Postmeiotic Nuclei Research Articles

Offspring analysis using two cleaved amplified polymorphic sequence (CAPS) markers reveals amphithallism in the edible mushroom Agaricus sinodeliciosus

ABSTRACTAgaricus sinodeliciosus is an edible wild mushroom known in northwest China. It belongs to Agaricus section Bivelares that includes several popular cultivated species, such as A. bisporus, the button mushroom. The life cycle of the latter species has been described as amphithallic because both homokaryotic (n) and heterokaryotic (n+n) spores are produced that lead to heterothallic and pseudohomothallic life cycles, respectively. The type of life cycle can impact population structures and breeding strategies. The main objective of this study was to identify the different categories of spores produced by A. sinodeliciosus. Using either a morphological approach based on the number of sterigmata per basidium or a genetic approach based on the genotypes of the progeny at two loci, the proportion of heterokaryotic spores was estimated at 6% and 15%, respectively. Two codominant markers were chosen from the mitochondrial intermediate peptidase gene (MIP) and the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. Genotypic analysis and mating tests confirmed that in A. sinodeliciosus, MIP is centromere-linked and tightly linked to the mating type locus as in A. bisporus and A. subrufescens. We conclude that A. sinodeliciosus has a unifactorial system of sexual incompatibility and an amphithallic life cycle that is predominantly heterothallic, and that its pseudohomothallism follows a nonrandom model with nonsister postmeiotic nuclei paired in the same spore, which give rise to a potentially fertile heterokaryon. This method of using two informative markers is reliable not only in selecting the homokaryotic offspring but also in classifying the homokaryons in two breeding stocks according to their mating type alleles.

The SpoMBe pathway drives membrane bending necessary for cytokinesis and spore formation in yeast meiosis

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membrane-bending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPI-anchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.

Involvement of a 25 kDa Tetrahymena Ca2+-binding Protein in Pronuclear Exchange

The Tetrahymena Ca2+-binding protein of 25 kDa (TCBP-25) is a calmodulin family protein containing four EF-hand type calcium-binding domains. TCBP-25 is localized in the whole cell cortex and around both the migratory and stationary pronuclei at the pronuclear exchange stage during conjugation. TCBP-25 is expected to play an important role in conjugation, though its function during sexual reproduction has not been elucidated. According to the localization of this protein and its timing, three possible roles of TCBP-25 are proposed. TCBP-25 may play a role in 1) differentiating the two functional pronuclei from the degenerative post-meiotic nuclei, 2) the process of pronuclear exchange and 3) pronuclear fusion. To test these hypotheses, the localization of TCBP-25 in conjugation mutants (cnj10, cnj7 and bcd2) was examined. The results ruled out the first and the third hypotheses and suggested that TCBP-25 may play a role in pronuclear exchange. In the next step we succeeded in reducing expression of the TCBP-25 gene using the antisense ribosome system, and we analyzed the phenotype of the transformants. The knock down of TCBP-25 function also suggests that TCBP-25 plays a role in the pronuclear exchange and in the maintenance of cell shape.

Intraplant Variation in Nuclear DNA Content in Laminaria saccharina and Alaria esculenta (Phaeophyceae)

Intraplant variation in nuclear DNA content was quantified in DAPI-stained cells of Laminaria saccharina and Alaria esculenta. In both species, meristoderm, cortical and medullary cells were examined from vegetative fronds. In addition, DNA was quantified during sporogenesis, in free-swimming zoospores and gametophytes of A. esculenta. Zoospores and gametophytes had 1C (= N) levels of nuclear DNA in all cells, but vegetative cells of sporophytes varied from 2C to 16C. In A. esculenta nuclear DNA was significantly correlated with cell length, total cell volume and nuclear volume, whereas in Laminaria saccharina these correlations were not apparent. During sporogenesis of Alaria esculenta, maximal nuclear DNA levels of 8C were present in post-meiotic nuclei. During four subsequent mitotic divisions (to form 64 zoospores per sporangium at the 1C level) only one cycle of DNA synthesis occurred. These results show that mitosis can be independent of DNA synthesis in Laminariales, and that endopolyploidy is a generalized feature of multicellular plants.

Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum.

Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.

Prospore membrane formation linked to the leading edge protein (LEP) coat assembly.

In yeast, the differentiation process at the end of meiosis generates four daughter cells inside the boundaries of the mother cell. A meiosis-specific plaque (MP) at the spindle pole bodies (SPBs) serves as the starting site for the formation of the prospore membranes (PSMs) that are destined to encapsulate the post-meiotic nuclei. Here we report the identification of Ady3p and Ssp1p, which are functional components of the leading edge protein (LEP) coat, that covers the ring-shaped opening of the PSMs. Ssp1p is required for the assembly of the LEP coat, which consists of at least three proteins (Ssp1p, Ady3p and Don1p). The assembly of the LEP coat starts with the formation of cytosolic precursors, which then bind in an Ady3p-dependent manner to the SPBs. Subsequent processes at the SPBs leading to functional LEP coats require Ssp1p and the MP components. During growth of the PSMs, the LEP coat functions in formation of the cup-shaped membrane structure that is indispensable for the regulated cellularization of the cytoplasm around the post-meiotic nuclei.

Nuclear phenomena during conjugation of Heliophrya erhardi (Ciliata, Suctoria). II. The orientation of postmeiotic and metagamic division spindles is organized by the cytoskeleton

Summary Whereas the spindles of the meiotic divisions appear in a random orientation within the co-conjugants of Heliophrya erhardi, the spindles of the postmeiotic and metagamic nuclei show a distinct fixed position within the conjugational bridge and in the cytoplasm of each partner’s cell body. In postmeiotic divisions, the spindle orientation results in a different functional determination of the division products. For their later fate, mostly a concentration gradient of “cytoplasmic factors” was postulated [29]. But above all, the question arises, what is the mechanistic basis for the spindle orientation? We followed the sequential steps of conjugation by time lapse microcinematography and by EM-observation. The result of the maturation divisions are 12 to 24 haploid nuclei [11] from which only one nucleus reaching the conjugational bridge survives and starts the postmeiotic division. The position of this gonal nucleus is fixed on the left marginal side of the bridge by a dense microfilament network surrounding the nucleus in a cage-like manner and anchoring it to the epiplasmic septum separating the co-conjugants. During the following division, the spindle extends parallel to the septum fixed by cytoskeletal elements during anaphasic spindle elongation. Additionally, at metaphase, a basal body appearing at the spindle pole proximal to the pellicle nucleates microtubules (MTs) which penetrate the microfilamentous cage and contact also the nuclear envelope. The fixation of the spindle axis parallel to the separating septum by cytoskeletal elements guarantees the positioning of one pronucleus as the future stationary nucleus on the left side, and the other — the presumptive migratory nucleus — on the right side of the conjugational bridge. After exchange of the migratory nuclei and subsequent fusion with formation of the diploid synkaryon, one metagamic division follows immediately in each partner cell. From meta- to telophase the spindles exhibit in most cases a strict central-peripheral orientation, at which the anterior daughter nucleus (oriented to the cell’s center) becomes the macronucleus anlage, while the posterior nucleus condenses to the micronucleus. Interestingly, this spindle orientation is held in position by aster-like organized microtubules radiating out to a microfilamentous cap situated at the anterior spindle pole. As an organizing center for this cytoskeletal structure again a basal body is responsible, which is localized between nuclear envelope and cap region from meta- to telophase. It is evident, that in both divisions, the orientation of the spindle axis and consequently the fate of the division products depends completely on the existence of a cytoskeletal organizing structure, which is comparable to the asters of mitotic spindles in some higher eukaryotes.

