_??_1988 by Cytologia, Tokyo Cytologia 53: 7-17 , 1988

Cytogenetic Studies in Calomys musculinus(Rodentia, Cricetidae)

Marcelo Adriana Ciccioli

Depto. Cs. Biologicas, Facultad de Ciencias Exactas y Naturales, (1428), Buenos Aires, Argentina Accepted December 11, 1986

The rodents of the genus Calomys are widely distributed in South America and many spe cies are represented in Argentina (Cabrera 1961). Among them, Calomys musculinus is one of the reservoirs of the "Arenavirus" agent of the Argentine Haemorrhagic Fever (Gardenal et al. 1977). The systematic position of some species of the group is not clear and brought about con troversies due to their morphological similitude. An example of this have been Calomys laucha and Calomys musculinus,which have often been described as only one species. Neverthe less, Massoia et al. (1968) confirmed C. musculinus as a good species based on studies of ex ternal characters, metric and craneal indexes, and chromosomic numbers. The mitotic karyotype, and the C and G bands have been described (Luna et al. 1979); the chromosomic numbers counted initially as 2n=38 for C. musculinusand 2n=64-62 for C. laucha (Massoia et al. 1968) were confirmed (Gardenal et al. 1977, Forcone et al. 1980, D'Aiutolo et al. 1981). Isoenzymatic studies (Gardenal et al. II, 1977) and research concerning the organi zation of the DNA in both species (Corach and Vidal Rioja 1982) were also carried out, pro ving in all the cases their distinctness. Based on the preliminary studies carried out on C. musculinus' meiosis (Ciccioli and Rahn 1984) the first stages of that process were analysed so as to observe and describe the diffuse stage, the frequency and distribution of chiasmata in relation to their possible terminalisation, and the formation of the synaptonemal complexes, in order to study the behavior of the sexual pair.

Material and methods

Fifteen males of Calomys musculinus which were kindly facilited from a colony pretaining to Laboratory of Virology (Depto. Quimica Biol6gica, FCEyE, UBA) were used. This colony was originated from eight couples provided by the Institute of Virology of Cordoba (Origin: Laguna Larga, Pcia. Cordoba). Animals were free of Junin and LCM viruses.

Conventional preparations Animals were chloroformed and immediatly dissected. Testes were kept in Hank's solu tion 1 M, while material was separated in four parts. The following procedures were used: a) 20 minutes in 2.2% Na citrate solution, fixation with glacial acetic acid, according to Welshons et al. (1962); b) 20 minutes in 0.1% formalin in 0.7% Na citrate and fixation in 3:1 (methanol: glacial acetic acid) according to Pathak and Hsu (1979); c) 20 minutes in 0.7% Na citrate hypotonic solution and further fixation with 3:1 (methanol: glacial acetic acid); 8 Marcela Adriana Ciccioli Cytologia 53

d) direct fixation in 3:1 (methanol: glacial acetic acid), without hypotonic treatment. Preparations were made as following: a) by squash of seminiferous tubes on slides previously washed with 3:1, and further staining

with propionic haematoxylin (Saez 1960); b) by dispersion and air drying. Some preparations were stained with 4% Giemsa solution in phosphate buffer, pH 6.8. Others were treated according to Klasterska (1976) for ob servation of the diffuse stage. In all cases slides were mounted in Depex.

Histological sections Testes were fixed directly in Zenker's fixative about 4-6hs and then were included in

paraffine at 54•Ž. The plugs obtained were cut into 2-6ƒÊm sections using a Minot microtome. Slides were stained with Regaud's haematoxylin and differentiation was made in ferric alum. Stain was

controlled under a light microscope and alcoholic eosine was used as a contrast stain. Slides were mounted in euparal.

Synaptonemal complexes at optic microscope (O. M.) Synaptonemal complexes were studied using the method of Solari (1983) for electron micro scopy, adapting the stain which was made with 50% (W/V) of NO3Ag in distilled water, at O. M. Two or three drops of silver solution were placed on slides previously air dried. Floating coverslips were put on slides which were incubated in a moist chamber at 60•Ž for 3-5hs. Stain was controlled under phase objetive observing yellowish pachytene nuclei, in which, snaptonemal complexes were visualized in dark-brown colour. The process was stopped washing with distilled water after removal of the coverslip. Slides were air-dried and mounted in Depex without xylol.

