Late Leptotene Research Articles

Meiotic chromosome behaviour of a newly recorded ant-like spider, Myrmarachne melanocephala MacLeay, 1839 from Manipur, India

Cytological and chromosomal studies of ant-like spiders Myrmarachne melanocephala MacLeay, 1839, were undertaken with 12 males captured alive in the months of January to July 2023 from three habitats. The haploid count of male specimens was observed to be 13: 4 acrocentric (including X chromosome), 6 subtelocentric, 2 submetacentric, and 1 metacentric and showed X0 sex determination mechanisms, so the diploid count of the species was 25 (13+12). The single sex chromosome occupied roughly 4-6 per cent of the nuclear volume prominently in the Interphase – Prophase I stage. The structure of X chromosome in interphase stage was circular-rectangular block. The peculiar shape of rod-shaped X chromosome was maintained from early pre-leptotene stage till the end of the division particularly the Prophase I. The synapsis started early from late leptotene and duration of zygotene was long enough to visualize the perfect ones in late zygotene. There were nine diplotene bivalents with interstitial chiasma.

INFECTED FRESHWATER SNAILS, BIOMPHALARIA ALEXANDRINA, THE INTERMEDIATE HOST OF SCHISTOSOMA MANSONI

The objective was to study the chromosomes of Biomphalaria alexandrina snails (class Gastropoda) in control and infected state which could be helpful in understanding how host-parasite relationships in feasible and effective control measures. The chromosomal changes were studied using the air-drying method. The results showed that B. alexandrina had a diploid chromosome number, 2n = 36. Also, the meiotic stages were detected as early- leptotene and late leptotene, zygotene, diplotene, metaphase I. The result also revealed the presence of a primitive sperm, with a conical head and a very long, uni-flagellate tail. A comparative meiotic chromosome analysis between the control and infected ones showed some significant differences, as pachytene and diplotene were more condensed.

Short periods of high temperature during meiosis prevent normal meiotic progression and reduce grain number in hexaploid wheat (Triticum aestivum L.)

Key messageExposure of wheat to high temperatures during male meiosis prevents normal meiotic progression and reduces grain number. We define a temperature-sensitive period and link heat tolerance to chromosome 5D.This study assesses the effects of heat on meiotic progression and grain number in hexaploid wheat (Triticum aestivum L. var. Chinese Spring), defines a heat-sensitive stage and evaluates the role of chromosome 5D in heat tolerance. Plants were exposed to high temperatures (30 or 35 °C) in a controlled environment room for 20-h periods during meiosis and the premeiotic interphase just prior to meiosis. Examination of pollen mother cells (PMCs) from immature anthers immediately before and after heat treatment enabled precise identification of the developmental phases being exposed to heat. A temperature-sensitive period was defined, lasting from premeiotic interphase to late leptotene, during which heat can prevent PMCs from progressing through meiosis. PMCs exposed to 35 °C were less likely to progress than those exposed to 30 °C. Grain number per spike was reduced at 30 °C, and reduced even further at 35 °C. Chinese Spring nullisomic 5D-tetrasomic 5B (N5DT5B) plants, which lack chromosome 5D, were more susceptible to heat during premeiosis–leptotene than Chinese Spring plants with the normal (euploid) chromosome complement. The proportion of plants with PMCs progressing through meiosis after heat treatment was lower for N5DT5B plants than for euploids, but the difference was not significant. However, following exposure to 30 °C, in euploid plants grain number was reduced (though not significantly), whereas in N5DT5B plants the reduction was highly significant. After exposure to 35 °C, the reduction in grain number was highly significant for both genotypes. Implications of these findings for the breeding of thermotolerant wheat are discussed.

Replication Protein A2c Coupled with Replication Protein A1c Regulates Crossover Formation during Meiosis in Rice

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.

Triploid Induction in Populus alba x P. glandulosa by Chromosome Doubling of Female Gametes

Abstract Efficient methods for obtaining unreduced female gametes are needed for triploid induction in Populus alba x P. glandulosa. A cytological study of MMCs and PMCs meiosis and staminate floral elongation revealed that there was a close association between MMCs meiotic stage and male bud phenology during development under the same environment conditions. Female buds of Populus alba x P. glandulosa were treated with 0.5% colchicine solution at the selected meiotic stages based on male bud phenology. The treated buds were pollinated with pollen of P. tomentosa. A high rate of unreduced female gametes occurred in late leptotene and pachytene stages of MMC, as evidenced by a relatively higher percentage of triploids in the progeny, when compared to progeny from colchincine application at other meiotic stages. The present results demonstrated that male bud phenology can be used to guide colchicine applications to female buds and cause a more efficient production of triploids.

ASK1, a SKP1 homolog, is required for nuclear reorganization, presynaptic homolog juxtaposition and the proper distribution of cohesin during meiosis in Arabidopsis

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the "synizetic knot", which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.

