Biol. Bull. 209: 31-48. (August 2005)
© 2005 Marine Biological Laboratory
Nutritive Phagocyte Incubation Chambers Provide a Structural and Nutritive Microenvironment for Germ Cells of Strongylocentrotus droebachiensis, the Green Sea Urchin
Charles W. Walker1,*,
Laura M. Harrington1,
Michael P. Lesser1 and
Wayne R. Fagerberg2
1 Department of Zoology, Marine Biomedical Research Group and Center for Marine Biology, University of New Hampshire, Durham, New Hampshire 03824
2 Department of Plant Biology, University of New Hampshire, Durham, New Hampshire 03824
* To whom correspondence should be addressed. E-mail: cwwalker{at}christa.unh.edu
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Abstract
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Here we characterize the germinal epithelia of both sexes of Strongylocentrotus droebachiensis, the green sea urchin, throughout its annual gametogenic cycle, using light and electron microscopy and cytochemistry. In both sexes, germinal epithelia include two interacting cellular populations: nutritive phagocytes (NPs) and germ cells. After spring spawning, NPs accumulate nutrients; amitotic oogonia and often mitotic spermatogonia occur in clusters beneath NPs; and subsequent gametogenic stages are residual or absent. During the summer, NP nutrients are mobilized for use in vitellogenesis by residual primary oocytes or to support limited spermatogenesis. In addition, some residual primary oocytes may degenerate and be phagocytized by NPs. Significant nutrient mobilization from NPs and substantial gonial cell mitoses (indicative of new gametogenesis) occur in the fall. In both sexes, all of these changes are facilitated by NPs that form basal incubation chambers near the gonadal wall and within which germ cells are surrounded by nutrients released from the NPs. In females, germ cells at several stages of gametogenesis may be housed in separate chambers in the same NP. Primary oocytes also carry out jelly coat formation, meiosis, and cortical granule translocation within NP incubation chambers. In males, many NPs cooperate to provide large continuous chambers that contain spermatogenic cells at diverse stages. In both sexes these chambers persist throughout the year and isolate gametogenesis from the gonadal lumen. NPs become slender and shorten as their nutrients are depleted. Ova or spermatozoa are stored in the gonadal lumen. Post-spawning, NPs phagocytize differentiated germ cells while simultaneously enclosing intact gonial and residual gametogenic cells in basal chambers near the gonadal wall. In light of our observations, we suggest investigating proteins that may be important in the structural, phagocytic, and nutritive functions of NPs and for which corresponding genes have already been identified in the genome of S. purpuratus, the closely related purple sea urchin.
Abbreviations: AO, amitotic oogonium • AS, amitotic spermatogonia • BPB, mercuric bromophenol blue • C, coelom • CG, cortical granules • DPO, degenerating primary oocyte • FGPO, fully grown primary oocyte • GHS, genital hemal sinus • GI, gonad index • LSS, later spermatogenic stages • MS, mitotic spermatogonia • MYP, major yolk protein • NP, nutritive phagocyte • NPIC, nutritive phagocyte incubation chamber • NS, new spermatozoa • NVPO, new vitellogenic primary oocyte • O, ovum • OL, ovarian lumen • PAS, periodic acid Schiff • RIC, residual NP incubation chamber • RVPO, residual vitellogenic primary oocyte • RSC, residual spermatogenic cells • TL, testicular lumen
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Introduction
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Most sea urchins have an annual reproductive cycle characterized by seasonal changes in gonad mass relative to total body mass. Uniquely among animals, sea urchin gonads are large both before and during gametogenesis (Walker et al., 1998, 2001). This is a result of extensive intragonadal nutrient storage by nutritive phagocytes (NPs). NPs are the only somatic cells in the germinal epithelia in both sexes (Pearse and Cameron, 1991; Walker et al., 2001). Initially, NPs incorporate a wide variety of nutrients within membrane-bound vesicles and in their cytoplasm (Unuma et al., 2003; Brooks and Wessel, 2003). Nutrients are later mobilized to provision adjacent germ cells as the latter enlarge during vitellogenesis or increase in number in both sexes. Following production of ova and spermatozoa, external fertilization occurs during broadcast spawning (Scheibling and Hatcher, 2001).
Despite several studies using paraffin sections, electron microscopy, and autoradiography (see reviews by Walker, 1982; Pearse and Cameron, 1991; and Walker et al., 2001), existing data do not provide detailed information about the structural interrelationships of NPs and germ cells in both sexes of any sea urchin throughout an entire gametogenic cycle. Lacking this structural context, it is impossible to address specific questions about the progress of gametogenesis in these fundamentally important model organisms. For instance, the physical mechanisms of nutrient delivery to, storage within, and release from NPs and subsequent nutrient uptake by cells at successive gametogenic stages are all virtually uninvestigated.
Given the unique nature of sea urchin gametogenesis, in which NPs play such a pivotal role, it is appropriate to reinvestigate the germinal epithelium in both sexes with the goal of defining the intimate structural relationships of NPs and germ cells throughout gametogenesis. To achieve this, monthly collections of the gonads of both sexes of Strongylocentrotus droebachiensis, the green sea urchin, were analyzed with light and electron microscopy and cytochemistry.
On the basis of the higher resolution provided by plastic sections, we refine the four gametogenic stages proposed by Walker et al. (2001) that simultaneously consider cytological features of germ cells and NPs. These stages begin after spawning and are (1) inter-gametogenesis and NP phagocytosis, (2) pre-gametogenesis and NP renewal; (3) gametogenesis and NP utilization; and (4) end of gametogenesis, NP exhaustion, and spawning.
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Materials and Methods
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Sea urchins were collected at 10 m near Lunging Island, Isles of Shoals, New Hampshire (42°59.29'N; 70°37.01'W) by scuba diving from April 1998 to March 1999. Urchins with test diameters larger than 30 mm were collected. Thirty to fifty urchins were returned to the University of New Hampshire Coastal Marine Laboratory, New Castle, New Hampshire. From these, 20 urchins were processed on the day of collection. For each urchin, gonad index was determined and samples of gonads were fixed for light and electron microscopy and cytochemistry. Photoperiod data were derived from the Farmer’s Almanac (1998–1999), and temperature at the site of collection was continually monitored using temperature-recording thermistors (HOBO Temps, Onset Computer Corp.).
