Presence of calcium in oocytes of the diplopod Rhinocricus padbergi Verhoeff (Spirobolida, Rhinocricidae)

Texto

(1)Acta Histochem. Cytochem. 37 (5): 301–306, 2004. Presence of Calcium in Oocytes of the Diplopod Rhinocricus padbergi Verhoeff (Spirobolida, Rhinocricidae) Carmem S. Fontanetti1 and Maria I. Camargo-Mathias1 1 Departamento de Biologia, Instituto de Biociências, UNESP, Avenida 24–A, 1515, CP 199, 13506–900 Rio Claro, SP Departamento de Biologia, Instituto de Biociências, UNESP, Avenida 24–A, 1515, CP 199, 13506–900 Rio Claro, SP, Brasil. Received June 29, 2004; accepted November 22, 2004. tures apparently formed by overlapping calcium layers. Some authors have suggested that these structures represent a type of reserve used for the calcification of the embryo exoskeleton, whereas others believe that calcium inclusions are a mechanism of organism detoxification as a result of excess calcium ingested by animals during soil turnover. We suggest in this paper that the first hypothesis could be occurring in R. padbergi since at the juvenile stages of the individuals the uptake of calcium is low and because the oocyte is a specialized cell not associated with detoxification.. In diplopods, the presence of calciumcontaining structures seems to be a common finding in some species, with its formation being similar to that observed for other intracellular mineralization systems. In the present study, using histochemistry and transmission electron microscopy, a large amount of calcium was observed in the oocytes of Rhinocricus padbergi. Calcium was detected in both less and well developed oocytes, i.e., the occurrence of calcium coincided with the beginning of vitellogenesis. Calcium was observed as fine granulation distributed within the cytoplasm or deposited in spherical struc-. Key words: millipedes, oogenesis, Diplopoda, biomineralization, calcium. I.. detail the occurrence of calcium in R. padbergi oocytes.. Introduction. Investigations on the female reproductive system of diplopods are rare and have been basically conducted on species from Asia, with morphology and oogenesis being the main objective. Studies on the chemical nature of the elements forming oocytes are also scarce in the literature [1, 2, 7–9]. The ovaries of Rhinocricus padbergi consist of two paired cords to which oocytes in different stages of development are attached [3]. As observed for other diplopod species, no formation of ovarian follicles (oocyte+nurse cell) occurs and these cells are therefore similar to the panoistic type found in some insect groups [3, 6]. The R. padbergi oocytes possess, besides the elements normally found in these structures, i.e., lipids, proteins and carbohydrates, a large amount of calcium from the beginning of oocyte development [1]. The objective of the present study was to analyze in. Adult females of R. padbergi were collected on the UNESP, IB, Rio Claro, SP, Brazil, in January 1998 by B. Tiritan and E. R. Fantazzini. The ovaries were placed in saline (7.5 g NaCl, 2.38 g Na2HPO4+2.72 g KH2PO4+1000 ml of distilled water), fixed in 4% paraformaldehyde and embedded in Leica resin for histological processing. The glass slides with the sections were stained with both hematoxylin/eosin and the Von Kossa method, a histochemical test for calcium detection [4]. The ovaries were also fixed in 2.5% glutaraldehyde in 0.2% sodium cacodylate buffer, pH 7.2, and processed for transmission electron microscopy.. Correspondence to: Carmem S. Fontanetti, Departamento de Biologia, Instituto de Biociências, UNESP, Caixa Postal 199, 13506–900 Rio Claro, SP, Brasil. E-mail: fontanet@rc.unesp.br. Calcium was detected in R. padbergi throughout oocyte development, i.e., from the initial stages, in which the cytoplasm is homogenous and still without chorion deposition,. II. Materials and Methods. III.. 301. Results.

