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a Istituto di Tecnologie Biomediche, CNR, 00161 Rome, Italy
b Istituto Superiore di Sanità, 00161 Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the last few years, we have studied the mechanism of interaction of sperm cells with DNA molecules of foreign origin and their subsequent delivery to the eggs during fertilization, in order to produce genetically transformed offspring ([1, 2]; reviewed in [3]). In the course of those studies, unexpected biochemical and molecular activities were depicted in spermatozoa, which revealed functions contradicting the view that sperm cells are only inactive vectors of the male genome. Our findings show that the binding of exogenous DNA to sperm cells is mediated by specific classes of proteins acting as DNA receptors on the surface of sperm cells [4], and that the binding is antagonized by glycoprotein component(s) of the seminal fluid [5]. They also indicate that a portion of the sperm-bound DNA is spontaneously internalized within nuclei and is embedded in the chromatin as determined by autoradiography on sections of spermatozoa [6]; the process of internalization is mediated by CD4 molecules present in sperm cells [7]. Finally, we have found that once the foreign DNA is internalized in sperm nuclei, it is subjected to extensive rearrangements and can be integrated into the sperm genome [8]. We have recently found that the rearrangement of exogenous DNA molecules in sperm nuclei is caused by sperm endogenous nucleases that are activated upon interaction with the foreign DNA [9]. In addition, we have observed that the activation of sperm nucleases also causes a partial degradation of the sperm chromosomal DNA, eventually leading to cell death [9]. That phenomenon closely resembles the events occurring during apoptotic degeneration of somatic cells. As previously reported in several somatic cell types [10], sperm nucleases can be blocked by the nuclease inhibitor drug aurintricarboxylic acid (ATA), thereby preserving the integrity of the sperm genome [9]. Apoptosis is not exclusively triggered by interaction with foreign DNA molecules; it can also spontaneously occur as a degenerative process, though at a much lower rate, in mature sperm cells after ejaculation or surgical removal from the epididymis.
While studying the effect of ATA on sperm cells, we observed that this drug to a certain extent preserved spermatozoa from the loss of fertilization power that usually occurs after long incubation periods and/or incubation with DNA. Here we report the results of systematic in vitro fertilization (IVF) assays in which sperm cells were preincubated with various amounts of ATA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were purchased from Charles River Italia (Calco, Italy) and were maintained at a 13L:11D light cycle. Spermatozoa for IVF were obtained from CD1 strain mice. Sperm cells were collected from the cauda epididymides of proven males that had not mated for at least 3 days but no longer than 1 wk. The sperm suspension was prepared by squeezing the terminal part of the vas deferens and puncturing the middle part of the epididymis in 1 ml of FM [11] supplemented with 4 mg/ml BSA (Fraction V; Miles, Tarrytown, NY, or Sigma, St. Louis, MO) overlaid with silicone oil (Aldrich, Milwaukee, WI). The sperm suspension was allowed to disperse by incubating the drop for 30 min at 37°C in 7% CO2 in air.
Eggs were collected from 6–9 superovulated B6D2F1 females, 3–6 wk old. Superovulation was induced by i.p. injection of 5 IU of eCG (Folligon; Intervet, Angers, France), followed by 5 additional IU of hCG (Corulon; Intervet) after 48 h. Thirteen hours after hCG injection, females were killed, and oviducts were removed and squeezed out into FM supplemented with BSA (4 mg/ml) and overlaid with silicone oil. Groups of two oviducts were squeezed into 1 ml of FM.
Aliquots of 1–2 x 106 spermatozoa were withdrawn and added to the egg-containing dishes. Dishes containing both sperm cells and eggs were incubated for 5 h. After incubation, nondegenerated eggs were transferred to dishes containing 1 ml of hypoxanthine-free Ham's F-10 medium (GIBCO BRL, Gaithersburg, MD) supplemented with NaHCO3 (2.106 g/L) and BSA (4 mg/ml) and overlaid with silicon oil.
The percentage of embryos at the 2-cell stage with respect to the total number of inseminated oocytes was routinely evaluated. To assess whether embryos obtained with ATA-pretreated sperm cells undergo a normal development, batches of 2-cell embryos were cultured in vitro for 5 days to the blastocyst stage or, in other experiments, were implanted into foster mothers to obtain born progeny.
Incubation of Spermatozoa, Oocytes, and Embryos with ATA
IVF experiments using ATA-treated sperm cells were performed essentially as described above, except that increasing concentrations of ATA (0.5–50 µM) were added to FM from the very early steps of spermatozoa isolation, i.e., squeezing and dispersal, and during the insemination of the eggs. Five hours after insemination, eggs were transferred to ATA-free Ham's F-10 medium and further cultured.
