Endopolyploidization patterns in non-generative antheridial cells in mono- and dioeciousChara spp. (Characeae) with different DNA C-values

Cytophotometric measurements of nuclear DNA contents and morphometric analyses in the antheridia of five species ofChara indicate that the level of endopolyploidy plays an important role in determining the maximum sizes that shield cells, manubria, and capitular cells attain at the final stage of spermiogenesis. Dioecious species with low DNA C-values—C. aspera andC. tomentosa—display higher values of endopolyploidy: their male sex organs are larger and the quantities of spermatozoids per antheridium are greater than those estimated for monoecious species—C. vulgaris, C. fragilis, andC. contraria. Two types of nucleotypic effects were found to synchronize developmental traits in germ line cells and non-generative component parts within the antheridia; both are discussed in relation to the biological productivity of male sex organs in mono- and dioecious species ofChara.     

Symplasmic isolation ofChara vulgaris antheridium and mechanisms regulating the process of spermatogenesis

Summary The antheridium ofChara vulgaris L. is connected by plasmodesmata with the thallusvia a basal cell. Prior to the initiation of spermatozoid differentiation these plasmodesmata are spontaneously broken, resulting in symplasmic isolation of the antheridium.Premature plasmolytically evoked symplasmic isolation of the antheridium leads to a 2–4 fold reduction in the length of antheridial filaments and the elimination of 1–2 cell cycles from the first stage of spermatogenesis.Autoradiographic and cytophotometric studies have shown that, as a result of induced symplasmic isolation of the antheridium, endomitotic DNA synthesis was blocked both in the young manubria (after 24 hours) and in the capitular cells (after 48 hours). In the antheridial filaments DNA synthesis was inhibited together with either elimination of divisions and induction of spermatid differentiation or developmental block. We propose that breakage of plasmodesmata connecting the antheridium with the thallus is a signal which releases, in all antheridia, mechanisms that (i) block endomitotic DNA synthesis in the manubria, (ii) restrict the growth rate and the divisions of antheridial filament cells, and (iii) induce spermiogenesis in these antheridia in which the manubria attained the sufficient level of polyploidy.This work is supported by the Polish Academy of Sciences within the project CPBP 04.01.5.05.     

GA3 content in antheridia of Chara vulgaris at the proliferative stage and in spermiogenesis estimated by capillary electrophoresis

In male sex organs of Chara vulgaris L., the gibberellic acid (GA3), was identified by capillary zone electrophoresis. The antheridia at cell division stage of antheridial filaments leading to formation of spermatids contain 0.09 microg GA3 per antheridium, i.e. 5.3 times more than antheridia at differentiation stage of spermatozoids (spermiogenesis). Spermiogenesis is not regulated by gibberellins.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. I. Manubria

During the development of an antheridium DNA content in manubria gradually increases to 8C-16C level. 3H-thymidine incorporation into the nuclei of the manubria lasts till the stage of quantitative predominance of the 16 celled antheridial filaments. The nucleus of the manubria is characterized by the low content of the condensed chromatin and the presence of nucleoli with the nucleolonema-like structure the number of which increases from 6-8 to 32-38 along with the increase of DNA content in a nucleus. In the cytoplasm of the manubria there are numerous secretive vesicles filled with fine-granular substance discharged outside plasmalemma, active Golgi apparatus, well-developed rough ER, numerous polysomes, mitochondria with the condensed structure and plastids with granar and inter-granar thylakoids as well as plastoglobules which increase in number and size along with the development of the antheridium. During spermiogenesis the cells are vacuolated, the number of the secretive vesicles decreases whereas the electron density of their content increases, smooth ER appears while rough ER is reduced. The manubria actively incorporate 3H-uridine, 3H-tryptophane and 3H-leucine. The increase of the incorporation activity is gradual in the period of increasing polyploidy of the manubria and rapid during the initiation of the spermatozoid differentiation. It has been suggested that the manubria should play an important role in the process of spermatogenesis and the induction of spermatozoid differentiation.     

Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin

Translocation of [¹⁴C]gibberellic acid into antheridial cells of Chara vulgaris L. was investigated in relation to the presence of symplasmic connections between the antheridium and the thallus. It was found that manubria, capitular cells, and antheridial filaments were about three-fold more strongly labelled in young antheridia connected to the thallus by plasmodesmata than in older antheridia in which spontaneous symplasmic isolation had occurred. Plasmolytically induced symplasmic isolation of young antheridia severely diminished the radioactivity of all the cells, down to the level characteristic for spontaneously isolated antheridia. It is concluded that plasmodesmata are the main channel of gibberellin transport into antheridia. The change in the character of symplasmic connections during the course of morphogenesis might, among other events, constitute a signal determining a shift of cell metabolism in a new direction, in response to a rapid change in gibberellin level.Abbreviations GA_(n) gibberellin (A_n) - GA₃ gibberellic acid - IAA indole-3-acetic acid This study was supported by the Polish Academy of Sciences research project CPBP 04.01.5.05.     

Seasonal patterns of cell-to-cell communication in Chara corallina Klein ex Willd. II. Cell-to-cell communication during the development of antheridia

Cell-to-cell communication has been studied in lateral branches and developing antheridia of male Chara corallina plants. The moving cytoplasm is specialized to include essentially separate ascending and descending cytoplasmic streams within the inter-nodes. The neutral line which demarcates the ascending from the descending stream is established by the divisions of the nodal initial, which gives rise to both the node and internode. The ascending stream is located beneath the first-formed node-cells and the descending stream beneath the last-formed cells. The cells destined to develop into antheridia were always located on the same side as the descending internodal stream, and thus, were derived from the cells last formed during divisions of the nodal initial. Three stages of anther idial development have been defined: (1) young antheridia from the initial division of a node-cell to the formation of an octant structure; (2) maturing antheridia where differentiation into shield, manubria and capitular cells has occurred, including antheridia where an internal cavity has formed but contains filaments of less than 32 cells; and (3) mature antheridia where filaments contain more than 32 cells and spermatid production commences. Internodal cells of branches bearing young antheridia had similar characteristics to spring branches, including high plasmalemma potential differences (-217·7±31·5mV, [K⁺]_o 0·5 mol m^?3; pH 7·6) and extensive cell-to-cell communication (frequency of intercellular transport of 6 carboxyfluorescein 86%). The small probe 6 carboxy fluorescein moved into the entire young antheridium in 100% of injections. The molecular exclusion limit for internodes and the nodal complex lay between 874 and 1678Da whereas the exclusion limit for the young antheridium was smaller (between 750 and 874Da). Internodal cells of branches bearing maturing antheridia had similarly high PDs (–221·7±40mV; [K⁺]_o 0·5 mol m^?3; pH 7·6). Cell-to-cell communication between internodes bearing maturing antheridia was extensive (frequency of intercellular transport of 6 carboxyfluorescein 100%). The shield cells were isolated from the symplast of the thallus at this stage since they did not admit 6 carboxyfluorescein. Internodal cells of branches bearing only mature antheridia showed different characteristics. Intercellular communication between internodes was restricted to a level similar to that found in winter (frequency of intercellular transport of 6 carboxyfluorescein = 57%). The mature antheridium was entirely isolated from the symplast of the thallus. A period of extensive cell-to-cell communication and high PDs in internodal cells commences in vegetative lateral branches in spring, immediately before reproductive structures are initiated. These features persist throughout summer whilst reproductive structures develop, until the antheridial filaments contain 32 or more cells (mature stage), at which point spermatid production commences and the antheridium is isolated from the thallus. In autumn, following the stage of mature antheridia, no further antheridia are initiated. Internodes are subsequently vegetative throughout winter and their lateral branches are characterized by restricted cell-to-cell communication, low internodal PDs, and little obvious growth, all features consistent with winter dormancy.     

Disappearance of 3H-leucine incorporation circadian rhythm induced by symplasmic isolation of Chara vulgaris antheridium from thallus.

