JOURNAL OF MORPHOLOGY 269:1412–1424 (2008)

Accumulation of Yolk in a ( ramaswamii) Oocyte: A Light and Transmission Electron Microscopic Study

Reston S. Beyo,1 Lekha Divya,1 Oommen V. Oommen,1* and Mohammad A. Akbarsha2

1Department of Zoology, University of , Kariavattom, Thiruvananthapuram, Kerala 695581, India 2Department of Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India

ABSTRACT There is a paucity of information on the lecithal (Balinsky and Devis, 1963; Hope et al., female reproductive biology of the caecilian 1964a,b). The major constituents of the when compared with the other vertebrate groups. yolk are phosvitin and lipovitellin. They are pro- Hence, the accumulation of nutrient reserves in the duced as a precursor material, vitellogenin, in the form of yolk and formation of yolk platelets were studied liver and transported in blood to the ovary for in Gegeneophis ramaswamii, adopting light microscopic histological and transmission electron microscopy analy- sequestration into the oocytes. Vitellogenin is pro- sis. Previtellogenic as well as vitellogenic follicles were teolytically cleaved into lipovitellin, phosvitin, and observed in appropriate preparations. On the basis of phosvette in the oocytes (Redshaw and Follett, the source and the routes of entry, we identified four 1971; Wiley and Wallace, 1981; Opresko and types of yolk precursor materials, precursors 1 to 4. The Karpf, 1987). This sequestration occurs through earliest material appearing in the oocyte consists of the follicle cells and/or via the intercellular spaces abundant lipid vesicles during the previtellogenic phase, between the follicle cells into the perivitelline i.e., much before the follicular epithelium is fully estab- space to be internalized by the oocyte (Hope et al., lished. This is a contribution from the oocyte mitochon- 1963; Dumont, 1978; Wallace, 1978; Lofts, 1984; dria, which we identified as yolk precursor material 1, Norris, 1997; Polzonetti-Magni, 2004). There is and it is autosynthetic. Once the follicle cell-oocyte inter- face is fully established, there is an accumulation of the also a view that the follicle cells do not merely principal component of the heterosynthetic yolk by translocate the heterosynthetic yolk but select and sequestration from the blood through the intercellular modify it (Uribe, 2003). spaces between follicle cells in a pinocytic process. This Yolk is a complex material and contains, in addi- we identified as yolk precursor material 2. There was tion to lipovitellin, phosvitin, and phosvette, sev- also an indication of a lipidic yolk material synthesis in eral other proteins, carbohydrates, and lipids the follicle cells sequestered from maternal blood (Albanese-Carmignani and Zaccone, 1977; Romek, through the follicle cells in an endocytic process in 1998; Buschiazzo et al., 2003; Buschiazzo and which the macrovilli of follicle cells and the microvilli of Alonso, 2005). It is generally believed that the lat- the oocyte play a role. This we identified as yolk precur- ter macromolecules are produced by the oocyte sor material 3. Contribution to the yolk of peptidic, gly- cosidic, and/or lipidic material synthesized in the vitello- itself, but a contribution from the follicle cells can- genic oocyte was also indicated. This we identified as not be excluded (Sanchez and Villecco, 2003; yolk precursor material 4. The sequential development Uribe, 2003). Thus, yolk as a complex entity has of intercellular associations and indications of synthesis/ contributions from i) the liver, ii) the follicle cells, sequestration of the yolk have been traced. Thus, we and iii) the oocyte. These various yolk constituents report the mechanistic details of synthesis/sequestration associate from small to large yolk platelets in the of the yolk materials in a caecilian. J. Morphol. 269: 1412–1424, 2008. Ó 2008 Wiley-Liss, Inc. Contract grant sponsor: Department of Zoology, University of KEY WORDS: ; oocyte; ovarian follicles; Kerala, Thiruvananthapuram (DST-FIST); Contract grant number: vitellogenesis; yolk UGC-SAP No: F-3-15/2007; Contract grant sponsor: Department of Animal Science, Bharathidasan University, Tiruchirappalli; Con- tract grant numbers: DST-FIST SR/FST/LSI-112/2002, UGC-SAP F.3-5/2007 (SAP-II). Vitellogenesis is the accumulation of yolk in the ooplasm. The amount of yolk accumulated depends *Correspondence to: Prof. Oommen V. Oommen, Department of upon the species in relation to the pattern of em- Zoology, University of Kerala, Kariavattom Campus, Thiruvanan- bryonic development and the quantity of nutrient thapuram, Kerala 695581, India. E-mail: [email protected] material required for the embryo. Most amphib- Published online 5 September 2008 in ians produce large eggs with abundant yolk and, Wiley InterScience (www.interscience.wiley.com) hence, are considered to be macrolecithal and telo- DOI: 10.1002/jmor.10670

