The fine structure of meiotic chromosome polarization and pairing in Locusta migratoria spermatocytes

At the leptotene stage of meiotic prophase in Locusta spermatocytes (2n=22 telocentric autosomes + X-chromosome), each chromosome forms an axial core. The 44 ends of the autosomal cores are all attached to the nuclear membrane in a small region opposite the two pairs of centrioles of the juxtanuclear mitochondrial mass. At later stages of meiotic prophase, the cores of homologous chromosomes synapse into synaptinemal complexes. Synapsis is initiated near the nuclear membrane, in the centromeric and the non-centromeric ends of the chromosomes. Homologous cores have their attachment points close together and some cores are co-aligned prior to synapsis. At subsequent stages of zygotene, the number of synaptinemal complexes at the membrane increases, while the number of unpaired axial cores diminishes. At pachytene, all 11 bivalents are attached to the membrane at both ends, so that there are 22 synaptinemal complexes at the membrane near the centrioles. Because each bivalent makes a complete loop, the configuration of the classic Bouquet stage is produced. The X-chromosome has a poorly defined single core at pachytene which also attaches to the nuclear membrane. These observations are based on consecutive serial sections (50 to 100) through the centriolar zone of the spermatocytes. Labeling experiments demonstrated that tritiated thymidine was incorporated in the chromatin of young spermatocytes prior to the formation of the axial cores at leptotene. It is concluded that premeiotic DNA synthesis is completed well in advance of pairing of homologous chromosomes, as marked by the formation of synaptinemal complexes.     

Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place. Received: 16 November 1999; in revised form: 29 December 1999 / Accepted: 3 January 2000     

Synaptonemal complexes of the A- and B-chromosomes of the spermatocytes from the East Asian mouse Apodemus peninsulae

The analysis of whole-mount preparations of synaptonemal complexes (SCs) from surface-spread spermatocytes of A. peninsulae (2n = 48A + 1, 2, ... 12 B) had revealed SCs of 23 autosomal bivalents, sex bivalent XY, axial cores and SCs of the B-chromosomes. The intercellular and interindividual variability of the number of B-chromosomes varied from 1 to 12 per cell. The SCs of autosomal bivalents were shown to have a typical structure. The structure and behaviour of SCs of sex bivalent throughout meiotic prophase I appeared to be similar to those observed in other species of this order. Mainly B-univalents and less frequently B-bivalents containing SCs were found to be formed in meiotic prophase I. The full homologues appear to be rarely seen among B-chromosomes of the East-Asiatic mouse. A tendency of forming clusters of B-univalents near the sex bivalent was found, in addition to B-bivalents with lateral elements, having the form of bi- and tri-stranded elements with rare synaptic fragments. Besides this, the SCs of the autosomes of pachytene cells were found to contain structures resembling the recombination nodules.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Trivalent behavior during prophase I in male mice heterozygous for three Robertsonian translocations: an electron-microscopic study

A synaptonemal complex (SC) analysis was carried out in male mice heterozygous (CHT/+) for three Robertsonian translocations. All pachytene preparations studied showed the presence of three trivalents. At early pachytene, the nonhomologous centromeric regions of the acrocentric chromosomes were unpaired. Heterosynapsis subsequently took place with complete pairing of the trivalents. Association between one of the three trivalents and the sex vesicle was observed in 30.4% of the nuclei. Association between the unpaired regions of two trivalents was present in 14.4% of the cells, suggesting that the relationship between unpaired regions of structural rearrangements and the X-Y bivalent may simply reflect the tendency of unpaired regions to establish end-to-end associations or heterosynapses among them, which are usually resolved during the pachytene stage of prophase I. Since the sex bivalent always has unpaired regions, these associations often affect the sex chromosomes.     

Immunochemical localization of contractile proteins in mammalian meiotic chromosomes

Summary Smooth muscle heavy myosin and actin have been detected in mouse and rat meiotic chromosomes, by indirect immunofluorescence performed on testis cryostat sections and isolated germ cells. Both contractile proteins are detectable in the nuclei of meiotic cells during the first prophase. The appearance and disappearance time of myosin and actin, however, is not synchronous. While actin is visible in small spots from resting to late diplotene spermatocytes, myosin appears as filaments in the primary spermatocytes from the zygotene to the early stage of diplotene. The number of myosin filaments in the pachytene spermatocytes corresponds to the number of bivalent chromosomes, whereas actin spots constantly outnumber the pairing chromosomes by two units. These immunochemical observations suggest that the two contractile proteins are associated with the synaptonemal complex (SC). Myosin seems to be associated with the central region of the SC, while actin is present in its basal knob which is in connection with the nuclear membrane. The difference in number between myosin filaments and actin spots appears to be related to the peculiar behaviour of the pairing sex chromosomes. The presence of contractile proteins in the nuclei of primary spermatocytes seems to suggest that they might play a role in the process of pairing of homologous chromosomes.     