To assess whether eggs were sensitive to the effect of ATA, eggs were suspended in FM containing ATA and inseminated with sperm cells suspended in ATA-free FM. After insemination, eggs were transferred to and grown in ATA-free Ham's F-10 as described above. In other groups of experiments, IVF was performed using both spermatozoa and eggs suspended in ATA-free FM, and 5 hours after insemination eggs were transferred to and grown in Ham's F-10 containing ATA.
To rule out the possibility that ATA induced parthenogenetic activation, eggs were collected and cumulus cells were removed with hyaluronidase [12]. The cells were then washed in PBSS and transferred to FM that contained 5 µM ATA to be further cultured for 24 h. Aliquots of sperm cells and oocytes were routinely withdrawn from each individual preparation and used to run control experiments without ATA pretreatment.
Assessment of the Rate of Sperm Penetration
Sperm penetration was assessed essentially as described by Fuller and Whittingham [13] with minor modifications. Briefly, eggs were fertilized in FM with either untreated or ATA-treated spermatozoa and, after 5 hours, transferred to Ham's F-10 to remove excess sperm cells. Inseminated eggs were fixed for 15 min at room temperature in a 300-µl PBS droplet containing 2% formaldehyde (Carlo Erba, Milano, Italy) and 4 mg/ml polyvinylpyrrolidone (PVP), and subsequently washed four times in 300-µl PBS droplets. Pronuclei were stained by incubating embryos for 15 min at 37°C in 300-µl PBS droplets containing 40 µg/ml Hoechst 33258 (Sigma). This step was performed under low-intensity light. After fixation, embryos were washed four times in 300-µl PBS droplets containing 4 mg/ml PVP. Small aliquots containing roughly 5 embryos were loaded onto microscope slides carefully prewashed with acetone and allowed to stand until excess buffer was evaporated. Embryos were covered with a drop of mounting medium (Sigma), then with a cover slide. Preparations were observed under a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with an epifluorescence attachment and a 02 filter set.
DNA Uptake
Sperm cells were counted after dispersal and then were incubated with the plasmid ß-lactoglobulin (pßLG) plasmid containing the ovine ß-lactoglobulin structural gene [14], as already described [1]. Briefly, DNA uptake was routinely performed in 1 ml FM [11] using 25–30 x 106 spermatozoa and pßLG plasmid at a ratio of 0.5 µg DNA:106 spermatozoa for 30 min at 37°C in 7% CO2 in air. After incubation, aliquots of 1–2 x 106 spermatozoa were withdrawn and used for IVF.
Statistical Analysis
Confidence intervals for a binomial proportion were calculated for proportions of fertilized eggs in each treatment. Data reported in Table 6 were also compared for statistical difference using Student's t-test. A p value less than 0.05 denoted the presence of a statistically significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous study, we noticed that ATA exerted an inhibitory effect on the apoptotic response induced in mammalian spermatozoa upon interaction with foreign DNA [9]. In the course of that study, we observed that preincubation of sperm cells with ATA stimulated the production of 2-cell-stage embryos in IVF experiments as compared to parallel experiments in which untreated spermatozoa were used. That observation prompted us to undertake a systematic study with the aim of characterizing the effect of the drug in IVF assays. Mouse epididymal sperm cells were collected by squeezing cauda epididymides into FM droplets containing various concentrations of ATA and subsequently used in IVF experiments. Results are shown in Table 1: as can be seen, preincubation of spermatozoa with 1 µM and 5 µM ATA markedly increased the percentage of inseminated eggs that developed into 2-cell-stage embryos to 81.4% and 84.4%, respectively, as compared to 64.8% obtained in control experiments using spermatozoa that had not been exposed to ATA. Treatment of sperm cells with 0.5 µM ATA caused only a modest increase in 2-cell embryos (76.5%), while at 10 µM the efficiency decreased to 65.3%; 25 µM ATA caused a further reduction, and at 50 µM development was almost abolished.
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To study the effect of ATA in more detail, the drug was tested on spermatozoa whose fertilization efficiency was artificially reduced by exposure to stressful conditions. To achieve this, spermatozoa were left in FM for 2.5–4 hours after squeezing before fertilization; we have observed that this "aging" treatment considerably decreases sperm fertility and yields a low proportion of 2-cell-stage embryos in IVF experiments (unpublished observations). Table 2 summarizes the results of several experiments performed under these conditions: the overall efficiency in IVF experiments using "aged" spermatozoa dropped to 23.6%, whereas the efficiency progressively increased when aged sperm cells were preincubated with increasing concentrations of ATA immediately after isolation (see Materials and Methods). The highest efficiency, 79%, was obtained with 5 µM ATA. The effect exerted by ATA on the overall rate of embryo production was specifically observed only when sperm cells were incubated with the drug. No increased production of embryos was observed when the eggs, but not the sperm cells, were preincubated with ATA before fertilization; as shown in Table 3, a similar percentage of eggs, either untreated or preincubated with 5 µM ATA, developed into 2-cell embryos after fertilization by untreated spermatozoa. In addition, the absence of further development in cultures of unfertilized eggs exposed to ATA enabled us to rule out the possibility that ATA might cause the parthenogenetic activation of eggs (not shown).
