Life cycle strategies in free-living unicellular eukaryotes: Diversity, evolution, and current molecular tools to unravel the private life of microorganisms.

An astonishing range of morphologies and life strategies has arisen across the vast diversity of protists, allowing them to thrive in most environments. In model protists, like Tetrahymena, Dictyostelium, or Trypanosoma, life cycles involving multiple life stages with different morphologies have been well characterized. In contrast, knowledge of the life cycles of free-living protists, which primarily consist of uncultivated environmental lineages, remains largely fragmentary. Various life stages and lineage-specific cellular innovations have been observed in the field for uncultivated protists, but such innovations generally lack functional characterization and have unknown physiological and ecological roles. In the actual state of knowledge, evidence of sexual processes is confirmed for 20% of free-living protist lineages. Nevertheless, at the onset of eukaryotic diversification, common molecular trends emerged to promote genetic recombination, establishing sex as an inherent feature of protists. Here, we review protist life cycles from the viewpoint of life cycle transitions and genetics across major eukaryotic lineages. We focus on the scarcely observed sexual cycle of free-living protists, summarizing evidence for its existence and describing key genes governing its progression, as well as, current methods for studying the genetics of sexual cycles in both cultivable and uncultivated protist groups.

Triparental inheritance in Dictyostelium.

Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes, although our knowledge of the molecular details of sexual inheritance is scant in several major lineages. In social amoebae, sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells, but for technical reasons, sexual progeny have been difficult to obtain and study. We report here genome-wide characterization of meiotic progeny in Dictyostelium discoideum We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the Spo11 enzyme that is normally required to initiate crossover formation. Fusions of more than two gametes to form transient syncytia lead to frequent triparental inheritance, with haploid meiotic progeny bearing recombined nuclear haplotypes from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. D. discoideum mitochondrial genomes are polymorphic, and our findings raise the possibility that some of this variation might be a result of sexual selection on genes that can promote the spread of individual organelle genomes during sex. This kind of self-interested mitochondrial behavior may have had important consequences during eukaryogenesis and the initial evolution of sex.

Putative Meiotic Toolkit in the Dinoflagellate Prorocentrum cordatum: Additional Evidence for Sexual Process from Transcriptome.

Prorocentrum cordatum (Ostenfeld) Dodge-is a planktonic armored dinoflagellate that is a bloom-forming, potentially toxic cosmopolitan species. The transition from vegetative reproduction to the sexual process has been recently shown for this organism. Here, we present the results of transcriptomic data analysis that uncovered one syngamy-associated and 16 meiosis-associated proteins in P. cordatum. We also detected an amino acid sequence homologous to bacterial MutS2 protein. The MutS2 presence and origin in dinoflagellates are discussed for the first time.

The molecular foundations of zygosis.

Zygosis is the generation of new biological individuals by the sexual fusion of gamete cells. Our current understanding of eukaryotic phylogeny indicates that sex is ancestral to all extant eukaryotes. Although sexual development is extremely diverse, common molecular elements have been retained. HAP2-GCS1, a protein that promotes the fusion of gamete cell membranes that is related in structure to certain viral fusogens, is conserved in many eukaryotic lineages, even though gametes vary considerably in form and behaviour between species. Similarly, although zygotes have dramatically different forms and fates in different organisms, diverse eukaryotes share a common developmental programme in which homeodomain-containing transcription factors play a central role. These common mechanistic elements suggest possible common evolutionary histories that, if correct, would have profound implications for our understanding of eukaryogenesis.

Did meiosis evolve before sex and the evolution of eukaryotic life cycles?

Biologists have long theorized about the evolution of life cycles, meiosis, and sexual reproduction. We revisit these topics and propose that the fundamental difference between life cycles is where and when multicellularity is expressed. We develop a scenario to explain the evolutionary transition from the life cycle of a unicellular organism to one in which multicellularity is expressed in either the haploid or diploid phase, or both. We propose further that meiosis might have evolved as a mechanism to correct for spontaneous whole-genome duplication (auto-polyploidy) and thus before the evolution of sexual reproduction sensu stricto (i.e. the formation of a diploid zygote via the fusion of haploid gametes) in the major eukaryotic clades. In addition, we propose, as others have, that sexual reproduction, which predominates in all eukaryotic clades, has many different advantages among which is that it produces variability among offspring and thus reduces sibling competition.

Direct cleavage during the first mitosis is a sign of abnormal fertilization in cattle.

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

Observing silkworm embryos at the fertilization stage using a tissue clearing reagent.

There are various reports on embryogenesis in silkworm, Bombyx mori, a model organism for lepidopteran insects. New tissue observation methods have been developed with the development of biological science. Applying these methods to silkworm eggs makes it possible to capture morphological and histological features that have not been observed until now. Tissue transparency technology is a method of observation that has advanced remarkably recently. This study emphasized the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) method. The tissue clearing reagent used in CUBIC is water-soluble, containing urea and amino alcohol, which easily and effectively clears animal tissue by immersing the sample in the reagent. This study showed that silkworm eggs are made transparent using the modified CUBIC method at the fertilization stage. Furthermore, we observed the behavior of egg nucleus, polar body nuclei, and sperm nuclei at the fertilization stage.

Updating embryonic ontogenesis in Araucaria angustifolia: from Burlingame (1915) to the present.

