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a Hofmann Center for Reproductive Studies, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania 19348
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels.
Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The compaction of the spermatozoal chromatin can be divided into two processes, condensation and stabilization [2]. The deposition of protamines is temporally associated with condensation of spermatozoal chromatin during spermiogenesis, whereas disulfide bonding involving protamine molecules is primarily responsible for the stabilization of spermatozoal chromatin in the epididymis [3]. Protamines are deposited on DNA during the spermatid stage, when the nucleus changes from a round, less compact form to an elongated compact form [4]. While histones are also present in the mature sperm, they probably do not have a role in condensation, but they may play a role in gene regulation after fertilization [5]. During stabilization, intramolecular and intermolecular disulfide bonds are formed in the epididymis between cysteine residues of protamine molecules.
The SCSA was introduced by Evenson et al. [6] to determine the in vitro susceptibility to denaturation of spermatozoal DNA. In addition, the flow cytometer has been applied to monitor the effects of chemotherapy [7, 8] and potential environmental toxicants on spermatogenesis, and to evaluate the head shape of mammalian sperm [9] and changes in spermatogenic cell populations in the testes [10–13]. The SCSA has also been used to determine the relationship between the susceptibility of the DNA to denaturation and fertility in cattle [14], humans and mice [6], and stallions [15, 16].
The objectives of this study were 1) to create a stallion model of heat stress to the testes and epididymides by insulating the scrotum of experimental animals to produce sufficient short-term heat stress to induce varying degrees of susceptibility to denaturation as determined by the SCSA; and 2) to measure protamine and disulfide bond levels from samples used for objective 1 and compare the results with the degree of denaturability as measured by the SCSA to determine whether either or both are related to an increased susceptibility of the DNA in chromatin to denaturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The testes of four pony stallions were heat-stressed by insulating the scrotum with a layer of wool next to the scrotal skin. The wool was covered by a plastic sheet to prevent air circulation and create an air-tight environment around the scrotum. Both of these layers were covered by a sling-jock strap apparatus that elevated the testes in the inguinal area. The sling was suspended by three ropes. One went between the hind legs; the other two went up either flank region and were tied over the dorsal midline. This apparatus was kept in place for 48 h [17]. Forty-eight hours was used as the time period after preliminary work using 36 h as the time of insulation resulted in no alterations in spermatozoal chromatin structure posttreatment. The temperature of the scrotal skin was monitored by a temperature probe (Electromedics Inc., Denver, CO). The animals were tied to limit movement and to minimize displacement of the apparatus.
Semen Collection
Before insulation of the testes, the ejaculated semen was collected and evaluated to determine baseline normal (control) values. The pretreatment evaluation consisted of 6 consecutive days of once-daily semen collections to stabilize extra gonadal sperm reserves.
After the 48-h heat treatment, semen was collected once daily from Days 1 to 64 after the start of treatment to cover one complete spermatogenic cycle.
The semen was aliquoted into plastic Eppendorf (Hamburg, Germany) tubes, which were frozen in a -70°C freezer, immediately after collection, for subsequent evaluation by sperm chromatin structure assay, gel electrophoresis, and sulphydryl quantitation.
Sperm Chromatin Structure Assay
This assay was performed as previously described [18]. Semen samples were collected, and immediately frozen and stored in either liquid nitrogen or in a -70°C freezer until analysis. The spermatozoal samples were handled individually and thawed in a 35–37°C water bath. The sample was covered with aluminum foil, placed in the flow cytometer (FACScan; Becton Dickinson, Mountain View, CA), and allowed to pass through the tubing for 2 min before evaluation of the cells. A cell flow rate of 100–200 cells/sec was used, and a total of 5000 sperm were evaluated for each sample. The flow cytometer was adjusted so that the mean green fluorescence was set at 500 channels (Fl-1 @ 500) and mean red fluorescence at 150 channels (Fl-3 @ 150) fluorescence. This results in a scattergram, schematically similar to Figure 1. Each dot represents the amount of green and red fluorescence emitted by an individual sperm. Sperm to the right of this main population represent those sperm that have an increase in the amount of red fluorescence and a decreased amount of green fluorescence when compared to those sperm in the main population.
