Cytologia 45:641-649, 1980

Sequential Analysis of Meiotic Prophase in

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 (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 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. So, Wilson (1925) described it as a general feature of the meiotic cycle, but with broad variation in the degree of condensation. Inouye (1929) observed in barley that "towards the end of pachytene stage the spireme gradually loses its chromatin and reaches the achromatic stage, the threads in the nucleus being nearly achromatic". In Neurospora, Singleton (1953) reported that" ... the di plotene nuclei are refractory subjects for detailed observations .... the chromo somes form a confussed mass of thickened rather fuzzy strands". Rossen and Westergaard (1966) observed in Neottiella a "disintegration stage". A great number of references concerning the oogenesis have been indicated by Davidson (1968) and phases similar to the diffuse stage being found at every taxa of the kingdom. It can be pointed out that the great part of these references are included within the lampbrush stages which we will refer to later. Nevertheless other authors refer to the diffuse stage as a special stage within the meiotic cycle (Moens 1964, Ghidoni 1967, Lu 1967, Pijnacker 1967, Peacock 1968, Nagl 1969, Whelan and Hornby 1969, Barry 1969, Lu and Raju 1970, Pogosianz 1970, Klasterska 1971, Lacadena and Vazquez 1971, Owens and Molder 1971, Kezer and Macgregor 1971, Koch et al., 1972, Baker and Franchi 1972, Zickler 1973, Jones 1973; Sotelo et al. 1973;... See reviews by Klasterska 1976, 1977). Not all the authors locate the diffuse stage between the same phases, although the majority place it between pachytene and diplotene. Peacock (1968) reported a diffuse stage earlier than the pachytene ("early pachytene") in the australiasae, although this author did not use any criterion of chronology to locate it. In rye it appears together with diplotene and diakinesis, even being associated with more advanced stages (Lacadena and Vazquez 1971). Some authors found it with the diplotene (Henderson, 1961, 1964, Owens and Molder 1971). Sotelo et al. (1973) mentioned it as a substage of the diplotene call ing it "lobulate stage" from a morphological point of view because the nuclear lobu lation was the feature more readily distinguished in the electron microscope. As similable to the diffuse stage could be the dictyotene found in human, mouse and rat oogenesis after diplotene and before diakinesis (Ohno et al. 1961, 1962). In our material it appears between pachytene and diplotene, as in the majority of the cases described. As indicated in Table 1, the diffuse stage represents a rather short period, about an 8% of the total length of the prophase I measured as the proportion of cysts at diffuse stage in relation to the other prophasic stages. The same can be found in other species such as tomato (Moens 1964), hamster (Pogosianz 1970), and Rosa canina (Klasterska 1971). However in other species the diffuse stage appears as a period of long duration (Barry 1969, Owens and Molder 1971), being exceptionally 646 A. M. Vazquez,C. Martinez and J. R. Lacadena Cytologia 45

long in the case of human dictyotene which could last many years. As to its significance, insufficient facts are available still now to explain it. Ac cording to some authors it can represent a period of activity related to crossing-over (Rossen and Westergaard 1966, Peacock 1968, Barry 1969). Nevertheless, the ex istence of species in which the diffuse stage is not located in pachytene but in later phases, leads one to think that this hypothesis has little probability. For other authors it could signify a phase of genetic activity (Lu and Raju 1970, Klasterska 1971, 1976, Owens and Molder 1971). In species where the meiosis stops to be restarted after a certain time, the resting phase is precisely the diffuse stage. This stop has been described in the spermato - genesis process of some (Wilson 1925) and during the oogenesis of many more (Tennent and Ito 1941, Ohno et al. 1961, 1962) as well as in the male gameto - genesis of some plants (Ekberg et al. 1968, Owens and Molder 1971). Perhaps these nuclei, being blocked for any time, could need a DNA uncoiling to synthesize some proteins if their were necessary for the life of the meiocyte or the achievement of any physiological process. Thus Owen and Molder (1971) found a lower fertil ity in the conifers lacking the diffuse stage. However, in some with a meroi stic oogenesis this stage cannot be observed (Bier 1967) but according to this author the RNA is synthesized into the nurse cells and transported into the oocytes through cytoplasmic bridges. In this way the impossibility of synthesis from the nuclei with shrunken chromosomes could be compensated by the external contribution of the RNA necessary at a given moment. Perhaps this could explain that not all the species show this particular phase since, for instance, Moens (1969) did not mention it to be present in the grasshopper Locusta migratoria. In this aspect of genetic activity could be mentioned the cases of nuclei with lampbrush chromosomes. The lampbrush stage could be a particular case of the diffuse stage and some authors consider it as such from the morphological point of of view (Lu and Raju 1969, Klasterska 1971, Henderson 1964, Kezer and Macgre gor 1971, Jones 1973). Koch et al. (1972) have found an extra DNA synthesis during the diffuse stage of Carausius morosus, being perhaps assimilable this process to the phenomena of amplification characteristic of the lampbrush stage. Bakken and McClanahan (1978) found that 3H-uridine incorporation is detect able in mouse oocyte meiotic prophase being diplotene and dictyotene the most heavily labelled stages. They noticed the relationship between chromosome struc ture (state of condensation) and function (transcription) As pointed out by Klasterska (1976), the occurrence of a diffuse stage during meiosis appears to be a rather common phenomenon. However with the exception of the much studied lampbrush chromosomes, not much attention has been paid to this stage up to now. Thus, the inclusion of these new views of meiotic phases in textbooks of genetics is rather exceptional (see for example, Lacadena 1976). In other occasions ambigous situations are produced: for instance Rieger et al. (1976) include the concepts of "diffuse stage" and "synizesis" as entries of their Glossary but they do not mentioned them when describing the meiotic process. On his hand, Strickberger (1976) when describing the meiotic process makes reference to the synizesis but does not mention the diffuse stage. 1980 Sequential Analysis of Meiotic Prophase in Grasshoppers 647

