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Ultrastructure of oogenesis of two oviparous demosponges: Axinella damicornis and Raspaciona aculeata (Porifera)

Ultrastructure of oogenesis of two oviparous demosponges: Axinella damicornis and Raspaciona aculeata (Porifera)
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  Tissue and Cell 41 (2009) 51–65 Contents lists available at ScienceDirect Tissue and Cell  journal homepage: www.elsevier.com/locate/tice Ultrastructure of oogenesis of two oviparous demosponges:  Axinella damicornis and  Raspaciona aculeata  (Porifera) Ana Riesgo ∗ , Manuel Maldonado Department of Marine Ecology, Advanced Studies Centre of Blanes (CSIC), c/Acces cala St. Francesc 14, Blanes 17300, Girona, Spain a r t i c l e i n f o  Article history: Received 1 April 2008Received in revised form 15 July 2008Accepted 17 July 2008Available online 16 October 2008 Keywords: GametogenesisReproductionOocytesVitellogenesisYolkPorifera a b s t r a c t Weinvestigatedthecytologyoftheoogeniccycleintwooviparousdemosponges,  Axinelladamicornis and Raspaciona aculeata , during 2 consecutive years both by light and electron microscopy. Oocytes of bothspeciesweresimilarintheirbasicmorphologicalfeaturesbutdifferenceswerenoticedintimerequiredtocomplete oocyte maturation and mechanisms of acquisition of nutritional reserves. The oogenic cycle of   A.damicornis extendedfor7–8monthsinautumn-spring,whilethatof  R.aculeata diditfor3–5monthsinsummer-autumn.Yolkof   A.damicornis waspredominantlyformedbyautosynthesis.Oocytesendocytosedbacteriaindividuallyandstoredthemingroupsinlargevesicles.Bacteriaweredigestedandlipidicmate-rial was added to the vesicles to produce a peculiar granular yolk hitherto unknown in sponges. Scarcecells carrying heterogeneous inclusions were observed in the perioocytic space, and were interpreted asputative nurse cells. Such cells were presumably releasing lipid granules to the perioocytic space. In con-trast, large numbers of nurse cells were found surrounding the oocytes of   R. aculeata . They transportedbothlipidgranulesandheterogeneousyolkbodiestotheoocytes. R. aculeata alsoproducedsomeoftheiryolk by autosynthesis. The involvement of nurse cells in the vitellogenesis of   R. aculeata  shortened theoocytematuration,whereasalargelyautosyntheticvitellogenesisin  A.damicornis prolongedthedurationof oogenesis.© 2008 Elsevier Ltd. All rights reserved. 1. Introduction Sexual reproduction in demosponges exhibits profuse vari-ation in terms of mode (oviparism/viviparism and gonocho-rism/hermaphroditism), dynamics (from very short gametogeniccycles to nearly continuous gametogenesis) and morphology of gametes (Fell, 1974; Reiswig, 1983; Simpson, 1984; Boury-Esnaultand Jamieson, 1999; Riesgo et al., 2007a,b; Riesgo and Maldonado,2008a). Origin of gametes in demosponges is also diverse. To datethereisnoevidenceofapredeterminedgermline,anddemospongegametes are known to derive from at least three types of somaticcells (archaeocytes, choanocytes, or storage cells), depending onthespecies(Fell,1974,1983;Reiswig,1983;Simpson,1984;Willenzand Hartman, 2004).Oocytes usually derive from archaeocytes (see Fell, 1983 and Simpson, 1984 f or reviews), although in few cases, choanocyteshave been suggested as the oocyte anlagen (Diaz, 1973a,b; Gainoet al., 1986). Oocytes usually develop relatively scattered throughthe sponge mesohyl (Fell, 1983; Simpson, 1984), though in some ∗ Corresponding author. Tel.: +34 972336101; fax: +34 972337806. E-mail address:  ariesgo@ceab.csic.es (A. Riesgo). species appear clustered or aggregated (e.g., Lévi, 1956; Diaz,1973a,b; Fell and Jacob, 1979; Fromont, 1994; Riesgo et al., 