＶ)ＤＮＡ的存在[1 ,2 ] 。为从形态上明确ＨＢＶＤＮＡ是否存在于男性慢性乙型肝炎患者成熟精子中及其分布情况 ,我们采用原位杂交法检测了 2 2例慢性乙型肝炎患者精子中ＨＢＶＤＮＡ。一、材料与方法1.标本收集 :精液采自 1990～ 1993年华西医科大学附属第一医院 2 2例男性慢性乙型肝炎患者 ,ＨＢｓＡｇ、ＨＢｅＡｇ均为阳性 ,年龄 19～ 2 5岁。为保持精子新鲜 ,采集后的标本尽快进行实验。2 .试剂 :Ｄｉｇｏｘｉｇｅｎｉｎ试剂盒购自西德Ｂｏｅｈｒｉｎ…     

小鼠卵细胞质内显微注入单精子受精的实验研究

为了探讨提高小鼠卵细胞质内单精子注入受精率的方法，选取鼠龄１２－１４周的健康昆明白小鼠作为精子和卵子的供体，受用ＩＣＳＩ技术，以受精后二细胞卵裂的形成率为指标，了解不同采卵时间，不同微注射针参数及不同培养液对细胞质单精子注入的影响。结果表明，ｈＣＧ注射后１８－１９ｈ采卵，用针尖内径为４－５μｍ，斜面角度为３５－４０度的微注射针进行ＩＣＳＩ操作受精后卵子置ＣＺＢ中培养可获得较多的２细胞胚，卵裂率明显     

微波局部照射小鼠睾丸对睾丸,附睾及精子的形态影响

微波作为环境污染物，其对人类的危害已逐渐受到人们的重视，尤其生殖细胞，不但对微波第三，而且在人类遗传学占有独特的。本实验应用２４５０ＭＨＺ微波局部照射ＢＡＬＢ／ｃ小鼠睾丸，于照后不同时间观察睾丸附睾和精子的形态学改变，结果表明，微波可引起各级生精细胞以浊肿为主的病理学改变，并存在不均一性，持久性，不同的细胞具有不同的敏感性等特点，精子的形态学改变表现为大头畸形增多（Ｐ＜０．０５－０．０１），超微结     

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.     

Glass wool filtration leads to a higher percentage of spermatozoa with intact acrosomes: an ultrastructural analysis

We investigated the possibility of ultrastructural damage to human spermatozoa induced by different sperm preparation techniques. Ejaculates from 20 normozoospermic men were divided into equal aliquots and processed by glass wool filtration, Percoll density gradient centrifugation, and a simple two-step centrifugation procedure which served as a control. The evaluation of 60 spermatozoa from each of 20 test subjects (in all, n = 1200) ensured that a sufficiently large number of spermatozoa were investigated. Ultrastructural damage was assessed by scanning electron microscopy. We investigated the state of the acrosome after sperm preparation and measured the percentage of intact spermatozoal structures compared with that of the control. Compared with Percoll density gradient centrifugation, glass wool filtration yielded a significantly increased proportion of intact acrosomes. However, both preparations gave significantly better results than the control. In conclusion, both glass wool filtration and Percoll centrifugation are efficient techniques for the accumulation of spermatozoa with intact acrosomes. Because of the significantly higher percentage of intact acrosomes, glass wool filtration appears to be the more appropriate method. The significance of the conspicuous bending of sperm tails after Percoll centrifugation is not yet known.      

Lectins binding on human sperm surface increase membrane permeability and stimulate acrosomal exocytosis