Ó 2008 WILEY-LISS, INC. YOLK ACCUMULATION IN CAECILIAN OOCYTE 1413 ooplasm, which have a differential distribution in the ovary in relation to the reproductive cycles, the cortex and medulla and also along the animal– adopting light microscopic tools. Therefore, to pro- vegetal axis (Sanchez and Villecco, 2003; Uribe, vide an in-depth knowledge about the aspects of 2003). ovarian changes in caecilians, taking advantage of The mechanisms underlying the synthesis of the the fairly abundant distribution of caecilians in yolk constituents and their formation into yolk pla- the of India (Oommen et al., 2000), telets in amphibians are known through investiga- an extensive study was conducted on Ichthyophis tions in anuran species, Xenopus laevis (Redshaw tricolor and Gegeneophis ramaswamii, using trans- and Follett, 1971; Dumont, 1972; Wallace and Ber- mission electron microscopy (TEM). We have gink, 1974; Wallace, 1985; Wall and Patel, 1987a,b; recently described in these caecilians about the Richter and Bauer, 1990; Wallace and Selman, stages in the female reproductive cycle and the fol- 1990), Rana esculenta (Wartenberg and Gesuk, licle cell-oocyte interface (Beyo et al., 2008), follicu- 1960; Albanese-Carmignani and Zaccone, 1977), logenesis (Beyo et al., 2007a), and the ultrastruc- Rana pipiens (Ward, 1978), Rana tigrina (Sretar- tural organization of previtellogenic follicles (Beyo ugsa et al., 2001), Flectonotus pygmaeus (Del Pino et al., 2007b). Because these two species do not dif- and Humphries, 1978), Bufo arenarum (Valdez fer much in ovarian cycles (Beyo et al., 2007a,b, Toledo and Pisano, 1980; Villecco et al., 1999; 2008), further studies were limited to G. ramaswa- Buschiazzo et al., 2003; Buschiazzo and Alonso, mii because it is more abundant than I. tricolor in 2005), and Ceratophrys cranwelli (Villecco et al., the collection area. In this article, we describe the 1999), and urodele species, Notophthalmus virides- accumulation of yolk and establishment of yolk cens (Hope et al., 1963, 1964a), Triturus cristatus platelets in the oocytes of G. ramaswamii. (Albanese-Carmignani and Zaccone, 1977), and Necturus maculosus (Kessel and Ganion, 1980). The caecilians are an enigmatic group of MATERIALS AND METHODS amphibians with discontinuous distribution lim- The methodology adopted was described (Beyo et al., 2007a,b, ited to the tropics. Their limbless nature, vermi- 2008). Briefly, monthly samples of medium-sized Gegeneophis form appearance, subterranean habitus, consider- ramaswamii (Caeciliidae) were collected from the Western able freedom from water, including for breeding Ghats of Kerala and Tamil Nadu, India. Each month three female were sacrificed using 25% MS-222 (tricaine and development, and internal fertilization, mak- methane sulfonate) anesthesia and dissected to expose the ing use of the eversible phallodeum as an intromit- female reproductive system. In accordance with Beyo et al. tant organ, are some of the features which chal- (2008), the ovarian follicles were assigned to stages. The ovaries lenge the biologists in understanding of the evolu- were removed, washed thoroughly in an amphibian saline solu- tion. Although the female reproductive biology of tion, and then fixed in 2.5% glutaraldehyde solution. After fixa- tion the tissue was washed in cacodylate buffer, postfixed in 1% anurans and urodeles has been studied rather osmium tetroxide, and embedded in methacrylate (Sigma extensively (see Sanchez and Villecco, 2003; Uribe, Chemical, St. Louis, MO). Semithin sections (1 lm thick) were 2003), corresponding studies in the caecilian are stained in toluidine blue O (TBO). Ultrathin sections, obtained scanty (see Exbrayat, 2006). The major contribu- with a Leica ultra-microtome (Jena, Germany), were stained with uranyl acetate and lead citrate and subjected to transmis- tions to the understanding of various basic aspects sion electron microscopic analysis using a Phillips 201C trans- of female reproductive biology of caecilians have mission electron microscope (Amsterdam, Holland). The light come from the work of Wake and her associates, microscopic preparations were observed with a Leica research in Dermophis mexicanus, Gymnopis multiplicata, microscope and the images were captured in a Pentium IV com- etc., (Wake, 1968, 1970a,b, 1972, 1977, 1980, puter using Q-Win software (Leica, Jena, Germany). 1993a,b, 1998; Wake and Dickie, 1998) and Exbrayat and his associates in Typhlonectes RESULTS compressicauda (Exbrayat and Collenot, 1983; Appearance, Accumulation, and Exbrayat and Delsol, 1985; Exbrayat and Laurent, Condensation of Yolk in the Oocyte 1986; Exbrayat et al., 1981, 1995; Exbrayat, 1983, Cytoplasm 1984a,b, 1985, 1986, 1992a,b, 1993, 1996, 2006). Sporadic contributions have also come from studies Some material for yolk formation appears in the of other species such as Ichthyophis glutinosus oocyte cytoplasm during stage II previtellogenic (Sarasin and Sarasin, 1887–1890), Hypogeophis follicles much before the follicular epithelium is rostratus (Tonutti, 1931), Uraeotyphlus menoni fully established and the vitelline envelope (VE) (Chatterjee, 1936), Uraeotyphlus oxyurus (Garg formed. The material appears as small vesicles and Prasad, 1962), Chthonerpeton indistinctum scattered throughout the cytoplasm individually or (Berois and De Sa`, 1988), Ichthyophis beddommei in small clusters. The appearance of these vesicles (Masood-Parveez and Nadkarni, 1991, 1993a,b; suggested that they contain a lipid material (Fig. Exbrayat et al., 1998). 1A). These vesicles are abundant around the ger- These studies are in general cursory, compared minal vesicle (Fig. 1B). In a magnified view, the to those of anurans and urodeles, describing content of these vesicles revealed an electron- female reproductive anatomy or gross changes in dense core surrounded by an electron-lucent mate-