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ATA Increased the Embryo Yield in IVF Experiments Using DNA-Loaded Sperm Cells
Earlier observations suggested that mouse eggs inseminated with epididymal sperm cells loaded with exogenous DNA show a reduced rate of 2-cell embryo production [1]. That observation offers an alternative system in which to test the effect of ATA in IVF experiments and might help to establish improved protocols for sperm-mediated gene transfer experiments. The results of these experiments are summarized in Table 4: fertilization experiments using sperm cells pretreated with 5 µM ATA and subsequently loaded with pßLG plasmid DNA yielded 66.7% of inseminated eggs that developed into 2 cell-stage embryos, compared to 26.2% obtained with untreated DNA-loaded spermatozoa. Preincubation with higher ATA concentrations, i.e., 10 µM and 25 µM, caused a lower efficiency, although still higher than that of the control.
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ATA Did Not Improve the Motility nor Increase the Rate of Fertilization of Treated Spermatozoa
In order to understand the role of ATA, it was necessary to first clarify whether the improvement of 2-cell embryo production obtained with sperm cells pretreated with ATA was the consequence of an increase in the fertilization efficiency, i.e., in the direct penetration of spermatozoa into the egg, or, alternatively, a result of an effect during subsequent steps during embryogenesis. We first checked the effect of ATA on sperm motility. Two epididymides from the same animal were separately squeezed either into basic FM or into FM containing 5 µM ATA, and sperm cells were collected and incubated for increasing lengths of time. Samples were withdrawn from the incubation mixtures at different times, and sperm motility was checked microscopically. No substantial alteration in motility was induced by exposure to ATA as assessed by direct observations under the microscope.
The efficiency of penetration of untreated and ATA-treated spermatozoa was determined in IVF experiments by staining the eggs with the Hoechst 33258 DNA dye 5–7 hours after insemination; the percentages of oocytes containing distinguishable male pronuclei, and of zygotes from the same batch that developed into 2-cell embryos, were then estimated. In brief, batches of eggs were inseminated and after 5–7 hours were divided in two subgroups: eggs in the first group were fixed with formaldehyde and stained with Hoechst 33258 to visualize pronuclei [13], while those in the second group were allowed to develop into 2-cell embryos. Observations made after that general approach were carried out under a variety of experimental conditions, i.e., using normal, aged, and DNA-loaded sperm cells; results are collectively reported in Table 5. Untreated and ATA-treated sperm cells had a similar ability (i.e., 55.6% and 58.7%, respectively) to penetrate the egg, as determined by Hoechst staining; however, the fraction of embryos developing to the 2-cell stage was significantly lower when untreated spermatozoa were used (40%), than when ATA-treated spermatozoa were used (61.2%). The latter value is comparable to that determined by staining the male pronuclei in fertilized eggs. These results lead to the conclusion that the increased yield of 2-cell embryos obtained using ATA-preincubated sperm cells in IVF experiments reflects a "protecting" effect of ATA on the first cell division in the zygote, exerted via sperm cells.
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Pretreatment of Sperm Cells with ATA Improved the Transition from Zygote to 2-Cell Embryos, but Not the Subsequent Developmental Stages
The finding that pretreatment of sperm cells with ATA improved the production of 2-cell embryos prompted us to ask whether that original effect was exerted only on the first embryonic cleavage or would also affect the later stages of the developing embryos. Eggs were fertilized with either untreated or 5 µM ATA-treated sperm cells; 2-cell embryos were then collected, transferred to ATA-free Ham's F-10 medium, and allowed to further grow to the blastocyst stage. The results of these experiments, summarized in Table 6, show that comparable percentages of 2-cell-stage embryos developed to the blastocyst stage using either untreated or 5 mM ATA-treated spermatozoa; the 6% difference observed between ATA-treated and control embryos was not significant, since the 95% confidence interval values were largely overlapping. The data were also processed by applying the t-test statistical analysis: the t value was 1.097, i.e., below the usual critical points, consistent with the hypothesis that statistically equivalent yields were obtained from the control and experimental groups. Therefore, these results indicate that the treatment of sperm cells with ATA, though clearly improving the rate of 2-cell embryo production, does not substantially affect later developmental stages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present work, we extended those observations and assessed whether, through sperm cells, ATA would also exert an effect on fertilization and/or early embryo development. The results reported here indicate an effect exerted by ATA on the early stages of embryogenesis. We found that exposure of mature sperm cells to as low a concentration of ATA as 5 µM significantly increased the rate of 2-cell embryo production in IVF assays. We cannot provide a straightforward explanation for that improving effect, yet it may be that those results reflect the anti-apoptotic action of ATA, although it is not straightforwardly correlated with the full inhibition of nucleases, which is achieved only when spermatozoa are exposed to higher concentrations [9].
