Ogielska, 1994. Nucleus-like bodies in gonial cells of Rana esculenta (Amphibia, Anura) tadpoles

Nucleus-like bodies in gonial cells of Rana esculenta (Amphibia, Anura) tadpoles – putative pathways of chromosome eliminationMaria OgielskaNucleus-like bodies in gonial cells of Rana esculenta (Amphibia, Anura) tadpoles - a putative way of chromosome elimination. Zoologica Poloniae (1994) 39/3-4: 461-474.Department of General Zoology. Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, PolandAbstractRana esculenta is a natural hybridogenetic hybrid of two parental species, R. lessonae and R. ridibunda. Owing to its particular hybridogenetic mode of reproduction, both sexes produce gametes carrying the haploid chromosome set of one parental genome while the other set is eliminated prior to meiosis. In order to detect the pathways of chromosome elimination, the gonads of tadpoles (both hybrid and parental forms) – i.e., the stages at which cells are most actively proliferating – were examined by electron, light, and fluorescence microscopy. Of the two chromosome elimination pathways – gradual loss during successive mitoses and budding from the interphase nucleus with formation of nucleus-like bodies (NLB) – the latter appears to be the more probable.IntroductionThe edible frog, Rana esculenta, is a hybrid whose genome consists of haploid sets from the parental species R. lessonae and R. ridibunda (BERGER 1983, GRAF and POLLS-PELAZ 1989). R. esculenta typically occurs in populations together with one of the parental species. Mixed esculenta-lessonae populations are the most widespread and stable; their genetic system is termed the E-L system (UZZELL and BERGER 1975). Individuals of R. esculenta within this system ordinarily transmit to their gametes only the haploid chromosome set of R. ridibunda, while the R. lessonae set is eliminated during gametogenesis. The hybrids thus propagate from generation to generation the genome of the parental species that is absent from the population. This mode of reproduction is called hybridogenesis (SCHULTZ 1969, TUNNER 1974).Our recent results (OGIELSKA and WAGNER 1993, WAGNER and OGIELSKA 1993) showed that the development and differentiation of the ovary in R. esculenta is a more protracted and delayed process compared with that of the parental species (OGIELSKA and WAGNER 1990, WAGNER and OGIELSKA 1990). In particular, the postponement of meiotic onset and the prolonged oogonial division stage are the most conspicuous features of ovarian development in hybrids. Moreover, distinctive structures termed nucleus-like bodies (NLB) were described in the cytoplasm of hybrid oogonia. NLB were not detected in either parental species, nor in Xenopus laevis (AL-MUKHTAR and WEBB 1971, COGGINS 1972) or Rana pipiens (MERCHANT-LAROIS and VILLALPANDO, 1981), the only anuran amphibians for which ultrastructural studies of tadpole gonadal differentiation had been conducted. The aim of the present study was to detect morphological markers of the eliminated chromosomes in the germline during the early stages of gonadal differentiation in hybrid tadpoles.Materials and MethodsTadpoles used in the study were obtained from crosses performed under laboratory conditions. Females were stimulated by injection of homogenized fresh or frozen frog pituitary glands. Eggs were artificially fertilized according to RUGH (1965). Males and females of R. esculenta and R. lessonae were collected from natural mixed E-L populations in the vicinity of Wroclaw, Poland (Kotowice, Zakrzow, Paniowice). R. ridibunda tadpoles and parental individuals were collected near Poznan, Poland and were kindly provided by LESZEK BERGER and MARIUSZ RYBACKI (POLISH ACADEMY OF SCIENCES IN POZNAN).Offspring from the following crosses were studied:7 esculenta x lessonae crosses (EL)2 ridibunda x lessonae crosses (RL)1 lessonae x ridibunda cross (LR)and control crosses:5 lessonae x lessonae crosses (LL)3 ridibunda x ridibunda crosses (RR).Tadpoles were examined from stage 25 (feeding tadpole) through stage 46 (end of metamorphosis), according to GOSNER (1960). Tadpoles were maintained in plastic containers and fed with boiled lettuce and fish food. At every 2nd–5th stage, individuals from each cross were subjected to microscopic analysis. Several juvenile individuals (aged 1 and 1.5 years) were also examined.For electron microscopy, gonads were dissected from tadpoles and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, followed by 1% OsO4 or a mixture (1:1) of 2% OsO4 with 1% sodium ferrocyanide (MCDONALD 1984).For cytochemical detection of DNA on paraffin sections fixed in glutaraldehyde, formaldehyde, or paraformaldehyde, Feulgen staining and fluorescent DAPI staining were employed.Serial paraffin sections, 5–7 μm thick, stained with iron haematoxylin after Zenker or Helly fixation, were used for analysis of mitoses.