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Regulation of Foreign DNA Uptake by Mouse Spermatozoa
Experimental Cell Research 262, 104 –113 (2001) doi:10.1006/excr.2000.5079, available online at http://www.idealibrary.com on
Regulation of Foreign DNA Uptake by Mouse Spermatozoa Rosa Carballada 1 and Pedro Esponda Centro de Investigaciones Biolo´gicas, CSIC, Vela´zquez 144, 28006 Madrid, Spain
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. © 2001 Academic Press
Key Words: DNA uptake; epididymis; prostate; sperm; sperm membranes; seminal plasma; seminal vesicles; transgenesis.
INTRODUCTION
In 1971, Brackett et al. [1] reported that rabbit spermatozoa were able to take up naked DNA present in the incubation medium. Years later, Lavitrano et al. [2] used this property to generate transgenic mice. The simplicity of the method prompted many labs to explore the ability of spermatozoa of other species to incorporate foreign DNA and there is already a variety of species analyzed. They include other mammals such as humans [3], bulls [3– 6], and pigs [3, 5–7], but also birds [5, 8], amphibians [9], fishes [5, 10, 11], and some invertebrates [4, 12, 13]. In many of these studies, the spermatozoa were used for insemination or in vitro 1 To whom reprint requests should 34-915627518. E-mail: [email protected].
0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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fertilization, giving rise to transgenic embryos or adults [6, 9 –14]. Although in most cases the spermatozoa are simply incubated in presence of the foreign DNA, sometimes electroporation or cationic liposomes are used to enhance the uptake [11, 13–16]. Even some viruses have been used as vectors [17]. Part of the DNA remains bound to the sperm head plasma membrane; however, part is internalized and in mammals it is consistently found in the postacrosome region of the sperm nucleus [3, 18]. The process is still poorly understood. Some authors have proposed that MHC II molecules and the antigen CD4, present in the sperm head membranes, are involved in the binding and internalization of the foreign DNA [19, 20]. Spermatozoa obtained from CD4 knockout mice are still capable of binding the DNA but unable to internalize it. In addition antibodies against CD4 can inhibit the latter process. Both MHC II molecules [19] and CD4 [20] have been described in the postacrosome region of the mouse sperm head and this localization could be determining the pattern of DNA incorporation. Once in the nucleus, the DNA becomes tightly associated to nuclear proteins [21, 22] and then it can be cleaved by sperm endonucleases and finally integrated in the genome [23]. On the other hand, there are also molecules that act as negative regulators of the process. Seminal plasma proteins are able to inhibit the DNA uptake [3, 24], a mechanism that could have evolved to protect sperm from foreign DNA present in the reproductive tract. While in rodents mature spermatozoa are routinely collected from the epididymis and therefore have never been in contact with the seminal plasma, in species such as dairy animals the researcher needs to work with ejaculated spermatozoa. This brings about the necessity to thoroughly wash the spermatozoa to be able to have a good transfection rate. Regarding the inhibitory factors so far analyzed, a 37-kDa protein present in sea urchin sperm and human seminal plasma has been shown to inhibit the DNA uptake [24] although the mechanism remains obscure. In addition, mouse whole seminal plasma displays a similar inhibitory effect and it has been proposed that some factors present in it, such as the glycosaminoglycans, could be binding the mouse sperm and blocking the receptors
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for DNA [3]. This hypothesis, however, has not been proved yet. In this study we have tried to go further in the analysis of the characteristics of the process of DNA uptake by the spermatozoon and its inhibition by seminal plasma. We have examined the minimum requirements in the incubation medium that allow transfection in mature sperm. In addition, we have also examined the differences among mature and immature sperm in their ability to bind DNA. We have performed assays in intact cells, in spermatozoa disrupted by sonication, and in isolated sperm nuclei to see the role of the different membranes and cellular compartments in this process. We have also tried to characterize some molecules that can be responsible for the differences observed. On the other hand, we have carried out transfection experiments in the presence of different accessory gland fluids to determine those that contain inhibitory activities. Finally, we have characterized the inhibitory activities found and the mechanism of inhibition. MATERIALS AND METHODS Animals and sperm collection. Male mice from the CD-1 strain bred in our colony were used as source of spermatozoa. All animals were sexually mature. For mature spermatozoa, the caudae epididymides from one or two males were dissected free of fat tissue and minced in 400 l of PBS (phosphate-buffered saline, pH 7.5) supplemented with 2 mM CaCl and 1 mM MgCl when indicated. For immature spermatozoa, caput epididymides were obtained from five males and minced in 200 l of PBS. The spermatozoa were washed twice in PBS (by centrifuging 1000g ⫻ 3 min) and resuspended in the chosen buffer (PBS, supplemented or not with Ca and Mg and containing or not EDTA). The concentration was estimated by the use of a hemocytometer and adjusted to 10 –15 ⫻ 10 6 sperm/ml. Motility was also routinely examined and whenever there was calcium in the incubation medium it was always ⬎90%. Samples poorly motile were discarded. Sperm transfection assays. The DNA used for transfection was a commercial plasmid, pGeneGrip (GTS, California) that encodes the green fluorescent protein under the control of hCMV IE promoter/ enhancer and is rhodamine labeled. This allowed a direct visualization of the DNA uptake by the use of fluorescence microscopy. For every experiment 250 ng of plasmid DNA was added to 50 l of the sperm suspension and incubated for 30 min at 37°C. After transfection, sperm were washed three times (at 1000g ⫻ 3 min), fixed for 1 h in 2% p-formaldehyde in PBS, washed again, and mounted with Vectashield (Vector Laboratories, Burlingame, CA) for observation using a fluorescence microscope (Labophot-2; Nikon). The cells were scored and the percentage of labeled spermatozoa was recorded. At least 200 cells were counted and a minimum of three different experiments for each of the conditions tested were carried out. Sperm sonication and removal of the sperm membranes. Spermatozoa, obtained from the caput and cauda epididymides as described above, were collected in 4 ml of cold PBS (containing Ca and Mg) in a glass Corex tube placed in ice. Sonication took place in a Branson sonifier (Model 450) at the lowest setting (No. 1, equivalent to 10 W) for 1 min. This treatment produced head detachment in ⬎95% of the cells. Heads were washed in PBS (plus Ca and Mg), counted, and diluted to give a final concentration of 10 –15 ⫻ 10 6/ml. Then, they were transfected under the same conditions described for intact cells.
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In another series of experiments, complete removal of the sperm membranes was carried out using a method adapted from that described by Balhorn et al. [25]. Briefly, the sonicated sperm were incubated for 10 min in 1% cetyldimethylethylammonium bromide (Sigma, St. Louis, MO) at room temperature. Then, the cells were washed three to five times in PBS (supplemented with Ca and Mg), their concentration was estimated and adjusted, and finally the cells were transfected as described above. Electron microscopy. Samples from the sonicated and demembranated cauda and caput spermatozoa were fixed in a solution of 2% glutaraldehyde, 1% p-formaldehyde, and 0.1 M sucrose in 0.5 M cacodylate buffer (pH 7.3). Samples were thoroughly washed with the buffer and postfixed in 2% osmium tetroxide in cacodylate buffer. Then, they were dehydrated in an alcohol series and embedded in an epoxy resin. Ultrathin sections were obtained using an LKB Ultratome and stained with uranyl acetate and lead citrate. Finally, they were observed and photographed in a Philips 400 electron microscope. Southwestern blot. Southwestern blot was carried out with protein extracts obtained by boiling either cauda or caput sperm in Laemmli buffer [26] or by incubating the spermatozoa in radioimmunoprecipitation assay (RIPA) buffer [27] for 1 h in ice. One million cells were loaded per lane and electrophoresed in 10% acrylamide gels according to Laemmli [26]. The proteins were transferred to nitrocellulose filters (0.2-m pore) using a Millipore semidry blotting apparatus according to the manufacturer’s specifications. The correct transfer was assessed by reversible staining of the filters using a solution of 0.2% Ponceau S (Sigma) in 3% trichloroacetic acid (TCA). Filters were destained in PBS and renatured overnight in buffer 1 described by Zani et al. [24] and then incubated in buffer 2 [24] containing 150 ng of digoxigenin (DIG)-labeled DNA (Roche, Basel, Switzerland). Positive bands were then revealed using the digoxigenin detection reagents and protocols provided by Roche. Briefly, the filter was rinsed in PBS and incubated for 30 min in blocking solution and then 2 h in anti-DIG antibody conjugated to alkaline phosphatase. The positive signal was revealed using a chemiluminescent substrate (CDP-Star; Roche) diluted 1:200 in AP buffer (100 mM Tris, 100 mM NaCl, and 50 mM MgCl 2; pH 9.5) and X-OMAT AR film (Kodak). Collection of accessory gland fluids and seminal plasma. Seminal vesicle fluid was obtained by isolating the glands and collecting some drops from the fluid in cold PBS (supplemented or not with Ca and Mg). The suspension was cleared by centrifuging at maximum speed for 5 min in a microfuge. The protein concentration was measured with the Bio-Rad protein assay (Bio-Rad, Hercules, CA) and then the fluid was diluted to give the desired value (for most experiments, 1 mg/ml protein) and stored at ⫺20°C until their use. Fluids from the ventral and dorsolateral prostates and coagulating glands were obtained by mincing the tissue from five animals in 0.5 ml of cold PBS. The fluids were also cleared and the protein concentration measured and standardized. They were all stored as described above. On the other hand, whole seminal plasma was obtained from the uterus of normal estrous females after copulation. Briefly, a male of proven fertility was allowed to mate and copulation was observed under red light. Ejaculation was assessed by the presence of a copulatory plug in the vagina of the female and the animal was then removed from the cage and killed by ether overdose. The uteri were exposed and instilled with 50 l of PBS using a tuberculin syringe and then the total contents were retrieved by aspiration. The spermatozoa were removed by centrifuging at maximum speed for 5 min in a microfuge, the supernatant was transferred to a clean Eppendorf tube, and the volume was measured. It was 100 l in all cases, which accounted for a dilution 1:2. Samples were aliquoted in Eppendorf tubes and stored at ⫺20°C until use. Test of the effect of seminal plasma and male accessory sex gland fluids on the sperm DNA uptake. Mature spermatozoa were collected from the caudae epididymides of adult males as described
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FIG. 1. DNA binding to mature spermatozoa. Sperm were collected from the cauda epididymis and incubated with plasmid DNA in different buffers under the conditions described in the text. (a, d) Phase-contrast and fluorescence images of spermatozoa incubated in PBS supplemented with Ca and Mg. Red fluorescence is restricted to the postacrosome region of the head. (b, e) Phase-contrast and fluorescence images of spermatozoa incubated in PBS supplemented with Ca and Mg and then treated with DNase I (0.1 mg/ml) to remove surface bound DNA. Although the fluorescence is less intense, at least two of the three sperm show a considerable amount of internalized DNA. (c, f) Phase-contrast and fluorescence images of spermatozoa incubated in PBS containing 200 mM EDTA. DNA binding is completely abolished. Bar, 7 m.