Meiotic studies in infertile domestic pig-babirusa hybrids

Mating of a babirusa (Babyrousa babyrussa) boar and a domestic sow (Sus scrofa) resulted in the birth of 5 live domestic pig-babirusa hybrid piglets. Chromosome analysis of one of the surviving males confirmed that they were domestic pig-babirusa hybrids by revealing the presence of a complete haploid set of 19 porcine chromosomes as well as a complete haploid set of 19 babirusa chromosomes in the karyotype. None of the surviving piglets, two males and one female, had shown signs of sexual maturity at age 27 months. Histological examination of gonadal biopsies from the 2 males revealed that both were azoospermatic. Immunostaining revealed SCP3-positive axial elements in the nuclei of primary spermatocytes, indicating that they were progressing through leptotene and zygotene of meiotic prophase. However, the presence of multiple short stretches of axial elements in pachytene nuclei indicated that this phase was blocked, probably due to aberrant chromosome pairing. Histological examination of the ovaries revealed follicular structures, but oocytes within them were generally degenerated. We conclude that both male and female pig-babirusa hybrids were infertile, most likely due to germ cell death resulting from abnormalities of chromosome pairing during meiotic prophase.     

Dense bodies in silver-stained spermatocytes of the chinese hamster: behavior and cytochemical nature

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, thread-like axes seen at midpachytene, and (3) multistranded, branched, and anastomosed axes seen at late pachytene.Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body.Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.     

Live cell analyses of synaptonemal complex dynamics and chromosome movements in cultured mouse testis tubules and embryonic ovaries

During mammalian meiotic prophase, homologous chromosomes connect through the formation of the synaptonemal complex (SC). SYCP3 is a component of the lateral elements of the SC. We have generated transgenic mice expressing N- or C-terminal fluorescent-tagged SYCP3 (mCherry-SYCP3 (CSYCP) and SYCP3-mCherry (SYCPC)) to study SC dynamics and chromosome movements in vivo. Neither transgene rescued meiotic aberrations in Sycp3 knockouts, but CSYCP could form short axial element-like structures in the absence of endogenous SYCP3. On the wild-type background, both fusion proteins localized to the axes of the SC together with endogenous SYCP3, albeit with delayed initiation (from pachytene) in spermatocytes. Around 40% of CSYCP and SYCPC that accumulated on the SC was rapidly exchanging with other tagged proteins, as analyzed by fluorescent recovery after photobleaching (FRAP) assay. We used the CSYCP transgenic mice for further live cell analyses and observed synchronized bouquet configurations in living cysts of two or three zygotene oocyte nuclei expressing CSYCP, which presented cycles of telomere clustering and dissolution. Rapid chromosome movements were observed in both zygotene oocytes and pachytene spermatocytes, but rotational movements of the nucleus were more clear in oocytes. In diplotene spermatocytes, desynapsis was found to proceed in a discontinuous manner, whereby even brief chromosome re-association events were observed. Thus, this live imaging approach can be used to follow changes in the dynamic behavior of the nucleus and chromatin, in normal mice and different infertile mouse models.     

Atm-dependent interactions of a mammalian Chk1 homolog with meiotic chromosomes

Background: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest.Results: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1^Hu/Mo has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22–23, a region marked by frequent deletions and loss of heterozygosity in human tumors.Conclusions: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22–23 indicates that the CHK1 gene is a candidate tumor suppressor gene.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

The structure and function of the synaptinemal complex inLilium longiflorum sporocytes

The development of meiotic prophase in pollen mother cells ofLilium longiflorum is presented through photomicrographs of squashes and sections and through electron micrographs of thick and thin sections. Emphasis is placed on the first appearance of axial cores, the participation of axial cores in the formation of synaptinemal complexes, the fine structure of the complex and the fate of the complex at the end of pachytene. It is shown that axial cores are formed in early meiotic prophase chromosomes and that the two axial cores of a set of homologous chromosomes participate in the formation of a synaptinemal complex. It is proposed that the transverse filaments of each axial core meet and interdigitate and so produce the transverse filaments of the complex. It is shown that the complex is axial to the pachytene bivalent and that the association of the complex with chromosomal material is terminated at the end of pachytene. The pairing affinity of the cores in homologous and non-homologous chromosome associations is discussed. The zygotene stage is defined in terms of the occurrence of synaptinemal complexes and the attachment of the nucleolus to the nuclear membrane during this stage is noted.     

