Cytochrome Oxidase and Lactate Dehydrogenase Activities in Peroxidized Rabbit Epididymal Spermatozoa/Cytochromoxydase- und Lactatdehydrogenase-Aktivität bei peroxydierten Kaninchen Spermatozoen aus dem Nebenhoden

We report a quantitative cytochemical study on cytochrome oxidase and lactate dehydrogenase activities on rabbit epididymal spermatozoa during spontaneous lipid peroxidation. Our data show that during aerobic incubation both in NTP and KTP media the sperm cytochrome oxidase activity undergoes a significant decrease. The lactate dehydrogenase activity shows different cytochemical patterns in comparison between the two media considered. Such activity significantly increases in rabbit spermatozoa suspended in NTP medium from the first until the sixteenth hour of incubation time. At the following times the lactate dehydrogenase activity significantly declines showing yet until the later times of incubation integrated optical density values fairly high. During the whole period of the aerobic incubation, the spermatozoa suspended in medium KTP show lactate dehydrogenase integrated optical density values which not significantly differ from those of the control in spite of an initial enhancement from the first until the thirteenth hour of the experimental treatment.     

Phospholipids in Guinea Pig Spermatozoa Before and After Capacitation In Vitro

Phospholipids of guinea pigs spermatozoa were examined using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Two types of spermatozoa were compared: fresh epididymal (uncapacitated) spermatozoa and epididymal spermatozoa preincubated in Ca2+-free medium for 17-18 hr (capacitated spermatozoa). Determination of lipid phosphorus revealed that the total phospholipid content of spermatozoa did not change significantly during capacitation of spermatozoa. HPLC analyses of choline and ethanolamine phosphatides, lysophosphatides, and sphingomyelin revealed that the relative amounts of these phospholipids remained unchanged during capacitation. In contrast to spermatozoa, phospholipids in the medium surrounding spermatozoa reduced by 20% during capacitation of spermatozoa, suggesting that spermatozoa may utilize extracellular phospholipids as possible energy sources during their capacitation.     

Effect of Various Prostaglandins on the Metabolism of Fructose by Epididymal and Ejaculated Ram Spermatozoa in Vitro

Die Auswirkungen einer Anzahl von Prostaglandinen auf den Fruktosestoffwechsel von Nebenhoden- und Ejakulatspermatozoen des Schafbocks wurden mit den üblichen Techniken nach Warburg untersucht. Spermatozoen aus beiden Quellen wurden gleich-zeitig mittels elektrischer Stimulierung gesammelt; die Nebenhodenspermatozoen wurden über eine Kanüle abgenommen, die ständig in ein vas deferens eingelegt war. So wurden die Spermatozoen nicht den Prostaglandinen der Samenflüssigkeit ausgesetzt. Im allgemeinen wurde der Fruktosestoffwechsel der Spermatozoen von einem weiten Bereich der Prostaglandine bei einer Konzentration von 20 μg/ml wenig beeinflußt. Von den im Samen vorhandenen Prostaglandinen hatten PGE₂, PGE₃ und PGF_2α keine signifikanten Effekte, wohingegen PGE₁ und PGF_1α die Laktatakkumulation signifikant stimulierten und das letztere die O₂ -Aufnahme erhöhte, in beiden Fällen ohne signifikante Veränderung der Fruktoseverwertung, der oxidierten Fruktose oder der CO₂-Produktion. Die 15-methyl-substituierten PGE₂ und PGF_2α sowie ihre entsprechenden Methylester veränderten den Fruktosestoffwechsel nicht und daher ist es unwahrscheinlich, daß die fehlende Wirkung von PGE₂ und PGF_2α darauf zurückzuführen ist, daß sie während der Inkubation von den Spermatozoen metabolisiert wurden. Von den untersuchten Abbauprodukten schienen PGA₁ und ih einem geringeren Ausmaß auch PGA₂ den Krebs-Zyklus zu hemmen; 15-keto 13,14-dihydro PGF_2α schien ihn zu stimulieren. Im allgemeinen reagierten die Nebenhodenspermatozoen in gleicher Weise auf die Prostaglandine wie die ejakulierten Spermatozoen. Die aus der Literatur entnommene Angabe, daß Spermatozoen eine verringerte Empfindlichkeit gegenüber Prostaglandinen in vitro haben, wenn sie einmal mit den Prostaglandinen der Samenflüssigkeit zusammengekommen waren, konnten wir daher nicht bestätigen.     

Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.     

