Experimental Biology Online - EBO ISSN 1430-3418

Exp. Biol. Online 2:12 (1997)

Nucleotide pool changes in coelomic cells (eleocytes) of the polychaete Nereis virens during sexual maturation U. Hoeger and C. Märker Institut für Zoologie, Universität Mainz, Saarstr. 21, D-55099 Mainz, Germany Correspondence to: U. Hoeger (email: [email protected]) Received: 23 June 1997 / Accepted: 19 August 1997 Abstract. Eleocytes (a type of coelomic cell) of the polychaete Nereis virens can store large amounts of adenine nucleotides at certain times. Since eleocytes have specific functions related to gametogenesis, we tested whether the presence of these large nucleotide stores in eleocytes is specific to gender or related to specific events during gametogenesis. Nucleotide pools in eleocytes isolated at different stages of sexual maturation from N. virens were analysed using high-performance liquid chromatography. Eleocytes from immature and male animals had extremely high concentrations of both AMP and ADP (each > 10 µmol/ml of packed cell volume). In eleocytes from male animals, the high nucleotide stores were maintained throughout the maturation phase and decreased at a late stage, while in female animals the nucleotides were degraded at an early stage of maturation. In male eleocytes, the decrease in the adenine nucleotide pool may be the result of its conversion to inosine which is then released by the eleocytes and reutilized by male germ cells for nucleic acid biosynthesis, as has been suggested previously. Our study shows that the time of degradation of the adenine nucleotide pool coincides with the period of spermatogonia proliferation which involves intense nucleic acid synthesis. ATP levels (0.4Œ1.5 µmol/ml packed cell volume) and the guanine nucleotide pool (GTP+GDP+GMP; 0.08Œ0.18 µmol/ml packed cell volume) were similar in both sexes, did not change during germ cell development and were decreased only in eleocytes from prespawning females. The GTP/GDP ratios were initially higher (up to 14) in eleocytes from females compared to ratios in eleocytes from immature (4Œ9) and male animals (up to 8), and decreased during the maturation phase of the animals. GTP levels were correlated with those of ATP; this correlation was much closer in eleocytes from females than from males. The results further support the hypothesis that the adenine nucleotide stores in the eleocytes are maintained as a supply of purine precursors for the growing germ cells. Key words: Coelomic cells Œ Eleocytes Œ Nereis Œ Nucleotides Œ Polychaetes Œ Sexual maturation Introduction Materials and Methods Animals Determination of maturation stage Isolation of coelomic cells Preparation of metabolite extracts Determination of the nucleotides Statistical treatment

Results and Discussion Adenine nucleotides Guanine nucleotides References

Introduction Eleocytes of polychaetous annelids represent a type of coelomic cell which has specialized functions related to gametogenesis (for recent reviews, see Fischer and Hoeger 1993; Fischer et al. 1996). Our studies of the physiological changes in eleocytes related to the sexual maturation of Nereis virens (Hoeger et al. 1995) revealed that there are extremely high concentrations of both ADP and AMP in these cells, each ranging between 10 and 20 µmol/ml of packed cell volume (pcv). 31 P nuclear magnetic resonance studies of living eleocytes (Hoeger et al. 1995) suggested that these high nucleotide concentrations are located in the large acidic vacuole which is characteristic of eleocytes, thus preventing these highly concentrated nucleotides from interfering with cellular metabolism. These nucleotide stores were found only in eleocytes isolated from animals before or at the onset of germ cell development and in eleocytes from male but not female animals. In light of the specific functions of eleocytes in gametogenesis, these high nucleotide concentrations raise the question of whether their presence is related to specific events during the growth and development of the germ cells. In this study, we compared the concentrations of these nucleotides within the rich stores of eleocytes from N. virens of both sexes, isolated at different stages of germ cell development. In addition, we compared adenine nucleotide pools in eleocytes with the levels of other cellular nucleotides to detect additional stage-specific differences in eleocytes during the course of the sexual maturation of N. virens.

