The staining of the synaptonemal complex for light microscopic study in the mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

Sequential study of synaptonemal complexes in mouse spermatocytes by light and electron microscopy

Synaptonemal complex (SC) studies in male mice from the beginning of meiosis to its completion (7–40 days of age) indicate: (1) the existence of a leptotene stage with fragmented and completely unpaired axial elements; (2) that synapsis begins at several different initiation points of the axial elements, resulting in X-shaped and Y-shaped configurations; (3) that interlocking of axial elements at zygotene is a normal phenomenon; and (4) that zygotene (presynaptic) and diplotene (postsynaptic) configurations are different from each other. This investigation received financial support from the special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization.     

Observations on the synaptonemal complex in Armenian hamster spermatocytes by light microscopy

Using the silver staining technique, in somatic and meiotic chromosomes of the Armenian hamster (Cricetulus migratorius), it is possible to stain synaptonemal complexes (SCs) and the nucleolus organizer regions (NORs) in early spermatocytes. There are five pairs of autosomes (Nos. 2, 4, 6, 7, and 8) which have terminally located NORs. Synaptonemal complexes and accessory structures present in the sex chromosomes within the sex vesicle can be easily observed using light microscopy.     

The effect of tequila in the synaptonemal complex structure of mouse spermatocytes.

The effect of tequila in the synaptonemal complex (SC) of mouse spermatocytes was determined. We tested 3 dosages (2.1, 4.2 and 8.4 g/kg) administered in a single intraperitoneal inoculation. The frequency of SC alterations was established in pachytenic nuclei 5 days after the administration using a silver impregnation technique. Three types of alterations were observed (desynapses, breaks and multiaxials) and the rate of each alteration was compared with that obtained with appropriate controls, including cyclophosphamide (CP) (150 mg/kg). The results showed a significant increase induced by tequila only in the frequency of desynapses. This damage began at the second highest dose (4.2 g/kg). The other SC alterations were in the control range. CP, however, induced a significant increase in all 3 types of SC alterations.     

Evolution of the synaptonemal complex in Helix aspersa spermatocytes

In spermatocytes of Helix aspersa, the structure of the synaptonemal complexes undergoes changes in the course of the pachytene, the lateral elements being transformed into wide bands of lesser density than the chromatin. By using the uranyl-EDTA-lead sequence, which preferentially stains RNA, the lateral elements can be made to appear positive in the early pachytene while the corresponding areas, which become wider and more diffuse, are positive during late pachytene. — Apparently, the lateral elements do not persist in the diplotene and remnants of the central element can occasionally be observed. Using the uranyl-EDTA-lead method reveals some positively stained material surrounding the chromatin, mostly granular in appearance, which is observed in late pachytene and attains its maximum amount during diplotene. Several aspects of these observations are here discussed.     

A sequential analysis of the development of the synaptonemal complex in spermatocytes of the mouse by electron microscopy using hydroxyurea and agar filtration

A method is presented for sequential analysis of the development and behaviour of the Synaptonemal Complex (SC) in primary spermatocytes of male mice, using agar filtration for electron microscope grid preparation. The mice were treated with hydroxyurea (HU) to produce a gap in the spermatogenic line. The front of surviving cells behind the gap was examined day by day. The first visible parts of unpaired axial elements, with some barely recognizable paired regions were found 9 days after the last HU injection i.e. directly after the last S-phase before meiosis. During mid zygotene and late zygotene the axes of the autosomes had a fuzzy ill-defined appearance with irregular regions of apparent thickening. The axes of the XY pair could be recognized only at late zygotene. During pachytene the SCs of the autosomal pairs did not show a significant change except for a slight increase in size of the attachment points of the axial elements. On the first day of pachytene the axes of the XY pair appeared thin and long. On the second day the axes of the XY pair showed maximal pairing of about 50% of the axis of the Y chromosome. From the third to the fifth day a decrease of the paired region of the sex chromosomes was found together with an increase in thickness of the axes, which reached its maximum on the fourth day. Diplotene could be easily recognized: the autosomal axes showed a sharp, well-defined outline with thick attachment points with deltoid structure, and desynapsis was very clear. The axes of the XY pair showed variation during diplotene but on the third day of diplotene a characteristic bulging could be seen. The axes of the autosomes disappeared at this time and in most cases only the attachment points remained visible. The duration of the prophase classes of meiosis I was found to be: zygotene approximately 2 days; pachytene a little more than 5 days and diplotene approximately 3 days. Leptotene could not be traced by the method used. If it exists at all, it must be a stage of very short duration.     