Determination of chiasma frequency a) Coefficients of terminalisation were determined (terminal associations/total chiasmata ta/tch) among autosomes of 51 pachytene nuclei, 65 diakinesis and 92 metaphase I. b) Results obtained in a), were compared to the interstitial chiasmata number (ich) and independently with terminal associations (ta) for three stages. Variance analysis was made considering as data: a) ta/tch; b) ich and c) ta; and Bon ferroni contrasts were made constructing confidence intervals of 5% (Scheffe 1959).

Results a) The meiosis in Calomys musculinus The meiotic sequence was studied starting from the observations already carried out in preparations stained with propionic haematoxylin or Giemsa. The leptotenic nuclei are small, like tangles where simple threads can be distinguished and the sexual vesicle is rately well defined (Fig. lax). Fig. lag shows a Sertoli cell where three chromocenters are clearly seen and a big nucleolus associated to them. The zygotene (Fig. lb) seems to have a short duration compared to the other meiotic stages, due to its low frequency of occurrence. More condensed zones were observed in the nucleus and the sexual vesicle is placed in its periphery, which is highly stained. Fig. lc-d shows early pachytenic nuclei, where the bivalents are highly condensated; the sexual pair is not easily distinguished from the autosomes. As midpachytene (Fig. ld) a visible 1988 CytogeneticStudies in Calomysmusculinus (Rodentia , Cricetidae) 9

increase of the nuclear volume is noticed, together with a partial despiralization of the chromatin. The ends of each pair of homologous seem to insert themselves in the nuclear membrane (Fig. le). From late pachytene to the beginning of diplotene, there is a diffuse stage (Fig. If). It is characterized by the increase of the nuclear volume compared to the mid-pachytene and the

Fig. 1. Meiosis of Calomys musculinus stained with propionic haematoxylin or Giemsa. al, leptotene. a2, Sertoli cell; (n) nucleolus, (c) chromocenters. b, zygotene. c-d, early pachytene. e, mid-pachytene. chromosome ends apparently insert themselves in the nuclear membrane (ar rowheads). f, diffuse stage. g, transitional nucleus. h, diplotene. i, diakinesis. j, metaphase I. (sv: sexual vesicle. loss of definition of the bivalents inside the nucleus. The high frequency of appearance of these nuclei in the conventional preparations in comparison to other stages, suggests a long duration for this period. On the other hand, it is possible to recognise within the diffuse stage some sub-stages characterized by the increase of the initial volume and the growing despiralization of the chromosomes. 10 MarcelaAdriana Ciccioli Cytologia53

The existence and chronological location of the diffuse stage in the male meiosis of Calomys musculinus have been proved in two different ways. First of all, with Klasterska' (1976) technique, as the nuclei which are observed in Fig. 2a -e. They show the development of the prophase I from the early pachytene (Fig. 2a) up to the metaphase I (Fig. 2e). The despiralization process accompanied by the increase of the nuclear volume advances till it reaches the diffuse stage (Fig. 2b-c), where the chromatin seems frayed. The presence of nuclei with greater condensation, where it is possible to identify bivalents where the repulsion of the homologous chromosomes is starting, establishes the end of the diffuse stage and the despiralization process. The bivalents are already seen with the typical configurations of the diplotene and metaphase I (Fig. 2d-e, respectively).

Fig. 2. Meiosis with Klasterska' (1976) technique. a, early pachytene. b-c, diffue stage. d, diplotene. e, metaphase I. sv; sexual vesicle.

