A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

Meiotic analysis of two human reciprocal X-autosome translocations

Two cases of human reciprocal X-autosome translocation, t(X;12) and t(X;2), are described in sterile males, along with meiotic findings. Each carrier had inherited the translocation from his mother. Both showed azoospermia and germ-cell maturation arrest at the primary spermatocyte level, with most cells being arrested at the pachytene stage. A few metaphase I (MI) divisions were found, with occasional metaphase II cells being seen in the t(X;2) carrier. MI air-dried preparations gave clear evidence of chain quadrivalent formation. In the t(X;2) heterozygote, the pairing characteristics of the quadrivalent at pachytene were also analyzed in electron microscopic spreads. Disturbance of pairing around the breakpoints characterized most quadrivalents, and there was evidence in about 20% of the cells that nonhomologous pairing had taken place between the translocated chromosomes and the normal chromosome 2. Comparisons are made with similar nonhomologous pairing configurations seen at pachytene in quadrivalents of male reciprocal X-autosome translocations of the mouse.     

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.     

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175 SEP1 arrest at pachytene during meiotic prophase

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175 ^SEP1 enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.     

Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins

The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase. Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing. Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes. These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene.     

Model of chromosome associations in Mus domesticus spermatocytes

Understanding the spatial organization of the chromosomes in meiotic nuclei is crucial to our knowledge of the genome's functional regulation, stability and evolution. This study examined the nuclear architecture of Mus domesticus 2n=40 pachytene spermatocytes, analyzing the associations among autosomal bivalents via their Centromere Telomere Complexes (CTC). The study developed a nuclear model in which each CTC was represented as a 3D computer object. The probability of a given combination of associations among CTC was estimated by simulating a random distribution of 19 indistinguishable CTC over n indistinguishable "cells" on the nuclear envelope. The estimated association frequencies resulting from this numerical approach were similar to those obtained by quantifying actual associations in pachytene spermatocyte spreads. The nuclear localization and associations of CTC through the meiotic prophase in well-preserved nuclei were also analyzed. We concluded that throughout the meiotic prophase: 1) the CTC of autosomal bivalents are not randomly distributed in the nuclear space; 2) the CTC associate amongst themselves, probably at random, over a small surface of the nuclear envelope, at the beginning of the meiotic prophase; 3) the initial aggregation of centromere regions occurring in lepto-zygotene likely resolves into several smaller aggregates according to patterns of preferential partitioning; 4) these smaller aggregates spread over the inner face of the nuclear envelope, remaining stable until advanced stages of the meiotic prophase or even until the first meiotic division.     

Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro

Chromosome breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 micrograms/ml of gossypol, while cytotoxic effects appeared at concentration of 20 micrograms/ml and were evident at 50 micrograms/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive test system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.     

Involvement in Meiotic Prophase of H1 Histone Kinase and p34cdc2 Homologues in Lily (Lilium longiflorum) Microsporocytes

We have taken advantage of the synchrony of meiotic prophase I in Lilium microsporocytes to investigate the presence and involvement in four stages of meiotic prophase I (leptotene, zygotene, pachytene, and diplotene) of the p34^cdc2 H1 histone kinase, a component of MPF and a key participant in division control in other eukaryotes. H1 kinase activity showed a peak pattern during meiotic prophase I with the highest kinase activity at pachytene. A monoclonal antibody directed against a highly conserved region of p34^cdc2 (termed the 'PSTAIR') recognized three major protein forms by immunoblotting. The highest level of the fastest-migrating form was observed at pachytene, coinciding with the highest activity of H1 kinase. Both the proteins recognized by the anti-PSTAIR antibody and H1 histone kinase activity were retained on beads conjugated with p13^suc1, a protein known to physically associate with p34^cdc2. These observations suggest that p34^cdc2 or protein(s) highly homologous to p34^cdc2 is a component of Lilium H1 histone kinase and plays a role in regulating meiotic prophase I.     

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.     

THE EFFECT OF X-RAYS ON CHROMOSOMES IN DIFFERENT STAGES OF MEIOSIS

1. Pollen mother cells exposed to low dosages of x-rays at various stages show different frequencies of chromosome abnormalities in the first meiotic anaphase. 2. Maximum frequencies of abnormalities were obtained in buds irradiated in the pachytene stage of the meiotic prophase and in the preceding mitosis. 3. These results are taken to indicate that the x-ray-sensitive portions of the chromonemata are closely approximated in pairs in pachytene and in the early mitotic prophase. The significance of this in relation to non-homologous pairing is indicated. 4. From the nature of the chromosome configurations observed it is concluded that chromonemata are two-parted when they synapse and that a chromonematic division occurs between pachytene and anaphase and during the mitotic prophase. 5. The frequencies of abnormalities show a linear relationship to dosage. 6. The diameter of the sensitive volume of the chromonema is calculated and found to approximate the diameter of some known protein molecules. 7. The linkage mechanism is found to make up about 90 per cent of the total sensitive volume which corresponds with the approximate reduction in length of the chromonema from pachytene to anaphase. 8. The relation of these sensitive volumes to the gene is discussed.     

