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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

1Departamento 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

In diplopods, the presence of calcium-

containing structures seems to be a

common finding in some species, with

its formation being similar to that ob-

served for other intracellular mineraliza-

tion systems. In the present study, using

histochemistry and transmission elec-

tron 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 vitello-

genesis. Calcium was observed as fine

granulation distributed within the cyto-

plasm or deposited in spherical struc-

tures apparently formed by overlapping

calcium layers. Some authors have sug-

gested 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 detoxifica-

tion as a result of excess calcium ingest-

ed by animals during soil turnover. We

suggest in this paper that the first hypo-

thesis could be occurring in R. padbergi

since at the juvenile stages of the indi-

viduals the uptake of calcium is low and

because the oocyte is a specialized cell

not associated with detoxification.

Key words: millipedes, oogenesis, Diplopoda, biomineralization, calcium

I. 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 develop-

ment are attached [3]. As observed for other diplopod spe-

cies, 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 begin-

ning of oocyte development [1].

The objective of the present study was to analyze in

detail the occurrence of calcium in R. padbergi oocytes.

II. Materials and Methods

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.

III. Results

Calcium was detected in R. padbergi throughout oocyte

development, i.e., from the initial stages, in which the cyto-

plasm is homogenous and still without chorion deposition,

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

Fontanetti and Camargo-Mathias302

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

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.

Calcium in Diplopod Oocyte 303

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 pre-

sented 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 struc-

tures (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 mate-

rial 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 con-

taining 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 carbo-

hydrates, which can occur in their free form or bound to

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.

Fontanetti and Camargo-Mathias304

other elements forming complexes.

In addition to these elements, mineralized bodies have

been observed in the ooplasm of some species but their func-

tion 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 mineral-

ization 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 accu-

mulate 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

Fig. 3. A, B. Concentric bodies in R. padbergi oocytes. cb, concentric bodies; m, membrane; mi, mitochondria; arrowheads, core.

Calcium in Diplopod Oocyte 305

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 homo-

genous 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 con-

stantly exposed to high mineral concentrations in the soil.

This hypothesis was based on the fact that mineral accumu-

lations 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

Fig. 4. A, B. Concentric bodies in R. padbergi oocytes. cb, concentric bodies; m, membrane; mi, mitochondria; l, lipid; arrowheads, core.

Fontanetti and Camargo-Mathias306

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 detoxi-

fication.

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 struc-

ture (Fig. 3A), or b) calcium deposition is due to the coales-

cence 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 diffi-

culties 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 collec-

tions, FAPESP and CNPq for financial support.

VI. References

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E. (2000) Histochemical studies of Rhinocricus padbergi Ver-

hoeff 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) Morfolo-

gia ovariana e desenvolvimento dos ovócitos de Rhinocricus

padbergi Verhoeff (Diplopoda, Spirobolida, Rhinocricidae). Rev.

Brasil. Biol. 53; 7–12.

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Básicas de Citologia e Histologia, Livraria Editora Santos, São

Paulo.

5. Kubrakiewicz, J. (1989) Deposition of calcium salts in oocytes

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6. Makioka, T. (1989) Ovarian structure and oogenesis in chelicer-

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1–11.

7. Petit, J. (1970) Sur la nature et l’accumulation de substances min-

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