Materials and Methods Animals N. virens were collected between 1989 and 1995 from the mudflats of the Oosterscheldt Bay at Yerseke, The Netherlands. The animals were transferred on ice to the laboratory within 8 h of capture and kept at 8Œ12°C under a natural light regime in 40-l trays with artificial seawater and a sandy bottom. Coelomic cells were isolated (see below) from immature (i.e. with no gamete cells) male and female animals at different stages of sexual maturation. In the Yerseke population, N. virens has a 3-year life cycle (A. Fischer and M. Lehmann, unpublished observation) and N. virens reaches sexual maturity during the last 16 months of the life cycle. Thus, animals at different stages of germ cell development co-exist. However, in a population of maturing animals, the growth and development of the germ cells is highly synchronized between individuals (see Fischer and Hoeger 1993), thus allowing precise identification of the different stages of germ cell development (see below). Eleocytes isolated at six different stages of germ cell development were analysed in this study (Table 1). The first set of samples represents immature individuals (collected in January), the next four sets comprise animals between the onset of sexual maturation and the prespawning stage of development. The last set represents animals which are ready to spawn. One set of samples may contain eleocyte samples from different years of collection. Table 1. Characterization of the eleocyte samples isolated from Nereis virens at different stages of germ cell development. Data for female animals are taken from Hoeger (1991). Data for developmental stages of male germ cells are taken from Geier and Hoeger (1997). Number in parentheses indicate number of samples analysed

Sample Nr. Time of eleocyte preparation Stage of sexual maturation 1

Jan. 1991

immature animals (7) (no germ cells present) female animals male animals (mean oocyte volume (germ cell stage) [10 6 µm 3 ]) a

2

June 1989

0.11 (9)

Spg stage I

3

Jul./Aug. 1989

0.19 (3)

Spg stage I (5)

4

Nov. 1989

0.94 (5)

Spermatocytes (6)

5

Jan/Feb. 1990,1991

2.55 (7)

Spz (immotile)

6

Apr. 1990,1991 (prespawning animals)

3.55 (6)

Spz (motile) (3)

a b

(12)

b

(6)

spg: spermatogonia spz: spermatozoa

Determination of maturation stage The gender and the stage of maturation were identified by the presence and the developmental stage of the germ cells in samples of coelomic fluid obtained by puncturing the coelomic cavity with a fine glass capillary. In females, the stage of maturation was characterized as the mean oocyte volume calculated from the diameters of 25Œ30 oocytes. Males at different stages of sexual maturation were identified by the presence of spermatogonia clusters, spermatocytes and spermatozoa, respectively (Geier and Hoeger 1997).

Isolation of coelomic cells Coelomic cells were isolated as described in detail previously (Hoeger 1991). In brief, the body cavity of the anaesthetized animals was opened and the coelomic cells were flushed out with Ca/Mg-free seawater. Eleocytes were separated from other coelomic cells by passing the suspension through nylon sieves with mesh sizes of between 40 and 150 µm. The eleocyte suspensions were further purified by layering onto a linear Ficoll gradient, and from the finally purified suspensions 50- or 100-µl aliquots of packed cells were frozen in liquid nitrogen until analysis.

Preparation of metabolite extracts To the frozen pellets, a ninefold volume of ice-cold perchloric acid (0.4 M) was added and the cells were broken up by three 5-s bursts (50 W) of an ultrasonic disintegrator. The homogenates were centrifuged for 3 min at 15,000 g in a refrigerated centrifuge and the extracts were recovered by puncturing the microcentrifuge tube near the bottom to separate the floating lipid layer. The perchloric acid extracts were neutralized by adding a twofold volume of 0.5 M N-trioctylamine in chloroform (1:3.55 v/v; Dobson et al. 1991). After vigorous mixing, the phases were separated by brief centrifugation and the aqueous (top) phase was removed. The aqueous phase was further purified by a brief centrifugation (1500 g, 1 min) through a 1-ml solid phase extraction column (Adsorbex RP 18, Merck) previously equilibrated with water and precentrifuged under the same conditions. The purified extracts were stored at -20°C for no longer than 2 weeks. The recovery of the purification procedure was determined using standard solutions for each of the compounds of interest. Depending on the compound, recoveries were between 77 and 97% (SD 4.6Œ6.5%, n=5).