[IMG_1]Fig. 1. Nucleus-like bodies in gonadal cells of Rana esculenta tadpoles. NLB – nucleus-like body; N – nucleus. A–C 11000×. D 6600×ResultsI. Nucleus-like bodies (NLB)During ultrastructural analysis of germline cells of tadpole gonads, spherical bodies 1.5–3.0 μm in diameter were detected in the cytoplasm of gonial cells. These bodies (NLB) were enveloped by a double membrane and contained material resembling nuclear chromatin (Fig. 1 A–D). NLB were found at varying distances from the “main” nucleus. Under the electron microscope these bodies were never detected in the parental species, R. ridibunda and R. lessonae. NLB were observed in interphase gonial cells (both oogonia and spermatogonia) beginning at developmental stage 28, when sexual differentiation commences. NLB were not detected in undifferentiated gonads (stages 25–27).The highest frequency of NLB was recorded in gonads (both testes and ovaries) at stages 28–41, i.e., between 30 and 80 days post-fertilization. In some individuals the number of NLB was high at the time of fixation, while in others it was low or NLB were entirely absent. During metamorphosis (stages 42–46) NLB were also detected, but less frequently than at earlier stages. They were observed exclusively in interphase cells as isolated spherical bodies situated at varying distances between the nucleus and the cell membrane. Certain electron micrographs permit reconstruction of the putative pathway of their formation: the nuclear membrane forms a protrusion, and first the inner, then the outer membrane fuses. The next step is the detachment of the NLB from the “main” nucleus (Fig. 2 A–D and II). In several cases atypical NLB were observed in tadpoles at stages 35–36.DAPI and Feulgen staining of serial paraffin sections revealed the presence of DNA in small spherical bodies in the cytoplasm of gonial cells (Fig. 3 and 4). The number of such bodies was 1–3 per cell. In hybrid gonads, germ cells bearing such bodies were numerous. Unexpectedly, such cells were also occasionally detected in the gonads of parental species, although their number was low.[IMG_2][IMG_3]Fig. 2. Reconstruction of the putative pathway of NLB formation in interphase nuclei of Rana esculenta tadpoles at stages 29–31. A – Low magnification (2300×) of an oogonium with two NLB. B – Formation of a protrusion of the “main” nucleus (N). The outer membrane has already fused. 19200×. C – Enlarged fragment of one NLB from Fig. 2A. Note the state of the “main” nucleus (N) indicated by the arrow. 16000×. D – NLB detached from the nucleus. Note the double membrane between the nucleus and the body (arrow). 22400×. E – An atypical NLB, possibly undergoing disintegration. 16000×.[IMG_4]Fig. 3 and 4. DAPI staining of an ovary (Fig. 3) and a testis (Fig. 4) at stage 31 of Rana esculenta tadpoles. In gonial cells the round nucleus contains more dispersed chromatin than somatic cells. Arrows indicate small spherical DNA-containing bodies. 700×.2. Mitotic division of gonial cells.One possible pathway of chromosome removal is their gradual elimination from the cell during successive mitotic divisions. This possibility was taken into account, and chromosome distribution during ana- and telophases was analyzed on serial paraffin sections. Another pathway – single-step elimination during one mitotic division through formation of a monopolar spindle – was evaluated by analysis of spindle morphology. The first mitotic division was observed in primordial germ cells (PGC) of gonadal anlagen in late stage 25 tadpoles (Fig. 5). The highest mitotic activity in parental species was observed at stages 26–29 (Fig. 6, 7, 8), and in hybrids at stages 29–41. All mitotic spindles, in both parental species and hybrids, were bipolar; no monopolar spindles were detected.Until stage 28 (undifferentiated gonads), mitoses in hybrids are regular and no differences between parents and hybrids are observed. With the onset of sexual differentiation of hybrid gonads (stages 28–29), anaphases and telophases occasionally appear with single chromosomes irregularly distributed within the spindle (Fig. 9 A–D, 10 A, B).[IMG_5]Fig. 5–8. 5. Prophase of a primordial germ cell (PGC) in the gonadal anlage of a late stage 25 tadpole. Y – yolk platelet. 1150×. 6, 7 and 8. Mitoses of spermatogonia at stages 29 and 33 of Rana lessonae tadpoles. 