For each monthly sample, wet weight (g) was used to calculate a gonad index for each urchin (GI = gonad wet weight/sea urchin total wet weight x 100). Portions of gonads from each urchin were excised and fixed in 3% glutaraldehyde buffered with 0.2 M sodium cacodylate and postfixed in 2% osmium tetroxide (Walker, 1979). Samples were embedded in Epon-Araldite, allowed to polymerize for 24–36 h at 60 °C, and sectioned at 1–1.5 µm (for light microscopy) with glass knives on a Reichert OM U3 ultramicrotome. Sections for light microscopy were mounted on glass slides coated with 3-aminopropyl-trimethoxysilane (Electron Microscopy Sciences) and stained for 1–2 min with buffered 0.2% azure B in 1% NaHCO3 at 60 °C. Sections for electron microscopy (gold/silver) were mounted on Formvar-coated 100-mesh copper grids, contrasted with uranyl acetate (3%) and Reynolds lead citrate, and viewed on a JEOL 100S electron microscope operated at 80 kV. For each urchin, NP lengths (µm) were determined with an ocular micrometer on a Zeiss Axiopan-2 light microscope at 40x. For each month, three nutritive phagocytes from each urchin (n = 20) were measured between their origin on the gonadal wall and their tips in the gonadal lumen. Representative sections from gonads of all monthly collections were digitized using a Spot camera mounted on a Zeiss Axioplan-2 microscope. Gametogenic stages were based on Walker et al. (1998, 2001).
Additional samples of gonadal tissue were fixed and dehydrated as above, omitting postfixation with osmium tetroxide. After dehydration, these gonads were embedded in JB- 4 methacrylate resin and allowed to polymerize at room temperature under vacuum. Samples were sectioned and mounted as described above. Two representative testes and ovaries from each of the four reproductive stages (Walker et al., 1998) were chosen for cytochemistry. Cytochemical stains, with appropriate controls, included periodic acid Schiff (PAS) and mercuric bromophenol blue (BPB) and were used to localize carbohydrates and proteins respectively (Humason, 1979).
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Results
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When data from males and females are combined, the average length of the nutritive phagocytes (NPs) and the gonad index (GI) demonstrate an inverse relationship (Fig. 1). As photoperiod decreases (days are becoming shorter and solar zenith angle is increasing) there is a lag of 2 months between the decrease in NP length and the increase in GI (Figs. 1 and 2). From May to August the GI remains low, while NP length is steadily increasing. As temperature and photoperiod both decrease, NP length decreases (Fig. 2). Generally, the length of NPs increases significantly from April to August and then decreases significantly from August to March. As temperature increases, the length of NPs increases. Temperature peaks in September, NP length peaks in August, and both decrease from August to March. NP length is consistently greater in the ovaries than in the testes.
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Figure 1. Monthly mean nutritive phagocyte lengths (µm) plotted with monthly mean gonad indices (GI) for combined data from both sexes of the green sea urchin Strongylocentrotus droebachiensis.
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Figure 2. Male and female nutritive phagocyte lengths (µm) plotted (± SD) with both photoperiod (length of daylight in hours) and temperature (°C) for Strongylocentrotus droebachiensis.
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During each gametogenic stage, the condition of the germinal epithelia varies among individuals. Different urchins in the same monthly sample may contain gonads at the beginning or at the end of a particular gametogenic stage. Also, each of the four stages persists for two to several months, and the boundaries of each stage may vary slightly in different years. We have selected what we consider to be an average appearance on the basis of observations of the gonads of 5–15 individuals per sex from each stage. In the following, we characterize the cellular structure of the germinal epithelium of both sexes during each gametogenic stage (Walker et al., 2001) and for the months indicated.
Ovaries
After spawning and during the inter-gametogenesis and NP phagocytosis stage (spring: February–April; Figs. 1–2, 3a–g; 11a), the ovarian germinal epithelium consists of somatic NPs and germ cells, including amitotic oogonia (AOs); irregularly or conically shaped residual vitellogenic primary oocytes (RVPOs); and a few large residual ova (Os) (Fig. 3a–b, d–e, g). Ova reside in the ovarian lumen and are either intact or are undergoing phagocytosis where they contact NPs (Fig. 3g). Cytological observations and changes in GI both suggest that all ovaries observed at this stage had spawned many of their stored ova (Figs. 1–3a). NPs line the ovarian wall and are short (40–60 µm in length; Fig. 3a, arrow) compared with their maximum dimensions during active oogenesis (
200 µm in length). NPs contain a single randomly positioned nucleus, small and either few or numerous membrane-bound, mostly round vesicles with contents that are PAS- and BPB-positive (Fig 3b–e). Empty vesicles are also present. In this and all subsequent stages, individual NPs may bear a single flagellum and may be interconnected apically by septate desmosomes (not observed here, but as seen by Takashima, 1968, and Reunov et al., 2004b).
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Figure 3. Ovary: Inter-Gametogenesis and NP Phagocytosis Stage. (a) Low-magnification view of an ovarian lobe collected in April, showing residual ova (O) free in the lumen and thin layer of nutritive phagocytes (NPs) (arrow); scale bar = 100 µm. (b) Enlarged view of a portion of the ovarian wall, showing the reduced layer of NPs (see the nuclei of several at arrows), a residual vitellogenic primary oocyte (RVPO) within a residual NP incubation chamber (RIC), and an amitotic oogonium (AO) and two ova (O) in the lumen; scale bar = 20 µm. (c) A single representative nutritive phagocyte showing the nucleus with a single nucleolus, few nutrient-containing vesicles (dark dots), and other empty vesicles; scale bar = 10 µm. (d) A portion of the ovarian wall showing PAS+ vesicles within NPs (dark dots); scale bar = 20 µm. (e) A portion of the ovarian wall showing BPB+ vesicles within NPs (dark dots); scale bar = 20 µm. (f) A cluster of amitotic oogonia with distinct nucleoli underneath NPs; scale bar = 10 µm. (g) NPs in contact with three degenerating ova; scale bar = 20 µm. C, coelom; OL, ovarian lumen.