On the other hand, histological sections of seminiferous tubes show the nuclei identified as diffuse associated with pachytenic and diplotenic nuclei. For the identification of different prophase stage, various histological and cytological criteria were used, such as the condensa tion degree of the chromatin, the nuclear volume and the characteristics visible configurations, as can be seen in the photographs and explanatory schemes which correspond to Fig. 3. This is consistent with the information that in mammals, maturation takes place in an helicoidal way from one end of the seminiferous tube to the other. This establishes a germinative gradient, which goes from the periphery to its interior, and in portions, laterally (Oakberg 1956). Fig. 1g shows the appearance of a transitional nucleus between the diffuse stage and the diplotene, stained with conventional techniques. There is a visible decrease of nuclear volume compared to the diffuse nucleus and a greater condensation of bivalents, some of which show chromomeric appearance and evidence that homologous chromosomes are beginning to pull apart. During diplotene (Fig. lh), there are eighteen autosomic bivalents and the sexual pair, the latter showing end-to-end association. During diakinesis the bivalents are more condensed, their double structure can n_??_ger 1988 CytogeneticStudies in Calomysmusculinus (Rodentia , Cricetidae) 11 be observed and the sexual pair keeps the end-to-end association (Fig. li). In metaphase I the contraction reaches its maximum; during this stage, the XY pair shows negative heteropyknosis (Fig. lj). The very low frequency of cells in more advanced stages of meiosis in relationshiop to the great number of nuclei in the already described stages, suggests that the meiotic process up to spermatids production is extremely rapid.

Fig. 3. Diffuse stage in histological transverse sections, a-b, microphotographs of portions of seminiferous tubes. c-d, explanatory schemes. (G) gonia; (P) pachytene; (Df) diffuse stage; (Dp) diplotene; (Ep) spermatids; (ext) external of seminiferous tube; (int) internal of seminiferous tube. b) Frequency and distribution of chiasmata Tables 1 and 2 show respectively the decrease of the mean of interstitial chiasmata (ich) per cell in diplotene, diakinesis and metaphase I; and the statistical study of variance applied to data obtained from 208 meiocytes. It includes the confidence intervals calculated through the Bonferroni' method. The comparison shows the existence of significant differences among the three analyzed stages. Similar results were obtained applying the same methodology for the analysis of data on the increase of terminal associations (ta) from diplotene up to metaphase I (Tables 3, 4). So far it was studied the variation of the mean of ich and to per cell and per stage, both 12 MarcelaAdriana Ciccioli Cytologia53

independently, and without to consider the nature of terminal associations. Besides the relationship terminal associations/total chiasmata number (ta/tch) was ana lyzed considering here terminal associations as chiasmatic, including them in the total chiasmata number per nucleus (Tables 5, 6). The analysis of the data showed the existence of global significant dieffrences between diplotene and metaphase I, and more specificallybetween diplotene and diakinesis, and between diakinesis and metaphase I. The statistical analysis would indicate the existence of a terminalization process, through which, the interstitial chiasmata (visualized in the point of their occurrence in diplotene) would

Fig. 4. Synaptonemal complexes at O. M. a, mid-pachytene, (n) nucleolus, (sv) sexual vesicle. b-g, pairing sequence of XY pair, (sc) synaptonemal complex, (x) X axis, (y) Y axis, (e) excres cences. move significantly during the prophase I up to the observation of some of them as terminal associations in diakinesis and metaphase I. c) Synaptonemal complexes at O. M. In pachytene nuclei stained with Ag nitrate, the synaptonemal complexes corresponding to the eighteen autosomic bivalents were visualized, together with the axes of the X and Y chromosomes, in which, the synapitc portion is of variable length according to the meiotic stage (Fig. 4b-g). 1988 CytogeneticStudies in Calomysmusculinus (Rodentia , Cricetidae) 13

In Fig. 4a, can be observed a nucleus in mid-pachytene , where five synaptic complexes clearly show they are associated to heavy silver containing deposits. These are interpreted as products of the nucleolar organizing activity of these autosomes. In most of the observed nuclei three of these deposits were associated, forming a single nucleolus. When it was analyzed the behavior of the sexual pair during pachytene , the following sequence of events was observed: 1. During the early pachytene there is a total synapsis of the axis of the Y chromosomes with a portion of the axis corresponding to the X chromosome. The number of cells observed during the early synapsis of X and Y was very low (Fig. 4b). This data was confirmed at E. M. (Cic cioli and Rahn 1984). 2. In early pachytene the desynapsis of the X and Y axes begins, which progressively advances while the autosomes remain completely synapsed till the diplotene (Fig. 4c-d); 3. During mid-pachytene (Fig. 4e), one can observe the formation of glomerular excrescences in the partially desynapsed axes of the X and the Y. Meanwhile, the desynapsis process con tinues until there only remains a very small portion of synaptonemal complex at the end of both X-Y axes. 4. Half-way through mid-pachytene, the excrescences become larger and numerous. Finally, they start to become detached staying around the sexual vesicle, very near the lateral axes of the X and the Y (Fig. 4f-g).

