Cytologia 45:641-649, 1980 Sequential Analysis of Meiotic Prophase in Grasshoppers A. M. Vazquez, C. Martinez and J. R. Lacadena Departamentode Genetica,Facultad de Biologia UniversidadComplutense, Madrid, Spain ReceivedJanuary 9, 1979 Prophase meiotic stages of difficult interpretation, namely synizesis and diffuse stage, have been noted since early descriptions of meiosis (Wilson 1925 , Inouye 1929). The lack of chronology criteria which might establish a proper sequence of the observed phases has been, perhaps, one of the reasons which have made the under standing difficult. It has also made difficult to locate both stages within the meiotic cycle. In recent years, several criteria of chronology have come to be used which per mit the real establishment of the prophase meiotic stages. Moens (1964) was the first to give a new interpretation to the prophase I in which he included some atypi cal stages, namely synizesis, schizonema and diffuse stage. The synizesis, described as a stage in which homologous chromosomes appear paired forming a compact tangle, has been placed at the beginning of the prophase (Moens 1964, Nagl 1969, Lacadena and Vazquez 1971, Owens and Molder 1971). The diffuse stage is a period of apparent decondensation of the chromosomes previously paired and contracted. This stage has been reported by several authors and generally placed close to pachytene or diplotene (see review by Klasterska 1976). Its significance is still to be explained being connected with crossing over by some authors (Rossen and Westergaard 1966, Barry 1969, Peacock 1970) or with special periods of genetic activity by others (Lu and Raju 1970, Klasterska 1971, 1976, Owens and Molder 1971). In the present work, the sequence of the prophase I in the male meiosis of two genus of grasshoppers has been studied. The sequence was determined and the presence of a diffuse stage found. For this study longitudinal sections of testis tubu les were used showing the presence of a maturity gradient along the tubules. Material and methods Three species of the genus Chortippus (C. longicornis, C. apicalis and C. bicolor and one of the genus Oedipoda (O. coerulenses coerulenses) were used. No signi ficant differences were found in the process of spermatogenesis in the species studied, so that the results are set out together. All specimens were collected in autumn in the campus of the University. The testicles were extracted by dissection and fixed for six hours in Bouin's fixative. Next, they were washed in an alcohol series from greater to lesser grada tion, beginning with ethanol of 70•Ž down to distilled water. At this stage the sepa ration of tubules was carefully carried out. 642 A. M. Vazquez, C. Martinez and J. R. Lacadena Cytologia 45 After dehydratiation, the tubules were embedded in paraffin and 5,U longitudi nal sections were cut. A double acetic-carmine and fast-green staining was carried out. The former 10 minutes at 40•Ž and the latter a rapid pass through an alcoholic solution of fast green. The sections were made permanent mounted in Euparal. Results Each tubule was found to be divided into cysts, the number of which fluctuated from seven to twelve. The meiocytes belonging to a cyst arise from only one cell and they divide synchronouslly (King 1967). It was observed that a maturity gradient exist along the tubule in such a way that the distant cysts are found in an earlier stage of division than those situated in the proximate part. After having checked the existence of a maturity gradient in side the tubule, sequential studies of meiotic stages were carried out. After the cysts whose cells were spermatogonia in division or in a resting phase, others appeared with a bulky nuleus in their cells. In some of these cells the existence of loops coming out of the interior of the chromatin clew was observed. The double nature of the loops was established through the study of preparations made by squash because it was difficult to do so from the sections. Two parallel filaments were ob served leaving a clearly visible space between them. This phase was considered com parable to the synizesis (Fig. 1). Following the cysts whose nuclei were at synizesis, cysts at zygotene stage were found. The compactness of their nuclei was smaller, being possible to discern the double nature of the bivalents. After zygotene, cysts with normal appearence of pachytene stage were found. Following the pachytene cysts, and on same occasions appearing associated with that stage, one can find cells whose nuclei were very rounded, of great size, and completely filled by a fairly compact weft of filaments which resembled a net work. These cells showed a darker stain and the nucleolus was seen with difficulty (see Fig. 2). Sometimes nuclei could be seen which were interpreted as a tran sition between the pachytene nuclei and those previously described. In these, the chromosomes start losing their definite outline becoming diffused and losing their individuality. It can be