抗精子抗体对人精子HOS试验及膜间Ca2+内流的影响

目的:探讨在正常精液中加入IgG类抗精子抗体阳性精浆对人精子膜功能完整性及压力敏感性Ca~(2+)内流的影响。方法:在门诊的不育患者中收集精浆IgG抗精子抗体阳性患者,通过离心的方法从其精液中分离出精浆,并同正常人精子孵育,与正常精浆及正常人精子、精子培养液及正常人精子孵育的对照组比较研究,分别对它们进行低渗肿胀(HOS)试验及测定精子细胞内钙离子浓度的变化。结果:与IgG抗精子抗体阳性精浆孵育的精子,其g形精子百分率和精子尾部总肿胀率均明显低于两个对照组,差异有统计学意义(P0.01);试验组的精子细胞内Ca~(2+)荧光强度差值明显低于两个对照组,差异有统计学意义(P0.01)。结论:IgG类抗精子抗体对人精子膜功能完整性及压力敏感性Ca~(2+)内流均可造成影响。     

213例不育男性抗精子抗体与精液有关参数的分析

我们检测了９００多例男性不育症患者的血清、精浆抗精子抗体（ＡｓＡｂ）和精液各项指标，对其中２１３例ＡｓＡｂ阳性患者的精液有关参数与ＡｓＡｂ的关系进行了分析，并与生育男性组作了比较。结果表明，ＡｓＡｂ阳性患者的精子密度、精子活率、精子顶体完整率、精浆免疫抑制物（ＳＰＩＭ）及α－糖苷酶均明显低于生育组（Ｐ＜０．０１），不育男性ＡｓＡｂ阳性组ＳＰＩＭ阳性率也明显高于ＡｓＡｂ阴性组（Ｐ＜０．０１）。此外，血清与精浆ＡｓＡｂ同为阳性组的精子活率明显低于单纯血清或精浆ＡｓＡｂ阳性组（Ｐ＜０．０１）。     

人精浆抗精子抗体与顶体酶活性关系的探讨

目的 :研究精浆抗精子抗体 (AsAb)对精子顶体酶活力的影响。　方法 :用间接血凝法测定 34 32例不育男性和 6 5例生育男性精浆的AsAb ,并对其中的 2 882例不育男性精子用比色法进行了顶体酶活性的测定。　结果 :34 32例不育者精浆AsAb阳性率为 10 .2 0 %,2 882例进行了顶体酶活性测定的不育者精浆AsAb阳性率为 9.37%,对照组精子顶体酶活性明显高于各不育组 (P <0 .0 0 1)。不育组AsAb阳性组与AsAb阴性组比较顶体酶活性没有明显差异 (P >0 .0 5 ) ,顶体酶活性正常与异常组AsAb阳性率比较差异无显著性 (P >0 .0 5 )。　结论 :精浆AsAb对精子顶体酶活性没有影响。     

植物凝集素荧光标记法检测精子顶体结构的临床意义

目的　探寻评估人精子授精能力的简便有效方法。　方法　采用络合异硫氰酸荧光素的植物凝集素 (FITC PSA)进行精子顶体荧光标记 ,分别测定 5 8例不育患者及 4 6例正常生育男子的精子顶体完整率 ,并对其中 14例精液标本 (不育组及正常组各 7例 )进行人卵透明带诱发的精子顶体反应实验 ,对精子顶体完整率与发生顶体反应的百分率的相关关系进行分析。　结果　正常生育组顶体完整率及发生精子顶体反应百分率显著高于不育组 (P <0 .0 1) ,精子顶体完整率与发生精子顶体反应百分率呈正相关 (r =0 .74 68)。　结论　FITC PSA荧光标记法操作简便 ,技术结果可靠 ,特异性强 ,可作为评价人精子授精能力的有效指标之一。     

人精子运动功能与腹腔液中干扰素-γ含量的关系

目的 观察子宫内膜异位症（EMs）患者腹腔液干扰素（IFN）-γ体外对精子运动功能的影响。方法 用双抗体夹心法，检测30例EMs不孕妇女，30例其他病因所致不孕妇女和20例生育力正常妇女腹腔液中IFN-γ含量，用精液自动分析系统检测与腹腔液共同孵育后的正常精液中精子活动力参数，荧光显微镜观察顶体反应发生率。结果 EMs组腹腔液IFN-γ含量（250．3±102．6）ng／L高于其他2组（P〈0．01）；EMs组腹腔液与正常精液共同孵育后，精子活动力显著降低（P〈0．01），并与IFN-γ含量呈负相关（r=-0．958，P〈0．01）。精子与EMs组腹腔液孵育16h后顶体反应发生率低于正常对照组（P〈0．01）。结论 EMs患者腹腔液IFN-γ对精子活动的抑制可能是导致不孕因素。     

