Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis.

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Cytological Monitoring of Meiotic Crossovers in Spermatocytes and Oocytes.

Crossing-over between homologous chromosomes is essential for accurate chromosome segregation at anaphase-I of meiosis. Defective crossing-over is associated with infertility, pregnancy miscarriage, and congenital disease. This chapter presents optimized protocols for the analysis of meiotic crossovers at the cytological level in spermatocytes and oocytes from mouse. The first approach employs immunocytology to detect MLH1, a DNA mismatch-repair protein that specifically marks crossover sites in the pachytene stage of meiotic prophase-I. These immunocytological methods have general utility for the analysis of other recombination steps, such as initiation and DNA strand exchange. The second approach visualizes chiasmata, the points of physical exchange between homologous chromosomes that are present during the diakinesis and metaphase-I stages. Both approaches are readily adaptable to the analysis of crossing over in other vertebrate species.

Cytological features of spermatogenesis in Opsariichthys bidens (Teleostei, Cyprinidae).

Spermatogenesis is important for male fertility, but has not been well-studied in Opsariichthys bidens, an economically important freshwater fish in China. In this study, there was investigation of the cytological features of spermatogenesis in O. bidens using light microscopy, transmission electron microscopy, and immunofluorescence detection of microtubules. O. bidens has tubular testis. Spermatogenesis in O. bidens is of the cystic type, in which the spermatogenic cells develop into spermatozoa in cysts. There was asynchronous development of primary spermatocytes within a single cyst. Spermiogenesis was classified as Type I, which develops into a Type I aquasperm with an oval nucleus, a small and simple midpiece, a flagellum and no acrosome. There was a nuage in spermatogonia, spermatocytes, and spermatids in different developmental stages of spermatids which may have important functions in fish spermatogenesis. Furthermore, microtubule dynamics may be involved in spermatid reshaping, material transport, and polar distribution of organelles during spermiogenesis.

Role of GPER-Mediated Signaling in Testicular Functions and Tumorigenesis.

Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor family, GPR30, which binds also with high affinity to estradiol and is responsible, in part, for the rapid non-genomic actions of estrogens. GPR30, renamed as GPER, was detected in several tissues including germ cells (spermatogonia, spermatocytes, spermatids) and somatic cells (Sertoli and Leydig cells). In our previous review published in 2014, we summarized studies that evidenced a role of GPER signaling in mediating estrogen action during spermatogenesis and testis development. In addition, we evidenced that GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth; however, the effects on cell survival and proliferation depend on specific cell type. In this review, we update the knowledge obtained in the last years on GPER roles in regulating physiological functions of testicular cells and its involvement in neoplastic transformation of both germ and somatic cells. In particular, we will focus our attention on crosstalk among GPER signaling, classical estrogen receptors and other nuclear receptors involved in testis physiology regulation.

LncRNA-Gm2044 is transcriptionally activated by A-MYB and regulates Sycp1 expression as a miR-335-3p sponge in mouse spermatocyte-derived GC-2spd(ts) cells.

Long noncoding RNAs (lncRNAs) have been shown to execute key roles in spermatogenesis. However, little is known about how lncRNAs gene expression is itself regulated in the germ cells of testis. We previously demonstrated that high expression of lncRNA-Gm2044 exists in spermatocytes and can regulate male germ cell proliferation. Here, the transcriptional regulation of lnRNA-Gm2044 expression in spermatocytes and the downstream signaling were further explored. A bioinformatics assessment predicted two potential binding-sites for the spermatocyte-specific transcription factor A-MYB in the promoter region of lncRNA-Gm2044. Our results proved that the transcription factor A-MYB promotes the expression of lncRNA-Gm2044 in mouse spermatocyte-derived GC-2spd(ts) cells. ChIP and luciferase assays verified that A-MYB mainly binds to the distal promoter region (-819 bp relative to the transcription start site) of lncRNA-Gm2044 and regulates lncRNA-Gm2044 expression through the -819 bp binding-site. In addition, we confirmed that lncRNA-Gm2044 functions as a miR-335-3p sponge to enhance the levels of miR-335-3p's direct target protein, Sycp1. Furthermore, A-MYB can up-regulate Sycp1 expression and down-regulate GC-2spd(ts) cell proliferation by activating its target, lncRNA-Gm2044. Overexpression of lncRNA-Gm2044 or knockdown of miR-335-3p can, at least partially, rescue the effects of A-MYB on Sycp1 expression and GC-2spd(ts) cell proliferation.Taken together, our results provide new information on the mechanistic roles of lncRNA-miRNA in transcription factor A-MYB regulation of spermatocyte function.

Single-cyst transcriptome analysis of Drosophila male germline stem cell lineage.

