FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

PRODUCTION OF MALE GAMETES AND AUXOSPORES IN THE CENTRIC DIATOMS CYCLOTELLA MENEGHINIANA AND C. CRYPTICA12

Vegetative cells of Cyclotella meneghiniana Kützing form male gametes and anxospores following transfer from a synthetic freshwater medium to a modified artificial seawater. Both spermatogenesis and auxospore formation are correlated with an increase in the Na⁺ concentration in the medium. Spermatogenesis can also be induced in C. cryptica Reimann, Lewin, and Guillard in artificial seawater with an adjusted sodium level.     

THE SEXUAL AND ASEXUAL LIFE CYCLE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The sexual and asexual portions of the life cycle of Glenodiniopsis steinii were examined at both the light and scanning electron microscopic levels. Asexual reproduction by cell division occurs every 2 to 3 d under optimal conditions. Gamete formation and sexual reproduction can be induced by either nitrogen deprivation or aging of cultures. Fusion of gametes peaks about 10 hr into the light cycle and requires about 10 hr for completion. Cells remain motile during fusion but the zygote loses motility. The cell then undergoes changes that give it a thick-walled, warty appearance.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

PHOTOINHIBITION OF PSII IN EMILIANIA HUXLEYI (HAPTOPHYTA) UNDER HIGH LIGHT STRESS: THE ROLES OF PHOTOACCLIMATION,PHOTOPROTECTION, AND PHOTOREPAIR1

The response of the coccolithophorid Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler to acute exposure to high photon flux densities (PFD) was examined in terms of PSII photoinhibition, photoprotection, and photorepair. The time and light dependencies of these processes were characterized as a function of the photoacclimation state of the alga. Low‐light (LL) acclimated cells displayed a higher degree of photoinhibition, measured as decline in F_v/F_m, than high‐light (HL) acclimated cells. However, HL cultures were more susceptible to photodamage but also more capable of compensating for it by performing a faster repair cycle. The relation between gross photoinhibition (observed in the presence of an inhibitor of repair) and PFD to which the algae were exposed deviated from linearity at high PFD, which calls into question the universality of current concepts of photoinhibition in mechanistic models. The light dependence of the de‐epoxidation state (DPS) of the xanthophyll cycle (XC) pigments on the timescale of hours was the same in cells acclimated to LL and HL. However, HL cells were more efficient in realizing nonphotochemical quenching (NPQ) on short timescales, most likely due to a larger XC pool. LL cells displayed an increase in the PSII effective cross‐section (σ_PSII) as a result of photoinhibition, which was observed also in HL cells when net photoinhibition was induced by blocking the D1 repair cycle. The link between σ_PSII and photoinhibition suggests that the population of PSII reaction centers (RCIIs) of E. huxleyi shares a common antenna, according to a “lake” organization of the light‐harvesting complex.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi

Allophanate lyase can be induced by urea or acetamied 20–40-fold within 4 h in NH₄⁺-deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

LIFE CYCLE OF A STEPHANODISCUS SP. (BACILLARIOPHYTA)1

Studies of the life cycle of a centric diatom, tentatively identified as Stephanodiscus neoastraea Håkansson & Hickel, showed that sexual reproduction occurred every year in a freshwater lake (Lough Neagh, Northern Ireland). Male and female gametes were produced in cells below 55% of the maximum diameter during a 3–4-week period in late summer, following the return of nitrate concentrations above 10 μM NO_3-N. The frequency of sexual reproduction was linked to the cycle of diameter size reduction and regeneration. The times of largest decreases in cell diameter were during nutrient stress in summer and low light conditions in late autumn, rather than during the main spring growth period. So, environmental conditions (combined with the limited life-spans of individual cells) affected the rate of diameter reduction and, therefore, the length of the life cycle (3–4 years).     

CHANGE OF UBIQUITINATED PROTEINS DURING THE CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYTA)1

Changes in the amount of heat shock-related ubiquitinated proteins in Chlamydomonas were investigated during the cell cycle and gamete induction. In a division-synchronized culture induced by periodic illumination, the amount of the 28-kDa ubiquitinated protein increased during the dark phase. This increase correlated with the increase of total DNA. Such an increase was repressed when nuclear DNA replication was inhibited with aphidicolin. These results suggest that ubiquitination to form the 28-kDa protein is involved in nuclear DNA replication or during the cell cycle. The amount of 31-kDa ubiquitinated protein gradually increased throughout the light phase and decreased in the dark phase. The amount of 28-kDa ubiquitinated protein also increased during gamete induction caused by nitrogen starvation, while that of the 31-kDa did not. These results suggest that the change of ubiquitination of 28-kDa protein mat play a fundamental role in the cell cycle and gamete induction in Chlamydomonas.     

GAMETOGENESIS AND GAMETE RELEASE OF ULVA MUTABILIS AND ULVA LACTUCA (CHLOROPHYTA): REGULATORY EFFECTS AND CHEMICAL CHARACTERIZATION OF THE “SWARMING INHIBITOR”1

Gametophytes of Ulva mutabilis Føyn and Ulva lactuca L. were artificially induced to form gametangia by removal of sporulation inhibitors. After this treatment, U. mutabilis gametes were ready for swarming on the third morning after induction, while U. lactuca gametangia needed 1–2 d longer for maturation. Release of gametes of U. lactuca was dependent solely upon exposure to the first light in the morning. Gametangia of U. mutabilis, however, also required sufficient dilution of the swarming inhibitor (SWI). SWI was excreted transiently by both Ulva species early during gametogenesis. While the SWI concentration in U. mutabilis medium remained above the inhibitory concentration until the gametangia were mature, the concentration of U. lactuca‐SWI dropped rapidly below this level. In the presence of sufficient SWI, mature gametes of U. mutabilis remained motionless within the gametangia despite light and open exit pores. However, using SEM, an additional seal was detected within these pores, which probably prevented premature swarming until dilution of SWI and exposure to light. Observations by time lapse microscopy and experiments with the myosin kinase inhibitor BDM suggest that the gametes may be either extruded by the gametangium or leave the exit pore by active gliding motion, driven by a myosin‐like motor protein. The SWIs were purified from both Ulva species, and mass spectral analysis showed their molecular masses (292 Da) were identical.     

