Analysis of meiotic prophase I in live mouse spermatocytes

Events occurring during meiotic prophase I are critical for the successful production of haploid gametes. Many prophase I events are mediated by a meiosis-specific structure called the synaptonemal complex. To date, the limited knowledge we have about the dynamics of these prophase I events in mice comes from fixed, two-dimensional preparations of meiotic cells making it impossible to study the three-dimensional (3D) arrangement of meiotic chromosomes. The current study involves the development of an imaging system to view prophase I events in live mammalian spermatocytes by generating a transgenic mouse, Sycp3-Eyfp ( 21HC ), expressing a fluorescently tagged synaptonemal complex protein, SYCP3. Using this live imaging system, the 3D structural arrangement of chromosomes in the different prophase I substages has been characterized in live spermatocytes, and aspects of the 3D architecture of spermatocytes have been observed that would not be possible with existing techniques. Additionally, chromosome movement in prophase I spermatocytes and meiotic progression from pachynema to diplonema were observed following treatment with the phosphatase inhibitor, okadaic acid (OA), which accelerates the progression of cells through late prophase I. These studies demonstrate that the Sycp3-Eyfp ( 21HC ) live imaging system is a useful tool for the study of mammalian prophase I dynamics.     

Cytogenetic analysis of unfertilized human oocytes

Cytogenetic studies were carried out on 180 oocytes that appeared unfertilized after in-vitro fertilization. The majority of the 135 that were informative had grossly haploid second meiotic metaphases, two were grossly diploid, and five had a variety of different abnormalities. Twenty-one oocytes were abnormally fertilized and included prematurely condensed sperm chromosomes. The frequency of this phenomenon varied according to the stimulation protocol, those oocytes maturing longer in vivo showing less propensity to abnormal fertilizations. Thirteen per cent of the analysable haploid metaphases were hyperhaploid but none contained extra whole chromosomes. The extra components were a single chromatid (one case), or two single chromatids replacing a whole chromosome (four cases). The data suggest that the chromatids arose as a result of premature centromere division at meiosis I, and that this may be a major mechanism for trisomy formation rather than non-disjunction of whole bivalents at meiosis I, as generally believed.     

Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects

BACKGROUND: The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. METHODS: Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids, and spermatozoa containing condensed chromatin. RESULTS: The flow cytometric method differentiated between cases of 'Sertoli cell-only' syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained from testis histology findings. CONCLUSIONS: The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.     

Haplodiploidy in Pharyngodonid (Oxyuroidea: Nematoda) parasites of Testudo graeca

Gametogenesis is described in Tachygonetria conica, T. dentata, T. macrolaimus, T. numidica, T. longicollis longicollis, T. l. pusilla, T. l. setosa, Mehdiella uncinata and M. microstoma, parasites of Testudo graeca from Tunisia. All species reproduced by haplodiploidy (males developed from unfertilized eggs and were haploid and females developed from fertilized eggs and were diploid). Metaphase of oogonial divisions contained ten rod-shaped chromosomes and meiosis in females resulted in two polar bodies and an egg pronucleus. Embryos in eggs in utero contained either five or ten chromosomes depending presumably on whether or not they developed from fertilized ova. Metaphase of spermatogonial divisions contained five rod-shaped chromosomes but divisions just anterior to the seminal vesicle contained five short round chromosomes similar to meiotic chromosomes but consisting of only two chromatids. Males of haplodiploid species have no homologous chromosomes and it is suggested that meiosis during spermatogenesis, rather than being replaced by mitosis as previously suggested (Adamson, 1981), simply begins at the second meiotic division.     

Meiotin-1, a Meiosis-enriched Protein Present in Normal Leptotene Chromosomes and Lacking in Precociously Condensed Leptotene Chromosomes

During mitotic prophase, chromosomes progressively compact to their metaphase length. In contrast, meiotic chromosomes condense moderately until late in prophase I, then they condense more dramatically (coil) to their fully condensed state. Meiotin-1 is a meiosis-enriched, chromosomal protein. We propose that it delays coiling until after reciprocal genetic exchange. We have used immunoblotting and immunocytochemistry with normal lily cells undergoing meiosis to demonstrate that meiotin-1 is present during the early portions of prophase I, but diminishes at the time when meiotic chromosomes begin to coil. Additionally, we have examined lily meiotic nuclei undergoing the reversible phenomenon of precocious leptotene chromosome condensation (precocious coiling). The leptotene chromosomes that are precociously condensed lack meiotin-1 immunostaining. Furthermore nuclei returning to the normal state of moderate prophase I condensation acquire meiotin-1.     