Journal of Morphology 1414 R.S. BEYO ET AL.

Fig. 1. Gegeneophis ramaswamii. Oocytes prior to establishment of the complex follicle cell-oocyte interface. TEM. A: Accumula- tion of yolk precursor 1 (YP1) among the profuse rough endoplasmic reticulum (RER) in a previtellogenic oocyte. B: These YP1 pre- cursors are present in the ooplasm up to the germinal vesicle (GV). NE, nuclear envelope; NU, nucleolus. C: The yolk precursor 1 (YP1) magnified, showing a dense concretion around the periphery of each mitochondrion (MI) and rough endoplasmic reticulum (RER). D: The prominent golgi apparatus (GA) of the oocyte. RER, rough endoplasmic reticulum. Scale bar in A 5 0.5 lm; in B 5 0.5 lm; in C 5 0.12 lm; in D 5 0.1 lm. rial (Fig. 1C). The cytoplasm of the oocytes at this pinching of these blebs (not shown). In such mito- stage is rich in rough endoplasmic reticulum chondria, the cristae are disturbed and have an (RER) and mitochondria (Fig. 1C). The Golgi appa- irregular pattern (Fig. 2B–D). This is followed by ratus (GA) is prominent (Fig. 1D). The vesicles the appearance of vesicles mentioned earlier, appear immediately after the breakdown of the mi- which at higher magnifications appeared as large tochondrial cloud and dispersion and proliferation vesicles dispersed among the distorted mitochon- of the mitochondria. A noticeable change in the dria (Fig. 3A,B). Subsequently, the content of these mitochondria at this stage is the appearance of vesicles undergoes condensation beginning from blebs of the outer membrane (Fig. 2A,B) and the the core (Fig. 3C) and proceeding towards the

Journal of Morphology YOLK ACCUMULATION IN CAECILIAN OOCYTE 1415

Fig. 2. Gegeneophis ramaswamii. Previtellogenic oocytes. TEM. A: The abundant mitochondria (MI), some of which produce blebs (arrowheads). B–D: The different manifestations of the mitochondrial blebs. The bleb has a content that is dense in the pe- riphery and amorphous in the core (arrowheads). The cristae of the mitochondria are atypical. RER, rough endoplasmic reticulum. Scale bar in A 5 1 lm; in B 5 0.1 lm; in C 5 0.1 lm; in D 5 0.1 lm.

periphery (Fig. 3D) to form a yolk precursor mate- This material is the heterosynthetic yolk precur- rial which is autosynthetic, produced in the oocyte sor, sequestered into the ooplasm across the follic- itself. We designated this substance as yolk precur- ular epithelium from the capillaries. We desig- sor material 1. nated these yolk precursors as yolk precursor ma- In the stage III oocyte the macro and microvilli terial 2. Large vesicles containing an amorphous are established and the VE is being established. material also appear in great numbers during this Additional material of the yolk appears in the cort- stage (Fig. 4A,B). This we designated as yolk pre- ical cytoplasm of the oocyte as small to large cursor material 3. vesicles, the content of which undergoes condensa- In the stage IVa oocyte, the yolk precursor mate- tion into one to several dense granules (Fig. 4A,B). rial 2 in the cortical cytoplasm undergoes conden-

Journal of Morphology 1416 R.S. BEYO ET AL.