From our studies we cannot reach any conclusion which clarify the meaning of the diffuse stage, although we can establish its presence in a definite period of the prophase I in the species studied . In our consider opinion, the environmental conditions, in their widest sense , could influence the appearance of the diffuse stage to such an extent that in some species it could exist or not due to them 'Thi . s is perhpas another reason that it has not been described in the case of some species.

Summary Longitudinal sections of testis tubules has been used to study the sequence of the prophase I in the male meiosis of grasshoppers of the genera Chortippus and Oedipoda. The sequence was determined and the presence of a diffuse stage found . An estimation of the duration of each phase in relation to the total duration of the prophase I has been made, namely synizesis 12.7%; zygotene, 16.7%; pachytene, 39.9%; diffuse stage, 8.2%; diplotene, 19.3%; and diakinesis 3.1%.

References

Baker, T. G. and Franchi. L. L. 1972. The fine structure of oogonia and oocytes in the rhesus mon - key (Macaca mulata). Z. Zellforsch Mikrosk. Anat. 126: 53-74. Bakken, A. H. and McClanahan, M. 1978. Patterns of RNA synthesis in early meiotic prophase oocytes from fetal mouse ovaries. Chromosoma 67: 21-40. Barry, E. G. 1969. The diffuse diplotene stage of meiotic prophase in Neurospora. Chromosoma 26: 119-129. Bier, K. 1967. Oogenese, das Wachstum von Riesenzellen. Naturwissenschaften 54: 189 Brown, W. V. and Stack, S. M. 1968. Somatic pairing as a regular preliminary to meiosis. Bull. Torrey Bot. Club 95 (4): 369-378. Chauman, K. P. S. and Abel, W. O. 1968. Evidence for the association of homologous chromo somes during premeiotic stages in Impatiens and Salvia. Chromosoma 25: 297-302. Davidson, E. H. 1968. Gene Activity in Early Development. Academic Press Inc. Ekberg, U. et al. 1968. Meiosis and pollen formation in Larix. Hereditas 59: 427-438. Feldman, M. 1966. The effect of chromosomes 513, 5D and 5A on chromosomal pairing in Triti cum aestivum. Proc. Nat. Acad. Sciences 55 (6): 1447-1453. Ghidoni, A. 1967. The diffuse stage in meiotic prophase I of maize PMC. Maize Genet. Coop. Newsletter 41: 84. Henderson, S. A. 1961. The chromosomes of the British Tetrigidae (). Chromosoma 12: 553-572.- 1964. RNA synthesis during male meiosis and spermiogenesis. Chromosoma 15: 345-366. Inouye, Ch. 1929. Studies on the development of chromosomes in Hordeum. Proc. Crop Sci. Soc. 1: 29-39. Jones, G. H. 1973. Light and electron microscope studies of chromosome pairing in relation to chiasma localization in Stethophyma grossum (Orthoptera: ). Chromosoma 42: 145-162. Kezer, J. and Macgregor, H. C. 1971. A fresh look at meiosis and centromeric heterochromatin in the red backed salamander, Plethodon cinereus cinereus (Green). Chromosoma 33: 146-166. King, R. C. 1967. Genetics. Oxford University Press Inc. Klasterska, 1. 1971. New phenomena during meiosis in the genus Rosa. Hereditas 67: 55-64.- 1976. A new look on the role of the diffuse stage in problems of plant and animal meiosis. 648 A. M. Varquez, C. Martinez and J. R. Lacadena Cytologia 45

