棕色田鼠睾丸和附睾胚后发育中的睾酮表达

应用免疫组织化学方法研究了产后1、10、25、45、60日龄(成体)5个发育阶段的棕色田鼠(Lasiopodomys mandarinus)睾丸和附睾组织内睾酮的免疫阳性反应.1日龄和10日龄,棕色田鼠睾丸生精小管内的前精原细胞胞质中有睾酮阳性表达.25日龄,有许多精子细胞产生,睾酮主要集中于精子细胞胞质表达.45日龄,精母细胞和精子中也有睾酮表达.成体精原细胞、精母细胞、精子细胞和精子中均有睾酮表达.1日龄至成体睾丸间质细胞和肌样细胞均有睾酮表达,25日龄时表达最强(P0.05).1日龄至成体附睾上皮细胞和连接组织有睾酮表达,成体附睾管内的大量精子有睾酮表达.这些结果说明,棕色田鼠从出生到性成熟过程中,在精子发生的各阶段,睾酮对生精细胞的分化增殖有直接的调控作用,这种调控作用随发育阶段不同具有可变性,同时,附睾的功能和精子的成熟也受到睾酮的调节.     

棕色田鼠睾丸和附睾雌激素α受体表达的增龄变化

应用免疫组织化学方法系统研究了初生（1日龄）到成体（60日龄）5个发育阶段各6只棕色田鼠睾丸和附睾内雌激素受体α（ERα）的表达。结果发现：①在生殖细胞中,1日龄组幼鼠的生殖母细胞和支持细胞有微弱的ERa阳性表达,10日龄组精原细胞中ERa有弱表达,25日龄组精子细胞出现了较强ERα阳性表达,发育到45日龄时精子细胞ERα阳性表达最强,成体组中各级生精细胞中均有ERα阳性表达。②在间质细胞中,1日龄组间质前体细胞有ERα阳性表达,10日龄表达减弱,而到25日龄增强,至45日龄达到峰值,而成体又减弱。③附睾中,1日龄附睾上皮细胞有ERα阳性表达,10和25日龄附睾管类肌细胞出现ERα阳性表达,45日龄和成体附睾管上皮细胞和类肌细胞中均有ERα阳性表达。上述结果表明,ERα可能作为一种特异性受体在棕色田鼠睾丸发育过程中影响睾丸间质细胞雄激素的分泌,进而调节生精过程和精子的发育成熟。     

棕色田鼠睾丸和附睾雄激素受体表达的增龄变化

应用免疫组织化学方法研究了1、10、25、45及60日龄（成体）5个发育阶段的棕色田鼠（Lasiopodomys mandarinus）睾丸和附睾中雄激素受体（androgen receptor,AR）的表达。结果发现：①睾丸间质细胞中：1日龄有AR表达,至10日龄和25日龄AR表达减弱,45日龄AR表达最强,至成体AR表达又减弱（P＜0.05）;②肌样细胞中：从1日龄至成体均有AR表达,25日龄AR表达最弱,45日龄AR表达最强,至成体AR表达又减弱（P＜0.05）。③1日龄前精原细胞偶有AR表达,10日龄精原细胞没有AR表达;25日龄精子细胞有AR表达,45日龄精子细胞和部分精母细胞有AR表达,成体精原细胞和精母细胞及精子中有AR表达。④支持细胞中：性成熟前AR表达不明显,成体有AR表达。⑤从1日龄到成体,附睾中均有AR表达。这些结果表明,雄激素在棕色田鼠睾丸间质细胞、肌样细胞和生精细胞的表达随个体的发育阶段而变化;雄激素可促进青春期棕色田鼠间质细胞的功能与分化,肌样细胞在精子发生过程中有重要作用;同时,雄激素对附睾功能有调控作用。     