2007b).Demospongeoocytesusuallydiffermorphologicallyinaspectssuchas oocyte size, type and abundance of yolk, presence of envelopingnurseorfollicularcells,andcollagenouscovers(seeFell,1974,1983;Simpson, 1984 f or reviews).Yolk formation (i.e., vitellogenesis) in poriferans, has beenreported to take place by: 1) autosynthesis, with or without usingpino-orendocytosedbasicprecursors(proteins,lipids,etc);2)het-erosynthesis,withyolkand/oryolkprecursorssuppliedbysomaticcells (nurse cells), or 3) both types simultaneously (Fell, 1974,1983; Simpson, 1984; Sciscioli et al., 1991). All these three typesofyokformationpatternshavebeenalsoreportedformostmarineinvertebrates(Nørrevang,1968;Anderson,1974;Eckelbarger,1994;RamírezLlodra,2002).Itispostulatedthatbasalinvertebratespre-dominantly form their yolk by the most “primitive” mechanism:autosynthesis(Eckelbarger,1994).Thediversepopulationsofinclu- sionstraditionallycalledyolkcanberoughlydividedintofattyyolk-lipid droplets- and proteid yolk -composed of protein and carbo-hydrates (Nørrevang, 1968; Anderson, 1974). Proteid yolk shows a remarkable uniformity throughout the animal kingdom, occurringas membrane-bound electron-dense bodies with a homogeneousstructure that, in some cases, possess a finely outlined dense core 0040-8166/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.tice.2008.07.004  52  A. Riesgo, M. Maldonado / Tissue and Cell 41 (2009) 51–65 Fig. 1.  Morphology and location of oocytes of   Axinella damicornis  and  Raspaciona aculeata  studied by light microscopy. Mature oocytes spread homogeneously within themesohyl of (A)  Axinella damicornis  and (B)  Raspaciona aculeata . Young (C) and mature (D) oocytes of   Axinella damicornis . Young (E) and mature (F) oocytes of   Raspacionaaculeata . embedded in a less dense matrix (Nørrevang, 1968; Anderson,1974; Adiyodi and Adiyodi, 1983). Only in some invertebrates,such as some molluscs, polychaetes, and echinoderms, yolk looksheterogeneous (e.g., ringed or vesiculated) (Anderson, 1974). In demosponges, yolk has been reported to have both homogeneous(e.g.,LéviandLévi,1976;GallissianandVacelet,1976;Scisciolietal.,1989, 1991, 2002; Riesgo et al., 2007a) and heterogeneous appear- ance (e.g., Diaz et al., 1975; Watanabe, 1978; Gaino et al., 1986;Gaino and Sarà, 1994; Lepore et al., 1995), but the latter is morecommon and abundant.Since vitellogenesis remains poorly documented in oviparousdemosponges, we selected two species belonging to differentorders:  Raspaciona aculeata  (Johnston, 1842) (order Poeciloscle-rida) and  Axinella damicornis  Esper, 1794 (order Halichondrida).The reproductive cycle of   Raspaciona aculeata  has been studiedby Riesgo and Maldonado (2008b), who reported the oogenesis to occurfromJulytoNovemberanddescribedthegametesusinglightmicroscopyonly.Similarly,littleisknownaboutthegametogenesisof   A.damicornis .Thedurationofthegametogeniccyclewasstudiedby Siribelli (1962) in the western coast of Italy and by Riesgo and Maldonado (2008b) in the northeastern coast of Spain. WhereasSiribelli (1962) reported the oogenesis to extend from Februaryto July, Riesgo and Maldonado (2008b) documented it from Octo- ber to May. In contrast, in the western coast of France, Lévi (1950)documented spawning of mature eggs of   A. damicornis  in Septem-ber. Thus, it appears that timing and duration of oogenesis in  A.damicornis  depend on the particular environmental characteristicsof each location. Again, all these studies described only the basicmorphology of gametes using light microscopy. Therefore, sinceultrastructural features of female gametes of   A. damicornis  and  R.aculeata arelargelyunknown,wedecidedtoinvestigatetheseooge-neses using light and electron microscopy to describe the processof oocyte maturation, with focus on the mechanisms of yolk pro-duction and storage. 