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BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

Spermiation is a multiple-step process involving profound cellular changes in both spermatids and Sertoli cells. We have observed spermiation defects, including abnormalities in spermatid orientation, translocation and release, in mice deficient in the retinoic acid receptor alpha (RARA) and upon treatment with a pan-RAR antagonist. To elucidate the role of retinoid signaling in regulating spermiation, we first characterized the time course of appearance of spermiogenic defects in response to treatment with the pan-RAR antagonist. The results revealed that defects in spermiation are indeed among the earliest abnormalities in spermatogenesis observed upon inhibition of retinoid signaling. Using fluorescent dye-conjugated phalloidin to label the ectoplasmic specialization (ES), we showed for the first time that these defects involved improper formation of filamentous actin (F-actin) bundles in step 8–9 spermatids and a failure of the actin-surrounded spermatids to move apically to the lumen and to disassemble the ES. The aberrant F-actin organization is associated with diminished nectin-3 expression in both RARA-deficient and pan-RAR antagonist-treated testes. An abnormal localization of both tyrosinated and detyrosinated tubulins was also observed during spermatid translocation in the seminiferous epithelium in drug-treated testes. These results highlight a crucial role of RAR receptor-mediated retinoid signaling in regulating microtubules and actin dynamics in the cytoskeleton rearrangements, required for proper spermiation. This is critical to understand in light of ongoing efforts to inhibit retinoid signaling as a novel approach for male contraception and may reveal spermiation components that could also be considered as new targets for male contraception., A crucial role of RAR receptor-mediated retinoid signaling in regulating microtubule and actin dynamics in cytoskeleton rearrangements during spermiogenesis is demonstrated.

Retinoic acid receptor alpha (RARA), a nuclear receptor protein, has been validated as a target for male contraception by gene knockout studies and also pharmacologically using a pan-retinoic acid receptor antagonist. Retinoic acid receptor alpha activity is indispensable for the spermatogenic process, and therefore its antagonists have potential as male contraceptive agents. This review discusses the effects of systematic dosing regimen modifications of the orally bioavailable and reversible pan-antagonist BMS-189453 as well as studies with the alpha-selective antagonists BMS-189532 and BMS-189614 in a murine model. We also provide an overview of structure–activity studies of retinoic acid receptor alpha antagonists that provide insight for the design of novel alpha-selective ligands., Fertility studies with retinoic acid receptor antagonists in male mice and the current state of RARA-selective antagonist development are reviewed.

In mammals, male gonocytes are derived from primordial germ cells during embryogenesis, enter a period of mitotic proliferation, and then become quiescent until birth. After birth, the gonocytes proliferate and migrate from the center of testicular cord toward the basement membrane to form the pool of spermatogonial stem cells (SSCs) and establish the SSC niche architecture. However, the molecular mechanisms underlying gonocyte proliferation, migration and differentiation are largely unknown. Cyclin A2 is a key component of the cell cycle and required for cell proliferation. Here, we show that cyclin A2 is required in mouse male gonocyte development and the establishment of spermatogenesis in the neonatal testis. Loss of cyclin A2 function in embryonic gonocytes by targeted gene disruption affected the regulation of the male gonocytes to SSC transition, resulting in the disruption of SSC pool formation, imbalance between SSC self-renewal and differentiation, and severely abnormal spermatogenesis in the adult testis.

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. Most of our understanding of their functions has been obtained from studies in single-cell organisms and mitotically proliferating cultured cells. In mammals, there are more than 20 cyclins and 20 CDKs. Although genetic ablation studies in mice have shown that most of these factors are dispensable for viability and fertility, uncovering their functional redundancy, CCNA2, CCNB1, and CDK1 are essential for embryonic development. Cyclin/CDK complexes are known to regulate both mitotic and meiotic cell cycles. While some mechanisms are common to both types of cell divisions, meiosis has unique characteristics and requirements. During meiosis, DNA replication is followed by two successive rounds of cell division. In addition, mammalian germ cells experience a prolonged prophase I in males or a long period of arrest in prophase I in females. Therefore, cyclins and CDKs may have functions in meiosis distinct from their mitotic functions and indeed, meiosis-specific cyclins, CCNA1 and CCNB3, have been identified. Here, we describe recent advances in the field of cyclins and CDKs with a focus on meiosis and early embryogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is expressed uniquely in the male germ line, is essential for spermatogenesis in the mouse, and binds to acetylated transcription start sites of genes expressed in meiosis and spermiogenesis. It has thus been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in regulating gene expression, we characterized its genome-wide distribution, in particular the features of the BRDT binding sites within gene units, by ChIP-Seq analysis of enriched fractions of spermatocytes and spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exon, and introns of genes that are highly transcribed during meiosis and spermiogenesis. Furthermore, in promoters, BRDT binding sites overlapped with several histone modifications and histone variants associated with active transcription, and were also enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE and RFX in round spermatids. Our analysis further revealed that BRDT-bound genes play key roles in diverse biological processes that are essential for proper spermatogenesis. Taken together, our data suggest that BRDT is involved in the recruitment of different transcription factors to distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

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.