粗糙沼虾精巢发育的组织学

利用光镜技术,对粗糙沼虾精巢发育进行了研究,根据精子发生过程中每种生殖细胞所占的比例和发生的次序,并结合精巢的形态特征,把精巢发育过程分为五个时期,即精原细胞期,精母细胞期,精细胞期,成熟精子期及退化期,精原细胞期,精巢小,透明乳白色,生精小管内的生殖细胞以精原细胞为主;精母细胞期;精巢体积增大,半透明乳白色,主要由处于初级精母细胞的次级精母细胞阶段的生殖细胞组成;精细胞期,精巢体积继续增大,颜色加深,生精小管内的生殖细胞以精细胞为主;成熟精子期,精巢体积可达最大,紫红色,生精小管内充满着成熟的精子,退化期;精巢体积减小,半透明乳白色,生精小管内的成熟精子几乎排空。     

生精细胞及精子赖氨酸含量的研究

本实验取１０只Ｗｉｓｔａｒ大鼠的睾丸和附睾,睾丸石蜡切片,附睾精子涂片后用苯胺蓝染色显示赖氨酸含量。结果是睾丸生精小管中精原细胞和精母细胞染色较深即赖氨酸含量较高,精子细胞和精子染色渐淡即赖氨酸含量降低,而附睾精子显示,在附睾头部的精子染色较深,附睾尾部的精子几乎不着色,应用显微分光光度计测定附睾精子,计算出头部的精子赖氨酸含量在１左右,尾部的精子赖氨酸含量接近于零。本实验还检测了１０例正常人及１０例不育者精子的赖氨酸,结果为正常人精子的赖氨酸含量较低,不育者精子赖氨酸含量高且畸形率也高。提示精子赖氨酸含量高是核蛋白转型异常的征象,可能是男性不育的一个重要原因。     

睾丸及附睾免疫环境与男性生殖

生精细胞在发育成熟过程中产生大量抗原性蛋白,但在睾丸内并不引起免疫反应;另一方面,一些机体系统性免疫反应可以破坏男性生育能力.这些现象的调控机理是男性生殖领域广泛关注的重要问题,对这些问题的深入认识可能为预防及治疗由炎症引起的男性生育障碍提供新线索.睾丸是精子发生的场所,而附睾是精子成熟的器官,二者均会发生影响男性生育能力的免疫反应.然而睾丸和附睾中的免疫反应存在很大区别,睾丸具有较强的免疫豁免能力,附睾比睾丸易发生免疫反应,附睾内的精子比睾丸中的生精细胞更容易被免疫系统损伤.而且离睾丸越远的附睾区域,产生的炎症反应及其对生殖的影响越大.深入研究其调控机制将有助于揭示男性生殖系统特殊的免疫环境.     

秦岭北坡中国林蛙精巢显微结构的年周期变化

用光镜观察了秦岭北坡中国林蛙(Ranachensinensis)精巢显微结构的年周期变化,结合精巢系数的变化探讨其生殖规律。结果显示,秦岭北坡中国林蛙的生精周期属于非连续型。精巢系数的变化与精子发生的活动周期相一致。精子发生从每年5月开始,翌年4月结束,历时1年。生精周期可划分为5个时期。Ⅰ期,精原细胞增殖期,5～7月,精巢系数最小,精原细胞进行有丝分裂;Ⅱ期,精母细胞成熟分裂期,8～9月,精巢系数最大,精原细胞、精母细胞和精子细胞在生精小管内共存;Ⅲ期,精子形成期,9～1 0月,精子细胞变态形成精子;Ⅳ期,成熟精子贮存越冬期,1 1月至翌年2月,成熟精子贮存在生精小管中;V期,精子排放期,翌年3～5月,精巢系数显著下降,成熟精子从生精小管脱离,通过输精管道排出体外。     