2. Materials and methods  2.1. Sampling  We studied two populations of the oviparous demosponges  Axinelladamicornis and Raspacionaaculeata ,inthesublittoralrockycommunities of the North-eastern Mediterranean coast of Spain,between the localities of Blanes and Tossa de Mar (41 ◦ 11  18  N,2 ◦ 45  2  W). A previous 2-year study based on repetitive monthly   A. Riesgo, M. Maldonado / Tissue and Cell 41 (2009) 51–65  53 Fig.2.  Youngoocyteof   Axinelladamicornis .(A)Generalviewofayoungoocyte.(B)Largepseudopodiumemittedbyayoungoocyte.(C)Filiformmicrovilliwithasmallvesiclein the tip (mv) displayed by the oolemma. The cytoplasm of the oocyte contained heterogeneous yolk (hv). (D) Higher magnification of the microvilli, in close proximity to abacteria (b). sampling of 5 tagged individuals and additional fortnightly sam-plingof25untaggedindividualsduringthemaximumreproductiveactivity revealed that males and females of both species producesynchronically oocytes and spermatic cysts during a brief periodof the year (Riesgo and Maldonado, 2008b). From that study, a sex ratio close to 1:1 was inferred for  Raspaciona aculeata  and close to2:1 for  Axinella damicornis .Thecurrentcytologicalstudyoftheoogenesiswasbasedonlyonthe tagged individuals ( n =5) of each species mentioned above. Allthese5individualswerelargeandpresumablymatureadults.Theywere sampled monthly during 2 consecutive years (2003–2005).Using scuba and surgical scissors, we collected a small tissue piece(approx.1cm × 0.5cm × 0.3cm)fromeachspongeateachsamplingtime. In no case tissue collection involved death or reproductiveinterruption in the injured sponges over the study period. Tis-sue samples were divided into two pieces, one assigned to lightmicroscopyandtheothertoelectronmicroscopy.Samplesforelec-tron microscopy were always pre-fixed in 2.5% glutaraldehyde in0.2MMillonig’sphosphatebuffer(MPB)and1.4Msodiumchloride,and stored until further examination.  2.2. Light microscopy Tissue samples for light microscopy were maintained in ambi-ent seawater for transportation to the laboratory and fixed within2h after collection in 4% formaldehyde in seawater for 24h. Then,samples were desilicified with 5% hydrofluoric acid for 5h, rinsedin distilled water, dehydrated through a graded ethanol series(70%, 96%, 100%), cleared in toluene, and embedded in paraffin tocut them into 5  m-thick sections with an Autocut Reichert-Jungmicrotome 2040. After deparaffining with xylene, sections werestained with Hematoxylin-PAS, and studied through a Zeiss Axio-plan II compound microscope connected to a Spot Cooled Colordigital camera. When light-microscopy sections revealed oogenic  54  A. Riesgo, M. Maldonado / Tissue and Cell 41 (2009) 51–65 activity in any of the species, we resumed processing for samplesfor electron microscopy.  2.3. Transmission electron microscopy After primary aldehyde fixation, samples were post-fixed in 2%osmium tetroxide in MPB, dehydrated in a graded acetone series,and embedded in Spurr’s resin. Ultrathin sections obtained withan Ultracut Reichert-Jung ultramicrotome were mounted on goldgrids and stained with 2% uranyl acetate for 30min, then with leadcitrate for 10min (Reynolds, 1963). Observations were conducted withaJEOL1010transmissionelectronmicroscope(TEM)operatingat80kVandprovidedwithaGatanmoduleforacquisitionofdigitalimages. 