Sex-specific behavioral traits in the Brd2 mouse model of juvenile myoclonic epilepsy.

Idiopathic generalized epilepsy represents about 30-35% of all epilepsies in humans. The bromodomain BRD2 gene has been repeatedly associated with the subsyndrome of juvenile myoclonic epilepsy (JME). Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/-) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased gamma-aminobutyric acid (GABA) markers in the direct basal ganglia pathway as well as in the neocortex and superior colliculus. Here, we tested male and female Brd2+/- and wild-type littermate mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) to identify whether Brd2 haploinsufficiency is associated with the human behavioral patterns, the so-called JME personality. Brd2+/- females but not males consistently displayed decreased anxiety. Furthermore, we found a highly significant dominance trait (aggression) in the Brd2+/- mice compared with the wild type, more pronounced in females. Brd2+/- mice of either sex did not differ from wild-type mice in spatial learning and memory tests. Compared with wild-type littermates, we found decreased numbers of GABA neurons in the basolateral amygdala, which is consistent with the increase in aggressive behavior. Our results indicate that Brd2+/- haploinsufficient mice show no cognitive impairment but have behavioral traits similar to those found in patients with JME (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of JME or it controls upstream regulators of individual phenotypes.

Retinoid signaling during spermatogenesis as revealed by genetic and metabolic manipulations of retinoic acid receptor alpha.

The importance of dietary retinol (vitamin A) and retinoid signaling for normal development and differentiation has been recognised for many years. Vitamin A deficiency results in a variety of abnormalities, most of which can be corrected by supplementing the diet with all-trans-retinoic acid (ATRA), with the exception of blindness and male sterility. ATRA, an active metabolite of vitamin A, functions primarily by binding to nuclear receptors of the steroid hormone superfamily, the retinoic acid receptors (RARs). Gene targeting studies revealed the importance of ATRA signaling through the RARs for spermatogenesis. Mice that are homozygous for a null mutation in the gene encoding RARalpha, Rara-/-, exhibit defects in spermatogenesis and male sterility. The abnormalities in these RARalpha-deficient testes have been examined in detail in a series of recent studies from our laboratory and will be summarised in this paper. We also review how dietary, pharmacologic and genetic strategies, alone or in combination, can be used to gain further insight into retinoid function in mammalian spermatogenesis.

Role of retinoid signaling in the regulation of spermatogenesis.

While the need for vitamin A for the normal progression of male germ cell differentiation has been known for many years, the molecular mechanisms underlying this requirement are poorly understood. This review will explore the aspects of the effects on spermatogenesis of dietary deprivation of vitamin A, in particular as to how they compare to the male sterility that results from the genetic ablation of function of the retinoid receptor RARalpha. The effects of other genes involved with retinoid synthesis, transport, and degradation are also considered. The possible cellular mechanisms that may be affected by the lack of retinoid signaling are discussed, in particular, cell cycle regulation and cell-cell interaction, both of which are critical for normal spermatogenesis.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Insights into regulation of the mammalian cell cycle from studies on spermatogenesis using genetic approaches in animal models.

The genetic hierarchy controlling mitosis and especially meiosis during gamete formation is not well understood, even in less complicated systems such as the yeasts. Meiotic divisions are obviously restricted to germ line cells and as such likely require mechanisms of cell cycle control that do not function and may not exist in somatic cells. While male and female germ cells have stages of cell cycle regulation in common, the timing of these events and the stage of development at which these events occur differ in the two sexes. Understanding the genetic program controlling the mitotic and meiotic divisions of the germ line represents a unique opportunity for providing insight into cell cycle control in vivo. Elucidating the key control points and proteins may also enhance our understanding of the etiology of infertility and provide new directions for contraception.

[Human infertility: meiotic genes as potential candidates]. / Infertilité humaine: des gènes de méiose comme candidats potentiels.