Nuclear and cortical regulation in doublets of Paramecium: I. Interactions between macronucleus and contractile vacuoles and their implication on the frequency of autogamy

Summary The macronucleus of ciliates is a huge “bag” of DNA whose mode of segregation during cell division is still not understood (no chromosome condensation, no spindle, no centrosomes or functional equivalents). We unexpectedly obtained some new hints on this process through the analysis of doublets of Paramecium . We had previously noted [32] that autogamy occurred at a seemingly higher frequency in regulating lines. In the present work, we studied this process in P. tetraurelia and in P. undecaurelia which has a long, more convenient, interautogamous interval. We confirmed a highly significant decrease in duration of the interautogamous interval in both species and tried to determine its causes. The process of regulation which concerns the return of doublet state to the singlet one was previously described in part [24. 61, 35]. We observed its course through successive vegetative and sexual cycles. We first described precisely the contractile vacuole (CV) systems, and their number, extension and relations with the nuclear division events, in normal cells and in tam mutant cells of P. tetraurelia which are known to show abnormal nuclear division [6, 58]. We deduced that cortical and nuclear events are correlated during division by means of the contractile vacuole system (immunocytology and TEM observations). In regulating doublets, the enlarged cell volume leads to an increase in the number of CVs on two meridians M1, M2 and to the changes of their positioning and extension. CVM2 disappears and the number of CVs decreases when the angle between both oral apparatuses decreases from 180° towards 90°. Our results strongly suggest that abnormal number, size and position of macronuclei in doublets are related to number, spreading and positioning of the CVs. During division of doublets, the macronuclei appear to be “quartered” between both dorsal surfaces bearing the CVMs, and this leads to gross asymmetries in the distribution of the macronuclear DNA to the two daughter doublets [8]. Abnormal numbers of micronuclei and macronuclear anlagen are observed along with the evolution of the regulation process and, in turn, appear to lead to numerous anomalies at autogamy, a process which also requires correct intracellular positioning of meiotic and postmeiotic nuclei. From such observations, we deduced that contractile vacuoles, through their microtubular rootlets, exert a major function in ensuring proper macronuclear segregation during amitosis, such as the role played by “atractophores” in other protists during extranuclear pleuromitosis [57].

α-Tubulin and F-actin distribution during microsporogenesis in a 2n pollen producer of Solanum

Spatial and temporal changes in configurations of microtubules (MTs) and microfilaments (MFs) were determined during microsporogenesis, using -tubulin immunolocalization and rhodamine-phalloidin staining of F-actin, in a 2n pollen producer of Solanum, to assess the anomalies in meiotic spindles and cytokinesis and their relationships to 2n pollen formation. InSolanum, MTs and MFs generally showed patterns of localization similar to those described in other dicotyledons with simultaneous cytokinesis. However, deviations in spatial configurations of both MTs and MFs that are related to 2n pollen were observed in meiosis II and in cytokinesis. MTs and MFs localized in spindles in parallel orientation at meiosis II. In contrast, in the majority of normal meiocytes, the two spindles were seen to be perpendicular to each other. The parallel spindles altered the position of the postmeiotic nuclei, causing a uniplanar instead of a tetrahedral arrangement. Subsequently, the formation of regular radial MT systems was suppressed and only two MT arrays, which lay parallel on one plane, formed between the nuclei at the end of meiosis. A single cell plate formed across the two MT arrays, giving rise to a dyad containing 2n microspores.Key words: cytokinesis, microfilaments, microtubules, spindle, unreduced pollen.