The direct observation under the microscope that ATA does not improve sperm motility, together with the results in Table 5 showing that the rates of fertilization obtained with untreated and ATA-treated spermatozoa were equivalent, makes it very unlikely that the high rate of embryo production reflects an improved ability of spermatozoa to penetrate the egg cell. At least two non-mutually exclusive explanations may be envisaged to reconcile the differences observed in the effectiveness of ATA against sperm cell DNA degradation and in IVF experiments. First, it may be that low concentrations of ATA, which proved insufficient to fully inhibit sperm nuclease activities as previously revealed by analyzing sperm DNA cleavages and rearrangements [9], may still interfere with other levels of the apoptotic cascade and be effective in blocking the process and preserving the viability and functions of sperm cells. That hypothesis is substantiated by the experiments using aged and "DNA-loaded" spermatozoa: both stressful conditions clearly caused a drastic reduction of 2-cell embryo production (Tables 2 and 4) as well as triggering significant biochemical and molecular events characteristic of apoptosis [9]. Both conditions act as comparable stimuli in inducing the responses; thus, the induction of DNA nuclease activities and the reduced fertilization ability can be reasonably assumed to be mutually related, suggesting that apoptosis is one of the causes, if not the cause, of the loss of fertility of mature sperm cells. Apoptosis can spontaneously occur in freshly squeezed epididymal spermatozoa, though at a much lower rate and restricted to a small proportion of the cell population; early exposure of sperm cells to ATA presumably results in blocking or slowing the degenerative process. ATA is a very versatile molecule whose differential, dose-dependent effects are compatible with the multitargeting features described in the literature: ATA has been reported to effectively interfere with the functions of a variety of enzymes,and has long been used as a general nuclease inhibitor; more recently, it has been employed as an anti-apoptotic agent [10] because of its inhibitory effect on nucleases responsible for the nucleosomal fragmentation of chromatin [15], as well as on proteases [16]. Moreover, ATA inhibits the sealing, but not the cleaving, activity of topoisomerase II in vitro [17], and it also interferes with the function of many enzymes of the Krebs cycle [18]. Given the ability of ATA to interfere with a variety of biochemical pathways, it may be envisaged that its protective effect on sperm cells is exerted by blocking apoptosis at various steps besides the nuclease level. Such multitargeting effects may also explain why preincubation of spermatozoa with higher doses of ATA, i.e., 25 and 50 µM (see Table 1), cause, respectively, a reduction and a block in embryo development. ATA has been described to essentially act as an enzyme inhibitor; high doses of the drug are therefore likely to cause a drastic inhibition of enzyme activities required in crucial biochemical and/or molecular pathways during early embryonic development.
A second interpretation of the role of ATA is also conceivable. It is possible that ATA plays a role in favoring molecular events that follow egg penetration by the sperm nucleus, such as the sperm chromatin transition from the nucleoprotamine to the nucleohistone organization, an early-occurring process that leads to the functional reorganization of the male pronucleus before the pronuclei fusion [19] and/or early in gene expression in the zygote [20–22]; both processes are known to be stimulated by kinase activities. ATA has been reported to stimulate the protein tyrosine-kinase cascade in cultured cells [23], which also contributes to preserve the cells from apoptotic degeneration. In that context, the observation that ATA pretreatment of spermatozoa increases the rate of survival of zygotes through the first mitotic division may be relevant, and may indicate a target process whose full characterization will require further studies. Interestingly, later developmental stages appear to be insensitive to the exposure of sperm cells to ATA. We have maintained under observation for several months as many as 65 mice born after IVF experiments using spermatozoa preincubated with 5–25 µM ATA: all animals looked normal and healthy, and under no circumstance did their behavior obviously diverge from that of animals obtained with untreated spermatozoa (data not shown). In conclusion, therefore, our data depict a temporal and functional window during fertilization, following penetration of the egg cytoplasm and extending to the 2-cell stage, during which the functional state of the sperm nucleus is sensitive to the action of ATA.
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FOOTNOTES |
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2 Correspondence: Corrado Spadafora, Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Via G.B. Morgagni 30/E, 00161 Rome, Italy. FAX: 39 6 49387120; cspadaf{at}tin.it
Accepted: August 6, 1998.
Received: April 20, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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