Hereditas 82: 193-204.- 1977. The concept of the prophase of meiosis. Hereditas 86: 205-210. Koch, P. et al. 1972. DNA reduplication during meiotic prophase in the oocytes of Carausius morosus (Insecta, Cheleutoptera). Chromosoma 36: 313-321. Lacadena, J. R. 1976. Genetica (2nd ed.). A. G. E. S. A., Madrid.- and Vazquez A. M. 1971. Sequential analysis of meiotic prophase in pollen mother cells of rye Secale cereale. Genetica Iberica 23: 95-116. Lu, B. C. 1967. The course of meiosis and centriole behaviour during the ascus development of the Ascomycete Gelasinospora calospora. Chromosoma 22: 210-226.- and Raju, N. B. 1970. Meiosis in Coprinus II. Chromosome pairing and the Iampbrush diplo tene stage of meiotic prophase. Chromosoma 29: 305-316. Maguire, M. P. 1968. Evidence on the stage of heat induced crossover effect in maize. Genetics 51: 23-40.- 1972. Premeiotic mitosis in maize: Evidence for pairing of homologues. Caryologia 25: 17-24.- 1974. A new model for homologous chromosome pairing. Caryologia 27: 349-357. Moens, P. B. 1964. A new interpretation of meiotic prophase in Lycopersicon esculentum (tomato) Chromosoma 15: 231-242.- 1969. The fine structure of meiotic chromosome polarization and pairing in Locusta migra toria spermatocytes. Chromosoma 28: 1-25. Nagl, W. 1969. The course of the first meiotic prophase in Beta procumbens and in the Fl between B. vulgaria and B. procumbens. Theor. Appl. Genet. 39: 356-360. Ohno, S. et al. 1961. Female germ cells of man. Experimental Cell Research. 24: 106-110. - et al . 1962. Human oogenesis. Cytogenetics 1: 42-51. Owens, J. N. and Molder M. 1971. Meiosis in conifers: prolonged pachytene and diffuse diplo tene stages. Can. J. Bot. 49: 2061-2064. Peacock, W. J. 1968. Chiasmata and crossing-over. In: (W. J. Peacock and R. D. Brock, edit.), Austral. Acad. Sci., Camberra, pp. 242-252.- 1970. Replication, recombination and chiasmata in Goniaea australasiae (Orthoptera: Acridi dae). Genetics 65: 593-617. Pijnacker, L. P. 1967. Oogenesis in the parthenogenetic stick Sipyloidea sipylus Westwood. (Orthoptera, Phasmidae) Genetica 38: 504-515. Pogosianz, H. E. 1970. Meiosis in Djungarian Hamster I. General pattern of male meiosis. Chromosoma 31: 392-403. Rieger, R. et al. 1976. A glossary of Genetics and Cytogenetics (4th ed.) George Allen & Unwing Ltd. London Springer-Verlag. Rossen, J. M. and Westergaard, M. 1966. Studies on the mechanism of crossing over II. Meiosis and the time of meiotic chromosome replication in the ascomycete Neottiella rutilans (Fr) Dennis. C. R. Trav. Lab. Carlsberg 35: 233-260. Singleton, J. R. 1953. Chromosome morphology and the chromosome cycle in the ascus of Neuro spora crassa. Amer. J. Bot. 40: 124-144. Smith, S. G. 1942. Polarization and progression in pairing II. Premeiotic orientation and the initiation of pairing. Can. J. of Res. D. 20: 221-229. Sotelo, J. R. et al. 1973. Serial sectioning study of some meiotic stages in Scapteriscus borrelli (Grylloiea) Chromosoma 42: 307-333. Stack, S. 1961. Premeiotic changes in Ornithogalum virens. Bull. of the Torrey Bot. Club. 98: 207-214.- and Brown, W. V. 1969. Somatic and premeiotic pairing of homologues in Plantago ovata. Bull. Torrey Bot. Club. 96: 143-149. Strickberger, M. W. 1976. Genetics. (2nd ed.) Macmillan Pub. Co. Inc. New York. Collier Mac millan Pub. London Swanson, C. P. 1958. Cytology and Cytogenetics (2nd. ed.). Macmillan & Co. Ltd. Tennent, D. H. and Ito, T. 1941. A study of the oogenesis of Mespilia globulus L. J. Morphol. 1980 Sequential Analysis of Meiotic Prophase in Grasshoppers 649

69: 347. Westergaard, M. 1964. Studies on the mechanism of crossing-over I. Theoretical consideration. C. R. Trav. Lab. Carlsberg 34: 359-405. Whelan, E. D. P. and Hornby, C. A. 1969. Meiotic prophase in Prunus avium. Can. J. Genet. Cytol. 11: 1813-1814. Wilson, E. B. 1925. The Cell in Development and Heredity (3th ed.). Mac-Millan, New York. Zickler, D. 1973. Fine structure of chromosome pairing in ten Ascomycetes: Meiotic and pre meiotic (mitotic) synaptonemal complexes. Chromosoma 40: 401-416.