Localization of the Mating Type Gene in Agaricus bisporus

The cultivated mushroom Agaricus bisporus is secondarily homothallic. Most basidia produce two basidiospores, each of which receives two of the four postmeiotic nuclei. Usually, the two packaged nuclei carry compatible mating types. Previous studies suggested that there may be only a single mating type locus in A. bisporus. In this study, we determined whether the mating type segregated as a single Mendelian determinant in a cross marked with 64 segregating molecular markers. To score mating types, each of the 52 homokaryotic offspring from this cross was paired with each of the two progenitor homokaryons. Compatible matings were identified by the formation of genetically stable heterokaryons which were verified by assay of restriction fragment length polymorphisms (RFLPs). Data for screening mycelial interactions on petri plates as well as fruit body formation were compared with the RFLP results. Mating types of 43 of the 52 homokaryotic offspring were determined on the basis of RFLP analysis. Our results indicate (i) there is a segregating mating type gene in A. bisporus, (ii) this mating type gene is on the largest linkage group (chromosome I), (iii) mycelial interactions on petri plates were associated with heterokaryon formation under selected conditions, (iv) fruit body formation was dependent upon the mating type gene, and (v) compatible mating types may not always be sufficient for fruiting.

Discrimination of cytoskeletal elements of Paramecium by heterologous antisera: A preliminary investigation on the presence of intermediate filament-related protein

The present study of the cytoskeleton of Paramecium employed a number of heterologous monoclonal antisera raised against intermediate filament proteins of higher organisms, and also an antiserum against a 49 K protein from Tetrahymena. Anti-49 K, and also monoclonal anti-bovine cytokeratin K 8.13 which has a broad cross-reaction spectrum for epithelial intermediate filaments, differentiated the microtubular systems of Paramecium into two categories. Those closely associated with surface membranes, such as basal bodies, contractile vacuole pores and the cytospindle, and the gullet also, were labeled by these antibodies. Others internal to the epiplasm are not labeled, such as transverse and postciliary microtubular bands associating with basal bodies, contractile vacuole canals, cytopharyngeal ribbons and postoral fibers. None of the heterologous anti-tubulins tested in this and previous studies generated such a pattern of discrimination of the microtubular systems of Paramecium, indicating that the discrimination by anti-49 K and by anti-cytokeratin K 8.13 was not based on the molecular composition (α- or β-tubulin) of the microtubular systems, their state of tyrosination, or acetylation. These two antibodies thus reveal a determinant that is associated exclusively with certain membrane-bound microtubular systems of Paramecium. This determinant appears to participate in the development of nascent structures like the cytospindle and the oral apparatus, since it co-distributed with these structures in their different stages of development. In addition, two monoclonal antibodies were superimposable specifically on certain cytoskeletal elements also residing close to surface membranes: anti-pig vimentin V 9 labeled the epiplasm underlying the inner alveolar membrane, while anti-human cytokeratin K 4.62 labeled the outer lattice lining the cortical ridges. On the other hand, anti-49 K labeled the micronucleus and its derivatives in all stages of sexual nuclear reorganization in Paramecium, unlike the specific labeling of postmeiotic and zygotic nuclei in Tetrahymena. The presence of intermediate filament-related protein in Paramecium is discussed.

Laccaria fraterna, a common ectomycorrhizal fungus with mono- and bi-sporic basidia and multinucleate spores: comparison with the quadristerigmate, binucleate spored L. laccata and the hypogeous relative Hydnangium carneum

Examination of Laccaria collections from Western Australia revealed that the most common species L. fraterna had basidia which were monosporic, bisporic or very occasionally trisporic, in contrast to L. laccata which was quadrisporic. For L. fraterna, all post-meiotic nuclei migrated into the spores leaving basidia anucleate. Spores of monosterigmate basidia had four nuclei, and bisterigmate basidia usually had two. Post-meiotic mitosis occurred after spores were delimited and prior to morphological maturation. Mature spores had up to eight nuclei. That most spores had compatible mating-type nuclei was indicated by most single germinated spores having clamped mycelium with dikaryotic cells. In Hydnangium carneum, a hypogeous relative of Laccaria, post-meiotic mitosis occurred in the basidium and two nuclei migrated into each of the four spores. Migration patterns indicated that some spores had compatible mating-type nuclei, as in L. fraterna. Allocation of compatible mating types to spores partly explains why L. fraterna ‘weed-like’ behaviour and is a pioneer in plantations of eucalypts on former farmland or rehabilitated mine sites. Mycorrhizas of these fungi have been synthesized aseptically with several species of Eucalyptus.

