大鼠精子在附睾成熟中精子膜变化的研究

我们运用Ｐｅｒｃｏｌｌ离心技术对ＳＤ大鼠附睾头、体、尾各段的精子分离纯化后，再用硫代巴比妥酸法、酶法、ＳＤＳ－ＰＡＧＬ电泳技术等对精手膜依次进行唾液酸、甘油－３－磷酸胆碱（ＧＰＣ）及蛋白质含量变化的检测。结果显示：精子膜唾液酸、ＧＰＣ量不断降低且有显著统计学意义（Ｐ＜０．０１）。附睾头、体、尾各段精子膜唾液酸和ＧＰＣ量分别为１０．１８±２．８２、８．４２±３．０７、７．８３±２．７９μｇ／１０８精子；１１２．３１±２８．１４、１０９．３３±３７．１６、７４．５０±２５．１３ｎｍｏｌ／１０８精子（－ｘ±ｓ）。膜蛋白变化主要是由大分子蛋白转变成较小分工的蛋白。大鼠精子附睾转运中精子膜的变化与精子成熟具有十分密切的关系。     

Contribution of epididymal factors to sperm maturation and storage

Summary.  In fertile men, the majority of epididymal spermatozoa acquire the potential to fertilize (assessed with sperm function assays) on passage into the corpus and cauda regions of the epididymis. Although secretions of the epididymal epithelium are clearly important for sperm maturation and survival, their role in this process has yet to be fully determined. Alterations in epididymal sperm membranes may result from the incorporation of protein, sugar and lipid determinants. Most probably, factors of epididymal origin act in concert with constitutional changes to spermatozoa, which together permit full sperm function. Epididymal spermatozoa incubated with epididymal epithelial cell cultures can undergo some maturation in vitro , which can lead to the development of sperm fertilizing capacity. Co-incubations of human sperm with epididymal epithelial cultures, at 37 °C with medium replenished every other day, led to 50% of spermatozoa retaining motility after 8 days. In one case, a few spermatozoa survived for 17 days, the inherent maximal survival time of human spermatozoa in situ. An important aspect of coculture experiments is that close interactions between spermatozoa and epithelial cells can be examined in detail. This coculture technique may yield important information related to epididymal sperm maturation and storage.     

大鼠、金黄地鼠附睾中精子及管腔液唾液酸含量变化

应用改良硫代巴比妥酸比色法测定了ＳＤ大鼠和金黄地鼠附睾不同节段精子及管腔液的唾液酸（ＳＡ）含量。发现附睾的头、体、尾三个不同节段，精子结合唾液酸含量逐渐降低，差异显著（Ｐ＜０．０ｌ）；而管腔液唾液酸含量则有增高趋势。这两种动物相对应的附睾不同节段之间唾液酸含量存在显著差异（Ｐ＜０．０１），但二者的变化规律是一致的。对附睾中唾液酸含量变化过程的干扰，可以影响精子的成熟。     

Seasonal and epididymal maturation of stallion spermatozoa

Summary. Epididymal sperm maturation in the stallion was analysed using eight epididymides and deferent ducts from healthy animals. Samples were obtained in June-July and October-November (resting and breeding periods, respectively). Epididymides were divided into head, body and tail. Sperm samples were submitted to a routine seminogram, chromatin decondensation test (Lung, 1972) and sperm velocity determination (Makler, 1980).
Results demonstrate that stallion spermatozoa achieve maturation in the transition between the head and body of the epididymis as revealed by chromatin decondensation. Objective and subjective motility and sperm velocity acquire maximal values in the cauda epididymis where they are similar to the values seen in the vas deferens. No differences were found in the proximal and medial segments of the epididymis regarding these parameters when sexually active and resting animals were compared.     