Fig. 3. Gegeneophis ramaswamii. Previtellogenic oocytes showing the appearance and condensation of the autosynthetic yolk precursor 1 (YP1). TEM. A: The autosynthetic yolk precursor 1 is abundantly present among the mitochondria (MI). B: A magni- fied view showing the mitochondria (MI) sandwiched among autosynthetic yolk precursors 1 (YP1). The content of the YP1 is partially condensed (arrowheads). C: The condensation is intensified, resulting in a dense core and an amorphous boundary (arrow- heads). D: The condensation is much more intensified, resulting in thinning of the amorphous periphery (arrowheads). Mitochon- dria (MI) are prominent. The uncondensed yolk particles 1 (asterisks) are also present. Scale bar in A 5 0.2 lm; in B 5 0.1 lm; in C 5 0.1 lm; in D 5 0.2 lm. sation with little, if any, corresponding condensa- sor material 2 undergoes much more condensation, tion in the medullary cytoplasm. In the latter, on becoming dark, dense, and abundant. The con- the contrary, there are vesicles (yolk precursor ma- densed yolk material is surrounded by a clear or terial 1) in a density much different from the corti- amorphous boundary. In the medullary cytoplasm, cal heterosynthetic yolk precursor material 2 (Fig. there are enormous lightly stained vesicles with a 5A,B). The cortical vesicles of yolk precursor mate- thin dense outer boundary and an almost clear rial 3 continue to persist (Fig. 5B). In the stage core, the yolk precursor material 1 (Fig. 6A,B). In IVb oocyte the heterosynthetic cortical yolk precur- the stage IVc oocyte, the cortical yolk precursor

Journal of Morphology YOLK ACCUMULATION IN CAECILIAN OOCYTE 1417 of yolk granules is almost similar to that in stage Va but the dark dense cortical yolk granules fur- ther increase in size, with smaller yolk precursors fusing with them to form the yolk platelets. There is also the addition and accumulation of an amor- phous material around the periphery of the con- densing yolk platelets. Localization of several vesicles in the cytoplasm of the follicle cells was observed during this stage (Fig. 9A). TEM observa- tion revealed these precursors to have a dense core and an amorphous boundary in an alveolar pat- tern (Fig. 9B). The contents of these vesicles are transferred to the oocyte through the long proc- esses of the follicle cells which intrude deep into the oocyte; it constitutes yolk precursor material 4. The condensation and assembly of the yolk ma- terial in the oocyte result in the formation of yolk platelets (Fig. 9C). The yolk precursor material 3, first appearing during stage IIIa, persists until stage Vb (Fig. 9C) and much beyond (not shown).

Fig. 4. Gegeneophis ramaswamii. An early vitellogenic fol- licle (stage III) showing accumulation of heterosynthetic yolk precursor. TEM. A: A low power micrograph showing theca (TH), basement membrane (BM) of the follicle cells (FC), perivi- telline space (PV), macrovilli and microvilli (arrowheads), ooplasm (OP), yolk precursor 2 (YP2) undergoing condensation and lipid droplets (LD). B: A portion enlarged showing the yolk precursors 2 (YP2), lipid droplets (LD) or yolk precursors 3 (YP3), and the mitochondria (MI). Scale bar in A 5 3 lm; in B 5 0.75 lm. material 2 vesicles are much more condensed. Those along the outer region are larger than the ones deeper in the cytoplasm. The yolk precursor material 3 is more widely distributed in the outer cortical cytoplasm (Fig. 7A,B). In the stage Va oocyte, the cortical yolk precur- sor 2 vesicles are further condensed into dense granules, many of which are quite large. These yolk granules are biphasic, with an amorphous thin periphery and a dense core. This amorphous material appears to be added to the yolk granules from the cytoplasm (Fig. 8A,B). Deeper in the med- ullary cytoplasm there are small yolk granules Fig. 5. Gegeneophis ramaswamii. A: Toluidine blue O- comparable in content to those in the cortical cyto- stained semithin section of a stage IVa oocyte. Note the abun- plasm but, in addition, there are larger vesicles dant large yolk precursors 2 (YP2) in the cortical cytoplasm (CC) and their relative absence in the medullary cytoplasm with staining properties different from those of the (MC). B: Cortical cytoplasm of stage IVa oocyte showing yolk dense yolk granules, the yolk precursor material 2 precursor 2 (YP2) and lipid droplets (LD). TEM. Scale bar in A (Fig. 8C). In the stage Vb oocyte, the organization 5 2.5 lm; in B 5 1.5 lm.

