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Effects of dimethyl sulfone (DMSO2) on early gametogenesis in caenorhabditis elegans: ultrastructural aberrations and loss of synaptonemal complexes from pachytene nuclei
ReproductiveToxicology,Vol.6, pp. 149-159, 1992
0890-6238/92$5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the U.S.A.All rightsreserved.
EFFECTS OF DIMETHYL SULFONE (DMSO2) ON EARLY GAMETOGENESIS IN CAENORHABDITIS ELEGANS: ULTRASTRUCTURAL ABERRATIONS AND LOSS OF SYNAPTONEMAL COMPLEXES FROM PACHYTENE NUCLEI PAUL GOLDSTEIN,* LISA MAGNANO,* a n d JAVIER ROJO t *Department of Biological Sciences and tDepartment of Mathematical Sciences, University of Texas at El Paso, Texas Abstract - - The free-living nematode Caenorhabditis elegans has been used extensively for studies in developmental and reproductive genetics. Recently, toxicologic studies have been initiated using specific sex chromosome mutations. In the present study, high incidence of male (him) mutants, him-5 and him-& were treated with dimethyl sulfone (DMSO2), the primary metabolite of dimethyl sulfoxide (DMSO). In addition to differential effects on X-chromosome nondisjunction, loss of viability and fertility were observed. Much lower concentrations of DMSO2 were required to elicit the same aberrational effects characteristic of DMSO (1); thus, the toxicity of the former was significantly more potent. The observed decrease in life span was associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens could not be differentiated. Aging in oocytes at pachytene is characterized by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm, and decrease in numbers of mitochondria. Increasing concentrations of DMSO2 resulted in a corresponding decrease in fertility and increased production of abnormal gametes. At DMSO2 concentrations higher than 1.0%, synaptonemal complexes (SC) were absent from pachytene nuclei; thus, effective pairing and segregation of homologous chromosomes was prohibited. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DMSO2-treated specimens. Key Words: dimethyl sulfone; gametogenesis; synaptonemal complex; pachytene; toxicology.
(8). In mammalian cell culture, it is a significantly more potent cytotoxic drug than DMSO, and its effects are less reversible (8). It has some different properties than DMSO, for example, it is not a free-radical scavenger and will not act as a vector in the transport of drugs through the skin, although it maintains its high permeability (Jacob, personal communication, 1990). Very few studies compare the effects of a drug and its metabolite on the pairing of homologous chromosomes at the pachytene stage of meiosis. The present ultrastructural analysis was initiated to observe the effects of DMSO2 on developing gametes, specificially at the pachytene stage since it is at this time that homologous chromosomes are in the process of pairing. The data presented here, combined with our previous study on the effects of DMSO (1), indicate that treatment with DMSO, resulting in the production of DMSO2, negatively affects the process of meiosis. We have chosen the free-living nematode Caenorhabditis elegans as a biologic model because it has a short life span (20 days), rapid development (egg to adult in 3.5 days), and can be easily cultivated under
INTRODUCTION
The biologic interactions of dimethyl sulfoxide (DMSO) are not limited to changes in membrane structure and DNA. Rather, DMSO can effectively react with every type of molecular species, from enzymes through microtubules, that occur in the mammalian body. The chemical nature of DMSO, a dipolar aprotic substance, is such that it acts as the acceptor of hydrogen bonds from water, thus, the resultant H-bonds are stronger than those between water-water (3). It has high cytotoxic and mutagenic activity, leading to increased aneuploidy (4) and single-stranded breaks in DNA (5). The primary metabolite of DMSO, after oxidation, is dimethyl sulfone (DMSO2). It has been isolated from human, rabbit, rat, and other mammalian urine (6,7) and persists in the blood for five times as long as DMSO after percutaneous application in man Address correspondence to Paul Goldstein, Department of Biological Sciences, University of Texas at El Paso, E1 Paso, T X 79968. Received 8 January 1991 ; Revision received 8 July 1991 ; Accepted 12 July 1991. 149
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controlled environmental conditions. Hermaphrodites have two X chromosomes, and XO males arise via X chromosome nondisjunction. The Caenorhabditis Genetics Center maintains thousands of genetically defined mutants and coordinates the genetic map. In addition, the pachytene karyotype has been well defined (2,9) in the wild-type and him mutants, and C elegans has previously been used as a biologic model for sensitive toxicologic assay (10). MATERIALS AND M E T H O D S Nematode worms used for this study were wildtype (N2, Bristol), him-5 and him-8 hermaphrodite Caenorhabditis elegans that were cultured as previously described (11) at a constant temperature of 16 °C. The mutant him-8 has a point mutation on Linkage Group IV and produces 38% male progeny while him-5 is a point mutation on Linkage Group V and produces 16% males. The wild-type produces only 0.1% males (12). Under these conditions, the average life span was 22 days, and hermaphrodites produced an average of 320 eggs. Development of a worm is rapid: egg to adult takes 3.5 days, youth and highest fertility values are observed at 4 to 5 days old, and middle-age with decreased fertility occurs by 11 days old. Three young (5 to 7-day-old) wild-type, him-5, and him-8 hermaphrodites were placed on separate bacteria-seeded petri dishes containing varying concentrations (0.5%, 1.0%, 2.0%, and 5.0%) ofdimethyl sulfone (DMSO2; Sigma Chemicals, St Louis, Mo), and the number of offspring were counted at 4, 7, and 11 days old. The culture medium also contained M9 agar with 5 ug/mL cholesterol. These concentrations are similar to the ones used for a study of the effects of DMSO on Drosophila (13) and of DMSO2 in mammalian cell culture (8). Worms were grown in these media for a maximum of three generations, and fecundity and viability were measured. Fecundity was determined by counting the number of eggs laid per hermaphrodite. The worms were selected at intervals (4, 7, and 11 days old) and processed for either light or electron microscopy. For light microscopy, Nomarski Interference optics were utilized to study active or anesthetized worms (in 1% Tricaine). Worms were examined at varying times after being placed on the DMSO2-agar plate and, if the worms remained viable, following generations were also examined. Worms were assayed for morphologic abnormalities and for the position of the pachytene nuclei in the gonad (which is normally midway along the gonad at the curved region) (14). Increased opacity of the body cavity and
Volume 6, Number 2, 1992
differences in the organization ofpachytene nuclei in the gonad, were noted that may have related to increasing concentration of DMSO2. Worms that were treated with higher concentrations of DMSO2 (5%), were paralyzed rapidly and did not have to be anesthetized. Chemotactic response of the worms was assessed by placing a 5-#L drop of Escherichia coli at the edge of a petri dish (with varying concentrations of DMSO2) and placing 10 young worms on the opposite edge. For electron microscopy, the worms were processed as previously described (15). Statistical analyses, consisting of notched box plots, linear regression, and P values, were done using the S-plus computer program and Figures 1 through 10 were generated with this program. Notched box plots for the number of offspring versus DMSO and DMSO2 concentrations were constructed to check the distribution of the number of offspring at 4, 7, and 11 days. These plots are shown in Figures 1 through 6. The notches provide an approximate 95% test of the null hypothesis that the true medians are equal. That is, if the notches overlap, then the null hypothesis is not rejected at the 5% level. In other words, the difference between the medians could be described as statistically significant at the 5% level when the notches do not overlap. RESULTS Initiation and maintenance of the pachytene stage of meiosis are sensitive to environmental factors such as temperature and chemical concentrations (16). For example, treatment with colchicine prior to pachytene results in the disruption of synaptonemal complexes at pachytene (16). In the present study, C elegans were grown in varying concentrations of
O "a
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Fig. 1. Distribution of offspring as a function of DMSO concentration after 4 days.