above. The spermatozoa were washed twice in PBS and the concentration was adjusted to 10 –15 ⫻ 10 6 sperm/ml. Aliquots of 50 l were placed in Eppendorf tubes and centrifuged at 3000 rpm for 2–3 min. The supernatant was discarded and replaced by the accessory fluid or seminal plasma at the desired concentration. In some experiments, seminal fluids were heated at 60°C for 15 min or at 95°C for 2 min or contained different concentrations of EDTA. The sperm were gently resuspended and then 250 ng of plasmid DNA was added and transfection was allowed to take place for 30 min at 37°C. Controls without seminal proteins were included for every experiment. After transfection, the spermatozoa were processed for fluorescence microscopy as described above. DNase assays. To evaluate putative DNase activities in the accessory fluids or seminal plasma, 5 g of DNA was added to 50 l of the diluted fluid and incubated for 30 min at 37°C. Controls with only uterine fluid, PBS, or PBS containing 0.1 mg/ml DNase I (Roche) were included. Ten microliters of the reaction volume was loaded in an agarose gel containing ethidium bromide and electrophoresed. The integrity of the DNA was evaluated under UV light. Some of the seminal plasma samples underwent also heating at 60°C for 15 min or at 95°C for 2 min. In addition, to analyze the calcium dependence of the enzyme(s) another series of experiments in which different concentrations of EDTA were included was performed. Isolation and characterization of DNA binding proteins from the ventral prostate fluid. Ventral prostate fluid from 30 adult males was collected in 1 ml of PBS (containing Ca and Mg) following the procedure described above. The total volume was loaded in a
DNA–Sepharose column (Pharmacia, Uppsala, Sweden) and eluted with 10 mM Tris (pH 7.5), 1 mM EDTA containing increasing concentrations of KCl (0.4, 0.6, 0.8, and 1 M). One-milliliter fractions were collected and the absorbance at 280 nm was estimated. From each elution peak, fractions were pooled and concentrated using a macrosolute concentrator (Minicom, Danvers, MA) and used for protein electrophoresis. The proteins were boiled 5 min in Laemmli sample buffer containing 5% -mercaptoethanol and loaded in SDS gels (10% acrylamide). They were run at 30 mA of constant current. All reagents and apparatus for protein electrophoresis were purchased from Bio-Rad. Gels were fixed in 40% methanol, 10% acetic acid for 1 h and then silver stained according to the protocol described elsewhere [28]. The silver-stained gels were then restained with Coomassie blue to enhance the contrast and finally photographed.
RESULTS
Influence of the Medium and the Maturational Status of the Sperm on the Transfection Rates Mouse sperm were successfully transfected using a simple buffer containing Ca and Mg. The fluorescence was restricted to the postacrosome region of the sperm head (Figs. 1a and 1d). The percentage of labeled cells was around 80 –95% in every experiment (91.6 ⫾ 4.4%; n ⫽ 5). This percentage is re-
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FIG. 2. Influence of the maturational status and sperm membrane integrity in the DNA uptake. Mouse spermatozoa (5–7.5 ⫻ 10 5) were incubated for 30 min (37°C) in presence of 250 ng of plasmid DNA in PBS supplemented with Ca and Mg. (a) Intact spermatozoa obtained from the caput epididymis. They do not bind DNA. (b) Spermatozoa from the same sample, after sonication. The sperm heads now show some labeling in the postacrosome region of the head. (c) Cauda sperm after sonication. DNA binding is not affected by sonication (compare with Fig. 1d). (d) Cauda sperm head after sonication and treatment with cetyldimethylethylammonium bromide. Fluorescence is weaker than in c and, in addition, no longer restricted to the postacrosome region of the head. Bar, 8 m. (e) Electron micrograph of cauda sperm after sonication. Sonication completely removes the sperm acrosome in all cells. Part of the plasma membrane is also disrupted. Bar, 1.5 m. (f) Electron micrograph of cauda sperm after sonication and treatment with cetyldimethylethylammonium bromide. The sperm nucleus is “naked.” All membranes including the nuclear envelope are removed. Bar, 1.5 m.
duced to about 50% (46.26 ⫾ 23%, n ⫽ 4) after 30 min of treatment with 0.1 mg/ml DNase I, indicating that not all the sperm did internalize the bound DNA (Figs. 1b and 1e). Similar values were obtained using PBS without Ca or Mg supplementation, although the fluorescence was less intense; however, the inclusion of 200 mM EDTA completely blocked the DNA binding (Figs. 1c and 1f). The inhibition was fully reversible, since once the EDTA was removed
and the spermatozoa resuspended in a Ca- and Mgcontaining buffer in presence of the plasmid DNA, 75% (75.23 ⫾ 1.3%, n ⫽ 3) of the cells were labeled. Nevertheless, immature spermatozoa obtained from the upper regions of the epididymis, in contrast to the observations in mature cells, did not take up DNA (Fig. 2a). Only a reduced number of cells (4.6 ⫾ 4.4; n ⫽ 5) did show a faint labeling, always restricted to the postacrosome region of the head.
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Role of the Sperm Head Membranes in Transfection Disruption of the membranes by sonication did not alter either the percentage of labeled heads or the pattern (Fig. 2c) in mature spermatozoa. When the sonicated sperm were observed by electron microscopy, most of the heads lacked the acrosome and several of the head membranes had been partially removed (Fig. 2e). This indicates that it is not the acrosome which is preventing the binding of DNA in anterior regions of the head. On the other hand, membrane disruption had dramatic effects on the immature cells. The heads, after treatment, did bind some DNA in the postacrosome region of the head (Fig. 2b), resembling the mature sperm. Almost all sperm heads displayed this labeling (95.17 ⫾ 2.75%, n ⫽ 3). The observation of the sperm heads by electron microscopy showed an effect similar to that found in the mature sperm. The treatment with cetyldimethylethylammonium bromide removed all sperm membranes from the head, including the nuclear envelope (Fig. 2f). Incubation of isolated nuclei showed that they still retain some ability for DNA incorporation, although the fluorescence was weak compared to that found in intact heads. Interestingly, not only the postacrosome region, but the whole nucleus, was labeled (Fig. 2d). Detection of DNA Binding Proteins by Southwestern Blot Several bands with different intensities were detected on the caput and cauda samples (Fig. 3). They correspond to the groups already described by Zani et al. [24]. The extraction with RIPA slightly modified the bands recognized. The protein pattern from caput and cauda in Laemmli buffer extracts was very similar with the exception of a 50-, a 60-, and a 100-kDa band (Fig. 3a, marked with an asterisk). In the RIPA buffer extracts, the 50-kDa protein was not present, but the other two showed the same differences among caput and cauda (Fig. 3b, see asterisks). These differences do not reflect major differences in total protein among caput and cauda spermatozoa, as judged by staining with Ponceau S (Figs. 3a and 3b). Inhibition of DNA Uptake by Seminal Plasma Whole seminal plasma completely inhibited the DNA uptake up to a dilution of 1:10, in which the percentage of stained cells dropped to 1.5 ⫾ 1.29% (n ⫽ 4). This inhibitory activity, however, was lost when the seminal plasma was heated at 65°C for 15 min (80.7 ⫾ 4.7% stained cells, n ⫽ 3) or at 95°C for 2 min (90 ⫾ 5.56% stained cells, n ⫽ 3) or by addition of 50 mM EDTA (83.25 ⫾ 9.42% stained cells, n ⫽ 4).