Cross-linked complexes formed between certain lectins and theirspecific multivalent carbohydrates and glycoconjugates on thesperm surface were studied for their ability to modify spermmembrane permeability and to induce the acrosome reaction. Wheatgerm agglutinin (WGA), concanavalin A (Con A) and peanut agglutinin(PNA) increased the proportions of human spermatozoa permeableto the impermeable propidium iodide (31.9 compared with 13.8%,38.4 compared with 18.4% and 72.7 compared with 18.9% respectively).Removal of sperm surface sialic acid by neuraminidase treatmentwas a prerequisite for Con A and PNA binding to the sperm surface.The percentage of permeable and acrosome-reacted spermatozoawas not affected by sperm treatment with 500 mlU/ml Arthrobacterureafaciens neuraminidase. WGA did not induce the acrosome reaction,whereas PNA induced the acrosome reaction regardless of thesperm capacitation status, allowing the proportion of acrosome-reactedspermatozoa to reach 27.7% of capacitated spermatozoa. However,the ability of Con A to induce the acrosome reaction was limitedto uncapacitated spermatozoa. To test the physiological relevanceof this study, uncapacitated human spermatozoa were incubatedwith human zonae pellucidae and the permeability of spermatozoabound to the zona surface was analysed according to the timepost-insemination. Two-thirds of spermatozoa bound to zona pellucidabecame permeable to propidium iodide in the first 30 min post-inseminationand almost all bound spermatozoa became permeable to the impermeabledye after 60 min. Our results show that molecular interactionsbetween human zona pellucida and sperm surface increase thepermeability of sperm membranes; the cross-linked complexesformed by PNA lectin and its specific multivalent carbohydratesand glycoconjugates on the sperm surface were also able to increasesperm membrane permeability and to induce the acrosome reaction.These results suggest a role for the saccharide moieties ofsperm surface glycoconjugates in the induction of the acrosomereaction. acrosome reaction/capacitation/lectins/peanut agglutinin/receptor aggregation     

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.     

Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacitated spermatozoa

Changes in the distribution of sialylglycoconjugates on thesurface of uncapacitated and in-vitro capacitated human spermatozoawere studied by means of two sialic acid specific lectins (Maackiaamurensis agglutinin and Sambucus nigra agglutinin). On theuncapacitated sperm surface, sialylglycoconjugates were foundto be localized from the post-acrosomal region of the spermhead to the tail middle piece, whereas after in-vitro capacitationthese molecules were only found in a small area of the post-acrosomalregion. The surface of capacitated human spermatozoa was alsoinvestigated by specifically radiolabelling its terminal sialicacid residues. A 20 kDa glycoprotein, which was partially purifiedby anion-exchange chromatography, was the main component ofthe sialylglycoconjugate pattern after in-vitro capacitation. capacitation/glycoconjugates/human spermatozoa/lectins/sialic acid     

Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity.

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.     

精子凝集素受体含量与体外受精率的关系

目的研究常规检查精液的精子膜麦芽凝集素受体(WGA)、精子畸形率和精子顶体酶的临床意义。方法收集单纯因输卵管梗阻行体外受精-胚胎移植(IVF-ET)并于促排卵周期获取成熟卵8个以上患者258例,在患者丈夫精液常规检查均正常后,进行精子WGA受体含量、精子畸形率和精子顶体酶含量的检测,并统计患者受精率。结果体外受精率<65%组64例,受精率≥65%组为194例,两组比较,WGA受体含量有显著性差异,顶体酶活性和精子畸形率差异不显著。结论精子膜麦芽凝集素受体的含量、精子畸形率与精子顶体酶均能影响体外受精,但WGA受体含量更能预见体外受精率。     

Molecular glass wool filtration as a new tool for sperm preparation

BACKGROUND: Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter. METHODS: Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody. RESULTS: Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS. CONCLUSIONS: The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.     

Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining

The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.      

The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods.

Different procedures were investigated for the dilution of human cryopreserved semen and the preparation of an enriched population of motile spermatozoa for assisted reproduction. The dilution of a 0.25 ml straw of cryopreserved human semen by addition of 2.0 ml Ham's F-10 buffer in one step caused a large decrease in the proportion of motile spermatozoa. This was due to osmotic stress because many of the diluted spermatozoa exhibited swollen tails. To a large extent the damage could be avoided by adding the buffer in 0.10-ml aliquots at 30-s intervals. Spermatozoa obtained after such dilution of cryopreserved human semen were subjected to the swim-up procedure, to centrifugation on two-step gradients of Nycodenz or Percoll, or to filtration through glass fibre paper and compared with respect to yield, motility parameters and penetrating ability in the hamster egg test. The swim-up procedure yielded spermatozoa with excellent motility but only 12% of the available motile spermatozoa were recovered. On both Nycodenz and Percoll gradients, greater than 40% of the available motile spermatozoa were recovered and the average velocity of the spermatozoa was not significantly less than for the swim-up technique. When A23187 was used to promote acrosome reactions in the hamster egg test, Percoll-prepared spermatozoa achieved an average of 8.6 decondensed sperm heads/egg compared to 1.9 for Nycodenz and 1.3 for the swim-up procedure. The yield from glass fibre paper filtration was only 12% and the velocity of the spermatozoa and their performance in the hamster egg test was significantly poorer than in all the other methods.(ABSTRACT TRUNCATED AT 250 WORDS)