INTRODUCTION

Toluidine Blue dye containing increasing con-centrations of Mg2+ ou Ca2+ can show loss ofmetachromacy at a certain concentration of theinorganic cation when staining DNA-proteincomplexes in vitro and in vivo. This process hasbeen named Critical Electrolyte Concentration(CEC) and is applied to the study of protein-nucleic acid complexes at different stages of chro-matin supra-organization (VIDAL and MELLO

1989; MELLO et al. 1993; MELLO and FALCO

1996).

The difference in the availability of phosphategroups in the DNA molecule for the electrostat-ic binding of cations has been demonstrated bythe differences in the values of CEC, when com-paring the DNA molecule on its own with theDNA-protein complex. In this case, the ToluidineBlue dye was used as a cationic dye and the Mg2+

ions as the inorganic cations. The values of CECwere then calculated as the Mg2+ concentration atwhich metachromacy is completely lost (VIDAL

and MELLO 1989).In the methodology proposed by VIDAL and

MELLO (1989), the Mg2+ ions are added to theexperimental dye solution and the concentrationof Mg2+ at which loss of the metachromacy occurshas been considered to be the value of CEC.

CARYOLOGIA Vol. 56, no. 4: 495-499, 2003

RNA reallocation during initial spermatic meiosis ofPseudonannolene tocaiensis Fontanetti, 1996

VANESSA CABREIRA DE FREITAS1; SEBASTIÃO ROBERTO TABOGA2 and CARMEM S. FONTANETTI1, *1 Departamento de Biologia, Instituto de Biociências, 13506-9000 - Av. 24A, 1515, Bela Vista, UNESP Rio Claro, São Paulo,

Brazil.2 Depto. de Biologia, Instituto de Biociências, UNESP São José do Rio Preto, São Paulo, Brazil.

Abstract - Toluidine Blue dye containing increasing concentrations of Mg2+ orCa2+ can show loss of metachromacy at a certain concentration of the inorganiccation when staining DNA-protein complexes in vitro and in vivo. This processhas been named Critical Electrolyte Concentration (CEC) and is applied to thestudy of protein-nucleic acid complexes at different stages of chromatin supra-organization. Male gametocytes of the species Pseudonannolene tocaiensis werestudied, observing a large amount of ribonucleoproteins in the gametocytescytoplasm throughout prophase I. The nucleolus is maintained during most of theprophase. The highly condensed region showing the bouquet formation appearedstained with the typical tonality for chromatin; this region corresponds to the con-stitutive heterochromatin. We also observed the presence of RNA all through thechromosomes in prophase I. The permanence of this material surrounding thechromosomes during male meiosis is difficult to explain, since a great reductionof the products of spermatogenesis occurs due to the fact that most of the mate-rial of the spermatozoids is not used during fecundation. However, in P. tocaien-sis this material is remains even at the spermatids. It is known that during thespermiogenesis of certain insects, RNA synthesis continues at the spermatid,being subsequently eliminated from the nucleus and then from the cell due to theelongation of the nucleus. Therefore, we could suggest that permanence of thismaterial (RNA) during meiosis has a function in the process of cell division.

Key words: Critical Electrolyte Concentration (CEC); Diplopoda; meiosis;metachromacy; RNA.

* Corresponding author: e-mail: vanessacabreira@hotmail.com

Based on the fact that the CEC point for RNAis always higher than that for DNA, a variation ofthe CEC method has been proposed. In this newmethod, the treatment with Mg2+ is not simulta-neous but followed by the Toluidine Blue staining(MELLO et al. 1993). Under these conditions,

while the DNA stains green, the RNA remainsstained metachromatically allowing a special dis-crimination of the rRNA, it evidences and pointsthe location of details of the nucleolus, and it ispossible to follow the RNA during the process ofcell division (MELLO et al. 1993; MELLO 1995).

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Fig. 1A-D – Histological sections of the testis of P. tocainesis subjected to the CEC technique. Spermatocytes in prophase I;c = chromatin; nu= nucleolus; v = vacuole; arrows = chromosomes. Scale Bars: A, C 50 µm; B, D 25 µm.

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Fig. 2A-E – Histological sections of the testis of P. tocainesis subjected to the CEC technique. Figs. A-C: spermatocytes inprophase I. Figs. D-E: spermatide. c = chromatin; s = spermatid; n = nucleus; nu = nucleolus; v = vacuole; arrows = vacuolizednucleolus. Scale Bars: A, B, D 50 µm; C, E 25 µm.

In the present work we studied the nuclei ofthe testicular cells of the species Pseudonannolenetocaiensis through the method of Critical Elec-trolyte Concentration (CEC).

MATERIALS AND METHODS

Males of Pseudonannolene tocaiensis were collect-ed at the Caverna da Toca in Itirapina, São Paulo State– Brazil, during the months of February 2000 and July2001 by Fontanetti et al. The specimens were kept ina periodically wet terrarium containing soil, woodpieces and leaves.