FIG. 3. Results from the Southwestern blot. Samples from caput (cp) and cauda (cd) spermatozoa were extracted with Laemmli buffer (a) or RIPA (b). Numbers on the left represent the molecular weight in kDa of the protein standards (Low and Broad Range Molecular Weight Prestained Standards, Bio-Rad). The lanes on the left (SW) represent the bands revealed after Southwestern blot. The lanes on the right (Ponceau S) show the pattern of proteins in caput and cauda samples after staining with 0.2% Ponceau S in 3% TCA. The bands present only in cauda samples are indicated by asterisks.
In addition, transfection assays carried out in presence of different accessory gland fluids at protein concentration of 1 mg/ml showed that both ventral prostate and seminal vesicle fluid contain inhibitory activities (Table 1). The inhibition for both is dose dependent (Fig. 4). However, in seminal vesicle fluid the inhibitory activity (Fig. 4a) is stronger than in ventral prostate fluid, which at concentrations as high as 3 mg/ml causes an inhibition of 60%, still far from 100% (Fig. 4b).
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TABLE 1 DNA Uptake by the Spermatozoa in the Presence of Fluids from the Male Accessory Sex Glands Fluid tested a
Percentage of labeled spermatozoa (mean ⫾ SD)
PBS Seminal vesicle fluid Ventral prostate fluid Dorsal prostate fluid Coagulating gland fluid
85 ⫾ 14.21 0 ⫾ 0* 49.25 ⫾ 17.34* 76.33 ⫾ 2.62 79.5 ⫾ 7.29
a All fluids were tested at a concentration equivalent to 1 mg/ml protein. Values represent the average of three experiments. * Statistically significant (P ⬍ 0.01).
Characterization of the Inhibitory Activities Present in the Seminal Plasma Both seminal vesicle and ventral prostate fluid were tested for the presence of DNases. The former showed a powerful DNase activity (Fig. 5a), dose and
FIG. 4.
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calcium dependent (Fig. 5b). As expected we found also DNase activity in the seminal plasma (Fig. 5c). This activity is dose dependent and is greatly diminished by adding 50 mM EDTA or by heating at 65°C for 15 min and completely abolished by heating at 95°C for 2 min (Fig. 5c). It is noteworthy that in both seminal plasma and seminal vesicle fluid, the addition of EDTA up to 50 mM did not suppress completely the DNase activity, suggesting the presence of some other DNases whose activities are not regulated by divalent cations. Ventral prostate fluid was fractionated in a DNA– Sepharose column giving the elution profile showed in Fig. 6a. A major peak that elutes at relatively low salt concentration (0.4 M KCl) was consistently obtained. A second peak eluting at 0.6 M KCl was sometimes also found. Fractions from both peaks were pooled separately and concentrated. Once electrophoresed and stained, peak I revealed a single band of about 25 kDa (Fig. 6b, lane I) distinct from the major bands present in the fluid (Fig. 6b, lane VP). Two minor bands of
Dose-dependent inhibition of DNA uptake by vesicular (a) and prostatic (b) fluids. Incubation conditions are provided in the text.
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iments made so far with mammalian spermatozoa have been carried out using complex capacitating media, so it was not clear which components were essential for the DNA binding and subsequent internalization. Our results have shown that this process can, in fact, take place in a very simple saline buffer containing calcium. The presence of calcium, even in traces, is needed for the binding step. An additional unexpected requirement is epididymal maturation. The traverse through the epididymis brings about many changes in the sperm: they acquire motility and their surface properties are modified [30 –32]. Many new proteins are gained and others are posttranslationally modified [33–36]. The fact that caput sperm do not bind DNA, but they can internalize it once sonicated, shows that it is the plasma membrane which lacks a functional receptor(s) for the DNA. This putative receptor must be either acquired or modified through epididymal transit. In contrast, all the cytoplasmic and nuclear machinery for the DNA uptake seems to be already present in immature sperm, because we saw no differences in the pattern of binding among the sonicated mature and immature sperm. We have also shown that it is not the nucleus which determines the pattern of binding. Isolated nuclei were still able to bind the foreign
FIG. 5. DNase activity in the male accessory gland fluids. Details of the digestion procedures are given in the text. (a) DNA gel to show the integrity of plasmid DNA incubated in PBS, seminal vesicle fluid (SVF), ventral prostate fluid (VPF), or coagulating gland fluid (CGF). All fluids contained 1 mg/ml protein. A control incubated with DNase (100 g/ml) is shown on the right. M represents the molecular weight markers (Molecular Weight Markers II; Roche). (b) Dose and calcium dependence of the seminal vesicle DNase. The numbers represent the protein concentration in mg/ml. M, molecular weight markers. (c) DNase activity in the seminal plasma. The gel on the left shows the dose dependence of the DNase activity. U, uterine fluid diluted 1:10 in PBS. Seminal plasma samples were diluted in PBS and the numbers above each lane indicate the dilution. The gel on the right shows DNase activity of seminal plasma diluted 1:10 and untreated (C), combined with 50 mM EDTA (EDTA), or heated at 95°C for 2 min (95°C) or at 65°C for 15 min (65°C).
about 50 and 80 kDa were also detected in peak II (Fig. 6b, lane II). DISCUSSION
The fact that the spermatozoon is capable of DNA uptake is now well documented [29]. However, the mechanisms involved in this process as well as its regulation are still poorly understood. All the exper-
FIG. 6. Detection of DNA binding proteins in the ventral prostate fluid of the mouse. (a) Elution profile of the fluid from the column of DNA–Sepharose. Two peaks (I, II) could be detected. (b) SDS– PAGE of the fractions eluted from the column. A major band of about 25 kDa is indicated by an asterisk. Two minor bands of 50 and 80 kDa are indicated by arrows. VP, input (unfractionated prostatic fluid). M, molecular weight markers (Broad Range; Bio-Rad).
DNA UPTAKE IN MOUSE SPERMATOZOA
DNA but they bound much less plasmid DNA and it did not accumulate in the postacrosome region, but homogeneously in the whole nucleus. This strongly argues in favor of a specific system at the membrane and cytoplasmic levels that is located in the postacrosome region of the head. While there is no clue as to the identity of the cytoplasmic components, two types of molecules have been related to the DNA transport through the sperm plasma membrane, MHC type II molecules and the antigen CD4 [19, 20]. We have tried to detect both of them in immature spermatozoa and compare to the situation in the mature cells (data not shown). Unfortunately, we have been unable to detect the I-A in mature or immature cells. These results, while contradicting those reported by Wu et al. [19], are in agreement with the results obtained by Lavitrano et al. [20]. Regarding CD4, we also found much lower levels than those previously reported [20], perhaps reflecting differences among the antibodies used. In addition, we found no significant differences among mature and immature cells. This suggests that other sperm-specific molecules would be the ones playing a major role in the DNA uptake. There are additional reasons to support this fact. First, both types of molecules are also present in other cell types, i.e., immune cells. Nevertheless, so far, there is no report indicating that these cells can take up naked DNA in the way the sperm do. Second, there are discrepancies among authors regarding to the presence and abundance of MHC II molecules and even though the knockouts have a reduced ability to incorporate DNA, they are still able to do it [20]. Looking for other candidates, Zani et al. [24] used Southwestern blot to detect DNA binding proteins and described four classes of proteins from the sperm capable of binding DNA. The 20-kDa class seems to represent protamines, so they focused on the others. They favor the 30- to 35-kDa proteins as the primary receptors for the DNA, although they do not show any proof that those are indeed membrane proteins. We have also performed Southwestern experiments using caput and cauda sperm extracts. According to our results the 30- to 35-kDa class should be either membrane bound or cytosolic, because it appears in Laemmli buffer extracts only after long exposure times, but is quite clear in RIPA extracts. Nevertheless, it is equally present in caput and cauda extracts, so it is not likely to be the primary membranebound DNA receptor of the sperm. Two candidates arise to fulfill this role, a 60-kDa band and a 100-kDa band. Both are either absent or consistently weaker in the immature sperm. The 30- to 35-kDa proteins would represent probably cytoplasmic components from the cascade that both types of spermatozoa share. It is clear, though, that more studies are
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needed to determine the nature of all these proteins. Their cloning could be very interesting also for their application in transfection analyses in other cell types. Regarding the modulation of DNA uptake by the seminal plasma, we have identified both the seminal vesicles and the ventral prostate as the sources of inhibitory activities. The nature of the activity is different, though, according to its origin. Seminal vesicle fluid possesses a strong DNase activity that is calcium and magnesium dependent. This finding is not surprising, since a DNase activity of similar characteristics has been already described in the seminal vesicles of the bull [37]. DNases are present in the ejaculate of many species of mammals [38] and they are likely to account for most of the inhibitory activity found in the seminal plasma. In fact, in our experiments with mouse seminal plasma, we have found a strong correlation between the DNase activity present in it and its ability to inhibit DNA uptake in transfection assays. Moreover, the inhibitory activity of the seminal plasma can be suppressed by heating at 95°C for 2 min, which suggests inactivation of a protein, and also by heating at 65°C for 15 min, which is likely to be related to the inactivation of an enzymatic activity, and since 50 mM EDTA has the same effect this allows us to think of an enzyme regulated by calcium. Importantly, in all these cases the DNase activity present in the seminal plasma is either completely absent or strongly reduced. It is also interesting that, in addition to the vesicular, the prostatic fluid exhibits a moderate inhibitory activity of its own. This is represented by the presence of DNA binding proteins. All of them are found in very low levels, specially those of 50 and 80 kDa. The most abundant has a molecular weight of 25 kDa and its affinity for the DNA is not very high since it elutes at relatively low salt concentrations. If it does play a physiological role at the physiological concentrations in which it is present in the rodent ejaculate is difficult to tell. In comparison to the vesicular activity, its contribution to the total inhibitory activity found in the seminal plasma must be irrelevant. Another authors [3, 24] have found activities different from those described here. We think that all of them, though, are insignificant compared to the DNase activity. A possibility, then, for transfection experiments using ejaculated spermatozoa, is to add some EDTA to the incubation medium. We have observed that EDTA at concentrations of 50 – 100 mM do not interfere with DNA binding, but do effectively block the vesicular DNase activity allowing DNA incorporation in presence of diluted seminal plasma (1:10) or seminal vesicle fluid (up to 1 mg/ml protein concentration). The chelator, of
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course, should be removed before using the spermatozoa for in vitro fertilization or insemination. In summary, DNA uptake by the spermatozoa is a highly regulated phenomenon. The molecular basis underlying this process is still only partially understood and needs to be thoroughly studied. All research in this area is essential to help researchers to improve sperm-mediated transgenesis.
