A Method for Visualizing the Acrosome by Light Microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol: acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies

The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.     

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a “cone”-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.     

Analysis of a centric shift in the S11 chromosome of Aiolopus strepens (Orthoptera: Acrididae)

A centric shift in the S₁₁ chromosome of Aiolopus strepens, previously described as a pericentric inversion (Cabrero & Camacho, 1982), was analyzed by using the C-banding procedure. The location of breakage points as well as the posibilities of pericentric inversion and three-break transposition are discussed. The relationship between C-bands and NOR location as revealed by both silver staining and a double procedure Ag-NOR C-banding is also discussed.     

A method for visualizing the acrosome by light microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol:acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Relationship between incomplete synapsis and chiasma localization

One of the subjects within the meiotic field that has been actively investigated in the recent years is the temporal and functional relationships between meiotic recombination, cohesin loading and synaptonemal complex (SC) assembly. Although the study of meiotic mutants has shed some light, many questions remain to be answered. Here, we have studied this topic in the orthopteran Paratettix meridionalis, a species with telocentric chromosomes, which shows two unusual cytological features: pairing and synapsis of homologues during prophase I are restricted to the non-centromeric distal regions and extremely distal chiasma localization in metaphase I bivalents. In order to determine whether there is a relationship between both phenomena, we have used: (1) a spreading technique for following the ultrastructure of SC assembly and (2) immunofluorescence for SMC3 and SMC1α cohesin subunits, which mark the development of the axial element (a SC component); the histone γ-H2AX, which mostly labels the sites of double-strand breaks; and the recombinase RAD51. Spermatocytes showed conspicuous polarization of both the maturation of cohesin axes and the initiation of meiotic recombination events. Consequently, it is proposed that maturation of cohesin axes, which begins in very distal regions, could drive the latter loading of recombinases to such regions. This restricted distribution of recombination events along homologues would finally be responsible for the incomplete pairing and synapsis observed in all autosomes of the complement and hence for chiasma localization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. All authors contributed equally to this work.     

Thiol status in human sperm

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol-disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence-activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% +/- 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P less than 0.05). However, within the normozoospermic group, no correlation was found between thiol-labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between "swim-up" and "whole semen" preparations.     

Non-random segregation during anaphase II in an individual of Arcyptera tornosi (Bol.) (Orthoptera) heterozygous for three supernumerary heterochromatic segments

An individual of Arcyptera tornosi heterozygous for distal heterochromatic segments affecting M₆, S₁₀ and S₁₁ chromosomes has been analyzed during all the meiotic stages in order to establish the pattern of meiotic segregation in anaphase I and II. S-bivalents invariably show an equational separation during anaphase I and the anaphase II separation is non-random, both chromatids with heterochromatic segments often segregating to the same pole. Differences are significant if compared with the expected segregation. Some aspects of this particular chromosome behaviour are briefly discussed.     

Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.