Determination of the nucleotides Nucleotides were separated by anion exchange HPLC at 55°C on a Nucleosil 5SB column (250 X 4.6 mm; Macherey-Nagel) using a phosphate gradient elution (buffer A: 25mM K-phosphate, pH 2.8, 10% methanol; buffer B: 600 mM K-phosphate, pH 2.8, 10% methanol; modified after Abe and Okuma 1991) and a flow rate of 1 ml/min. Elution of the nucleotides was monitored by simultaneous UV detection at 254 and 280 nm. Compounds were identified by comparison with the retention times of standard solutions chromatographed under identical conditions and their characteristic absorbance ratios at 254 and 280 nm. The concentration was calculated from the area of the peak and corrected for recovery of the purification procedure (see above).

Statistical treatment Mean nucleotide concentrations and GTP/GDP ratios were compared using Student’s t-test after log transformation of the data. ATP and GTP concentrations were correlated using the Spearman’s rank sum test.

Results and Discussion Adenine nucleotides Our previous work (Hoeger et al. 1995) suggested that the majority of total cellular AMP and ADP is stored in the large acidic vacuole that is characteristic of eleocytes. For this reason, the sum of AMP and ADP given in Fig. 1a reflects the large intra-vacuolar nucleotide store. The sum of AMP and ADP concentrations reached an extremely high value of up to 50 µmol/ml pcv in eleocytes of immature and male animals. Considering their location in the vacuolar compartment of the eleocyte, their intra-vacuolar concentrations can be assumed to be almost twice as high taking a vacuole volume of 40Œ50% of the cell volume into account (Hoeger et al. 1995). Such high concentrations of AMP and ADP have not been reported to occur in any other invertebrate tissues, while the ATP levels are comparable to those of other non-muscular tissues of both vertebrates and invertebrates (Beis and Newsholme 1975; Traut 1994). Fig. 1 Seasonal changes in the concentrations (expressed as µmol/ml of packed cell volume) of ADP+AMP (a) and of ATP (b) in eleocytes from immature (green symbols), female (red symbols) and male (blue symbols) Nereis virens isolated at different stages of sexual maturation. For explanation of maturation stages see Table 1. Asterisks indicate significant differences between male and female eleocyte samples (unpaired t-test; P < 0.002). Hatched squares below the abscissa designate the time at which eleocyte samples were prepared. Numbers 1Œ6 correspond to the samples as characterized in Table 1. (pcv Packed cell volume)

After germ cells have formed and developed in the coelomic cavity (samples 2Œ5, see Table 1), the concentration of both AMP and ADP decreased in female eleocytes to the much lower levels (< 0.5 µmol/ml pcv; separate values for AMP and ADP not shown) commonly observed in animal tissues. Male eleocytes on the other hand maintained the high adenylate concentrations for a prolonged period (Fig. 1a). In contrast, ATP levels (0.4Œ1.5 µmol/ml pcv; Fig. 1b) remained unchanged in eleocytes of both sexes until the later phases of sexual maturation during which they decreased to levels below 0.2 µmol/ml pcv. The decrease in the adenylate stores observed between November and January in male eleocytes (samples 4 and 5 see Fig. 1a) may be related to the increase in the intracellular levels of inosine, which is a main breakdown product of adenine nucleotides, found during this period (Hoeger et al. 1996). The concentrations of inosine monophosphate (IMP), which is a potential intermediate in the breakdown of adenine nucleotides, were not found to be increased when the AMP+ADP concentration was decreased (data not shown); however, this could be the result of rapid further conversion to inosine. Inosine is released by eleocytes in culture, at a much higher rate by male eleocytes compared with female eleocytes (Hoeger et al. 1996). Labelled inosine, in turn, has been shown to be taken up by both male (Geier and Hoeger 1997) and female (G. Geier and U. Hoeger, in preparation) germ cells, where it is utilized for anabolic pathways such as nucleic acid biosynthesis. Interestingly, the increase in the inosine concentration in male eleocytes coincides with the period of spermatogonia proliferation in male animals. This is accompanied by a sharp increase in the rate of inosine uptake by the spermatogonia cells in vitro, reflecting increasing utilization of exogenous inosine for DNA synthesis (Geier and Hoeger 1997). Although our data indicate that this increase in intracellular inosine concentration takes place between August and November, and thus precedes the observed degradation of adenylate nucleotides between November and January (see Fig. 1a), the conversion of adenine nucleotides to inosine may not be initially visible as a decrease in their concentrations due to the much larger size of the adenine nucleotide pool (20Œ50 µmol/ml pcv) compared to that of inosine (< 2 µmol/ml pcv). In female eleocytes, such a correlation between adenine nucleotide breakdown (observed between January and May; see Fig. 1a) and the time of highest inosine uptake rates by the female germ cells (measured in August; Geier and Hoeger 1997) was not found. Furthermore, our (unpublished) data show only a transient increase in the intracellular inosine levels in female eleocytes during this time. Further studies are needed to determine the fate of the degraded adenine nucleotides in female eleocytes.