14.
Gagne, M. B., Pothier, F., and Sirard, M. A. (1991). Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev. 29, 6 –15.
15.
Bachiller, D., Schellander, K., Peli, J., and Ruther, U. (1991). Liposome-mediated DNA uptake by sperm cells. Mol. Reprod. Dev. 30, 194 –200.
16.
Patil, J. G., and Khoo, H. W. (1996). Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation. J. Exp. Zool. 274, 121–129.
17.
Farre, L., Rigau, T., Mogas, T., Garcia-Rocha, M., Canal, M., Gomez-Foix, A. M., and Rodriguez-Gil, J. E. (1999). Adenovirusmediated introduction of DNA into pig sperm and offspring. Mol. Reprod. Dev. 53, 149 –158.
18.
Francolini, M., Lavitrano, M., Lamia, C. L., French, D., Frati, L., Cotelli, F., and Spadafora, C. (1993). Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol. Reprod. Dev. 34, 133–139.
19.
Wu, G. M., Nose, K., Mori, E., and Mori, T. (1990). Binding of foreign DNA to mouse sperm mediated by its MHC class II structure. Am. J. Reprod. Immunol. 24, 120 –126.
20.
Lavitrano, M., Maione, B., Forte, E., Francolini, M., Sperandio, S., Testi, R., and Spadafora, C. (1997). The interaction of sperm cells with exogenous DNA: A role of CD4 and major histocompatibility complex class II molecules. Exp. Cell Res. 233, 56 – 62.
21.
Magnano, A. R., Giordano, R., Moscufo, N., Baccetti, B., and Spadafora, C. (1998). Sperm/DNA interaction: Integration of foreign DNA sequences in the mouse sperm genome. J. Reprod. Immunol. 41, 187–196.
22.
McCarthy, S., and Ward, W. S. (2000). Interaction of exogenous DNA with the nuclear matrix of live spermatozoa. Mol. Reprod. Dev. (Suppl.) 56, 235–237.
We thank Marı´a Angeles Ollacarizqueta for all the assistance in taking and processing the fluorescence images. This work was partially supported by Grant PB96-0808 from DGICYT (Spain).
REFERENCES 1.
Brackett, B. G., Baranska, W., Sawicki, W., and Koprowski, H. (1971). Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci. USA 68, 353–357.
2.
Lavitrano, M., Camaioni, A., Fazio, V. M., Dolci, S., Farace, M. G., and Spadafora, C. (1989). Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57, 717–723.
3.
Camaioni, A., Russo, M. A., Odorisio, T., Gandolfi, F., Fazio, V. M., and Siracusa, G. (1992). Uptake of exogenous DNA by mammalian spermatozoa: Specific localization of DNA on sperm heads. J. Reprod. Fertil. 96, 203–212.
4.
Atkinson, P. W., Hines, E. R., Beaton, S., Matthaei, K. I., Reed, K. C., and Bradley, M. P. (1991). Association of exogenous DNA with cattle and insect spermatozoa in vitro. Mol. Reprod. Dev. 29, 1–5.
5.
Castro, F. O., Herna´ndez, O., Uliver, C., Solano, R., Milane´s, C., Aguilar, A., Pe´rez, A., De Armas, R., Herrera, L., and De la Fuente, J. (1991). Introduction of foreign DNA into the spermatozoa of farm animals. Theriogenology 34, 1099 –1110.
23.
Maione, B., Pittoggi, C., Achene, L., Lorenzini, R., and Spadafora, C. (1997). Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol. 16, 1087–1097.
6.
Sperandio, S., Lulli, V., Bacci, M. L., Forni, M., Maione, B., Spadafora, C., and Lavitrano, M. L. (1996). Sperm mediated DNA transfer in bovine and swine species. Anim. Biotech. 7, 59 –77.
24.
Zani, M., Lavitrano, M., French, D., Lulli, V., Maione, B., Sperandio, S., and Spadafora, C. (1995). The mechanism of binding of exogenous DNA to sperm cells: Factors controlling the DNA uptake. Exp. Cell Res. 217, 57– 64.
7.
Horan, R., Powell, R., McQuaid, S., Gannon, F., and Houghton, J. A. (1991). Association of foreign DNA with porcine spermatozoa. Arch. Androl. 26, 83–92.
25.
Balhorn, R., Gledhill, B. L., and Wyrobek, A. J. (1977). Mouse sperm chromatin proteins: Quantitative isolation and partial characterization. Biochemistry 16, 4074 – 4080.
8.
Nakanishi, A., and Iritani, A. (1993). Gene transfer in the chicken by sperm-mediated methods. Mol. Reprod. Dev. 36, 258 –261.
26.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.
9.
Habrova, V., Takac, M., Navratil, J., Macha, J., Ceskova, N., and Jonak, J. (1996). Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization. Mol. Reprod. Dev. 44, 32– 42.
27.
10.
Khoo, H. W., Ang, L. H., Lim, H. B., and Wong, K. Y. (1992). Sperm cells as vectors for introducing foreign DNA into zebrafish. Aquaculture 107, 1–19.
Kamps, M. P., and Sefton, B. M. (1988). Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2, 305– 315.
28.
Carballada, R., and Esponda, P. (1991). Electrophoretic pattern of rodent seminal vesicle proteins as revealed by silver staining. Int. J. Androl. 14, 52–57.
29.
Spadafora, C. (1998). Sperm cells and foreign DNA: A controversial relation. BioEssays 20, 955–964.
30.
Cooper, T. G. (1986). “The Epididymis, Sperm Maturation and Fertilization,” Springer-Verlag, Berlin.
31.
Bedford, J. M., and Hoskins D. D. (1992). The mammalian spermatozoon: Morphology, biochemistry and physiology. In “Marshall’s Physiology of Reproduction” (G. E. Lamming Ed.), 2nd ed., Vol. 2, Churchill Livingstone, London.
11.
Sin, F. Y., Walker, S. P., Symonds, J. E., Mukherjee, U. K., Khoo, J. G., and Sin, I. L. (2000). Electroporation of salmon sperm for gene transfer: Efficiency, reliability, and fate of transgene. Mol. Reprod. Dev. (Suppl.) 56, 285–288.
12.
Arezzo, F. (1989). Sea urchin sperm as a vector of foreign genetic information. Cell Biol. Int. Rep. 13, 391– 404.
13.
Tsai, H. J. (2000). Electroporated sperm mediation of a gene transfer system for finfish and shellfish. Mol. Reprod. Dev. (Suppl.) 56, 281–284.
DNA UPTAKE IN MOUSE SPERMATOZOA 32.
Kirchhoff, C., Pera, I., Derr, P., Yeung, C. H., and Cooper T (1997). The molecular biology of the sperm surface. Post-testicular membrane remodelling. Adv. Exp. Med. Biol. 424, 221– 232. 33. Anakwe, O. O., Sharma, S., Hoff, H. B., Hardy, D. M., and Gerton, G. L. (1991). Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains. Mol. Reprod. Dev. 29, 294 –301. 34. Kirchhoff, C., and Hale, G. (1996). Cell-to-cell transfer of glycosylphosphatidyl inositol-anchored membrane proteins during sperm maturation. Mol. Hum. Reprod. 2, 177–184. Received June 7, 2000 Revised version received September 29, 2000
113
35.
Lum, L., and Blobel, C. P. (1997). Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev. Biol. 191, 131–145.
36.
Kirchhoff, C. (1999). Gene expression in the epididymis. Int. Rev. Cytol. 188, 133–202.
37.
Tanigawa, Y., Yoshihara, K., and Koide, S. S. (1975). Endonuclease activity in bull semen, testis and accessory sex organs. Biol. Reprod. 12, 464 – 470.
38.
Mann, T., and Lutwak-Mann, C. (1981). “Male Reproductive Function and Semen,” Springer-Verlag, Berlin.