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Data were acquired in list-mode and translated by Oswego software (Oswego Software Inc., Oswego, IL) from Hewlett-Packard- to PC-compatible files. Quantitation of DNA denaturation is determined by the term alpha-t [12], which is defined as the ratio of red/red + green fluorescence and which was measured for each sperm cell represented in the scattergram (Fig. 1). Alpha-t values were calculated for individual sperm using the red and green fluorescence values represented in the scattergram (Fig. 1) using Listview software (Phoenix Flow Systems, San Diego, CA). These alpha-t values were then represented as a histogram (Fig. 2). The mean of alpha-t (Mean
t) reflects the degree of DNA denaturation of the whole spermatozoal population. The SDt indicates the extent of denaturation or how far individual sperm deviate from the main population. Percent COMPt is the percentage of cells outside the main population, determined by selecting those cells to the right of the main population (Fig. 2), and represents the number of cells outside the main population as a percentage of the total number of sperm evaluated.
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Disulfide Bond Quantitation
Monobromobimane (mBBr; Thiolyte, cat. #596105, Calbiochem, San Diego, CA) was used as the fluorescent label (excitation between 360–400 nm; emission at wavelengths > 440–450 nm) that is specific for thiol groups. This nonradioactive label easily penetrates live-intact cells at a physiological pH at room temperature, binds specifically with thiol groups, and results in a complex that is biochemically stable until excited [19].
The sperm samples were thawed at room temperature, and 30 x 106 sperm were combined into with PBS, pH 7.4 (cat. #1000-3, Sigma, St. Louis, MO) for a total of 1.0 ml. The sample was centrifuged (Eppendorf microcentrifuge #5415 C) for 10 min at 600 x g (3000 rpm). The pellet was saved, and 1.0 ml of a 10 mM N-ethylmaleimide-PBS solution was added to block reactive thiols. This was incubated for 30 min at room temperature and washed twice in PBS. The pellet was combined with dithiothreitol (1 mM) to break the remaining disulfide bonds and incubated for 10 min at room temperature; it was then washed twice in PBS, and the pellet was resuspended to 1.0 ml in PBS. The mBBr was added to a final concentration of 0.5 mM (10 µl mBBr stock solution/ml of resuspension). The mBBr stock solution was prepared as a 50 mM solution (14 mg mBBr/ml acetonitrile) as previously described [20]. This solution was incubated for 10 min at room temperature and washed twice in PBS. Total fluorescence of the sample was determined by fluorimetry using the TKO 100 (Hoefer Scientific Instruments, San Francisco, CA).
The changes in all the variables measured were compared to a scale based on the time period, in days, for the formation of the different cell types in spermatogenesis—specifically the spermatogonia, early spermatocytes, late spermatocytes, and spermatids—and the epididymal transit time [21].
Means and standard errors for all measures on all stallions (n = 4) for each day were determined. Pearson's correlations were determined between the SCSA values (Meant, SDt, COMPt) and the fluorescent measures for each individual stallion as well as the mean values for each day.
Protamine Isolation and Gel Electrophoresis
A technique described previously by Swierstra et al. [22] was used. To standardize the number of sperm involved in the extraction process, 200 x 106 sperm were used. This was done in an effort to determine quantitative (absolute) as well as qualitative (relative) changes in nucleoprotein content.
The spermatozoal protein was dissolved in acetic acid, 2-mercaptoethanol, and sucrose and was analyzed by electrophoresis on acid-urea gels.
The nucleoproteins of 20 different spermatozoal samples were isolated from each stallion. The samples were chosen to represent the different plateaus of denaturability as determined by the SCSA as described in the Results section.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this report (Figs. 3 and 4), the transition of spermatogenic cell types (spermatogonia, primary spermatocytes, spermatids) through the spermatogenesis are used as a reference point to which the changes in spermatozoal values are compared.
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A spermatogenic cycle in the stallion is approximately 37 days, not including the spermatogonia stage, and is followed by epididymal transit, which is approximately 9 days [21]. The mean values for all features of the four stallions are given in Figure 3, while a representative individual stallion value is presented in Figure 4.
A representative scattergram from one stallion shows the changes that occur in SCSA throughout the treatment period (Fig. 5). This sequence shows an increase in the number of sperm to the right of the main population, which resulted in an increase in the SCSA values for this stallion (Fig. 4).
