Meiotic Spermatocytes Research Articles

Nuclear Architecture of Mouse Spermatocytes: Chromosome Topology, Heterochromatin, and Nucleolus

The nuclear organization of spermatocytes in meiotic prophase I is primarily determined by the synaptic organization of the bivalents that are bound by their telomeres to the nuclear envelope and described as arc-shaped trajectories through the 3D nuclear space. However, over this basic meiotic organization, a spermatocyte nuclear architecture arises that is based on higher-ordered patterns of spatial associations among chromosomal domains from different bivalents that are conditioned by the individual characteristics of chromosomes and the opportunity for interactions between their domains. Consequently, the nuclear architecture is species-specific and prone to modification by chromosomal rearrangements. This model is valid for the localization of any chromosomal domain in the meiotic prophase nucleus. However, constitutive heterochromatin plays a leading role in shaping nuclear territories. Thus, the nuclear localization of nucleoli depends on the position of NORs in nucleolar bivalents, but the association among nucleolar chromosomes mainly depends on the presence of constitutive heterochromatin that does not affect the expression of the ribosomal genes. Constitutive heterochromatin and nucleoli form complex nuclear territories whose distribution in the nuclear space is nonrandom, supporting the hypothesis regarding the existence of a species-specific nuclear architecture in first meiotic prophase spermatocytes.

KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice.

The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.

Microscopy Methods for Analysis of Spindle Dynamics in Meiotic Drosophila Spermatocytes.

The spindle is a microtubule-based structure whose remodeling is required for partitioning the chromosomes and cytoplasm during meiosis. Characterizing microtubule behavior is fundamental to understanding how these tubulin polymers contribute to successful cell division. Here, a procedure is described for the imaging and analysis of spindle microtubule dynamics in cultures of living Drosophila melanogaster primary spermatocytes expressing tubulin tagged with enhanced green fluorescent protein. It employs time-lapse scanning confocal microscopy and the photobleaching of fiduciary marks onto fluorescently tagged microtubules. These labels are subsequently used to determine the sites and rates of kinetochore fiber growth and shrinkage during metaphase. This method can be readily applied to different microtubule populations, meiotic stages, and genetic backgrounds.

ROR2 in the adult mouse testis

Sertoli cells in the adult testis serve a secretory function and have a key structural role in the blood testis barrier (BTB). The BTB prevents meiotic spermatocytes from entering the blood and mounting an immune response. Spermatogonial maturation proceeds from the periphery of the seminiferous tubules towards the lumen, while maintaining constant contact with Sertoli cells. This article is protected by copyright. All rights reserved

Multiple roles of FOXJ3 in spermatogenesis: A lesson from Foxj3 conditional knockout mouse models.

The transcription factor FOXJ3 (Forkhead box J3) is highly expressed in spermatogonia and meiotic spermatocytes within mouse testes. Here, we addressed how FOXJ3 might participate in spermatogenesis using two conditional knockout mouse models in which Foxj3 was deleted from either spermatogonia or meiotic spermatocytes. Both models exhibited complete male sterility, but distinct etiologies: Deleting FOXJ3 from spermatogonia using Foxj3flox/flox , Mvh-Cre mice caused Sertoli-cell-only syndrome in males. Foxj3-deficient spermatogonia were lost as early as postnatal Day 4, partially due to the accumulation of DNA double-stranded breaks. In contrast, loss of FOXJ3 in spermatocytes using Foxj3flox/flox , Stra8-Cre mice led to meiotic arrest. Indeed, the mRNA abundance of meiotic arrest-related proteins (Rad51, Dmc1, Brca1, Brca2, Brit1, Eif4g3, Hop2, Hormad1, and Rnf212) was significantly reduced in Foxj3flox/flox , Stra8-Cre spermatocytes. Thus, we conclude that FOXJ3 is required for the survival of spermatogonia and participates in spermatocyte meiosis. Mol. Reprod. Dev. 83: 1060-1069, 2016. © 2016 Wiley Periodicals, Inc.

PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility.

Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility.

FOXJ2 controls meiosis during spermatogenesis in male mice.