Autoradiographic research with the use of 3H-leucine demonstrates that circadian rhythm of protein synthesis characteristic of manubria at the proliferative phase of spermatogenesis in Chara vulgaris disappears after symplasmic isolation of antheridium from thallus during the time preceding block of DNA endoreplication in manubria following initiation of spermiogenesis insensitive to light.     

Effect of GA3 Treatment on the Number of Spermatozoids and Endopolyploidy Levels of Non-Generative Cells in Antheridia of Chara vulgaris L.

The youngest nodes located under an apical bud of Chara vulgariswere isolated and cultivated in the presence or absence of 10^–5M GA₃ under laboratory conditions to form spermatozoids. GA₃increased the DNA C-value in manubria by 20% and increased thenumber of spermatozoids per antheridium over 2-fold. (Received April 27, 1998; Accepted October 8, 1998)     

Plasmodesmal changes are related to different developmental stages of antheridia of Chara species

Summary During the development of the antheridia of Chara species, dynamic changes in the occurrence and ultrastructure of plasmodesmata are observed which are closely correlated to particular developmental phases and presumably regulate the morphogenetic events in the antheridia. The disappearance of plasmodesmata between shield cells and between shied cells and the basal cell leads to a cessation in symplasmic transport around the antheridum and determines its concentric or centrifugal character via centrally situated capitular cells. Unplugged plasmodesmata are present between fully synchronously developing antheridial filament cells and obviously coordinate the development of the cells. In the middle phase of spermiogenesis, rough endoplasmic reticulum in antheridial filaments passes uncompressed through wide plasmodesmata and provides an additional transport pathway for developmental control factors. Plugged plasmodesmata link cells of different types or cells of the same type which are at different phases of cell cycle and guarantee their individual development. The plugging of plasmodesmata is a reversible process that depends on the morphogenetic situation. Plasmodesmata connecting the basal cell and the subbasal cell as well as the basal cell and capitular cells are transformed successively from the simple into the complex type and might be the pathways for an import of gibberellins and nutrients into the strong sink tissues of the developing antheridium. There is a symplasmic connection between the antheridum and the thallus via a basal cell. Prior to the initiation of spermatozoid differentiation (spermiogenesis), plasmodesmata connecting the basal cell with a subbasal cell and the basal cell with capitular cells are spontaneously broken, resulting in symplasmic isolation of the antheridium that is probably a signal which triggers the induction of spermatozoid differentiation. Premature plasmolytically evoked symplasmic isolation of the antheridium leads to the elimination of 1 to 2 cell cycles from the proliferative stage of spermatogenesis. Autoradiographic studies demonstrate that both natural and induced symplasmic isolation drastically decreases the entry of isotopically labeled gibberellic acid into antheridia of Chara species that may be the consequence of the elimination of the hormone's transport through plasmodesmata.Correspondence and reprints: Department of Cytophysiology, University of ód, ulica Pilarskiego 14, 90-231 ód, Poland.Received March 11, 2002; accepted September 19, 2002; published online August 26, 2003     

SCANNING ELECTRON MICROSCOPY OF ANTHERIDIA AND ARCHEGONIA OF ANEMIA MEXICANA KLOTZSCH

Antheridia and archegonia of the fern Anemia mexicana were viewed with scanning electron microscopy (SEM). The mature antheridium is composed of a cap, ring, and basal cell with spermatozoids inside. The archegonium neck is composed of a neck canal cell surrounded by four rows of neck cells. The ventral canal cell and egg were not observed. The neck bends toward the notched meristem. The neck cells usually are uniform in shape and arrangement, but in some archegonia, shape and arrangement of neck cells was irregular. The apex of these archegonia often appeared swollen because of the random cell arrangement. In the presence of water, the antheridium cap is partially detached and the spermatozoids emerge. At this time, the neck cells open at the end of the archegonium in preparation for fertilization. The basic morphology of the antheridia and archegonia is similar to previous reports, although SEM provides more structural detail and a better three-dimensional visualization of these reproductive structures.     