Up to now, the identification of gene mutations causing infertility in humans remains poorly investigated. Temporal progression through meiosis and meiosis specific genes had been extensively characterized in yeast. Recently some mammalian homologous were found. The molecular mechanisms regulating entry into and progression through meiosis in mammals are still unknown. However, disruption of some meiotic genes in mouse showed an essential role of them in meiotic chromosome synapsis and gametogenesis. Moreover, the phenotype of gonads in null mutant mice for some meiotic genes (failure to initiate or blockage in meiosis, lack of gametes or small size of gonads...) could be strikingly similar to clinical observations found in human infertility. The aim of this study was to identify putative mutations in 5 meiotic genes of several clinically well-characterized patients who present unexplained infertility (normal karyotype, women with premature ovarian failure, men with azospermia and without Y micro-deletion). For this purpose, the exons of these 5 genes (DMC1, SPO11, MSH4, MSH5, CCNA1) were all amplified by PCR with specific primers and each amplified-exon was sequenced. Sequences were aligned in comparison to the human corresponding gene available in Genbank. Many heterozygous mutations were found in different genes. Two homozygous mutations were found in MSH4 and DMC1 genes in a young man presenting a testis vanishing syndrome and a woman presenting a premature ovarian failure, respectively. Consequences of such mutations will be examined and verified in model organisms (yeast, mouse) to check the relevance of the mutations in clinical setting.

Characterization of D1Pas1, a mouse autosomal homologue of the human AZFa region DBY, as a nuclear protein in spermatogenic cells.

OBJECTIVE: To gain insight into the function of D1Pas1 in spermatogenesis. DESIGN: The cellular and subcellular distribution of D1Pas1 protein were examined. SETTING: Academic research laboratory. ANIMALS: Swiss Webster and C57B1/6J mice. INTERVENTION(S): Antibodies were generated against a D1Pas1 fusion protein. Immunoblot analysis was performed on lysates of testicular cells separated into enriched populations of spermatogenic cells and fractionated into nuclear and cytoplasmic compartments. Immunohistochemistry was performed on histological sections of testis from adult and postnatal day 17 mice. MAIN OUTCOME MEASURE(S): D1Pas1 protein distribution. RESULT(S): D1Pas1 was expressed in germ cells, and its expression was developmentally regulated because it was detected specifically in the meiotic and postmeiotic haploid stages of spermatogenesis. D1Pas1 protein was predominantly localized in the nucleus, with weak cytoplasmic staining. CONCLUSION(S): Nuclear localization of D1Pas1 in the testis and its sequence homology to putative RNA helicases suggests a role of D1Pas1 in pre-mRNA processing during spermatogenesis. Germ cell expression of D1Pas1 and homology to the Y chromosome gene DBY, which is located in an area deleted in azoospermia, suggests a potential role for an autosomal gene in the regulation of spermatogenesis.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury.

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

Altered myelopoiesis and the development of acute myeloid leukemia in transgenic mice overexpressing cyclin A1.

A mammalian A-type cyclin, cyclin A1, is highly expressed in testes of both human and mouse and targeted mutagenesis in the mouse has revealed the unique requirement for cyclin A1 in the progression of male germ cells through the meiotic cell cycle. While very low levels of cyclin A1 have been reported in the human hematopoietic system and brain, the sites of elevated levels of expression of human cyclin A1 were several leukemia cell lines and blood samples from patients with hematopoietic malignances, notably acute myeloid leukemia. To evaluate whether cyclin A1 is directly involved with the development of myeloid leukemia, mouse cyclin A1 protein was overexpressed in the myeloid lineage of transgenic mice under the direction of the human cathepsin G (hCG) promoter. The resulting transgenic mice exhibited an increased proportion of immature myeloid cells in the peripheral blood, bone marrow, and spleen. The abnormal myelopoiesis developed within the first few months after birth and progressed to overt acute myeloid leukemia at a low frequency ( approximately 15%) over the course of 7-14 months. Both the abnormalities in myelopoiesis and the leukemic state could be transplanted to irradiated SCID (severe combined immunodeficient) mice. The observations suggest that cyclin A1 overexpression results in abnormal myelopoiesis and is necessary, but not sufficient in the cooperative events inducing the transformed phenotype. The data further support an important role of cyclin A1 in hematopoiesis and the etiology of myeloid leukemia.

Hoxa4 expression in developing mouse hair follicles and skin.

We have examined the expression of the Hoxa4 gene in embryonic vibrissae and developing and cycling postnatal pelage hair follicles by digoxigenin-based in situ hybridization. Hoxa4 expression is first seen in E13.5 vibrissae throughout the follicle placode. From E15.5 to E18.5 its expression is restricted to Henle's layer of the inner root sheath. Postnatally, Hoxa4 expression is observed at all stages of developing pelage follicles, from P0 to P4. Sites of expression include both inner and outer root sheaths, matrix cells, and the interfollicular epidermis. Hoxa4 is not expressed in hair follicles after P4. Hoxb4, however, is expressed both in developing follicles at P2 and in catagen at P19, suggesting differential expression of these two paralogous genes in the hair follicle cycle.