Journal of Morphology 1418 R.S. BEYO ET AL. of the heterosynthetic yolk precursor material 3 by the oocyte appears to occur through these mem- brane specializations, viz., coated pits, coated vesicles, and pinocytic vesicles, resulting in endo- cytic vesicles (Fig. 10B). When a section of an oocyte is cut in a diagonal plane it looks as though the macrovilli are engulfed by the oocyte (Fig. 10D). There is a second route for material to reach the oocyte reflected in the continuous passages formed at the intercellular spaces between the follicle cells. This is in spite of the desmosomes, since a thorough passage is still maintained (Fig. 11A). Certain of the heterosynthetic yolk precursors, in all likelihood yolk precursor type 2, would reach the perivitelline space from the thecal capillaries through these passages. This mode is further strengthened by certain areas where a follicle cell layer is extremely thin so that the perivitelline space is prominent with fewer micro and macro- villi (Fig. 11B). There is a wide gap between two follicle cells (Fig. 11C) so that the perivitelline

Fig. 6. Gegeneophis ramaswamii. A,B: Toluidine blue O- stained semithin sections of stage IVb oocyte showing the differ- ence in the organization of the yolk in the cortical (CC) and medullary (MC) cytoplasm. CY, cortical yolk, MY, medullary yolk; LD, lipid droplets. Scale bar in A 5 2.5 lm; in B 5 1.5 lm.

Source and Routes of Sequestration of the Yolk Precursors The follicle cell-oocyte interface, with special ref- erence to macrovilli of follicle cells, microvilli of oocytes and different kinds of membrane associa- tions between the two, provides for transfer of the heterosynthetic yolk precursor material from the follicle cells to the oocyte (Beyo et al., 2008). Gap and tight junctions that appear in the perivitelline space between macro and microvilli facilitate pino- cytic uptake of heterosynthetic yolk precursor mate- rial 2 from the follicle cells to the oocyte (see Fig. 10). Long cytoplasmic processes of follicle cells extend up to and even project into the oocyte, pro- viding for formation of coated pits, which pinch off to become coated vesicles (Fig. 10A). There are also invaginations of the oolemma where follicle cell Fig. 7. Gegeneophis ramaswamii. A: Toluidine blue O- processes are not seen (Fig. 10A). With the estab- stained semithin section of a stage IVc oocyte showing the highly condensed cortical yolk (CY); follicle cells (FC) are also lishment of the VE (Fig. 10B), the macro and micro- shown. B: Cortical cytoplasm showing cortical yolk (CY) and villi come to associate only through tunnels in the lipid droplets (LD) or yolk precursor 3. TEM. Scale bar in A 5 VE (Fig. 10C,D). The acquisition of a major portion 2.5 lm; in B 5 0.5 lm.

Journal of Morphology YOLK ACCUMULATION IN CAECILIAN OOCYTE 1419 the anuran and urodele species so far studied (Sanchez and Villecco, 2003; Uribe, 2003). The inferences that emerge are as follows:

1. In one route, the major yolk precursor (vitello- genin) passes through the basal lamina and the underlying follicular epithelium and localizes within the intercellular spaces between the fol- licular cells and also the pores in the vitelline envelope (Wallace, 1985; Opresko and Karpf, 1987; Richter, 1987; Villecco et al., 1999; San- chez and Villecco, 2003). It is transferred to the oocyte through pinocytosis (Brummett and Dumont, 1977; Wallace, 1985; Richter, 1987; Opresko and Karpf, 1987; Villecco et al., 1999; Sanchez and Villecco, 2003; Uribe, 2003). This is yolk precursor material 2 in this study.