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Figure 11. Nutritive phagocytes (NPs) in ovaries (a) and testes (b) at each gametogenic stage with germ cells removed from NP incubation chambers (not to scale). For ovaries, the shapes and dimensions of representative, individual NPs are shown, as well as the positions of various germ cell types within discrete NP chambers. For testes, which have spermatozoa still in the lumen, the positions of germ cells relative to groups of NPs are revealed, especially the continuous incubation chamber that eventually forms. AO, amitotic oogonia; AS, amitotic spermatogonia; NVPO, new vitellogenic primary oocyte; O, ovum; LSS, later spermatogenic stages; RVPO, residual vitellogenic primary oocyte; NP, nutritive phagocyte; NS, new spermatozoa; MS, mitotic spermatogonia.
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During this stage, NPs have begun to enlarge as they store nutrients (increasing from 5 to 60 µm in length since spawning). Many residual NP incubation chambers (RICs) are present basally, and these may contain RVPOs near the ovarian wall (Fig. 3b). The RICs appear to be remnants of basal NP incubation chambers occupied by ova that were released earlier into the ovarian lumen. Conical RVPOs in such chambers contain new nutrients, as indicated by the presence of vacuoles stained with PAS (not shown). Clusters of AOs are often present (5–6 gonia/cluster) in small incubation chambers at the bases of the same NPs (Fig. 3f). AOs have one or two nucleoli, do not contain PAS- or BPB-positive vacuoles, and are directly in contact with the ovarian wall (Fig. 3f). Apically, several NPs may be phagocytising single residual, unspawned ova (Fig. 3g).
During the pre-gametogenesis and NP renewal stage (summer: May–August; Fig. 4a–e; 11a), the ovarian germinal epithelium consists of somatic NPs and germ cells, including AOs; conical or gum drop-shaped RVPOs; and, occasionally, residual degenerating primary oocytes (DPOs). Ova are absent from the lumen (Fig. 4a). NPs are round or elongated (average length is 150 µm). By the end of the summer, the NPs extend into the lumen, are full of PAS- and BPB-positive vacuoles, and occupy most of the ovary (Fig. 4a, d–e). As the summer progresses, oocytes continue to enlarge (75 µm), and the number of yolk platelets in their cytoplasm increases. At the end of the summer, RVPOs (<100 µm) have grown to fill the basal RICs already observed in the previous stage (Figs. 3b; 4b, d). PAS-positive and a few BPB-positive yolk platelets are present within the cytoplasm of these oocytes (Fig. 4b, d–e). Clusters of about 6 AOs (12.5 µm) are enclosed in chambers beneath NPs and directly contact the connective tissue of the ovarian wall.
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Figure 4. Ovary: Pre-Gametogenesis and NP Renewal Stage. (a) Low-magnification view of an ovarian lobe collected in July, showing substantial growth of nutritive phagocytes (NP) and residual vitellogenic primary oocytes (darker conical objects near the ovarian wall) within basal NP incubation chambers; scale bar = 100 µm. (b) Enlarged view of a portion of the ovarian wall, showing a residual vitellogenic primary oocyte (RVPO) and also a degenerating primary oocyte (DPO), each within separate residual NP incubation chambers. An amitotic oogonium (AO) is also present underneath NPs in contact with connective tissue of the ovarian wall; scale bar = 50 µm. (c) A single NP containing residual vitellogenic (RVPO) and previtellogenic primary oocytes (arrow) within discrete basal NP incubation chambers; scale bar = 50 µm. (d) A portion of the ovarian wall showing reddish-pink PAS+ vesicles filling NPs. Several NP incubation chambers contain residual vitellogenic primary oocytes that are incorporating PAS+ nutrients within yolk platelets; scale bar = 50 µm. (e) A portion of the ovarian wall showing blue BPB+ vesicles within NPs and several NP incubation chambers housing residual vitellogenic primary oocytes that contain a few BPB+ yolk platelets; scale bar = 50 µm. C, coelom; OL, ovarian lumen.
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During the gametogenesis and NP utilization stage (fall: September–December; Fig. 5a–h; 11a), the ovarian germinal epithelium consists of somatic NPs and germ cells, including amitotic (AO) and mitotic oogonia (Fig. 5h, arrow); RVPOs (187–200 µm), and smaller new vitellogenic primary oocytes (NVPOs) (<100 µm), the latter resulting from recent oogonial mitosis (Figs. 5a–h). During this stage, both generations of oocytes grow and become distinctly conical within NPICs. Both RVPOs and NVPOs also contain large, membrane-bound clear vesicles with unknown contents (Fig. 5b, arrows; c, arrow in RVPO). As oocytes increase in length, NPs progressively occupy less space while they appear to increase in length (Figs. 2; 5a, d). The size and number of vesicles within the NPs have decreased, and oocytes that already contained PAS-positive yolk platelets in the previous stage (RVPOs), as well as new oocytes (NVPOs), have become increasingly BPB-positive (Fig. 5b, d–f). New oocytes derived from this year’s gametogenesis are conical or rounded, are contained in basal NPICs, and have many pseudopodia that contact NPs (Fig. 5g). Clusters of amitotic (Fig. 5b, AO) and mitotic oogonia (Fig. 5h, arrow) are present near the gonadal wall.