This study addresses gaps in our understanding of pre-fertilization and archegonia development and reinterprets embryonic ontogenesis from Burlingame (Bot Gaz 59:1-39, 1915) to the present based on timescale and structural features allowing us to determine functionally and developmentally accurate terminology for all these stages in A. angustifolia. Different from previous reports, only after pollination, pre-fertilization tissue development occurs (0-13 months after pollination (MAP)) and gives rise to a mature megagametophyte. During all this period, pollen is in a dormant state at the microphyla, and pollen tube germination in nucellus tissue is only observed at the stage of archegonia formation (13 MAP) and not at the free nuclei stage as reported before. For the first time, 14 months after pollination, a fertilization window was indicated, and at 15 MAP, the polyzygotic polyembryony from different archegonia was also seen. After that, subordinated proembryo regression occurs and at least three embryonic developmental stages of dominant embryo were characterized: proembryogenic, early embryogenic, and late embryogenic (15-23 MAP). Along these stages, histochemical and ultrastructural analyses suggest the occurrence of cell death in suspensor and in cap cells of dominant embryo that was not previously reported. The differentiation of meristems, procambium, pith, and cortex tissues in late embryogenic stage was detailed. The morphohistological characterization of pre-fertilization and embryonic stages, together with the timescale of megastrobili development, warranted a referential map of female reproductive structure in this species.

Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically evolving lineages viable by rescuing the incipient eukaryotic lineage from Muller's ratchet. The origin of mitochondria was, in this view, the decisive incident that precipitated symbiosis-specific cell biological problems, the solutions to which were the salient features that distinguish eukaryotes from prokaryotes: A nuclear membrane, energetically affordable ATP-dependent protein-protein interactions in the cytosol, and a cell cycle involving reduction division and reciprocal recombination (sex).

Comparación entre el clivaje temprano y el estadio pronuclear como parámetros de referencia para inducir el embarazo en pacientes ovoreceptoras en Cali (Colombia) / Comparing early cleavage and pronuclear state as reference parameters for inducing pregnancy in embryo implantation patients in Cali, Colombia

Introducción: algunos autores han propuesto métodos no invasivos que se centran en el clivaje temprano del estadio de dos células a las 25 y 27 horas luego de ocurrida la inseminación, los cuales son aplicables a los programas de fertilización in vitro que aumentan la probabilidad de implantación y embarazo. Objetivo: comparar la tasa de embarazo en pacientes ovoreceptoras con embriones provenientes de clivaje temprano con respecto a la del estadio pronuclear. Metodología: se realizó un estudio de cohorte retrospectivo, en el cual se incluyeron a las pacientes ovoreceptoras que asistieron entre abril de 2004 y diciembre de 2007 al Centro de Biomedicina -FECUNDAR en Cali (Colombia) por presentar baja respuesta al ciclo de estimulación ovárica controlada, factor edad, falla ovárica o por ser pacientes subrogadas o gestacionales. El tamaño de la muestra se dividió de la siguiente manera: el porcentaje de embarazo esperado para el grupo de clivaje temprano-singamia (grupo 1) fue 79% (36 pacientes) mientras que el porcentaje de embarazo para el grupo pronuclear (grupo 2) llegó a ser 21% (9 pacientes). A partir de la obtención de estos datos, se determinaron las tasas de implantación y de embarazo y se compararon los dos grupos por medio de la prueba Chi-cuadrado (X2). Resultados: de un total de 289 cigotos, se incluyeron 183 que cumplieron los parámetros propuestos, entre embriones provenientes de clivaje temprano (CT), singamia (S) y estadio pronuclear (PN) a las 25 y 27 horas posinseminación. De este total, 79% provenían del estadio clivaje temprano-singamia y 21% del estadio pronuclear. El promedio de embriones transferidos a las pacientes ovoreceptoras fue tres embriones, la edad promedio fue 38 años, la tasa de embarazo obtenida al transferir los embriones provenientes de clivaje temprano fue 44% en comparación con 41% para la transferencia de los embriones no clivados a las 25 horas posinseminación. Finalmente, no se encontraron diferencias significativas en la obtención del embarazo. Conclusión: bajo las condiciones del estudio, el resultado muestra que no hay diferencias en la tasa de éxitos para ambos procedimientos.

Introduction: some authors have proposed that non-invasive embryo implantation methods (based on early cleavage or pronuclear stage for two cells 25 and 27 hours post-insemination) are applicable to in vitro fertilisation programmes, thereby increasing the probability of implantation and pregnancy. Objective: comparing pregnancy rates in patients implanted with embryos originating from early cleavage to those from the pronuclear stage. Methodology: a retrospective cohort study was carried out. Embryo implantation patients were included who had been attending the FECUNDAR -Biomedicine Centre in Cali, Colombia, from April 2004 to December 2007 who presented a poor response to the controlled ovarian stimulation cycle, age factor and/or ovarian failure and pregnancy substitute patients. The sample consisted of 45 patients; 36 patients (79%) were expected to become pregnant in the early-syngamy and cleavage group (group 1) and 9 patients (21%) in the pronuclear group (group 2). Implantation and pregnancy rates were determined and the Chi-square (X2) test was used for comparing both groups. Results: 183 of the 289 zygotes complied with the proposed parameters (i.e. embryos from early cleavage, syngamy and pronuclear stage 25 and 27 hours post-insemination); 79% of these 183 came from the early cleavage and syngamy stage and 21% from the pronuclear stage. Three embryos on average were transferred to embryo implantation patient; average age was 38, the pregnancy rate obtained when transferring early cleavage embryos was 44% compared to 41% for non-cleaved embryo transfer 25 hours post-insemination. No statistically significant differences were found regarding how pregnancy was induced. Conclusion: equal success rates were obtained for both procedures in the given study conditions.