Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

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。     

Regulation of foreign DNA uptake by mouse spermatozoa

We have studied some features of DNA uptake in both mature and immature mammalian spermatozoa. Mature sperm collected from the cauda epididymis are able to incorporate foreign DNA in a buffer containing only salts and calcium. Immature spermatozoa, however, are unable to bind DNA. This seems to be caused by the lack of a functional receptor in the sperm membrane since once this membrane is disrupted by sonication, DNA can be detected in the postacrosome region of the sperm nucleus, matching the distribution of the mature spermatozoa. Comparison between the DNA binding proteins of mature and immature spermatozoa allowed us to identify two bands that could be part of the putative membrane receptor for the DNA. On the other hand, DNA uptake in mature sperm is prevented by the seminal plasma. We have identified two components of the seminal plasma, a calcium-dependent DNase present in the seminal vesicle fluid and several DNA binding proteins secreted by the ventral prostate, that could account for the inhibitory activity. Taken as a whole, our results indicate that DNA uptake by the mammalian spermatozoa is a very specific and highly regulated phenomenon.     

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.     

Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

DNA fibre autoradiography, after incorporation of high specific activity 3H-thymidine and 3H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) or G1 (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however, without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G1 cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white-tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37 °C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t(0)) and after 4 hr (t(4)) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t(0) and t(4) were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1-to-protamine 2 ratios.     

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.     

First attempt to characterize 3H-fucose acrosomal label in spermatoza

Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-N-alpha-benzoyl-L-agrinine ethyl ester splitting), akin to that exhibited by acrosin.     

The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry with tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd. The use of BrdUrd allowed the visualization of a population of S-phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.     

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。     

First attempt to characterize 3H-fucose acrosomal label in spermatozoa

Summary Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-Nalpha-benzoyl-l-arginine ethyl ester splitting), akin to that exhibited by acrosin.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

Abstract. We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine ([³H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5·8 ± 0·3 h), a high S phase efflux and an initially low influx of cells from G_: into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 ± 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15–16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300–1400 h, efflux rate falls from high (19–20 cells %/h) to low (4–8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Reproduction of chicken chromosomes at the end of the DNA synthesis period]

Reduplication of chicken chromosomes at the end of S-period in the cells of bone marrow was studied, using the method of prolonged labelling with 3H-thymidine and autoradiography. It is shown that during the last hour of the S-period the DNA synthesis in macrochromosomes ends primarily in the centromere region and the regions adjacent to it. Sex chromosomes (ZZ) of cocks accomplish the reproduction simultaneously.     

Cell proliferation kinetics of six xenografted human cervix carcinomas: comparison of autoradiography and bromodeoxyuridine labelling methods

Cell kinetic and histologic parameters of six xenografted tumours with volume doubling times ranging from 6 to 43 d were investigated in order to obtain kinetic information on a panel of tumours to be used in radiobiological studies. The six tumours covered a range of histologies and their DNA indices varied from 2.7 to 1.4. The length of the cell cycle (Tc), potential doubling time (Tpot) and labelling index (LI) were determined by continuous labelling with [3H]TdR and autoradiography in three tumours, Tc varied from 30 to 40 h. Determinations of the length of the S phase (Ts) were found to be less reliable by this method. Data on Ts and LI were also determined in all six tumours using bromodeoxyuridine (Brd) labelling and the single sample method: values of Tpot were slightly longer than those obtained via the autoradiographic method. In addition, multiple samples were taken after BrdU labelling. Tc was determined by fitting the data obtained from mid-S, mid-G2 and mid-G1 windows to curves described by a damped oscillator. Data obtained via the mid-S window were found to be most reliable. Generally, cell cycle times obtained by the BrdU method were longer than those observed with the autoradiographic method. Differences between the two methods could be explained by inaccuracies in the determination of Ts, LI and Tc and differences in the experimental approach. We consider the BrdU labelling method to be a suitable alternative for the time-consuming autoradiography, if data on Ts or Tpot are sufficient. Due to difficulties in the reproducibility of the immunofluorescence staining and asynchronization of cells approximately 10 h after labelling, the method of windows analysis was affected by similar problems to those observed in interpretation of percentage labelled mitosis (PLM) curves. However, the method may serve as an alternative to determine cell cycle times in vitro and, if improved technically, in vivo. Careful comparison of the data obtained from mid-S, mid-G1 and mid-G2 windows may increase the reliability of the determination of cell kinetic parameters.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.