葫芦藓精子发生过程中胞间连丝与胞质桥的超微结构

从超微结构水平上对葫芦藓(Funaria hygrometrica Hedw.)精子发生过程中胞间连接系统的结构及其变化动态进行了研究.结果表明,同一区中的相邻生精细胞由大量胞质桥相连,而不同区的细胞之间则不存在胞质桥.胞间连丝存在于套细胞之间以及套细胞与生精细胞之间,但它在生精细胞间不存在.在精子器发生的后期,当精子细胞壁开始降解时,同一个精子器中所有的精子细胞似乎都由扩大的胞质桥相互连接.胞质桥一直保持到精子分化的后期,最终精子细胞同步分化成精子.胞间连丝与胞质桥具有不同的内部结、分布以及生物发生机制,这表明它们在精子器的发育过程中可能扮演着不同的角色.     

Differentiated reaction of different types of antheridial cells in Chara vulgaris to the changes in light conditions during 24 hours.

Circadian rhythm of activity of 3H-leucine incorporation into antheridial cells of Chara vulgaris, in natural photoperiod was compared with changes in mitotic activity of antheridial filament cells which form spermatozoids. Three types of cells functionally connected with each other i.e. manubria, capitular cells and antheridial filaments indicate high amplitude (80-90%) changes in circadian translational activity and some similarities in their course. The shield cells are characterized by small circadian changes in translational activity in the range of 15-30% and their different rhythm. Manubria, which are the secretive cells indicated the highest dependence of the dynamic of translational activity on the time of day. Their high activity overlaps light phase, low activity--dark phase. The reaction of capitular cells to day/night change is delayed in comparison with the reaction of manubria, and that of antheridial filaments is delayed in comparison with the capitular cells reaction. The assumption was set forth that manubria play the role of oscillators (starttercells) which induce the wave of changes translocating to the other cells functionally and spatialy connected with them. The course of the wave of antheridial filaments mitotic activity suggests that a distinct drop in MI in the morning may be the result of the lack of the factors necessary for initiation of the mitosis, dependent on light-induced high translational activity of antheridial cells.     

Different Patterns of Intercellular Transport of Lucifer Yellow in Young and Mature Antheridia of Chara vulgaris L.

Intra- and intercellular movement of the fluorochrome Lucifer Yellow (LY), microinjected into the shield cells of male sex organs, was examined at different stages of spermatogenesis in Chara vulgaris L. Two distinct stage-associated types of probe movement were noted: (I) a fast and uniform spreading of fluorescence within the shield cell injected, followed by centripetal transport of LY into the adjoining manubrium and the capitular cells, and subsequent redistribution into the other manubria and shield cells in young antheridia (up to the stage of 4-cellular filaments), and (II) limited spreading of fluorescence, restricted to the triangle-shaped area of the impaled shield cell in more mature antheridia with multicellular filaments. These results confirm earlier electron microscopic observations on plasmodesmatal connections indicating an intensive solute movement between all cell types in young antheridia of Chara. Developmental changes appearing at later stages, during differentiation of sperm cells, strongly reduce the capacity for intercellular symplasmic transport.     

Do motile spermatozoids limit the effectiveness of sexual reproduction in bryophytes? Not in the liverwort Marchantia polymorpha

To test the premise that fertilization distances in bryophytes are limited by spermatozoid motility, we measured the distances between over 80 000 male and female thalli of Marchantia polymorpha on heathland over 2 successive years after a major fire and recorded the incidence of sporophytes. The first comparative data on spermatozoid numbers per antheridium across bryophytes and pteridophytes were calculated from antheridial sizes and spermatid diameters and cross‐checked with the likely numbers produced by successive antheridial mitoses. Individual antheridia of Marchantia produce over 200 000 spermatozoids. Extrapolating from individual antheridia to the numbers of mature antheridia per antheridiophore and then to individual male plants, we calculated that a single flooding event on a male thallus with 10–12 antheridiophores will result in the release of over 50 million spermatozoids. Assuming radial dispersal in surface water films by lipids released from the dehiscing antheridia, spermatozoids can reach distances exceeding 20 m from the parent plants, in line with our finding of 100% fertilization in female plants of Marchantia up to 19 m from the nearest males, far beyond published fertilization distances in dioicous bryophytes. We attribute this to (i) much greater spermatozoid production and numbers in Marchantia than in other bryophytes and (ii) highly effective sperm transport both within the antheridiophores and archegoniophores via their overlapping scales and grooves in the stalks and between male and female thalli via surface water films. These features, coupled with the massive production of small spores, explain the success of Marchantia as the primary colonist of open habitats.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris L. III. Shield cells

