Metaphase-specific cell death in meiotic spermatocytes in mice

Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ(-)+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.     

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.     

The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction in Drosophila melanogaster females

The ability of the rodent carcinogens 1,4-dioxane (DX) and thiourea (TU) to induce meiotic non-disjunction (ND) was assessed in 3- and 6-day-old Drosophila melanogaster females. The chemicals were administered orally and three 24 h and one 48 h broods were obtained after mating, to sample oocytes treated in increasingly earlier stages of development. The broods represent mainly mature oocytes (brood I), nearly mature oocytes (brood II), early oocytes (brood III) and very early oocytes (brood IV). The toxicity of DX increased with dose (1% (not toxic), 1.5, 2, 3, 3.5%) as well as a reduction in fecundity which was moderate. Induction of ND in mature oocytes was positive with 2, 3 and 3.5% concentrations and was not related to dose. In immature oocytes it was also positive though already at the lowest concentration tested (1%), suggesting a sensitivity higher than that of mature oocytes. TU at 0.10-10%, did not affect viability, but since fecundity was seriously impaired at high doses, ND was not assessed beyond the 1.5% concentration. TU also induced ND in mature and in immature oocytes; neither a threshold nor a dose effect was detected. The response of mature oocytes was lower than that of immature oocytes. TU induced increases of ND in the earliest cells tested in a more consistent fashion than DX. The data clearly show that both chemicals induced ND in mature oocytes and in the three subsets in which immature oocytes were fractionated. Though toxicity may play a significant unspecific role in the induction of chromosome malsegregation by DX and TU, the induction of ND at low doses, moderately toxic to the oocytes, suggests that the interaction with specific targets contributed to the results obtained.     

Parental origin and meiotic stage of non-disjunction in 139 cases of trisomy 21

The parental origin and the meiotic stage of non-disjunction have been determined in 139 Down syndrome patients with regular trisomy 21 and in their parents through the analysis of DNA polymorphism. The meiotic error is maternal in 91.60% cases and paternal in 8.39% of cases. Of the maternal cases, 72.41% were due to meiosis I errors (MMI) and 27.58% were due to meiosis II errors (MMII). Of the paternal cases, 45.45% were due to meiosis I (PMI) and 54.54% were due to meiosis II (PMII). The mean maternal ages were 31.6 +/- 5.3 (+/- SD) years in errors from MMI, 32.3 +/- 6.4 years in errors from MMII, 31.4 +/- 4.6 years in errors from PMI and 29.5 +/- 2.7 years in errors from PMII. No significant statistical differences were observed between maternal and paternal errors, further supporting the presence of a constant chromosome 21 non-disjunction error type.     

Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules] extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6° C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 m diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.     

The spatial relationship of the X and Y chromosomes during meiotic prophase in mouse spermatocytes

The ultrastructure of whole X-Y pairs has been reconstructed by serial sectioning and model building. Seven X-Y pairs were completely reconstructed and the lengths of the cores of the sex chromosomes were measured. These X-Y pairs corresponded to zygonema, early, middle and late pachynema. Special regions of the X-Y pair were reconstructed from thinner sections. — It has been shown that two cores exist in the sex pair during the cited stages, and that their lengths and morphology are rather constant in specific stages. The long core averages 8.9 in length and the short core is 3.5 long. Both cores have a common end region in which a synaptonemal complex is formed from zygonema up to midpachynema. This synaptonemal complex shortens progressively up to mid-pachynema and at late pachynema becomes obliterated. Each core has a free end touching the nuclear membrane. During mid-pachynema an anomalous synaptonemal complex is developed on most of the length of the long core. This complex is asymmetric and disappears at late pachynema. The meaning of the cores and the complexes are discussed, and the existence of a homologous region in the X-Y pair of the mouse is interpreted to be proved.     

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Background

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Results

In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.     

