精子介导的转基因技术

精子介导的转基因技术是近十几年发展起来的一种新技术.从各个种的实验证明,精子有瞬间吸收外源DNA的能力.这一过程由一系列因子起重要调节作用.一种特异的DNA结合蛋白介导了外源DNA与精子的结合,同时精浆中存在一种因子起抑制两者结合的拮抗作用.CD4分子与外源基因的内化有关.内化的DNA可能与精子核支架结构(nuclear scaffold)结合,并整合或重排.但仍需要大量实验数据进一步证明是否产生真正可遗传的转基因后代,及如何提高转基因效率,以使这一方法得到普遍的推广及应用.     

Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa

The uptake of exogenous DNA by mouse and rat spermatozoa was analyzed using in vitro and in vivo methods. Two DNA constructs were used, one containing the Growth hormone (GH) gene and the other the c-myc oncogene linked to the αA-crystallin promoter (CPV-1 plasmid). For the in vitro approach, washed epididymal spermatozoa were incubated for 2 hr in the presence of linearized DNA. For in vivo experiments, DNA was injected into the proximal region of the vas deferens, and spermatozoa were recovered 6 hr later. In situ hybridization employing fluorescent markers and electron microscopy were used to localize the exogenous genes in spermatozoa. The precise localization of the foreign DNA in spermatozoa was visualized by tridimensional reconstructions using a confocal laser microscopy. Uptake of exogenous DNA occurred in 60–70% of the spermatozoa after in vitro or in vivo treatments. A positive signal was detected in the sperm nucleus and was not affected by DNase treatments. Incorporation of exogenous DNA was also evaluated by slot blot and PCR techniques using the DNA isolated from the sperm nuclei and the corresponding labelled probes. Comparison of a nucleotide sequence between the DNA isolated from in vivo treated spermatozoa and CPV-1 plasmid showed a 98.6% identity. These results show the in vivo capacity of spermatozoa to incorporate exogenous DNA, the ability of this DNA to reach the nucleus, and also demonstrate that epididymal and vas deferens secretions do not block these capacities. Mol. Reprod. Dev. 51:42–52, 1998. © 1998 Wiley-Liss, Inc.     

Sperm cells and foreign DNA: a controversial relation

Sperm cells from a variety of species share the spontaneous ability to take up foreign DNA. That feature has been exploited to generate genetically modified animals with variable efficiency in different species. An unexpectedly large set of factors appears to modulate the interaction of sperm cells with exogenous DNA. The binding is mediated by specific DNA-binding proteins and is antagonized by an inhibitory factor in the seminal fluid. A portion of sperm-bound DNA is internalized in nuclei, a process mediated by CD4 molecules. Sperm interaction with foreign DNA triggers endogenous nuclease(s) that cleaves both the exogenous and the genomic DNA, eventually leading to a cell death process which resembles apoptosis. Internalized foreign DNA sequences reach the nuclear matrix and undergo recombination with chromosomal DNA. From these studies, a surprising network of metabolic functions is beginning to emerge in mature spermatozoa, which are normally repressed and are specifically activated upon exposure to appropriate stimuli. BioEssays 20:955–964, 1998. © 1998 John Wiley & Sons, Inc.     

精子结合外源DNA的特征及影响因素

外源DNA与精子相互作用后的定位及内化率是精子载体法制备转基因动物的关键环节。实验以标记的DNA片段为示踪材料,就精子与外源DNA相互作用的基本特征及影响因素进行了研究。结果表明：山羊精子可自发性结合外源DNA,外源DNA最初结合于顶体后区质膜外表面,随后部分内化进入细胞内。精子对外源DNA的结合和内化能力随供体的不同而差异明显,在实验所检查的35只公羊中,结合率（DNaseⅠ消化前）波动于4.6% ~ 62.4%,内化率（DNaseⅠ消化后）波动于2.1% ~ 53.8%,个体间差异显著（P<0.01）。对于同一供体的精子而言,阻止DNA结合的最主要因素是精浆,与射出的原精液相比,洗涤后精子的结合率和内化率分别提高了3倍和5倍;其次精子获能也将导致结合率和内化率降低（P<0.01）。死精子不能完成外源DNA的内化过程,但反复冻融导致质膜破裂的死精子具有更高的结合率,而且阳性率与动物个体无关。上述结果提示,筛选合适的精子供体,采用优化的转染处理方法是提高精子载体方法效率的前提和保证。     

The interaction between exogenous DNA and sperm cells.

Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.     

Association of rabbit sperm cells with exogenous DNA

The ability of rabbit spermatozoa to bind exogenous DNA during sperm-mediated gene transfer (SMGT) was tested in our study. Fresh collected semen, or fully capacitated sperm cells, was co-cultured with plasmid DNA labeled with tetramethyrodamine-6-dUTP. Fluorescent spermatozoa were counted before and after DNaseI treatment. Results showed that fluorescent-labeled plasmid DNA could be taken up by capacitated rabbit sperm cells. 66% spermatozoa carried exogenous DNA in the presence of lipofectin. Bovine serum albumin could block this process effectively. Associated DNA was mainly located in the posterior area of the sperm head. In order to verify whether exogenous DNA was carried into the embryo and expressed in the offspring, further SMGT experiments were carried out using the pHM-CR plasmid which contains LacZ and Neomycin genes. beta-galactosidase was expressed in different stages of embryo development and in the tissues of young rabbit as detected by using X-gal staining. Large portion of embryos survived under the selection pressure in G418 containing medium, after SMGT. Transgene integration was further verified by PCR analysis. These results confirmed the ability of rabbit sperm cells to carry transgene into the embryo during in vitro fertilization.     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

外源基因对精子的影响及其在山羊早期胚胎中的表达

摘要: 在前期实验中发现, 山羊精子可自发结合外源DNA, 但结合能力在不同动物个体之间差异显著。挑选结合能力明显不同的3只公羊, 进一步探讨了外源DNA对精子的影响及其在早期胚胎中的表达, 结果发现: 外源基因与精子共同孵育后, 精子的活率、顶体反应发生率和受精能力均呈下降态势, 其降幅与精子的结合能力密切相关。利用与DNA共育后的精子进行体外受精, 外源基因可被导入卵母细胞并在早期胚胎中获得表达, 但胚胎阳性率因精子供体不同而差异显著(P<0.05); 其中来源于高、中结合能力供体生产的胚胎, 分别有16.2%(25/154)和5.3%(4/76)可检测到外源基因存在, 但表达仅见于高结合能力供体生产的早期胚胎, 表达率为6.5%(10/154); 低结合能力供体生产的胚胎无外源基因。研究表明, 在以精子载体方法生产转基因动物的实验过程中, 筛选对DNA结合能力较强的精子供体是提高转基因效率的前提, 但需要考虑外源DNA对精子受精能力的影响。     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

The negative effects of exogenous DNA binding on porcine spermatozoa are caused by removal of seminal fluid

Sperm-mediated gene transfer (SMGT) might become the most efficient and cost effective technique to generate transgenic animals, which will significantly increase their application in biomedical research and in commercial production. Despite some successes, the technique has remained controversial for almost 20 years and despite number of studies the reasons for poor reproducibility of this promising technology has not been understood. We suggest that the reason for poor reproducibility is the presence of natural defences against exogenous DNA invasion acting in spermatozoa or in embryo. Based on previous reports we have investigated the effect of foreign DNA binding on spermatozoa by monitoring motility, viability and genomic DNA damage. Evaluation of DNA binding in sperm collected from 16 boars demonstrated that 28-45% of the added pEGFP plasmid was bound to spermatozoa with 9-32% being internalized in sperm nucleus. In agreement with previous reports, our results demonstrated that the pEGFP-treated sperm show an average a 2-fold decrease in motility (p<0.05), 5-fold decrease in progressive motility (p<0.05), and 1.4-fold increase in number of sperm with highly damaged DNA (p<0.05) as detected by Comet assay. In contrast with previous reports, we demonstrate that all such changes were associated with the removal of seminal plasma during the washing step and not with foreign DNA binding per se. We suggest that poor reproducibility of SMGT most likely result from selection against DNA-loaded sperm at later stages of fertilization.     