ENCYSTMENT OF THE DINOFLAGELLATE GYRODINIUM UNCATENUM: TEMPERATURE AND NUTRIENT EFFECTS1

Sexual reproduction and encystment of the marine dinoflagellate Gyrodinium uncatenum Hulburt were induced in nitrogen and phosphorus-limited batch cultures. Sexuality did not occur under nutrient-replete conditions even when growth rate was reduced by non-optimal temperatures. Growth was optimal over a broader temperature range than encystment and virtually no cysts were produced at some low and high temperatures where growth occurred. Most cells initiated sexuality as intracellular pools of each limiting nutrient reached minimum or subsistence levels as much as four days after extracellular nutrients were exhausted. High nitrogen cell quotas during the phosphorus experiment indicate that sexuality was induced by a shortage of phosphorus and not by an indirect effect on nitrogen uptake. Total cyst yield corresponded to successful encystment of 9–13% of the motile populations, yet 60–85% of the plateau-phase motile cells were planozygotes (swimming zygotes formed from fusing gametes). Batch culture studies monitoring total cyst yield may thus seriously underestimate the extent of sexuality. More importantly, the number of cysts produced in a dinoflagellate population may be significantly reduced by environmental factors acting on the cells after sexual induction and fusion.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.     

A STUDY OF THE SPERMATOZOIDS OF DICHOTOMOSIPHON TUBEROSUS (CHLOROPHYCEAE)1

Spermatozoids of the siphonous green alga Dichotomosiphon tuberosus (A. Br.) Ernst are specialized gametes which differ in many respects from other green algal motile cells, but whose microanatomy nevertheless indicates its chlorophycean affinities. Each cell is anteriorly biflagellate and contains an irregularly shaped nucleus attached to the flagellar bases by a complex support apparatus. There is a single reduced chloroplast in each spermatozoid and numerous (50–100) minute spherical mitochondria, only 0.3 μm diam. These move vigorously in the living cell and when viewed with the light microscope they bear a striking resemblance to bacteria. Rather unexpectedly, no contractile vacuoles could be detected, even though the gametes are naked freshwater cells. Daring spermatogenesis the nucleoli of the vegetative cells disperse and are replaced by a large dense body presumably formed from either nucleolar material or condensed chromatin. The flagellar apparatus includes a cruciate flagellar root system, a feature now known to be characteristic of most green algae, exceptions being those putative ancestors of the higher plants and bryophytes. Discharge of spermatozoids from the antheridia is extremely rapid and the whole process may be finished in 30 sec. The antheridium lacks a pore apparatus, but at maturity bursts open explosively at the apex. Phyletic affinities are discussed and it is concluded that the ultrastructure of the motile cells does not, at this time, support the separation of the siphonous green algae from other green algae into a separate class.     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

Cell Cycle Regulation by Light in Prochlorococcus Strains

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m⁻² s⁻¹, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G₁ phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G₂ phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

THE EFFECTS OF REPLACEMENT OF CALCIUM BY STRONTIUM ON THE REPRODUCTION OF CHLOROCOCCUM ECHINOZYGOTUM

In liquid inorganic axenic cultures of Chlorococcum echinozygotum containing 20 ppm CaCl₂, which was optimal for growth, motile cells were released in abundance; gametic fusion occurred commonly and the resulting zygospores reached a maximum of 31% of the total cell population. In a Sr-replaeement medium, less growth, not quite equivalent to that with 5 ppm CaCl₂, occurred, and more than 99% of the cells were non-motile vegetative cells. Old Sr-replacement cultures contained much-enlarged vegetative cells which, upon transfer to fresh Sr-medium, produced entrapped motile cells equivalent in number to those produced and released upon transfer to fresh Ca-medium. Release of these entrapped cells, after their production in Sr, was induced in 4 hr by transferring them to Ca (25 ppm CaCl₂).     

ANALYSIS OF GROWTH OF TETRAPLOID NUCLEI IN ROOTS OF VICIA FABA

Growth of nuclei of a marked population of cells was determined from G₁ to prophase in roots of Vicia faba. the cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G₁ and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. of the marked population of cells, about 65% had completed a cell cycle 14–15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

THE LIFE CYCLE OF PEDIASTRUM SIMPLEX1

A sexual cycle and a newly discovered mode of asexual reproduction by azygotes arc described for Pediastrum simplex. In the sexual cycle isogametes from colony cells fuse in pairs, develop into zygotes, which grow and divide, producing motile cells which become angular polyeders. Polyeders may divide internally to form 4-celled colonies, or they enlarge, cleave, and produce zoospores or nonmotile cells. Zoospores are released in vesicles, swarm, adhere to each other, and form, flat colonies I cell thick. The nonmotile cells adhere to each other, enlarge, break out of the polyeder, and become spheroidal colonies. In the asexual cycle, nonfusing gametes, and motile cells derived from colony cells, become 4-spined azygotes which enlarge, divide internally, and release motile cells developing into polyeders. These polyeders develop further like those from zygotes.