Alterations in protein profile of rat spermatozoa during maturation

Alterations in the polypeptide pattern of rat spermatozoa during epididymal transit were studied by SDS-PAGE and compared with that of epididymal cytosol and luminal fluid. The total number of cytosol and luminal fluid polypeptides increase from caput to cauda epididymidis but sperm associated polypeptides decrease during epididymal transit. Changes in polypeptide pattern of spermatozoa are due to their acquisition, loss or modification. Spermatozoa acquire seven polypeptides, of which six are acquired in corpus (MW 16.5, 38, 41, 72, 75 and 100 Kdal) and one (MW 28.5 Kdal) in cauda epididymidis. Spermatozoa lose one polypeptide of MW 72.5 Kdal in caput and two polypeptides of MW 70 and 115 Kdal in cauda epididymidis. Four polypeptides of MW 18.5, 19.5, 64 and 67.5 Kdal disappear from cauda spermatozoa without appearing in the luminal fluid. Polypeptide of MW 62.5 Kdal is observed only in spermatozoa and luminal fluid from cauda epididymidis.     

Histophysiological and morphometric studies of the post-natal development of rat vas deferens

Summary. The post-natal growth and differentiation of the vas deferens was studied in rats aged 1–180 d by light, and with transmission electron microscopy in parallel with a radioimmunoassay of serum testosterone. During the first week and the major part of the second week of the post-natal life the vas deferens presented embryological features. Differentiation of the proximal and distal segments of the vas started 12 d after birth. At 25 d, the epithelium of the mucosa in the distal segment already showed some adult histologic characteristics. The weight notably increased between 1 and 45 d and the higher rates of growth of their histologic components occurred until 45 or 60 d, in parallel to testosterone serum levels. These results suggest that growth and differentiation of the vas deferens are androgen-dependent events and precede the end of the first spermatogenic cycle, assuring the viability of spermatozoa that will enter the duct.     

大鼠睾丸扭转复位后附睾唾液酸含量变化及意义

目的 :探讨大鼠睾丸扭转 2h和 4h复位后 2 4h附睾唾液酸含量的变化和意义。　方法 :用 2 4只雄性SD大鼠建立左侧睾丸扭转复位模型 ,分为对照组、扭转 2h组和 4h组 ,每组 8只。 5 甲基苯二酚法检测扭转侧附睾唾液酸的含量。　结果 :睾丸扭转 2h复位后 2 4h扭转侧附睾唾液酸含量 [(2 3.385± 9.2 2 0 )mg/mgprot]改变不明显 ;睾丸扭转 4h复位后 2 4h附睾唾液酸含量 [(13.72 5± 7.80 1)mg/mgprot]下降明显 (P <0 .0 5 )。　结论 :睾丸扭转 2h复位后 2 4h附睾分泌唾液酸功能不受影响 ,扭转 4h复位后 2 4h附睾分泌唾液酸功能下降 ;附睾耐受缺血再灌注损伤的时间可能较长。     

Effect of flutamide on the physiological status of epididymis and epididymal sperms

Flutamide, a pure antiandrogen, increases the levels of plasma luteinizing hormone but antagonizes the biological expression of androgen on target organs. Flutamide was administered to rats to study the effect of altered availability of hormones on the functional status of epididymis. The weights of ventral prostate, seminal vesicles and epididymis showed antiandrogenic effects of flutamide. However, increased activity of kidney beta-glucuronidase reflected increased availability of testosterone. The concentrations of protein and DNA along with the activities of acid phosphatase and hyaluronidase decreased in flutamide-treated rats. The activities of acid phosphatase and hyaluronidase in epididymal sperms along with protein concentration increased in flutamide-treated rats. Alteration of epididymal function by treatments affecting lysosomal stability was indicated.     