Effects of DMSO2 on garnetogenesis • P. GOLDSTEIN ET AL.
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DMSOz. Since it has been shown that DMSO also has a colchicine-like effect on tissues (17), it follows that its first metabolite would have a similar effect. Although disruption of the meiotic chromosome segregation mechanism in the organism may occur after treatment with either DMSO or DMSO2, the concentrations at which such an inhibitory response would be first observed could be significantly different. With increasing concentration of DMSO2, there were corresponding nuclear and chromosomal changes.
Effects of varying concentrationsof DMS02: Fecundity and viability Increasing concentrations of DMSO2 had a drastic effect on the viability and fecundity of treated worms. Worms grown in 0.5% DMSO2 were essentially normal and produced normal quantities of eggs. At 1.0% DMSOE, there was a marked decrease in the
0.5 1 2 DMSO2 Concentration
5
Fig. 4. Distribution of offspring as a function of DMSO2 concentration after 7 days.
number of offspring with loss of viability near day 4, and certainly by day 11 of incubation (Figures 1 through 6). Thus, DMSO2 has a larger adverse effect than DMSO at concentrations greater than 1.0%. After 7 days, DMSO had an effect on the population when compared with the control group (see Figure 3), although no statistically significant difference exists between the 0.5% and the 1.0% concentrations. Similarly, Figure 4 shows the strong (statistically significant) effect that DMSO2 had on population size. When compared with the DMSO effect, it was clear that DMSO2 had a stronger effect at the lower concentrations. At 11 days (see Figures 5 and 6) a pattern similar to that at 7 days (see Figures 3 and 4) was observed; and namely, DMSO2 had a stronger adverse effect on the population size than DMSO. It is important to note that Figures l, 3, and 5, show no statistically significant differences between 0.5% and 1.O%.
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Fig. 3. Distribution of offspring as a function of DMSO concentration after 7 days.
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Fig. 5. Distribution of offspring as a function of DMSO concentration after I l days.
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Volume6, Number 2, 1992
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Fig. 6. Distribution of offspring as a function of DMSO2 after 11 days. To study the relationship between number of offspring and time of incubation, at fixed concentrations, regressions were performed at 0.0%, 0.5%, 1.0%, and 2.0% concentrations of DMSO and DMSO2. Figures 7 through 10 show the regression lines for these data. As Figure 7 shows, at 0.0% concentration (the control), the population seems to grow linearly as a function of time (R 2 = 0.9708, slope = 160.82, P = 0). Figure 8 shows that at 0.5%, the linear fit is strong for both DMSO and DMSO2 (R 2 = 0.93 and R 2 = 0.937, respectively). One may conclude from Figure 8 that a 0.5% concentration of either chemical still allows the population to grow over time, although growth is at a slower rate (DMSO, slope = 57.44, P = 0; DMSO2, slope = 56.97, P = 0) than that of the control group. At a concentration of 1.0%, however, the behavior of the size of the population, as a function of time, is quite different for DMSO and DMSO2 (Figure 9). While the size of the population increases for DMSO (slope = 57.98, P = 0), it decreases for DMSO2 (slope = - 24.94, p = 0). The R 2 values are 0.841 and 0.812, respectively. Finally, for 2.0% concentrations (Figure 10), the size of the population increased for DMSO (R 2 = 0.916), while it decreased for DMSO2. The rate of decrease for population treated with 2.0% DMSO2 (slope = 11.30) was lower than at the 1.0% concentration, although, as shown by Figure 2, the median of the number of offspring at 4 days is statistically significantly higher at 1.0% compared with 2.0%. A marked delay in development was noted in worms treated with 2% DMSO2, such that the normal development time from egg to adult of 3.5 days was increased to 10 to 15 days. This apparently represented a delay in development as the life span was increased proportionally to a mean of 27 days. Worms treated in 5.0% DMSO2 all died within one hour.
4
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Fig. 7. Regression lines for the number of offspring versus time--0.0% concentration.
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Fig. 8. Regression lines for the separate and combined chemicals; number of offspring versus time--0.5% concentration.
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Fig. 9. Regression lines for the separate and combined chemicals; number of offspring versus time--l.0% concentration.
Effects o f DMSO2 on gametogenesis • P. GOLDSTEIN ET AL. 0
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Fig. 10. Regression lines for the separate and combined chemicals; number of offspring versus time--2.0% concentration.
Wild-type hermaphrodite A brief review of the wild-type morphology is presented to permit comparison with DMSO~-associated aberrations. In the C elegans wild-type hermaphrodite, the oocyte pachytene nuclei are arranged peripherally around a central rachis (Figure 1 la) (15). Each oocyte is connected to the noncellular, nonnutritive rachis via a cytoplasmic bridge that accounts for the observed synchrony of all pachytene nuclei in that restricted area of the gonad. The peripheral arrangement of the nuclei is characteristic only of the pachytene stage, and at all other stages the meiotic nuclei are present in a honeycomb pattern (18). Within the pachytene nucleus, a single, large, nucleolus is present, which is not fragmented and has no nucleonemata (Figure 11a). Synaptonemal complexes (SC) are present between homologously paired chromosomes (Figure 11 b) and SC-associated structures, termed "Disjunction Regulator Regions" (DRR), are attached to the SC through the lateral or central element (9,19-21).
Light microscopy observations DMSO2 has little effect on the worms at a concentration of 0.5%. Opacity of the body, muscular control and behavioral responses (chemotaxis, touch sensitivity) were all normal. At concentrations of 1.0% and 2.0%, significant changes were noted in the worms, including loss of movement. In 5% DMSO2, 75% of body movement was lost after 10 min of treatment, resulting in paralysis. All pharyngeal and vulval movements were absent within 15 min. The body cavity was only 50% translucent and death occurred within 15 min after exposure. In addition, the organization of the gonad was altered such that pachytene
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nuclei could not be observed in the curve of the gonad, or anywhere else. From these light microscopic observations, inhibition and possible destruction of muscle tissue, or inhibition of neurons might explain the paralysis of the worms. However, these structrues were normal after ultrastructural analysis (see Figure 13b).The control study for nematode chemotactic response, without DMSO2 added to the agar, showed that within 6 h all the worms had migrated directly to the food source. This was also true for worms treated with 0.5 and 1% DMSO2. However, worms treated with 2% DMSO2 showed impaired chemotactic response such that after 24 h only half the worms had moved to the nutrient source. In both of the two high incidence of male mutants used in this study, him-5 and him-8, the amount of X-chromosome nondisjunction, as measured by production of males, was decreased signifcantly, over 90% in him-8 and over 80% in him-5, compared with complete inhibition in DMSO whereby no males were produced(1).
