Molecular Human Reproduction, Vol. 5, No. 9, 825-830,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of spermatogenesis |
Relationship between abnormal sperm chromatin packing and IVF results
1 Department of Molecular Radiobiology and Biophysics, St Petersburg Nuclear Physics Institute Rassian AS, 188350, Gatchina, and 2 D.O.Ott Institute Obstetric and Gynecology Rassian AMS, Mendeleyevskaya Linia 3, 199034, St Petersburg, Russia
Abstract
This study was initiated to determine the relationship between the fertilizing potential of spermatozoa and abnormalities in the compact packing of their chromatin which occurs in the final stage of male germ cell differentiation. Chromatin packing involves disulphide bridge covalent cross-linking. The degree of packing was determined from the accessibility of DNA to a fluorescent dye, ethidium bromide, following detergent treatment of the spermatozoa. The amount of dye bound was determined by flow cytometry in the presence or absence of heparin, a polyanion which removes only non-disulphide bridge-linked proteins. The results of a number of different sperm samples were compared with their results following in-vitro fertilization, and a relationship between disordered sperm chromatin packing and rates of embryo cleavage was observed. This study suggests that abnormal chromatin packing in spermatozoa may contribute to male fertility.
chromatin packing/ethidium bromide/fertility disorder/flow cytometry/human spermatozoa
Introduction
Methods of predicting a decrease in the potential of sperm fertilizing ability for the most part evaluate a reduction in sperm number, changes in sperm morphology (Kruger and Acosta, 1989
), or sperm motility (Liu and Baker, 1992), ability to adhere to the zona pellucida (Burkman et al., 1988), etc. Detrimental changes in these factors may pose an obstacle to germinal cell fusion. If all problems associated with male infertility were characterized by defects of this kind, they would be readily overcome by increasing the number of spermatozoa added to the oocyte culture medium (Oehninger et al., 1988), since the majority of sperm samples usually contain some cells that are normal in the above respects. In any case, using in-vitro fertilization (IVF), especially the microinjection of a sperm nucleus into the ovum (ICSI), could tackle all the problems. However, despite the fact that ICSI is able to completely overcome the problem of cell fusion, it frequently does not achieve fertilization and the desired pregnancy. It is, therefore, likely that disturbances in the organization of the genetic material may hinder developmental processes that occur after fusion. The quality of the sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected to fertilize an oocyte (Golan et al., 1997). In the literature there is some evidence for the existence of a correlation between disturbances in the organization of sperm nuclei and the potential fertilizing ability of spermatozoa (Bedford et al., 1973; Evenson et al., 1980; Tejada et al., 1984; Jeulin et al., 1986; Auger et al., 1990; Foresta et al., 1992).
We have attempted to develop a simple method to evaluate the intact organization of the nuclear material in spermatozoa and to test its predictive ability by comparison with IVF results. The underlying concept was that compact packing of sperm chromatin is possible only if there is no significant damage to the genetic material. It focused on the key stage of packaging, the effective cross-linking of protamines by disulphide bonds. In the presence of significant damage, compact packing in the final stage of sperm differentiation becomes impossible.
To evaluate the effectiveness of chromatin packing in sperm nuclei we used the capacity of chromatin proteins to hinder the binding to DNA of the fluorescent dye, ethidium bromide. If primary proteins are extracted from the chromatin by treatment with polyanions (heparin) leaving the disulphide bonds intact, those proteins which are not cross-linked by covalent disulphide bridges can be removed. As a result, the accessibility of DNA to ethidium bromide staining increases. Thus, binding of ethidium bromide to cell DNA can be measured by flow cytofluorometry, and can serve as a measure of the level of sperm chromatin packing. We studied this parameter in semen samples of different donors, and compared the data obtained with the outcome of IVF.