计算机辅助分析人、家兔、大鼠和小鼠附睾精子运动能力

本研究应用计算机辅助精子分析（ＣＡＳＡ）定量分析了人、家兔、大鼠和小鼠精子附睾成熟过程中，精子运动能力的发生和发展。同时对这几种动物和人进行了系统分析和比较。结果表明：在不同种属之间，其运动的发生和发展具有一定的差异；各种不同种属动物精子在各自附睾成熟过程中，其运动能力的两个方面参数，运动速度和运动方式的发展是不平行的；附睾尾部精子的运动能力（包括运动速度和直线程度）最强。     

Changes during puberty in chromatin condensation, morphology and fertilizing ability of epididymal spermatozoa of the golden hamster

Summary. Male golden hamster sperm acquire complete fertilizing ability at about 48 days of age. In this study hamsters, 27–130 days of age were killed and their male reproductive tracts examined. Sperm were found in the caudae epididymides from 37 days onward. None of the sperm from animals younger than 41 days were capable of fertilizing ova when placed in the uteri of superovulated females. Using flow cytometry of acridine-orange-stained cells, the chromatin condensation in cauda epididymal sperm was investigated. It was seen that DNA from sperm from the younger animals (under 40 days of age) was less tightly bound to protamine than that obtained from mature animals. In summary, the earliest sperm produced by pubertal hamsters were immature with regard to chromatin condensation, morphology, motility, and ability to fertilize ova, and they developed mature characteristics in the period between 40–48 days of age.     

Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction

A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X-100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (10 micrograms ml-1) caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 microM) 16% increase of enzyme activity could be detected.     

Intracytoplasmic sperm injection using surgically retrieved epididymal and testicular spermatozoa in cases of obstructive and non-obstructive azoospermia

This was a retrospective study of 115 patients who underwent 124 cycles of ICSI using surgically retrieved spermatozoa. The objective was to compare the results of ICSI in patients with obstructive azoospermia using epididymal spermatozoa (36 cycles) or testicular spermatozoa (58 cycles) with ICSI in patients with non-obstructive azoospermia using testicular spermatozoa (30 cycles). When epididymal spermatozoa were used for ICSI, the fertilization rate per injected metaphase-II oocyte and the clinical pregnancy rate per ICSI cycle were 60.4 and 25%, respectively. When testicular spermatozoa were used in obstructive cases, the fertilization rate and pregnancy rate were 57.9 and 34.5%. In non-obstructive cases the fertilization and pregnancy rates were 41.2 and 16.6%. When patients with obstructive azoospermia were regrouped according to the cause of obstruction, the fertilization and pregnancy rates were 59.1 and 35.1% in acquired obstruction and 58.7 and 24.3% in congenital obstruction. The fertilization and pregnancy rates were not statistically different ( p  > 0.05) when testicular or epididymal spermatozoa were used in obstructive cases; neither was statistically different ( p  > 0.05) when compared in patients with congenital and acquired obstruction. On the other hand, the fertilization and pregnancy rates in cases with non-obstructive azoospermia were significantly lower ( p  < 0.05) than in obstructive cases.     

Kinetic activity of spermatozoa from fertile subjects and asthenozoospermic infertile patients

Summary. The motility characteristics of spermatozoa from asthenozoospermic semen were investigated and compared to the same parameters in fertile semen. The motility characteristics assessed by the CellSoft semen analyser (CRYO Resources Ltd, NY) were the following: curvilinear velocity (VCL), straight line velocity (VSL), amplitude of lateral head displacement (ALH), linearity (%LIN), and beat cross frequency (BCF).
Analysis of the data indicated a decreased kinetic activity in the spermatozoa from the astheno-zoospermic group which is expressed as a highly significant decrease ( P < 0.002) in the VCL and VSL compared to velocities from normospermic samples. Moreover, percentage linearity and ALH were also statistically lower ( P <0.05) in this group. However, no difference was evidenced for the BCF.     