Electron microscopic observations Treatment of C elegans with 0.5% DMSOz had little or no effect on the ultrastructure of the worm, gonad, or pachytene nuclei and associated structures. The nuclei retained their peripheral arrangement around the central rachis and the synaptonemal complex (SC) was tripartite, consisting of two lateral and one central element (Figure 11c). However, SC-associated structures, termed "Disjunction Regulator Regions" (DRR) (9) (not shown here) were not present, although they were present at lower treatment levels of DMSO (1). The nuclear envelope appeared bipartite and was comprised of well-defined inner and outer membranes that were contiguous with the cytoplasm (Figure 11c). Within each pachytene nucleus there was one large nucleolus that showed characteristic fibrillar and granular regions (Figure 1 It). All other bodily structures and tissues appeared normal. With increasing concentrations of DMSOz, changes were observed in the gonad and pachytene nuclei. Worms grown in 1.0% DMSO2 manifested changes in gonadal structure, particularly in the distribution of the nuclei around the central rachis, which itself was compromised (Figure 12a). The nucleus was also affected such that synaptonemal complexes (and assocated DRRs) were absent and the chromatin was slightly disorganized (Figures 12b and 12c). The nuclear envelope was not always continguous with the cytoplasm, and the ultrastructure of the nucleolus was essentially normal (Figure 12b). Worms grown in 2.0% DMSO2 lacked a wellformed central rachis, and the relationship of the nu-
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Fig. 1 l.a. The wild-type hermaphrodite of the nematode Caenorhabditis elegans contains a gonad in which the pachytene nuclei (N) are arranged peripherally around a central rachis (R). Tripartite synaptonemal complexes (bracket in panel b) contain two lateral elements and a striated central element (C). Worms treated with 0.5% DMSO2 have identical morphology of the gonad, and the synaptonemal complexes are normal (panel c). Nucleolus (Nu). Nuclear envelope (NE). Intestine (I). Muscle (M). Bar equals 0.5, 0.1, and 0.2 um, respectively.
clei was compromised (Figure 13a). There were no SCs in the nuclei, the chromatin was not completely organized, and the nucleoplasm appeared extremely dense (Figure 13c), similar to that of senescent cells. The nucleolus was more condensed than normal. In addition, manifestation o f senescence was indicated by decreasing numbers of mitochondria. The nuclear envelope was not always contiguous with the cytoplasm (similar to Figure 12b), which may represent loss of integrity o f the outer nuclear m e m b r a n e with
a subsequent decrease in nucleocytoplasmic interaction. These are not fixation artifacts, since the changes in the nucleus can be observed in vivo with the light microscope. Although the worms showed poor chemotropic response to food and were paralyzed after exposure to DMSO2, the muscle structure was not affected (Figure 13b). DMSO2 concentrations as high as 5% were extremely toxic to C elegans. Death started to occur within 15 min o f exposure, and after I h all the worms
Effectsof DMSO2on gametogenesis• P. GOLDSTE|NETAL.
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Fig. 12.a. The peripheral arrangement of nuclei (N) around the rachis (star) was compromised in nematodes treated with 1.0% DMSO2 b,c. The chromatin (CH) within the nucleus was slightly disorganized, synaptonemal complexes were absent, and the nuclear envelope (NE) was not always contiguous with the cytoplasm (arrow). Nucleolus (Nu). Intestine (I). Cuticle (C). Bar equals 0.5, 0.2, and 0.2 um, respectively.
had died. Severe structural aberrations were also apparent such that the worms appeared to be burst apart, that is, the intestine was bloated, the muscles were torn, the cuticle stressed, etc. There was no obvious structure to the gonad at pachytene, and the nuclei contained tightly condensed, apparently inactive,
nucleoli. Synaptonemal complexes were not present, and the chromatin was decondensed (Figures 14a and b). The nuclear envelope was usually not contiguous with the cytoplasm (Figure 20). The morphology of all these structures were identical to those from worms treated with 10% D M S O (Figure 14c).
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Fig. 13.a. Significant morphologic changes were apparent in worms treated with 2.0% DMSO2. Peripheral arrangement of nuclei around the poorly formed rachis was lost. b. Although worms treated with this concentration of DMSO2 were paralyzed within a short time and showed poor chemotropic response to food, movement of the worm was not related to muscle (M) structure as this was normal, c. Chromatin (CH) was not completely organized, and synaptonemal complexes were absent. Nuclear envelope (NE). Cuticle (C). Intestine (I). Bar equals 0.5, 0.2, and 0.2 urn, respectively.
DISCUSSION
Toxicity of DMSO compared with DMSOz Organisms developing in the presence of DMSO2 exhibit greater abnormalities, under less concentration of DMSO2, than those that are grown in DMSO. Thus, 5% DMSO2 elicits the same response as 10% D M S O but takes a little longer (1). The two chemicals have different properties that m a y account for this ob-
servation: 1) D M S O (CH3SOCH3) is a free-radical scavenger while DMSO2 is not, as evidenced by its chemical structure (CH3802CH3) (Jacob, personal c o m m u n i c a t i o n , 1990). Perhaps some of the effects attributed to D M S O are ameliorated since it can scavenge the hydroxyl radical. On the other hand, any m e m b r a n e interaction involving DMSO2 releases free radicals that can not be immediately sequestered; 2) DMSO2 persists in the blood five times longer than
Effects of DMSO2 on gametogenesis • P. GOLDSTEIN ET AL.
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Fig. 14. Treatment of Caenorhabdit&eleganswith 5.0% DMSO2 resulted in paralysis and death for most of the worms within 15 min. b. The chromatin (star) was characteristic of interphase, and synaptonemal complexes were not present (panels a and b). The nucleoli (Nu) were tightly condensed and apparently inactive. The nuclear envelope (NE) usually was not contiguous with the cytoplasm (arrow heads), c. Severe structural aberrations were apparent and identical to those worms treated with 10.0% DMSO. The intestine (I) was bloated (also see Figure 13a) and the entire organization of the gonad (in brackets) was disrupted. Nucleus (N). Mitochondria (m). Bar equals 0.2, 0.2, and 0.5 um, respectively.
DMSO (8); 3) D M S O 2 is a more potent inhibitor of cell growth than DMSO, and its growth inhibition is less reversible than that produced by DMSO (8); and 4) DMSO penetrates membranes more quickly than DMSO2, indicating a difference in membrane interaction. In addition, DMSO can be used as an agent in transferring dissolved drugs through membranes, whereas DMSO2 apparently lacks this ability (Jacobs, personal communication).
Effects of DMS02 on synaptonemal complexes The toxicity of DMSO2 on gametogenesis is evidenced by its effect on the production of synaptonemal complexes (SC) during pachytene such that SCs are not present in worms that have been exposed to DMSO2 concentrations exceeding 0.5%. In addition, the response of worms to elevated levels of DMSO2 is drastic: alterations in chromosome structure, loss of movement and chemotactic response, and failure of chromosomes to pair during meiosis with resultant segregational problems. It has been suggested that one
of the functions of the SC is the regulation of the segregation of homologous chromosomes after recombination. In the event an SC is not formed between two homologous chromosomes, aneuploid gametes would be produced (22,23). Thus without the proper machinery for effective meiotic pairing and segregation, normal gametes cannot be produced, and fecundity and viability will be decreased. The inhibition of SC formation can be understood from two different viewpoints; l) Formation of an SC is highly regulated and demands specific protein-DNA interactions (16). Since DMSO, and probably DMSO2 as well, interact with DNA and protein configuration (5), the formation of the SC would thus be compromised; and 2) DMSO, and probably DMSO2, act in a similar way as colchicine in the disruption of microtubules and interfere with the morphology of actin-containing structures (17,24,25). Thus, absence of SCs may also be the result of these colchicine-like effects of D M S O 2 , since it has been shown that cells subjected to colchicine at premeiotic
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interphase have greatly decreased levels of recombination (16). The SCs of C elegans are very short (15), so the colchicine effect may have an overwhelming influence on the behavior of the meiotic chromosomes.