Fig. 8. Gegeneophis ramaswamii. Toluidine blue O-stained semithin sections of stage Va oocytes. A: Cortical cytoplasm (CC) with abundant cortical yolk (CY). B: A magnified view of cortical yolk (CY). Arrowhead points to addition of new material to the yolk precursor. C: Medullary cytoplasm showing medul- lary yolk (MY) among lipid droplets (LD). Scale bar in A 5 2.5 lm; in B 5 1.5 lm; in C 5 1.5 lm. space reaches the basal lamina of the follicle cells. This material is acquired by the oocyte. The dense material, with an alveolar boundary, present in the cytoplasm of follicle cells (Fig. 9B), suggests synthetic activity in the follicle cells for contribu- tion to the yolk as yolk precursor material 4. This is supported by the presence of abundant RER, mi- tochondria (Fig. 11D), and dense vesicles (Fig. 9B) in the cytoplasm of these cells. This material would arrive at the oocyte through the long contin- uous processes formed from the follicle cells and intrude deep into the oocyte. Fig. 9. Gegeneophis ramaswamii. A: Toluidine blue O- stained semithin sections of a stage Vb oocyte showing the DISCUSSION outer cortical region relatively free from yolk. The deeper corti- cal ooplasm has small to large cortical yolk platelets (CYP). The ultrastructural organization of the ovarian Note a dense material in the follicle cell (FC) cytoplasm (arrow- follicles of Gegeneophis ramaswamii, with special heads). B: Electron-dense material (DE) in the follicle cell cyto- plasm containing a dense core and an amorphous (AM) periph- reference to the appearance and accumulation of ery in an alveolar pattern. TEM. C: The heterogeneous nature yolk precursors and their fabrication into yolk pla- of the yolk platelets (YP) and also lipid droplets (LD). TEM. telets, is similar in most respects to that in most of Scale bar in A 5 2.5 lm; in B 5 0.2 lm; in C 5 0.2 lm.

Journal of Morphology 1420 R.S. BEYO ET AL.

Fig. 10. Gegeneophis ramaswamii. Follicle cell-oocyte interface at different stages of vitellogenesis. TEM. A: Stage III follicle showing follicle cell (FC), macrovilli (MAV), microvilli (MIV), perivitelline space (PV), coated vesicle (CV), coated pit (CP), and ooplasm (OO). A follicle cell process (arrowhead) reaches up to the oocyte and projects into it (arrow). B: Stage IVc follicle showing VE, with tunnels (TU), and endocytic vesicles (VS). C: Same as Figure 10B, showing the tunnels (TU) in a diagonal section. The vitelline envelope (VE), coated pits (CP), coated vesicles (CV), and multivesicular bodies (MVB) are also shown. D: A portion magni- fied, showing macrovilli (MAV), apparently surrounded by the oolemma (OL); microvilli (MIV) and perivitelline space (PV) are also shown. Scale bar in A 5 0.5 lm; in B 5 0.5 lm; in C 5 0.3 lm; in D 5 0.2 lm.

2. In the second route, the yolk precursor material in all likelihood is the yolk precursor material 3 would pass through the follicle cells and reach of this study and would constitute the hetero- the oocyte through the macro and microvilli, synthetic lipids and/or glycogen. especially the areas of membrane associations 3. One or more of the yolk precursors may be syn- as in the anurans Ceratophrys cranwelli, Bufo thesized by the follicle cells themselves, which arenarum (Villecco et al., 1999; Sanchez and is/are acquired by the oocyte through the long Villecco, 2003), and Xenopus laevis (Dumont, processes of the follicle cells that project into 1978) and the urodeles Ambystoma dumerilii the oocyte (Hope et al., 1963). This is yolk pre- and Ambystoma mexicanum (Uribe, 2003). This cursor material 4 of this study.

Journal of Morphology YOLK ACCUMULATION IN CAECILIAN OOCYTE 1421 during the vitellogenic period, 80–90% of total oocyte protein is derived from the specific uptake of vitellogenin from the maternal blood stream, which means that the remaining 10–20% is syn- thesized in the oocyte itself. Electron microscopic studies of the Necturus maculosus oocyte indicated that the primary proteinaceous yolk arises within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appeared to result from the activity of the Golgi complexes, which form multivesicular and granular–vesicular bodies, the limiting membrane of which is at times incom-

Fig. 11. Gegeneophis ramaswamii. A: A highly magnified transmission electron micrograph of intercellular junction between two follicle cells (FC) showing a through passage along the desmosome (arrowheads) connecting to the perivitelline space (PV). B: A thin follicle cell (arrowhead) overlying a spa- cious perivitelline space (PV). TEM. C: A portion of follicular epithelium where there is a wide gap (arrowhead) between two follicle cells (FC). TEM. D: A portion of a follicle cell (FC) rest- ing on the basement membrane (BM). TEM. The cytoplasm of the cell contains mitochondria (MI), rough endoplasmic reticu- lum (RER), and vesicles containing a material (arrowhead). NU, nucleus. Scale bar in A 5 0.2 lm; in B 5 0.8 lm; in C 5 0.8 lm; in D 5 0.8 lm.