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Figure 5. Ovary: Gametogenesis and NP Utilization Stage. (a) Low-magnification view of an ovarian lobe collected in December, showing substantial growth of primary oocytes within basal nutritive phagocyte (NP) incubation chambers; scale bar = 100 µm. (b) Enlarged view of a portion of an ovary from October, showing growth of both smaller new (NVPO) and larger residual vitellogenic primary oocytes (RVPO) within basal NP incubation chambers. A cluster of amitotic oogonia (AO) is also present in contact with connective tissue of the ovarian wall; scale bar = 20 µm. (c) An electron micrograph, showing a residual vitellogenic primary oocyte (RVPO) with yolk platelets and also clear vacuoles (arrow) in relationship to an adjacent NP that contains rough endoplasmic reticulum (arrow); scale bar = 20 µm. (d) A single representative NP showing the distension of an NP incubation chamber as a residual vitellogenic primary oocyte (RVPO) continues to elongate within. Basally, the same NP houses a small new vitellogenic primary oocyte (NVPO) and an amitotic oogonium (AO); scale bar = 20 µm. (e) A portion of the ovarian wall showing extensive PAS+ vesicles (dark dots) within NPs and several NP incubation chambers containing residual (RVPO) and new (NVPO) vitellogenic primary oocytes, all of which contain PAS+ nutrients within yolk platelets; scale bar = 20 µm. (f) A portion of the ovarian wall showing NP, that contain BPB+ contents (dark dots) of numerous vesicles within NPs and within yolk platelets in vitellogenic primary oocytes; arrow indicates a new vitellogenic primary oocyte (NVPO); scale bar = 20 µm. (g) Extensions from the surface of a new vitellogenic primary oocyte (NVPO) and either endocytotic or secretory vesicles in relation to the surrounding NP incubation chamber; scale bar = 20 µm. (h) A mitotic oogonium (arrow), adjacent prophase oogonia (to left), and a residual vitellogenic primary oocyte (RVPO); scale bar = 10 µm. C, coelom; NP, nutritive phagocyte; NPIC, nutritive phagocyte incubation chamber.
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During the end of gametogenesis, NP exhaustion, and spawning stage (winter: January–March; Fig. 6a–f, 11a), the ovarian germinal epithelium consists of somatic NPs and germ cells, including AOs; previtellogenic, vitellogenic, and fully grown primary oocytes (FGPOs); and ova (O). NPICs are maximally distended laterally as oocytes change in shape from elongated to nearly round and as they secrete the jelly coat (Fig. 6a–e). In living tissues, the jelly coat appears as a clear space around oocytes (Fig 6b, inset). PAS- and BPB-positive vesicles are present in the cytoplasm of these distended NPs (Fig. 6d–e). Each NP may simultaneously contain an FGPO or an ovum (O) as well as previtellogenic primary oocytes (Fig. 6b, single arrow) and AOs (Fig. 6b, double arrow) in discrete incubation chambers. The latter two germ cell types always occur basally. Intermediate-sized vitellogenic primary oocytes have a thick cortical layer underlain by vesicles. They extend filamentous PAS-positive pseudopodia into the surrounding jelly coat (Figs. 6b–c). Within NPICs, oocytes complete first and second meiotic divisions, releasing two polar bodies (the first of which does not complete second meiosis); all cytoplasmic extensions are withdrawn from the jelly coat; and cortical granules (CG) are translocated from the general to the cortical cytoplasm (Fig. 6b, f; Wessel et al., 2002).
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Figure 6. Ovary: End of Gametogenesis, NP Exhaustion, Spawning Stage. (a) Low-magnification view of an ovarian lobe collected in February, showing fully grown primary oocytes in expanded nutritive phagocyte incubation chambers (NPICs); scale bar = 100 µm; C = coelom. (b) Enlarged view of a portion of an ovary from January, showing intact, maximally expanded NP incubation chambers (NPIC), a fully grown primary oocyte (FGPO), and an ovum (O), each of which is contained in separate NPICs; the single arrow indicates a new, previtellogenic primary oocyte, and the double arrow a cluster of amitotic oogonia, each within discrete incubation chambers of the same NP; scale bar = 50 µm. (c) An intermediate-sized vitellogenic primary oocyte (IVPO) in relationship to the NPIC in which it is contained. Notice pseudopodial extensions from the oocyte surface, the thick cortical layer underlain by clear vesicles and yolk platelets in cytoplasm; also note the lack of clear vesicles that are present in the previous stages; scale bar = 50 µm. (d) A portion of the ovarian wall showing reddish-pink PAS+ vesicles within distended NPs and several NP incubation chambers containing FGPOs that have extensive PAS+ yolk platelets; scale bar = 20 µm. (e) A portion of the ovarian wall showing NPs that contain blue BPB+ vesicles and considerable BPB+ cytoplasm of FGPOs; scale bar = 20 µm. (f) Arrow indicates polar body released from an adjacent ovum (O) in a March ovary; cortical granules can be seen as a layer of dark dots just below the oolemma (CG); scale bar = 20 µm. C, coelom.
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Testes
After spawning and during the inter-gametogenesis and NP phagocytosis stage (spring: April–May; Figs. 1–2, 7a–d, 11b), the testicular germinal epithelium consists of somatic NPs and germ cells, including amitotic spermatogonia (ASs); residual spermatogenic cells (RSCs); and limited or numerous residual spermatozoa (Fig. 7c, arrows). NPs are about 40 µm in length, shortened from their maximum length, about 100 µm, although they have already begun to accumulate nutrients in membrane-bound vesicles and to increase in length, by 20–30 µm, from the previous stage. NPs contain a nucleus with a single large nucleolus (Fig. 7b, arrows). Apically, they may also contain a large central membrane-bound vesicle (Fig. 7c). As seen in ovaries at this and all subsequent stages, individual NPs may bear a single flagellum and are interconnected apically by septate desmosomes (not observed here, but as seen by Takashima, 1968, and Reunov et al., 2004b). It is difficult to trace individual NPs from their bases to their tips. This suggests that NPs may be intertwined near their bases or that they may have multiple connections to the testicular wall. Apically, many NP vesicles contain one to several spermatozoa, apparently engulfed from the testicular lumen (Fig. 7b, d). Membrane-bound vesicles that contain spermatozoa are called heterophagosomes by Reunov et al. (2004a, Reunov et al.). Amitotic spermatogonia (AS) with one or two nucleoli occur in clusters (2–3 per cluster) in tiny basal NP chambers near the testicular wall (Figs. 7b, 11b). Spermatogonia directly contact the testicular wall and each other without intervening extensions from NPs.
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Figure 7. Testis: Inter-Gametogenesis and Phagocytosis Stage. (a) Low-magnification view of a testicular lobe collected in April, showing spermatozoan-free lumen (TL) and nutritive phagocytes (NPs) that have begun to store nutrients and increase in size after spawning. Other samples from the same collection were identical morphologically but contained many residual spermatozoa; scale bar = 50 µm; C, coelom. (b) Enlarged view of a portion of the testicular wall, showing the layer of NPs (see the nuclei of several at arrows) with newly formed nutrient-containing vesicles. Near the testicular wall, amitotic spermatogonia occur in clusters (AS), as do residual spermatogenic cells (RSC); scale bar = 20 µm. (c) Cross-sections of several NPs at midlevel along their axes, showing a large central vacuole surrounded by cytoplasm; scale bar = 20 µm. (d) Tips of several NPs showing vesicles with sub-granules and also heterophagosomes containing remnants of spermatozoa (arrows); scale bar = 5 µm; TL, testicular lumen.