Exogenous DNA uptake by South American catfish (Rhamdia quelen) spermatozoa after seminal plasma removal

Sperm from different species shows biological differences, determining the success or failure of the sperm-mediated gene transfer (SMGT) technique. There is evidence that exogenous DNA uptake by the spermatozoa is a species-specific and highly regulated phenomenon. Problems involving SMGT procedures might be related to activation of defenses in spermatozoa and in seminal plasma such as DNase enzymes. The objective in the present study was to transfect South American catfish spermatozoa after seminal plasma removal. Seminal plasma had a strong DNase activity that is reduced after sperm washes in isosmotic solution, in which Western blot analysis demonstrated a reduction in the DNase content after washes and Southern blot evaluations show the presence of plasmid after sperm washes. The seminal plasma DNase digests exogenous DNA in a few minutes and has an optimal activity at 43°C. Also, EDTA at 30 mM concentration inhibits the DNase activity. Using PCR the pEGFP vector was internalized by sperm cells even at lesser concentrations (5-40 ng/10(6) spermatozoa) without motility loss after seminal plasma removal. Conversely, using greater pEGFP concentrations (100 ng/10(6) spermatozoa), there were no motile cells, suggesting toxicity of exogenous DNA for sperm cells. These results are interpreted to provide information that can improve the protocol for generation of transgenic South American catfish.     

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.     

Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.     

DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012. © 2012 Wiley Periodicals, Inc.     

Sperm-mediated gene transfer into oocytes of the golden hamster: assessment of sperm function

The possibility of sperm as a vehicle to deliver foreign DNA to oocytes was tested in hamsters. Epididymal spermatozoa, incubated with linearized plasmid DNA encoding ovine growth hormone (pCMXoGH), showed a spontaneous tendency to interact with DNA. Kinetics of sperm uptake of DNA was determined by using [32P]-labeled DNA. Spermatozoa took up the added DNA by 15-30 min and the uptake was inhibited by human seminal fluid in a dose dependent manner. Addition of DNA did not affect the functional competence of spermatozoa, in terms of their ability to undergo capacitation and acrosome reaction (34.5% +/- 2.2 vs 35% +/- 1.5). The fertilizing ability of DNA treated-spermatozoa from hamsters and humans was assessed by zona-free hamster egg penetration assay. Number of sperm penetrated per oocyte were 23 +/- 4.5 and 1.4 +/- 1.3 for hamster and human spermatozoa, respectively. Penetrated oocytes harbored sperm-treated DNA both with hamster (30.2 cpm/oocyte) and human (19.2 cpm/oocyte) spermatozoa. These results show that the hamster and human spermatozoa have a strong tendency to interact with exogenous (foreign) DNA and are able to transfer DNA to oocytes. Sperm may be used as a vector for DNA transfer and this approach has potential in the production of transgenic animals.     

REVIEW: Spermatozoa,DNA binding and transgenic animals

The idea that sperm cells could be used as an effective tool for introducing exogenous DNA into an oocyte at fertilization is generally regarded with scepticism. However, in recent years, several investigators have been working on different aspects of this intriguing research topic. In the present review, their results are summarised and discussed. Sections have been dedicated to the way DNA molecules bind to spermatozoa of different species, to the events regulating such binding, to the fate of the DNA within sperm cells, and to the attempts made to produce transgenic animals with this method. The data available on the interaction between DNA and spermatozoa begin to explain how this event takes place and how it is regulated. However, the stable integration of exogenous genes into the genome of adult animals mediated by sperm cells is a very rare event, although several reports describe forms of partial success. Available evidence suggests that changes to the DNA molecules, oc curring mostly within the oocyte, represents the limiting step in the production of transgenic animals using spermatozoa as vectors of exogenous genes. At present there are not enough data to understand what happens to sperm-associated DNA upon its entrance into the oocyte at fertilization. Therefore, it has not yet been resolved whether sperm-mediated gene transfer is a possible way to manipulate the genome or if evolution has imposed some unsurpassable barriers to its use     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.