一种新的大鼠隐睾模型的建立

目的改善大鼠隐睾模型的制作方法,提高隐睾模型的质量,并对新模型的稳定性进行研究。方法28只大鼠随机分为对照组（ctrl）和模型组（modl）采用模拟失重大鼠模型,对大鼠进行3周尾部悬吊进行造模,随后模型组大鼠解悬吊恢复8周观察该模型的稳定性。结果经过3周的尾部悬吊,模型组所有大鼠睾丸均滑入腹腔,同时和对照组相比,睾丸和附睾的重量出现极显著的降低（P〈0.01）。HE染色发现对照组大鼠睾丸的生精小管结构排列紊乱,精原细胞消失,附睾尾中成熟精子消失。经过8周的恢复,大鼠的睾丸及附睾仍未恢复到正常水平（P〈0.01）,HE染色显示其生精小管结构和精原细胞数量并未出现明显的改善。结论尾吊法所建立的大鼠隐睾模型效果稳定,可以有效模拟大鼠隐睾时的睾丸温度变化情况,同时对大鼠的伤害较小,操作比较简单。     

中国石龙子雄性生殖腺的年周期变化

20 0 1年 3月至 2 0 0 2年 2月期间 ,通过每月捕捉浙江丽水中国石龙子 (Eumeceschinensis)雄性成体 ,解剖动物、观测性腺的形态和组织学特征 ,研究雄性生殖周期。睾丸重量和体积、附睾重、输精管重和曲细精管直径有显著的季节变化。睾丸 3月份最重 ,5-9月最轻。睾丸体积和重量的年周期变化规律一致。附睾 3月份最重 ,8-9月份最轻。输精管 4月最重 ,8-10月最轻。生精活动始于 9月下旬 ,翌年 4月最为活跃。 3月下旬曲细精管直径达全年最大值 ,管腔中开始出现呈穗状排列的精子。从基膜到管腔 ,各级生精细胞依次排列。 4月份生精上皮的生精活动最为活跃 ,5月下旬生精活动已近停止 ,7-8月份曲细精管管壁仅由精原细胞 (其间夹有支持细胞 )构成。根据曲细精管生精上皮的年周期变化规律 ,中国石龙子 8月份睾丸生精活动处于第Ⅰ期 ,9月至次年 2月份第Ⅱ期 ,3月上、中旬为Ⅲ期 ,3月底至 4月为Ⅳ期 ,5-6月份为Ⅴ期 ,7月份为Ⅵ期。 4月下旬附睾管腔中有大量的成熟精子 ,7月附睾管腔中已无精子。中国石龙子属于关联型繁殖周期     

双峰驼睾丸和隐睾细胞外基质相关蛋白的组织化学特征及超微结构比较

为探索细胞外基质相关蛋白在隐睾双峰驼的分布情况及其组织化学特征,应用电镜技术和多种组织化学方法比较了隐睾和正常睾丸的超微结构,组织化学特点及层粘连蛋白（LN）、Ⅳ型胶原（Col Ⅳ）和硫酸乙酰肝素糖蛋白（HSPG）的分布特征。结果显示：(1)与正常睾丸间质结构相比,光镜下隐睾生精小管发育不全,间质内胶原纤维稀疏,网状纤维分布明显,间质血管及生精小管固有膜PAS及AB-PAS阳性反应较弱。电镜下,隐睾生精上皮基膜明显增生,外围I型胶原纤维较少,管周肌样细胞不典型;间质毛细血管及Leydig细胞周围纤维细胞多见,而正常睾丸在间质毛细血管及Leydig细胞周围多分布有成纤维细胞。(2) 免疫组织化学染色显示,正常睾丸组织的Col Ⅳ、LN及HSPG在Leydig细胞内均为强阳性表达,Col Ⅳ和LN在毛细血管内皮细胞强阳性表达,后者在Sertoli细胞的表达尤为明显,HSPG在精原细胞无表达;隐睾时Col Ⅳ、LN及HSPG在Leydig细胞内阳性表达均明显减弱,Col Ⅳ、LN在管周肌样细胞及毛细血管内皮细胞阳性表达也减弱明显,HSPG在精原细胞较强阳性表达,且在精子细胞呈强阳性表达。免疫组织化学图像分析结果显示,双峰驼正常睾丸组织中Col Ⅳ和LN的分布显著高于隐睾组织（P<0.05）,HSPG检测结果在正常睾丸与隐睾之间无统计学差异（P>0.01）。该研究表明,双峰驼隐睾生精小管发育异常,间质组织中合成胶原纤维的能力下降,睾丸细胞外基质的重要成分Col Ⅳ,LN与正常组差异显著与生精小管及Leydig细胞异常发育有关,而HSPG在隐睾生精上皮的强阳性表达与精原细胞发育不成熟密切相关。     

