超声波对大鼠睾丸曲细精管上皮影响的超微结构研究

用超声波处理大鼠睾丸10分钟(1瓦/厘米~2 800千周连续波),睾丸温度升至约38.5℃。0.5～1小时后部分曲细精管上皮已出现明显的超微结构损伤,至60天仍未恢复。精子细胞、精母细胞及B 型精原细胞受损,主要是内质网扩张、线粒体肿胀、核膜间隙局部扩大、核内基质空化及核膜破裂,最终细胞崩解。精子细胞的内质网和核膜改变及中段线粒体肿胀出现最早。顶体系统有异常改变。A 型精原细胞及间质细胞无明显改变。本文还讨论了超声波所致超微结构改变的作用机制。     

人类成纤维细胞培养X染色质的吖啶橙荧光法

本文作者报导了应用吖啶橙(Acridine o-range)荧光染色,能选择性地对培养的成纤维细胞的X染色质着色,从而能明确鉴定性别。此种染色在X染色质的鉴别上较焦油紫(Cresyl violet)或氮芥喹吖因(Quinacrine mustard)染色优越。吖啶橙荧光染色后,细胞核的X染色质呈黄色荧光,而核仁发红色荧光,细胞核的其余部份和核膜对荧光染色的亲和力差。男性成纤维细胞的Y染色质对吖啶橙无亲和力,因此能精确地鉴定X染色质。用焦油紫染色的细胞核,其成份之间差别很小,用氮芥喹吖因染色的特点为染色质及核仁都呈发亮的荧光,故核仁可能影响X染色质的观察,而吖啶橙可根据颜色,大小,和荧光强度来辨别。用     

NOR的临床应用

所谓核小体形成区(NOR)即染色体上形成核小体的区域,用银染色可以识别。此嗜银性物质并非DNA本身,而是位于其周围的蛋白质。由NOR形成核小体,从某种意义上讲就是核糖体的细胞内工厂,且核糖体顺反子(基因)只存在于核小体形成区。位于核糖体顺反子周围的多种蛋白质于细胞分裂间期参与核糖体RNA的转录合成,其中的酸性蛋白质被银染色而识别(Ag-NOR)。     

微波照射对兔曲细精管上皮超微结构的影响

以2450兆周的微波照射健康成年雄兔的阴囊及睾丸,使阴囊皮肤温度升至41—42℃。维持20分钟。用电镜观察照射后不同时间(30分钟至4个月)的曲细精管上皮的改变。照射后30分钟至1小时的曲细精管上皮,见初级精母细胞、早期和晚期精子细胞有明显的形态学改变,3—4个月后则普遍恢复正常。精原细胞于照射12小时后见有改变,60天后恢复正常。初级精母细胞和精子细胞的超微结构损伤较精原细胞严重。精原细胞、初级精母细胞和早期精子细胞的超微结构损伤均表现为滑面内质网与核膜间隙扩张、线粒体肿胀、核膜局部破坏,最终细胞崩解。精子细胞的改变主要表现在顶体系统、核、尾部的线粒体鞘和纤维鞘上。文章对微波损伤生精细胞的作用机理进行了讨论。     

微波子宫内膜去除术中组织热损伤的观察

目的探讨微波子宫内膜去除术（MEA）中热效应对组织结构的影响,寻求适宜的内膜薄化方法和微波作用方式。方法对离体和在体子宫分别行全面刮宫薄化内膜后的MEA（刮宫组）和早卵泡期（未刮宫）直接MEA（早卵泡期组）。术中将微波探头移动方式设为“z”形和“z＋w”形两种方式,同时测量宫底、两侧宫角、子宫后壁及前壁下段的子宫浆膜面温度。并将作用后的子宫标本切片行HE染色、尼克酰胺腺嘌呤核苷酸-黄递酶（NADH-d）染色,光学显微镜（光镜）、电子显微镜（电镜）下观察组织热损伤后的形态学改变及损伤深度。结果（1）光镜下见子宫内膜腺体扭曲、细胞界限消失,核浓染,间质中大量急性炎性细胞浸润;浅肌层细胞核固缩、浓染,胞质浓缩,细胞排列密集;深层肌细胞无改变。NADH-d染色可见损伤后的子宫内膜层及部分浅肌层为无色区;宫壁组织热损伤的范围清晰可辨。电镜下坏死部分的平滑肌细胞核染色质、核膜、细胞膜被破坏;线粒体肿胀、膜破裂、嵴消失,细胞器被大量破坏;坏死与正常平滑肌移行区的平滑肌细胞核染色质轻度破坏;核膜及细胞膜存在,线粒体高度水肿,粗面内质网轻度扩张、脱颗粒。（2）离体子宫与在体子宫的最高温度均位于子宫后壁,分别为50．9oC和37．6oC,两者比较,差异有统计学意义（P〈0．01）。（3）离体子宫的宫体组织热损伤深度为4．0～8．8mill,颈体交界处为1．6～3．8mill。在体子宫的宫体组织热损伤深度为4．1～6．6mm,颈体交界处为0—2．8mm。子宫后壁的损伤深度最深,与宫底、两侧宫角及前壁下段等部位比较,差异均有统计学意义（P〈0．01）。离体子宫及在体子宫的对应部位之间损伤深度比较,差异均有统计学意义（P〈0．05）。刮宫组和早卵泡期组的组织热损伤深度比较,差异无统计学意义（P〉0．05）;微波探头以“z＋w”形移动时,组织损伤深度明显深于“z”形移动方式,两者比较,差异有统计学意义（P〈0．05）。结论全面刮宫后和早卵泡期施行MEA均可有效去除子宫内膜,其热损伤深度可控。     