3. Results  3.1. Axinella damicornis First evidence of oogenesis was detected in the sponge tissue inOctober-November(dependingonyear).Oogenesiswassynchronicin the studied population, and extended through April-May (7–8months). Most females (2 out of three) that produced oocytes in2004yeardiditalsoin2005.Oocytesconsistentlylocatedscatteredthroughout the mesohyl (Fig. 1A, C and D), and were very similar in morphology to archaeocytes, even showing a similar affinity forstains. Youngest oocytes, found in October and November, mea-sured approximately 30  m (Fig. 2A) and were lobate because of  the formation of pseudopodia (Figs. 1C and 2A and B). They had a nucleolatenucleusmeasuringapproximately10  m(Fig.1C).They Fig. 3.  Inclusions of the young oocyte of   Axinella damicornis . (A) Homogeneous yolk inclusion. Note the numerous vesicles (arrow head) surrounding the yolk body. (B)Vesicle containing fibrous material in the cytoplasm of the oocyte. (C) Vacuoles showing bacteria in different digestion stages (db) and numerous vesicles (arrow heads)with a fine-grained content in the cytoplasm. (D) Glycogen rosettes (g), heterogeneous yolk inclusions (hv), and multiple vesicles (arrow heads) within the cytoplasm of theoocyte.   A. Riesgo, M. Maldonado / Tissue and Cell 41 (2009) 51–65  55 displayed great number of filiform microvilli with a small vesiclein the tip (Fig. 2C and D). In early oocytes, vitellogenesis involved obvious changes inmost of the ooplasm, except for the perinuclear space. The oocytecytoplasm progressively filled with different types of inclusions:heterogeneous and homogeneous yolk bodies (Figs. 2C and 3A, respectively),smallvesicleswithfibrouscontent(Fig.3B),andvesi- cles with bacteria in different stages of digestion (Fig. 3C and D). Bacteria contained in these vesicles have previously been endocy-tosed from the mesohyl of the sponge (Figs. 2D and 4A) and stored individually in small vesicles (Fig. 4B–D). Later, such small vesi- cles fused together and showed evident signs of bacteria digestion(Figs. 3C and D and 4A). The ooplasm also showed great number of small electron-clear vesicles (Fig. 3A, C and D), which were par- ticularly abundant in the periphery of the oocyte (Fig. 2C and D). Glycogen rosettes located scattered within the ooplasm (Fig. 3D). Lipid droplets were scarce (not shown).Oocyte maturation progressed synchronously within andbetween individuals during both years of study. During oocytegrowth, both cytoplasm and nucleus increased in size. Matureoocytes became round, attaining approximately 120–150  m(Fig. 1A and D). The nucleolated nucleus (15  m in diameter)contained fine-grained chromatin and several chromatin masses(Fig.5AandB).Anarrowarea(approx.3  m)withscarceyolkinclu-sionsandhighlyvesiculatedsurroundedthenucleus(Fig.5AandB). Multipledictyosomeslocatedwithinthisperinucleararea,withthelamellaeorientedparalleltothenuclearmembraneandsmallvesi-clesdetachingfromtheirends(Fig.5B).Mitochondriawerehardto seebecauseofthehighdensityofinclusionsintheooplasm,aswellas endoplasmic reticulum and free ribosomes.In mature oocytes, different types of yolk inclusions wereobserved in the ooplasm, presumably being correlative stages of yolk formation: (1) heterogeneous membrane-bound compositescontainingsemi-digestedbacteria(Fig.5C–F),(2)largevesiclescon- Fig.4.  Endocytosisofbacteriain  Axinelladamicornis .(A)Microvilli(mv)oftheoolemmaclosetoafreebacteria(b).Thecytoplasmoftheoocyteshowednumerousmicrovesicles(v)andlargevesicleswithbacteriaindifferentstagesofdigestion(db).(BandC)Bacteria(b)endocytosedbyamid-stageoocyte.Notetheoolemma(ol)andthegreatnumberof microvesicles (v) in the periphery of the cytoplasm of the oocyte. (D) Small vesicles containing single bacteria (b) in the cytoplasm of an oocyte. Note the proximity to theoolemma (ol).

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