Spermatogenesis is a highly complex cell differentiation process necessary for production of haploid spermatozoa. Central to this unique process is spermatocyte meiosis. FOXJ2 (Forkhead box J2), a FOX transcription factor, is specifically expressed in meiotic spermatocytes in adult mouse testes, so we used a germ cell specific conditional knockout model (Foxj2(flox/flox) , Mvh-Cre) to explore its role in spermatogenesis. Loss of FOXJ2 in the male germ line led to meiotic arrest and complete infertility. Although, DNA double-strand breaks (DSBs) were initiated, Foxj2-deficient spermatocytes failed to form chromosomal synapses and perform DSB repair. Furthermore, Foxj2-deficient spermatocytes contained significantly less mRNA encoding DSB repair-associated factors (Rad18, Rad51, Brca1, Brca2, and Tex15) and meiotic arrest-related proteins (Fzr1, Hsp70-2, Spata22, Eif4g3, and Zpac); in contrast, no change was observed in the expression of spermatogonia markers (Gfra1, Zbtb16, and c-Kit) and germ cell markers (Dazl, Mvh, and Tra98). Taken together, FOXJ2 appears to promote meiotic progression in male mice by a mechanism that needs further investigation. Mol. Reprod. Dev. 83: 684-691, 2016 © 2016 Wiley Periodicals, Inc.

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus.

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development.

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.

Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells.

Testicular phagocytosis by Sertoli cells (SCs) plays an essential role in the efficient clearance of apoptotic spermatogenic cells under both physiological and pathological conditions. However, the molecular mechanism underlying this unique process is poorly understood. Herein, we report for the first time that α-taxilin protein (TXLNA), a binding partner of the syntaxin family that functions as a central player in the intracellular vesicle traffic, was dominantly expressed in SCs. Induction of apoptosis in murine meiotic spermatocytes and haploid spermatids by busulfan treatment stimulated a significant increase of TXLNA in SCs at day (d) 14 and d 24 after busulfan treatment, respectively. Consistently, TXLNA expression was steadily upregulated when SCs were co-cultured with apoptotic germ cells (GCs). Moreover, using siRNA treatment, we found that ablation of endogenous TXLNA significantly impaired the phagocytotic capacity of SCs and thereby resulted in defective spermiogenesis and reduced fertility during the late recovery after testicular heat stress. Mechanistically, upregulation of TXLNA expression by apoptotic GCs was associated with the stabilization of ATP-binding cassette transporter 1 (ABCA1), a transporter-mediated lipid efflux from SCs and influencing male fertility. TXLNA acted as an upstream suppressor of ABCA1 ubiquitination and thus promoted ABCA1 stability and accumulation following GC apoptosis. We further provide in vitro evidence that epidermal growth factor receptor (EGFR)-mediated phosphorylation regulated ABCA1 ubiquitination and was enhanced by TXLNA deficiency during testicular phagocytosis. Taken together, the TXLNA/ABCA1 cascade may serve as an important feedback mechanism to modulate the magnitude of subsequent phagocytotic process of SCs in response to testicular injury.

Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling.

Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4(flox/flox); Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into Kit(W/W-v) recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth.

Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes.

Mechanistic target of rapamycin (mTOR) is a signal transduction pathway that modulates translation initiation in several animals including mammals. Rapamaycin, an allosteric inhibitor of mTOR pathway, is often used as an immunosuppressive drug following kidney transplantation and causes gonadal dysfunction and defects in spermatogenesis. The molecular mechanism behind rapamycin-mediated testicular dysfunction is not known. We have therefore explored the contribution of rapamycin in mTOR regulation and microRNA (miRNA) expression in mouse spermatocytes, the intermediate stage of spermatogenesis, where meiosis takes place. In the present study, we optimized the isolation of highly pure and viable spermatocytes by flow sorting, treated them with rapamycin, and investigated the expression of mTOR and downstream effector molecules. Western blot and immunocytochemical analysis confirm that rapamycin treatment suppresses mTOR and phopsphorylated P70S6 kinase activities in spermatocytes, but not that of phosphorylated 4E-binding protein 1. Also, rapamycin treatment modulates the expression of several spermatocyte-specific miRNAs. To complement these finding an invivo study was also performed. In silico prediction of target genes of these miRNAs and their functional pathway analysis revealed that, several of them are involved in crucial biological process, cellular process and catalytic activities. miRNA-transcription factor (TF) network analysis enlisted different TFs propelling the transcription machineries of these miRNAs. In silico prediction followed by quatitative real-time PCR revealed two of these TFs namely, PU.1 and CCCTC binding factor (CTCF) are down and upregulated, respectively, which may be the reason of the altered expression of miRNAs following rapamycin treatment. In conclusion, for the first time, the present study provides insight into how rapamycin regulates mTOR pathway and spermatocyte-specific miRNA expression which in turn, regulate expression of target genes post-transcriptionally.

Focus On Light Microscopy.