The testes were fixed in 4% paraformaldehydeduring approximately 24 h in the refrigerator. Next,the material was dehydrated in a standard ethanolseries during 40 minutes for each bath and then trans-ferred to a solution of embedding resin (JB-4-PolaronInstruments/BIO RAD) during 72 h at 4 °C. Thematerial was then placed in a mold previously filledwith the resin containing a catalyzer. The molds weresealed with the aid of metallic supports for microtomyand the material was sectioned with a Sorvall JB-4microtome (BIO RAD). The sections were thenhydrated and arranged on slides. Once dry, the slideswere stained using the variant technique for CEC(Critical Electrolyte Concentration) of nucleoli andribonucleoproteins according to MELLO et al. (1993).The slides were hydrated for about 10 minutes, stainedwith Toluidine Blue pH 4.0 for 20 minutes, washed,dried, and submerged in an aqueous solution of 0.05 MMgCl2 for 6 minutes. Next, the slides were washed indistilled water, air-dried, submerged in Xylol andmounted in Permount.

RESULTS AND DISCUSSION

The variant CEC technique for nucleoli andribonucleoprotein allows the observation of real-location of RNA during part of the meiosis. Weobserved a large amount of ribonucleoproteins inthe gametocytes cytoplasm during all prophase I.

The nucleolus remains noticeably in the“bouquet formation” (Fig. 1C, D) during mostpart of the prophase (Fig. 1A-D). The highlycondensed region in the bouquet formationappeared clearly differentiated by the techniqueused, showing the typical tonality for chromatin(bluish green – Fig. 1C, D). This region corre-sponds to the constitutive heterochromatin,which was detected by FONTANETTI (1990) whendescribing the process of meiotic prophase inmillipedes.

The chromosomes in prophase I (arrows inFig. 1C, D) did not show the typical colorationexpected for chromatin, evidencing the presenceof RNA all through the chromosomes.

As the process of meiosis advances, the hete-rochromatin begins to disperse (Fig. 2A) togeth-er with the nucleolus (Fig. 2B, C). During thisphase, we observed the vacuolization of thenucleolus in some cells, which is a clear indicationof nucleolar disassociation (arrow in Fig. 2C).

After this phase, it is typical to observe inthese animals a diffuse stage in which the hete-rochromatin remains heteropicnotic and theeuchromatin becomes diffuse; this process accel-erates after this phase, difficulting the observationof other phases of the meiosis (FONTANETTI,1990).

The spermatids presented a clear and homo-geneous nucleus (Fig. 2D, E), ribonucleoproteinsin the cytoplasm, and a very characteristic vac-uole.

During the meiosis, it has been reported thatpart of the nucleolar material dissolves in thecytoplasm during prometaphase, returning after-wards for the development of the nucleolus dur-ing telophase. A second part of the nucleolarmaterial remains associated to the NORs and, athird part, will join the perichromosomal sheathof condensing chromosomes and then returns tothe nucleolus with the chromosomal deconden-sation during telophase (MELLO, 1995).

Results similar to the ones observed in thiswork were described by MELLO (1995) for cellsundergoing mitosis. According to this author, thelarge amount of RNA observed during mitosissupports the idea of a great complexity in thearchitecture of chromosomes. This global archi-tecture might be substituting the protection roleperformed by the laminae of the nuclear enve-lope, besides acting as a site of storage for RNAmaturation factors and contributing to the dis-tribution of nuclear and nucleolar proteins, aswell as particular types of RNA among daughtercells. The permanence of this material surround-ing the chromosomes during male meiosis is dif-ficult to explain, since a great reduction of theproducts of spermatogenesis occurs due to thefact that most of the material of the spermato-zoids is not used during fecundation. However, inP. tocaiensis this material remains even in thespermatids, as observed in Fig. 2E. It is knownthat in the spermatozoids of certain insects, RNAsynthesis continues in the spermatid, being sub-

498 FREITAS, TABOGA and FONTANETTI

sequently eliminated from the nucleus and thenfrom the cell, due to the elongation of the nucle-us (MUCKENTHALER 1964). Therefore, we couldsuggest that permanence of this material (RNA)during meiosis has a function in the process ofcell division.

REFERENCES

FONTANETTI C.S., 1990 – Meiotic prophase inDiplopoda. Revista Brasileira de Genética, 13:697-703.

MELLO M.L.S., 1995 – Realocation of RNAmetachromasy of mitosis. Acta HistochemicalCytochemical, 28: 149-154.

MELLO M.L.S. and FALCO J.R.P., 1996 – Criticalelectrolyte concentration of DNA-protein com-plexes in spermatozoal and somatic cell nuclei of

the honey bee. Insect Biochem. Mol Biol., Elms-ford, 26: 793-795.

MELLO M.L.S., VIDAL B.C., 1989 – Critical elec-trolyte concentration of heterochromatin andeuchromatin of Triatoma infestans. Cytobios, 59:87-93.

MELLO M.L.S., VIDAL B.C., DANTAS M. M., MON-TEIRO A.L.P., 1993 – Discrimination of the nucle-olus by a critical eletrolyte concentration method.Acta Histochemical Cytochemical, 26: 1-3.

MUCKENTHALER F.A., 1964 – Autorradiographicstudy of nuclei acid synthesis during spermatoge-nesis in grasshopper Melanoplus differentialis.Experimental Cell Research, 35: 531-596.

VIDAL B.C., MELLO M.C.S., 1989 – Criticaleletrolyte concentration of DNA and nucleopro-tein complexus in vitro. Acta HistochemicalCytochemical, 22: 471-478.

Received March 10, 2003; accepted June 17, 2003

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