4. The material(s) acquired by the oocyte take a pinocytic pathway in the first case and an endo- Fig. 12. Gegeneophis ramaswamii. A schematic illustration cytic pathway in the second and third. of the origin and pathways of the various constituents of the yolk platelets. 1, mitochondrial blebbing and discharge of lipid 5. There is evidence for autosynthesis of certain of material to form yolk precursor 1; 2, sequestration of the major the yolk constituents, including yolk precursor heterosynthetic (yolk precursor 2) from capillaries, through the material 1, as discussed in detail. intercellular spaces between the follicle cells, and its acquisition by the oocyte thro ugh pinocytosis; 3, acquisition of yolk precur- In Gegeneophis ramaswamii the ultimate yolk sor (yolk precursor 3) synthesized in the follicle cells; 4, auto- synthesis of yolk precursor (yolk precursor 4), other than that constituents that form the yolk platelets are heter- in 1 above, in the oocyte itself. CA, capillary; CT, connective tis- ogeneous in the sense that i) vitellogenin, lipids, sue strand; DS, desmosome; FC, follicle cells; GA, Golgi appara- and glycogen from the liver, ii) a contribution from tus; LD, lipid droplet; LY, lysosome; MVB, multivesicular the follicle cells, and iii) materials synthesized in bodies; MAV, macrovilli; MIV, microvilli; MI, mitochondria; OE, ovarian epithelium; RER, rough endoplasmic reticulum; PL, the oocyte itself, are assembled into small to large yolk platelet; VE, vitelline envelope; YP1, yolk precursor 1; yolk platelets (see Fig. 12). According to Opresko YP2, yolk precursor 2; YP3, yolk precursor 3 (lipid droplet); and Wiley (1987), in the Xenopus laevis oocyte YP4, yolk precursor 4. (Not to scale).

Journal of Morphology 1422 R.S. BEYO ET AL. plete. During differentiation, the yolk precursor the anurans Ceratophrys cranwelli and Bufo arena- sacs contained small vesicles similar in size to rum (Villecco et al., 1999). Thus, our data suggest Golgi vesicles, larger vesicles similar to vesicular that the pattern of sequestration and accumulation elements of the granular endoplasmic reticulum of heterosynthetic yolk precursors in the caecilian and, on occasion, portion of a mitochondrion. The examined appears be similar to that in the anurans interior of these sacs becomes granular, perhaps and urodeles studied. by dissolution of the components just described Uribe (2003) has suggested that the contribution and, soon becomes organized into a crystalline con- from the follicle to the yolk is lipid. As seen in this figuration (Kessel and Ganion, 1980). study, lipid inclusions are abundant among the According to Kress (1982), the oocytes of Ambys- yolk vesicles in the vitellogenic oocyte of Gegeneo- toma mexicanum indicate a small but distinct con- phis ramaswamii. The abundant lipid in the tribution of the oocyte itself towards yolk forma- oocyte, vis a vis yolk platelets, may be an adapta- tion. This process is called autosynthesis and tion to provide for development of the embryo to starts before the onset of the heterosynthetic activ- the juvenile stage (Mu¨ ller et al., 2005) unlike in ities. The cell organelles possibly involved in this most anurans and urodeles in which the egg yolk precursor formation are the annulate lamel- hatches as tadpole. Characterization of full-grown lae, endoplasmic reticulum, Golgi complex, RER, Bufo arenarum oocytes revealed varied lipid and mitochondria. In Gegeneophis ramaswamii, at classes viz., saturated phospholipids (phosphatidyl- least the substance of the yolk accumulating prior choline and phosphatidylethanolamine as the to establishment of the complex follicle cell-oocyte major constituents), unsaturated phospholipids interface is the product of autosynthesis and we (phosphatidylinositol enriched in arachidonic acid designate it as yolk precursor material 1. The as the principal constituent), triacylglycerols, and major contribution to this substance appears to polar and neutral lipids to be present in the yolk come from the oocyte mitochondria. We have sug- (Buschiazzo et al., 2003; Buschiazzo and Alonso, gested previously that mitochondria contribute to 2005). The total amount of phospholipids in stage the previtellogenic lipid synthesis in the caecilian IV oocytes is around 50% higher than in stage III oocyte (Beyo et al., 2007b). A recent study in the oocytes, indicating an increase in lipid content as fishes Acipenser gueldenstaedtii and Polydon spa- the oocyte progresses in growth (Bruzzone et al., thula by Zelazowska and Kilarski (2007) adds 2003). Growing evidence indicates that lipids are strength to our inference that this material is involved in the maturation process in Bufo arena- lipid. The early dictyotene and previtellogenic rum full-grown arrested oocytes. They are mainly oocyte mitochondria, with distorted and fused cris- located in yolk platelets, the principal organelles of tae, in these fishes contain and release material amphibian oocytes. The yolk platelet lipids are morphologically similar to that of lipid droplets. involved in resumption of the meiotic cell cycle, This process of mitochondrial transformation is suggesting that these organelles participate in a accompanied by an accumulation of lipid droplets dynamic role during amphibian development located in the vicinity of oocyte nucleus. This is (Buschiazzo and Alonso, 2005). The increase of lip- further strengthened by the observation in Bufo ids during oogenesis is consistent with the puta- arenarum that triacylglycerols, phosphatidylcho- tive use of these lipids as a source of energy in line, free fatty acids, phosphatidylethanolamine, embryo development (Buschiazzo et al., 2003). etc., are localized in the oocyte mitochondrial frac- This study reveals the accumulation of glycogen tion (Gili and Alonso, 2004). in the oocyte of Gegeneophis ramaswamii. In the In Necturus maculosus oocytes, an extensive amphibian oocytes, labeled glucose is readily incor- micropinocytotic activity is elaborated throughout porated into glycogen, which is the primary end the period of vitellogenesis and constitutes the pri- product of glucose metabolism. Frog oocytes incor- mary mechanism of yolk protein formation. Numer- porate glucose units into glycogen by so-called ous coated and smooth-surfaced vesicles, as well as direct (through UDP-glucose) and, preferentially, electron-dense and electron-lucent ones, fuse in the indirect (through lactic acid) pathways (Kessi cortical ooplasm to form progressively larger yolk et al., 1996; Ureta et al., 2001; Ba´ez et al., 2003; platelets (Kessel and Ganion, 1980). In vitellogenic Preller et al., 2007). In growing oocytes of Rana Xenopus oocytes, coated pits apparently fuse with esculenta and Triturus vulgaris, a carbohydrate coated vesicles and the latter fuse with each other fraction consisting of neutral mucopolysaccharides in the outermost cortical cytoplasm. Vesicles, is distributed in the superficial layer of each plate- depleted of their clathrin coat, fuse with cortical tu- let (Albanese-Carmignani and Zaccone, 1977). In bular endosomes and discharge their contents into Bufo arenarum oocytes, cytoplasmic feature evi- yolk endosomes. These endosomes are the direct dent during nuclear maturation is the gathering of precursors of the yolk organelles. Endocytic vesicles glycogen granules in clusters, some phagocytosed fuse only with primary yolk precursors to become by empty vesicles. Some vesicles were observed in the fully-grown yolk platelets (Richter and Bauer, close proximity to the oolemma and others were 1990). A similar situation has been described for freely suspended in the perivitelline space,