人精子顶体反应的检测及其与穿卵试验的相关性

选择正常生育男子（生育组）、输精管结扎再吻合（吻合组）、不育症患者（不育组）各２５例的精液进行人精子顶体蛋白酶及酸性磷酸酶测定,顶体组织化学三色染色和人精子穿透去透明带金黄仓鼠卵试验,比较不同生育条件下精子功能和生育能力的相关性。结果显示,精子获能后顶体反应率明显高于获能前（Ｐ＜０．０１）。生育组顶体反应率和穿透试验高于吻合组和不育组（Ｐ＜０．０１）。结果表明,精子穿卵试验结合精子顶体反应测定可作为客观评价人类精子受精能力的依据     

流式细胞计检测人精子顶体及其与精液其他参数的关系

运用流式细胞计及人精子顶体特异性标记物结合异硫氰酸荧光素的花生凝集素检测了３４例精液精子顶体结构的完整性。其中正常生育精液１２例，弱精症精液１２例，畸形弱精症精液１０例。并且进一步检测了２０例精液在体外诱导下精子发生顶体反应的能力，其中正常生育精液１０例，不育精液１０例。结果表明人精子顶体结构的完整性与精于正常形态百分率，存活率，前向运动百分率显著正相关（Ｐ＜０．００１）。不育组精子在体外诱导下发生顶体反应的能力明显低于生育组（Ｐ＜０．０１），说明流式细胞计能用于预测生育力。     

213例不育男性抗精子抗体与精液有关参数的分析

我们检测了９００多例男性不育症患者的血清，精浆抗精子抗体和精液各项指标，对其中２１３例ＡｓＡｂ阳性患者的精液有关参数与ＡｓＡｂ的关系进行了分析，并与生育男性组作了比较。结果表明，ＡｓＡｂ阳性患者的精子密度精子活率，精子顶体完整率，精浆免疫抑制物及α－糖苷酶均明显人氏于生育组，不育男性ＡｓＡｂ阳性组ＳＰＩＭ，阳性率也明显高天ＡｓＡｂ阳性率也明显高于ＡｓＡｂ阴性组。此外，血清与精浆ＡｓＡｂ同为阳性组的     

解脲支原体与男性免疫性不育关系的探讨

本文对１５０例男性不育患者进行了精浆抗精子抗体检测与解脲支原体培养，发现抗精子抗体阳性组解脲支原体检出率为６６．７％，抗精子抗体阴性组解脲支原体检出率为４０．２％，两组之间存在显著性差异（Ｐ＜０．００５）。对抗精子抗体、解脲支原体均为阳性组进行了两种治疗方法的疗效对比，结果表明，使用抗生素组抗体转阴率为７０％，未使用抗生素组抗体转阴率为３６．４％，两组疗效存在显著性差异（Ｐ＜０．０５）。提出解脲支原体可能是导致男性免疫性不育的病因之一。认为积极治疗生殖道感染有助于降低抗体滴度，阻止抗体形成，对免疫性不育的治疗有重要意义。     

抗精子抗体对人精子表面ConA受体影响的研究

本研究运用刀豆蛋白-A(ConA)-胶体金分子探针,对经兔抗人精子抗血清处理的正常人精子和免疫性不育患者的精子做分子水平的定位观察。结果表明,经兔抗人精子抗血清处理的精子和免疫性不育症患者的精子,其顶体反应的囊泡上和顶体后区胶体金的结合均明显低于正常,提示抗精子抗体能封闭精子顶体后区与卵细胞识别的位点,从而影响精子的受精能力。     

