Flow Cytometry and Sizing for Routine Andrological Analysis

Summary: Flow fluorometry and Coulter type sizing analysis of sperm have been applied separately in order to improve human semen analysis. Different methods of sample preparation were evaluated and a protocol involving prestaining pepsin treatment of sperm samples is proposed for fluorometric analysis. The data obtained with fluorometry and sizing analysis result in different kinds of information: Coulter counting allows to automate sperm counting and fluorometry yields more datailled information about normozoospermia and oligozoospermia by determining the proportion of mature spermatozoa and immature germ-cells. These two methods, together with light microscopy, may help to explore the correlation of fertility and pathology of spermatozoa. The aim of these investigations is to yield the preconditions for simultaneous two-parameter analysis of DNA content and cellular size distributions. Zusammenfassung: Durchflußcytophotometrie und Größenmessung für andrologische Routineuntersuchungen von Spermatozoen Durchflußanalyse und Größenanalyse nach dem Coulter-Prinzip wurden angewandt, um die Begutachtung menschlichen Spermas zu verbessern. Unterschiedliche Methoden der Probenaufbereitung wurden ausprobiert, und ein Protokoll mit Pepsinbehandlung der Spermatozoenproben für fluorometrische Analysen wird vorgeschlagen. Die mit der Cytofluorometrie und Größenanalyse erhaltenen Daten ergeben unterschiedliche Informationen: Das Coulter-Prinzip erlaubt die Automatisierung der Spermatozoenzählung, und die Fluorometrie ergibt detailliertere Erkenntnisse über Normozoo-spermie und Oligozoospermie, da die Unterscheidung von reifen Spermatozoen und unreifen Keimzellen ermöglicht wird. Diese beiden Methoden können in Kombination mit der Lichtmikroskopie helfen, die Korrelation zwischen Fertilität und Pathologie von Spermatozoen zu erkunden. Das Ziel dieser Untersuchungen ist es, die Voraussetzungen für gleich-zeitige Zweiparameteranalysen des DNA-Gehaltes und der zellulären Größenverteilungen zu liefern.     

Combined Human Chorionic Gonadotropin (HCG) and Human Menopausal Gonadotropin (HMG) Treatment in Gonadotropin-Deficient Males with Pituitary Dwarfism

The effects of hCG-hMG treatment in 13 boys with pituitary dwarfism associated with gonadotropin deficiency, were assessed.
No patients except one showed signs of puberty at a bone age of 13 years or above. The one patient with some signs of puberty did not become fully mature. The hCG-hMG was started at a mean age of 20.4 years. The hCG at a dose of 5,000 IU was injected intramuscularly twice a week and the hMG at a dose of 75 IU was given once a week at first. During treatment, the frequency of hMG injections was increased to twice a week in six patients who still had not produced normal sperm counts. After a mean duration of 19.23 months, spermatozoa appeared in eight patients, of whom four showed more than 20 × 10⁶ sperm/ml. Among six patients who did not have normal sperm counts and had increased hMG injections, one produced a pregnancy and four achieved sperm counts of more than 35 × 10⁶/ml. One patient had refractory azoospermia. In 13 boys with growth hormone and gonadotropin deficiency, hCG-hMG treatment produced normal spermatogenesis in nine patients, one of whom fathered a girl. Thus, hCG-hMG treatment, especially twice-a-week injections of both hCG and hMG, appears to be effective for gonadotropin deficiency in males.     

中华蚱蜢精子发生和减数分裂染色体形态变化的观察

介绍了中华蚱蜢Acrida Chinensis(Westw)精子发生细胞减数分裂的标本制作方法,此法效果满意,并用照片详述了精子发生过程中染色体形态的连续变化。     

Evidence of a Wave of Spermatogenesis in Human Testis*

Hinweis auf das Vorliegen einer Welle der Spermatogenese im menschlichen Hoden In Samenkanälchen von zwei alten Männern war eine ungewohnt klare Gliederung des Keimepithels in Stadien erkennbar. An flächenhaften Rekonstruktionen der Wand von Samenkanälchen konnte die Existenz wendelförmig angeordneter Stadiensequenzen festgestellt werden. Ähnliche Befunde ließen sich auch bei einem jüngeren Patienten erheben. Die vorliegenden Ergebnisse weisen darauf hin, daß es auch im menschlichen Samenkanälchen eine Welle der Spermatogenese gibt. Es ist denkbar, daß die besondere lokale Organisation des Keimepithels beim Menschen auf einem spezifischen Modus der Spermatogoniogenese beruht.     