A role for cyclin A1 in the activation of MPF and G2-M transition during meiosis of male germ cells in mice.

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2--M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases.

Specific expression of soluble adenylyl cyclase in male germ cells.

The cAMP signaling pathway is an important mediator of extracellular signals in organisms from prokaryotes to higher eukaryotes. In mammals two types of adenylyl cyclase synthesize cAMP; a ubiquitous family of transmembrane isoforms regulated by G proteins in response to extracellular signals, and a recently isolated soluble enzyme insensitive to heterotrimeric G protein modulation. Using the very sensitive reverse transcription-polymerase chain reaction (RT-PCR), soluble adenylyl cyclase (sAC) expression is detectable in almost all tissues examined; however, Northern analysis and in situ hybridization indicate that high levels of sAC message are unique to male germ cells. Elevated levels of sAC mRNA are first observed in pachytene spermatocytes and expression increases through spermiogenesis. The accumulation of high levels of message in round spermatids suggests sAC protein plays an important role in the generation of cAMP in spermatozoa, implying possible roles in sperm maturation through the epididymis, capacitation, hypermotility, and/or the acrosome reaction.

Murine Myak, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system.

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary. Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos.

Regulation of the Hoxa4 and Hoxa5 genes in the embryonic mouse lung by retinoic acid and TGFbeta1: implications for lung development and patterning.

We have previously described a 5; cis-acting retinoic acid response element that is required for a subset of Hoxa4 expression, including the midgestation mouse lung. As both retinoids and Hox genes have been implicated in lung development and patterning, we have examined Hoxa4 expression in the developing mouse lung and extended our work on its regulation. At E12.5, a Hoxa4/lacZ transgene is expressed in the mesenchymal compartment of the lung. Later in development expression is restricted to the proximal mesenchyme and is also observed in smooth muscle cells, subepithelial fibroblasts, and alveolar cells. We show that both Hoxa4 and Hoxa5 are upregulated when cultured in the presence of all-trans retinoic acid. In addition, retinoic acid extends the domain of Hoxa4 and Hoxa5 expression to the periphery of the explants where the distal epithelia are developing. Interestingly, the effect of retinoic acid on Hoxa5 expression was not observed in a Hoxa4 mutant background. In contrast, TGFbeta1 was found to downregulate both Hoxa4 and Hoxa5 expression in cultured lung explants. We also establish that retinoic acid has the effect of proximalizing the mouse lung when cultured in a serum-free medium, as evidenced by reduced expression of the distal marker surfactant protein-C. Lungs from Hoxa4 mutant embryos exhibited a similar response to retinoic acid, suggesting that Hoxa4 alone is not required for the proximalizing effect. Based on their retinoid-dependent expression, we conclude that members of the group 4 and/or group 5 Hox genes are likely to be involved in patterning of the mouse lung. Dev Dyn 2000;217:62-74.

Biochemical properties, tissue expression, and gene structure of a short chain dehydrogenase/ reductase able to catalyze cis-retinol oxidation.

We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9-cis- but not all-trans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid. There is little information regarding either the biochemical properties of cRDH or how its 9-cis-retinol substrate is formed. We now report studies of the properties and expression of human and mouse cRDH and of the characteristics and location of the murine cRDH gene. Additionally, we report mouse hepatic 9-cis-retinol concentrations and demonstrate that 9-cis-retinol is formed in a time- and protein-dependent manner upon incubation of all-trans -retinol with cell homogenate. Human and mouse cRDH display similar substrate specificities for cis-isomers of retinol and retinaldehyde. Moreover, human and mouse cRDH show marked sensitivity to inhibition by 13-cis-retinoic acid, with both being inhibited by approximately 50% by 0.15 microm 13-cis-retinoic acid (for substrate concentrations of 10 microm). Lesser inhibition is seen for 9-cis- or all-trans-retinoic acids. Immunoblot analysis using antiserum directed against human cRDH demonstrates cRDH expression in several tissues from first trimester human fetuses, indicating that cRDH is expressed early in embryogenesis. Adult mouse brain, liver, kidney, and to a lesser extent small intestine and placenta express cRDH. The murine cRDH gene consists of at least 5 exons and spans approximately 6 kb of genomic DNA. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10. Taken together, these data extend our understanding of the properties of cRDH and provide additional support for our hypothesis that cRDH may play an important role in 9-cis-retinoic acid formation.