Inheritance of restriction fragment length polymorphisms in Agaricus brunnescens.

The cultivated mushroom, Agaricus brunnescens, is secondarily homothallic; most basidia produce only two basidiospores, each of which receives two of the four post meiotic nuclei. The segregation of restriction fragment length polymorphisms (RFLPs) detected by four plasmid probes carrying single-copy nuclear DNA of Agaricus was followed in seven parental strains including commercial, wild-collected, and artificially synthesized heterokaryons. Of a total of 367 single-spore progeny examined, 351 (95.6%) were heteroallelic at all RFLP loci heteroallelic in the respective parents. Of the 16 segregant isolates, ten (2.7% of the total) were homoallelic at all segregating loci assayed, suggesting that these isolates were most probably derived from rare spores that had received only a single postmeiotic nucleus. Some of these ten isolates had recombinant genotypes. Only five isolates (1.4% of the total) showed homoallelism at one of the loci heteroallelic in the parent, while remaining heteroallelic at other loci. These five genotypes suggest that a crossover had occurred between a marker locus and its respective centromere. Taken together, the results suggest that meiosis in A. brunnescens is accompanied by low levels of recombination and that nonsister nuclei are preferentially incorporated into basidiospores after meiosis II.

Evolutionary Aspects of Ascospore and Conidium Ontogeny in Scoleconectria cucurbitula

Morphological changes and nuclear behavior during ascospore ontogeny in a collection of Scoleconectria cucurbitula with two-spored asci were characterized, and asexual states observed in artificial culture. Following meiosis, two single-celled, uninucleate ascospores were formed within the developing ascus; the other two post-meiotic nuclei did not take part in ascospore development. Ascospores progressed through two-celled, four-celled, and multi-celled stages, finally becoming highly elongate and transversely septate, with numerous uninucleate cells. While within the ascus, ascospore cells differentiated into phialides and produced numerous conidia. Synanamorphs formed in culture included a phialidic hyphomycetous state, a pycnidial state, and a state producing spores within hyphal cells by a process resembling free cell formation. It is suggested that intra-ascal germination obviates the need for greater numbers of ascospores, and that the two-spored form of S. cucurbitula likely arose from the four-spored form. Intra-ascal germination appears correlated with loss of ascus rings and possibly with loss of forcible spore discharge in certain Nectria species. The possible role of heterochrony in the evolution of phialidic ascospore germination in S. cucurbitula and allied fungi is discussed.

Evolutionary Aspects of Ascospore and Conidium Ontogeny in Scoleconectria Cucurbitula

Morphological changes and nuclear behavior during ascospore ontogeny in a collection of Scoleconectria cucurbitula with two-spored asci were characterized, and asexual states observed in artificial culture. Following meiosis, two single-celled, uninucleate ascospores were formed within the developing ascus; the other two post-meiotic nuclei did not take part in ascospore development. Ascospores progressed through two-celled, four-celled, and multi-celled stages, finally becoming highly elongate and transversely septate, with numerous uninucleate cells. While within the ascus, ascospore cells differentiated into phialides and produced numerous conidia. Synanamorphs formed in culture included a phialidic hyphomycetous state, a pycnidial state, and a state producing spores within hyphal cells by a process resembling free cell formation. It is suggested that intra-ascal germination obviates the need for greater numbers of ascospores, and that the two-spored form of S. cucurbitula likely arose from the four-spored form. Intra-ascal germination appears correlated with loss of ascus rings and possibly with loss of forcible spore discharge in certain Nectria species. The possible role of heterochrony in the evolution of phialidic ascospore germination in S. cucurbitula and allied fungi is discussed.

The role of the micronucleus in stomatogenesis in sexual reproduction of Paramecium tetraurelia: laser microbeam irradiation of the micronucleus.