Regulation of Foreign DNA Uptake by Mouse Spermatozoa Rosa Carballada 1 and Pedro Esponda Centro de Investigaciones Biolo´gicas, CSIC, Vela´zquez 144, 28006 Madrid, Spain
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. © 2001 Academic Press
Key Words: DNA uptake; epididymis; prostate; sperm; sperm membranes; seminal plasma; seminal vesicles; transgenesis.
INTRODUCTION
In 1971, Brackett et al. [1] reported that rabbit spermatozoa were able to take up naked DNA present in the incubation medium. Years later, Lavitrano et al. [2] used this property to generate transgenic mice. The simplicity of the method prompted many labs to explore the ability of spermatozoa of other species to incorporate foreign DNA and there is already a variety of species analyzed. They include other mammals such as humans [3], bulls [3– 6], and pigs [3, 5–7], but also birds [5, 8], amphibians [9], fishes [5, 10, 11], and some invertebrates [4, 12, 13]. In many of these studies, the spermatozoa were used for insemination or in vitro 1 To whom reprint requests should 34-915627518. E-mail: [email protected].
0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
be
addressed.
Fax:
fertilization, giving rise to transgenic embryos or adults [6, 9 –14]. Although in most cases the spermatozoa are simply incubated in presence of the foreign DNA, sometimes electroporation or cationic liposomes are used to enhance the uptake [11, 13–16]. Even some viruses have been used as vectors [17]. Part of the DNA remains bound to the sperm head plasma membrane; however, part is internalized and in mammals it is consistently found in the postacrosome region of the sperm nucleus [3, 18]. The process is still poorly understood. Some authors have proposed that MHC II molecules and the antigen CD4, present in the sperm head membranes, are involved in the binding and internalization of the foreign DNA [19, 20]. Spermatozoa obtained from CD4 knockout mice are still capable of binding the DNA but unable to internalize it. In addition antibodies against CD4 can inhibit the latter process. Both MHC II molecules [19] and CD4 [20] have been described in the postacrosome region of the mouse sperm head and this localization could be determining the pattern of DNA incorporation. Once in the nucleus, the DNA becomes tightly associated to nuclear proteins [21, 22] and then it can be cleaved by sperm endonucleases and finally integrated in the genome [23]. On the other hand, there are also molecules that act as negative regulators of the process. Seminal plasma proteins are able to inhibit the DNA uptake [3, 24], a mechanism that could have evolved to protect sperm from foreign DNA present in the reproductive tract. While in rodents mature spermatozoa are routinely collected from the epididymis and therefore have never been in contact with the seminal plasma, in species such as dairy animals the researcher needs to work with ejaculated spermatozoa. This brings about the necessity to thoroughly wash the spermatozoa to be able to have a good transfection rate. Regarding the inhibitory factors so far analyzed, a 37-kDa protein present in sea urchin sperm and human seminal plasma has been shown to inhibit the DNA uptake [24] although the mechanism remains obscure. In addition, mouse whole seminal plasma displays a similar inhibitory effect and it has been proposed that some factors present in it, such as the glycosaminoglycans, could be binding the mouse sperm and blocking the receptors
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for DNA [3]. This hypothesis, however, has not been proved yet. In this study we have tried to go further in the analysis of the characteristics of the process of DNA uptake by the spermatozoon and its inhibition by seminal plasma. We have examined the minimum requirements in the incubation medium that allow transfection in mature sperm. In addition, we have also examined the differences among mature and immature sperm in their ability to bind DNA. We have performed assays in intact cells, in spermatozoa disrupted by sonication, and in isolated sperm nuclei to see the role of the different membranes and cellular compartments in this process. We have also tried to characterize some molecules that can be responsible for the differences observed. On the other hand, we have carried out transfection experiments in the presence of different accessory gland fluids to determine those that contain inhibitory activities. Finally, we have characterized the inhibitory activities found and the mechanism of inhibition. MATERIALS AND METHODS Animals and sperm collection. Male mice from the CD-1 strain bred in our colony were used as source of spermatozoa. All animals were sexually mature. For mature spermatozoa, the caudae epididymides from one or two males were dissected free of fat tissue and minced in 400 l of PBS (phosphate-buffered saline, pH 7.5) supplemented with 2 mM CaCl and 1 mM MgCl when indicated. For immature spermatozoa, caput epididymides were obtained from five males and minced in 200 l of PBS. The spermatozoa were washed twice in PBS (by centrifuging 1000g ⫻ 3 min) and resuspended in the chosen buffer (PBS, supplemented or not with Ca and Mg and containing or not EDTA). The concentration was estimated by the use of a hemocytometer and adjusted to 10 –15 ⫻ 10 6 sperm/ml. Motility was also routinely examined and whenever there was calcium in the incubation medium it was always ⬎90%. Samples poorly motile were discarded. Sperm transfection assays. The DNA used for transfection was a commercial plasmid, pGeneGrip (GTS, California) that encodes the green fluorescent protein under the control of hCMV IE promoter/ enhancer and is rhodamine labeled. This allowed a direct visualization of the DNA uptake by the use of fluorescence microscopy. For every experiment 250 ng of plasmid DNA was added to 50 l of the sperm suspension and incubated for 30 min at 37°C. After transfection, sperm were washed three times (at 1000g ⫻ 3 min), fixed for 1 h in 2% p-formaldehyde in PBS, washed again, and mounted with Vectashield (Vector Laboratories, Burlingame, CA) for observation using a fluorescence microscope (Labophot-2; Nikon). The cells were scored and the percentage of labeled spermatozoa was recorded. At least 200 cells were counted and a minimum of three different experiments for each of the conditions tested were carried out. Sperm sonication and removal of the sperm membranes. Spermatozoa, obtained from the caput and cauda epididymides as described above, were collected in 4 ml of cold PBS (containing Ca and Mg) in a glass Corex tube placed in ice. Sonication took place in a Branson sonifier (Model 450) at the lowest setting (No. 1, equivalent to 10 W) for 1 min. This treatment produced head detachment in ⬎95% of the cells. Heads were washed in PBS (plus Ca and Mg), counted, and diluted to give a final concentration of 10 –15 ⫻ 10 6/ml. Then, they were transfected under the same conditions described for intact cells.
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In another series of experiments, complete removal of the sperm membranes was carried out using a method adapted from that described by Balhorn et al. [25]. Briefly, the sonicated sperm were incubated for 10 min in 1% cetyldimethylethylammonium bromide (Sigma, St. Louis, MO) at room temperature. Then, the cells were washed three to five times in PBS (supplemented with Ca and Mg), their concentration was estimated and adjusted, and finally the cells were transfected as described above. Electron microscopy. Samples from the sonicated and demembranated cauda and caput spermatozoa were fixed in a solution of 2% glutaraldehyde, 1% p-formaldehyde, and 0.1 M sucrose in 0.5 M cacodylate buffer (pH 7.3). Samples were thoroughly washed with the buffer and postfixed in 2% osmium tetroxide in cacodylate buffer. Then, they were dehydrated in an alcohol series and embedded in an epoxy resin. Ultrathin sections were obtained using an LKB Ultratome and stained with uranyl acetate and lead citrate. Finally, they were observed and photographed in a Philips 400 electron microscope. Southwestern blot. Southwestern blot was carried out with protein extracts obtained by boiling either cauda or caput sperm in Laemmli buffer [26] or by incubating the spermatozoa in radioimmunoprecipitation assay (RIPA) buffer [27] for 1 h in ice. One million cells were loaded per lane and electrophoresed in 10% acrylamide gels according to Laemmli [26]. The proteins were transferred to nitrocellulose filters (0.2-m pore) using a Millipore semidry blotting apparatus according to the manufacturer’s specifications. The correct transfer was assessed by reversible staining of the filters using a solution of 0.2% Ponceau S (Sigma) in 3% trichloroacetic acid (TCA). Filters were destained in PBS and renatured overnight in buffer 1 described by Zani et al. [24] and then incubated in buffer 2 [24] containing 150 ng of digoxigenin (DIG)-labeled DNA (Roche, Basel, Switzerland). Positive bands were then revealed using the digoxigenin detection reagents and protocols provided by Roche. Briefly, the filter was rinsed in PBS and incubated for 30 min in blocking solution and then 2 h in anti-DIG antibody conjugated to alkaline phosphatase. The positive signal was revealed using a chemiluminescent substrate (CDP-Star; Roche) diluted 1:200 in AP buffer (100 mM Tris, 100 mM NaCl, and 50 mM MgCl 2; pH 9.5) and X-OMAT AR film (Kodak). Collection of accessory gland fluids and seminal plasma. Seminal vesicle fluid was obtained by isolating the glands and collecting some drops from the fluid in cold PBS (supplemented or not with Ca and Mg). The suspension was cleared by centrifuging at maximum speed for 5 min in a microfuge. The protein concentration was measured with the Bio-Rad protein assay (Bio-Rad, Hercules, CA) and then the fluid was diluted to give the desired value (for most experiments, 1 mg/ml protein) and stored at ⫺20°C until their use. Fluids from the ventral and dorsolateral prostates and coagulating glands were obtained by mincing the tissue from five animals in 0.5 ml of cold PBS. The fluids were also cleared and the protein concentration measured and standardized. They were all stored as described above. On the other hand, whole seminal plasma was obtained from the uterus of normal estrous females after copulation. Briefly, a male of proven fertility was allowed to mate and copulation was observed under red light. Ejaculation was assessed by the presence of a copulatory plug in the vagina of the female and the animal was then removed from the cage and killed by ether overdose. The uteri were exposed and instilled with 50 l of PBS using a tuberculin syringe and then the total contents were retrieved by aspiration. The spermatozoa were removed by centrifuging at maximum speed for 5 min in a microfuge, the supernatant was transferred to a clean Eppendorf tube, and the volume was measured. It was 100 l in all cases, which accounted for a dilution 1:2. Samples were aliquoted in Eppendorf tubes and stored at ⫺20°C until use. Test of the effect of seminal plasma and male accessory sex gland fluids on the sperm DNA uptake. Mature spermatozoa were collected from the caudae epididymides of adult males as described
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FIG. 1. DNA binding to mature spermatozoa. Sperm were collected from the cauda epididymis and incubated with plasmid DNA in different buffers under the conditions described in the text. (a, d) Phase-contrast and fluorescence images of spermatozoa incubated in PBS supplemented with Ca and Mg. Red fluorescence is restricted to the postacrosome region of the head. (b, e) Phase-contrast and fluorescence images of spermatozoa incubated in PBS supplemented with Ca and Mg and then treated with DNase I (0.1 mg/ml) to remove surface bound DNA. Although the fluorescence is less intense, at least two of the three sperm show a considerable amount of internalized DNA. (c, f) Phase-contrast and fluorescence images of spermatozoa incubated in PBS containing 200 mM EDTA. DNA binding is completely abolished. Bar, 7 m.