Immunolocalization of Smad4 protein in the testis of domestic fowl (Gallus domesticus) during postnatal development

The transforming growth factor beta (TGF-β) superfamily exerts a wide range of effects on biological events, including spermatogenesis. Smad proteins are downstream signal mediators, which transduce TGF-β signals from the cell surface to the nucleus. Smad4 protein is the common transducer of the TGF-β superfamily that participates in the signaling of all the members of TGF-β superfamily. Smad4 is expressed in the mammalian testis and is believed to play an important role during testicular development and spermatogenesis. Information about Smad4 distribution and function in the testis of birds, including the domestic fowl, is still unclear. In the current study, our objective was to clarify the signal transduction pathway of the TGF-β superfamily in the regulation of testicular development and spermatogenesis by investigating the expression of Smad4 protein in the testis of newborn, prepuberty, puberty and adult domestic fowl. Cellular localization of Smad4 was determined by immunohistochemistry. Our study revealed that the Smad4 was widely expressed in the fowl testis, mainly immunolocalized in the cytoplasm of Sertoli cells, Leydig cells and germ cells. The presence of Smad4 protein in these testicular cells provides molecular and morphological evidence for TGF-β signal transduction during testicular development and spermatogenesis.     

DEAD box家族蛋白与精子生成的研究进展

精子的发生和成熟过程不但需要多种相关基因正确表达,而且还需要多种RNA的参与.因此,这些RNA需要较好的稳定性以保证其功能的正确执行.DEAD-box家族蛋白是一个ATP依赖的RNA解旋酶家族,参与RNA的各种代谢过程如RNA二级结构变换,转录起始,线粒体RNA剪接,核糖体和剪接体装配、mRNA降解以及维持mRNA的稳定性等.最近的一些研究显示这个家族的一些成员如DDX3,DDX4,DDX25等与精子生成有着密切的关系.     

Hormonal approaches to male contraception: approaching reality

The ‘pre-testicular’ suppression of gonadotrophins is the most likely approach for reversible therapeutic male fertility control to reach imminent clinical application. Maintenance of spermatogenesis depends on adequate gonadotrophin and intratesticular testosterone concentrations. Hormonal contraception for men interrupts this physiological axis by various means of gonadotrophin suppression; this interferes with spermatogonial differentiation and meiosis entry resulting in reversible azoospermia or severe oligozoospermia in virtually all men. Clinical trials have confirmed that high contraceptive efficacy, similar to female hormonal contraceptives, can be reliably attained with few side effects. However, the simultaneous suppression of Leydig cell steroidogenesis mandates the requirement for testosterone replacement in hormonal male contraception. Combination regimens of new synthetic progestins and androgens at various stages of development are being investigated with the lead products poised to go into phase III trials. Heterogeneity in response to spermatogenesis suppression has been observed within and between population; the mechanisms are unclear. This new method of reversible and effective contraception has registered high acceptability in surveys of both men and women. The recent entry of pharmaceutical companies into this area of research and development has considerably enhanced the prospects of translating years of academic efforts into new products which provide added family planning choice for many couples.     

青春期SD大鼠单侧睾丸扭转后FAS/FASL系统变化的研究

目的初步研究青春期一侧睾丸扭转SD大鼠性成熟后对侧睾丸的FAS/FASL系统的表达与对侧睾丸生精功能的变化情况的相互关系.方法建立青春期SD大鼠一侧睾丸扭转模型5组,A组为假手术对照组;B组为睾丸扭转组;C组为睾丸扭转+甲强龙组;D组为扭转侧睾丸切除组;E组为扭转侧睾丸切除+甲强龙组.于扭转后24h给予睾丸切除或/和注射甲强龙处理,术后1月处死,取对侧睾丸组织,HE染色检查,用免疫组织化学染色法检测睾丸生精细胞,支持细胞的FAS及FASL的表达情况,并对FAS及FASL的表达作定量分析.用放射免疫法检测血清中FSH,LH,T的值并做分析.结果 B组对侧非扭转睾丸的形态有病理性改变.各组睾丸组织同时表达FAS和FASL,B组的FAS和FASL的表达强度较其他各组有明显的升高.各组血清中FSH,LH,T的值没有统计学差别.结论青春期SD大鼠单侧睾丸扭转后对侧睾丸生精细胞发生FAS和FASL的高表达,从而导致细胞的凋亡增加,生育力下降.皮质激素可能通过抑制FAS和FASL的表达,降低生精细胞的凋亡,维持生精功能的稳定.     

小鼠精子发生过程中雄激素受体对靶基因Septin7的表达调控

目的:探讨雄激素受体(AR)对睾丸Sertoli细胞中Septin 7基因表达的影响。方法:分别采用实时荧光定量PCR及Western blot,检测睾丸Sertoli细胞AR特异性敲除(S-AR-/y)和野生型小鼠(WT)睾丸Sertoli细胞中SEPTIN 7的表达,并观察雄激素对过表达AR的Sertoli细胞系TM4中SEPTIN 7表达的影响。结果:与WT小鼠相比,S-AR-/y中Septin 7 mRNA表达水平升高(P0.05);雄激素显著降低TM4中SEPTIN 7表达(P0.05)。结论:Septin 7可能是睾丸Sertoli细胞中AR调控的靶基因。该研究对阐明雄激素及其受体调节精子发生的分子机理具有重要意义。