ABCG2 is expressed in late spermatogenesis and is associated with the acrosome

An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72 kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.     

五唇兰精细胞的分离

五唇兰(Doritis pulcherrima)具二胞花粉,授粉后1 d即有花粉开始萌发,授粉后5 d开始有生殖细胞完成有丝分裂形成一对精细胞。通过人工授粉使花粉管在子房内发育,再利用花粉管直接爆破,成功分离出五唇兰的精细胞。成对的2个精细胞在直径大小、荧光强弱上均显示出较大差异,预示2个精细胞具有不同的前途。     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Organization of the Y chromosome in testis cells of fetal,subadult and adult mice as determined by in situ hybridization

In order to reveal the time-course of decondensation of the Y chromosome in Sertoli cells, testes preparations of fetal, subadult and adult laboratory mice in different developmental stages were hybridized in situ with biotinylated probe pY353/B, which binds along the entire long arm of the mouse Y chromosome. All fetal and subadult testicular cells exhibited tightly compacted hybridization signals, indicating highly condensed Y chromosomes. Diffuse signals, indicating decondensation of the Y chromosome were found for the first time in the structurally differentiated Sertoli cells of 35 to 40 day old animals. Since this coincides with the appearance of mature sperm nuclei, a correlation between decondensation of the Y chromosome and its activity in sperm maturation and/or sperm motility can be presumed.     

A light- and electron-microscopic investigation of gametogenesis in Typosyllis pulchra (Berkeley and Berkeley) (polychaeta: Syllidae)

Summary Typosyllis pulchra reproduces by the production of three to four gamete-bearing stolons (schizogamy) during consecutive 30-day periods. Although gonads are found in a large number of segments, only those in the posterior-most segments produce gametes and become incorporated into the developing stolon. The more anterior gonads remain undifferentiated and probably sexually undetermined until they are needed in future stolonizations. Gonial cells, which will eventually become either male or female, are ultrastructurally identical at the onset of each stolonization period. Spermatogenesis is marked by a short proliferative period followed by differentiation and spermiogenesis. The first ultrastructural signs of spermatogenesis were found in coelomic spermatogonia on day 10 of stolon formation. Spermatogonia are joined by intercellular bridges, which are maintained until the early spermatid stage. Synaptonemal complexes mark the onset of meiosis, which is apparently synchronized in the syncytial clusters of primary spermatocytes. Spermiogenesis occurs during the final 10 days of stolonization and a variety of stages is present within a single animal. All sperm mature by the time the stolon detaches. Acrosome formation and nuclear condensation are described in addition to the ultrastructure of mature sperm.     

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.     

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.     

Post-ejaculatory changes in the metabolic status of rat spermatozoa as measured by GC-MS

Analysis of low molecular weight or metabolic compounds can provide deeper insights into the biochemical pathways regulating normal cell function. Here we report, for the first time, a method to extract a large number of metabolites from rat spermatozoa, which were analysed using gas chromatography mass spectrometry. Based on the retention time index and at least 3–5 fragment qualifier ions, we positively identified 71 compounds, 2 of which we could not find any match to the database. Several different classes of metabolic compounds including amino acids, sugars, fatty acids, sterols and lipids were found. In order to gain insight into sperm function, we extracted metabolites from sperm cells that were in the initial stages of the post-testicular sperm maturation process known as capacitation, and compared the relative intensity of each compound to non-capacitated spermatozoa through the use of an internal standard. We could clearly demonstrate significant down regulation of cholesterol, a hallmark of capacitating cells, being less abundant in the more mature cells. In addition, several monosaccharides including glucose, fructose, sorbitol, galactose and the polyol myo-Inositol decreased in their abundance as sperm begin to capacitate. Interestingly, galactose was able to support sperm motility and an increase in the level of tyrosine phosphorylation, however this came at the expense of longevity of these cells when compared to glucose.     

