The Development of Nuclear Vacuoles during Meiosis in Plants Elizabeth Sheffield ~, A.H. Cawood 2, P.R. Bell s, and H.G. Dickinson 3 1 Department of Botany and Microbiology, University College, Gower Street, London WC1E 6BT, 2 Department of Agricultural Botany, and 3 Department of Botany, Reading University, Whiteknights, Reading RG6 2AH, U.K.
Abstract. Vacuoles formed by the invagination of the inner m e m b r a n e of the nuclear envelope have been observed during meiotic prophase in a wide range of plants. In the angiosperm Lycopersicon their formation was found to coincide with the completion of synaptonemal complex formation, and this timing is analogous to that observed during this stage in the silkworm Bombyx. The implications of this activity in relation to the process of chromosome movement are discussed. In the gymnosperm Pinus, the heterosporous fern Marsilea and h o m o s p o r o u s ferns Pteridium and Dryopteris the formation of nuclear vacuoles begins much earlier, coinciding with the condensation of chromatin during leptotene. They enlarge and become more elaborate as meiosis proceeds, and may eventually become detached from the nuclear envelope. It is therefore thought unlikely that they fulfil functions connected with c h r o m o s o m e movement in the manner proposed for the silkworm and the tomato. During diplotene/diakinesis they contain electron-opaque granules and fibrils, and the possible origin and significance of this material is discussed. Key words: Nuclear envelope - Nuclear vacuoles Meiosis - Prophase - Sporogenesis.
Introduction Dilatations of the perinuclear space caused by invagination of the inner m e m b r a n e of the nuclear envelope have been observed during meiotic prophase in animals (see, for example, Meek and Moses, 1961 ; Danilova and Vereiskaya, 1970; Danilova, 1973a). These lacunae have been termed 'nuclear vacuoles' by Rasmussen (1976) who followed their development in the meiocytes of Bombyx mori. The presence of such nu-
clear vacuoles has not previously been reported during meiosis in plants. Blebbing of the outer m e m b r a n e of the nuclear envelope has been observed by Godward and Peristianis (1976) during the pachytene stage in Lilium martagon, and similar evaginations occur in post-meiotic microspores of Podocarpus macrophyllus (Aldrich and Vasil, 1970). Neither of these reports, however, contain any mention of comparable inwardly directed activity associated with the inner membrane. Nuclear vacuoles were noted in the prophase meiocytes of the h o m o s p o r o u s fern Pteridium aquilihum (see also Sheffield and Bell, in press). It was at first thought, in view of the absence of any descriptions of similar structures in prophase nuclei of other genera, that their occurrence was peculiar to this plant. The examination of micrographs has revealed, however, that the phenomenon is a general one, and that meiosis in other h o m o s p o r o u s ferns, and in certain heterosporous ferns, gymnosperms and angiosperms is also regularly accompanied by the development of nuclear vacuoles.
Materials and Methods Fertile portions of the fronds of Pteridium aquilinum and Dryopteris borreri; megasporangia of Marsilea vestita and microsporangia of Pinus banksiana, were excised from the plants and fixed in 3% glutaraldehyde in 0.05 M phosphate buffer, pH 6.9 for 3 h at room temperature. The material was then washed twice and left overnight in ice cold buffer. Post fixation was in 2% osmium tetroxide (aq.) for 2 h at 4~ C and dehydration in acetone. The material was embedded in Epon or Araldite and sectioned with a glass or diamond knife. Sections were mounted on formvar-coated nickel grids and stained successivelywith nranyl acetate (7% aq.) and Reynold's lead citrate. They were examined in a Hitachi HS9 or Siemens Elmiskop I electron microscope. Anthers of Lycopersicon esculentum were fixed in 2.5% glutaraldehyde in 0.03 M phosphate buffer, pH 6.8 for 4 h at 4~ C. They were washed in two changes in buffer overnight at 4~ C.
E. Sheffield et al. : Nuclear Vacuoles during Meiosis in Plants
E. Sheffieldet al. : Nuclear Vacuoles during Meiosis in Plants Post fixation was in 2% osmium tetroxide (aq.) for 3 h at 4~ and dehydration in acetone. The samples were embeddedin Spurr's resin, sectioned with a diamond knife and mounted on formvarcoated copper grids. They were stained with uranyl acetate (7% aq.) and lead citrate and examined in a Jeol 100B electron microscope.
