Aprendizaje significativo

Normally during oocyte development, the nurse cells undergo apoptosis and are finally phagocytosed by the oocyte (McCall and Steller, 1998; Technau et al., 2003) for instance in C. elegans half of the female germ cells are engulfed by the surrounding sheath cells (Gumienny et al., 1999). Apoptosis is involved in maintenance of germ line homeostasis because full-size oocytes cannot form in the apoptosis-deficient mutant’s ced-3 and ced-4 for instance in C. elegans (Matova and Cooley, 2001; Gumienny et al., 1999). Moreover, in Drosophila, the caspase-3-deficient mutant dcp-1 cannot form fertile oocytes (McCall and Steller, 1998; Technau et al., 2003). Apoptosis of nurse cells in

Drosophila is involved in regulating the intracellular rearrangements of the actin

cytoskeleton, which are important for the transfer of cytoplasmic components into the oocyte (De Cuevas and Spradling, 1998).

B A

Figure 11 shows a schematic comparison of oogenesis among Hydra, C. elegans and Drosophila. The common feature is that multiple germ cells contribute cytoplasm to the oocyte. For instance, in C. elegans, nuclei proliferate in a syncytium and their gene products contribute to form a common mass of cytoplasm. Apoptosis occurs in the half of these nuclei and the remaining nuclei enclose units of the common cytoplasm to form oocytes (Gumienny et al., 1999). In Drosophila germ cells in the ovariole transfer cytoplasm through ring canals to the growing oocyte (Mahajan-Miklos and Cooley, 1994) and in the Hydra egg patch, 4000 germline cells contribute to the growing oocyte. These cells fuse with the oocyte and transfer cytoplasm to it before undergoing apoptosis. The

Hydra oocyte has a size of nearly 600 µm, Drosophila oocyte is roughly 150 µm and a

C. elegans oocyte is about 50 µm in diameter. These differences between oocyte sizes

correlated with the number of germ cells contributing to each oocyte. Therefore, alimentary oogenesis represents a strategy to rapidly produce egg cytoplasm by utilizing multiple germline nuclei to synthesize it.

In the case of nurse cells differentiation, they are derived from germline cells, which undergo a premeiotic S-phase prior to differentiation. Some of these nurse cells in Hydra (GCIV) and two cells in Drosophila initiate meiosis (De Cuevas et al., 1997), while all nuclei in C. elegans reach meiotic pachytene. At this stage, nurse cells undergo apoptosis and are phagocytosed. At this time point, MAPK pathway activation is involved in C. elegans and mutants in MAPK do not exit pachytene and do not undergo apoptosis (Gumienny et al., 1999). It is not clear yet, if similar signaling pathway regulates apoptosis in Hydra and Drosophila. The fate of nurse cells is different in these mentioned invertebrates. As shown in Figure 11, C. elegans and Drosophila nurse cells

(Gumienny et al., 1999; Nezis et al., 2000) but in Hydra are phagocytosed by the oocyte itself (Alexandrova et al., 2005).

Fig. 11 Comparation of oogenesis among hydra, fruit fly and C. elegans

(Alexandrova et al., 2005).

In Hydra, Oocyte precursors are derived from interstitial stem cells (Bosch and David,

1986) and Oocyte development begins in a so-called egg patch in the ectoderm layer of body column and progressively thickens over 6 days and rapidly contracts to form the oocyte (Fig. 12). Every egg patch contains 4000 germ cells but only one oocyte is formed per egg patch and the rest of germ cells differentiate as nurse cells (Miller et al., 2000; Alexandrova et al., 2005). Nurse cells directly transfer cytoplasm through specialized ring canals to the developing oocyte and then undergo apoptosis and are phagocytosed by the oocyte (Nezis et al., 2000; Alexandrova et al., 2005).

Fig. 12 Stages of oogenesis in Hydra vulgaris

(from Alexandrova et al., 2005).

Progressive stages of differentiation in Hydra germ cells (GC) during oogenesis are shown with GCI, GCII, GCIII and GCIV according to their morphology (Fig. 13) (Alexandrova et al., 2005). GCI cells are in a postmitotic G1 phase and turn into GCII cells when entering a pre-meiotic S-phase and increasing their cytoplasmic and nuclear volume. GCII cells complete DNA replication and turn into GCIII cells, which have 4n DNA content. GCIII cells increase dramatically in size with the synthesis of numerous cytoplasmic vacuoles and granules (Aizenshtadt, 1975; Honegger et al., 1989) there after about 1% of GCIII cells enter prophase I of meiosis (leptotene to pachytene) thus becoming GCIV cells and only Two to three GCIV cells develop into diplotene oocytes. These cells increase dramatically in size from 100 µm to 600 µm due to transfer of cytoplasm from adjacent nurse cells and then they fuse into one cell. The surrounding

The single remaining oocyte undergoes two meiotic divisions and the egg is ready for fertilization.

Fig. 13 Germ cell (GC) differentiation scheme in

Fig. 14 (A) Schematic representation of germ cells in Hydra oogenesis; (B) TUNEL assay on germ cells in macerates (stages indicated in the picture). It is shown that germ cells in stages 2–4 are positive for TUNEL. Scale bar: 20µm (Böttger and Alexandrova, 2007).

As described above, the remains of nurse cells are phagocytosed by the oocyte and the morphological features of their nuclei suggest apoptosis. The fascinating thing is that nurse cells are not further degraded after phagocytosis by the oocyte and instead they are kept by oocyte enclosed to the phagocytic vacuoles even after fertilization, which is termed as an arrested apoptosis (Technau et al., 2003). During embryonic development they are distributed to the endodermal cells of the embryo and only get degraded after the new polyp has hatched. After hatching of the polyp, apoptosis is resumed and the nurse cells are degraded within 3 days. Persistent peroxidase activity in nurse cells detected throughout oogenesis and embryogenesis which have been implicated as anti-

responsible for preventing nurse cells from completing apoptosis until hatching and high level of peroxidase activity was found in the nurse cells from early stages of oogenesis on throughout embryogenesis (Habetha and Bosch, 2005) (Fig. 15). In newly hatched polyp peroxidase activity decreases within following 1–3 days, this decreasing is started at the apical end of the polyp and scattered over the whole body. This pattern of decrease in peroxidase activity is correlated with the pattern of degradation and precedes the disappearance of nurse cells after hatching, suggesting a role for peroxidase in preventing apoptosis in nurse cells until hatching (Habetha and Bosch, 2005) (Fig. 15).

Fig. 15 Expression of HvAPX1 (Hydra viridis plant related ascorbate peroxidase) activity in hermaphroditic H. viridis (A-C) Whole-mount in situ hybridisation. (D-E) Cellular in situ hybridisation. (D) Early oocyte; (E) I-cells from the ovary region showing HvAPX1 expression in one of the interstitial cells. Scale bars: 100µm (D) and 5µm (E) (Habetha and Bosch, 2005).