Spermatogenesis in XY, XYSxra and XOSxra mice: a quantitative analysis of spermatogenesis throughout puberty

Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one-half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.     

Genetic and cytological characterization of the recombination protein RAD-51 in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis>

We investigated the role of Caenorhabditis elegans rad-51 during meiotic prophase. We showed that rad-51 mutant worms are viable, have no defects in meiotic homology recognition and synapsis but exhibit abnormal chromosomal morphology and univalent formation at diakinesis. During meiosis RAD-51 becomes localized to distinct foci in nuclei of the transition zone of the gonad and is most abundant in nuclei at late zygotene/early pachytene. Foci then gradually disappear from chromosomes and no foci are observed in late pachytene. RAD-51 localization requires the recombination genes spo-11 and mre-11 as well as chk-2, which is necessary for homology recognition and presynaptic alignment. Mutational analysis with synapsis- and recombination-defective strains, as well as the analysis of strains bearing heterozygous translocation chromosomes, suggests that presynaptic alignment may be required for RAD-51 focus formation, whereas homologous synaptonemal complex formation is required to remove RAD-51 foci.     

Analysis of highly expressed genes in the late zygotene to pachytene stages of meiotic prophase I in david lily

Plant meiotic prophase I is a complex process involving the late zygotene and pachytene stages, crucial for both completing synapsis and recombination. Using David lily (Lilium davidii var. Willmottiae) as research material, we performed suppressive subtractive hybridization to construct expessed sequence tag (EST) library of anthers at various stages of development by the pollen mother cells. From this library, we identified 34 genes with significantly enhanced expression during the late zygotene to pachytene stages. The cDNA fragment sequences were compared with data in GenBank by BLASTN and BLASTX, and 18 unique ESTs were shown to exhibit significant homology to the data in GenBank. They were classified into eight different groups: metabolism, protein modification, signal transduction, etc. Through the study of classification and functions of these highly expressed genes during the late zygotene to pachytene stages, we obtained much information about the complex biological progress of meiotic prophase I, especially during chromosome synapsis and recombination.     

A pachytene analysis of two male-fertile paracentric inversions in chromosome 1 of the mouse and in the male-sterile double heterozygote

Meiotic studies have been made at pachytene on two paracentric inversions in chromosome 1 of the mouse. Surface-spread preparations of primary spermatocytes have been analysed at the light microscope level in males heterozygous for the inversions In(1)1Rk and In(1)12Rk and in the double heterozygote In(1)1RK/In(1)12Rk. In singly heterozygous form, neither inversion produces any serious effect on male fertility. In the double heterozygote, spermatogenesis is arrested in the majority of cells at the spermatocyte stage and males are rendered totally sterile by azoospermia. In the double heterozygote, a complex loop, indicating the inversion bivalent, is found in 90% of pachytene cells analysed. In the In(1)1Rk/+ heterozygote, a looped bivalent was seen in 47 per cent of pachytene cells but in In(1)12Rk/+ no cells containing loops could be found. -80% of pachytene spermatocytes from the In(1)1Rk/In (1)12Rk double heterozygote showed apposition of the inversion bivalent to the sex bivalent. Such an association was rarely seen in pachytene cells of either of the fertile single heterozygotes. Spermatogenic failure in the double heterozygote may be related to interference, by the inversion bivalent, with X chromosome inactivation at meiotic prophase.     

A quantitative analysis of mitochondria during fetal mouse oogenesis

A quantitative analysis of mitochondrial populations during the meiotic prophase of mouse oogenesis was carried out. The mean absolute area occupied by mitochondria and the mean number of mitochondria per cell increases in a linear fashion from pachytene through dictyate. The mean area occupied by mitochondria increases at pachytene and thereafter. Both small and large aggregations of mitochondria are seen, particularly during the later stages of prophase. Vacuolated mitochondria are present from preleptotene through pachytene. Mitochondria show major dynamic changes throughout fetal mouse oogenesis, which may suggest significant functional activities yet to be elucidated.