The expression of BRDT, a member of the BET sub-family of double bromodomain-containing proteins, is restricted to the male germ line, specifically to pachytene-diplotene spermatocytes and early spermatids. We previously showed that loss of the first bromodomain of BRDT by targeted mutagenesis (Brdt(ΔBD1) ) resulted in sterility and abnormalities in spermiogenesis, but little is known about BRDT's function at the molecular level. As part of studies designed to identify BRDT-interacting proteins we stably introduced a FLAG-tagged BRDT cDNA into 293T cells, which do not normally express BRDT. Affinity-purification of FLAG-tagged BRDT complexes indicated that BRDT has novel interactions with the histone deacetylase HDAC1, the arginine-specific histone methyltransferase 5 PRMT5, and the Tripartite motif-containing 28 protein TRIM28. Immunofluorescent microscopy revealed that BRDT co-localized with each of these proteins in round spermatids and co-immunoprecipitation of testicular extracts showed that these proteins interact with BRDT. Furthermore, they bind the promoter of H1t, a putative target of BRDT-containing complexes. This binding of H1t was lost in mice expressing the Brdt(ΔBD1) mutant protein and concomitantly, H1t expression was elevated in round spermatids. Our study reveals a role for BRDT-containing complexes in the repression of gene expression in vivo that correlates with dramatic effects on chromatin remodeling and the progression of spermiogenesis.

It remains poorly understood how the haematopoietic stem/progenitor cells (HSPC) are attracted to their niches and the functional consequences of such interaction. In the present study, we show that the cell cycle regulator cyclin A1 in association with vascular endothelial growth factor receptor 1 (VEGFR1), is required for HSPC and their niches to maintain their function and proper interaction. In the absence of cyclin A1, the HSPC in the BM are increased in their frequency and display an increased migratory and homing ability. Concomitantly, the ability of the endosteal and central BM niche zones to attract and home the wild-type HSPC is significantly reduced in cyclin A1-null mice as compared to the wild-type controls. The impaired proliferation and homing of HSPC in the BM of cyclin A1-null mice are attributed to the increased density of microvessels in the endosteal and central BM niche zones, which is associated with the increased VEGFR1 expression. Thus, modulation of cyclin A1 and VEGFR1 in HSPC and their niches may provide new insights into therapeutic approaches.

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.

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. Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/−) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased 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, so-called juvenile myoclonic epilepsy personality. Brd2+/− females but not males consistently displayed decreased anxiety. Further, we found a highly significant dominance trait (aggression) in the Brd2+/− mice compared to 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 to wild type littermates, we found decreased numbers 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 juvenile myoclonic epilepsy (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of juvenile myoclonic epilepsy or it controls upstream regulators of individual phenotypes.

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.

Oral administration of a retinoic acid receptor (RAR) pan-antagonist reversibly inhibits spermatogenesis. Given the importance of RARα in regulating spermatogenesis, we identified two RARα-selective antagonists by transactivation and transactivation competition assays and asked whether they effectively inhibit spermatogenesis. Although these two antagonists were potent in vitro, they displayed poor in vivo activity in mice when administered orally. Testicular weights were normal, and morphological analysis revealed normal spermatid alignment and sperm release. In vitro drug property analyses were performed with one of these antagonists and compared with the pan-antagonist. We showed that the discrepancies may be explained by several factors, including high plasma protein binding, faster hepatic metabolism relative to the pan-antagonist, and only moderate permeability. The conclusion of poor oral bioavailability was supported by more pronounced defects in mice when the antagonist was administered intraveno...

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDT(ΔBD1)) and observed disrupted spermiogenesis and male sterility. To determine whether BRDT(ΔBD1) protein results in altered transcription, we analyzed the transcriptomes of control versus Brdt(ΔBD1/ΔBD1) round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3'-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3'-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3'-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.

The human BRD2 gene has been linked and associated with a form of common epilepsy and electroencephalographic abnormalities. Disruption of Brd2 in the mouse revealed that it is essential for embryonic neural development and that viable Brd2(+/-) heterozygotes show both decreased GABAergic neuron counts and increased susceptibility to seizures. To understand the molecular mechanisms by which mis-expression of BRD2 might contribute to epilepsy, we examined its regulation at multiple levels. We discovered that BRD2 expresses distinct tissue-specific transcripts that originate from different promoters and have strikingly different lengths of 5' untranslated regions (5'UTR). We also experimentally confirmed the presence of a highly conserved, alternatively spliced exon, inclusion of which would result in a premature termination of translation. Downstream of this alternative exon is a polymorphic microsatellite (GT-repeats). Manipulation of the number of the GT-repeats revealed that the length of the GT-repeats affects the ratio of the two alternative splicing products. In vitro translation and expression in cultured cells revealed that among the four different mRNAs (long and short 5'UTR combined with regular and alternative splicing), only the regularly spliced mRNA with the short 5'UTR yields full-length protein. In situ hybridization and immunohistochemical studies showed that although Brd2 mRNA is expressed in both the hippocampus and cerebellum, Brd2 protein only can be detected in the cerebellar Purkinje cells and not in hippocampal cells. These multiple levels of regulation would likely affect the production of functional BRD2 protein during neural development and hence, its role in the etiology of seizure susceptibility.