考马斯亮蓝染色法检测人精子形态和顶体反应

目的 :探讨考马斯亮蓝G2 5 0染色法在人精子畸形率、顶体完整率和顶体反应检测中的应用。　方法 :用瑞 吉染色法和考马斯亮蓝G2 5 0染色法分别对获能前后和孕酮诱导顶体反应后的人精子进行涂片染色 ,观察其形态 ,并计算精子畸形率、顶体完整率及顶体反应率。　结果 :瑞 吉染色法和考马斯亮蓝G2 5 0染色法在检测人精子畸形率和顶体完整率上差异无显著性 (P >0 .0 5 )。孕酮诱导顶体反应后的精子用考马斯亮蓝G2 5 0染色后呈现两种类型 :顶体区染成紫蓝色和不着色或染成淡紫色 ,后者随诱导时间延长而增加 ,可能代表了已发生顶体反应的精子。诱导 1h的顶体反应率为 (75 .1± 3.8) %。　结论 :考马斯亮蓝G2 5 0染色法可作为检测人精子形态和顶体反应的可靠而实用的方法     

SPERM ACROSOME STATUS AND SPERM ANTIBODIES IN INFERTILITY

Purpose

We studied whether the spermatozoa from sperm autoimmune infertile men undergo premature acrosomal loss and whether this relates to the presence of sperm antibodies in wives.

Materials and Methods

We evaluated acrosome status of live washed native and overnight capacitated spermatozoa from 17 sperm nonautoimmune fertile and 23 sperm autoimmune infertile men using an immunofluorescent peanut lectin binding assay. We used cytotoxic and immunobead binding assays to prescreen the serum and seminal plasma of these men, and serum and cervical mucus of the wives for immunological infertility. We performed immunofluorescent sperm antibody assays on all study samples to ascertain sperm antibody isotype levels in each sample. Levels of acrosomal loss in husband native and capacitated spermatozoa were correlated with levels of IgG, IgA and IgM sperm antibodies in the study samples.

Results

Sperm autoimmune infertile men had a significantly larger percentage of sperm (p <0.0001) that had lost the acrosome and a lower percentage of sperm with intact acrosome (p <0.0001) in native and capacitated preparations in contrast to those of fertile controls. Levels of cytotoxic and IgA antibodies, especially in seminal plasma and cervical mucus, correlated significantly with percentages of sperm with a total loss of acrosome in native and capacitated sperm preparations (p [less than =] 0.01).

Conclusion

Infertile men with sperm antibodies in serum and seminal plasma undergo premature acrosome loss. This loss may expose the reproductive tract immune system, especially that involving IgA, in autoimmune infertile men and the wives to high immunogenic levels of sperm acrosome membrane antigens, thereby rendering them immunologically infertile.     

Uteroglobin and transglutaminase modulate human sperm functions

During the process of capacitation, spermatozoa undergo significant changes in membrane composition, including removal of decapacitating factors (DFs), which are present in seminal plasma, that lead to increased sensitivity to physiological stimuli of the acrosome reaction. In the present study we investigated the presence, localization, and effects on human spermatozoa of 2 proteins of seminal plasma origin, uteroglobin (UG) and transglutaminase (TG). These 2 proteins interact with one another because TG promotes covalent links of UG to sperm surface proteins. We found that UG is localized around the entire surface of ejaculated human sperm, whereas TG is predominantly localized in the neck. FACScan analysis confirmed the surface localization of both antigens and demonstrated that swim-up selection of spermatozoa was associated with a significant reduction in the contents of the 2 substances when compared with unselected samples. Western blot analysis of UG in total sperm lysates confirmed the lower content of the protein in swim-up-selected sperm. Swim-up-selected sperm were characterized by their ability to undergo a spontaneous, time-dependent increase of capacitation-characteristic chlortetracycline pattern of fluorescence and increase in responsiveness to progesterone. Such changes were not observed in unselected sperm. Exogenous addition of TG, together with recombinant rabbit UG, prevented the spontaneous increase in responsiveness to progesterone (acrosome reaction and intracellular calcium) at 24 hours in swim-up-selected sperm, suggesting the occurrence of a capacitation-inhibiting activity of the 2 substances. In addition, we found that endogenous UG and TG contents, as determined by FACScan analysis, were negatively correlated (P < .0001) with sperm motility and that exogenous addition of the 2 substances resulted in a substantial reduction of progressive motility (P < .01). Collectively, these data indicate that TG and UG represent 2 DFs, and contribute to understanding the biochemical mechanisms that characterize the process of capacitation.     