above. The spermatozoa were washed twice in PBS and the concentration was adjusted to 10 –15 ⫻ 10 6 sperm/ml. Aliquots of 50 l were placed in Eppendorf tubes and centrifuged at 3000 rpm for 2–3 min. The supernatant was discarded and replaced by the accessory fluid or seminal plasma at the desired concentration. In some experiments, seminal fluids were heated at 60°C for 15 min or at 95°C for 2 min or contained different concentrations of EDTA. The sperm were gently resuspended and then 250 ng of plasmid DNA was added and transfection was allowed to take place for 30 min at 37°C. Controls without seminal proteins were included for every experiment. After transfection, the spermatozoa were processed for fluorescence microscopy as described above. DNase assays. To evaluate putative DNase activities in the accessory fluids or seminal plasma, 5 g of DNA was added to 50 l of the diluted fluid and incubated for 30 min at 37°C. Controls with only uterine fluid, PBS, or PBS containing 0.1 mg/ml DNase I (Roche) were included. Ten microliters of the reaction volume was loaded in an agarose gel containing ethidium bromide and electrophoresed. The integrity of the DNA was evaluated under UV light. Some of the seminal plasma samples underwent also heating at 60°C for 15 min or at 95°C for 2 min. In addition, to analyze the calcium dependence of the enzyme(s) another series of experiments in which different concentrations of EDTA were included was performed. Isolation and characterization of DNA binding proteins from the ventral prostate fluid. Ventral prostate fluid from 30 adult males was collected in 1 ml of PBS (containing Ca and Mg) following the procedure described above. The total volume was loaded in a
DNA–Sepharose column (Pharmacia, Uppsala, Sweden) and eluted with 10 mM Tris (pH 7.5), 1 mM EDTA containing increasing concentrations of KCl (0.4, 0.6, 0.8, and 1 M). One-milliliter fractions were collected and the absorbance at 280 nm was estimated. From each elution peak, fractions were pooled and concentrated using a macrosolute concentrator (Minicom, Danvers, MA) and used for protein electrophoresis. The proteins were boiled 5 min in Laemmli sample buffer containing 5% -mercaptoethanol and loaded in SDS gels (10% acrylamide). They were run at 30 mA of constant current. All reagents and apparatus for protein electrophoresis were purchased from Bio-Rad. Gels were fixed in 40% methanol, 10% acetic acid for 1 h and then silver stained according to the protocol described elsewhere [28]. The silver-stained gels were then restained with Coomassie blue to enhance the contrast and finally photographed.
RESULTS
Influence of the Medium and the Maturational Status of the Sperm on the Transfection Rates Mouse sperm were successfully transfected using a simple buffer containing Ca and Mg. The fluorescence was restricted to the postacrosome region of the sperm head (Figs. 1a and 1d). The percentage of labeled cells was around 80 –95% in every experiment (91.6 ⫾ 4.4%; n ⫽ 5). This percentage is re-
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FIG. 2. Influence of the maturational status and sperm membrane integrity in the DNA uptake. Mouse spermatozoa (5–7.5 ⫻ 10 5) were incubated for 30 min (37°C) in presence of 250 ng of plasmid DNA in PBS supplemented with Ca and Mg. (a) Intact spermatozoa obtained from the caput epididymis. They do not bind DNA. (b) Spermatozoa from the same sample, after sonication. The sperm heads now show some labeling in the postacrosome region of the head. (c) Cauda sperm after sonication. DNA binding is not affected by sonication (compare with Fig. 1d). (d) Cauda sperm head after sonication and treatment with cetyldimethylethylammonium bromide. Fluorescence is weaker than in c and, in addition, no longer restricted to the postacrosome region of the head. Bar, 8 m. (e) Electron micrograph of cauda sperm after sonication. Sonication completely removes the sperm acrosome in all cells. Part of the plasma membrane is also disrupted. Bar, 1.5 m. (f) Electron micrograph of cauda sperm after sonication and treatment with cetyldimethylethylammonium bromide. The sperm nucleus is “naked.” All membranes including the nuclear envelope are removed. Bar, 1.5 m.
duced to about 50% (46.26 ⫾ 23%, n ⫽ 4) after 30 min of treatment with 0.1 mg/ml DNase I, indicating that not all the sperm did internalize the bound DNA (Figs. 1b and 1e). Similar values were obtained using PBS without Ca or Mg supplementation, although the fluorescence was less intense; however, the inclusion of 200 mM EDTA completely blocked the DNA binding (Figs. 1c and 1f). The inhibition was fully reversible, since once the EDTA was removed
and the spermatozoa resuspended in a Ca- and Mgcontaining buffer in presence of the plasmid DNA, 75% (75.23 ⫾ 1.3%, n ⫽ 3) of the cells were labeled. Nevertheless, immature spermatozoa obtained from the upper regions of the epididymis, in contrast to the observations in mature cells, did not take up DNA (Fig. 2a). Only a reduced number of cells (4.6 ⫾ 4.4; n ⫽ 5) did show a faint labeling, always restricted to the postacrosome region of the head.
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Role of the Sperm Head Membranes in Transfection Disruption of the membranes by sonication did not alter either the percentage of labeled heads or the pattern (Fig. 2c) in mature spermatozoa. When the sonicated sperm were observed by electron microscopy, most of the heads lacked the acrosome and several of the head membranes had been partially removed (Fig. 2e). This indicates that it is not the acrosome which is preventing the binding of DNA in anterior regions of the head. On the other hand, membrane disruption had dramatic effects on the immature cells. The heads, after treatment, did bind some DNA in the postacrosome region of the head (Fig. 2b), resembling the mature sperm. Almost all sperm heads displayed this labeling (95.17 ⫾ 2.75%, n ⫽ 3). The observation of the sperm heads by electron microscopy showed an effect similar to that found in the mature sperm. The treatment with cetyldimethylethylammonium bromide removed all sperm membranes from the head, including the nuclear envelope (Fig. 2f). Incubation of isolated nuclei showed that they still retain some ability for DNA incorporation, although the fluorescence was weak compared to that found in intact heads. Interestingly, not only the postacrosome region, but the whole nucleus, was labeled (Fig. 2d). Detection of DNA Binding Proteins by Southwestern Blot Several bands with different intensities were detected on the caput and cauda samples (Fig. 3). They correspond to the groups already described by Zani et al. [24]. The extraction with RIPA slightly modified the bands recognized. The protein pattern from caput and cauda in Laemmli buffer extracts was very similar with the exception of a 50-, a 60-, and a 100-kDa band (Fig. 3a, marked with an asterisk). In the RIPA buffer extracts, the 50-kDa protein was not present, but the other two showed the same differences among caput and cauda (Fig. 3b, see asterisks). These differences do not reflect major differences in total protein among caput and cauda spermatozoa, as judged by staining with Ponceau S (Figs. 3a and 3b). Inhibition of DNA Uptake by Seminal Plasma Whole seminal plasma completely inhibited the DNA uptake up to a dilution of 1:10, in which the percentage of stained cells dropped to 1.5 ⫾ 1.29% (n ⫽ 4). This inhibitory activity, however, was lost when the seminal plasma was heated at 65°C for 15 min (80.7 ⫾ 4.7% stained cells, n ⫽ 3) or at 95°C for 2 min (90 ⫾ 5.56% stained cells, n ⫽ 3) or by addition of 50 mM EDTA (83.25 ⫾ 9.42% stained cells, n ⫽ 4).