Materials and methods
Patients
A total of 176 IVF cycles in 171 unselected patients were analysed in order to determine the influence of the state of sperm chromatin on IVF outcome. Semen analysis was performed for each patient and those samples with a sperm concentration of <20x106 were classified as oligozoospermic, while those samples where <50% of spermatozoa showed forward progression were assessed as asthenozoospermic.
IVF procedure
Routine protocols for ovulation induction were used with administration of clomiphene citrate (153 IVF cycles) or gonadotrophin-releasing hormone (GnRH) agonist (23 IVF cycles) in combination with human menopausal gonadotrophin (HMG).
Oocytes were collected by ultrasound-guided transvaginal follicular aspiration ~34 h after administration of human chorionic gonadotrophin (HCG). They were cultured in Ham's F-10 medium, containing synthetic serum substitute (Medi-Cult) at 37°C in atmosphere of 5% CO2 in air. For IVF, oocytes were inseminated with 100 000 motile spermatozoa obtained by mini-Percoll gradient centrifugation.
Fertilization was assessed by the presence of pronuclei ~14–18 h after insemination and embryo development was recorded on days 2 and 3. The number of blastomeres was counted, together with the degree of fragmentation. Embryos were transferred into the uterus on days 2 or 3.
The data obtained were analysed using the following criteria: formation of two pronuclei, occurrence of cleavage, occurrence of anomalous blastomere fragmentation and pregnancy rate. IVF cycles with normal and abnormal standard sperm characteristics were analysed separately: group 1 (normozoospermia), group 2 (oligozoospermia, asthenozoospermia or oligoasthenozoospermia). Each of these groups was divided in two subgroups, according to the state of the sperm chromatin.
Ethidium bromide staining, heparin treatment and flow cytometry
An aliquot (0.2–0.5 ml) of each semen sample was washed twice in 150 mmol/l phosphate-buffered saline (PBS), pH 7.0, by centrifugation for 10 min at 300 g. The washing procedure caused no significant loss of spermatozoa, but ensured a more standardized staining technique. The resulting pellet was resuspended in the same solution to a final concentration 106 spermatozoa/ml and divided into two aliquots. Triton X-100 (final concentration 0.1%) was added to permeabilize cell membranes and to allow entry of ethidium bromide into the cell. The cells were then stained with ethidium bromide (Sigma, St Louis, MO, USA) at a concentration of 25 µg/ml. One aliquot was also mixed with heparin solution (final concentration 1 mg/ml). For one typical sperm sample, a third aliquot was treated with both heparin and 2-mercaptoethanol (final concentration 10 mmol/l).
After 60 min at room temperature, ethidium bromide-stained cells were analysed in a flow cytofluorimeter equipped with a 488 nm argon laser as the light source. This device was our version of a standard flow cytofluorimeter constructed in Petersburg Nuclear Physics Institute. A 620 nm band pass filter was used to collect the fluorescence emitted by ethidium bromide complexed to nucleic acids. Sheath and sample pressures (20 ml/min) were kept constant. The analytical rate was maintained at ~500 events/s by adjusting the flow rate of the sample.
Results
Permeabilized spermatozoa were prepared and stained with ethidium bromide and subjected to flow cytofluorometry. Figure 1 shows typical flow-cytofluorometric histograms. Heparin treatment caused the staining intensity to increase by a factor of approximately two, as illustrated in Figure 1b. Treatment with ß-mercaptoethanol and heparin was accompanied by a more than five-fold increase in staining (Figure 1c).
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When histograms from different donors were compared, it was apparent that, although the majority of the spermatozoa had densely packed nuclei (Figure 2
) which gave a narrow peak (coefficient of variance, 3–7%), a variable number of spermatozoa were represented by a tail extending toward higher staining intensities, suggesting incomplete or disordered chromatin packing. The fraction of spermatozoa exhibiting this phenomenon varied among the donors studied. Abnormally packaged spermatozoa were defined as those with a fluorescence intensity 10% greater than the average intensity of the main peak. Heparin extraction increased the level of inter-sample variation.