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During the pre-gametogenesis and NP renewal stage (summer: June–August; Fig. 8a–d, 11b), the testicular germinal epithelium consists of somatic NPs and germ cells, including amitotic (Fig. 8b, black arrow, right) and occasionally mitotic (Fig. 8d) spermatogonia; new spermatogenic cells (dashed line arrow to left); and minimal new spermatozoa (black on white arrows). Residual spermatozoa are absent. Testicular NPs grow to nearly their maximum length during this stage (average 100 µm), but are shorter than those in the ovary at the same time (average 180 µm) (Figs. 8a–b). Apically, NPs contain PAS- and BPP-positive membrane-bound vesicles (Fig. 8c). Spermatogonia and subsequent stages of spermatogenesis are PAS negative and exist as nonstaining cells in basal NP incubation chambers formed by several adjacent NPs (Figs. 8c, 11b). Spermatogonial mitoses are often observed (Fig 8d). As new spermatozoa are formed, NPs can actually shorten by 10–15 µm during this stage (Fig. 2).
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Figure 8. Testis: Pre-Gametogenesis and NP Renewal Stage. (a) Low-magnification view of a testicular lobe collected in July, showing an increase in the size of nutritive phagocytes (NPs) over the previous stage (which was represented at 50 µm); scale bar = 100 µm; C, coelom. (b) Enlarged view of a portion of the testicular wall, showing an increase in size and number of nutrient-containing vesicles and substantial growth of NPs. Limited groups of new spermatogenic cells (dashed black arrow to left) and amitotic spermatogonia (black arrow at right) are present in basal NP incubation chambers; a few new spermatozoa can be seen between NPs (white on black arrows); scale bar = 20 µm. (c) A portion of the testicular wall showing extensive PAS+ vesicles within NPs (gray dots) and several NP incubation chambers containing PAS– spermatogonia (unstained at arrows); scale bar = 50 µm. (d) An electron micrograph of a mitotic spermatogonium within a basal NP chamber; scale bar = 20 µm.
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During the gametogenesis and NP utilization stage (fall: September–February; Fig. 9a–f; 11b), the testicular germinal epithelium consists of somatic NPs and germ cells, including a prominent, nearly continuous layer of mitotic spermatogonia in a common basal NP incubation chamber (Fig. 9a, arrow; b, inset, arrows). As this stage progresses, an increasingly thick, continuous layer of mitotic spermatogonia and all subsequent spermatogenic stages fill this common NP incubation chamber (Fig. 9b–c, 11b). Throughout this stage, new spermatozoa accumulate in the testicular lumen (Fig. 9a–c). NPs shorten as spermatogenesis proceeds, ultimately losing nearly 100 µm in length. They become thin columns of tissue extending between spermatogenic cells to the testicular wall (Figs. 9a–d). Primary spermatocytes, secondary spermatocytes, spermatids, and differentiating spermatozoa extend, in that order, from the gonadal wall luminally between NPs (Fig. 9b). Apically, PAS-positive vesicles (some with sub-vesicles) are abundant within the NPs (Figs. 9d–e), as are vesicles containing spermatozoa (Fig. 9f, arrow).
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Figure 9. Testis: Gametogenesis and NP Utilization Stage. (a) Low-magnification view of a testicular lobe collected in October, showing the development of a continuous layer of spermatogonia within a common basal NP incubation chamber (arrow). Nutritive phagocytes (NPs) are obvious as a layer of vesicle-containing cells above the spermatogonia; their tenuous connections with the testicular wall are not obvious. New spermatozoa (NS) have been accumulating in the testicular lumen; scale bar = 100 µm; C, coelom. (b) Enlarged view of a portion of the testicular wall from an October individual, showing the increase in size of the continuous basal NP incubation chamber at the expense of NP mass; scale bar = 50 µm. Inset shows mitotic spermatogonia (arrows). (c) Low-magnification view of a testis collected in December, showing decrease in size and vesicle content of NPs (dark tissue at arrow) and increase in size of the common, basal NP incubation chamber containing lighter spermatogenic cells (X); scale bar = 100 µm; C, coelom. (d) Two isolated NPs presented to scale: the NP on the left is typical of testes in Fig. 9a. Above the horizontal line, the NP contains extensive nutrient vesicles (PAS+ staining), while below the line, the NP maintains a thread-like connection surrounded by lighter spermatogenic cells to the testicular wall. The NP on the right is typical of the testis shown in Fig. 9c; it has shortened considerably since October and consists mostly of a thin thread of cytoplasm (arrows) surrounded by darker spermatogenic cells; scale bar = 30 µm. (e) A portion of the testicular wall showing PAS+ apical vesicles (dark dots) within NPs and a continuous, basal NP incubation chamber (NPIC) that contains PAS– spermatogenic cells; scale bar = 50 µm. (f) An electron micrograph, showing cross-sections of the tips of several NPs containing vesicles with sub-vesicles and one with a spermatozoan in a heterophagosome (arrow); scale bar = 2 µm.
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During the end of gametogenesis, NP exhaustion, and spawning stage (winter: March–April; Fig. 10a–c, 11b), the testicular germinal epithelium consists of somatic NPs and germ cells, including amitotic (AS) and mitotic (arrows) spermatogonia; primary and secondary spermatocytes that are dividing meiotically; spermatids; and differentiating spermatozoa. New spermatozoa (NSs) fill the testicular lumen. During this stage, testicular NPs decrease to their shortest length (10–60 µm) and contain the smallest and fewest vesicles (Fig. 10b–c). They also may have one to several apparently empty, membrane-bound vesicles (Figs. 10b–c). As this stage progresses, and prior to spawning, fewer primary and secondary spermatocytes are present, and testes contain the maximum numbers of spermatozoa for the year.