Guanine nucleotides Considering the intra-sample variability, guanine nucleotides (GMP+GDP+GTP; Table 2) did not change significantly over the time course of maturation, with pool sizes between 0.08 and 0.3 µmol/ml pcv, and there were no gender-specific differences, except for prespawning animals where females contained much less (0.015Œ0.04 µmol/ml pcv) than males. GTP levels (Table 2) ranged between 0.02 and 0.15 µmol/ml pcv, with no apparent change or gender-specific differences. Only at the end of the maturation period did the GTP concentration drop to levels between 0.002 and 0.015 µmol/ml pcv; this decrease took place earlier in males (sample 4) than in females (sample 6). For samples 4Œ6, the GTP/GDP ratios (Fig. 2) in each sample group were significantly higher (unpaired t-test; P < 0.025) in female compared to male eleocytes. At the end of sexual maturation of N. virens (data for sample groups 5 and 6 combined), the GTP/GDP ratios were significantly lower (unpaired t-test; P < 0.025) than in either of the preceding sample groups (1Œ4) in male and female eleocytes, respectively. Table 2. Concentrations of guanine nucleotides (given as mmol/ml of packed cell volume; means ± SD) in eleocytes of Nereis virens isolated from immature and female (a) and from male animals (b). Number of determinations is given in parentheses. For small sample size (n = 3), the range of determinations is given. For explanation of maturation stages, see Table 1 a Immature (sample 1) and female (samples 2Œ6) animals

Sample no. 1(Immature) 2

3

4

5

6

GMP

0.058 ± 0.012 (7)

0.066 0.031 0.069 0.068 0.024 ± 0.024 (10) (3)0.019Œ0.039 ± 0.035 (6) ± 0.029 (8) ± 0.008 (6)

GDP

0.011 ± 0.002 (7)

0.007 0.011 0.011 0.018 0.002 ± 0.003 (10) (3)0.009Œ0.012 ± 0.006 (6) ± 0.006 (8) (3)0.001Œ0.002

GDP+GMP

0.069 ± 0,013 (7)

0.074 0.042 0.084 0.087 0.026 ± 0.022 (10) (3)0.031Œ0.048 ± 0.031 (6) ± 0.031 (8) (3)0.013Œ0.037

GTP

0.069 ± 0.019 (7)

0.057 ± 0.028 (7)

0.094 0.079 0.060 0.005 (3)0.064Œ0.125 ± 0.032 (6) ± 0.042 (6) ± 0.003 (4)

GTP+GDP +GMP 0.138 ± 0.029 (7)

0.139 ± 0.042 (7)

0.136 0.162 0.150 0.032 (3)0.112Œ0.155 ± 0.056 (5) ± 0.049 (7) (3)0.013Œ0.045

b Male eleocytes Sample no. 2

3

4

5

6

GMP

0.078 0.074 0.056 0.105 0.132 ± 0.058 (12) ± 0.024 (5) ± 0.021 (6) ± 0.033 (8) (3)0.086Œ0.185

GDP

0.018 0.020 0.013 0.016 0.012 ± 0.013 (12) ± 0.005 (4) ± 0.007 (6) ± 0.005 (6) (3)0.008Œ0.015

GDP+GMP

.0.096 0.092 0.069 0.118 0.144 ± 0.053 (12) ± 0.026 (4) ± 0.019 (6) ± 0.033 (6) (3)0.094Œ0.198

GTP

0.069 0.069 0.038 0.013 0.013 ± 0.037 (12) ± 0.023 (5) ± 0.013 (6) ± 0.011 (7) (3)0.010Œ0.015

GTP+GDP +GMP

0.165 0.169 0.106 0.130 0.157 ± 0.046 (12) ± 0.032 (5) ± 0.024 (6) ± 0.035 (5) (3)0.110Œ0.212 Fig. 2 Seasonal changes in the GTP/GDP ratios in eleocytes from immature (green symbols), female (red symbols) and male (blue symbols) Nereis virens isolated at different stages of sexual maturation. For explanation of the maturation stages see Table 1. Asterisks indicate significant differences between male and female eleocyte samples (unpaired t-test; P < 0.025). Hatched squares below the abscissa designate the time at which eleocyte samples were prepared. Numbers 1Œ6 correspond to the samples as characterized in Table 1.