Biochemical and ultrastructural analyses of the synaptonemal complex in mammalian spermatocytes

In order to study the molecular organization of synaptonemal complex (SC), a preparative method for isolation of relatively purified SC from rat, mouse and hamster testes was elaborated which involves isolation of SC-containing (pachytene) nuclei, their lysis, DNAase digestion of DNA and fractionation of nuclear elements by the discontinuous sucrose density gradient centrifugation. Electron microscopy revealed a rather good preservation of the SC structure after the isolation procedure. Effects of the dissociating agents on the SC structural integrity were studied. It has been demonstrated that the treatment with 2M NaCl, Triton X-100, sodium deoxycholate, 6M urea, and with a buffer containing 2% SDS and 5% mercaptoetthanol does not lead to a complete SC dissociation, though it results in some structural chanes. Possible reasons of the high resistance of SC to dissociating treatment are discussed.     

Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes

The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene glycol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.     

Development and behavior of synaptonemal complexes in human spermatocytes by light and electron microscopy

Summary We describe in this paper the human male synaptic cycle using light and electron microscopy and the distribution of cells in the different stages of prophase I. The pattern of chromosome pairing and synapsis is an important tool to determine accurately whether a given synaptic behavior in infertile or sterile men is really abnormal or not. The relationship of prepachytene to pachytene cells is also important for the diagnosis of the different types of meiotic arrest at the primar spermatocyte level.     

Damage to synaptonemal complex structure and peculiarities of selection of mouse spermatocytes I at response to drug administration

Using immunocytochemistry methods, the structure of synaptonemal complexes (SC) of chromosomes in spread nuclei of primary spermatocytes of mice at 1, 10, and 36 days after the 10-day intraperitoneal administration of antibacterial preparations of three pharmacological groups: furacilin, an antiseptic derivative of nitrofuran; cifran, an antibiotic from the group of fluoroquinolones; and sextaphage, a polyvalent piobacteriophage was investigated. The maximal number of damages in the structure and behavior of synaptonemal complex was revealed on the first day after the end of preparation administration. On days 10 and 36, the total number of damages in SC structure decreased gradually. On the first day after the end of the administration of cifran and sextaphage in 41.8 and 25% of nuclei, respectively, the fragmentation of synaptonemal complexes was revealed and, in males to whom furacilin had been administered, the fragmentation of synaptonemal complexes was identified in 100% of nuclei. Multiple chromosome fragmentation is a meiotic catastrophe and results in the degeneration of cells without enabling the mechanism of pachytene arrest. The features of pachytene arrest were revealed in the nuclei of primary spermatocytes with the violation of chromosomes pairing. After the administration of sextaphage, circle structures released from the lateral elements of SC and are dyed with antibodies to SCP3 protein.     

A method for producing synaptonemal complex complements in lily and mouse

A whole-mount procedure for producing pachytene synaptonemal complex complements of Lilium longiflorum was developed. The method involves swelling of the meiotic nuclei followed by nonionic detergent lysis of the nuclear envelope. This technique adequately spreads out the long lily chromosomes while producing only minimal distortion of the chromosomal axes. The ultrastructure of the synaptonemal complex is normal, and the chromatin remains closely associated with the synaptonemal complex. The procedure also was used successfully to produce pachytene synaptonemal complex preparations of mouse chromosomes. In the mouse, the centromeric heterochromatin remains associated with the synaptonemal complex, but the euchromatin is more widely dispersed.     