HemT-3, an alternative transcript of mouse gene HemT specific to male germ cells.

A number of genes are known to be expressed primarily in hematopoietic cells and testis and are thought to function in the control of both blood cell and male germ cell differentiation. We have recently identified a mouse gene, HemT, that encodes two alternatively spliced transcripts specific to hematopoietic cells (HemT-1 and HemT-2) and kidney (HemT-2). We have now isolated a third HemT transcript, HemT-3, that is found only in testis by Northern blot analysis and RT-PCR. HemT-3 is alternatively spliced and may be initiated differently from HemT-1 and HemT-2. RNA in-situ hybridization of testis from wild-type and germ-cell-deficient adult mice, as well as from mice at different developmental stages, indicates that HemT-3 is expressed only in early spermatocytes. HemT-3 cDNA has a major open reading frame related to a human glycosylphosphatidylinositol (GPI)-anchored protein, GML. Using an antibody generated against a peptide derived from the HemT-3 open reading frame, we have detected a testis-specific 22kDa protein by Western blot analysis.

Novel human and mouse homologs of Saccharomyces cerevisiae DNA polymerase eta.

The Saccharomyces cerevisiae RAD30 gene encodes a novel eukaryotic DNA polymerase, pol eta that is able to replicate across cis-syn cyclobutane pyrimidine dimers both accurately and efficiently. Very recently, a human homolog of RAD30 was identified, mutations in which result in the sunlight-sensitive, cancer-prone, Xeroderma pigmentosum variant group phenotype. We report here the cloning and localization of a second human homolog of RAD30. Interestingly, RAD30B is localized on chromosome 18q21.1 in a region that is often implicated in the etiology of many human cancers. The mouse homolog (Rad30b) is located on chromosome 18E2. The human RAD30B and mouse Rad30b mRNA transcripts, like many repair proteins, are highly expressed in the testis. In situ hybridization analysis indicates that expression of mouse Rad30b occurs predominantly in postmeiotic round spermatids. Database searches revealed genomic and EST sequences from other eukaryotes such as Aspergillus nidulans, Schizosaccharomyces pombe, Brugia malayi, Caenorhabditis elegans, Trypanosoma cruzi, Arabidopsis thaliana, and Drosophila melanogaster that also encode putative homologs of RAD30, thereby suggesting that Rad30-dependent translesion DNA synthesis is conserved within the eukaryotic kingdom.

Developmentally regulated expression of cyclin D3 and its potential in vivo interacting proteins during murine gametogenesis.

To begin to assess the function of the cell cycle regulator cyclin D3 during gametogenesis, the present study examined its expression, interacting partners, and associated kinase activity in the murine testis and ovary. In the early stages of postnatal testicular development, cyclin D3 protein was detected in spermatogonia and Leydig cells. In the adult testis, cyclin D3 was also expressed in terminally differentiating spermatids. In the embryonic ovary, detection of cyclin D3 was limited to somatic cells. In the postnatal ovary, its localization was predominantly in the nuclei of oocytes in primordial and small follicles, a localization that diminished with oocyte growth. Cdk4 and p27 were expressed in a similar subset of testicular and ovarian cells, suggesting that they may regulate cyclin D3 function during testicular and ovarian development in a cell type-specific manner. Cyclin D3-associated kinase activity was detected in immature, but not adult, testes and ovaries. These observations suggest unique roles for cyclin D3 in the control of cell division and differentiation in the germ line and the differential regulation of mitotic and meiotic cell cycles during male and female gametogenesis.

The cellular distribution and kinase activity of the Cdk family member Pctaire1 in the adult mouse brain and testis suggest functions in differentiation.

Pctaire1, a member of the family of cyclin-dependent kinases, has been shown to be particularly abundantly expressed in differentiated tissues such as testis and brain. However, very little is known about the cellular and subcellular distribution and function of Pctaire1 protein(s), which is the focus of this study. We show that Pctaire1 encoded two major proteins of M(r) approximately 62,000 and approximately 68,000, found predominantly in testis and brain. Within these two tissues, Pctaire1 was most abundant in the cytoplasm of terminally differentiated cells, notably, the pyramidal neurons in brain and elongated spermatids in testis. Immunoprecipitation experiments further showed that a kinase activity toward myelin basic protein was associated with Pctaire1 in the adult testis and brain and that its activity was potentially regulated through association with regulatory partner(s). These results suggest that Pctaire1 kinase might have an important role in differentiated cells such as postmitotic neurons and spermatogenic cells.