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Analysis of several hundred samples (data not shown) has shown that, together with the most frequently occurring normal samples for which heparin treatment increased the intensity of staining by a factor of two for the majority of cells, in many samples an appreciable number of cells were stained more intensely. Chromatin-defective sperm samples were subsequently defined as those in which >50% of heparin-treated spermatozoa exhibited enhanced staining.
The results obtained following IVF using 176 sperm samples are shown in Table I. In group 2 (oligo-, astheno-, or oligoasthenozoospermia) sperm samples with packing defects were observed more often than in group 1 (normozoospermia) (63 and 37% respectively, P < 0.05). In subgroups of normozoospermic patients with normal and abnormal sperm chromatin the fertilization rate and the proportion of cycles without fertilization were similar (66 and 62%; 7 and 10% respectively). The percentage of cleaved zygotes and embryos without fragmentation also did not differ (95 and 98%; 54 and 53% respectively) but the pregnancy rate per cycle or per embryo transfer was significantly higher in the subgroup with normal sperm chromatin packing (23 and 6%; 24 and 7% respectively, P < 0.05). Results in the group of oligozoospermia, asthenozoospermia or oligoasthenozoospermic patients (group 2) were similar to those of the normozoospermic males, but the fertilization rate was higher in the subgroup with normal sperm chromatin (61 and 33%, P < 0.05).
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Considerable differences were noted in the rates of embryo cleavage. After 24 h culture (Figure 3
), 15% of embryos developed to the >4-cell stage in the subgroup with normal sperm chromatin compared with 8% in the subgroup with sperm packing defects (P < 0.05). After 48 h culture (Figure 4), the percentage of embryos which had reached the 8-cell stage was 37 and 25% respectively in these subgroups (P < 0.05).
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) and abnormal (
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Discussion
At the final stage of spermatogenesis, chromatin reorganization takes place in the nuclei of male sex cells in which histone is replaced by protamine. During epididymal maturation of sperm S–S bonds between protamine molecules are formed and chromatin packing becomes denser and more stable. A great deal of evidence suggests that these processes are vital for male fertility, and that complete chromatin packing is essential for normal sperm functioning (Kosower et al., 1992). Thus, it has been shown that incomplete replacement of histones by protamines (Auger et al., 1990), aberrant ratios of protamine 1 to protamine 2 (Balhorne et al., 1988), or high concentrations of non-oxidized SH groups in protamine molecules are associated with male subfertility. The presence of specific mRNA in the nuclei of mature mammalian spermatozoa has led to the notion that the `sleeping' genome may not be so quiescent after all (Kramer and Krawetz, 1997), but may indicate another potential role of chromatin packaging in the mature sperm nucleus.
Although at present the influence of sperm chromatin condensation on male fertility is incompletely understood numerous groups have shown that patients with male factor infertility possess hidden anomalies in the composition of their sperm nuclei, displaying larger amounts of abnormally packaged chromatin (Evenson et al., 1980; Ibrahim et al, 1988; Foresta et al., 1992). It has been shown that semen with defective chromatin packing has a significantly lower fertilization capacity following subzonal insemination (Bianchi et al., 1996) or IVF (Auger et al., 1990). Poor chromatin packaging may also contribute to failure of sperm decondensation after ICSI (Sakkas et al., 1996) and may be responsible for slow and partial decondensation of sperm nuclei in cytoplasmic extracts from unfertilized Xenopus laevis oocytes (Griveau et al., 1992). The pregnancy rate may be influenced by the state of sperm chromatin. A low pregnancy rate was observed in cows following insemination with spermatozoa with abnormal chromatin (Sailer et al., 1996). Anomalies in sperm chromatin may also be associated with spontaneous abortion in the first trimester (Ibrahim et al., 1988).