白念珠菌体外感染对人精子运动和超微结构的影响

目的: 体外研究白念珠菌(Ca)对人精子的运动功能有无直接影响并观察精子超微结构变化,对其影响机制作初步探讨。　方法: 将从念珠菌性阴道炎患者的分泌物中分离纯化的Ca制成活菌悬液,对 10例健康青年男性手淫获得并经上游优化处理的精子进行体外感染。按细菌与精子比例不同分成A组 ( 1∶1 )、B组 ( 1∶10 )、C组(1∶100)、D组(1∶1 000)、E组(1∶10 000)、F组(空白对照)分别孵育,于 0、1、2、4h取样,计算机辅助精子分析系统(CASA)检测人精子运动参数(前向性运动百分率、直线速度、曲线速度、平均路径速度、头部侧摆幅度 )、精子存活率、精子形态及凝集情况。透射电镜观察孵育 4h后的各组精子的超微结构变化。　结果: Ca与精子体外孵育后,前向运动百分率所受影响最大,且与菌体密度及时间密切相关;其他参数与对照组相比也相继出现显著性差异。观察到精子黏附于菌体和凝集现象。精子超微结构产生变化:精子核空泡增多,顶体破裂,质膜破损,线粒体排列紊乱。　结论: Ca体外可显著降低精子存活率和抑制精子运动功能,其机制可能与Ca对人精子的黏附作用和超微结构的损伤有关。     

Testicular Steroid Metabolism In Vitro in an XX-Male

XX-males are known to display dysgenetic gonads with failing sperm production. Since defective male gametogenesis has in certain instances been proposed to be related to an aberrant intratesticular steroid biosynthesis (Steinberger et al. 1974), we have analysed the metabolism of [³H] progesterone in vitro in an XX-male.
The 33-year-old patient was sterile and demonstrated high levels of gonadotrophic hormones in the peripheral circulation, though he displayed ordinary male secondary sex characteristics. Testicular biopsy specimens were incubated with [³H] progesterone. This radiolabelled precursor was found to be avidly metabolized along the so-called "Δ⁴-pathway" towards biologically active C₁₉-androgens in this XX-male, as in testicular tissue derived from adult control subjects.
Furthermore, the relative metabolism of [³H] progesterone per mg of testicular tissue incubated, was observed to be significantly higher in the dysgenetic gonadal tissue derived from the present XX-male as compared to the metabolism in the control subjects.
The amount of [³H] 17α-hydroxyprogesterone synthesized in the XX-male was very high, and also the ratio of [³H] 20α-dihydroprogesterone:[³H]-17α-hydroxyprogesterone was found to be remarkably small, i. e. a pattern repeatedly observed in other hypergonadotrophic individuals (Kjessler & Berg 1976b).
The present investigation thus demonstrated that intratesticular steroidogenesis along the Δ⁴-pathway in vitro may be excellent in dysgenetic gonadal tissue with an XX-chromosomal constitution.     

Testosterone Production in Vitro in Human Testicular Tissue

The biosynthesis of testosterone within the testis has been shown to take place along different metabolic pathways in different species. In the testis from elderly men with prostatic carcinoma the delta 5-metabolic pathway has been shown to be preferred. Less is known about the preferred pathway in younger adults. In this study, testicular biopsy specimens from 33 adults (ranging from 27 to 35 years of age) were incubated with both tritiated pregnenolone and progesterone. The production of testosterone from these precursors was determined in vitro and was found to be significantly higher from pregnenolone than from progesterone. This indicates that pregnenolone was initially mainly converted along the delta 5-metabolic pathway to 17 alpha-hydroxypregnenolone and probably further to dehydroepiandrosterone before it was subsequently transformed to a delta 4-metabolite. Similar results were obtained in incubation studies performed with testicular tissue from eleven elderly men (ranging from 60 to 83 years of age). In conclusion, studies of the testosterone production in vitro suggest that the delta 5-metabolic pathway is preferred in the testis from younger as well as elderly men.     

Effect of Long Term Treatment with Gonadotrophic Hormones on In Vitro Metabolism of [3H]Progesterone in Human Testicular Tissue

Gonadotrophic hormones appear to influence the preferred pathways for intratesticular androgen biosynthesis. Individuals with low levels of gonadotrophin in the peripheral circulation, as e. g. prepubertal boys and a hypophysectomized adult male, have previously been shown to metabolize [³H]progesterone in vitro mainly to 20α-dihydroprogesterone (20α-DH-P) and less to 17α-hydroxyprogesterone (17α-OH-P), i. e. an "immature metabolic pattern" (Berg et al. 1976; Kjessler & Berg 1976b).
With physiologically increasing amounts of circulating gonadotrophins the preferred metabolic pathway in vitro shifts in favour of 17α-OH-P, i. e. a "mature metabolic pattern". Such an alteration in the preferred in vitro metabolic pattern of [³H]progesterone has also been obtained by exogenous administration of gonadotrophins to a 47, XYY-male with an initially immature metabolic pattern (Berg & Kjessler 1976).
We have sequentially analysed the metabolism of [³H]progesterone in vitro in testicular incubates derived from a chromosomally normal male before and after 16 weeks of substitution therapy with gonadotrophic hormones.
The patient originally displayed an "immature" pattern of progesterone metabolites, i. e. the ratio 20α-DH-P/17é-OH-P was 3.04. After treatment with human menopausal gonadotrophins and hCG, the metabolic activity in total had increased, and the ratio 20é-DH-P/17é-OH-P had switched to 0.39.
These results confirm and extend the concept that gonadotrophic hormones may have a regulatory function on androgen biosynthesis by stimulating the oxidative metabolic pathway from progesterone to biologically active androgens via 17α-hydroxyprogesterone.     

Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress

Cytochrome c oxidase (CcO) plays a key role in cellular respiration and energetic metabolism. The latter is a prerequisite for osmotic and synthetic function, motility and the maintenance of cell structure. The objective of this study was to develop and assess a facile microscopic technique that would demonstrate CcO activity insitu, both at the microscopic and sub-microscopic levels. The cytochemical technique proposed is based on oxidation of 3,3'-diaminobenzidine (DAB) by the cytochrome c complex (including CcO), in a chain reaction in which the reagent is polymerized and deposited at the reaction sites. The deposit can be identified by its colour under the light microscope and by osmiophilia under the electron microscope. The reaction is restricted to mitochondria at the light microscope level, and to the outer face of the inner mitochondrial membrane at the ultrastructural level. The activity is inhibited promptly by greater than or equal to 0.5 mM KCN, a specific inhibitor of CcO, and by sodium azide or heat (70 degrees C/5 min). The technique was validated on a number of domestic and laboratory animals, using sperm that were ejaculated or epididymal in origin, cryopreserved or treated. The data obtained displayed activity profiles of individual cells, ejaculates or donors and emphasized differences among species. This technique depicts spontaneous CcO decline during ageing and changes induced by various physical or chemical treatments.     

Testicular Steroid Metabolism In Vitro and Spermatogenesis in a Male with an XYY-Karyotype

Males with a 47, XYY-chromosomal constitution have been reported to generally suffer from an impaired gametogenesis, and the reason for this spermatogenetic failure is unknown.
The concentration of gonadotrophic hormones and testosterone in the peripheral circulation of XYY-males have not been found to differ significantly from normal control subjects, but so far there is a lack of information on the intratesticular steroid conversion patterns in such individuals.
We have repeately analysed the metabolism of [³H] progesterone in vitro in incubated testicular biopsy specimens derived from an XYY-male before and after gonadotrophic substitution therapy. In addition hormone levels in peripheral blood samples, gametic output and testicular histopathology were investigated.
The present patient was a 34-year-old healthy, but infertile male with a 47.XYY-karyotype. His gonads were reduced in volume (6–8 ml) and displayed a heterogenous morphological picture. The appearance of the tubules varied from normal to complete destitution of germinal cells. The Leydig cell apparatus appeared normal. Serum levels of FSH, ICSH (LH) and testosterone were repeatedly within normal limits. The seminal fluid contained less than 10 mill. sperm per ml.
Incubated testicular biopsy specimens metabolized [³H] progesterone in vitro mainly to 20α-dihydroprogesterone (20α-DH-P) and to a less extent to 17α-hydroxyprogesterone (17α-OH-P). Thus, the ratio 20α-DH-P:17α-OH-P was found to be remarkably high, i.e. an immature pattern often observed in prepubertal testicular tissue (Berg et al. 1976). By exogenous administration of gonadotrophic hormones to the present patient the steroid metabolic pattern in vitro changed in favour of an increased production of 17α-OH-P, concomitantly with an observed rise in gametic output.     

Studies on the Distribution of Feulgen-DNA Content in Normal and Pathological Human Spermatozoa

Untersuchungen über die Verteilung des Feulgen-DNA-Gehaltes in normalen und pathologischen menschlichen Spermatozoen
Die Entwicklung eines Scan-Tisches mit einer Schrittgröße von 0.25 μm erlaubte es uns, genauere Untersuchungen über die Feulgen-DNS-Verteilung in normalen und pathologischen menschlichen Spermatozoen durchzuführen.
Die Art der Verteilung der Feulgen-DNS ließ keine Unterschiede zwischen normalen und pathologischen Spermatozoen erkennen.
Die Wahl der Fixierung, die Hydrolysedauer und die Passage von Cervixmucus führten jedoch zu signifikanten Unterschieden der Feulgen-DNS-Werte und ihrer Verteilung in normalen und pathologischen Spermatozoen.
Das Verteilungsmuster der Feulgen-DNS vermag wahrscheinlich Informationen über die Sekundär- und Tertiärstruktur der Spermatozoen-DNS zu vermitteln.     

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.     

