Meiotic failure in cyclin A1-deficient mouse spermatocytes triggers apoptosis through intrinsic and extrinsic signaling pathways and 14-3-3 proteins

Cyclin A1 (Ccna1), a member of the mammalian A type cyclins, is most abundantly expressed in spermatocytes and is essential for spermatogenesis in the mouse. Ccna1- deficient spermatocytes arrest at late meiotic prophase and undergo apoptosis. To further delineate the mechanisms and key factors involved in this process, we have examined changes in expression of genes involved in both intrinsic and extrinsic signaling pathways that trigger apoptosis in the mutant spermatocytes. Our results show that both pathways are involved, and that the factors involved in the intrinsic pathway were expressed earlier than those involved in the extrinsic pathway. We have also begun to identify in vivo Ccna1- interacting proteins, using an unbiased biochemical approach, and identified 14-3-3, a key regulator of apoptosis, as a Ccna1-interacting protein. Expression levels of 14-3-3 proteins remain unchanged between wild type and mutant testes but there were differences in the subcellular distribution. In wild type control, 14-3-3 is detected in both cytosolic and nuclear fractions whereas it is restricted to the cytoplasm in mutant testes. This differential distribution of 14-3-3 may contribute to the induction of apoptosis in Ccna1-deficient spermatocytes. These results provide insight into the apoptotic mechanisms and pathways that are triggered when progression through the meiotic cell cycle is defective in male gametogenesis

Identification of an IL-4-Inducible Gene Expressed in Differentiating Lymphocytes and Male Germ Cells

Interleukin 4 (IL-4) is a cytokine that is involved in the differentiation of B and T lymphocytes. In this report, we describe the identification of a novel gene, N.52, which was cloned from the murine pre-B cell line R8205 grown in the presence of IL-4 for 48 hr. Although N.52 expression is detectable at low levels in unstimulated R8205 cells, the level of N.52 dramatically increases after only .4 hr exposure to IL-4 and remains at a high .level up to 48 hr. Although N.52 expression is low or absent in normal spleen B and T cells, its expression can be induced by the differentiation signals delivered by LPS in B cells and by Con A in T-cell hybrids. While N.52 mRNA is absent in all highly differentiated organs, it is detectable in stem cell harboring lymphoid tissues such as bone marrow, fetal liver, and thymus. Furthermore, N.52 mRNA is expressed at strikingly high levels in the testis, specifically in differentiating male germ cells. It is induced by differentiation signals triggered by the combination of cyclic AMP and retinoic acid in teratocarcinoma F9 cells. Taken together, these data suggest that N.52 is a developmentally regulated gene whose expression in cells of the immune and reproductive systems may be controlled by stimuli that induce differentiation

Mammalian E-type Cyclins Control Chromosome Pairing, Telomere Stability and CDK2 Localization in Male Meiosis

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis

Cyclin-dependent kinase control of motile ciliogenesis

Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication

Canonical and Atypical E2Fs Regulate the Mammalian Endocycle

SUMMARY The endocycle is a variant cell cycle consisting of successive DNA synthesis and Gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using novel lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals

GABAergic Neuron Deficit As An Idiopathic Generalized Epilepsy Mechanism: The Role Of BRD2 Haploinsufficiency In Juvenile Myoclonic Epilepsy

Idiopathic generalized epilepsy (IGE) syndromes represent about 30% of all epilepsies. They have strong, but elusive, genetic components and sex-specific seizure expression. Multiple linkage and population association studies have connected the bromodomain-containing gene BRD2 to forms of IGE. In mice, a null mutation at the homologous Brd2 locus results in embryonic lethality while heterozygous Brd2+/− mice are viable and overtly normal. However, using the flurothyl model, we now show, that compared to the Brd2+/+ littermates, Brd2+/− males have a decreased clonic, and females a decreased tonic-clonic, seizure threshold. Additionally, long-term EEG/video recordings captured spontaneous seizures in three out of five recorded Brd2+/− female mice. Anatomical analysis of specific regions of the brain further revealed significant differences in Brd2+/− vs +/+ mice. Specifically, there were decreases in the numbers of GABAergic (parvalbumin- or GAD67-immunopositive) neurons along the basal ganglia pathway, i.e., in the neocortex and striatum of Brd2+/− mice, compared to Brd2+/+ mice. There were also fewer GABAergic neurons in the substantia nigra reticulata (SNR), yet there was a minor, possibly compensatory increase in the GABA producing enzyme GAD67 in these SNR cells. Further, GAD67 expression in the superior colliculus and ventral medial thalamic nucleus, the main SNR outputs, was significantly decreased in Brd2+/− mice, further supporting GABA downregulation. Our data show that the non-channel-encoding, developmentally critical Brd2 gene is associated with i) sex-specific increases in seizure susceptibility, ii) the development of spontaneous seizures, and iii) seizure-related anatomical changes in the GABA system, supporting BRD2's involvement in human IGE

Effects of Space Flight-Associated Stimuli on Development of Murine and Medaka Sensory-Motor Systems

The major goal of these studies was to continue investigations into the influence of altered gravitational fields on the development and function of the vertebrate brain and nervous system. Of major focus during the 18-month finding period of this award was the maintenance of the animals used in the experimental mouse and medaka model paradigms. The experiments focused on characterization of stress-sensitive periods in neural development and immediate or delayed effects on gene expression, physiology and behavior. The hypothesis under investigation was that the environment of space will have biologically significant effects on the development and function of the vertebrate nervous system. We have postulated that these effects will be more significant on certain neural compartments, such as the vestibular-motor system, and that these effects will have greater impact at particular stages of embryonic and post-natal development of the animal. Development of the central nervous system is well known for its vulnerability and sensitivity to environmental stimuli, although the effects of gravitational influences are poorly understood. The long-term goals of this research effort, initiated previously and continued in limited capacity during this interim period, were to provide important new information on the effects of altered environments during these critical periods