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.     

Liposome-mediated DNA uptake by sperm cells.

To investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.     

Expression of the rabbit sperm protein Sp17 in cos cells and interaction of recombinant Sp17 with the rabbit zona pellucida

This study extends our analysis of rabbit recombinant Sp17 (rSp17) by examining whether rSp17 synthesized in transfected COS cells will show a particular localization within the cell and whether the COS cell will bind with zona pellucida. We show, using the cross-linking, reagent DSS that rSp17 can bind to rabbit zona glycoprotein R45 or R55. © 1995 Wiley-Liss, Inc.     

Relationships between bacteriospermia,DNA integrity,nuclear protamine alteration,sperm quality and ICSI outcome

The Aim of this study was to evaluate the effects of bacteriospermia on human sperm parameters, nuclear protamines, DNA integrity and ICSI outcome in patients enrolled for ICSI treatment. 84 unselected couples consulting in infertility and obstetrics clinic and enrolled for ICSI treatment were included in this study. The semen specimens were screened bacteriologically; semen and sperm parameters were also evaluated according to WHO guidelines. DNA integrity, protamines concentration and protamine deficiency were estimated by TUNEL assay, AU-PAGE and Chromomycin (CMA3) respectively. The results of this study revealed that 34.52% of studied semen samples were infected with bacteria. The isolated bacteria were identified as Staphylococcus aureus, Staph. epidermidis, Staph. haemolyticus, Escherichia coli, Enterococcus faecalis and Streptococcus agalactiae. Bacteriospermia had a significant (p?<?.010) negative effect on sperm parameters; concentration, motility, progressive motility and chromatin condensation. Moreover, high DNA fragmentation with low P1 and P2 concentrations were noticed in infected patients in comparison to non-infected patients but non-significant. Also, the fertilization rate decreased significantly (p?<?.05) with infected patients. In conclusion: bacteriospermia has significant negative effect on sperm quality and fertilization rate in patients who underwent ICSI treatment.     

Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs

C1q is a component of the classical complement pathway that can react with the Fc-fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona-free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm-oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 micrograms/ml concentration was 90 +/- 35 vs. 29 +/- 7 for the control (P less than 0.001). At 1 microgram/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P less than 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 micrograms/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti-C1qR monoclonal antibodies (Mabs), was demonstrated both on zona-free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody-free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I-C1q.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major morphological sperm abnormalities in the bull are related to sperm DNA damage

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the “so called” major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.     

Hereditary variability of plants under the action of exogenous DNA

Summary Injection of exogenous barley donor DNA into grains of barley recipient plants at the milk maturity stage, with a specially designed syringe, led to the appearance of transformed plants. The transformation (in rare cases) was caused by the unsheared DNA since the DNA passing through the syringe needle remained relatively stable (10⁶ to 10⁷ daltons) as was confirmed by DNA sedimentation analysis.14 plants grown from seeds injected with highly polymeric DNA containing close to 30 per cent protein had transformed pollen grains. In the 2nd generation only 2 plants from the 8 studied preserved these changes. In the progeny of these two plants, i.e., in the 3rd seed generation after injection, 82.1 per cent of plants preserved the transformed characters. The next, 4th generation, preserved a transformed phenotype in 89.6 per cent of plants.It was also shown that reversion to a recipient-like state was not always constant. We found the reversion of transformed properties (i.e., normal starch and two-rowed spikes) in 40 per cent of the 4th generation descendants of one of the plants which had lost the phenotypical expression of these properties in the 3rd generation but had them in the 2nd generation.The study of the morphological properties of transformed plants showed that with respect to phenotypic expression some characters were changed towards the donor type, some remained as in the recipients and some were of the intermediate type.     

Association of exogenous DNA with cattle and insect spermatozoa in vitro

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina), and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.     

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R² value = 0.949; P < 0.01; n = 16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n = 6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 °C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P = 0.001) in DNA damage, as soon as 4 h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6 h of incubation and revealed individual animal variation. Freshly collected and incubated (37 °C) rhinoceros (n = 3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.     

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.     

Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes

Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that (1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M-phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. (2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed. © 1994 Wiley-Liss, Inc.     

Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa

Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5′ and 3′ flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with 3′ and 5′ untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist.     

Production of mice through intracytoplasmic injection of sperm or spermatogenic cells

Summary Direct injection of spermatozoa or spermatogenic cells into oocytes bypasses many normal fertilization processes, yet it is a powerful tool to analyze basic principles/mechanism underlying normal fertilization and prefertilization processes. Birth of normal, fertile mouse offspring following injection of round spermatid nuclei into oocytes suggests that all the postmeiotic modifications in male gametes evolved as processes solely dedicated for the delivery of their nuclei into the eggs. Birth of normal mouse offspring following injection of infertile spermatozoa with grossly misshaped heads indicates that structurally abnormal spermatozoa are not necessarily genomically abnormal. The spermatozoa with disrupted plasma membranes are generally considered dead, but they are capable of producing normal offspring by injection, suggesting that cell death and nucleus death are not synonymous at least for the spermatozoa.     

Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than −80 °C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and −80 °C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.     

Cell biology and functional dynamics of the mammalian sperm surface

Theriogenology has now a 40-year rich history on covering sperm biological aspects with a special emphasis on farm and husbandry animals. The major and most influential of these contributions will be placed into an evolutionary perspective of ongoing and intriguing progresses made in this field. Although many molecular details have been published, it is more the aim of this contribution to provide a guide through the main established aspects and concepts of sperm surface biology and refer only to major molecular players and mechanisms involved in sperm physiology. Those interested in more molecular details and in-depth knowledge can easily access the most relevant literature which is included here for reference purposes. With this approach, a logical and easy to follow buildup can be made of the general picture of sperm surface dynamics and of the ergonomics of sperm physiology and their function in mammalian fertilization. Understanding the ins and outs of sperm surface biology and the dynamics thereof, might challenge future researchers to design novel generation of better sperm-handling procedures. This could be beneficial for assisted reproductive technology and animal breeding industries.     

Relationship between double fertilization and the cell cycle in male and female gametes of tobacco

Nuclear DNA content of male and female gametes of tobacco was determined using 4,6-diamindino-2-phenylindole and quantitative microfluorimetry. Pollen grains are released with generative cells containing 2C DNA. Mitotic division occurs in the pollen tube 8–12 h after germination. The resulting sperm cells have 1C DNA content during pollen tube elongation in the style. Sperm cells deposited in the degenerated synergid have a DNA content between 1C and 2C, indicating that sperm are in S-phase in the synergid. Concomitant with pollen tube arrival, the egg cell increases in DNA quantity from 1C to between 1C and 2C at 48 h after pollination. In the absence of pollination, S-phase in the egg cell is delayed by up to 36 h. Newly formed zygotes contain nuclear DNA concentrations of 4C at karyogamy and remain at 4C until zygote division. Tobacco displays cell fusion after the completion of S-phase, apparently during G₂. Failure to achieve an optimized system for in vitro fertilization in Nicotiana may reflect the challenges of achieving cell cycle synchrony in gametes isolated from pollen tubes. Receptive gametes are presumably those that pass through the protracted S-phase, reaching G₂ receptivity and cell cycle congruity before fusion.     

Preferential extrachromosomal localization of exogenous DNA in transgenic silkworm Bombyx mori L.

Summary Transgenic silkworms (Bombyx mori L.) were obtained by microinjection of plasmid pPrC-LTR1.5, which carris 1.5 DNA copies of Rous sarcoma virus (RSV) long terminal repeats (LTRs) inserted in the vector pBR322. The transgene was transmitted over the three generations obtained up to now. Most of the exogenous DNA failed to integrate into the genome and persisted as an extrachromosomal element that is subject to rearrangements. Plasmids carrying only part of the input DNA together with fragments of silkworm DNA were rescued from the transgenic animals. One of the rescued plasmids contained a sequence which belongs to a family of evolutionarily conserved repeated sequences.     

线粒体DNA多态性在海洋动物群体遗传结构研究中的应用

线粒体DNA( mtDNA)分析在揭示物种亲缘关系、遗传比较、系统进化和遗传结构等领域的研究中得到了广泛的应用,尤其是在海洋动物的遗传结构研究中发挥了重要的作用.介绍线粒体DNA的结构特征、多态性研究方法,并对其在海洋动物群体遗传结构研究中的应用进行了综述.     

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P = 0.493). However, when thawed semen was subsequently incubated at 37 °C for periods of between 2 and 48 h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ² = 158.6; DF = 3; P < 0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48 h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.     

Antibodies to flowering-plant sperm

Summary The mechanisms by which sperm cells recognize and fuse with the egg and central cell during double fertilization in flowering plants are unknown. To identify membrane surface molecules that might function in fertilization, we immunized mice with isolated sperm ofBrassica campestris and screened the polyclonal sera and monoclonal hybridoma supernatants by immunocytochemistry for binding to isolated sperm cells. We identified three cell lines producing hybridoma supernatants which bind to sperm cell surfaces inB. campestris and further analyzed the properties of one of these, BRSP1. The molecular mass of the epitope to BRSP1 was 54 kDa, and was not glycosylated. Although the antibodies were immunoglobulin M, neither removal of carbohydrates nor competition with antibodies which recognize arabinogalactan decreased binding. BRSP1 recognized sperm ofPlumbago zeylanica, Nicotiana tabacum. Arabidopsis thaliana, andEruca vesicaria and generative cells ofLilium longiflorum and ofNarcissus tazetta but did not recognize sperm ofHelianthus annuus, Gerbera jamesonii, orZea mays. Antibodies to plant sperm allow us to probe sperm membranes for functional components.