Physiological action of oestradiol on the acrosome reaction in human spermatozoa

The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l⁻¹); oestradiol plus progesterone (oestradiol at 840 pmol l⁻¹ and progesterone at 10.1 nmol l⁻¹), oestradiol (840 pmol l⁻¹) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction ( P  <   0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.     

Differential activities of malate and isocitrate NAD(P)-dependent dehydrogenases are involved in the induction of capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm catabolic processes produce energy for capacitation and acrosome reaction induction required for oocyte fertilization. The aim was to determine metabolic enzymes' activities and their participation in the supply of energy and generation of the redox state to acquire fertilizing capacity. Capacitation was induced with heparin and quercetin, and the acrosome reaction with progesterone. Enzymatic activities were determined spectrophotometrically. The chlortetracycline and differential-interferential contrast microscopy/tryptan blue techniques were used to evaluate capacitation and acrosome reaction, acrosomal integrity and sperm viability respectively. A 2 : 1 and 3 : 1 ratio were obtained for isocitrate dehydrogenase (IDH)-NADP/NAD and malate dehydrogenase (MDH)-NADP/NAD activities respectively. MDH-NADP activity remained constant with different treatments, unlike MDH-NAD activity, which diminished with both capacitation inducers and in acrosome-reacted spermatozoa previously treated with heparin (P < 0.05). IDH-NADP decreased its activity 50% in spermatozoa capacitated with heparin and acrosome reacted with progesterone (P < 0.05). Capacitation and acrosome reaction processes induced with heparin and progesterone, respectively, involve a differential oxidative metabolism, with the participation of MDH-NAD(P) and IDH-NAD(P) enzymes, whose activities would be linked to the malate-aspartate, lactate-pyruvate and isocitrate cytosolic-mitochondrial shuttles. These enzymes play a major role in supplying reduction equivalents and/or energy required for capacitation and acrosome reaction in cryopreserved bovine spermatozoa.     

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties

In previous reports, it has been demonstrated that progesterone (P) stimulates capacitation, hyperactivation of human sperm motility and initiates the acrosome reaction (AR). This last effect has been related to the presence of non-genomic receptors for the steroid, localized on the sperm head plasma membrane. These receptors can be detected after treating spermatozoa with the non-permeable conjugate Progesterone - 3-(O-carboxymethyl) oxime: bovine serum albumin-fluorescein isothiocyanate (P-BSA-FITC). In the present study, the presence of progesterone receptors was determined in a selected sperm population with normal morphology and high progressive motility. In addition, other parameters such as the AR, hypo-osmotic swelling test, stability of chromatin and capacitating effect of P were evaluated. The percentage of P-BSA-FITC positive-spermatozoa present in the selected sperm population was higher than in total seminal spermatozoa. Furthermore, spermatozoa incubated with P showed a higher percentage motility and AR than did control spermatozoa. The HOS test indicated that membrane integrity of P-treated spermatozoa was not different to that found in the control sperm suspensions. Unexpectedly, the total sperm population treated with P showed a marked susceptibility to nuclear decondensation with reducing agents. According to these results, the selected sperm population of this study, able to respond to P, may be similar to that with good motility and normal morphology selected in the female reproductive tract, before fertilization.     

不育症患者精浆IL-1β、IL-4、IL-10含量测定及临床意义

目的 :观察男性不育症患者精浆中白细胞介素 1β(IL 1β)、白细胞介素 4 (IL 4 )、白细胞介素 10 (IL 10 )含量 ,及其与精子的各项功能指标之间的相互关系。　方法 :应用放射免疫分析 (RIA)技术 ,对 12 6例男性不育症和 2 0例正常生育者精浆中IL 1β、IL 4、IL 10含量进行检测。根据精子密度将不育症患者分为A组 (精子密度≥ 2 0× 10 6/ml)、B组 (精子密度 <2 0× 10 6/ml)和C组 (无精子症者 ) 3组 ;根据精子活动力、活动率将A组分别分为精子活动力正常组和不良组 ,精子活动率正常组和下降组 ;根据不育症患者血清抗精子抗体 (AsAb)检测结果、精液中WBC多少分为AsAb阳性组和阴性组 ,WBC精液组和非WBC精液组。根据生育组检测结果 ,将不育A组和B组分为精子穿透力正常组和下降组 ,精子顶体完整率正常组和下降组 ,精子尾部肿胀率正常组和下降组。　结果 :不育症组精浆IL 1β含量显著高于生育组 (P <0 .0 1) ,IL 4、IL 10含量显著低于生育组 (P <0 .0 1)。不育症组精浆中IL 1β、IL 4、IL 10含量在WBC精液组与非WBC精液组、血清AsAb阳性组与阴性组之间差异均有显著性 (P <0 .0 5或P <0 .0 1) ;IL 4含量在不育症组精子活动力、活动率、精子穿透力、顶体完整率、尾部肿胀率正常与减少之间差异均有显著性 (P <0     