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The spermatozoal chromatin structure values for Meant, SDt, and COMPt each consistently had three consecutive plateaus for each of the stallions. The first rise and plateau was a small, abrupt rise in SCSA values at approximately Day 10 post-heat treatment, which lasted until Day 24; this timing implies that the cell was in the spermatid stage at the time of the heat stress (Figs. 3 and 4). While the day of the initial rise varied between individuals, it was consistent within a range of 1–2 days. This initial rise was followed by a more pronounced second rise at approximately Day 24, lasting until Day 33, which corresponds with the late primary spermatocyte stage, at which time there was a drop for several days and then a third rise in the SCSA values from Day 38 to Day 46, which implies that the cell was in the early primary spermatocyte stage at the time of the heat stress. There was individual stallion variation with respect to the day of onset and the magnitude of the changes, but the overall pattern was consistent and repeatable.
The correlations between the mean values for the SCSA variables and fluorescence measurements were similar, ranging from -0.31 to -0.37 (p < 0.05) for Meant and COMPt, respectively. These r values, however, tended to be higher within individual stallions, with the Meant having the highest correlations (-0.46 to -0.59).
Acid-urea gel electrophoresis of isolated spermatozoal nucleoproteins showed no relative change (ratio) in the amount of protamines (P1 and P2) or histone content (Fig. 6). While some lanes show variation in protein line density, there was no temporal trend that would support an association with changes in SCSA variables or disulfide bonding. Conclusions about absolute amounts of nucleoprotein could not be inferred because of the between gel-line density variation. In addition, there was no change in protein migration that could be interpreted as conformational alterations in the pattern or retention of protamine precursors.
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The between-animal variation in response to the heat stress was primarily confined to the magnitude of the response to the heat stress, but the temporal changes of all variables occurred in a very consistent pattern with respect to time and length of occurrence after treatment (Fig. 4) as described for the mean values (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The level of disulfide bonding was inversely correlated with the susceptibility of the chromatin to denaturation as determined by the SCSA variables, such that when the level of disulfide bonding is lowest, the chromatin is most easily denatured. Therefore, as the number of disulfide bonds between protamine molecules decreases, the chromatin becomes more susceptible to denaturation.
Even though disulfide bonding between and within protamine molecules occurs in the epididymis, it appears that the elevated scrotal temperature does not alter the formation of these bonds in sperm residing in the epididymis at the time of the scrotal insulation, since the chromatin stability of sperm collected 1–10 days after heat exposure was not altered. Rather, those sperm most susceptible to denaturation would have been at the primary spermatocyte stage of development at the time of the heat stress. This is particularly interesting since only histones, not protamines, are associated with the DNA at this stage of development, well in advance of the synthesis and deposition of protamines. Thus, neither the sulphydryl groups nor their substrate present in the mature sperm were present at the time of thermal heat stress. This leads to the suggestion that the production of a factor(s) in the primary spermatocyte, which is involved in the formation of protamine configuration or disulfide bonds later in spermiogenesis or during epididymal transit, has been stopped or altered, resulting in a decrease in the formation of disulfide bonds and less stable chromatin, rather than the breakage of disulfide bonds in the epididymis following their normal formation. Alternatively, the quantity of free sulfhydryls may be similar between the control and heat-stressed samples, but the condensation status of the chromatin is reduced such that DNA is more easily accessed by the mBBr.
Relative amounts (ratio) of nucleoproteins (P1, P2, and histones) in the spermatozoal chromatin did not change in this particular model at any day after treatment (Fig. 6). This does not rule out, but renders unlikely, a proportional decrease in the amounts of all the nucleoproteins. Even though an attempt was made to quantitate absolute amounts of nucleoproteins by standardizing the amount of sperm included in the extraction process, the results contained considerable variation within the pretreatment samples, and therefore meaningful conclusions about posttreatment samples are precluded.
There was no change in the migration pattern of the protamines and histones, suggesting that there was no alteration in the biochemical structure of these proteins that would alter the overall charge of the protein molecule and result in a different migration pattern. Thus, it appears that, biochemically, the nucleoproteins were correctly formed during spermiogenesis, and they were formed in the correct amounts and deposited in the nucleus. However, it appears that once the protamines are in the nucleus, they lack the potential to form a full complement of disulfide bonds, resulting in an increase of susceptibility of the chromatin to denaturation.