Discusion Although in general lines the meiosis of Calomys musculinus is similar to the one of the other mammals, the analysis of the diffuse stage, the frequency and distribution of chiasmata and the study of the synaptonemal complexes at O. M., present very interesting aspects.

a) Diffuse stage In 1925 Wilson described the existence of a "confuse" or diffuse stage which took place in the meiosis of both animals and plants, pointing out that the chromososome decondensation in the ovogenesis was much more pronounced that during the spermatogenesis. More recently, the diffuse stage was described in male meiosis of many organisms (Moens 1964, Peacock 1968, Barry 1969, Pogosianz 1970, Klasterska 1976, 1977, 1978, Cardoso et al. 1981, Dutra and Cardoso 1984). Due to the use of squashing techniques, the above mentioned authors were not able of chronologically correlating with certainty the diffuse stage. There are doubts as to their tem poral location in the development of the meiotic prophase I, which is variable (Klasterska 1977). Lacadena and Vazquez (1971) used histological sections of rye anthers and established the chronological occurrence of the diffuse stage. In C. musculinus,the evidences obtained through sections excluded the hypothesis that the diffuse stage was observed due to technical artifacts for deficient fixation. The appearance of nuclei in other meiotic stages is the one which corre sponds to a normal fixation (Fig. 3). On the other hand, the visible increase of the nuclear size from the middle pachytene to the diffuse stage, may not be attributed to the action of hypotonic treatment. The same graduation and relationship of measures were observed in non-hy potonized material. Finally, it is stated that the diffuse stage in Calomys musculinus covers the end of the pachy tene till the initiation of diplotene exclussively, due to two types of evidences. In preparations carried out through dispersion or squash, transitional nuclei were founded between pachytene and diffusse, and between diffuse stage and diplotene. In different portions of the transverse sections of the seminiferous tubes, the association of nuclei in diffuse stage with pachytenes and 14 MarcelaAdriana Ciccioli Cytologia53 diplotenes was observed. The physiological meaning of the diffuse stage has not yet been elucidated, anyhow, several researchers have presented different hypothesis on the subjet. Among them, Monesi (1967), detected an intense synthesis of RNA during the diffuse stage in mouse, Klasterska (1977) said that he was unable to correlate it with the long period of cell growth which took place during the ovogenesis. In Goniaea australasiae Peacock (1968) detected a variation in chiasma frequency when subjected the cells to thermic shocks during the early pachytene, which is diffuse. Owens and Molder (1971) observed in Conifers, a prolonged diffuse stage which covers part of the pachytene and all diplotene, during which an active enzymatic synthesis took place. This diffuse stage lasted the winter months, hence reducing the percentage of sterile pollen. b) Synaptonemal complexes at O. M. The visualization of the synaptonemal complexes to C. musculinus at O. M. enabled to study problems related to pairing of homologue chromosomes in a simple manner. Further more, in this particular case, the behavior of the sexual pair itself and its type of association was observed. These represent another data about the question of terminalisation of chiasmata. The behavior of the sexual pair in C. musculinus,the sequence of pairing, early desynapsis and formation of excrescences in the unpaired axes, coincide with the ones described by Moses (1977) in the Chinese hamster and by Solari (1980) in man. It is supposed that the duration of the complete pairing of the X and Y chromosomes is very short, due to the low number of nuclei where this complete pairing was observed. The nature and function of the glomerular excrescences is not known, and they may be the product of the relocation of the axial material after desynapsis (Moses et al. 1979). c) Frequency and distribution of chiasmata The terminalisation of chiasmata was proposed to explain the apparently terminal associ ation of chromosomes in metaphase I (Darlington 1937). As a consequence of this process, the chiasmata change positions from the point of its first observation in diplotene. Taking into account the case of organisms which have chromosome complements with heterochromatic ends (e.g. rye and some orthopterans) and assuming that chiasmata do not form in heterochromatic segments (John 1976). There are three possible theoretic explanations for the observation of terminal associations: 1. Chismata invariably arise proximal to the terminal C-bands and in the greater majority of cases terminalise through them; 2. Chiasmata that occur at euchromatin-heterochromatin junctions may become absorbed into the structure of the bivalent as a result of contraction, then it may be converted in apparent terminal association. Jones (1978) refers to this process as pseudoterminalisation. 3. Terminal associations do not have a chiasmatic origin, and they represent a distinct and non-chiasmatic mode of chromatid association, such-as described by John and King (1985) for the orthopterans Cryptobothrus chrysophorus and Heteropternis obscurella. In the case of organisms that do not have terminal blocks of heterochromatin, the question is more complicate because it is not possible to discard chiasma production in such regions and also that interstitial chiasmata do not to terminalise through these ends. Tease and Jones (1978) proved in Locusta migratoria non terminalisation, because in the different stages of meiotic prophase they observed that chiasmata always coincided with the recombination point. They visualized these points of recombinations by means of differential staining. Imai and Moriwaki (1982) re-examined the problem of recombination in the house mouse, 1988 CytogeneticStudies in Calomysmusculinus (Rodentia , Cricetidae) 15