Shield cells form the antheridium envelopes. At the first stage of spermatogenesis they grow intensively in the tangential direction, which is stopped during the period of spermatozoid differentiation. The increase in shield cell volumes is associated with the increase in DNA level in the nucleus up to 16-32 C. 3H thymidine incorporation occurs in about 30% of shields at younger developmental stages and lasts until the stage in which 16 celled antheridial filaments predominate. At first stage of spermatogenesis the intensity of 3H leucine incorporation increases as DNA amount in the nuclei increases, reaching the maximum value at the end of this period. During spermiogenesis it gradually decreases. Shield nuclei are characterized by low content of condensed chromatin, the presence of numerous nucleoli with nucleolonema-like structure as well as the occurrence of bands of intranuclear microtubules. It has been suggested that these microtubules are associated with cyclical changes in the shapes of nuclei. During DNA replication the nuclei have the form of flat discs which between successive endoreplication cycles become ring shaped. Peripheral zone of shield cells is compartmentalized through incomplete walls. They support the radial walls of shields increasing the contact surface of plasmalemma with a cell wall. During spermiogenesis the increase in plasmalemma surface results from the growth of shields in the radial direction. The shield cells contain plastids placed close to each other at the inner tangential wall. They are orange in colour and have fully formed system of grana and intergrana thylakoids, like the plastids of the thallus. The number and sizes of the plastoglobules increase as the anteridium develops. Dictiosomes are surrounded with numerous smooth and coated vesicles. Mitochondria exhibit poorly condensed structure. Microbodies adjoining the plastids are sporadically encountered. It has been assumed that changes in structural organization as well as growth character of shield cells constitute the factor regulating the exchange with external environment, determine light spectrum penetrating to the antheridium and the volume of antheridial space.     

Cell-specific endopolyploidy in developing Artemia

Summary Cells in developing Artemia franciscana SFB demonstrated tissue-specific differences in DNA content, as determined by fluorescence intensity of bisbenzimide-stained nuclei and by nuclear area. The general epidermis comprised proliferating diploid (2C) cells. The setal cells had 4C–8C DNA content and did not divide during the first two instars. Salt gland cells were polyploid (>8C) and also did not undergo mitosis. Neural cells in the brain were diploid and were replicating. Cells in the thorax region of the gut had a 4C–8C DNA content and were proliferating. The muscle cells in the cephalic appendages contained 2C non-replicating nuclei. Only diploid epidermal cells were involved in segment morphogenesis. There was no difference in number of chromosomes (n=42) in the epidermal cells and the gut cells, indicating that the tissue-specific endopolyploidy was due to endoreduplication.     

Cytochemical studies on the antheridial mucilage and changes in its concentration and amount during the spermatogenesis in Chara vulgaris L.

The internal space of the antheridium in Chara vulgaris L. is filled with the PAS-positive mucilage which is of pectic nature. Morphometric and cytophotometric measurements on the semithin sections indicate that the concentration and amount of PAS-positive polysaccharides: 1) increase during the time of antheridial growth accompanying the phase of antheridial filament divisions, 2) these parameters have the maximum after spermatid formation and at the beginning of their differentiation, i.e. spermiogenesis, 3) both concentration and amount of this substance decrease at the end of spermiogenesis. A decrease in mucilage concentration is also observed in the young antheridia after 3 days of continuous darkness. The results suggest that PAS-positive mucilagenous material is a nutritive substance, accumulated in the first phase of antheridial development and utilized mainly in spermiogenesis. These substances may also be used up in the young antheridia during the lack of energy supply. The autoradiographic studies with the use of a 3H-glucose and 3H-galactose mixture seem to confirm these suggestions.     