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Figure 10. Testis: End of Gametogenesis, NP Exhaustion, Spawning Stage. (a) Low-magnification view of a testicular lobe collected in March, showing the reduced layer of vacuolated nutritive phagocytes (NPs) (arrow) and the accumulation of new spermatozoa (NS); scale bar = 100 µm; C, coelom. (b) Enlarged view of a portion of the testicular wall from a March testis, showing the reduction in size and vacuoles in NPs and spermatogenic cells. As spermatogenesis concludes, new spermatozoa are stored within the testicular lumen (NS); an amitotic spermatogonium is indicated at AS, and mitotic spermatogonia are indicated by black arrows; scale bar = 20 µm. (c) A single representative NP showing only a few nutrient-containing vesicles, the single nucleus (arrow), and several apparently empty vesicles of unknown origin; scale bar = 7 µm.
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Discussion
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The intimate structural relationships between nutritive phagocytes (NPs) and germ cells have never been described for both sexes of any sea urchin during an entire annual gametogenic cycle. Thanks to the use of plastic sections, the images of the structure of somatic and germinal cells composing the germinal epithelia are of substantially higher resolution than in earlier investigations of the same and other sea urchin species (Walker, 1982; Pearse and Cameron, 1991; Walker et al., 2001).
We have also detailed, for the first time, that NPs in the gonads of both sexes of Strongylocentrotus droebachiensis enclose gametogenic cells within basal incubation chambers throughout gametogenesis (Fig. 11a–b). This structural relationship persists until ova or spermatozoa are released for storage in the gonadal lumen. On the basis of micrographs in several publications, we conclude that many (e.g., Miller and Smith, 1931; Tennet et al., 1931; Takashima and Takashima, 1965; Pearse, 1969b, 1970; Bal, 1970; Nicotra and Serafino, 1988), but perhaps not all (Harvey and Gage, 1984; Piatigorsky, 1975) species of sea urchins possess these chambers and that their existence and involvement in gametogenesis has not been fully appreciated. Many of the unique features of sea urchin gametogenesis may be explained by understanding the nature of NP incubation chambers and their relationship to germ cells. In the following sections, we reinterpret several aspects of gametogenesis in sea urchins in light of the existence of such chambers. In particular, we consider the structural, phagocytic, and nutritive functions of NPs that have been identified in previous literature. We also provide suggestions for tackling long-standing questions about sea urchin gametogenesis by using information that is becoming available from the sea urchin genome project.
Structural function of sea urchin nutritive phagocytes
In sea urchin ovaries, follicle cells like those in the ovaries of most other organisms, including echinoderms such as sea stars, do not exist or are highly modified and unrecognizable. In the ovary, each NP encloses a single, growing vitellogenic oocyte in a basal incubation chamber where it may be enveloped by dissolved or particulate nutrients (Fig. 11a). NPs containing oocytes were first illustrated for ovaries by Miller and Smith (1931) and Tennet et al. (1931) in Echinometra lacunter and later noted by (Pearse 1969a,Pearse 1969b; 1970) in several species of sea urchins. For the first time, we report that the same NP may simultaneously contain additional germ cells at several earlier stages of oogenesis within smaller, discrete basal chambers. In testes (Fig. 11b), many NPs cooperate to provide large basal incubation chambers that ultimately become continuous and that simultaneously supply nutrients to enormous numbers of spermatogenic cells at diverse stages. The existence of these large incubation chambers in males was first implied for several species of sea urchins by Holland and Giese (1965) and by (Pearse 1969a,Pearse b, and 1970).
Use of NP chambers for gametogenesis has significant consequences for both sexes of all sea urchins that possess them. As mentioned, the nature of NP chambers differs between sexes. Because of differences in structure, the way nutrients are supplied to germ cells must also differ. The delivery of specific nutrients to particular gametogenic stages is easily achieved in ovaries since different germ cell stages are separately housed in discrete incubation chambers and could interact uniquely with the same NP. After continuous incubation chambers are formed in testes, it is impossible for individual germ cells to be targeted in this way. Gamete output is also affected by this structural difference in NP incubation chambers, since a few million ova are produced, but many million spermatozoa.
It is also important to point out that oogonial mitoses were identified in this study only at the beginning of the gametogenesis and NP utilization stage, but spermatogonial mitoses were observed at low levels throughout the year and at maximum levels at the same time that oogonia undergo mitosis. Although no mechanism is obvious to explain why oogonial mitoses are limited to this short time, perhaps the individualized treatment of oogonial cells that can occur within discrete NP incubation chambers in females is involved. It may be possible to resolve this issue, since ovarian NP incubation chambers can be explanted from ovaries and treated in vitro with growth factors present in female sea urchins (Walker, unpubl. data).
Another result of the use of NP incubation chambers by sea urchins is the difference in response of sea star and sea urchin oocytes to treatment with the meiosis-inducing molecule 1 methyl adenine (1-MeAde). In sea stars, it has been proposed that 1-MeAde is released from follicle cells at the tips of obvious, elongated pseudopodial extensions that contact the membrane of each primary oocyte where receptors are presumably located (Schroeder, 1981). A signal transduction cascade is then initiated that promotes breakdown of the follicular envelope; meiosis of the primary oocyte; contraction of the muscles in the gonad wall; and finally, spawning of most ova (Shirai and Walker, 1988).
Much of what happens in sea stars during spawning seems impossible for sea urchins because of the structure of NP incubation chambers. Direct connections between NPs and primary oocytes do not exist, and transfer of 1-MeAde by the mechanism proposed for sea stars is impossible. Also, since meiosis of sea urchin primary oocytes does not occur at one discrete time as it does in sea stars, but rather continues over a period of several months, 1- MeAde cannot simultaneously initiate meiosis and contraction of the ovarian wall as it does in sea stars. In some sea urchins, males and females both increase production of 1-MeAde during the gametogenesis and NP utilization stage (Kanatani, 1974). Although available 1-MeAde could be involved in oocyte maturation, its function in inducing gonad contraction in both sexes must be repressed until spawning; alternatively, another mechanism may be involved. The gonadal wall of sea urchins does not have an outer layer of circularly arranged muscle cells, and it is possible that this muscle layer in sea stars is responsive to 1-MeAde (Walker, 1982).