Volume 6, Number 2, 1992 Acknowh'dgmenls - - We thank Dr. S.W. Jacob for his discussions. This project was supported by NIH Grant No. SO6 GM08012-21 to P.G and J.R. Nematodes used for this study were obtained from the Caenorhabditis Genetics Center.
REFERENCES
Effects on nuclear structure The pachytene cells in wild-type hermaphrodite C elegans have a unique peripheral arrangement around a central rachis in the gonad (18). There are gaps (bridges) that connect the cell membrane with the rachis, thus, accounting for the physical basis of synchronous development of all pachytene cells in the gonad (14,26). In other regions of the gonad, where the rachis is not present, different stages in meiosis may occur adjacent to each other. Within these cytoplasmic bridges, microtubules (MT) are present, however, in DMSO2-treated specimens, the MTs and rachis are partially disintegrated, which may account for the observed loss of synchrony. Nucleolar changes were also observed after treatment with 2% or greater concentrations of DMSO2. The nucleolus in C elegans occupies greater than 40% of the nuclear volume (12) and fibrillar and granular regions are clearly delineated. With increasing levels of DMSO2, the nucleolus became tightly condensed such that fibrillar and granular regions were not observed. This may affect the production of ribosomal RNA precursors (27) and result in reduced viability.
Relationship of DMSO: and X-chromosome nondisjunction After exposure to DMSO2, males (XO) were not observed in any of the thousands of offspring examined from wild-type, thus indicating an interaction with the X chromosome (since males are produced as a result of X-chromosome nondisjunction). The him8 mutation does not produce any male offspring after being treated with DMSO ( 1); however, under the influence of DMSO2, there is only a 90% inhibition. This is also true of the him-5 mutation, but not to the same degree. The response of the X-chromosome may be different in the two mutant forms. In him-5, autosomal nondisjunction occurs at the same rate as X-chromosome nondisjucntion (12), so the point mutation responsible for the mutant may affect the segregational properties of all the chromosomes. This would account for the lower viability and fecundity observed in this study. Production of males in him-8 would be more severely affected since the X-chromosome is specifically involved (12). Thus, DMSO2 directly influences the expression of these point mutations, most probably via interaction with chromosomal proteins and D N A (28).
1. Goldstein P, Magnano L. Effects ofdimethyl sulfoxide on early gametogenesis in Caenorhabditis elegans: ultrastructural aberrations and loss ofsynaptonemal complexes from pachytene nuclei. Cytobios. 1988;56:45-57. 2. Goldstein P, Curis M. Age-related changes in the meiotic chrom o s o m e s of the nematode Caenorhabditis elegans. Mech Ageing Develop. 1987;40:115-30. 3. Kharasch N, Thyagarajan B. Structural basis for biological activities of dimethylsulfoxide. Ann NY Acad Sci. 1983;411:392-402. 4. Fulton AM, Bond DJ. Dimethylsulfoxide induces aneuploidy in a fungal test system. Mol Gen Genet. 1984;197:347-9. 5. Walles SA, Erixon K. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique. Carcinogenesis. 1984:5:319-23. 6. Williams IH, Burstein S, Layne D. Metabolism of dimethyl sulfoxide, and dimethyl sulfone in the rabbit. Arch Biochem Biophys. 1966;117:84-7. 7. Hucker H, Hoffman E. A new method for assay of radioactive dimethyl sulfoxide and its metabolite, dimethyl sulfone. Experientia. 1966;12:855-6. 8. Layman DL. Growth inhibitory effects ofdimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro. In Vitro Cell Devel Biol. 1987;23:422-8. 9. Goldstein P. The synaptonemal complexes of Caenorhabditis eh,gans: pachytene karyotype analysis of the Dp 1 mutant and Disjunction Regulator Regions. Chromosoma. 1985;93:17782. 10. Goldstein P. Nuclear aberrations and loss of synaptonemal complexes in response to diethylstilbestrol (DES) in Caenorhabditis elegans hermaphrodites. Mutat Res. 1986;174:99107. 11. Brenner S. The genetics of Caenorhabditis eh,gans. Genetics. 1974;77:71-94. 12. Hodgkin JA, Horvitz H, Brenner S. Nondisjunction mutants of Caenorhabditis eh,gans. Genetics. 1979;91:67-94. 13. Traut H. The solvent dimethylsufoxide (DMSO) does not induce aneuploidy in oocytes of Drosophila melanogaster. Environ Mutagen. 1983;5:273-7. 14. Hirsch D, Oppenheim D, Klass M. Development of the reproductive system of Caenorhabditis eh,gans. Dev Biol. 1976;49:200-19. 15. Goldstein P, Slaton DE. The synaptonemal complexes of Caenorhabditis elegans: comparison of wild-type and mutant strains and pachytene karyotype analysis of wild-type. Chromosoma. 1982;84:585-90. 16. Moens PB. The onset of meiosis. In: Cell biology. New York: Academic Press; 1977:93-108. 17. Vanni GL, Poli F. Binucleation and abnormal chromosome distribution in Euglena gracilis cells treated with dimethylsulfoxide. Protoplasm. 1983;114:62-6. 18, Goldstein P. The synaptonemal complexes of Caenorhabditis eh,gans: pachytene karyotype analysis of male and hermaphrodite wild-type and him mutants. Chromosoma. 1982;86:577-93. 19. Goldstein P. Triplo-X hermaphrodite of Caenorhabditis elegans: pachytene karyotype analysis, synaptonemal complexes and pairing mechanisms. Can J Genet Cytol. 1984;26:13-17. 20. Goldstein P. Sterile mutants in Caenorhabditis elegans: the synaptonemal complex as an indicator of the stage-specific effect of the mutation. Cytobios. 1984;39:101-8. 21. Goldstein P. The synaptonemal complexes of Caenorhabditis
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elegans: pachytene karyotype analysis of the rad-4 radiation sensitive mutant. Mutat Res. 1984;129:337-43. 22. Goldstein P. Aneuploidy in the normal life cycle of the nematode Caenorhabditis elegans. In: Vig B, Sanberg A, eds. Aneuploidy, Part A: Incidence and etiology. New York: A.R. Liss; 1987:189-204. 23. Maguire M. Evidence for a role of the synaptonemal complex in provision for normal disjunction at meiosis II in maize. Chromosoma. 1982;84:675-86. 24. Robinson J, Engelborghs Y. Tubulin polymerization in dimethylsulfoxide. J Biol Chem. 1982;257:5367-71.