Immunofluorescent localization of cytoskeletal tubulin and actin during spermatogenesis inPteridium aquilinum (L.) Kuhn

Summary Details concerning the appearance and behaviour of blepharoplasts during spermatogenesis, and the assembly of the cytoskeletal motile apparatus of spermatids were elucidated by immunofluorescence microscopy using antibodies to tubulin and actin, applied to material prepared from antheridia of the fernPteridium aquilinum (L.) Kuhn. Blepharoplast immunofluorescence with antitubulin first appears as spheres at the future spindle poles prior to the last spermatogenous division. Developing spermatids each have one blepharoplast, which gives rise to a triangular layer corresponding to the incipient microtubule ribbon. Compared to the ribbon, immunoreactivity of the multilayered structure is relatively weak. Intensely fluorescing basal bodies appear, increase in number, and become arranged in rows along two edges of the microtubule ribbon as it widens and elongates. Along the dorsal edge is a dense file of basal bodies spaced at about 0.3 m intervals, parallel to each other and oriented at 145° to the multilayered structure. This spacing and orientation is maintained throughout spermatid development. Basal bodies at the opposite edge are initially oriented at 115° to the multilayered structure but become rearranged into small groups that rotate so that the angle is reduced to 55–70° by the time the assembly of flagella commences on both sets of basal bodies. By this stage the microtubule ribbon has encircled about 2/3 of the nuclear circumference and the nucleus is assuming a crescent shape. In fully developed spermatozoids the groups of basal bodies are oriented at 25° to the multilayered structure, parallel to the long body of the now helical nucleus. Immunofluorescence using antiactin showed that towards the completion of nuclear shaping, actin forms a strip along the helical multilayered structure. Detergent-extraction of mature spermatozoids revealed that actin is associated also with the flagellar band, particularly with basal bodies.Abbreviations MLS multilayered structure - MT microtubule     

Cytochemical studies on histone-type and protamine-type proteins during spermiogenesis in Chara vulgaris and Chara tomentosa

Comparative studies concerning detection of histone-type and protamine-type proteins were carried out on Chara species (C. vulgaris, C. tomentosa). Analysis of antheridia during spermiogenesis (stages I-X) of both Charta species showed very similar staining patterns obtained after reactions revealing the examined proteins. Cytochemical studies showed a replacement of lysine-rich histone proteins by more basic arginine-rich ones during medium spermiogenesis (st. VI-VIII) in two Charta species, while late spermiogenesis (st. IX) and mature spermatozoids (st. X) were characterised by the presence of protamine-like proteins only.     

Characterization of sexual reproductive processes in Chara braunii (Charales,Charophyceae)

Chara braunii is distributed worldwide and is the most common charalean species in Japan. This species is monoecious and produces numerous sets of sex organs, each of which consists of one antheridium and one oogonium, under laboratory culture conditions. In this study, we report that light intensity strongly affected the vegetative phase and sexual reproductive phase of this species. Under high‐light conditions (70.0 μmol photons m^?2 s^?1), thalli grew but did not form reproductive organs. Under a low‐light intensity (10.0 μmol photons m^?2 s^?1), algal bodies formed many reproductive organs. In addition, antheridia without the corresponding oogonia (lone antheridia) were observed under low‐light conditions. The absence of oogonium primordia adjacent to the lone antheridium was confirmed by several microscopic approaches. The addition of liquid fertilizer increased the total number of sex organs and growth; however, the number of lone antheridia decreased with increasing fertilizer concentrations. Exogenously applied gibberellin did not affect the number of lone antheridia. These results suggest that regulatory mechanisms for the appropriate allocation of resources exist in this alga, similar to those reported in some land plants.