Primary oocytes undergo meiosis in NP incubation chambers to become ova (Fig. 6b). Berg and Wessel (1997) point out that cortical granules are translocated from the general to the cortical cytoplasm early in oocyte maturation (Berg and Wessel, 1997), and so this process must also occur in NP incubation chambers. Before meiosis, the large vitellogenic oocyte within each NP incubation chamber secretes the jelly coat (Jondeung and Czihak, 1982). The space surrounding fully grown vitellogenic primary oocytes is the transparent jelly coat (Fig. 6b) and is not an artifact of fixation. Pseudopodia that stain with PAS extend from oocytes into the jelly coat during its synthesis; these are withdrawn before or during meiosis (Fig. 6b, c). It is unclear whether the many vesicles seen below the oolema in intermediate-sized vitellogenic primary oocytes are involved in endocytotic uptake of nutrients into the oocyte, in secretion of the jelly coat, or in both processes.
Finally, it is instructive to compare how oocytes are nourished in other classes of the Echinodermata. Whereas sea stars utilize typical follicle cells, other echinoderms (crinoids, brittle stars, sea cucumbers, and now urchins) isolate oocytes in incubation chambers where they can be individually supplied with nutrients (Holland, 1971; Walker, 1982; Smiley, 1990; Eckelbarger and Young, 1992; Byrne, 1989, 1999). Except in sea urchins, incubation chambers are elaborations of the nutrient-containing genital hemal sinus (GHS), a connective tissue component located just below the germinal epithelium (Walker, 1982; Walker et al., 2001). As pointed out by Byrne (1999), vitellogenic oocytes in sea urchins rest directly on the connective tissue of the GHS and may derive nutrients from this source. However, the GHS of sea urchin gonads does not become engorged with stored PAS-positive nutrients as it does in all other classes of echinoderms (Walker, 1982).
Since NPs have apparently taken on the nutrient storage and transfer functions of the GHS in euechinoids as well as in cidaroids, it is likely that NP incubation chambers arose early in echinoid evolution. What led to the development of this unconventional method of nutrient storage in sea urchins is unclear. Nutritive phagocytes are probably derived from cells that give rise to follicle cells in sea stars. Houk and Hinegardner (1980) comment on the presence of cells with recognizable NP characteristics in the genital primordium of the sea urchin Lytechinus pictus soon after metamorphosis and note their migration along with primordial germ cells into the gonad. This is also the way follicle cells arise in sea stars (Walker, 1982). On the other hand, Liebman (1950) suggests that NPs in Arbacia punctulata are derived from coelomocytes. This is an intriguing idea, since both cellular populations produce and contain major yolk protein (MYP; Unuma et al., 2001; Brooks and Wessel, 2002), but it lacks any obvious structural evidence for coelomocyte transfer to the germinal epithelium of either sex.
Phagocytic function of sea urchin nutritive phagocytes
After spawning, NPs may phagocytize residual ova or spermatozoa and may recycle nutrients derived in this way (Pearse, 1969b). Masuda and Dan (1977) proposed that residual ova are too large to be directly phagocytosed by single NPs. On the basis of an increase in acid phosphatase activity, these authors conclude that after major spawning events, such ova are autophagic, thus aiding in their own degeneration. We have also observed several NPs associated with single degenerating ova, suggesting that NPs may cooperate in oocyte digestion (see Fig. 3g).
Phagocytosis of spermatozoa and of residual bodies discarded by maturing spermatids has been described in detail for NPs in Anthocidaris crassispina (Reunov et al., 2004a, Reunov et al., 2004b). In this process, single spermatozoa are endocytosed by NPs into structures called heterophagosomes. Heterophagosomes then aggregate into multi-spermatozoan-containing heterophagosomes that fuse with other vesicles and finally become remnant bodies. (Reunov et al. 2004a, Reunov et al., 2004b) argue from ultrastructural evidence that NPs control their own reduction in size by becoming autophagic through the action of "cell-size-reducing autolysosomes." This autophagic mechanism deserves experimental analysis.
NPs must be able to identify gametogenic cells at different stages and react appropriately to them. Errors in this recognition system, especially regarding gonial cells, would lead to sterile gonads. In both sexes, recognition of germ cells that are destined for disposal may depend upon mechanisms that other organisms are known to use for identifying apoptotic cells. An example is the movement of phosphotidyl serine to the outer leaflet of the cell membrane in apoptotic spermatogenic cells as a identification signal for phagocytosis by rat Sertoli cells (Shiratsuchi et al., 1997; Nakanishi and Shiratsuchi, 2004).
Phagocytosis of earlier spermatogenic stages and of atretic, residual oocytes within NP incubation chambers can also occur during the pre-gametogenesis and NP renewal stage. Gamete digestion of this kind has been reported by several investigators (see Pearse and Cameron, 1991) and indicates the versatility of NP chambers. The present study also demonstrates that smaller residual primary oocytes from the previous year’s gametogenesis can resume vitellogenesis as NPs begin to accumulate nutrients in the pre-gametogenesis and NP renewal stage. Our observations correspond with those made for S. purpuratus by Holland and Giese (1965, pages 251–52), who also recognized resumption of vitellogenesis in residual oocytes. In ovaries, resurrecting residual oocytes may be a mechanism for maximizing oocyte production. In males, new spermatozoa may be produced prematurely, but they cannot fertilize ova, which will not be available for several months; such spermatozoa may be phagocytized by NPs.
Because the nutritional state of individual sea urchins is vital to the progress of gametogenesis (Meidel and Scheibling, 1998; Garrido and Barber, 2001), the provisioning of residual primary oocytes and the premature production of spermatozoa should also depend upon nutrition. In poorly fed individuals, most nutrients are used for somatic growth (probably including that of the NP), and gametogenesis itself may not occur (Thompson, 1982). The fact that some residual primary oocytes may die and undergo phagocytosis within residual NP incubation chambers where they might otherwise be provided with nutrients appears to support this view. A nutrient-sensing mechanism like that in the fat bodies of Drosophila melanogaster (Colombani et al., 2003) must exist in the sea urchin—perhaps in the NPs themselves. Such a system could monitor nutrient availability and lead to resumption of vitellogenesis by residual oocytes or premature production of spermatozoa (when nutrients are abundant) or to their phagocytosis (when nutrients are limiting).