小鼠曲细精管的扫描电镜观察——用DMSO冷冻割断法制备样品

本文用DMSO 冷冻割断法常规处理小鼠的睾丸组织,用HCP-2型临界点干燥器干燥样品,再用IB-3型离子镀膜机在样品割断面上喷镀一薄层金属膜,最后用日立S-450型扫描电子显微镜观察小鼠睾丸曲细精管各种细胞成分表面和内部的超微结构。观察到:在被割断的精子头部暴露出细胞内部的顶体及细胞核,有些成熟精子尾部中段的线粒体呈螺旋状排列,线粒体表面附着一层小颗粒;精子细胞头部和尾部中段插入支柱细胞(Sertoli cell)顶突内。同时也观察到:早期精子细胞插入到支柱细胞顶突的现象;支柱细胞顶突呈泡状膨胀,泡腔内有横断或纵断的精子尾部中段;有的细胞膜被剥离,有的却仍然包裹着一层细胞膜。     

枸橼酸对人精子杀伤作用的超微结构研究

为研制高效、无毒、价格低廉的新型杀精子剂，我们从传统中草药中筛选出杀精效果较好（１ｍｉｎ内使精子瞬间失活）的中药乌梅，经化学提纯、分析，确定其杀精有效成分为枸橼酸，其最有效杀精浓度为０．０９％（０．９ｍｇ／ｍｌ）。本文经透射电镜观察枸橼酸杀伤精子的主要靶结构在于精子顶体、质膜、核膜及线粒体。     

孕妇血浆中胎儿游离DNA测序在无创性产前检测中的应用

目的:探讨孕妇血浆胎儿游离DNA高通量测序(DNA测序)在无创产前检测中的应用价值。方法:对7561例唐氏筛查高风险孕妇行DNA测序,并行羊水细胞染色体核型分析确诊。结果:在7561例孕妇中,DNA测序异常40例,羊水细胞核型结果异常37例,DNA测序误诊3例。其中DNA测序检测到21-三体高风险25例,羊水细胞核型确诊24例;18-三体均为12例。DNA测序对于21-三体检测的敏感性和特异性分别为100.00%和99.98%,18-三体检测的敏感性和特异性均为100.00%。DNA测序的假阳性率为0.04%,假阴性率为0。结论:孕妇血浆胎儿游离DNA高通量测序是检测21和18-三体一种敏感性高和特异性强的新方法,在无创产前检测中有重要的实用价值。     

条件基因敲除技术在卵泡发育调控机制研究中的应用进展

卵泡发育是受内分泌、旁分泌及基因调节的复杂生理过程。条件基因敲除能选择去除调控特定发育阶段特定细胞或器官的基因,是在体基因功能研究的主要手段。近年来该技术被广泛应用于卵泡发育调控机制的研究。本文综述了条件基因敲除技术在始基卵泡激活、卵母细胞成熟、排卵、黄体形成及早期胚胎发育等机制研究中的应用进展。     

精子细胞发育过程中高尔基体的超微结构变化

本实验用电镜观察大鼠睾丸不同发育阶段精子细胞高尔基体的超微结构。结果表明,在精子细胞发育过程中,高尔基体经历一系列的超微结构变化。GERL 只存在于高尔基期和顶帽期精子细胞的高尔基体中,其主要功能是参与形成顶体系统和多泡体;顶体期精子细胞高尔基体的GERL 退化,但高尔基体可能仍具有一定功能;成熟期精子细胞中,高尔基体逐层退化,最后消失。     