First observations on enzymatic activity and protein content of vesicles separated from rat epididymal fluid

Fluid of rat cauda epididymidis was obtained by flushing the duct with 0.25 mol l-1 sucrose in 0.01 mol l-1 Tris-HCl buffer pH 7.4. The fluid was centrifuged at 600 x g for 15 min and the sperm free supernatant was centrifuged at 47,000 x g for 1 h. The sediments observed with the electron microscope consisted of a heterogeneous population of membrane-bound vesicles similar to those seen in the intact organ. In the sediment containing the vesicles the activity of beta-galactosidase was mostly unavailable for the substrate showing a high degree of latency: the activity became soluble after a treatment with 0.5% saponin. The activity of N-acetyl-galactosaminadase instead, was mainly available for the substrate and soluble in buffer containing 0.6 mol l-1 KCl. It was then inferred that beta-galactosidase is located inside vesicles with no or little affinity for the membrane, while N-acetylglucosaminadase is bound to the external surface of vesicles. Supernatants and precipitates from suspensions of vesicles in buffered 0.5% saponin were analysed for proteins by gel electrophoresis. The electrophoretic patterns of the sediments were very different from those of supernatants and showed a number of bands greater than that of the latter. The vesicles are believed to arise from the epididymal epithelium, but their physiological role is unknown.     

Changes in the lectin binding sites on the testicular, epididymal, vas, and ejaculated spermatozoon surface of dog

Summary. Changes in the localization of sperm surface glycocomponents of testicular, epididymal, vas deferens, and ejaculated spermatozoa of dog ( Canis domesticus ) were studied employing fluorescein isothiocyanate conjugated lectins viz., Concanavalin A (ConA), Triticum vulgaris (WGA), Maclura pomifera (MPA), and Arachis hypogaea (PNA) agglutinins. The plasma membrane clothing the acrosome of the testicular, epididymal, and vas deferens spermatozoa shows reactivity with all the lectins used. However, in the ejaculated spermatozoa, the entire sperm surface shows reactive sites for ConA, WGA, and PNA. Variation in the labelling of the cytoplasmic droplet in different stages of spermatozoon transit in the epididymis has been discussed.     

Visco-elasticity of seminal fluid in relation to the epididymal and accessory sex gland function and its impact on sperm motility

Seminal viscopathy was shown to be associated with male infertility. However, our knowledge about the regulatory mechanism of this process is still limited. In semen samples from 411 men attending for fertility assessment, traditional semen parameters including visco-elasticity were assessed according to the World Health Organization guidelines. Sperm motility was evaluated by use of computer aided sperm analysis (CASA). Seminal activity of neutral alpha-glucosidase (NAG) and concentrations of prostate-specific antigen (PSA), zinc, and fructose were measured. The activity of NAG, and the concentrations of PSA and zinc were significantly lower in hyper-visco-elastic semen samples (medians: 5 vs. 8 mU/mL; 741 vs. 924 mg/L; 1 vs. 2 mM/L), than in those with normal visco-elasticity (p = 0.004, 0.005 and 0.011, respectively). When comparing the total amounts, only for seminal fructose there was a difference between samples with high visco-elasticity as compared with those of normal visco-elasticity (median: 74 vs. 53 microM/ejaculate, p = 0.007) This seminal marker was the only significant independent parameter in predicting seminal visco-elasticity in a multiple logistic regression analysis (odds ratio for the highest quartile = 4.67). Hyper-visco-elasticity was associated with a lower percentage of motile spermatozoa (43 vs. 50%, p = 0.045). Similar trend was found for the CASA motility characteristics curvilinear velocity (VCL), average path length (VAP), amplitude of lateral head displacement (ALH) (p = 0.008, 0.038 and 0.020, respectively). Our study demonstrated the interplay between the regulatory effect of post-testicular organs on semen visco-elasticity. Hyper-visco-elasticity was associated with asthenozoospermia and lower levels of VCL, VAP and ALH.     

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

Summary.  This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotinstreptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.     

Comparison between swim-up and glass bead column techniques for the separation of human spermatozoa

The glass bead column filtration method was compared with the swim-up technique regarding spermatozoa recovery in normal and pathological human semen. The general characteristics of the semen and the spermatic ATP concentration were measured in 67 patients. Samples processed with glass bead columns provided a good amount of motile forms which however, showed less velocity and linearity than in the case of using the swim-up technique. Additionally, the ATP determination in spermatozoa was included.