The Drosophila male germline stem cell (GSC) lineage provides a great model to understand stem cell maintenance, proliferation, differentiation and dedifferentiation. Here, we use the Drosophila GSC lineage to systematically analyze the transcriptome of discrete but continuously differentiating germline cysts. We first isolated single cysts at each recognizable stage from wild-type testes, which were subsequently applied for RNA-seq analyses. Our data delineate a high-resolution transcriptome atlas in the entire male GSC lineage: the most dramatic switch occurs from early to late spermatocyte, followed by the change from the mitotic spermatogonia to early meiotic spermatocyte. By contrast, the transit-amplifying spermatogonia cysts display similar transcriptomes, suggesting common molecular features among these stages, which may underlie their similar behavior during both differentiation and dedifferentiation processes. Finally, distinct differentiating germ cell cyst samples do not exhibit obvious dosage compensation of X-chromosomal genes, even considering the paucity of X-chromosomal gene expression during meiosis, which is different from somatic cells. Together, our single cyst-resolution, genome-wide transcriptional profile analyses provide an unprecedented resource to understand many questions in both germ cell biology and stem cell biology fields.

m6A-dependent biogenesis of circular RNAs in male germ cells.

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.

Bovid microRNAs involved in the process of spermatogonia differentiation into spermatocytes.

The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak.

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 µl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 µl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 µl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

Increasing ERK phosphorylation by inhibition of p38 activity protects against cadmium-induced apoptotic cell death through ERK/Drp1/p38 signaling axis in spermatocyte-derived GC-2spd cells.

Many studies report that cadmium chloride (CdCl2)-induces oxidative stress is associated with male reproductive damage in the testes. CdCl2 also induces mitochondrial fission by increasing dynamin-related protein 1 (Drp1) expression as well as the mitochondria-dependent apoptosis pathway by extracellular signal-regulated kinase (ERK) activation. However, it remains unclear whether mechanisms linked to the mitochondrial damage signal via CdCl2-induced mitogen-activated protein kinases (MAPK) cause damage to spermatocytes. In this study, increased intracellular and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆Ψm) depolarization, and mitochondrial fragmentation and swelling were observed at 5 µM of CdCl2 exposure, resulting in increased apoptotic cell death. Moreover, CdCl2-induced cell death is closely associated with the ERK/Drp1/p38 signaling axis. Interestingly, SB203580, a p38 inhibitor, effectively prevented CdCl2-induced apoptotic cell death by reducing ∆Ψm depolarization and intracellular and mitochondrial ROS levels. Knockdown of Drp1 expression diminished CdCl2-induced mitochondrial deformation and ROS generation and protected GC-2spd cells from apoptotic cell death. In addition, electron microscopy showed that p38 inhibition reduced CdCl2-induced mitochondrial interior damage more effectively than N-acetyl-L-cysteine (NAC), an ROS scavenger; ERK inhibition; or Drp1 knockdown. Therefore, these results demonstrate that inhibition of p38 activity prevents CdCl2-induced apoptotic GC-2spd cell death by reducing depolarization of mitochondrial membrane potential and mitochondrial ROS levels via ERK phosphorylation in a signal pathway different from the CdCl2-induced ERK/Drp1/p38 axis and suggest a therapeutic strategy for CdCl2-induced male infertility.

A novel, dynein-independent mechanism focuses the endoplasmic reticulum around spindle poles in dividing Drosophila spermatocytes.

In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, we hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, our results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells.

The REGγ-Proteasome Regulates Spermatogenesis Partially by P53-PLZF Signaling.

Development of spermatogonia and spermatocytes are the critical steps of spermatogenesis, impacting on male fertility. Investigation of the related regulators benefits the understanding of male reproduction. The proteasome system has been reported to regulate spermatogenesis, but the mechanisms and key contributing factors in vivo are poorly explored. Here we found that ablation of REGγ, a proteasome activator, resulted in male subfertility. Analysis of the mouse testes after birth showed there was a decreased number of PLZF+ spermatogonia and spermatocytes. Molecular analysis found that REGγ loss significantly increased the abundance of p53 protein in the testis, and directly repressed PLZF transcription in cell lines. Of note, allelic p53 haplodeficiency partially rescued the defects in spermatogenesis observed in REGγ-deficient mice. In summary, our results identify REGγ-p53-PLZF to be a critical pathway that regulates spermatogenesis and establishes a new molecular link between the proteasome system and male reproduction.

Stem Cells as a Resource for Treatment of Infertility-related Diseases.

Worldwide, infertility affects 8-12% of couples of reproductive age and has become a common problem. There are many ways to treat infertility, including medication, intrauterine insemination, and in vitro fertilization. In recent years, stem-cell therapy has raised new hope in the field of reproductive disability management. Stem cells are self-renewing, self-replicating undifferentiated cells that are capable of producing specialized cells under appropriate conditions. They exist throughout a human's embryo, fetal, and adult stages and can proliferate into different cells. While many issues remain to be addressed concerning stem cells, stem cells have undeniably opened up new ways to treat infertility. In this review, we describe past, present, and future strategies for the use of stem cells in reproductive medicine.

How to become a successful invasive tapeworm: a case study of abandoned sexuality and exceptional chromosome diversification in the triploid carp parasite Atractolytocestus huronensis Anthony, 1958 (Caryophyllidea: Lytocestidae).