Results Meiotic prophase was characterized, in each of the plants examined, by the invagination of the inner membrane of the nuclear envelope. This process was followed closely throughout prophase in Pteridium, and examination of isolated stages during early meiosis in Dryopteris and Marsilea confirmed that a closely comparable sequence of events occurred in both these ferns. At the onset of leptotene, isolated portions of the inner membrane of the nuclear envelope moved away from the outer membrane and began to protrude into the karyoplasm. This activity occurred at numerous sites over the nuclear envelope (Fig. 1) and folding within the vacuoles so formed occasionally led to profiles showing several membranes (Fig. 2). The vacuoles increased in size during leptotene, and ramified through the karyoplasm between the synaptonemal complexes during mid prophase (see Sheffield and Bell, in press). Towards the end of prophase, during the diplotene/diakinesis period, the vacuoles had come to occupy a large proportion of the nuclear volume, and there were indications of dispersed, granular, electron-opaque material within them (Fig. 3). Although this material resembled the ground cytoplasm seen at this time in appearance (Fig. 3, inset), no interruptions were seen in the outer envelope and no continuities were detected between the vacuoles and the cytoplasm. The disintegration of the nuclear envelope at metaphase was accompanied by the disappearance of the membranes within the nucleus, and the contents of the vacuoles dispersed to become indistinguishable from the meiocyte cytoplasm. The development of nuclear vacuoles in Pinus and Lycopersicon, although less spectacular than that seen in the ferns, was nevertheless a regular feature of
599 meiotic prophase. Nuclear vacuoles develop during the late zygotene/pachytene period in Lycopersicon, their appearance coinciding with the completion of synaptonemal complex formation (Fig. 4). Their development has not yet been followed during the diplotene/diakinesis interval. In Pinus at this stage the vacuoles were found to contain diffuse, granular, electron-opaque material (Fig. 4, inset).
Discussion It has been proposed that the development of nuclear vacuoles and the tubulation of the inner membrane of the nuclear envelope in certain spermatocytes are associated with degenerative processes. The formation of tubular outgrowths from the inner membrane into the perinuclear space of the envelope of crayfish spermatocytes accompanies the decay of cells not utilized during the mating period (Meek and Moses, 1961). In atypical nuclei of Bombyx mori, common in polyploids, Danilova (1973 b) found that "large distended vesicles or vacuoles" replaced the lamellar bodies (which were initiated in the same manner) present in normal nuclei. She suggested that vacuoles were formed only when intranuclear pressure was reduced, and that lamellar bodies seen in normal nuclei resulted from the pressure of the "nuclear sap" on similar structures causing them to collapse and fold. No such lamellar bodies were seen in the plant meiocytes examined, but neither were there any indications that the formation of nuclear vacuoles was associated with a degenerative process. Material remaining on the plants completed normal sporogenesis. Nuclear vacuoles thus appear to be a normal feature of meiotic prophase and there is, in this respect, a striking parallel with the observations of Rasmussen (1976) on female Bombyx. Furthermore, in both Lycopersicon and Bombyx the initiation of nuclear vacuoles coincides with the completion of synaptonemal complex formation. In Bombyx, Rasmussen (1976) proposed that additional inner membrane was synthesized in the region of synaptonemal complex attachment, the excess membrane being taken up by the formation of vacuoles at the opposite pole of
Fig. 1. Early prophase meiocyteof Dryopteris borreri showing the developmentof three nuclear vacuoles (arrows). x 13,000 Fig. 2. Early prophase meiocyteof Marsilea vestita illustrating the invagination of the inner membrane of the nuclear envelope to produce a nuclear vacuole (arrow), and a vacuole with several membrane profiles (NV). x 56,000 Fig. 3. Meiocyteof Pteridium aquilinumat diplotene/diakinesisshowing a large nuclear vacuole (NV) containing electron-opaquematerial. x 7,500. Inset. Border between such a vacuole and the cytoplasm, illustrates the similarity in appearance of the vacuolar contents and the cytoplasm. C, cytoplasm, NE, nuclear envelope, NV, nuclear vacuole. • 27,000 Fig. 4. Pachytenemeiocyteof Lycopersicon esculentumshowing the invagination of the inner membrane of the nuclear envelope to produce nuclear vacuoles (arrows). SC, synaptonemal complex. • Inset. Late prophase meiocyte of Pinus banksiana where the nuclear vacuole contains electron-opaquematerial. 35, I00
the nucleus. The movement of telomeres over the nuclear envelope which was associated with these dilatations was, in the view of Rasmussen, brought about by a flow of the inner membrane. Although no polarity of vacuole distribution was observed in Lycopersicon, the coincidence between the appearance of these vacuoles and the redistribution of the chromosomes after synapsis suggests that the two phenomena are causally related. In ferns, however, the initiation of nuclear vacuoles is considerably earlier, at the onset of chromatin condensation at leptotene. The vacuoles enlarge and ramify throughout the nucleus before chromosome pairing begins, and it therefore seems unlikely that the significance of these formations lies entirely with chromosome movement in the ferns. It is noteworthy in this respect that there are indications that the vacuoles may eventually become detached from their