FIG. 3. Results from the Southwestern blot. Samples from caput (cp) and cauda (cd) spermatozoa were extracted with Laemmli buffer (a) or RIPA (b). Numbers on the left represent the molecular weight in kDa of the protein standards (Low and Broad Range Molecular Weight Prestained Standards, Bio-Rad). The lanes on the left (SW) represent the bands revealed after Southwestern blot. The lanes on the right (Ponceau S) show the pattern of proteins in caput and cauda samples after staining with 0.2% Ponceau S in 3% TCA. The bands present only in cauda samples are indicated by asterisks.
In addition, transfection assays carried out in presence of different accessory gland fluids at protein concentration of 1 mg/ml showed that both ventral prostate and seminal vesicle fluid contain inhibitory activities (Table 1). The inhibition for both is dose dependent (Fig. 4). However, in seminal vesicle fluid the inhibitory activity (Fig. 4a) is stronger than in ventral prostate fluid, which at concentrations as high as 3 mg/ml causes an inhibition of 60%, still far from 100% (Fig. 4b).
DNA UPTAKE IN MOUSE SPERMATOZOA
TABLE 1 DNA Uptake by the Spermatozoa in the Presence of Fluids from the Male Accessory Sex Glands Fluid tested a
Percentage of labeled spermatozoa (mean ⫾ SD)
PBS Seminal vesicle fluid Ventral prostate fluid Dorsal prostate fluid Coagulating gland fluid
85 ⫾ 14.21 0 ⫾ 0* 49.25 ⫾ 17.34* 76.33 ⫾ 2.62 79.5 ⫾ 7.29
a All fluids were tested at a concentration equivalent to 1 mg/ml protein. Values represent the average of three experiments. * Statistically significant (P ⬍ 0.01).
Characterization of the Inhibitory Activities Present in the Seminal Plasma Both seminal vesicle and ventral prostate fluid were tested for the presence of DNases. The former showed a powerful DNase activity (Fig. 5a), dose and
FIG. 4.
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calcium dependent (Fig. 5b). As expected we found also DNase activity in the seminal plasma (Fig. 5c). This activity is dose dependent and is greatly diminished by adding 50 mM EDTA or by heating at 65°C for 15 min and completely abolished by heating at 95°C for 2 min (Fig. 5c). It is noteworthy that in both seminal plasma and seminal vesicle fluid, the addition of EDTA up to 50 mM did not suppress completely the DNase activity, suggesting the presence of some other DNases whose activities are not regulated by divalent cations. Ventral prostate fluid was fractionated in a DNA– Sepharose column giving the elution profile showed in Fig. 6a. A major peak that elutes at relatively low salt concentration (0.4 M KCl) was consistently obtained. A second peak eluting at 0.6 M KCl was sometimes also found. Fractions from both peaks were pooled separately and concentrated. Once electrophoresed and stained, peak I revealed a single band of about 25 kDa (Fig. 6b, lane I) distinct from the major bands present in the fluid (Fig. 6b, lane VP). Two minor bands of
Dose-dependent inhibition of DNA uptake by vesicular (a) and prostatic (b) fluids. Incubation conditions are provided in the text.
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iments made so far with mammalian spermatozoa have been carried out using complex capacitating media, so it was not clear which components were essential for the DNA binding and subsequent internalization. Our results have shown that this process can, in fact, take place in a very simple saline buffer containing calcium. The presence of calcium, even in traces, is needed for the binding step. An additional unexpected requirement is epididymal maturation. The traverse through the epididymis brings about many changes in the sperm: they acquire motility and their surface properties are modified [30 –32]. Many new proteins are gained and others are posttranslationally modified [33–36]. The fact that caput sperm do not bind DNA, but they can internalize it once sonicated, shows that it is the plasma membrane which lacks a functional receptor(s) for the DNA. This putative receptor must be either acquired or modified through epididymal transit. In contrast, all the cytoplasmic and nuclear machinery for the DNA uptake seems to be already present in immature sperm, because we saw no differences in the pattern of binding among the sonicated mature and immature sperm. We have also shown that it is not the nucleus which determines the pattern of binding. Isolated nuclei were still able to bind the foreign
FIG. 5. DNase activity in the male accessory gland fluids. Details of the digestion procedures are given in the text. (a) DNA gel to show the integrity of plasmid DNA incubated in PBS, seminal vesicle fluid (SVF), ventral prostate fluid (VPF), or coagulating gland fluid (CGF). All fluids contained 1 mg/ml protein. A control incubated with DNase (100 g/ml) is shown on the right. M represents the molecular weight markers (Molecular Weight Markers II; Roche). (b) Dose and calcium dependence of the seminal vesicle DNase. The numbers represent the protein concentration in mg/ml. M, molecular weight markers. (c) DNase activity in the seminal plasma. The gel on the left shows the dose dependence of the DNase activity. U, uterine fluid diluted 1:10 in PBS. Seminal plasma samples were diluted in PBS and the numbers above each lane indicate the dilution. The gel on the right shows DNase activity of seminal plasma diluted 1:10 and untreated (C), combined with 50 mM EDTA (EDTA), or heated at 95°C for 2 min (95°C) or at 65°C for 15 min (65°C).
about 50 and 80 kDa were also detected in peak II (Fig. 6b, lane II). DISCUSSION
The fact that the spermatozoon is capable of DNA uptake is now well documented [29]. However, the mechanisms involved in this process as well as its regulation are still poorly understood. All the exper-
FIG. 6. Detection of DNA binding proteins in the ventral prostate fluid of the mouse. (a) Elution profile of the fluid from the column of DNA–Sepharose. Two peaks (I, II) could be detected. (b) SDS– PAGE of the fractions eluted from the column. A major band of about 25 kDa is indicated by an asterisk. Two minor bands of 50 and 80 kDa are indicated by arrows. VP, input (unfractionated prostatic fluid). M, molecular weight markers (Broad Range; Bio-Rad).
DNA UPTAKE IN MOUSE SPERMATOZOA
DNA but they bound much less plasmid DNA and it did not accumulate in the postacrosome region, but homogeneously in the whole nucleus. This strongly argues in favor of a specific system at the membrane and cytoplasmic levels that is located in the postacrosome region of the head. While there is no clue as to the identity of the cytoplasmic components, two types of molecules have been related to the DNA transport through the sperm plasma membrane, MHC type II molecules and the antigen CD4 [19, 20]. We have tried to detect both of them in immature spermatozoa and compare to the situation in the mature cells (data not shown). Unfortunately, we have been unable to detect the I-A in mature or immature cells. These results, while contradicting those reported by Wu et al. [19], are in agreement with the results obtained by Lavitrano et al. [20]. Regarding CD4, we also found much lower levels than those previously reported [20], perhaps reflecting differences among the antibodies used. In addition, we found no significant differences among mature and immature cells. This suggests that other sperm-specific molecules would be the ones playing a major role in the DNA uptake. There are additional reasons to support this fact. First, both types of molecules are also present in other cell types, i.e., immune cells. Nevertheless, so far, there is no report indicating that these cells can take up naked DNA in the way the sperm do. Second, there are discrepancies among authors regarding to the presence and abundance of MHC II molecules and even though the knockouts have a reduced ability to incorporate DNA, they are still able to do it [20]. Looking for other candidates, Zani et al. [24] used Southwestern blot to detect DNA binding proteins and described four classes of proteins from the sperm capable of binding DNA. The 20-kDa class seems to represent protamines, so they focused on the others. They favor the 30- to 35-kDa proteins as the primary receptors for the DNA, although they do not show any proof that those are indeed membrane proteins. We have also performed Southwestern experiments using caput and cauda sperm extracts. According to our results the 30- to 35-kDa class should be either membrane bound or cytosolic, because it appears in Laemmli buffer extracts only after long exposure times, but is quite clear in RIPA extracts. Nevertheless, it is equally present in caput and cauda extracts, so it is not likely to be the primary membranebound DNA receptor of the sperm. Two candidates arise to fulfill this role, a 60-kDa band and a 100-kDa band. Both are either absent or consistently weaker in the immature sperm. The 30- to 35-kDa proteins would represent probably cytoplasmic components from the cascade that both types of spermatozoa share. It is clear, though, that more studies are
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needed to determine the nature of all these proteins. Their cloning could be very interesting also for their application in transfection analyses in other cell types. Regarding the modulation of DNA uptake by the seminal plasma, we have identified both the seminal vesicles and the ventral prostate as the sources of inhibitory activities. The nature of the activity is different, though, according to its origin. Seminal vesicle fluid possesses a strong DNase activity that is calcium and magnesium dependent. This finding is not surprising, since a DNase activity of similar characteristics has been already described in the seminal vesicles of the bull [37]. DNases are present in the ejaculate of many species of mammals [38] and they are likely to account for most of the inhibitory activity found in the seminal plasma. In fact, in our experiments with mouse seminal plasma, we have found a strong correlation between the DNase activity present in it and its ability to inhibit DNA uptake in transfection assays. Moreover, the inhibitory activity of the seminal plasma can be suppressed by heating at 95°C for 2 min, which suggests inactivation of a protein, and also by heating at 65°C for 15 min, which is likely to be related to the inactivation of an enzymatic activity, and since 50 mM EDTA has the same effect this allows us to think of an enzyme regulated by calcium. Importantly, in all these cases the DNase activity present in the seminal plasma is either completely absent or strongly reduced. It is also interesting that, in addition to the vesicular, the prostatic fluid exhibits a moderate inhibitory activity of its own. This is represented by the presence of DNA binding proteins. All of them are found in very low levels, specially those of 50 and 80 kDa. The most abundant has a molecular weight of 25 kDa and its affinity for the DNA is not very high since it elutes at relatively low salt concentrations. If it does play a physiological role at the physiological concentrations in which it is present in the rodent ejaculate is difficult to tell. In comparison to the vesicular activity, its contribution to the total inhibitory activity found in the seminal plasma must be irrelevant. Another authors [3, 24] have found activities different from those described here. We think that all of them, though, are insignificant compared to the DNase activity. A possibility, then, for transfection experiments using ejaculated spermatozoa, is to add some EDTA to the incubation medium. We have observed that EDTA at concentrations of 50 – 100 mM do not interfere with DNA binding, but do effectively block the vesicular DNase activity allowing DNA incorporation in presence of diluted seminal plasma (1:10) or seminal vesicle fluid (up to 1 mg/ml protein concentration). The chelator, of
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CARBALLADA AND ESPONDA
course, should be removed before using the spermatozoa for in vitro fertilization or insemination. In summary, DNA uptake by the spermatozoa is a highly regulated phenomenon. The molecular basis underlying this process is still only partially understood and needs to be thoroughly studied. All research in this area is essential to help researchers to improve sperm-mediated transgenesis.