Since GDP acts as a competitive inhibitor of GTP binding to several factors involved in protein synthesis (Pall 1985), the cellular GTP/GDP ratio is considered to be a regulatory factor in protein synthesis (Pall 1985; Land et al. 1993). In this light, the higher GTP/GDP ratios in female eleocytes would favour the rate of protein synthesis compared to males, which is consistent with the capacity of female eleocytes to synthesize vitellogenin. GTP and ATP concentrations were also much more closely correlated in female (Spearman’s rank correlation; r s =0.95; P < 0.001) than in male eleocytes (r s =0.71; P < 0.001; see Fig. 3), indicating a tighter coupling of GTP regeneration by ATP, a reaction mediated by the enzyme nucleoside diphosphate kinase. Activity levels of this enzyme are similar in eleocytes of either sex (U. Hoeger, unpublished). However, the overall decline in the GTP/GDP ratios, suggestive of a gradual decrease in protein synthesis, is much in contrast to our expectations since female eleocytes continuously increase their rate of both vitellogenin synthesis and secretion into the medium during the course of gametogenesis in N. virens (Heil 1995). On the other hand, nucleotide pools are compartmentalized in the cell and may differ in their composition depending on the cellular compartment, as was found for rat liver Golgi vesicles (Fleischer 1981). Some of the GDP (as well as GMP) may be generated inside the Golgi apparatus by the breakdown of sugar nucleotides involved in glycosylation reactions (Capasso and Hirschberg 1984, Abeijon et al. 1993). Thus, the observed changes in the apparent GTP/GDP ratios could also reflect differences in the degree of GDP sequestration in these subcellular compartments and do not necessarily reflect the cytosolic GTP/GDP ratios for protein synthesis. The range of GTP/GDP ratios measured in this study (0.5Œ16) compares well with the GTP/GDP ratios found for different mammalian cell types (0.5Œ12; calculated from Traut 1994).The possible localization of some of the GDP (and GMP) in the eleocyte vacuole seems less likely since no correlation with the high AMP+ADP levels was found. Fig. 3 Correlation between the concentrations of ATP and GTP (expressed as µmol/ml of packed cell volume) in eleocytes of female (red symbols) and male (blue symbols) eleocytes. Data from all sample groups were combined. The correlations were significant at the p = 0.01 level. r s ; spearman’s rank correlation coefficient; n; number of samples.

The concentrations of nucleotides determined in this study fluctuated considerably within the different sample groups. Nucleotide concentrations in eleocyte samples isolated from animals at the same stages of germ cell development but collected in different years (see Table 1) were similar and did not significantly add to the observed sample variability. Likewise, differences in the time intervals between capture of the worms and eleocyte isolation (i.e. 2Œ20 days) were not found to contribute to the variation in the nucleotide levels. Apparently, the nucleotide levels are not tightly regulated in eleocytes. For comparison, the ATP content of the body wall musculature of another polychaete, Arenicola marina, is in a much narrower range (2.1Œ2.4 µmol/g tissue) and these values do not change during several hours of metabolic stress (i.e. anoxia; Schöttler et al. 1984). Eleocytes are not directly involved in vital functions such as movement and the functioning of the nervous system, and in such cell types the organism may allow a much greater tolerance for metabolically unfavourable situations such as low ATP levels and low GTP/GDP ratios, since this may not be a factor critical for the immediate survival of the animal. Acknowledgements. This study was supported by the grants Ho 889/3-1 and Ho 889/4-1 from the Deutsche Forschungsgemeinschaft, the Naturwissenschaftlich-Medizinisches Forschungszentrum and the Feldbausch Foundation at the University of Mainz. We thank Dr. G. Wegener and Dr. A. Fischer for helpful comments on the manuscript.

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