Acrosome reaction in human spermatozoa

Human spermatozoa were allowed to undergo the acrosome reaction in vitro by incubation in media with or without reagents known to accelerate the onset of the acrosome reaction. The first observable change before the acrosome reaction was a partial decondensation of the acrosomal matrix. This was followed by invaginations of the outer acrosomal membrane alone or of both the plasma and outer acrosomal membranes, which resulted in formation of many vesicles within the acrosomal cap. Subsequently, the plasma and outer acrosomal membranes fused, but the fusion was seldom seen in the acrosomal cap region. On the other hand, fusion of the two membranes was observed consistently at the anterior end of the equatorial segment of the acrosome. Soon after the membranes over the acrosomal cap disappeared, many vesicles, which were originally within the cap, were seen on or in the vicinity of the inner acrosomal membrane. These vesicles dispersed eventually, leaving the inner acrosomal membrane completely exposed. Thus the acrosome reaction in human spermatozoa seems to proceed in a way somewhat different from that in spermatozoa of most other species, although the end result of the reaction is the same.     

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.     

Distribution and removal of the acrosin of bull spermatozoa.

The effects of Hyamine 2389, Triton X-100, Nadeoxycholate, acetic acid and hypertonic KCl and MgCl2 as well as freezing and thawing and sonication were studied on the solubilization of acrosin from washed bull spermatozoa, from Hyamine-pretreated spermatozoa (devoid of cell and outer acrosome membrane and of acrosomal material) and from isolated acrosomal caps and vesicles. Concurrent ultrastructural changes were observed. Hyamine, Triton, KCl, and acetic acid effectively solubilized acrosin from whole spermatozoa but MgCl2 had a poor effect. The outer acrosome membrane and acrosomal material extracted by Hyamine contained about 35-40% of the total acrosin activity, and three quarters of it was soluble. The rest of acrosin situated in the innter acrosome membrane or equatorial segment was best extracted by hypertonic KCl and MgCl2, but the detergents were ineffective in this case indicating that acrosin is bound differently to the outer and inner parts of acrosome. The opposite effect of MgCl2 on the acrosin activities extracted from these two parts could even be a suggestion of multiple forms of acrosin. The increase of the total acrosin activity during the Hyamine treatment indicates that acrosin is partly in an inactive form. due either to steric hindrance, inhibitor complex of existence of a proacrosin.     

Inhibitors of acrosomal proteinase as antifertility agents. A problem of acrosomal membrane permeability (author's transl)

In vitro studies were performed to investigate the accessibility of acrosin to various proteinase inhibitors inside the intact acrosome of testicular, ejaculated, and uterine human spermatozoa. As test system, the gelatin plate assay was used. For this assay, it was shown formerly that a correlation exists between the size of the digested lysis areas (halo formation) and acrosin activity estimated with synthetic substrates. In addition, saturation of the gelatin substrate membranes with acrosin inhibitors including highly specific ones before application of spermatozoa completely prevented halo formation indicating that the gelatinolytic activity of human spermatozoa is caused exclusively by acrosin. When human spermatozoa were incubated with various acrosin inhibitors (concentration: 1 mmol/1) prior to application to the gelatine membrane, reduction of halo formation could not be observed, however. This result indicates that most of the tested acrosin inhibitors (9 naturally occurring protein inhibitors, 2 microbial peptide inhibitors, 19 synthetic inhibitors) were unable to penetrate the acrosomal membranes of testicular, ejaculated, and uterine human spermatozoa. Only 2 inhibitors caused moderate to complete inhibition of the gelatinolytic activity of the spermatozoa if applied in concentrations between 1-10 mmol/l: the proteinase inhibitor aprotinin and the synthetic inhibitor NPGB (4-nitrophenyl 4-guanidinobenzoate). Obviously, human acrosomal membranes seem to be especially impenetrable to proteins, polypeptides, and synthetic agents. Those acrosin inhibitors penetrating the human sperm head membranes are either too toxic or the local concentration necessary for effective acrosin inhibition in vivo cannot be achieved within the male or female genital tract secretions. Therefore, acrosin inhitibors cannot be used for human contraception at present. Thus, it is mandatory to continue the search for suitable acrosin inhibitors with low toxicity easily penetrating into the intact sperm acrosome.     

Ultrastructure of human sperm acrosome and determination of acrosin activity under conditions of semen preservation

Electron microscopical studies of cryopreserved human spermatozoa show considerable ultrastructural changes of the acrosome in spite of the use of a cryoprotective agent (10% glycerol), as compared with fresh semen and with glycerolated semen before freezing. A nearly complete loss of the plasma membrane and a partial removal of the outer acrosomal membrane with depletion of the acrosomal content is observed whereas the inner acrosomal membrane and the equatorial segment remain intact. Simultaneous determinations of the activity of the proteolytic penetration enzyme acrosin (EC 3.4.21.10) before and after addition of glycerol and immediately after freezing and thawing reveal a significant increase of the extractable acrosin activity in glycerol-treated and in cryopreserved spermatozoa compared with the fresh material. The results indicate an association of the acrosin with the inner acrosomal membrane and/or the equatorial segment.     