From spermatocytes to sperm: meiotic behaviour of human male reciprocal translocations

BACKGROUND: Human male translocation carriers may present alterations in the meiotic process due to the presence of the translocated chromosomes. The aim of this work was to study the mechanisms that affect meiotic segregation in translocation carriers by analysing different stages of the meiotic process. METHODS: Meiotic studies using fluorescence in-situ hybridization on both spermatocytes and sperm nuclei were performed in two translocation carriers, t(11;17)(q13.1;p11.2) and t(10;14)(q24;q32). RESULTS: A ring configuration was the main type of quadrivalent found in metaphase I. Overall chiasma frequency was significantly decreased in the t(11;17) carrier. In the t(10;14) carrier, chiasma frequency within the interstitial region of chromosomes 10 and 14 was increased and the recombination pattern was modified. As expected from the frequencies of interstitial chiasmata found in metaphase I in the two subjects, the incidence of asymmetric dyads was sporadic in t(11;17) and very high in t(10;14). In both carriers, segregation frequencies observed at metaphase II were not different from the segregation data obtained in decondensed sperm nuclei. CONCLUSIONS: The concordance observed among results obtained in different spermatogenic stages indicates an absence of cellular selection based on chromosomal imbalances. Results obtained in the aneuploidy assay have not provided any evidence for an interchromosomal effect.     

Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation

In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs). We disrupted the mouse gene encoding TF protein Sycp1 to analyze the role of TFs in meiotic chromosome behavior and recombination. Sycp1(-/-) mice are infertile, but otherwise healthy. Sycp1(-/-) spermatocytes form normal AEs, which align homologously, but do not synapse. Most Sycp1(-/-) spermatocytes arrest in pachynema, whereas a small proportion reaches diplonema, or, exceptionally, metaphase I. In leptotene Sycp1(-/-) spermatocytes, gammaH2AX (indicative of DNA damage, including double-strand breaks) appears normal. In pachynema, Sycp1(-/-) spermatocytes display a number of discrete gammaH2AX domains along each chromosome, whereas gammaH2AX disappears from autosomes in wild-type spermatocytes. RAD51/DMC1, RPA, and MSH4 foci (which mark early and intermediate steps in pairing/recombination) appear in similar numbers as in wild type, but do not all disappear, and MLH1 and MLH3 foci (which mark late steps in crossing over) are not formed. Crossovers were rare in metaphase I of Sycp1(-/-) mice. We propose that SYCP1 has a coordinating role, and ensures formation of crossovers. Unexpectedly, Sycp1(-/-) spermatocytes did not form XY bodies.     

Premature separation of sister chromatids in human male meiosis

BACKGROUND: Meiotic disorders result in meiotic arrest and aneuploid spermatozoa,leading to male infertility, spontaneous abortions or affectedoffspring. We carried out meiotic studies in an infertile maleto detect meiotic nondisjunction mechanisms leading to aneuploidyin spermatogenesis. METHODS AND RESULTS: Meiotic studies were performed in testicular and semen samplesfrom a 38-year-old teratozoospermic male with normal somatickaryotype and a history of spontaneous abortions. We analysed262 spermatocytes (69 pachytene cells, 106 metaphases I (MI),87 metaphases II (MII)) by multiplex-fluorescence in situ hybridizationand 20 193 spermatozoa by multicolour-FISH with probes for chromosomes9, 10, 15, 21, X and Y. The results indicate high increase of21 and XY disomies, as well as diploidy in both spermatocytesat MII and spermatozoa (P < 0.0001). Achiasmate segregationof sex chromosomes was found in 3.4% of spermatocytes II, precededby early-dissociated XY bivalent at MI (41.5% of cells). Wealso detected premature separation of sister chromatids (PSSC)in 4.6% of MII. CONCLUSIONS: This individual presents high levels of numerical abnormalitiesin germ cells, caused by two different nondisjunction mechanismsduring meiosis I. To our knowledge, this work represents thefirst time that PSSC has been demonstrated in human male germcells.     

Meiotic behaviour of holocentric chromosomes: orientation and segregation of autosomes in Triatoma infestans (Heteroptera)

The meiotic behaviour of the holocentric chromosomes of the heteropteran species Triatoma infestans has been analysed by means of orcein staining and C-banding on squashed spermatocytes. We have focused our analysis on chromosome 3, which shows a large distal heterochromatic band at one of the ends of both homologues. At metaphase I,and independently of the chiasma position, two alternative orientations have been observed: either the hetero-chromatic or the euchromatic ends of both homologues are directed to opposite poles. At anaphase I, the kinetic activity is restricted to the same chromosome end (euchromatic or heterochromatic) of each homologue. The frequencies of these two alternatives are not random and differ significantly among the five individuals analysed. However, the euchromatic ends present kinetic activity at a higher frequency than the heterochromatic ends. At metaphase II, half-bivalents also show the kinetic activity restricted to either of the chromosome ends (euchromatic orheterochromatic). The frequencies of each alternative are inverted in anaphase II compared with those scored in anaphase I. Accordingly, those ends that present kinetic activity at anaphase I segregate reductionally during the first meiotic division and equationally during the second meiotic division. These results provide sound evidence on the meiotic behaviour of holocentric chromosomes, as regards the absence of chiasma terminalization and the modes of orientation and segregation. This revised version was published online in August 2006 with corrections to the Cover Date.