Nuclear packaging in spermatozoa causes effective shielding of DNA by chromatin proteins, hindering the binding of fluorescent dye (Clausen et al., 1982; Xavier and Odile, 1991; Engh et al., 1992). We assessed sperm chromatin packaging quality as a function of the amount of fluorescent dye (ethidium bromide) which bound to DNA. Ethidium bromide was chosen as the DNA probe, as it is a small molecule and its mechanism of binding to DNA is relatively simple and well studied, involving mere intercalation between the nucleic acid bases with no other interactions.
It was discovered that although the majority of spermatozoa had densely packed nuclei, a variable number had nuclei with disordered packing. We defined as defective those sperm samples in which the number of abnormally-packaged spermatozoa was >30%; this was in line with a previous definition (Sakkas et al., 1996), which showed that in normal semen <30% of spermatozoa were stained by chromomycin A3. In some cases, sperm chromatin anomalies may be revealed only after additional treatment. Treating sperm nuclei with heparin appears to improve the sensitivity of the analysis of packing, as it reveals packing defects due to the reduced effectiveness of S–S bond cross-linking of shielding proteins, rather than to the lack of these proteins. Normally, the intensity of nuclei fluorescence is doubled following extraction of proteins by heparin. Semen samples were defined as abnormal when >50% of heparin-treated spermatozoa exhibited more than a doubling of staining intensity.
In this study, we attempted to determine the influence of sperm chromatin packing quality on IVF results. The fertilization rate, percentage of cleaved zygotes and embryos without fragmentation were similar in patients with normal and abnormal sperm chromatin, except that sperm samples with both morphological defects and abnormal chromatin, displayed a reduced fertilization rate. However, the pregnancy rate differed. This may be related to delayed embryo cleavage in vitro which was observed in patients with poor packing of sperm nuclei. These results suggest that there may be a mechanism which terminates development at an early stage, when the organization of genetic material in male germinal cells suffers distortion, manifesting itself in a chromatin packing disorder.
Factors responsible for disorders of chromatin packing in spermatozoa remain unclear. However the following suggestions may be made: (i) the high frequency of occurrence of these defects indicates that presumably the most common reason is associated with disruptions of spermatogenesis, resulting from some pathological or adverse environmental effects, rather than from direct hereditary genetic changes; (ii) these factors may lead either to disturbances of the maturation process and, consequently, to either an increase in the fraction of immature forms or to chromatin packing disorder in fully mature spermatozoa. Although the former of these may play a role, our results suggest the predominance of the latter, for the simple reason that immature sperm forms would be unable to participate in fertilization, whereas, according to our experiments, chromatin packing disorder correlates with developmental disturbances, rather than with a decrease in fertilization; and (iii) that disorders in sperm chromatin packaging themselves do not lead to disorders in fertility: it has been shown that ICSI using round spermatids, and even polar bodies, can produce full fertility, which means that complete chromatin packaging itself is not an absolute prerequisite (Sasagawa et al., 1998, Yanagimachi, 1998). Hence, we can assume that disorders of chromatin packaging detected in sub-fertile and infertile spermatozoa are the indirect results of a more severe disturbance of genetic material.
The relationship between the disorganization of genetic material in male sex cells and fertility disturbances is both a universal biological problem, and an important medical problem, since the existence of a mechanism rejecting during early development, embryos with deviations in the organization of their genetic material, could act as a filter to preclude severe developmental anomalies. The significance of this (at present hypothetical) mechanism in the understanding of ontogenesis and population genetics is difficult to overestimate. The medical importance of this problem results from the knowledge that the above relationship could provide a means of revealing disturbances in sperm fertility. These may be associated not only with abnormalities of sperm morphology or behaviour, which can be analysed easily (oligozoospermia, teratozoospermia, reduced adhesion to ovum envelopes, etc), but also with deviations in the organization of the genetic material.
Notes
3 To whom correspondence should be addressed 
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Submitted on April 29, 1998; accepted on March 9, 1999.
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