starting from the fact that the terminal associations observed during the meiotic prophase I are not chiasmatic. They concluded that there was no significant migration of the interstitial chiasmata from the point of their first visualization in diplotene . In Calomys musculinus it was not possible to determine whether the terminal associations were chiasmatic or not due to the small size of the chromosomes . The statistical estimates were carried out, on one hand, with the relationship of ta/tch , considering the terminal associations as chiasmatic, except the one of the sexual pair. On the other hand, considering only the vari ation in the number of ich and ta, independently between stages to as to separate the results of considering erroneously the nature of terminal associations. Both studies coincided in demonstrating the existence of significant differences between diplotene and diakinesis, and between the latter and metaphase I. The results would show that in C. musculinus there is terminalisation of chiasmata. Nevertheless, one may not leave aside as a possible source of error, that, due to the small size of bivalents and the variations of their contraction in prophase I, they may not be correctly evaluated. The XY association was considered achiasmatic because the complete pairing period be tween the lateral axes of the X and the Y is extremely short, and the desynapsis is almost total in early pachytene. This was confirmed through E. M. (Ciccioli and Rahn 1984). Since the absence of the synaptonemal complex implies that there is not recombination, it must be suppose that it is not probable that crossing-over occurs in the minimal terminal portion of the complex in the sexual bivalent. Accesory evidence was obtained observing at O. M. that in diplotene the sexual pair was generally end-to-end associated, and in the cases when this association failed, the X and Y were observed as univalents or medially associated, probably due to a precocious separation of the same, in absence of chiasmata formation.

Summary In Calomys musculinus (Rodentia, Cricetidae) the meiosis was studied. Diffuse stage was described with conventional and special techniques, and its chronological location was certainty determined through histological sections. Analysing the distribution and frequency of chiasmata a process of terminalisation was detected from diplotene up to metaphase I. Studying separately the decrease of the mean of interstitial chiasmata (ich), the increase of the mean of terminal associations (ta), and the de velopment of the relationship ta/tch (terminal associations/total chiasmata number) between diplotene-diakinesis and between diakinesis-metaphase I, significant differences were founded. Sequential events of pairing of the XY pair through the formation of their synaptonemal complex in pachytene at O. M. were analyzed. Data obtained allowed to consider the associ ation between X and Y chromosomes as achiasmatic.

Acknowledgement

I wish to express my gratitude to the Laboratory of Virology (Depto. Quimica Biologica, FCEyN, UBA) for the specimens of Calomys musculinus. I am greatly indebted to Professor Doctor Lidia Poggio and Professor Doctor Carlos A. Naranjo for their encouragement during the course of this investigation and their critical re ading of the manuscript. Special thanks go to Professor Doctor J. H. Hunziker and Professor Doctor N. O. Bianchi for their several valuable suggestions on this work. Marcela A. Ciccioli is "Becaria de Iniciacion, CONICET,Argentina". 16 Marcela Adriana Ciccioli Cytologia 53

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