Journal of Morphology YOLK ACCUMULATION IN CAECILIAN OOCYTE 1423 extruded from the oocyte (Ramos et al., 1999). Dumont JN. 1972. Oogenesis in Xenopus laevis (Daudin). I. Therefore, it is suggested that in G. ramaswamii Stages of oocyte development in laboratory maintained ani- mals. J Morphol 136:153–179. oocytes the glycogen that accumulates is derived Dumont JN. 1978. Oogenesis in Xenopus laevis (Daudin). VI. from the follicle cells. The route of injected tracer transport in the follicle and devel- Thus, this study describes the mechanistic details oping oocyte. J Exp Zool 204:193–217. of vitellogenesis in a caecilian, most of which appear Exbrayat JM. 1983. Premieres observations sur le cycle annuel to be similar to those in the anurans and urodeles de l’ovaire de Typhlonectes compressicaudus (Dume´ril et Bibron. 1841). Batracien Apode vivipare. CR Acad Sci Paris but some, such as mitochondrial blebbing, appear- 296:493–498. ance of yolk precursor 1 as a product of autosynthe- Exbrayat JM. 1984a. Cycle sexual et reproduction chez un sis, accumulation of great amounts of lipids and gly- Amphibien Apode Typhlonectes compressicaudus (Dume´ril et cogen, etc., indicate aspects unique to the caecilian. Bibron. 1841). Bull Soc Herp Fr 32:31–35. Exbrayat JM. 1984b. Quelques observations sur l’e´volution des Further, four types of yolk precursors identified voies genitals femelles de Typhlonectes compressicaudus based on the source and route of accumulation may (Dume´ril et Bibron. 1841). Amphibien Apode vivipare, au be the characteristic of caecilian oocytes. cours du cycle de reproduction. CR Acad Sci Paris 298:13–18. Exbrayat JM. 1985. Cycle des canaux de Mu¨ller chez le male adulte de Typhlonectes compressicaudus (Dume´ril et Bibron. ACKNOWLEDGMENTS 1841). Amphibien Apode. CR Acad Sci Paris 301:507–511. Exbrayat JM. 1986. 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