14.
Gagne, M. B., Pothier, F., and Sirard, M. A. (1991). Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev. 29, 6 –15.
15.
Bachiller, D., Schellander, K., Peli, J., and Ruther, U. (1991). Liposome-mediated DNA uptake by sperm cells. Mol. Reprod. Dev. 30, 194 –200.
16.
Patil, J. G., and Khoo, H. W. (1996). Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation. J. Exp. Zool. 274, 121–129.
17.
Farre, L., Rigau, T., Mogas, T., Garcia-Rocha, M., Canal, M., Gomez-Foix, A. M., and Rodriguez-Gil, J. E. (1999). Adenovirusmediated introduction of DNA into pig sperm and offspring. Mol. Reprod. Dev. 53, 149 –158.
18.
Francolini, M., Lavitrano, M., Lamia, C. L., French, D., Frati, L., Cotelli, F., and Spadafora, C. (1993). Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol. Reprod. Dev. 34, 133–139.
19.
Wu, G. M., Nose, K., Mori, E., and Mori, T. (1990). Binding of foreign DNA to mouse sperm mediated by its MHC class II structure. Am. J. Reprod. Immunol. 24, 120 –126.
20.
Lavitrano, M., Maione, B., Forte, E., Francolini, M., Sperandio, S., Testi, R., and Spadafora, C. (1997). The interaction of sperm cells with exogenous DNA: A role of CD4 and major histocompatibility complex class II molecules. Exp. Cell Res. 233, 56 – 62.
21.
Magnano, A. R., Giordano, R., Moscufo, N., Baccetti, B., and Spadafora, C. (1998). Sperm/DNA interaction: Integration of foreign DNA sequences in the mouse sperm genome. J. Reprod. Immunol. 41, 187–196.
22.
McCarthy, S., and Ward, W. S. (2000). Interaction of exogenous DNA with the nuclear matrix of live spermatozoa. Mol. Reprod. Dev. (Suppl.) 56, 235–237.
We thank Marı´a Angeles Ollacarizqueta for all the assistance in taking and processing the fluorescence images. This work was partially supported by Grant PB96-0808 from DGICYT (Spain).
REFERENCES 1.
Brackett, B. G., Baranska, W., Sawicki, W., and Koprowski, H. (1971). Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci. USA 68, 353–357.
2.
Lavitrano, M., Camaioni, A., Fazio, V. M., Dolci, S., Farace, M. G., and Spadafora, C. (1989). Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 57, 717–723.
3.
Camaioni, A., Russo, M. A., Odorisio, T., Gandolfi, F., Fazio, V. M., and Siracusa, G. (1992). Uptake of exogenous DNA by mammalian spermatozoa: Specific localization of DNA on sperm heads. J. Reprod. Fertil. 96, 203–212.
4.
Atkinson, P. W., Hines, E. R., Beaton, S., Matthaei, K. I., Reed, K. C., and Bradley, M. P. (1991). Association of exogenous DNA with cattle and insect spermatozoa in vitro. Mol. Reprod. Dev. 29, 1–5.
5.
Castro, F. O., Herna´ndez, O., Uliver, C., Solano, R., Milane´s, C., Aguilar, A., Pe´rez, A., De Armas, R., Herrera, L., and De la Fuente, J. (1991). Introduction of foreign DNA into the spermatozoa of farm animals. Theriogenology 34, 1099 –1110.
23.
Maione, B., Pittoggi, C., Achene, L., Lorenzini, R., and Spadafora, C. (1997). Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol. 16, 1087–1097.
6.
Sperandio, S., Lulli, V., Bacci, M. L., Forni, M., Maione, B., Spadafora, C., and Lavitrano, M. L. (1996). Sperm mediated DNA transfer in bovine and swine species. Anim. Biotech. 7, 59 –77.
24.
Zani, M., Lavitrano, M., French, D., Lulli, V., Maione, B., Sperandio, S., and Spadafora, C. (1995). The mechanism of binding of exogenous DNA to sperm cells: Factors controlling the DNA uptake. Exp. Cell Res. 217, 57– 64.
7.
Horan, R., Powell, R., McQuaid, S., Gannon, F., and Houghton, J. A. (1991). Association of foreign DNA with porcine spermatozoa. Arch. Androl. 26, 83–92.
25.
Balhorn, R., Gledhill, B. L., and Wyrobek, A. J. (1977). Mouse sperm chromatin proteins: Quantitative isolation and partial characterization. Biochemistry 16, 4074 – 4080.
8.
Nakanishi, A., and Iritani, A. (1993). Gene transfer in the chicken by sperm-mediated methods. Mol. Reprod. Dev. 36, 258 –261.
26.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.
9.
Habrova, V., Takac, M., Navratil, J., Macha, J., Ceskova, N., and Jonak, J. (1996). Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization. Mol. Reprod. Dev. 44, 32– 42.
27.
10.
Khoo, H. W., Ang, L. H., Lim, H. B., and Wong, K. Y. (1992). Sperm cells as vectors for introducing foreign DNA into zebrafish. Aquaculture 107, 1–19.
Kamps, M. P., and Sefton, B. M. (1988). Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2, 305– 315.
28.
Carballada, R., and Esponda, P. (1991). Electrophoretic pattern of rodent seminal vesicle proteins as revealed by silver staining. Int. J. Androl. 14, 52–57.
29.
Spadafora, C. (1998). Sperm cells and foreign DNA: A controversial relation. BioEssays 20, 955–964.
30.
Cooper, T. G. (1986). “The Epididymis, Sperm Maturation and Fertilization,” Springer-Verlag, Berlin.
31.
Bedford, J. M., and Hoskins D. D. (1992). The mammalian spermatozoon: Morphology, biochemistry and physiology. In “Marshall’s Physiology of Reproduction” (G. E. Lamming Ed.), 2nd ed., Vol. 2, Churchill Livingstone, London.
11.
Sin, F. Y., Walker, S. P., Symonds, J. E., Mukherjee, U. K., Khoo, J. G., and Sin, I. L. (2000). Electroporation of salmon sperm for gene transfer: Efficiency, reliability, and fate of transgene. Mol. Reprod. Dev. (Suppl.) 56, 285–288.
12.
Arezzo, F. (1989). Sea urchin sperm as a vector of foreign genetic information. Cell Biol. Int. Rep. 13, 391– 404.
13.
Tsai, H. J. (2000). Electroporated sperm mediation of a gene transfer system for finfish and shellfish. Mol. Reprod. Dev. (Suppl.) 56, 281–284.
DNA UPTAKE IN MOUSE SPERMATOZOA 32.
Kirchhoff, C., Pera, I., Derr, P., Yeung, C. H., and Cooper T (1997). The molecular biology of the sperm surface. Post-testicular membrane remodelling. Adv. Exp. Med. Biol. 424, 221– 232. 33. Anakwe, O. O., Sharma, S., Hoff, H. B., Hardy, D. M., and Gerton, G. L. (1991). Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains. Mol. Reprod. Dev. 29, 294 –301. 34. Kirchhoff, C., and Hale, G. (1996). Cell-to-cell transfer of glycosylphosphatidyl inositol-anchored membrane proteins during sperm maturation. Mol. Hum. Reprod. 2, 177–184. Received June 7, 2000 Revised version received September 29, 2000
113
35.
Lum, L., and Blobel, C. P. (1997). Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev. Biol. 191, 131–145.
36.
Kirchhoff, C. (1999). Gene expression in the epididymis. Int. Rev. Cytol. 188, 133–202.
37.
Tanigawa, Y., Yoshihara, K., and Koide, S. S. (1975). Endonuclease activity in bull semen, testis and accessory sex organs. Biol. Reprod. 12, 464 – 470.
38.
Mann, T., and Lutwak-Mann, C. (1981). “Male Reproductive Function and Semen,” Springer-Verlag, Berlin.