1150×.The later the stage, the more numerous the abnormal mitoses, although normal ones always constitute the majority. In hybrid females, oogonial mitoses are also observed after metamorphosis, which is not characteristic of the parental species. After stage 35 in R. ridibunda and stage 33 in R. lessonae, the number of oogonia rapidly decreases and the ovaries consist of an increasing proportion of cells at the diplotene stage. In hybrids, diplotene-stage oocytes are absent or scarce, and the majority of the ovarian cortex consists of oogonia. Mitotic activity in the testes after metamorphosis was not analyzed.[IMG_6]Fig. 9–10. 9. A–D. Irregular bipolar telophase of an oogonium at stage 20 of a Rana esculenta tadpole, visible in three consecutive sections (B and C are the same section at different focal planes). 1150×; 10. A and B. Bipolar mitosis of an oogonium at stage 30 of a Rana esculenta tadpole with one chromosome outside the metaphase plate. 1150×.DiscussionThe hybridogenesis model proposed by TUNNER (1974), although it explains well the genotypes of R. esculenta offspring, was not corroborated by cytological evidence of the elimination of one of the chromosome sets. In 1973 GUNTHER described spermatogenesis in adult males of R. esculenta but did not detect any signs of chromosome elimination. On the other hand, HEPPICH et al. (1982) and BUCCI et al. (1990) demonstrated that proliferating spermatogonia of adult male R. esculenta carry only R. ridibunda chromosomes. However, VINOGRADOV et al. (1990) found several adult male R. esculenta whose spermatogonia harbored the genomes of both parental species. Similar results were obtained for adult females. Experimental gynogenesis (development of an egg without the male pronucleus) of R. esculenta oocytes produced ridibunda offspring (GRAF and MULLER, 1979), and the cytoplasm of R. esculenta oocytes at the diplotene stage contained translation products of R. ridibunda genes (CHEN and STUMM-ZOLLINGER, 1986). Recently BUCCI et al. (1990) described bivalents of R. esculenta diplotene-stage oocytes composed of two ridibunda chromosomes.The data summarized above indicate that elimination of R. lessonae chromosomes must occur at early developmental stages and prior to meiosis, as first proposed by UZZELL et al. (1980). TUNNER and HEPPICH (1981) and TUNNER and HEPPICH-TUNNER (1991) developed a double-staining technique using fluorescence Hoechst-Actinomycin D, which allows distinction between ridibunda and lessonae chromosomes. They examined female R. esculenta shortly before metamorphosis, when dividing oogonia are still present, and described three classes of oogonia: diploid ridibunda, haploid ridibunda, and aneuploid with predominantly ridibunda chromosomes. It appears that elimination of lessonae chromosomes was gradual and may have begun at early developmental stages. Detailed studies of ovarian development in R. ridibunda, R. lessonae, and R. esculenta demonstrated that intensive oogonial proliferation in parental species ends before metamorphosis (stage 42), whereas in hybrids this process was observed after metamorphosis as well (OGIELSKA and WAGNER, 1990, 1993; WAGNER and OGIELSKA 1990, 1993). Furthermore, transformation of oogonia into oocytes was delayed, and in most juvenile females no diplotene-stage oocytes were present. These data are consistent with the occurrence of NLB: they were detected at stages 29–41, but also before and after metamorphosis – i.e., at those stages for which TUNNER and HEPPICH (1981) and TUNNER and HEPPICH-TUNNER (1991) described the gradual loss of lessonae chromosomes from germline cells. These data suggest that NLB may be the carriers of the eliminated chromosomes.NLB were also detected in spermatogonia during the early stages of testicular development in tadpoles. Studies of spermatogenesis were conducted by HEPPICH et al. (1982) and BUCCI et al. (1990), but these concerned adult males only. The genotype of spermatogonia in adults was exclusively ridibunda. This suggests that lessonae chromosomes during spermatogenesis are eliminated only before or immediately after metamorphosis. However, no detailed studies of spermatogenesis and testicular development exist.During electron microscopic analysis, NLB were detected exclusively in R. esculenta tadpoles and never in parental species. On the other hand, cytochemical analysis by light microscopy revealed small spherical bodies containing DNA in gonial cells of the parental species. If NLB and DAPI-positive bodies are the same structures, we must consider the possibility that parental species are also capable of extruding chromosomes from germ cells, although this phenomenon is rare in them. This may be viewed as the evolutionary basis of hybridogenesis in hybrids: hybridogenesis is possible because the parental species possess cytological mechanisms of chromosome elimination. The principal question in this study that requires an answer is by what means chromosomes are eliminated from the germline. Two possible pathways, schematically depicted in Fig. 12, are considered in the discussion:1. Chromosome elimination occurs during interphase. A possible mechanism is a form of enzymatic chromatin degradation, after which chromatin is expelled from the interphase nucleus in the form of NLB. This hypothesis is supported by the similarity of NLB to structures observed during apoptosis (programmed cell death) (see CLARKE, 1990). This probable pathway was also mentioned in the work of VINOGRADOV et al. (1990).