The Arabidopsis synaptonemal complex protein ZYP1 is required for chromosome synapsis and normal fidelity of crossing over

The duplicated Arabidopsis genes ZYP1a/ZYP1b encode closely related proteins with structural similarity to the synaptonemal complex (SC) transverse filament proteins from other species. Immunolocalization detects ZYP1 foci at late leptotene, which lengthen until at pachytene fluorescent signals extending the entire length of the fully synapsed homologs are observed. Analysis of zyp1a and zyp1b T-DNA insertion mutants indicates that the proteins are functionally redundant. The SC is not formed in the absence of ZYP1 and prophase I progression is significantly delayed suggesting the existence of an intraprophase I surveillance mechanism. Recombination is only slightly reduced in the absence of ZYP1 such that the chiasma frequency at metaphase I is approximately 80% of wild type. Moreover cytological analysis indicates that chiasma distribution within zyp1 bivalents is indistinguishable from wild type, providing evidence that the SC is not required for the imposition of interference. Importantly in the absence of ZYP1, recombination occurs between both homologous and nonhomologous chromosomes suggesting the protein is required to ensure the fidelity of meiotic chromosome associations.

Crossover/Noncrossover Differentiation, Synaptonemal Complex Formation, and Regulatory Surveillance at the Leptotene/Zygotene Transition of Meiosis

Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression. At 33°C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective. Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC. By implication, crossover interference does not require SC formation. We suggest that SC formation may require interference. Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC. zmm mutant phenotypes differ strikingly at 33°C and 23°C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.

Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase.

To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

Three stages of meiotic homologous chromosome pairing in wheat: cognition, alignment and synapsis

Chromosome painting enabled the study of homologous chromosome behaviour prior to and during meiosis. Total genomic DNA from rye, used as a probe for in situ hybridization, identified the rye chromosome arm in a wheat-rye translocation line (T5AS·5RL) at meiotic prophase and the preceding interphase. Accurate staging of the development of the meiocytes was attained by parallel studies of chromatin morphology, nucleolar behaviour and synaptonemal complex formation in electron microscopy thin sections and silver-stained surface spreads. Three stages of pairing were identified for the large cereal genomes that are organized in a Rabl configuration: first, cognition occurs during the long interphase before leptotene, bringing the homologous chromosome domains into close proximity and possibly starting at the centromere; second, homologous chromosome segments align at late leptotene; and third, zygotene synapsis initiates near the telomere, although it was also observed to occur near the centromere. A pairing model is proposed for wheat, with a genome size of 17000 Mbp, that shows prallels to and notable differences from yeast and mammalian models of meiosis.

Meiotic behaviour of the pollen mother cells in cultured anthers of rye excised at early meiotic prophase

Anthers of rye were excised during leptotene and zygotene and cultured in vitro on media with and without indole-3-acetic acid to study the behaviour of pollen mother cells during meiosis. Percentages of pollen sacs in which pollen mother cells continued meiosis increased from approximately 20% to 60–70% when anthers were excised at early and late leptotene. The highest values were about 90% when the excision was during zygotene. The results were similar on both media. The timing of pollen mother cells through meiosis on the hormone-free medium was similar to that described in vivo. The mean numbers of chiasmata per cell at metaphase I from the cultured pollen sacs were compared with those from a random population of pollen sacs grown in the field. A significant decrease was observed when anthers were excised during leptotene and cultured on both media, as well as when anthers were excised at early zygotene and cultured on the hormone-supplemented medium. We concluded that at early zygotene there are processes related to chiasma formation that seem to be influenced by hormone balance.Key words: Secale cereale, anther culture, meiosis development, chiasma formation, indole-3-acetic acid.

Topography of the rat blood-testis barrier after intratubular administration of intercellular tracers

Intratubular injection of electron-opaque tracers (lanthanum hydroxide, peroxidase) by micromanipulation showed that the intercellular spaces of the adluminal compartment are in continuity with the lumen of the rat seminiferous tubule at all stages of the spermatogenic cycle. This continuity involves the intercellular spaces which surround zygotene spermatocytes and late leptotene spermatocytes in stages X–XI. The present observations would seem to cast doubt on the real existence of a third compartment, or intermediate compartment, in the seminiferous epithelium. In the material which, in addition to intratubular administration, had been treated with intravascular tracer perfusion, we found the presence of a parabasal region of interSertoli junctions where the tracer stopped whatever its direction. Freeze-fracture replicas exhibit interSertoli tight junctions arranged, from basal lamina to lumen, in three well-defined patterns: in the most basal area, which is the zone of free penetration of tracers from the peritubular environment, junctions are very sparse or absent; a belt of closely apposed continuous and parallel junctions, also parallel to the basal lamina, is the exclusion zone which prevents tracer penetration from either direction; the most apical membrane areas display irregularly arranged, loosely spaced and frequently interrupted rows of particles, most tending to be perpendicular to the basal lamina. This area corresponds to the territory where the intraluminally injected tracer permeates the interSertoli space.

Structure, development, and cytochemical properties of the nucleolus-associated "round body" in rat spermatocytes and early spermatids.