(2) 302. Fontanetti and Camargo-Mathias. Fig. 1. Von Kossa histochemical test in Rhinocricus padbergi oocytes. cb, concentric bodies; gv, germinal vesicle; y, yolk spheres; arrows, von Kossa-positive granules; *, central core of concentric bodies.. to the completely developed stages showing cytoplasm filled with yolk spheres and deposited chorion. Although calcium was present throughout the cytoplasm (Fig. 1A), a stronger. positivity to the Von Kossa test was observed in the central region of the oocyte (Fig. 1A, B). Calcium occurred in two forms: a) as a fine granulation.

(3) Calcium in Diplopod Oocyte. 303. Fig. 2. Electronic micrographs of R. padbergi oocytes. A, B. Peripheral portion. ca, calcium fine granules; ch, chorion; cb, concentric bodies; mi, mitochondria; arrows, junction of different concentric bodies; arrowheads, core of concentric bodies.. distributed between yolk spheres (arrows in Fig. 1C, D), or b) as spherical structures (concentric bodies) apparently formed by deposition in layers (Fig. 1C, D). These spherical structures often showed a strongly positive central region (asterisks in Fig. 1C, D) and several layers concentrically placed around the nucleus with different positivity. TEM techniques showed that the concentric bodies presented the same morphology both at the periphery (Fig. 2A, B) and in the central region of the oocyte. Each concentric body appeared to be contained in a vacuole surrounded by a membrane where calcium was deposited in a lamellar arrangement (Figs. 3A, B; 4A, B). Mitochondria varying in shape and size were found to be associated with these structures (Figs. 2, 3 and 4B). In some cases, the concentric bodies were probably the result of incorporation of smaller bodies (Figs. 2A, B) containing the same type of material. (arrows in Fig. 2A). Ultrastructural details revealed different arrangements of the concentric bodies in terms of the deposition of material inside them (compare Figs. 3A, B and 4A, B). Some bodies showed a single central core (Fig. 3A), while in others more than one core could be noted, with those containing three cores (arrowheads in Figs. 2A, B, C; 3B and 4) being most frequently observed, i.e., those with a tripartite morphological structure (Figs. 3B, 4A). These structures occur in both peripheral and central oocyte portions.. IV. Discussion During vitellogenesis, the yolk forming the oocytes of most animals normally consist of lipids, proteins and carbohydrates, which can occur in their free form or bound to.

(4) 304. Fontanetti and Camargo-Mathias. Fig. 3.. A, B. Concentric bodies in R. padbergi oocytes. cb, concentric bodies; m, membrane; mi, mitochondria; arrowheads, core.. other elements forming complexes. In addition to these elements, mineralized bodies have been observed in the ooplasm of some species but their function has yet to be established. Calcium-containing structures seem to be a common finding in diplopod oocytes, with their formation being similar to that of other intracellular mineralization systems [5]. Crane and Cowden [2] found cytoplasmic inclusions in the oocytes of four diplopod species, which were interpreted as consisting of organic matrix with crystallized calcium salts. These structures were called concentric ring bodies. (CRB) and, according to these authors, represent a calcium reserve for the formation of the exoskeleton of the future embryo. These bodies have also been observed by other researchers [9] but were interpreted as being of a protein nature, representing a form of yolk. Later, other author [7] found that CRB consist mainly of phosphates and calcium carbonates, are associated with a protein network and accumulate in the cisternae of the rough endoplasmic reticulum. Petit [7] commented that concretions in the oocytes of Polydesmus complanatus coincide with the deposition of lipid globules and, thus occur before protein deposition. Our.

(5) Calcium in Diplopod Oocyte. Fig. 4.. 305. A, B. Concentric bodies in R. padbergi oocytes. cb, concentric bodies; m, membrane; mi, mitochondria; l, lipid; arrowheads, core.. data suggest an occurrence of these structures shortly before lipid vitellogenesis since they are already observed in less developed oocytes, i.e., oocytes presenting a still homogenous cytoplasm [1]. The function of these structures in millipedes is still controversial. Studies have suggested [2, 7] that calcium salt accumulation represents a particular type of reserve used for the calcification of the embryo exoskeleton. These inclu-. sions may be a mechanism of mineral detoxification of the organism, since animals possessing these structures are constantly exposed to high mineral concentrations in the soil. This hypothesis was based on the fact that mineral accumulations are found in several other diplopod tissues, including the somatic ovarian tissue. The detoxification of diplopod oocytes and somatic tissue is a mainly intracellular process, but it cannot be ruled out that part of the excess mineral is.