Sequential analysis of synaptonemal complexes in the repopulating spermatocytes of Rattus norvegicus after restricting the germ cell population to spermatogonia by gossypol treatment

Synaptonemal complexes of the repopulating spermatocytes of male rats were analyzed day by day using silver-stained surface spread nuclei between 8 and 25 days after restricting the germ cell population to spermatogonia by treatment of gossypol acetic acid at 30 mg/kg body weight/day for 70 days. The method allowed sequential analysis of male meiotic prophase on successive days after the last day of treatment. The leptotene cells appeared on day 11 and were characterized by a network of lateral elements and large nucleolar bodies in a diffuse mass. On day 13 the unpaired lateral elements and short stretches of synaptonemal complexes characteristic for zygotene could be seen. Pachytene nuclei showing 20 autosomal synaptonemal complexes and XY axes appeared on day 15. The diplotene cells were defined on day 22 by the loss of a complete synaptonemal complex set and by the appearance of disjoined lateral elements and persistent segments of synaptonemal complexes.     

Ultrastructural studies of late meiotic prophase nuclei of spermatocytes in Ascaris suum

Post pachytene stages of meiotic prophase in males of Ascaris suum have been analyzed with the electron microscope. No synaptonemal-like polycomplexes (PCs) have been observed in the nucleoplasm or cytoplasm during the period from pachytene to diakinesis. From Serially sectioned diplotene nuclei it was found that the bivalents are located near the periphery of the nuclei, the central part of the nuclei being vacant. Each nucleus contains one nucleolus. Up to 1 m long stretches of unpaired lateral elements (LEs) are found in some of the diplotene bivalents. These LEs are morphologically similar to unpaired LEs in early zygotene nuclei. Partial 3-dimensional reconstruction of two nuclei shows that the bivalents contain some small stretches of synaptonemal complex (SC) up to 1.9 m long. Some bivalents at diakinesis show remnants of SCs. At this stage chromosomes are fibrous, condensed, attached to the nuclear envelope and mostly with a rounded profile in cross section. The synchronous development of the spermatocytes and small bivalents at diplotene in A. suum make this system a good object for the study of localization of SC remnants.     

Colchicine effects on meiosis in the male mouse

Antimitotic agents administered at the time of synapsis (leptotene/zygotene) have been shown to induce synaptic abnormalities visible during pachytene in the male mouse. The object of this study was to test the hypothesis that cells with relatively large amounts of colchicine-induced damage to the synaptonemal complex (SC) are eliminated from prophase whereas cells with relatively small amounts of SC damage proceed through to the end of prophase. Male mice were injected with tritiated thymidine to mark a cohort of spermatocytes at premeiotic S-phase for tracking through pachytene. Forty-eight hours later, when those cells were at leptotene/zygotene, colchicine was administered intratesticularly. Whole-mount SC spreads were made from animals sacrificed at various times following colchicine administration, and prepared for autoradiography. The marked cells were examined by light and electron microscopy and the kind and number of synaptic abnormalities were scored throughout pachytene. Colchicine-induced SC damage included single axial elements (univalents), together with partially synapsed and nonhomologously synapsed SCs. The amount of SC damage (amount and type per cell and frequency of cells with damage) scored at early pachytene exceeded by three- to fivefold the amount at late pachytene. This is consistent with spermatogenic cell loss from the seminiferous tubule via colchicine-induced destruction of Sertoli cell microtubules. The presence of spermatocytes with no more than four autosomal univalents at late pachytene indicates that some cells with low amounts of synaptic damage progress to the end of pachytene. The loss of the most severely damaged cells may represent a meiotic checkpoint at early pachytene in the male mouse. Received: 24 April 1996; in revised form: 29 August 1996 / Accepted: 11 March 1997     

Synaptonemal complex and recombination nodules in rye (Secale cereale)

Meiotic prophase in rye was investigated by serial-section reconstruction of pollen mother cell nuclei. In the mid-late zygotene nucleus, all lateral elements were continuous from telomere to telomere, and 9–20 pairing initiation sites per bivalent were observed. Chromosome and bivalent interlockings detected during zygotene were resolved at early pachytene when pairing was completed. In the three pachytene nuclei, the relative synaptonemal complex (SC) lengths and arm ratios were found to be in good correlation with light microscopic data of pachytene bivalents. Spatial tracing of the bivalents showed that they occupy separate areas in the nucleus. Three types of recombination nodules were observed: large, ellipsoïdal and small nodules at early pachytene and irregularly shaped nodules mainly associated with chromatin at late pachytene. Their number and position along the bivalents correlated well with the number and distribution of chiasmata. The classification of the seven bivalents was based on arm ratio and heterochromatic knob distribution.