Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Chelicerae as male grasping organs in scorpions: sexual dimorphism and associated behaviour

Specialised structures that enable males to grasp females during sexual interactions are highly susceptible to selection and thus diverge relatively rapidly over evolutionary time. These structures are often used to test hypotheses regarding sexual selection such as sexually antagonistic co-evolution and sexual selection by female choice. In the present study, we determine whether there is a relationship between a novel record of scorpion sexual dimorphism, the sexual dimorphism of chelicerae (CSD), and the presence of the mating behaviour termed “cheliceral grip” (CG). The presence of both traits in the order Scorpiones is also reviewed from a phylogenetic perspective. The results confirm a strong relationship between CSD and the presence of CG. The morphological and behavioural patterns associated with “CSD–CG” are opposed to the predictions postulated by the hypothesis of sexually antagonistic co-evolution. However, if the female shows resistance after the deposition of the spermatophore, the possibility that the male exerts pressure as a “cryptic form” of coercion to prevent the interruption of mating cannot be ruled out completely. Female choice by “mechanical fit” could be another explanation for some aspects of the CG's contact zone. The possibility that the “CG–CSD” complex has evolved under natural selection in order to ensure sperm transfer is also considered.     

黑鲷精子的超低温冻存及DNA损伤的SCGE检测

以0.5 mL的麦细管为冻存管和DMSO为抗冻剂进行超低温冷冻黑鲷精子,对冻精核DNA的损伤情况进行单细胞凝胶电泳（SCGE）检测,其结果表明,以Cortland溶液为稀释液,5%、10%、15%及20%DMSO为抗冻剂的超低温冻存的黑鲷精子活力、受精率与鲜精无显著差异。其中以10%DMSO为抗冻剂的冻存效果最佳,冻精的激活率、运动时间、寿命及受精率分别达（92.91±1.25）%、（39.90±2.70）min、（53.82±2.84）min及（89.35±1.99）%;而以25%及30%DMSO为抗冻剂时,冻精活力及受精率显著下降。SCGE检测结果显示,DMSO浓度为5%、10%、15%及20%时,黑鲷冻精与鲜精的彗星率及损伤系数差异不显著;DMSO浓度为25%及30%时,冻精与鲜精的彗星率及损伤系数差异显著;冻精的彗星率与抗冻剂DMSO浓度成正相关。黑鲷鲜精及冻精核的DNA损伤主要为轻度和中度损伤,重度损伤比例较低,完全损伤仅存在于25%及30%DMSO为抗冻剂的冻精中,且比例低。分析认为,较高浓度的DMSO是引起冻精核DNA损伤的主要原因。     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

正常人及不育者精子核DNA的定量研究

用流式细胞术,Feulgen显微分光光度法,荧光显微分光光度法测定正常人精子核DNA相对含量:结果表明,上述方法所测得的精子核DNA相对含量稳定,变化范围小。同时用Feulgen显微分光光度法测定不育者精子核DNA相对含量。显示不育者精子核DNA相对含量高于正常人,提示精子核DNA核蛋白复合物异常可能是某些男性不育症的原因。此项研究为诊断男性不育症提供了新方法,在男性学精子核分子研究方面提供了依据。     

菲菊头蝠冬眠期的精子储存

菲菊头蝠是分布于亚热带和热带的翼手目种类,在中国大陆南部和海南岛有广泛分布。为探讨热带菲菊头蝠是否具有冬眠期(12 月至翌年2 月)储精现象及生殖腺组织结构的变化,对海南岛的菲菊头蝠成年雄蝠和成年雌蝠生殖系统在冬眠期间的变化进行了石蜡切片观察。结果显示:成年雄蝠在冬眠期间附睾内储存大量精子,推测其储存时间超过2 个月;冬眠期间曲细精管横截面积、精子细胞数量和间质细胞数量在冬眠期逐月显著性减少,精原细胞和精母细胞的数量在12 月与翌年1 月间均无显著性变化,而在冬眠末期的2 月显著增多。在雌蝠子宫和卵巢内均未发现精子储存现象,但卵巢内具有初级卵泡和次级卵泡,雄蝠在冬眠期间曲细精管逐月萎缩,但生精上皮精母细胞的数量在冬眠末期明显上升。     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

Sperm competition games: sperm selection by females

We analyse a co-evolutionary sexual conflict game, in which males compete for fertilizations (sperm competition) and females operate sperm selection against unfavourable ejaculates (cryptic female choice). For simplicity, each female mates with two males per reproductive event, and the competing ejaculates are of two types, favourable (having high viability or success) or unfavourable (where progeny are less successful). Over evolutionary time, females can increase their level of sperm selection (measured as the proportion of unfavourable sperm eliminated) by paying a fecundity cost. Males can regulate sperm allocations depending on whether they will be favoured or disfavoured, but increasing sperm allocation reduces their mating rate. The resolution of this game depends on whether males are equal, or unequal. Males could be equal: each is favoured with probability, p, reflecting the proportion of females in the population that favour his ejaculate (the 'random-roles' model); different males are favoured by different sets of females. Alternatively, males could be unequal: given males are perceived consistently by all females as two distinct types, favoured and disfavoured, where p is now the frequency of the favoured male type in the population (the 'constant-types' model). In both cases, the evolutionarily stable strategy (ESS) is for females initially to increase sperm selection from zero as the viability of offspring from unfavourable ejaculates falls below that of favourable ejaculates. But in the random-roles model, sperm selection decreases again towards zero as the unfavourable ejaculates become disastrous (i.e. as their progeny viability decreases towards zero). This occurs because males avoid expenditure in unfavourable matings, to conserve sperm for matings in the favoured role where their offspring have high viability, thus allowing females to relax sperm selection. If sperm selection is costly to females, ESS sperm selection is high across a region of intermediate viabilities. If it is uncostly, there is no ESS in this region unless sperm limitation (i.e. some eggs fail to be fertilized because sperm numbers are too low) is included into the model. In the constant-types model, no relaxation of sperm selection occurs at very low viabilities of disfavoured male progeny. If sperm selection is sufficiently costly, ESS sperm selection increases as progeny viability decreases down towards zero; but if it is uncostly, there is no ESS at the lowest viabilities, and unlike the random-roles model, this cannot be stabilized by including sperm limitation. Sperm allocations in the ESS regions differ between the two models. With random roles, males always allocate more sperm in the favoured role. With constant types, the male type that is favoured allocates less sperm than the disfavoured type. These results suggests that empiricists studying cryptic female choice and sperm allocation patterns need to determine whether sperm selection is applied differently, or consistently, on given males by different females in the same population.     

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.