New insights into placozoan sexual reproduction and development

Abstract

Unraveling animal life cycles and embryonic development is basic to understanding animal biology and often sheds light on phylogenetic relationships. A key group for understanding the evolution of the Metazoa is the early branching phylum Placozoa, which has attracted rapidly increasing attention. Despite over a hundred years of placozoan research the life cycle of this enigmatic phylum remains unknown. Placozoa are a unique model system for which the nuclear genome was published before the basic biology (i.e. life cycle and development) has been unraveled. Four organismal studies have reported the development of oocytes and one genetic study has nourished the hypothesis of sexual reproduction in natural populations at least in the past. Here we report new observations on sexual reproduction and embryonic development in the Placozoa and support the hypothesis of current sexual reproduction. The regular observation of oocytes and expressed sperm markers provide support that placozoans reproduce sexually in the field. Using whole genome and EST sequences and additional cDNA cloning we identified five conserved sperm markers, characteristic for different stages in spermatogenesis. We also report details on the embryonic development up to a 128-cell stage and new ultrastructural features occurring during early development. These results suggest that sperm and oocyte generation and maturation occur in different placozoans and that clonal lineages reproduce bisexually in addition to the standard mode of vegetative reproduction. The sum of observations is best congruent with the hypothesis of a simple life cycle with an alternation of reproductive modes between bisexual and vegetative reproduction.

Figure 1. Progress of Placozoa sp. H2 oocyte maturation and early embryogenesis.

Shown are light microscopy (A–D) and SEM (E, F) images of Placozoa sp. H2 oocytes and embryos. Typically, one oocyte with a large nucleus starts growing in a flat animal without any signs of degeneration (A). Accompanied by the generation of yolk droplets (B) the animal enters the degeneration phase (D-phase) after 5–6 weeks when the population density reaches its maximum. Occasionally several oocytes are found in a single degenerating animal. We found one animal with nine maturing oocytes (C). The D-phase starts with the lifting of the upper epithelium followed by condensing the lower epithelium until forming a hollow sphere (“brood chamber”) containing the embryo (D, compare Fig. 3a1). The oocyte grows until reaching a varying final size of 50–120 µm by incorporating extensions from fiber cells through pores. One ‘connection pore’ of a maturing oocyte is shown in (E) (arrow). After fertilization the protective ‘fertilization membrane’ (eggshell) is built around the zygote (D), which starts total equal cleavage. Often formerly nursing fiber cells are still attached to the fertilization membrane (D, F). n = nucleus, o = oocyte, y_o = yolk outside oocyte, fm = fertilization membrane, e = embryo, fc = fiber cells, dma = degenerating mother animal.

Figure 2. Cell counting in Placozoa sp. H2 embryos.

Shown are embryos at the zygote-, 2-cell, 8-cell and 64-cell stage inside the fertilization membrane under light microscopy (A–D). Cleavage is total and equal. Nuclear staining with DAPI shows a direct correlation of blastomer number and fluorescent signals under standard fluorescent microscopy (F–H; 1, 8 and 64 cells, respectively). The same was seen with propidium iodide staining in confocal laser scanning images (J–L; 1, 8 and 120 cells, respectively). Red signals at the surface of the fertilization membrane in K and L derive from attached bacteria and algae to the fertilization membrane of the free drifting embryos. Positive controls for the staining procedure with adult animals showed clear nuclear signals for both fluorescent dyes (E, I). Maturing oocytes and zygotes show a large nucleus compared to somatic cells of the mother animal (arrow in E; F). (F) shows a zygote with a large, regular shaped interphase nucleus (arrow). Note that cells from the mother animal are still attached to the embryo (arrowhead). Metaphase chromosome clumps were regularly found in fluorescent staining, indicating normal cell cycle (arrow in H, K and L; compare Figure 3d2). The scale bars of A, G and J also apply to B–D, H and K–L, respectively.

Figure 3. Ultrastructural analyzes of developing Placozoa sp. H2 oocytes and embryos.

Shown are toluidine stained semi-thin sections (left panels) and TEM images (middle and right panels) of maturing oocytes (a) and embryos in different stages (b, c, d). Yolk material inside and outside maturing oocytes and embryos is clearly visible in dark blue in toluidine stained sections (a1, b1, c1) and as moderately electron dense material in TEM images (a2, b2). The early ‘fertilization membrane’ is made up of two layers (b1, b2), whereas three layers are distinguishable in later stages (c1, c2). Additional features not reported before are glycogen granules (a3) and lipid droplets in the oocyte (b1, b2, c1). In some sections nuclei (d1) and chromosomes (d2) were found in blastomers, indicating a normal cell cycle. We identified a putative sperm cell (e) with a retracted flagellum (arrow in e). o = oocyte, y_o = yolk outside oocyte, y_i = yolk inside oocyte, fc = fiber cell, ex = fiber cell extensions, cg = cortex granulum, gl = glycogen, li = lipid droplet, fm = fertilization membrane, sl = striped layer, gs = ground substance, dgs = dense ground substance, bl = blastomer, n = nucleus, nl = nucleolus, c = metaphase chromosomes, sc = putative sperm cell, ue = upper epithelium.