N. V. Dorogova

The role of Drosophila Merlin in spermatogenesis

BackgroundDrosophila Merlin, the homolog of the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and receptor endocytosis. Male flies carrying a Mer3 allele, a missense mutation (Met177→Ile) in the Merlin gene, are viable but sterile; however, the cause of sterility is unknown.ResultsTestis examination reveals that hemizygous Mer3 mutant males have small seminal vesicles that contain only a few immotile sperm. By cytological and electron microscopy analyses of the Mer3, Mer4 (Gln170→stop), and control testes at various stages of spermatogenesis, we show that Merlin mutations affect meiotic cytokinesis of spermatocytes, cyst polarization and nuclear shaping during spermatid elongation, and spermatid individualization. We also demonstrate that the lethality and sterility phenotype of the Mer4 mutant is rescued by the introduction of a wild-type Merlin gene. Immunostaining demonstrates that the Merlin protein is redistributed to the area associated with the microtubules of the central spindle in telophase and its staining is less in the region of the contractile ring during meiotic cytokinesis. At the onion stage, Merlin is concentrated in the Nebenkern of spermatids, and this mitochondrial localization is maintained throughout sperm formation. Also, Merlin exhibits punctate staining in the acrosomal region of mature sperm.ConclusionMerlin mutations affect spermatogenesis at multiple stages. The Merlin protein is dynamically redistributed during meiosis of spermatocytes and is concentrated in the Nebenkern of spermatids. Our results demonstrated for the first time the mitochondrial localization of Merlin and suggest that Merlin may play a role in mitochondria formation and function during spermatogenesis.

RADIAL SPINDLE AND THE PHENOTYPE OF THE MAIZE MEIOTIC MUTANT, DV

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.

The role of Drosophila hyperplastic discs gene in spermatogenesis

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [ Martin et al. (1977) Dev Biol 55, 213–232; Mansfield et al. (1994) Dev Biol 165, 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [ Dorogova et al. (2008) BMC Cell Biol 9, 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)‐binding protein C‐terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.

ABNORMALITIES OF SPINDLE AND CYTOKINE BEHAVIOR LEADING TO THE FORMATION OF MEIOTIC RESTITUTION NUCLEI IN INTERGENERIC CEREAL HYBRIDS

Mobile stages of meiosis have been analysed by visualizing the spindle in fertile cereal F1 hybrids. We describe four different mechanisms of the formation of restitution nuclei in meiotic division: (1) centripetal migration of telophase chromosome groups from the poles of a curved spindle at early telophase; (2) centripetal migration of the chromosome groups at late telophase when cell plate formation has failed; (3) preferable migration of univalents to one of the poles although spindle appearance is morphologically normal; and (4) in the absence of chromosome segregation where kinetochore fibers have failed to form.

P elements and the determinants of hybrid dysgenesis have different dynamics of propagation in Drosophila melanogaster populations

Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.

GAGA protein is essential for male germ cell development in Drosophila

The Drosophila Trithorax‐like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. In this study, we identify a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy, and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm. genesis 52:738–751, 2014.

A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The function of the gene gilgamesh (89B9‐12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh–GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh–GFP concentration in the nucleus co‐localizes with γ‐tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

Cellular aspects of gonadal atrophy in Drosophila P-M hybrid dysgenesis

Gonadal atrophy is the most typical and dramatic manifestation of intraspecific hybrid dysgenesis syndrome leading to sterility in Drosophila melanogaster dysgenic progeny. The P-M system of hybrid dysgenesis is primarily associated with germ cell degeneration during the early stages of Drosophila embryonic development at elevated temperatures. In the present study, we have defined the phase of germ cell death as beginning at the end of embryogenesis immediately following gonad formation. However, the temperature-dependent screening of germ cell developmental patterns in the dysgenic background showed that early germ cells are susceptible to the hybrid dysgenesis at any Drosophila life-cycle stage, including in the imago. Electron microscopy of germ cells after dysgenesis induction revealed significant changes in subcellular structure, especially mitochondria, prior to cellular breakdown. The mitochondrial pathology can promote the activation of cell death pathways in dysgenic germ cells, which leads to gonadal atrophy.

Structural organization and dynamics of the endoplasmic reticulum during spermatogenesis of Drosophila melanogaster: Studies using PDI-GFP chimera protein

An important property of the endoplasmic reticulum (ER) is its ability to change morphology and intracellular localization during the cell cycle and differentiation. Visualization of the ER membranes using the protein disulphide isomerase (PDI) GFP chimeric protein makes it possible to trace the dynamics of all these processes and expose transitional forms and intermediate configurations. In this article the results of the study of the ER morphology during spermatogenesis of D. melanogaster are presented. It was shown that ER membranes retain high level of GFP-fluorescence through all stages of spermatogenesis, so that revealing of the stage-specific features of the ER organization was possible. The ER network has distinctive reticular morphology during the interphase and early prophase. Right before the cell division, this morphology changes and ER forms a system of branchless filamentous membranes. In prometaphase, these membranes form concentric circles adjacent to the nuclear membrane; from metaphase to telophase, they lengthen along the axis of cell division and resemble a spindle. Later, in the next interphase, this configuration transforms to a reticular structure seen previously. At the beginning of spermatid differentiation, the ER encompasses nebenkern and nucleus and elongates adjacent to them. During the latest stages of spermatogenesis, the ER network dissociates into separate membranous granules that are eliminated from the cyst with the individualization complex. Possible mechanisms of the ER dynamics and reorganization are discussed.

Role of the porcupine gene in the development of the wing imaginal disk of Drosophila melanogaster

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.