人工材料作为细胞体外培养载体的观察实验研究

目的：在体外培养的条件下了解人工材料与细胞的相容性以及细胞在材料上的生长情况。方法：将冻存复苏后的传代细胞在碳纤维、头发、涤纶、几丁质四种材料联合培养，进行光镜、扫描电镜观察。结果：在Ｆ－１２培养基中，碳纤维与成纤维细胞、肌腱细胞相容性均好，有良好的吸附性；眷属材料次之；涤纶材料上两种细胞生长均少，可能与其表面结构有关；几丁质则明显抑制这两种细胞生长。同时发现两种细胞在碳纤维上坐落的数量都明显优于     

Investigations on the Motility of Human Spermatozoa in a Defined Medium in the Presence of Metabolic Inhibitors and of Carnitine

When washed human sperm were incubated in a modified Krebs-Ringer buffer solution in the absence of exogenous metabolizable substrates at 30 degrees C they maintained progressive motility for at least six hours. Under these conditions the spermatozoa apparently utilize endogenous substrates but addition of exogenous substrates (glucose, fructose, acetate, short-chain fatty acids, branched chain amino acids) did not affect the % progressive motility or % total motility of the cells. The phospholipase inhibitors, quinacrine and Upjohn No. 1002, inhibited progressive motility when added to sperm utilizing endogenous substrate, and subsequent addition of oxidative or glycolytic substrates did not reverse the inhibition. In contrast, the inhibition by KCN of progressive motility based upon utilization of endogenous substrate was reversed upon addition of glycolyzable compounds (glucose or fructose). The addition of carnitine or its acetyl-, propionyl-, isobutyryl-, valeryl- or isovaleryl esters did not consistently affect progressive or total motility of sperm samples. The inhibitor, octylsulfobetaine, inhibited sperm motility at a concentration higher than that required for inhibition of carnitine acetyltransferase or translocase activity. On the basis of these results it does not appear that exogenous carnitine has an effect on the motility of human sperm incubated under the conditions described here.     

阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

雌二醇对不同性质的乳腺原代细胞增殖影响的体外研究

目的了解在体外条件下不同浓度的雌二醇对不同性质的乳腺组织原代细胞增殖的影响。方法用胶原酶消化法获取乳腺癌患者的各类乳腺组织原代细胞，并用MTT法检测乳腺癌组织、癌旁组织和正常乳腺组织原代细胞在体外培养下，给予不同浓度雌二醇时细胞的生长增殖情况。结果雌激素在体外对雌激素受体（ER）表达阳性的乳腺癌组织及癌旁组织原代细胞有明显的刺激生长作用；对正常乳腺组织细胞的促生长作用相对较弱，而且与肿瘤组织的ER表达情况无关。结论当体内的雌二醇水平≥10μmol／L时，乳腺癌的发病风险和复发风险会显著增加。     

Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer

The aim of this study was to evaluate the efficacy of swim-up, PureSperm gradient centrifugation and glass-wool filtration methods for semen preparation and to assess the possible enhancement of the quality of the subpopulation of spermatozoa in terms of sperm concentration, morphology and chromatin condensation. Moreover, to determine the effect of this semen processing technique on the clinical outcome after in vitro fertilization embryo transfer (IVF-ET). A total of 180 semen samples of patients' husbands who were undergoing IVF therapy were prepared by swim-up (G1, n = 60), PureSperm gradient centrifugation (G2, n=60) or glass-wool (G3, n=60) methods. Chromatin condensation was assessed by Chromomycin (CMA3), whereas sperm morphology was evaluated according to strict criteria. In all three semen processing methods, the percentage of chromatin condensed and morphologically normal spermatozoa was higher after semen processing in comparison with native semen samples. The proportion of normal chromatin condensed spermatozoa prepared in glass-wool filtration was significantly higher than that in swim-up (G.I, p=0.02) or PureSperm (G.II, p=0.001). In addition semen processing with PureSperm yields significantly a higher percentage of morphologically normal spermatozoa than swim-up (p < 0.001) or glass-wool method (p < 0.002). However, the fertilization, implantation and pregnancy rates, in turn were similar in all semen preparation methods. In conclusion, PureSperm gradient centrifugation yields a higher percentage of morphologically normal spermatozoa than shown in traditional swim-up or glass-wool filtration. However, the percentage of chromatin condensed spermatozoa was significantly higher after semen processing via glass-wool in comparison with the other two methods. Nevertheless, there were no significant difference in the fertilization, implantation and pregnancy rates of sperm prepared by means of swim-up, PureSperm or glass-wool filtration. Therefore, glass-wool filtration should be recommended as the first choice for semen preparation for Intracytoplasmic sperm injection (ICSI) technique as the natural selection is bypassed. Whereas, swim-up and PureSperm should be used for semen processing in IVF programme.