considered these cells as being in a diffuse stage. As the meiosis advanced, cells appeared in which the chromosomes started to be seen again and the weft of filaments became less compact. It is a state which could be described as an emergence of the diffuse stage chromosomes toward diplotene chromosomes. At diplotene it is possible to distinguish the bivalents quite clearly as shorter structures in which the chiasmata are visible. On some occasions cysts showing both classic diplotene and diffuse stage cells were found (Fig. 2) With the appearance of cysts in diakinesis following the diplotene ones, the prophase I was completed. It was attempted to establish the relative duration of each phase in relation to 1980 Sequential Analysis of Meiotic Prophase in Grasshoppers 643 Table 1. Duration of each phase in relation to the total duration of the prophase I Figs. 1-2. 1, cyst showing meiocytes at synizesis. a, magnification •~3600. b, magnification. •~ 5800. 2, cyst showing meiocytes at diffuse (df) and diplotene (dp) stages. •~1650. 644 A, M. Vazquez, C. Martinez and J. R. Lacadena Cytologia 45 the total duration of the prophase I. Assuming that the number of cells per cyst is constant, a survey was made of the number of cysts occupied by each phase in rela tion to the number of cysts with cells in prophase I. The results are shown in the Table 1. Discussion The appearance of a maturity gradient along the seminal tubule of the male grasshoppers of the Chortippus and Oedipoda genera, permits us to use the cysts arrangements as a criterion of chronology in order to establish the proper sequence of the prophase I of these species. Based on this arrangement, we have established as the first phase the synizesis, a meiotic stage in which the homologous chromosomes appear apparently paired. This stage has been previously described by other authors and it was also placed at the beginning of meiosis (Swanson 1958, Moens 1964, Nagl 1969, Lacadena and Vazquez 1971, Owens and Molder 1971, Maguire 1972). The fact that in this phase the homologous chromosomes lay one beside another parallelly does not necessarily mean that this pairing is similar to that of pachytene as Moens (l.c.) suggested, but rather a close spatial arrangement which would allow the meiotic pairing with a greater facility since the homologous chromosomes would not be situated at random in the leptotene clew. This idea, previously suggested by Feldman (1966), was also indicated by Maguire (1972, 1974) who stated that, as a premeiotic homologous pairing exists, the synapsis during the meiotic prophase would require very little movement in relation to the diameter of the nucleus. Already in 1925 Wilson and in 1942 Smith referred to a pairing of homologous chromosomes in the last premeiotic telophase. Westergaard (1964) and Rossen and Westergaard (1966) also referred to a "precocious synapsis". More recent studies (Feldman 1966, Maguire 1968, Brown and Stack 1968, Chauman and Abel 1968, Stack and Brown 1969) support this hypothesis. In 1971, Stack studied the changes which were produced in the successive mitosis during the development of the plant Ornithogalum virens and defined them as premeiotic changes which gave the mito sis a gradual meiotic appearance, indicating that the homologous chromosome pairing, at least in this species, is a progressive phenomenon. We consider this hypo thesis of the premeiotic pairing to facilitate the synapsis and it is what really would explain the presence of the synizesis in the early prophase. It could be thought that the double structure observed in synizesis represents the two sister chromatids of one single chromosome instead of the two homologous chromosomes of one bivalent. If it were the real event it must be observed later the beginning of pairing, nevertheless one can see the same configuration in the fol lowing states although the chromosomes form a less compact mass. After the synizesis we situated the zygotene and later the pachytene. The pair ing continues increasing gradually from one to another, there not being possible to establish the presence of the schizonema stage as observed in other species (Moens 1964, Nagl 1969, Whelan and Hornby 1969, Lacadena and Vazquez 1971). As indicated in the results, the diffuse stage was located after pachytene. Never- 1980 SequentialAnalysis of Meiotic Prophase in Grasshoppers 645 theless, it is worth mentioning that cells at diffuse stage could appear together with pachytene cells in the same cyst. A similar situation was found with diplotene cells. These facts can be interpreted as the progressive changes from pachytene to diffuse stage or from diffuse to diplotene stages. Moreover the presence of nuclei with inter mediate aspect between the mentioned phases corroborates our interpretation. One can find plenty of references concerning this phase (see reviews by Klasterska 1976, 1977) although it does not always appear defined as such, but the descriptions given coincide with this stage.