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25. Sanger JW, Sanger JM, Kreis T, Jockusch B. Reversible translocation of cytoplasmic actin into the nucleus caused by dimethylsulfoxide. Proc Natl Acad Sci USA. 1980;77:5268-72. 26. Starck J. Radioaudiographic study of RNA synthesis in Caenorhabditis elegans oogenesis. Biol Cellulaire. 1977;30:181-2. 27. Goessens G. Nucleolar structure. Int Rev Cytol. 1984;87:10758. 28. Reboulleau C, Shapiro H. Chemical inducers of differentiation cause conformational changes in chromatic and deoxyribonucleic acid of murine erythroleukemia cells. Biochemistry. 1983;22:4512-17.
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EFFECTS OF DIMETHYL SULFONE (DMSO2) ON EARLY GAMETOGENESIS IN CAENORHABDITIS ELEGANS: ULTRASTRUCTURAL ABERRATIONS AND LOSS OF SYNAPTONEMAL COMPLEXES FROM PACHYTENE NUCLEI PAUL GOLDSTEIN,* LISA MAGNANO,* a n d JAVIER ROJO t *Department of Biological Sciences and tDepartment of Mathematical Sciences, University of Texas at El Paso, Texas Abstract - - The free-living nematode Caenorhabditis elegans has been used extensively for studies in developmental and reproductive genetics. Recently, toxicologic studies have been initiated using specific sex chromosome mutations. In the present study, high incidence of male (him) mutants, him-5 and him-& were treated with dimethyl sulfone (DMSO2), the primary metabolite of dimethyl sulfoxide (DMSO). In addition to differential effects on X-chromosome nondisjunction, loss of viability and fertility were observed. Much lower concentrations of DMSO2 were required to elicit the same aberrational effects characteristic of DMSO (1); thus, the toxicity of the former was significantly more potent. The observed decrease in life span was associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens could not be differentiated. Aging in oocytes at pachytene is characterized by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm, and decrease in numbers of mitochondria. Increasing concentrations of DMSO2 resulted in a corresponding decrease in fertility and increased production of abnormal gametes. At DMSO2 concentrations higher than 1.0%, synaptonemal complexes (SC) were absent from pachytene nuclei; thus, effective pairing and segregation of homologous chromosomes was prohibited. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DMSO2-treated specimens. Key Words: dimethyl sulfone; gametogenesis; synaptonemal complex; pachytene; toxicology.
(8). In mammalian cell culture, it is a significantly more potent cytotoxic drug than DMSO, and its effects are less reversible (8). It has some different properties than DMSO, for example, it is not a free-radical scavenger and will not act as a vector in the transport of drugs through the skin, although it maintains its high permeability (Jacob, personal communication, 1990). Very few studies compare the effects of a drug and its metabolite on the pairing of homologous chromosomes at the pachytene stage of meiosis. The present ultrastructural analysis was initiated to observe the effects of DMSO2 on developing gametes, specificially at the pachytene stage since it is at this time that homologous chromosomes are in the process of pairing. The data presented here, combined with our previous study on the effects of DMSO (1), indicate that treatment with DMSO, resulting in the production of DMSO2, negatively affects the process of meiosis. We have chosen the free-living nematode Caenorhabditis elegans as a biologic model because it has a short life span (20 days), rapid development (egg to adult in 3.5 days), and can be easily cultivated under
INTRODUCTION
The biologic interactions of dimethyl sulfoxide (DMSO) are not limited to changes in membrane structure and DNA. Rather, DMSO can effectively react with every type of molecular species, from enzymes through microtubules, that occur in the mammalian body. The chemical nature of DMSO, a dipolar aprotic substance, is such that it acts as the acceptor of hydrogen bonds from water, thus, the resultant H-bonds are stronger than those between water-water (3). It has high cytotoxic and mutagenic activity, leading to increased aneuploidy (4) and single-stranded breaks in DNA (5). The primary metabolite of DMSO, after oxidation, is dimethyl sulfone (DMSO2). It has been isolated from human, rabbit, rat, and other mammalian urine (6,7) and persists in the blood for five times as long as DMSO after percutaneous application in man Address correspondence to Paul Goldstein, Department of Biological Sciences, University of Texas at El Paso, E1 Paso, T X 79968. Received 8 January 1991 ; Revision received 8 July 1991 ; Accepted 12 July 1991. 149
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controlled environmental conditions. Hermaphrodites have two X chromosomes, and XO males arise via X chromosome nondisjunction. The Caenorhabditis Genetics Center maintains thousands of genetically defined mutants and coordinates the genetic map. In addition, the pachytene karyotype has been well defined (2,9) in the wild-type and him mutants, and C elegans has previously been used as a biologic model for sensitive toxicologic assay (10). MATERIALS AND M E T H O D S Nematode worms used for this study were wildtype (N2, Bristol), him-5 and him-8 hermaphrodite Caenorhabditis elegans that were cultured as previously described (11) at a constant temperature of 16 °C. The mutant him-8 has a point mutation on Linkage Group IV and produces 38% male progeny while him-5 is a point mutation on Linkage Group V and produces 16% males. The wild-type produces only 0.1% males (12). Under these conditions, the average life span was 22 days, and hermaphrodites produced an average of 320 eggs. Development of a worm is rapid: egg to adult takes 3.5 days, youth and highest fertility values are observed at 4 to 5 days old, and middle-age with decreased fertility occurs by 11 days old. Three young (5 to 7-day-old) wild-type, him-5, and him-8 hermaphrodites were placed on separate bacteria-seeded petri dishes containing varying concentrations (0.5%, 1.0%, 2.0%, and 5.0%) ofdimethyl sulfone (DMSO2; Sigma Chemicals, St Louis, Mo), and the number of offspring were counted at 4, 7, and 11 days old. The culture medium also contained M9 agar with 5 ug/mL cholesterol. These concentrations are similar to the ones used for a study of the effects of DMSO on Drosophila (13) and of DMSO2 in mammalian cell culture (8). Worms were grown in these media for a maximum of three generations, and fecundity and viability were measured. Fecundity was determined by counting the number of eggs laid per hermaphrodite. The worms were selected at intervals (4, 7, and 11 days old) and processed for either light or electron microscopy. For light microscopy, Nomarski Interference optics were utilized to study active or anesthetized worms (in 1% Tricaine). Worms were examined at varying times after being placed on the DMSO2-agar plate and, if the worms remained viable, following generations were also examined. Worms were assayed for morphologic abnormalities and for the position of the pachytene nuclei in the gonad (which is normally midway along the gonad at the curved region) (14). Increased opacity of the body cavity and
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differences in the organization ofpachytene nuclei in the gonad, were noted that may have related to increasing concentration of DMSO2. Worms that were treated with higher concentrations of DMSO2 (5%), were paralyzed rapidly and did not have to be anesthetized. Chemotactic response of the worms was assessed by placing a 5-#L drop of Escherichia coli at the edge of a petri dish (with varying concentrations of DMSO2) and placing 10 young worms on the opposite edge. For electron microscopy, the worms were processed as previously described (15). Statistical analyses, consisting of notched box plots, linear regression, and P values, were done using the S-plus computer program and Figures 1 through 10 were generated with this program. Notched box plots for the number of offspring versus DMSO and DMSO2 concentrations were constructed to check the distribution of the number of offspring at 4, 7, and 11 days. These plots are shown in Figures 1 through 6. The notches provide an approximate 95% test of the null hypothesis that the true medians are equal. That is, if the notches overlap, then the null hypothesis is not rejected at the 5% level. In other words, the difference between the medians could be described as statistically significant at the 5% level when the notches do not overlap. RESULTS Initiation and maintenance of the pachytene stage of meiosis are sensitive to environmental factors such as temperature and chemical concentrations (16). For example, treatment with colchicine prior to pachytene results in the disruption of synaptonemal complexes at pachytene (16). In the present study, C elegans were grown in varying concentrations of
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Fig. 1. Distribution of offspring as a function of DMSO concentration after 4 days.