Acrosome reaction in fertile and subfertile boar sperm

The main purpose of sperm evaluation is to predict its fertilizing ability. However, basic sperm test results show a low correlation with fertilizing ability. The purpose of this study was to determine whether there is an association between acrosome reaction (AR) and the incidence of subfertility of normal sperm boar. The production records of 22 farms were analyzed to identify boars with low fertility and/or prolificity, classified as subfertile. Twenty-two subfertile boar semen samples were analyzed and compared with 51 samples of fertile boars. Sperm were capacitated during 4 h at 39 degrees C. viability was determined by bisbenzimide (Hoechst-33258) staining. Acrosome reaction was assessed with fluorescein isothiocyanate conjugated Pisum sativum agglutinin. The percentage of spontaneous acrosome reaction (SAR) was not significantly different in fertile (4.5%) and subfertile boars (4.75%) (p > .05). Nevertheless, the percentage of progesterone-induced acrosome reaction (IAR) was significantly lower in subfertile boars (5.75%) as compared with fertile boars (10%) (p < .01). These results suggest that assessment of IAR in vitro may be a useful parameter to identify subfertility in boars.     

Progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on noncapacitated cryopreserved bovine spermatozoa

An increase in intracellular calcium is essential to trigger capacitation and the acrosome reaction. The aim of this study was to determine the progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on heparin-capacitated and noncapacitated spermatozoa. Protein kinase C was activated by 1-oleoyl-2-acetyl glycerol, a membrane-permeant diacyl-glycerol, and inhibited by GF-109203X. The percentage of true acrosome reaction was evaluated using differential-interferential optical contrast microscopy and trypan blue stain. The calcium concentration was evaluated by FURA-2AM and methoxyverapamil was used as a voltage-dependent calcium channel inhibitor. A rapid calcium increase and acrosome reaction were induced by progesterone in capacitated and noncapacitated spermatozoa, a higher intracellular calcium increase being observed in capacitated than in noncapacitated samples (P < 0.05). The calcium increase and acrosome reaction were blocked significantly by GF-109203X in noncapacitated and capacitated spermatozoa by the addition of progesterone and/or 1-oleoyl-2-acetylglycerol. Methoxyverapamil blocked calcium influx in samples treated with progesterone and heparin/progesterone, but not in those treated with 1-oleoyl-2-acetyl glycerol. Progesterone induces the acrosome reaction in noncapacitated cryopreserved bovine spermatozoa through intracellular mechanisms dependent on protein kinase C and the voltage-dependent calcium channel.     

A comparative study of two cooling protocols on stallion sperm cryosurvival

There are many protocols for horse sperm cryopreservation, but results are inconsistent; sperm survival after freeze‐thawing is usually poor; in consequence, fertility is low. The objective of this work was to see whether slow cooling before freezing to minus 3 °C instead of +5 °C, the traditional target temperature, could improve horse sperm cryosurvival, capability to carry out capacitation and the acrosome reaction induced by progesterone. Spermatozoa from five stallions were packaged in straws and slowly cooled to +5 °C. Half of the straws were frozen directly and the other half was further cooled to ?3 °C before freezing. Progressive motility, viability, plasma membrane integrity, acrosome integrity and capacitation status were assessed. After thawing, there were no differences between cooling treatments on motility, viability, acrosome integrity and capacitation status; however, there was difference (P < 0.05) regarding plasma membrane integrity. Acrosome integrity decreased as incubation, without or with progesterone (2 μg ml^?1), progressed, but there were no differences between cooling treatments regardless of progesterone. Both capacitated and acrosome‐reacted spermatozoa increased as incubation progressed, but there were no differences between cooling treatments regardless of progesterone. Slow cooling to ?3 °C before freezing did not improve horse sperm cryosurvival or capability to undergo the acrosome reaction.