The secretion of two sperm maturation-related glycoproteins in BALB/c mouse epididymis

Summary A well-developed Golgi apparatus and rough and smooth endoplasmic reticulum in the principal cells of the mouse epididymis indicate active protein synthesis. Studies have shown that epididymal secretions are essential for sperm maturation. In a previous study, two wheat-germ agglutinin (WGA)-binding glycoproteins, GP-49 and GP-83, were identified on the surface of mature mouse sperm. In this study, synthesis and secretion of these two glycoproteins were investigated. Apparent WGA-binding was found on the stereocilia and in the apical region of principal cells in the corpus and cauda of epididymis. Post-fixation and pre-embedding cytochemical localization revealed that WGA-binding sites were situated in the Golgi apparatus, multivesicular bodies and stereocilia of principal cells. GP-49 and GP-83 were identified in the Nonidet P-40 homogenates of corpus and cauda epididymidis. In the epididymides of which ductuli efferentes had been ligated for more than 4 weeks, no sperm were found in the lumina of epididymal tubules. WGA-binding sites were present in the corpus and cauda; GP-49 and GP-83 were identified in tissue homogenates of the corpus and cauda as well. These findings suggest that GP-49 and GP-83 of mature sperm may be secreted by the principal cells of the corpus and cauda. These two molecules apparently conjugate to sperm whilst sperm transit through the epididymis.     

Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates

It is widely accepted that sperm morphology is a good indicator of fertility and it has been proposed that sperm quality may be related to subtle changes in sperm head morphology. However, a precise estimation of the morphology of ram sperm would be very useful to improve reproductive success in ovine. Computer-assisted morphometric analysis and clustering analysis have been important tools to study sperm subpopulations in domestic animals. However, to the best of our knowledge, no data exist studing morphometric differences regarding to sperm subpopulations within the ovine ejaculate. The aim of this study was to test the presence and distribution of sperm morphometric subpopulations in cryopreserved ejaculates from yearling and mature rams using an objective method by computer analysis system and to establish the relationship between the distribution of the subpopulations found and sperm quality in each individual ram. Principal component analysis revealed that three principal components for yearlings and four components for mature rams that represented more than 84% of the cumulative variance in both cases. After cluster analysis, three sperm morphometric subpopulations for yearlings (CLY) and four for mature (CLM) rams were identified with defined sperm dimensions and shapes. CLY1 included big, round and short sperm (37%), CLY2 included average size and slightly elliptical and elongated sperm (48%), CLY3 included small, long, elliptical and elongated sperm cells (15%). CLM1 consisted of average size and moderate elliptical and elongated (26%), CLM2 consisted of small, long, elliptical and elongated (31%), CLM3 consisted of small and round (32%) and CLM4 included big, short and round (8%) spermatozoa respectively. There were significant differences in the distribution of the three subpopulations (P < 0.001) as well as in the sperm concentration, total motility (%), sperm viability (%) and the overall (P < 0.05) in the ejaculates among the four yearling rams tested. Same results were found for the four subpopulations and the different sperm quality parameters in the ejaculates among the four mature rams tested. In conclusion, cryopreserved ram semen showed a specific structure with regard to sperm morphometric subpopulations. In addition, the distribution of these subpopulations seems to be related to stud maturity age and the ejaculate quality which would be a very important indicator of sperm function. Thus, analysis of sperm morphometric subpopulation structure together with functional tests could provide valuable information to assess the cryoresistence of ram spermatozoa.