Fifteen amicronucleate cell lines and 22 cell lines with defective micronuclei were obtained following selective laser microbeam irradiation of the micronucleus. The amicronucleate cell lines showed reduced growth rate and formed abnormal oral apparatuses in asexual reproduction, and failed to produce any oral apparatus in autogamy, in agreement with previous observations on amicronucleate cells obtained by micropipetting. The 22 cell lines with defective micronucleus exhibited various abnormalities of the oral apparatus newly formed during autogamy. These abnormalities included the arrest of membranelle assembly, reduction in the length of the buccal cavity and oral membranelles, disruption of the organization of the membranelles, quadrulation of the dorsal peniculus, and failure of addition of membranellar basal body rows. Hence the micronucleus plays multiple roles in sexual stomatogenesis, deciding early steps of oral membranelle assembly and affecting their subsequent patterning. Our results agree with the notion that the micronucleus acts during a critical period between the second meiotic division and up to the formation of the zygotic nucleus to control the early stage of oral membranelle assembly. Laser microbeam irradiation might have created recessive mutations and/or chromosomal aberrations, which were expressed during this critical period with the formation of abnormal postmeiotic nuclei.

Electron microscopic studies on a unique cytokinetic structure in tetrasporocytes of the red algaHarveyella sp. (Cryptonemiales, Choreocolaceae)

A unique type of cytokinesis is described in tetrasporocytes of the alloparasitic red alga,Harveyella sp. Cytokinesis takes place immediately after four post-meiotic nuclei are formed and may result from coalescence of endoplasmic reticulum cisternae. As a result of this cisternal fusion, a double-membraned cleavage channel is formed. This channel lacks wall material and is usually oriented tetrahedrally. Membranes of the peripheral portions of the cleavage channel fuse with the existing tetrasporocyte plasmalemma, delimiting four tetraspores. Subsequently, wall material is secreted within the preformed cleavage channels to form a continuous tetraspore wall between adjacent tetraspores. Wall secretion usually occurs in a centrifugal direction, beginning at the juncture of the cleavage channels, but it also may be random or centripetal. Dictyosome activity is absent during the first wall secretion stage but contributes to secondary wall deposition.

Auxotrophic markers and the cytogenetics of Filobasidiella neoformans.

In the sexual phase of Cryptococcus neoformans four post-meiotic nuclei give rise to the nuclei of four long chains of basidiospores. If there is fixed relationship between one post-meiotic nucleus and one chain the result should be genetic homogeneity within single chains; random composition of chains would represent the opposite case. We tested for homogeneity in single chains from crosses of niacin-auxotrophs to the wild type by dissecting single chains of basidiospores from mature fruiting bodies. Out of 14 chains, 11 were homogeneous; the probability of obtaining this result by random processes was less than 0.01. These results suggest that mitotic products of a particular post-meiotic nucleus may be channeled into a particular basidiospore chain.

Fine structure of shiitake, Lentinus edodes. VI. Cytoplasmic microtubules in relation to nuclear movement

In the course of electron microscopic observations on basidiosporogenesis of Lentinus edodes (Berk.) Sing., the cytoplasmic microtubules, which are about 24 nm in diameter and associate closely with the postmeiotic nuclei, were observed to be common during movement of nuclei from a basidium into the respective basidiospores through sterigmata. These microtubules orient striately from the spindle-pole body (SPB), situated at the leading portion of a migrating nucleus, into the cytoplasm of a nearly mature basidiospore and appear to precede the migrating nucleus. The association of microtubules radiating from the SPB with the interior of a migrating nucleus was particularly indicated as being a characteristic event observed during nuclear movement of L. edodes. After completion of nuclear movement, these microtubules disappear completely. It is suggested that the microtubules associated with SPB may play an essential role in nuclear movement from the basidium to the basidiospore.

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation

Variability in DNA content to testis cells and sperm from F 1 hybrids between the laboratory mouse ( M. muscullus) and the tobacco mouse ( M. poschiavinus), has been determined by flow cytometry (FMC). The F 1 hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F 1 hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f 1 hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F 1 hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.