Nutritive function of sea urchin nutritive phagocytes
Studies probing the mechanisms of nutrient delivery to NPs (Varaksina, 1985; Brooks and Wessel, 2003) and from them to germ cells are limited (Unuma et al., 1998, 2003; Brooks and Wessel, 2002). The molecular structure of MYP, the most abundant protein stored in yolk platelets (80%), has recently been determined. This protein is also a major component of the membrane-bound vesicles in NPs and is identical in both sexes. Molecular analysis of MYP indicates that it is not similar to the vitellogenin component of yolk seen in most other animals; instead, it is a transferrin-like protein that can bind iron (Unuma et al., 2001; Brooks and Wessel, 2002). MYP is synthesized in the gut and coelomocytes and then imported to NPs (Shyu et al., 1986; Harrington and Ozaki, 1986); it can also be synthesized locally within them (Unuma et al., 2001). No one has yet suggested why (or if) iron binding by MYP is involved in gametogenesis in either sex. Iron binding could, however, be important when eggs are fertilized and use the enzyme ovoperoxidase to form the fertilization membrane, a process that generates hydrogen peroxide as a by-product (Shapiro, 1991). Although the egg has systems that scavenge hydrogen peroxide (Shapiro, 1991), chelation of transition metals, especially iron, would be essential to prevent highly reactive hydroxyl radicals from being formed via Fenton chemistry (Fridovich, 1998).
In testes, it is assumed that MYP from NPs is utilized during spermatogenesis but that it is metabolized and unrecognizable (Unuma et al., 1998, 2003). This assumption rests upon the observation that an antibody identifies MYP in vesicles in testicular NPs but no proteins in spermatogenic cells (Unuma et al., 1998, 2003). Nonetheless, the amount of MYP and other nutrients stored in vesicles within male NPs progressively diminishes, and a considerable supply of nutrients must be available to support the annual production of millions of spermatozoa.
During the summer, vitellogenesis of residual oocytes seems to depend on diffusional or pinocytotic uptake of carbohydrate nutrients released from NPs (Takashima and Takashima, 1965; Tsukahara and Sugiyama, 1969; Tsukahara, 1970). The observations of Brooks and Wessel (2003) are relevant here in relation to ours. These authors indicate that previtellogenic and smaller (< 50 µm) vitellogenic primary oocytes do not incorporate MYP, while larger (> 50 µm) vitellogenic primary oocytes do. They also point out that endocytosis by oocytes increases 2-fold and that specificity of oocyte uptake for MYP increases 10-fold in summer. They suggest that sea urchin primary oocytes are "bathed" in MYP at this time. The ovarian NP chambers we identified provide an explanation for how oocytes can be surrounded by dissolved nutrients. Even after isolation from the ovary, late vitellogenic primary oocytes can endocytose MYP using a dynamin-dependent mechanism (Brooks and Wessell, 2004). Our observations also suggest that residual oocytes in the pre-gametogenesis and NP renewal stage incorporate mostly PAS-positive nutrients, while BPB-positive nutrients are not added in a major way until the gametogenesis and NP utilization stage. Whether this sequence of events is also true for new vitellogenic primary oocytes generated during this stage is unclear.
Information of this kind is vital for those interested in the aquaculture of particular urchin species. An understanding of which nutrients are available to and can be incorporated by cells at particular gametogenic stages should make it possible to develop diets that contain these nutrients and that can be fed sequentially to urchins during gametogenesis. Most existing diets have been developed without much attention to the nutrient requirements of gametogenic cells over time and instead emphasize maximizing the size and color of the gonads as commercial products.
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Conclusions
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The structural context provided by the present study and the many outstanding questions regarding the dynamics of NP interactions with germ cells emphasize the need for a through molecular analysis of sea urchin gametogenesis. It is remarkable that so much is known about the molecules involved in fertilization and early development in this important model organism and that virtually nothing is known about the proteins expressed at different times during gametogenesis in either sex.
We have already benefited from data provided by the Sea Urchin Genome Project (based on Strongylocentrotus purpuratus, http://sugp.caltech.edu). A number of proteins potentially involved in processes carried out by NPs or germ cells of sea urchins during gametogenesis are transcribed from genes now known to be in the sea urchin genome. However, knowing what genes are present is a long way from understanding what their expressed proteins do during a particular biological process like gametogenesis.
Experimental analyses of jelly coat formation, germ-line stem cell mitosis, autophagy, and phagocytosis are logical starting points for understanding the molecules and mechanisms involved in specific aspects of sea urchin gametogenesis. We now know that proteins active in other organisms in similar processes are also present in strongylocentrotid sea urchins. These include (1) a number of sialic-acid-rich jelly coat glycoproteins (Kitazuma et al., 1994); (2) activin and c-myc, respectively an inducer and an indicator of the initiation of mitosis of gonial cells (Walker et al., 1992, 2001; Nagahama et al., 1995); (3) TOR, a nutrient-dependent inhibitor of autophagy (Hay and Sonnenberg, 2004; Klionsky, 2005); and (4) dynamin, Bcl2, and Bax, all involved in apoptosis-dependent phagocytosis (Brooks and Wessel, 2004; Ratts et al., 1995; Rucker et al., 2004). To understand more complex processes—for example, nutrient storage within and mobilization from NPs—functional genomic, proteomic, and metabolomic surveys need to be made during transitions between the precisely defined gametogenic stages elaborated in the present study (especially the transition from the pre-gametogenesis and NP renewal stage to the gametogenesis and NP utilization stages).
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Acknowledgments
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This study was supported by grants from Sea Grant (R/FMD-146; R/FMD-166) to CWW and MPL; a National Research Initiative Competitive Grant (no. 2002-35206-11631) to CWW from the USDA Cooperative State Research, Education, and Extension Service; and a Northeast Regional Aquaculture Center Grant (04-15) to CWW. We thank Drs. John M. Lawrence, Gary M. Wessel, Arkadiy A. Reunov, and especially John S. Pearse, for their excellent reviews of earlier versions of this manuscript. Finally, we thank Danielle M. Perrone for her cytochemical studies.
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Footnotes
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Received 10 January 2005; accepted 25 April 2005.
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Abstract
Introduction