These results are different from those of previous workers, who reported changes in the P1/P2 ratios in humans [24], an increase in the histone fraction of the nucleoprotein extract [25], or the appearance of completely different proteins [26]. These workers did not rule out the possibility that the presence of shed spermatogenic cells or leukocytes, commonly present in the human ejaculate, could have contributed different proteins, such as histones, or altered the P1/P2 ratio. In the present study, the contribution of these cell types was minimal (0–4%). It is also possible that the temperature elevation of 2–3°C (from 34–37°C) in the present study was too low to evoke a change in the type or amount of nucleoprotein. It has been determined in vitro that heat-shock proteins are produced in primary spermatocytes exposed to 44°C temperatures for as little as 10 min [27]. The human testis is more likely than other species to be exposed to this type of stress due to disease (pyrexia) or recreational activities (hot tubs, etc.). This long-term stress to the testis may invoke alterations in nucleoprotein composition that short-term stresses do not. In addition, the chromatin of human sperm is more susceptible to denaturation than is that of other species; thus, even subtle stresses may have a more dramatic effect on fertility. It is important to emphasize that the brief heat stress used induced a decrease in disulfide bonding and chromatin stability, yet protamine synthesis and deposition remained intact.
The greatest susceptibility to denaturation occurred in the late primary spermatocytes (pachytene stage of prophase I), resulting in the second plateau, which lasted for 12 days. The third plateau was 8–10 days, was intermediate in severity, involved early primary spermatocytes in the leptotene and zygotene stages of prophase I, and marked the tail end of DNA synthesis, resulting in cells with a tetraploid DNA content. Alteration of the high metabolic activity during this time could result in changes that affect chromatin formation in a later cell type. The increased susceptibility to denaturation at this stage is probably related to the fact that the transition from the zygotene to the pachytene stage is known to be particularly susceptible to stress, although the mechanism for this susceptibility is not understood [28]. The initial and lowest plateau occurred just before sperm entered the epididymis and lasted for 15 days, which corresponds approximately to the development time of the round (9 days) and elongated (10 days) spermatids. The period from Days 9 to 16 post-heat treatment (elongated spermatid) showed a very consistent rise in denaturation, while Days 17–24 post-heat treatment (round spermatid) had more variation in the range of data points. The consistency of the first days may reflect increased resistance of the chromatin resulting from condensation and formation of the shape of the spermatozoal head, while the increase in variation that followed may represent a less resistant chromatin state in the round spermatid.
The dramatic decrease in the Meant during Days 38–39 post-heat treatment involved cells in the transition from early to late primary spermatocytes. This transition involves a change in the chromatin from an eccentric location (probably more dense packing) to a more centralized dispersed chromatin pattern, possibly making it more susceptible to heat stress. The 2-day period between these two plateaus may reflect a particularly resistant chromatin structure.
In this study, consistent changes in spermatozoal nuclear stability following mild heat stress to the testes could be identified when sperm were collected on a daily basis. The most distinct changes were the plateaus for the SCSA values that were induced by heat stress when the cells were at specific stages and occurred at time intervals corresponding to stages of the spermatogenic cycle. This indicated that the spermatozoal chromatin varies in its susceptibility to heat stress depending on the spermatogenic cell type affected at the time of the stress. This time-dependent variation in susceptibility allowed the use of heat stress to model degrees of altered spermatozoal chromatin. On the basis of these varying degrees of chromatin stability, it was possible to demonstrate that decreases in disulfide bonding, but not protamine content, accompanied abnormal SCSA results. Neither the types of protamine nor their structure as revealed on gels was altered regardless of the susceptibility of the chromatin to denaturation.
While epididymides were also subject to the temperature elevation in this study, no effect of the heat stress was evident on sperm exposed during epididymal transit (0–10 days posttreatment; Figs. 3 and 4). This indicates that while disulfide bonds are formed in the epididymis, the events responsible for the decrease in the numbers of disulfide bonds occurred before their entering the epididymis. These results also support the contention that protamines protect the chromatin of mature sperm from heat stress-related DNA damage.
An increased susceptibility to denaturation of spermatozoal DNA has been associated with decreased fertility in men [6] and bulls [14]. A possible mechanism for this reduced fertility could involve changes in the level of the intra- and intermolecular disulfide bonds in the protamine molecules. These levels may be critical during the decondensation process immediately following fertilization, at which time the spermatozoal chromatin is decondensed within the milieu of the oocyte cytoplasm. This process is dependent upon the reduction of disulfide bonds [29]. In addition, the timing of decondensation is dependent on the number of disulfide bonds within the spermatozoal nucleus [30], and this timing may be related to the type of protamine present [31]. The decondensation process is believed to be necessary for the male and female genomes to combine and form the zygote [32]. Whether the changes in disulfide bonding induced by elevated scrotal temperatures in this study affects fertility remains to be determined.
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FOOTNOTES |
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Accepted: October 13, 1998.
Received: March 16, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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