initiation sites and come to lie free in the karyoplasm. Since, in the later stages of prophase, the vacuoles were invariably found to lie within a nucleus bounded by a continuous envelope of two membranes (Fig. 3 and inset), it seems unlikely that they could be involved in the process of chromosome movement during this time, The presence of this barrier between the vacuoles and the cytoplasm, and the apparent absence of any continuity between the two also suggest that the resemblance between the electronopaque granules and strands within the vacuoles and the ground cytoplasm in Pteridium is purely accidental. This suggestion is strengthened by the evidence from Pinus, where the vacuolar contents resemble those of Pteridiurn (compare Fig. 3, inset and Fig. 4, inset) but where the cytoplasm presents an altogether different appearance. The question therefore arises of the nature and origin of the electron-opaque contents of the vacuoles. Two very different origins are possible for this material: the first being the ingress of substances from the cytoplasm via the perinuclear space, and the second, the accumulation of material diffusing in from the karyoplasm across the vacuolar membranes. The endoplasmic reticulum is known to be in continuity with the outer membrane of the nuclear envelope, and such continuity would clearly allow the passage of material in from the cytoplasm via the interlamellar space of the endoplasmic reticulum. Two further possibilities then present themselves. The first is that the incoming material is that which is being preserved by its inclusion in the nucleus at a time when other elements of the cytoplasm are being subjected to considerable elimination and possible reorganization (Sheffield and Bell, in press). The second, that the vacuoles are involved in the uptake of materials which
E. Sheffield et al. : Nuclear Vacuoles during Meiosis in Plants
are then subjected to degradation within the nucleus. Evidence relating to these alternatives is not yet available, but it should be noted that connections between the endoplasmic reticulum and the nuclear envelope were not conspicuous in the meiocytes examined, and the opportunity for flow into the nucleus may therefore be limited. The possibility that the content of the vacuoles is derived originally from nuclear material again gives rise to the problem of whether this material is thereby preserved of degraded. In incompatible fusions of plasmodia of Physarum polycephaIum, it has been found that 'blebbing' within the nuclear envelope accompanies the elimination of the nucleus of the sensitive strain (Birgitte Lane and Carlile, 1979). The lamellar bodies formed during spermatogenesis in Bombyx have been similarly implicated in the elimination of nuclear material (Danilova and Vereiskaya, 1970). It is therefore conceivable that the accumulations within the vacuoles represent nuclear material which is undergoing degradation or reorganization in the plants examined in the present study. This is a particularly attractive hypothesis in view of the events occurring in the cytoplasm at this time, where it has been shown that the reorganization occurring is accompanied by the elimination of ribosomes (see, for example, Dickinson and Heslop Harrison, 1977; Sheffield and Bell, in press) and possibly also other organelles (Pettitt, 1978). The restandardization of the non-genetic components of the nucleus in preparation for the gametophyte generation might therefore involve the elimination of those associated specifically with sporophytic growth. The development of nuclear vacuoles during prophase can, from the evidence presented, be taken as a regular feature of the meiotic stage of homosporous sporogenesis, megasporogenesis and microsporogenesis. The establishment of the identity of the contents of these vacuoles therefore presents an urgent cytochemical problem, relevant both to the mechanism of meiosis, and the associated change in the phase of growth.
References Aldrich, H.C., Vasil, I.K. : Ultrastructure of the postmeiotic nuclear envelope in microspores of Podocarpus macrophytlus. J. Ultrastruct. Res, 32, 307-315 (1970) Birgitte Lane, E., Carlile, M.J. : Post-fusion somatic incompatibility in plasmodia ofPhysarumpolycephaIum. J. Cell Sci. 35, 339-354 (1979) Danilova, L.V.: Electron microscopic study of meiosis in diploid male silkworms. Ontogenez 4, 40-48 (1973a)
E. Sheffield et al. : Nuclear Vacuoles during Meiosis in Plants Danilova, L.V. : An electron microscope study of meiosis in oligopyrene spermatocytes of the silkworm. Ontogenez 4, 281-287 (1973b) Danilova, L.V., Vereiskaya, V.N.: Lamellar bodies in spermatogenesis in the silkworm Bombyx mori. Ontogenez 1, 360-367 (1970) Dickinson, H.G, Heslop Harrison, J.: Ribosomes, membranes and organelles during meiosis in angiosperms. Philos. Trans. R. Soc. London, Ser. B 277, 327 342 (1977) Godward, M.B.E., Peristianis, G.C. : An electron microscope autoradiographic investigation of the uptake of [3H]thymidine into the pachytene nucleus. In: Current Chromosome Research. pp.. I85-194, Jones, K., Brandham, P.E., eds. North Holland: Elsevier 1976
601 Meek, G.A., Moses, M.J.: Microtubulation of the inner membrane of the nuclear envelope. J. Biophys. Biochem. Cytol. 10, 121-131 (1961) Pettitt, J.M. : Regression and elimination of cytoplasmic orga~elles during meiosis in Lycopodium. Grana 17, 99-105 (1978) Rasmussen, S.W. : The meiotic prophase in Bombyx mori females analysed by three-dimensional reconstructions of synaptonemal complexes. Chromosoma 54, 245-293 (1976) Sheffield, E., Bell, P.R.: Ultrastructural aspects of sporogenesis in a fern, Pteridium aquilinum (L.) Kuhn. Ann. Bot. (in press)
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