It would be hard to argue that light microscopy has not played a highly influential role in science since its invention in 1595. However, the past 35 years have seen impressive developments in light microscopy and its associated analytical techniques. The modern light microscope is now a highly evolved opto-electromechanical device. The “hottest” developments have occurred over a relatively short time frame, the most notable being the development of several new “flavors” of super-resolution microscopy for which the Nobel Prize was awarded recently. These advances have allowed the light microscope to be used as a powerful quantitative research tool. In this issue, there are 4 articles that give readers a sense of the breadth of modern light microscopy. These range from a mostly mathematical paper, A Quantitative Measure of Field Illumination, to a review of the current state of confocals, Any Way You Slice It—A Comparison of Confocal Microscopy Techniques. There is a research paper on spindle microtubules called Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes. Rounding out the group is a paper on genetics that uses imaging in a novel way: Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression. Brief summaries of the papers are indicated as follows: A Quantitative Measure of Field Illumination is a paper that produced the first mathematical algorithm for scoring the evenness of the illumination in any imaging device. Adaptive learning was used to construct the algorithm that was coded in MatLab. There were 89 test scopes, which were collected during an Association of Biomolecular Resource Facilities-sponsored project. To verify the algorithm, 33 additional scopes of various modalities were used. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes addresses the quantitative characterization of kinetochore microtubule flux during metaphase and anaphase A in Drosophila male meiois. The method includes transgenic expression of GFP-tubulin, Drosophila melanogaster spermatocytes primary culturing, confocal laser scanning-based photobleaching/image acquisition, and image analysis using the open source platform NIH ImageJ. Any Way You Slice It—A Comparison of Confocal Microscopy Techniques provides an up-to-date guide detailing the various modes of single photon confocal microscopes, which currently are available commercially. Newbies to confocal microscopy, who are in the market for a system or are trying to determine which platform is most suitable for their project, will find this review most useful in guiding their decision. The “New Developments” section brings the reader quickly up to date with the latest technological innovations and improvements. Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression used a novel method to follow angiogenesis in mice. Three-dimensional fluorescently (nuclei)-labeled Matrigel-embedded explants were collected and analyzed. Composite images were processed with large spectral filters and a watershed routine to distinguish overlapping nuclei. Semiautomatic image processing and object tracking procedures were developed, which are applicable to other explant assays. This assay was chosen with the aim of increasing sensitivity to detect angiogenesis response alleles by decreasing the number of genetic factors that might affect the measured phenotype.

Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

Figla Favors Ovarian Differentiation by Antagonizing Spermatogenesis in a Teleosts, Nile Tilapia (Oreochromis niloticus).

Figla (factor in the germ line, alpha), a female germ cell-specific transcription factor, had been shown to activate genetic hierarchies in oocytes. The ectopic expression of Figla was known to repress spermatogenesis-associated genes in male mice. However, the potential role of Figla in other vertebrates remains elusive. The present work was aimed to identify and characterize the functional relevance of Figla in the ovarian development of Nile tilapia (Oreochromis niloticus). Tissue distribution and ontogeny analysis revealed that tilapia Figla gene was dominantly expressed in the ovary from 30 days after hatching. Immunohistochemistry analysis also demonstrated that Figla was expressed in the cytoplasm of early primary oocytes. Intriguingly, over-expression of Figla in XY fish resulted in the disruption of spermatogenesis along with the depletion of meiotic spermatocytes and spermatids in testis. Dramatic decline of sycp3 (synaptonemal complex protein 3) and prm (protamine) expression indicates that meiotic spermatocytes and mature sperm production are impaired. Even though Sertoli cell (dmrt1) and Leydig cell (star and cyp17a1) marker genes remained unaffected, hsd3b1 expression and 11-KT production were enhanced in Figla-transgene testis. Taken together, our data suggest that fish Figla might play an essential role in the ovarian development by antagonizing spermatogenesis.