XY pair associates with the synaptonemal complex of autosomal male-sterile translocations in pachytene spermatocytes of the mouse (Mus musculus)

Analysis of the chromosome behaviour at pachytene has been performed by means of the silver staining technique visualizing the synaptonemal complexes (SCs) in male mice heterozygous for the male-sterile translocations T(5;12)31H, T(16;17)43H and T(7;19)145H, respectively. The T(9;17)138Ca male heterozygotes and T43H/T43H homozygous males were used as fertile controls. The sterile mice displayed a high frequency (about 60%) of pachytene spermatocytes with autosomal translocation configuration located in close vicinity of the XY pair. The dense round body (XAB), normally located near the X-chromosome axis in fertile males, exhibited abnormal affinity to translocation configuration in the sterile translocation heterozygotes. The incomplete synapsis of autosomes involved in translocation configuration was observed in more than 70% of the pachytene spermatocytes with the male-sterile translocations but in less than 20% of the cells from T138Ca fertile male.s. A hypothesis relating the spermatogenic arrest of carriers of male-sterile rearrangements to the presumed interference with X chromosome inactivation in male meiosis is discussed.     

Localization of the synaptonemal complex under the light microscope

The use of osmium tetroxide fixation followed by postreatment with p-phenylenediamine gives an opportunity of locating the synaptonemal complex (SC) under the light microscope in mouse testes and Allium cepa anthers. When semi-thin sections from these materials were observed under phase contrast optics or dark field microscopy, fine threads in the pachytene nuclei were clearly visible. Post-staining of semi-thin sections with ammoniacal silver increased the contrast of the SC and allowed for observations using a bright field illumination. Ultrathin sections of osmium tetroxide/ p-phenylenediamine treated material showed that, under the electron microscope, this technique stains preferentially elements of the synaptonemal complex, while the surrounding chromatin remains unstained.     

Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy

When Locusta migratoria spermatocytes are surface-spread on various salines, the axial element of leptotene and zygotene chromosomes, and the synaptonemal complex of pachytene chromosomes are well-preserved, although, in most instances, virtually denuded of chromatin. A complex association of chromosome ends with the nuclear membrane is apparent as early as leptotene, and, as pairing is initiated, the nuclear attachment points of the partner half-bivalents fuse, apparently incorporating additional membrane material between them. The meiotic kinetochore originates in association with the axial element during early prophase, and prior to synaptonemal complex formation and chromosome condensation.     

Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study.

The endocrine control of yolk deposition in Drosophila melanogaster was studied by ligation and transplantation techniques. Endocrine events associated with the initiation of vitellogenesis were found to be synchronized with eclosion rather than the completion fo adult development. Decapitation experiments showed that a cephalic event occurring at about the time of eclosion is necessary for each animal to initiate vitellogenesis. The morphogenetic effect of the head could be replaced by a juvenile hormone analog (JHA). In addition to the cephalic event, a thoracic factor is required for each follicle to initiate vitellogenesis, since preparation of isolated abdomens before 16 hours after eclosion prevented vitellogenesis. In abdomens isolated after this time, no early vitellogenic stages were formed. The suppression of vitellogenesis in isolated abdomens was reversed by implanting corpora allata or by treating these preparations with JHA, but not by implanting corpora cardiaca. Ovaries that were artificially induced to mature by treating isolated abdomens with JHA still displayed the normal complement of ovarian proteins after electrophoresis in polyacrylamide gels. These results show that a circadian clock triggers vitellogenesis via a cephalic signal at eclosion, which in turn triggers events in the thorax or abdomen. The cephalic signal can be superseded by juvenile hormone, whose presence is necessary for each follicle to become vitellogenic.     

Immunocytochemical localization of DNA in synaptonemal complexes of rat and mouse spermatocytes,and of chick oocytes

The distribution of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes was investigated by immunogold electron microscopy. Except for a few specific sites, DNA was not immunolocalized in the space between lateral elements of the complex. Some labeled fibrils connecting the lateral elements with the central element were observed associated with recombination nodules or near them. However, other labeled fibrils in the space between lateral elements did not appear to present any relationship to recombination nodules. The immunocytochemical approaches used here confirmed the presence of significant amounts of DNA in the lateral elements as previously indicated by preferential DNA staining methods. Furthermore, our findings support the view that recombination nodules are the site of chiasma formation.