BACKGROUND: A cytogenetic analysis of the new local triploid population of the caryophyllidean tapeworm Atractolytocestus huronensis, a unique parthenogenetic species with the ability to colonise new regions, was performed to understand the inner structure of its chromosome complement. METHODS: A karyotype analysis was carried out using classical Giemsa staining and C-banding combined with fluorescent DAPI staining. A hypothesis that triplets are composed from three homologue chromosomes of approximately the same length and same centromere position was tested statistically for multiple dependent variables using a non-parametric Friedman's ANOVA. The chromosomal location of ribosomal DNA clusters within the nucleolar organization region (NORs) and telomeric (TTAGGG)n sequences were detected by fluorescent in situ hybridization (FISH). Chromosomes were subjected to AgNO3 staining in order to determine whether the rDNA sites represent active NORs. RESULTS: The cytogenetic analysis confirmed the karyotype composed from eight chromosome triplets (3n = 24) as well as the existence of a pair of NORs located on each chromosome of the second triplet. Six NORs varied their activity from cell to cell, and it was reflected in the numbers of nucleoli (from 1 to 5). A huge morphological diversification of homologue chromosomes was originally detected in six out of eight triplets; the homologue elements differed significantly either in length and/or morphology, and some of them carried discernible interstitial telomeric sequences (ITSs), while the end telomeres were minute. The heterochromatin bands with high AT content varied irregularly, and the course of aberrant spermatogenesis was evident. CONCLUSIONS: Diversification of homologues is a unique phenomenon very likely caused by the long-term absence of a recombination and consequential accumulation of chromosome rearrangements in the genome of A. huronensis during species evolution. Unalterable asexual reproduction of the tapeworm, along with international trade in its host (carp), is facilitating its ongoing spread.

Ectopic miR-975 induces CTP synthase directed cell proliferation and differentiation in Drosophila melanogaster.

CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster, only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly's testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. Here, we report that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC. Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, we found that ectopic miR-975 concurrently increases both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D. melanogaster, and further reaffirms the enzyme's close ties to germline cells overgrowth.

Changes in the immunohistochemical localization of estrogen receptor alpha and in the stereological parameters of the testes of mature and aged chickens (Gallus domesticus).

The aim of this study was to detect through immunohistochemistry the presence of the estrogen receptor alpha (ER-α) in different cell subpopulations, as well as to evaluate the histological and stereological changes of the testes of mature and aged chickens. Histological results demonstrated several changes in the tubular compartment and interstitial tissue in aged chicken testis as compared to mature chicken testis. Stereological results revealed that the mature chicken testis increases in volume as well as the total volume of the tubular compartment, whereas the total volume of the interstitial tissue and the total volume of Leydig cells diminishes as compared to aged chicken testis. The increase in the total volume of Leydig cells shown in aged chicken testis is due to the increase of cellular volume of Leydig cells, and not in their number, which decreases in aged chicken testis. Results also revealed positive ER-α immunostaining in mature and aged chicken testes, but cellular distribution of ER-α immunostaining changed according to the animal's age. In mature chicken testis, the ER-α was localized in the nuclei of germ and somatic cells. In contrast, in the aged chicken testis, only scarce spermatogonia presented ER-α immunoreactivity. This differential expression of ER-α may contribute to regulate the reproductive function in the mature chicken or the cessation of reproductive function in aged chickens.

Type 1 diabetes upregulates metastasis-associated protein 1- phosphorylated histone 2AX signaling in the testis.

Under the sustained hyperglycemic state, oxidative stress induces irreparable DNA double-strand breaks resulting in germ cell death and testicular atrophy. Although molecular mechanisms underlying DNA damage repair in testicular cells are gradually getting unraveled, the effects on DNA double-strand breaks sensing are not precisely known. In this study, using streptozotocin-induced type 1 diabetic rats, we report that hyperglycemic state for one month or three months does not increase the levels of ataxia telangiectasia mutated (ATM) protein- an upstream kinase responsible for the phosphorylation of histone 2AX (Ɣ-H2AX)- after the formation of DNA double-strand breaks. The ATM expression is seminiferous epithelial stage-dependent in spermatogonia and primary spermatocytes, and the pattern of stage-dependent expression varies in diabetic rats, especially after three-month-long diabetes. However, the levels of metastasis-associated protein-1 (MTA1), an essential protein for ATM function, increase although not in a time-dependent manner. The amount of DNA double-strand breaks increases in a time- and stage-dependent manner as indicated by increased Ɣ-H2AX levels, especially in spermatogonia and primary spermatocytes, and in late spermatids in some tubular stages. Although ATM levels do not increase in diabetic rats, protein is expressed more or less in same testicular cells in which Ɣ-H2AX is expressed indicating that ATM might play a vital role in the phosphorylation of the histone. We conclude that diabetes upregulates MTA1-Ɣ-H2AX signaling in diabetic rat testis as a response to time-dependent increases in DNA double-strand breaks.

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila.

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

Phenotypic characterization of diamond (dind), a Drosophila gene required for multiple aspects of cell division.

Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.