(6) 306. Fontanetti and Camargo-Mathias. removed by extracellular deposition, as occurs during the development of the exoskeleton [5]. The fact that we observed calcium in R. padbergi oocytes during the initial developmental stages, i.e., during pre-vitellogenesis, corroborates other data [5]. However, the reserve hypothesis cannot be ruled out since the uptake of calcium at the beginning of development of the juvenile stages is low and because the oocyte is a specialized cell usually not associated with the function of organism detoxification. The present results also suggest that the incorporation and/or formation of concentric bodies occurs in a centripetal way since they were observed at high concentrations in the central region of the oocytes (Fig. 1A). This observation explains the association of these structures, which require energy for their transport from the peripheral to the central region, with a large number of mitochondria. The difference in the Von Kossa test positivity between concentric bodies located at the periphery (Fig. 1C, D) and those located in the central region (Fig. 1B) may indicate a higher calcium concentration in the latter. Our data therefore suggest two different processes for the formation of these structures: a) calcium deposition occurs in layers starting from a central core, resulting in a concentric lamellar structure (Fig. 3A), or b) calcium deposition is due to the coalescence of small bodies leading to larger structures with a variable, mostly tripartite, morphology (Figs. 3B, 4A). Ultrastructural descriptions of mineralized spherocytes and granules in different animal groups are rare due to difficulties in the fixation techniques and sample sectioning. In millipedes, detailed studies need to be performed to confirm the origin, destination and real nature of the structures present in the oocytes, fat body and midgut of these animals.. V. Acknowledgments We thank Gerson Mello Souza, Mônika Iamonte, Carmen Silvia Mengardo, Lucila de L. Segalla Franco and Cristiane Marcia Miléo for technical assistance, Evandro Fantazzini and Bianca Tiritan for help with the collections, FAPESP and CNPq for financial support.. VI.. References. 1. Camargo-Mathias, M. I., Fontanetti, C. S. and Micó-Balanguer, E. (2000) Histochemical studies of Rhinocricus padbergi Verhoeff ovaries (Diplopoda, Spirobolida, Rhinocricidae). Cytobios 94; 169–184. 2. Crane, D. F. and Cowden, R. R. (1968) A cytochemical study of oocyte growth in four species of milipedes. Z. Zellf. 90; 414–431. 3. Fontanetti, C. S. and Staurengo da Cunha, M. A. (1993) Morfologia ovariana e desenvolvimento dos ovócitos de Rhinocricus padbergi Verhoeff (Diplopoda, Spirobolida, Rhinocricidae). Rev. Brasil. Biol. 53; 7–12. 4. Junqueira, L. C. U. and Junqueira, M. M. S. (1983) Técnicas Básicas de Citologia e Histologia, Livraria Editora Santos, São Paulo. 5. Kubrakiewicz, J. (1989) Deposition of calcium salts in oocytes and ovarian somatic tissue of millipedes. Tissue Cell 21; 443– 446. 6. Makioka, T. (1989) Ovarian structure and oogenesis in chelicerates and other arthropods. Proc. Arthrop. Embryol. Soc. Jpn. 23; 1–11. 7. Petit, J. (1970) Sur la nature et l’accumulation de substances minerales dans les oocytes des Polydesmus complanatus (Myriapoda, Diplopoda). Compte Rendu Hebd des Seances de l’Acad. Sci. 270; 2107–2110. 8. Sareen, M. C. and Adiyodi, K. G. (1983) Arthropoda-myriapoda. In “Reproductive Biology of Invertebrates”, ed. by K. G. Adiyodi and R. G. Adiyodi, John Wiley & Sons, i, Chichester, pp. 497– 520. 9. Sharma, G. and Chhotani, D. B. (1957) The milliped egg. Res. Bull. Punjab. Univ. 103; 241–250..

(7)