[IMG_7]Fig. 11. Formation of NLB by budding from the interphase nucleus of a gonial cell. See also Fig. 2 A–D2. Chromosome elimination occurs during mitosis. This hypothesis is supported by the numerous meta-, ana-, and telophases in which individual chromosomes were irregularly distributed within the spindle. Such single chromosomes (or chromosomal fragments) may form their own nuclear envelope, which is characteristic of chromatin appearing in the cytoplasm – as, for example, in the case of viral DNA introduced into the cytoplasm of an egg (FORBES et al., 1983; SHIOKAWA et al. 1987), the sperm pronucleus penetrating the egg and reconstructing its envelope prior to fusion (LONGO and ANDERSON 1968), or karyomeres, when individual chromosomes form their own nuclear envelope during telophase (ITO et al., 1981). The same is true for the fish Hydrolagus colliei, in which a reduction of chromosome number in spermatogonia has been described (STANLEY et al. 1984). In hybrid R. esculenta this may, however, be a secondary effect of the reduction in interphase chromosome number during subsequent cell divisions. It is difficult to say which of the two hypotheses is correct. However, on the basis of morphological characteristics, the first appears more probable.[IMG_8]Fig. 12. Two putative pathways of NLB formationWhatever the pathway of chromosome removal (diminution), this phenomenon is generally rare in germline cells. Among vertebrates it is known in the fish Poeciliopsis lucida-monacha (CIMINO, 1972) and Hydrolagus colliei (STANLEY et al. 1984). P. lucida-monacha form diploid all-female populations for which hybridogenesis has been described (SCHULTZ, 1969). Single-step elimination of one parental chromosome set was observed during oogonial mitoses. Its cytological mechanism involves the formation of a monopolar spindle to which only the maternal chromosomes are attached, while the paternal chromosome set remains in the cytoplasm. During spermatogenesis of H. colliei, a portion of chromosomes is eliminated at metaphase I. The compact mass of eliminated chromosomes is not incorporated into the nucleus but forms separate bodies (CDC – chromatin diminution bodies) surrounded by a double membrane. The CDC remains in the cytoplasm of one of the daughter cells until the spermatid stage, then moves into the cytoplasm of the Sertoli cell, where it is degraded.R. esculenta is the third known vertebrate species in which chromosome diminution has been described. Both R. esculenta and P. lucida-monacha reproduce by hybridogenesis, whereas H. colliei does not have a specific reproductive mode and the role of CDC remains unknown.Chromosome diminution in the germline has also been described in invertebrates. During spermatogenesis of Sciara coprophila, paternal chromosomes are eliminated into the cytoplasm at metaphase I. They remain heterochromatinized and are subsequently expelled from the cell. Only the maternal chromosomes remain attached to the mitotic monopolar spindle (METZ, 1933; ABBOT et al. 1981). Another example is Metaseiulus occidentalis (Acarina), in which paternal chromosomes eliminated during spermatogenesis are first heterochromatinized within the nucleus and then expelled from the cell (NELSON-REES et al., 1980). Recently, hybridogenesis and genome elimination were described in certain populations of stick insects Bacillus rossius-grandii benazzi from Sicily (TINTI and SCALI, 1992). In this case, one of the parental chromosome sets undergoes heterochromatinization prior to metaphase I, then moves to a polar body and is eliminated.Neither monopolar spindle formation nor chromosome heterochromatinization was observed in the R. esculenta studied. The most probable pathway of elimination of one chromosome set is the formation of NLB during interphase. However, the mechanism of this phenomenon remains unresolved.REFERENCESABBOT, A.G., HEES, J.E. and GERBI. S.A., 1981: Spermatogenesis in Sciara coprophila. I. Chromosome orientation on the monopolar spindle of meiosis I. 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