Radiation-induced meiotic autosomal non-disjunction in male mice The effects of low doses of fission neutrons and X-rays in meiosis I and II of a Robertsonian translocation heterozygote

Male mice, heterozygous for the Rb(11.13)4Bnr translocation, were irradiated for 14.5 min with either a dose of 15-rad fission neutrons or 60-rad X-rays. Animals of this karyotype are known to show high levels of spontaneous autosomal non-disjunction (20–30%) after anaphase I. The effects of the irradiation on this process were determined after 2 and 3 in air-dried preparations.The length of the period from the end of meiosis I till the end of meiosis II was assessed autoradiographically, with the aid of cells showing a labelled Y chromosome only and appeared to last less than 3 h. Inter-mouse variation with regard to the duration of the period “last premeiotic S-phase till diakinesis/ methaphase I” prevented a more accurate estimate.On the basis of this 3-h datum, the induced effects were studied at intervals of 2 and 3 h after the start of the irradiation. The influence of irradiation was assessed by scoring: (1) univalents in primary spermatocytes, (2) deletions, aneuploid chromosome counts and precocious centromere separation in secondary spermatocytes, and (3) chromatid gaps and breaks in both cell types. Both radiation types induced comparable levels of chromosomal damage. A neutron-X-rays RBE value for these parameters was calculated to be 5.4 for the MI stage and 3.3 for the MII stage. The significantly higher incidence of cells showing damage at MII than at diakinesis/MI is not believed to indicate a difference in radiation sensitivity, but is believed to be merely the consequence of the different chromosomal processes taking place during the irradiation-fixation time interval.     

Absence of satellite DNA synthesis during meiotic prophase in mouse and human spermatocytes

Mouse spermatocytes were labelled in situ with ³H-thymidine at successive stages of meiosis. Isolated mouse as well as human spermatocytes were similarly labelled under in vitro conditions. DNA synthesis was followed either by tracking radioactivities in Cs₂SO₄ gradients or by measuring reassociation kinetics. Mouse satellite DNA and the 3 satellites of human DNA are labelled during S-phase but not during pachytene. In the mouse genome, there is a preferential labelling of regions containing foldbacks (human spermatocytes were not analyzed in this respect). The absence of detectable pachytene synthesis in satellite DNA is consistent with genetic evidence on the absence of crossing-over in constitutive heterochromatin.     

Induction of meiotic disturbances in spermatocytes I by pheromones as an inhibiting mechanism of male reproductive function in house mice

The influence of pheromons on reproduction and other important physiological characteristics has been reported for many mammalian species. However, mechanisms of this action at the level of target cells still remain unclear. A study was made of the influence of non-identified pheromones from adult males and a female pheromone 2,5-dimethylpyrazine on germ cells of CBA inbred strain mice. Cytogenetic analysis shows a significant increase in such meiotic disturbances as multivalent associations and autsomal univalents 24 h after exposure to pheromonal cues. Results of in situ hybridization show that the level of c-fos and c-jun expression is significantly higher 3.5 h after exposure to pheromones of adult males. It is likely that destabilization of chromosomal apparatus in dividing meiotic cells forms the basis of some reproductive effects of murine pheromones. Possible mechanisms of pheromone influence on reproduction are discussed.     

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.     

The regular divisions of the spermatocytes as related to a meiotic lysine-rich protein fraction

Lepidopteran primary spermatocytes are bipotential leading first to regular (eupyrene) and later to irregular (apyrene) meiotic divisions. The kinetics of the lysine-rich proteins during this dichotomous meiosis was studied using the fluorescent dye sulfoflavine. Throughout the spermatogonial divisions, the chromatin fluoresces while the cytoplasm remains unstained. Reversely, during the meiotic prophase, the cytoplasm fluoresces strongly while the nuclei show only a few weakly fluorescing structures. From premetaphase to telophase the meiotic chromosomes fluoresce strongly again. But during this period, only in the eupyrene cells the cytoplasm remains strongly fluorescent; the fluorescence vanishs in the cytoplasm of the apyrene spermatocytes. Thus, the regular (eupyrene) meiotic divisions and the presence of a lysine-rich protein fraction in the cytoplasm of the dividing spermatocytes of Lepidoptera, are probably related.     

Direct estimation of the non-disjunction rate at first meiotic division in the human male

Summary Chromosomal analysis of 100 second metaphases from 19 men attending an infertility clinic for various reasons was carried out to estimate the rate of non-disjunction occurring at first meiotic division. Second metaphases were selected on the basis of the good spread of their chromosomes. Karyotypes were performed using the relative length of the chromosomes and the centromeric index. Among aneuploid cells, only those containing a hyperhaploid complement (24) were regarded as informative. Of the 100 MII cells, two were hyperhaploid. The frequency of aneuploid MII cells following non-disjunction at first meiotic division is compared to the rate of aneuploid spermatozoa observed after using fertilization of zona-free golden hamster eggs.     

Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: Evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro

Summary Dispersed testicular cells prepared from 14-d-old rats were cultured on type 1 collagen gels using a medium composed of a 1∶1 mixture of Ham’s F12 medium and Leibovitz’s L15 medium (F12-L15 medium) containing 10% (vol/vol) fetal bovine serum. The viability of the spermatogenic cells was facilitated by supplementing a rat adrenal extract into the medium. The effective substance(s) (the survival factor) was purified from acid extracts of adrenals by molecular sieve high performance liquid chromatography and identified as epinephrine and norepinephrine. Both epinephrine and norepinephrine promoted the survival of the spermatogenic cells with a half saturating dose of 10 ng/ml. The spermatogenic cells, which could be cultured for 2 wk on a collagen gel by supplementing with the survival factor (epinephrine or norepinephrine), were subjected to Giemsa staining and to DNA flow cytometry. The following results were obtained: a) The spermatogenic cells from 14-d-old rats did not contain spermiogenic cells (lc-cells). b) During a culture period of 2 to 7 d the ratio of meiotic prophase spermatocytes (4c-cells) to premeiotic cells (2c-cells) increased. On Day 7, more than 90% of the surviving cells were meiotic prophase spermatocytes. c) On Day 10, spermatids (lc-cells) appeared for the first time. The time of the first appearance of spermatids in the culture was consistent with that in vivo. These results suggest that both epinephrine and norepinephrine facilitated the viability of meiotic prophase spermatocytes and that a part of the meiotic prophase spermatocytes completed the meiotic divisions in the testicular cell culture.     

Bacterial translocation from the gastrointestinal tracts of mice receiving immunosuppressive chemotherapeutic agents

Specific pathogen-free (SPF) mice were treated with certain classes of immunosuppressive chemotherapeutic agents to determine if they would promote bacterial translocation from the gastrointestinal tract to the mesenteric lymph node, spleen, or liver. The antimetabolites methotrexate, 5-fluorouracil, and cytosine arabinoside were injected once intraperitoneally into SPF mice, and the mice were tested for bacterial translocation from the gastrointestinal tract. When total organs from the treated mice were compared with the total organs from the control mice, the alkylating agent cyclophosphamide promoted bacterial translocation when injected once intraperitoneally at doses of 100–400 mg/kg. Increasing the number of injections of cyclophosphamide did not increase the incidence of bacterial translocation. The steroid prednisone also promoted bacterial translocation after one intraperitoneal injection of 10–150 mg/kg. Prednisone and cyclophosphamide at various doses appeared to be more effective in promoting bacterial translocation from the gastrointestinal tract than the antimetabolites. The aerobic and facultatively anaerobic bacteria translocating to the various organs were identified asLactobacillus acidophilus, Escherichia coli, Klebsiella pneumoniae, Streptococcus faecalis, Staphylococcus aureus, andProteus mirabilis. Groups of SPF mice also were injected once intraperitoneally with the minimal dose of each chemotherapeutic drug that induced bacterial translocation, and then tested for immune responsiveness toE. coli vaccination. Each of the chemotherapeutic agents at the minimal doses promoting bacterial translocation also suppressed the serum antibody responses to antigens of indigenousE. coli. However, other toxic manifestations of these chemotherapeutic agents also may be involved in promoting bacterial translocation. The promotion of bacterial translocation from the gastrointestinal tract by these chemotherapeutic agents has important implications for the pathogenesis of infectious disease in patients receiving these drugs.     

An improved method for cytogenetic analysis of meiotic aneuploidy in rodent and frog spermatocytes

Methods are described for the attachment of isolated spermatocytes to glass slides and the subsequent hypotonic swelling and gradual fixation of the metaphase I and metaphase II cells. The methods minimize cell loss and cell disruption and meiotic metaphase chromosomes become spread within residual cytoplasm thus reducing artefactual chromosome loss. Metaphase II complements from mouse, rat and frog spermatocytes prepared by these procedures had relatively low frequencies of hypoploidy (0.5-1.6%). Bivalent loss was not detected in 916 metaphase I complements. Injection of 0.1 mg/kg demecolcine into mice increased the incidence of metaphase II hypoploidy 8-fold. The hypoploid and hyperploid frequencies here increased equally. The results suggest that the methods described may be useful for the analysis of mechanisms of meiotic aneuploidy including aneuploidy resulting from chromosome loss during meiosis I.