The "round body," a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 micron in leptotene to 1.6 micron in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8. Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.

Synaptonemal complexes with modified lateral elements in Sordaria humana: development of and relationship to the ?recombination nodules?

Meiotic prophase in Sordaria humana has been analyzed by three-dimensional reconstructions of 3 leptotene, 2 zygotene, 10 pachytene and 3 diplotene nuclei. Several notable features emerged. The lateral components of the synaptonemal complexes (SC) are hollow tubes which show dilations of variable sizes from late leptotene to early diplotene. These bulges occur before pairing. Their number decreases as soon as the SC are completely formed, but their mean size increases. Bulges can be present in all parts of the lateral components including telomeres and nucleolar organizer region, but their distribution along bivalents is not random. The remarkably uniform width of the SC central region, normally observed in other species is not observed in S. humana. Although as a general rule the bulges rarely affect the homologous components at identical sites, they often either fill or partially cover the central region. The “recombination nodules” are not clearly connected with the bulges. This work provides also additional insight into the development of both SC and the nodules. At late leptotene, the homologues are aligned before SC formation. One case of interlocking has been observed at early pachytene. Nodules are present from zygotene to diplotene. They are not evenly distributed along the bivalents during pachytene. The mean number of nodules, constant from late pachytene to diplotene, is equal to the mean number of chiasmata.

Further Observations on the Blood-Testis Barrier in Monkeys

Tight junctions uniting adjacent Sertoli cells subdivide the monkey seminiferous epithelium into 1) basal and 2) adluminal compartments (Dym, 1973). Electron-opaque tracer studies demonstrate that types A and B spermatogonia and preleptotene and leptotene spermatocytes reside in the basal compartment of the seminiferous epithelium and that the more differentiated germ cells are located in the adluminal compartment. Late leptotene or early zygotene spermatocytes ascend in the epithelium from one compartment to the next and this occurs in stages IX-X of the cycle of the seminiferous epithelium. New Sertoli cell junctional complexes reform beneath these migrating germinal elements prior to the dissolution of the existing junctions above the cells. This temporal difference serves to seal the barrier during the germ cell migration. These results indicate that the initiation of meiosis occurs while the early spermatocytes (preleptotene and leptotene) are still in the basal compartment of the epitheium and may not require the special microenvironment provided by the Sertoli cells in the adluminal compartment.

Formation of cytoplasmic synaptonemal-like polycomplexes at leptotene and normal synaptonemal complexes at zygotene in Ascaris suum Male Meiosis.

A thread-like (more than 70 cm long) testis of Ascaris suum, when examined under the light and electron microscope, reveals the linear succession of meiotic stages. Beginning from, at least, late leptotene, the spermatocytes are synchronous in their development. Thus within each transverse section of the testis all the spermatocytes are in the same stage. The spermatocytes at each stage of prophase I occupies several (4 to 10) cm of the whole testis length. — At leptotene, synaptonemal-like polycomplexes of lateral and central stacked elements are formed in the cytoplasm of spermatocytes. At late leptotene, the polycomplexes are attached to the external nuclear membrane. The polycomplexes disappear at zygotene. Slightly discernable axial cores are observed in the late leptotene chromosomes. The synaptonemal complexes (SCs) are formed at the zygotene stage, their structure being characteristically tripartite. The SCs disappear from the nuclei at the diffuse stage of prophase I. In other organisms completely developed polycomplexes of stacked lateral and central elements were never found during the presynaptic period of meiosis, although single or two parallel layers of aggregated central regions of SC were found in Neottiella meiocytes at the stage prior to chromosome pairing (Westergaard and von Wettstein, 1970, 1972). — First appearance of the polycomplexes in the cytoplasm insetead of the nucleus is also a novel fact. It is concluded that the polycomplexes at leptotene are formed by a self-assembly of the SC molecular material precociously synthesized in the cytoplasm. Two hypotheses regarding possible function and the further fate for leptotene polycomplexes are discussed.

Development of the synaptonemal complex and the "recombination nodules" during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw.

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one “fixed” and one randomly attached telomeres. The “fixed” end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of “fixed” telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.

Genetic recombination in Coprinus: III. Inlluence of gamma-irradiation and temperature treatments on meiotic recombination

Non-lethal doses of gamma-irradiation (5 krad) increased meiotic recombination in Coprinus lagopus when treatments were given at the beginning of karyogamy. The division stage at this time was judged to be late leptotene and the duration of the sensitive period was assessed to be 3–4 h. In C. lagopus the radiation-sensitive stage is distinct from the cold-sensitive stage (pachytene). The additive effect of irradiation at early karyogamy followed by cold treatment in pachytene suggested that the two factors influenced different steps in the recombination process. On the other hand, irradiation followed by heat treatment did not significantly alter recombination frequency as compared to single treatments. It was surmised that radiation and high temperature act on the same factor(s) or at the same steps to bring about a similar net result. It was suggested that irradiation at leptotene may cause single-strand breaks in DNA which eventually participate in exchange.