Effects of DMSO2 on garnetogenesis • P. GOLDSTEIN ET AL.
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DMSOz. Since it has been shown that DMSO also has a colchicine-like effect on tissues (17), it follows that its first metabolite would have a similar effect. Although disruption of the meiotic chromosome segregation mechanism in the organism may occur after treatment with either DMSO or DMSO2, the concentrations at which such an inhibitory response would be first observed could be significantly different. With increasing concentration of DMSO2, there were corresponding nuclear and chromosomal changes.
Effects of varying concentrationsof DMS02: Fecundity and viability Increasing concentrations of DMSO2 had a drastic effect on the viability and fecundity of treated worms. Worms grown in 0.5% DMSO2 were essentially normal and produced normal quantities of eggs. At 1.0% DMSOE, there was a marked decrease in the
0.5 1 2 DMSO2 Concentration
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Fig. 4. Distribution of offspring as a function of DMSO2 concentration after 7 days.
number of offspring with loss of viability near day 4, and certainly by day 11 of incubation (Figures 1 through 6). Thus, DMSO2 has a larger adverse effect than DMSO at concentrations greater than 1.0%. After 7 days, DMSO had an effect on the population when compared with the control group (see Figure 3), although no statistically significant difference exists between the 0.5% and the 1.0% concentrations. Similarly, Figure 4 shows the strong (statistically significant) effect that DMSO2 had on population size. When compared with the DMSO effect, it was clear that DMSO2 had a stronger effect at the lower concentrations. At 11 days (see Figures 5 and 6) a pattern similar to that at 7 days (see Figures 3 and 4) was observed; and namely, DMSO2 had a stronger adverse effect on the population size than DMSO. It is important to note that Figures l, 3, and 5, show no statistically significant differences between 0.5% and 1.O%.
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Fig. 3. Distribution of offspring as a function of DMSO concentration after 7 days.
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Fig. 5. Distribution of offspring as a function of DMSO concentration after I l days.
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Fig. 6. Distribution of offspring as a function of DMSO2 after 11 days. To study the relationship between number of offspring and time of incubation, at fixed concentrations, regressions were performed at 0.0%, 0.5%, 1.0%, and 2.0% concentrations of DMSO and DMSO2. Figures 7 through 10 show the regression lines for these data. As Figure 7 shows, at 0.0% concentration (the control), the population seems to grow linearly as a function of time (R 2 = 0.9708, slope = 160.82, P = 0). Figure 8 shows that at 0.5%, the linear fit is strong for both DMSO and DMSO2 (R 2 = 0.93 and R 2 = 0.937, respectively). One may conclude from Figure 8 that a 0.5% concentration of either chemical still allows the population to grow over time, although growth is at a slower rate (DMSO, slope = 57.44, P = 0; DMSO2, slope = 56.97, P = 0) than that of the control group. At a concentration of 1.0%, however, the behavior of the size of the population, as a function of time, is quite different for DMSO and DMSO2 (Figure 9). While the size of the population increases for DMSO (slope = 57.98, P = 0), it decreases for DMSO2 (slope = - 24.94, p = 0). The R 2 values are 0.841 and 0.812, respectively. Finally, for 2.0% concentrations (Figure 10), the size of the population increased for DMSO (R 2 = 0.916), while it decreased for DMSO2. The rate of decrease for population treated with 2.0% DMSO2 (slope = 11.30) was lower than at the 1.0% concentration, although, as shown by Figure 2, the median of the number of offspring at 4 days is statistically significantly higher at 1.0% compared with 2.0%. A marked delay in development was noted in worms treated with 2% DMSO2, such that the normal development time from egg to adult of 3.5 days was increased to 10 to 15 days. This apparently represented a delay in development as the life span was increased proportionally to a mean of 27 days. Worms treated in 5.0% DMSO2 all died within one hour.
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Fig. 7. Regression lines for the number of offspring versus time--0.0% concentration.
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Effects o f DMSO2 on gametogenesis • P. GOLDSTEIN ET AL. 0
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Wild-type hermaphrodite A brief review of the wild-type morphology is presented to permit comparison with DMSO~-associated aberrations. In the C elegans wild-type hermaphrodite, the oocyte pachytene nuclei are arranged peripherally around a central rachis (Figure 1 la) (15). Each oocyte is connected to the noncellular, nonnutritive rachis via a cytoplasmic bridge that accounts for the observed synchrony of all pachytene nuclei in that restricted area of the gonad. The peripheral arrangement of the nuclei is characteristic only of the pachytene stage, and at all other stages the meiotic nuclei are present in a honeycomb pattern (18). Within the pachytene nucleus, a single, large, nucleolus is present, which is not fragmented and has no nucleonemata (Figure 11a). Synaptonemal complexes (SC) are present between homologously paired chromosomes (Figure 11 b) and SC-associated structures, termed "Disjunction Regulator Regions" (DRR), are attached to the SC through the lateral or central element (9,19-21).
Light microscopy observations DMSO2 has little effect on the worms at a concentration of 0.5%. Opacity of the body, muscular control and behavioral responses (chemotaxis, touch sensitivity) were all normal. At concentrations of 1.0% and 2.0%, significant changes were noted in the worms, including loss of movement. In 5% DMSO2, 75% of body movement was lost after 10 min of treatment, resulting in paralysis. All pharyngeal and vulval movements were absent within 15 min. The body cavity was only 50% translucent and death occurred within 15 min after exposure. In addition, the organization of the gonad was altered such that pachytene
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nuclei could not be observed in the curve of the gonad, or anywhere else. From these light microscopic observations, inhibition and possible destruction of muscle tissue, or inhibition of neurons might explain the paralysis of the worms. However, these structrues were normal after ultrastructural analysis (see Figure 13b).The control study for nematode chemotactic response, without DMSO2 added to the agar, showed that within 6 h all the worms had migrated directly to the food source. This was also true for worms treated with 0.5 and 1% DMSO2. However, worms treated with 2% DMSO2 showed impaired chemotactic response such that after 24 h only half the worms had moved to the nutrient source. In both of the two high incidence of male mutants used in this study, him-5 and him-8, the amount of X-chromosome nondisjunction, as measured by production of males, was decreased signifcantly, over 90% in him-8 and over 80% in him-5, compared with complete inhibition in DMSO whereby no males were produced(1).