Germline/meiotic genes in cancer: new dimensions

Intergenerational passage of genetic information in humans is dependent on a complex sexual program which is orchestrated by a large cohort of tissue-specific germline genes. These genes drive a range of germline-specific pathways including spermatogenesis in males and the reductional chromosome segregation of meiosis. In somatic tissues these genes are transcriptionally silenced, but can become activated in cancerous tissue.1-3 Initial interest in these genes was driven by the need to identify new cancer-specific biomarkers that could be used for diagnostics, prognostics and/or immunotherapeutics, and the first group to be discovered became widely known as the cancer/testis antigen (CTA) genes as they were expressed only in the testis and cancerous tissues, but not in healthy somatic cells. Latterly, many of these gene have also been referred to as the cancer germline genes.1,2 Despite the early interest in these genes as clinical biomarkers or targets, recent works have started to reveal another important cancer-associated role, one which has undergone remarkably limited scrutiny. It has emerged from various studies that germline genes can contribute to oncogenesis and can also contribute to tumor maintenance and drug resistance,1,2 potentially revealing this family of proteins as potent new drug targets. The genes that drive and maintain the male germline have a range of distinct functional roles, including germline cell maintenance in mitotically proliferating spermatagonial cell populations, cellular differentiation during spermatogenesis and the chromosomal reduction of meiosis. To date, many of the CTA genes that have been identified are encoded by the X chromosome and their expression is restricted to the spermatagonial germline cells, becoming transcriptionally inactive during the differentiation to meiotic spermatocytes. A recent screen for new cancer germline genes based on the human orthologues of mouse meiotic spermatocyte genes revealed a large number of autosomally encoded cancer germline genes that were transcriptionally activated in a wide range of cancer types.4,5 A number of these human genes have been shown to encode meiosis-specific functions, including the meiosis-specific inter sister chromatid cohesion protein gene RAD21L and the transcriptional activator / meiotic recombination hot spot regulator gene PRDM9.4,5 From this, it was speculated that the activation of such genes could drive oncogenesis and tumor evolution by interfering with normal equational mitotic chromosome segregation programmes, thus driving oncogenic genome instability.4,5 However, while germline genes have been implicated in a number of oncogenic processes, until recently no direct evidence had been offered to implicate meiosis-specific chromosome regulators in oncogenesis or tumor maintenance. This changed when it was discovered that the ALT telomere maintenance pathway used by some telomerease deficient cancer cells to maintain their telomeres was dependent upon 2 meiosis-specific factors, Hop2 and Mnd1, both of which normally drive the mechanism that biases meiotic recombination to establish the inter homolog connections required for correct reductional chromosome segregation during meiosis I.6 This discovery adds a new class of factors to the complex mix of genes that become activated during oncogenesis and highlights the need to explore the functional activity of other human meiotic genes that may contribute to cancer formation, maintenance and evolution.4,5 In addition, it places further importance on the study of basic molecular mechanisms of the meiotic program. Remarkably, given the emerging importance of germline genes and meiosis-specific genes in cancer, very little is known about their transcriptional regulation. Some studies imply that many of these genes are under a similar regulatory pathway, so that when this becomes dysregulated, functionally related groups of genes become active driving inappropriate functional germline/meiotic modules.3 The epigenetic regulation of previously well characterized CTA genes has been relatively well documented with all studied genes being controlled at some level by the methylation of DNA transcriptional regulatory regions. These genes are activated when cells are treated with the DNA methyltransferase inhibitor 5-AzaC. However, a new layer of complexity to this activation has been revealed by a recent study that demonstrated that not all human cancer germline genes are activated by inhibition of DNA methyltransferases, indicating a complex hierarchy of germline, and possibly meiotic, gene activation.7 In this study it was shown that not only did some cancer germline genes fail to become activated upon inhibition of DNA methyltransferase activity, a number were only transiently silenced during exposure to the demethylating agents while others remained more robustly activated following removal of the demethylation agent.7 Altogether, the finding that meiosis-specific factors can contribute to oncogenesis and that the normal somatic silencing of germline genes is controlled by unique, as yet uncharted factors, makes the study of meiotic and germline genes in cancer an emerging and important field, one which impinges directly on a range of clinical applications including diagnostics, patient stratification/prognostics and new therapeutic and drug targeting strategies.

RNA binding protein Musashi-1 directly targets Msi2 and Erh during early testis germ cell development and interacts with IPO5 upon translocation to the nucleus.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function.

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size.

A detailed cytogenetic analysis of the aspidogastrean fluke Aspidogaster limacoides revealed a karyotype consisting of six medium-sized chromosome pairs. The first and the last pairs were two-armed while four remaining were one-armed; 2n = 12, n = 1 m + 1 m - sm + 4a. Fluorescence in situ hybridization with 18S ribosomal DNA (rDNA) probe detected a single cluster of ribosomal genes (NOR) located in pericentromeric regions of the long arms of the third chromosome pair in a site of secondary constriction apparent in meiotic prophase, especially in diplotene. The silver nitrate staining showed only a single active NOR site on one of homologous chromosomes in the majority of spermatogonia and spermatocyte divisions. A course of meiosis corresponded to standard schemes. The nucleolus was apparent in early meiotic spermatocytes and disintegrated by the end of pachytene. For the first time in Aspidogastrea, the genome size was determined. The flow cytometry showed 1.21 pg DNA per haploid nucleus in A. limacoides which is in accordance with relatively low genome sizes of other flukes and tapeworms (Neodermata). A comparison of cytogenetic data available to date in the fluke sister groups Aspidogastrea and Digenea suggests that the lower chromosome number of Aspidogastrea might represent an ancestral condition and their split might have been accompanied by an increase in chromosome number via either chromosome fissions or paleopolyploidy.

Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility.

What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes.

BackgroundThe 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.ResultsIn 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.ConclusionsThe 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.