Molecular events that regulate mammalian fertilization

Mammalian fertilization is the net result of a highly programmed sequence of molecular events that collectively result in the union of two radically different looking haploid cells, sperm and egg, to form a diploid zygote. For successful fertilization, sperm cells undergo continuous modifications during their formation in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only capacitated acrosome-intact spermatozoa are capable of binding to the egg's extracellular coat, the zona pellucida (ZP) in a receptor-ligand manner. The species-specific irreversible binding of the opposite gametes elevates intrasperm Ca2+ and triggers a signal transduction cascade that results in the fusion of the sperm plasma membrane and outer acrosomal membrane at multiple sites (i.e., induction of the acrosomal reaction) and the secretion of acrosomal contents. The hydrolytic action of the acrosomal enzymes (i.e., glycohydrolases, proteinases etc.) released at the site of sperm-egg binding along with the hyperactivated beat pattern of the bound spermatozoon, are important factors that regulate its penetration of the ZP and fertilization of the egg. In this article, we intend to discuss data from this and other laboratories that provide useful insights into biology underlying sperm development in the testis, maturation in the epididymis, capacitation in the female genital tract, sperm-egg interaction, and induction of the acrosome reaction (AR) before the acrosome reacted sperm can fertilize an egg. Our intention is also to discuss how Ca2+ signaling cascades regulate sperm functions and male fertility. Finally, we will discuss sperm molecules that are under intensive research to regulate male fertility.     

Round-headed human spermatozoa.

Ultrastructural investigation of two cases of round-headed spermatozoa from human ejaculates revealed the existence of two different pathomorphogenetic types. Case 1 represented round-headed spermatozoa of the Schirren and Holstein type caused by loss of the abnormally formed acrosome during spermiogenesis and the missing nuclear transformation. In case 2 a primary maturing inhibition was responsible for the round-headed feature of the spermatozoa; the normally flattened, conical nucleus and acrosomal cap were surrounded by huge droplets of ample cytoplasm. Secondary degenerative changes contributed to the decreased motility of these round-headed spermatozoa. Therapeutic trials for this oligospermic patient showed that an increase in cell count to values in the low-normal range and a drastic reduction in the percentage of round-headed cells (from 80% to 47%) could be achieved. The Schirren and Holstein type, however, must be regarded as absolutely infertile due to the absence of an acrosome and its intrinsic enzymes.     

新型杀精子剂SM-3对人精子超微结构的影响

目的:探讨SM-3杀精、抗生育作用机理。方法:用透射电镜观察SM-3对体外人精子超微结构的影响。结果:SM-3引起精子顶体区质膜肿胀、破溃、顶体酶流失,线粒体空泡样变性。结论:SM-3主要破坏精子顶体及线粒体,从而达到杀精、避孕目的。     

新型杀精子剂SM-3对人精子超微结构影响

目的:探讨SM-3杀精、抗生育作用机理。方法:用透射电镜观察SM-3对体外人精子超微结构的影响。结果:SM-3引起精子顶体区质膜肿胀、破溃、顶体酶流失,线粒体空泡样变性。结论:SM-3主要破坏精子顶体及线粒体,从而达到杀精、避孕目的。     

VAMP/synaptobrevin as an acrosomal marker for human sperm.

OBJECTIVE: To determine the possible use of the mammalian acrosomal marker vehicle-associated membrane protein (VAMP)/synaptobrevin to detect acrosome abnormalities in human sperm. DESIGN: Analysis of human sperm after fixation and staining with an anti-VAMP antibody. SETTING: An academic research institution. PATIENT(S): Semen samples from consenting patients who were participating in an infertility treatment program. INTERVENTION(S): Human sperm samples were fixed, permeabilized with detergent, and examined by immunocytochemistry. MAIN OUTCOME MEASURE: Immunostaining. RESULT(S): Observation of sperm from patients with no obvious sperm morphological defects revealed normal looking acrosomes, as assessed by VAMP immunostaining. However, severe acrosome malformations were detected in other cases. The observations registered varied from the absence of a fully formed organelle in samples of patients with globozoospermia to abnormal VAMP staining in samples from patients with acrosomal defects. CONCLUSION: VAMP/synaptobrevin may be a useful marker for the functional assessment of acrosomal status in human sperm.