Electron microscopic observations Treatment of C elegans with 0.5% DMSOz had little or no effect on the ultrastructure of the worm, gonad, or pachytene nuclei and associated structures. The nuclei retained their peripheral arrangement around the central rachis and the synaptonemal complex (SC) was tripartite, consisting of two lateral and one central element (Figure 11c). However, SC-associated structures, termed "Disjunction Regulator Regions" (DRR) (9) (not shown here) were not present, although they were present at lower treatment levels of DMSO (1). The nuclear envelope appeared bipartite and was comprised of well-defined inner and outer membranes that were contiguous with the cytoplasm (Figure 11c). Within each pachytene nucleus there was one large nucleolus that showed characteristic fibrillar and granular regions (Figure 1 It). All other bodily structures and tissues appeared normal. With increasing concentrations of DMSOz, changes were observed in the gonad and pachytene nuclei. Worms grown in 1.0% DMSO2 manifested changes in gonadal structure, particularly in the distribution of the nuclei around the central rachis, which itself was compromised (Figure 12a). The nucleus was also affected such that synaptonemal complexes (and assocated DRRs) were absent and the chromatin was slightly disorganized (Figures 12b and 12c). The nuclear envelope was not always continguous with the cytoplasm, and the ultrastructure of the nucleolus was essentially normal (Figure 12b). Worms grown in 2.0% DMSO2 lacked a wellformed central rachis, and the relationship of the nu-
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Fig. 1 l.a. The wild-type hermaphrodite of the nematode Caenorhabditis elegans contains a gonad in which the pachytene nuclei (N) are arranged peripherally around a central rachis (R). Tripartite synaptonemal complexes (bracket in panel b) contain two lateral elements and a striated central element (C). Worms treated with 0.5% DMSO2 have identical morphology of the gonad, and the synaptonemal complexes are normal (panel c). Nucleolus (Nu). Nuclear envelope (NE). Intestine (I). Muscle (M). Bar equals 0.5, 0.1, and 0.2 um, respectively.
clei was compromised (Figure 13a). There were no SCs in the nuclei, the chromatin was not completely organized, and the nucleoplasm appeared extremely dense (Figure 13c), similar to that of senescent cells. The nucleolus was more condensed than normal. In addition, manifestation o f senescence was indicated by decreasing numbers of mitochondria. The nuclear envelope was not always contiguous with the cytoplasm (similar to Figure 12b), which may represent loss of integrity o f the outer nuclear m e m b r a n e with
a subsequent decrease in nucleocytoplasmic interaction. These are not fixation artifacts, since the changes in the nucleus can be observed in vivo with the light microscope. Although the worms showed poor chemotropic response to food and were paralyzed after exposure to DMSO2, the muscle structure was not affected (Figure 13b). DMSO2 concentrations as high as 5% were extremely toxic to C elegans. Death started to occur within 15 min o f exposure, and after I h all the worms
Effectsof DMSO2on gametogenesis• P. GOLDSTE|NETAL.
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Fig. 12.a. The peripheral arrangement of nuclei (N) around the rachis (star) was compromised in nematodes treated with 1.0% DMSO2 b,c. The chromatin (CH) within the nucleus was slightly disorganized, synaptonemal complexes were absent, and the nuclear envelope (NE) was not always contiguous with the cytoplasm (arrow). Nucleolus (Nu). Intestine (I). Cuticle (C). Bar equals 0.5, 0.2, and 0.2 um, respectively.
had died. Severe structural aberrations were also apparent such that the worms appeared to be burst apart, that is, the intestine was bloated, the muscles were torn, the cuticle stressed, etc. There was no obvious structure to the gonad at pachytene, and the nuclei contained tightly condensed, apparently inactive,
nucleoli. Synaptonemal complexes were not present, and the chromatin was decondensed (Figures 14a and b). The nuclear envelope was usually not contiguous with the cytoplasm (Figure 20). The morphology of all these structures were identical to those from worms treated with 10% D M S O (Figure 14c).
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Fig. 13.a. Significant morphologic changes were apparent in worms treated with 2.0% DMSO2. Peripheral arrangement of nuclei around the poorly formed rachis was lost. b. Although worms treated with this concentration of DMSO2 were paralyzed within a short time and showed poor chemotropic response to food, movement of the worm was not related to muscle (M) structure as this was normal, c. Chromatin (CH) was not completely organized, and synaptonemal complexes were absent. Nuclear envelope (NE). Cuticle (C). Intestine (I). Bar equals 0.5, 0.2, and 0.2 urn, respectively.
DISCUSSION
Toxicity of DMSO compared with DMSOz Organisms developing in the presence of DMSO2 exhibit greater abnormalities, under less concentration of DMSO2, than those that are grown in DMSO. Thus, 5% DMSO2 elicits the same response as 10% D M S O but takes a little longer (1). The two chemicals have different properties that m a y account for this ob-
servation: 1) D M S O (CH3SOCH3) is a free-radical scavenger while DMSO2 is not, as evidenced by its chemical structure (CH3802CH3) (Jacob, personal c o m m u n i c a t i o n , 1990). Perhaps some of the effects attributed to D M S O are ameliorated since it can scavenge the hydroxyl radical. On the other hand, any m e m b r a n e interaction involving DMSO2 releases free radicals that can not be immediately sequestered; 2) DMSO2 persists in the blood five times longer than
Effects of DMSO2 on gametogenesis • P. GOLDSTEIN ET AL.
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Fig. 14. Treatment of Caenorhabdit&eleganswith 5.0% DMSO2 resulted in paralysis and death for most of the worms within 15 min. b. The chromatin (star) was characteristic of interphase, and synaptonemal complexes were not present (panels a and b). The nucleoli (Nu) were tightly condensed and apparently inactive. The nuclear envelope (NE) usually was not contiguous with the cytoplasm (arrow heads), c. Severe structural aberrations were apparent and identical to those worms treated with 10.0% DMSO. The intestine (I) was bloated (also see Figure 13a) and the entire organization of the gonad (in brackets) was disrupted. Nucleus (N). Mitochondria (m). Bar equals 0.2, 0.2, and 0.5 um, respectively.
DMSO (8); 3) D M S O 2 is a more potent inhibitor of cell growth than DMSO, and its growth inhibition is less reversible than that produced by DMSO (8); and 4) DMSO penetrates membranes more quickly than DMSO2, indicating a difference in membrane interaction. In addition, DMSO can be used as an agent in transferring dissolved drugs through membranes, whereas DMSO2 apparently lacks this ability (Jacobs, personal communication).
Effects of DMS02 on synaptonemal complexes The toxicity of DMSO2 on gametogenesis is evidenced by its effect on the production of synaptonemal complexes (SC) during pachytene such that SCs are not present in worms that have been exposed to DMSO2 concentrations exceeding 0.5%. In addition, the response of worms to elevated levels of DMSO2 is drastic: alterations in chromosome structure, loss of movement and chemotactic response, and failure of chromosomes to pair during meiosis with resultant segregational problems. It has been suggested that one
of the functions of the SC is the regulation of the segregation of homologous chromosomes after recombination. In the event an SC is not formed between two homologous chromosomes, aneuploid gametes would be produced (22,23). Thus without the proper machinery for effective meiotic pairing and segregation, normal gametes cannot be produced, and fecundity and viability will be decreased. The inhibition of SC formation can be understood from two different viewpoints; l) Formation of an SC is highly regulated and demands specific protein-DNA interactions (16). Since DMSO, and probably DMSO2 as well, interact with DNA and protein configuration (5), the formation of the SC would thus be compromised; and 2) DMSO, and probably DMSO2, act in a similar way as colchicine in the disruption of microtubules and interfere with the morphology of actin-containing structures (17,24,25). Thus, absence of SCs may also be the result of these colchicine-like effects of D M S O 2 , since it has been shown that cells subjected to colchicine at premeiotic
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interphase have greatly decreased levels of recombination (16). The SCs of C elegans are very short (15), so the colchicine effect may have an overwhelming influence on the behavior of the meiotic chromosomes.
Volume 6, Number 2, 1992 Acknowh'dgmenls - - We thank Dr. S.W. Jacob for his discussions. This project was supported by NIH Grant No. SO6 GM08012-21 to P.G and J.R. Nematodes used for this study were obtained from the Caenorhabditis Genetics Center.
REFERENCES
Effects on nuclear structure The pachytene cells in wild-type hermaphrodite C elegans have a unique peripheral arrangement around a central rachis in the gonad (18). There are gaps (bridges) that connect the cell membrane with the rachis, thus, accounting for the physical basis of synchronous development of all pachytene cells in the gonad (14,26). In other regions of the gonad, where the rachis is not present, different stages in meiosis may occur adjacent to each other. Within these cytoplasmic bridges, microtubules (MT) are present, however, in DMSO2-treated specimens, the MTs and rachis are partially disintegrated, which may account for the observed loss of synchrony. Nucleolar changes were also observed after treatment with 2% or greater concentrations of DMSO2. The nucleolus in C elegans occupies greater than 40% of the nuclear volume (12) and fibrillar and granular regions are clearly delineated. With increasing levels of DMSO2, the nucleolus became tightly condensed such that fibrillar and granular regions were not observed. This may affect the production of ribosomal RNA precursors (27) and result in reduced viability.
Relationship of DMSO: and X-chromosome nondisjunction After exposure to DMSO2, males (XO) were not observed in any of the thousands of offspring examined from wild-type, thus indicating an interaction with the X chromosome (since males are produced as a result of X-chromosome nondisjunction). The him8 mutation does not produce any male offspring after being treated with DMSO ( 1); however, under the influence of DMSO2, there is only a 90% inhibition. This is also true of the him-5 mutation, but not to the same degree. The response of the X-chromosome may be different in the two mutant forms. In him-5, autosomal nondisjunction occurs at the same rate as X-chromosome nondisjucntion (12), so the point mutation responsible for the mutant may affect the segregational properties of all the chromosomes. This would account for the lower viability and fecundity observed in this study. Production of males in him-8 would be more severely affected since the X-chromosome is specifically involved (12). Thus, DMSO2 directly influences the expression of these point mutations, most probably via interaction with chromosomal proteins and D N A (28).
1. Goldstein P, Magnano L. Effects ofdimethyl sulfoxide on early gametogenesis in Caenorhabditis elegans: ultrastructural aberrations and loss ofsynaptonemal complexes from pachytene nuclei. Cytobios. 1988;56:45-57. 2. Goldstein P, Curis M. Age-related changes in the meiotic chrom o s o m e s of the nematode Caenorhabditis elegans. Mech Ageing Develop. 1987;40:115-30. 3. Kharasch N, Thyagarajan B. Structural basis for biological activities of dimethylsulfoxide. Ann NY Acad Sci. 1983;411:392-402. 4. Fulton AM, Bond DJ. Dimethylsulfoxide induces aneuploidy in a fungal test system. Mol Gen Genet. 1984;197:347-9. 5. Walles SA, Erixon K. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique. Carcinogenesis. 1984:5:319-23. 6. Williams IH, Burstein S, Layne D. Metabolism of dimethyl sulfoxide, and dimethyl sulfone in the rabbit. Arch Biochem Biophys. 1966;117:84-7. 7. Hucker H, Hoffman E. A new method for assay of radioactive dimethyl sulfoxide and its metabolite, dimethyl sulfone. Experientia. 1966;12:855-6. 8. Layman DL. Growth inhibitory effects ofdimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro. In Vitro Cell Devel Biol. 1987;23:422-8. 9. Goldstein P. The synaptonemal complexes of Caenorhabditis eh,gans: pachytene karyotype analysis of the Dp 1 mutant and Disjunction Regulator Regions. Chromosoma. 1985;93:17782. 10. Goldstein P. Nuclear aberrations and loss of synaptonemal complexes in response to diethylstilbestrol (DES) in Caenorhabditis elegans hermaphrodites. Mutat Res. 1986;174:99107. 11. Brenner S. The genetics of Caenorhabditis eh,gans. Genetics. 1974;77:71-94. 12. Hodgkin JA, Horvitz H, Brenner S. Nondisjunction mutants of Caenorhabditis eh,gans. Genetics. 1979;91:67-94. 13. Traut H. The solvent dimethylsufoxide (DMSO) does not induce aneuploidy in oocytes of Drosophila melanogaster. Environ Mutagen. 1983;5:273-7. 14. Hirsch D, Oppenheim D, Klass M. Development of the reproductive system of Caenorhabditis eh,gans. Dev Biol. 1976;49:200-19. 15. Goldstein P, Slaton DE. The synaptonemal complexes of Caenorhabditis elegans: comparison of wild-type and mutant strains and pachytene karyotype analysis of wild-type. Chromosoma. 1982;84:585-90. 16. Moens PB. The onset of meiosis. In: Cell biology. New York: Academic Press; 1977:93-108. 17. Vanni GL, Poli F. Binucleation and abnormal chromosome distribution in Euglena gracilis cells treated with dimethylsulfoxide. Protoplasm. 1983;114:62-6. 18, Goldstein P. The synaptonemal complexes of Caenorhabditis eh,gans: pachytene karyotype analysis of male and hermaphrodite wild-type and him mutants. Chromosoma. 1982;86:577-93. 19. Goldstein P. Triplo-X hermaphrodite of Caenorhabditis elegans: pachytene karyotype analysis, synaptonemal complexes and pairing mechanisms. Can J Genet Cytol. 1984;26:13-17. 20. Goldstein P. Sterile mutants in Caenorhabditis elegans: the synaptonemal complex as an indicator of the stage-specific effect of the mutation. Cytobios. 1984;39:101-8. 21. Goldstein P. The synaptonemal complexes of Caenorhabditis
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elegans: pachytene karyotype analysis of the rad-4 radiation sensitive mutant. Mutat Res. 1984;129:337-43. 22. Goldstein P. Aneuploidy in the normal life cycle of the nematode Caenorhabditis elegans. In: Vig B, Sanberg A, eds. Aneuploidy, Part A: Incidence and etiology. New York: A.R. Liss; 1987:189-204. 23. Maguire M. Evidence for a role of the synaptonemal complex in provision for normal disjunction at meiosis II in maize. Chromosoma. 1982;84:675-86. 24. Robinson J, Engelborghs Y. Tubulin polymerization in dimethylsulfoxide. J Biol Chem. 1982;257:5367-71.
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25. Sanger JW, Sanger JM, Kreis T, Jockusch B. Reversible translocation of cytoplasmic actin into the nucleus caused by dimethylsulfoxide. Proc Natl Acad Sci USA. 1980;77:5268-72. 26. Starck J. Radioaudiographic study of RNA synthesis in Caenorhabditis elegans oogenesis. Biol Cellulaire. 1977;30:181-2. 27. Goessens G. Nucleolar structure. Int Rev Cytol. 1984;87:10758. 28. Reboulleau C, Shapiro H. Chemical inducers of differentiation cause conformational changes in chromatic and deoxyribonucleic acid of murine erythroleukemia cells. Biochemistry. 1983;22:4512-17.