This article provides evidence that basic science research and education should be key priorities for global health training, capacity building, and practice. Currently, there are tremendous gaps between strong science education and research in developed countries (the North) as compared to developing countries (the South). In addition, science research and education appear as low priorities in many developing countries. The need to stress basic science research beyond the typical investment of infectious disease basic service and research laboratories in developing areas is significant in terms of the benefits, not only to education, but also for economic strengthening and development of human resources. There are some indications that appreciation of basic science research education and training is increasing, but this still needs to be applied more rigorously and strengthened systematically in developing countries.

The double bromodomain and extra-terminal domain (BET) proteins are critical epigenetic readers that bind to acetylated histones in chromatin and regulate transcriptional activity and modulate changes in chromatin structure and organization. The testis-specific BET member, BRDT, is essential for the normal progression of spermatogenesis as mutations in the Brdt gene result in complete male sterility. Although BRDT is expressed in both spermatocytes and spermatids, loss of the first bromodomain of BRDT leads to severe defects in spermiogenesis without overtly compromising meiosis. In contrast, complete loss of BRDT blocks the progression of spermatocytes into the first meiotic division, resulting in a complete absence of post-meiotic cells. Although BRDT has been implicated in chromatin remodeling and mRNA processing during spermiogenesis, little is known about its role in meiotic processes. Here we report that BRDT is an essential regulator of chromatin organization and reprograming during prophase I of meiosis. Loss of BRDT function disrupts the epigenetic state of the meiotic sex chromosome inactivation in spermatocytes, affecting the synapsis and silencing of the X and Y chromosomes. We also found that BRDT controls the global chromatin organization and histone modifications of the chromatin attached to the synaptonemal complex. Furthermore, the homeostasis of crossover formation and localization during pachynema was altered, underlining a possible epigenetic mechanism by which crossovers are regulated and differentially established in mammalian male genomes. Our observations reveal novel findings about the function of BRDT in meiosis and provide insight into how epigenetic regulators modulate the progression of male mammalian meiosis and the formation of haploid gametes., Author summary BRDT, a testis-specific member of the bromodomain and extra-terminal (BET) subfamily of epigenetic reader proteins, is essential for the generation of male gametes. In post-meiotic cells, BRDT is involved in chromatin organization and transcriptional regulation through its first bromodomain motif, as loss of the BD1 results in a truncated BRDT protein that fully interrupts the differentiation of the germ cells during the process of spermiogenesis. Complete loss of BRDT function results in an arrest during meiotic prophase with no cells progressing into post-meiotic stages. However, neither the specific role of BRDT in meiosis nor the pathways affected by its depletion are known. We investigated how BRDT controls meiosis by examining its subcellular localization during prophase I as well as the meiotic consequences observed with the loss of BRDT function. BRDT localizes throughout the chromatin of autosomes and sex chromosomes in a dynamic pattern during pachynema and diplonema. Loss of BRDT severely disrupts the epigenetic reprograming and silencing of transcription of the sex chromosomes, the global and regional chromatin configuration, and the formation and localization of crossovers in spermatocytes. Thus, BRDT regulates key meiotic processes that determine the genetic and epigenetic homeostasis of the male gamete.

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.

Brdt is a testis-specific member of the distinctive BET sub-family of bromodomain motif-containing proteins, a motif that binds acetylated lysines and is implicated in chromatin remodeling. Its expression is restricted to the germ line, specifically to pachytene and diplotene spermatocytes and early spermatids. Targeted mutagenesis was used to generate mice carrying a mutant allele of Brdt, Brdt(Delta)(BD1), which lacks only the first of the two bromodomains that uniquely characterize BET proteins. Homozygous Brdt(Delta)(BD1/)(Delta)(BD1) mice were viable but males were sterile, producing fewer and morphologically abnormal sperm. Aberrant morphogenesis was first detected in step 9 elongating spermatids, and those elongated spermatids that were formed lacked the distinctive foci of heterochromatin at the peri-nuclear envelope. Quantitative reverse transcription (RT)-PCR showed threefold increased levels of histone H1t (Hist1h1t) in Brdt(Delta)(BD1/)(Delta)(BD1) testes and chromatin immunoprecipitation revealed that Brdt protein, but not Brdt(DeltaBD1) protein, was associated with the promoter of H1t. Intracytoplasmic sperm injection suggested that the DNA in the Brdt(Delta)(BD1) mutant sperm could support early embryonic development and yield functional embryonic stem cells. This is the first demonstration that deletion of just one of the two bromodomains in members of the BET sub-family of bromodomain-containing proteins has profound effects on in vivo differentiation.

Mammalian myocardial infarction is typically followed by scar formation with eventual ventricular dilation and heart failure. Here we present a novel model system in which mice constitutively expressing cyclin A2 in the myocardium elicit a regenerative response after infarction and exhibit significantly limited ventricular dilation with sustained and remarkably enhanced cardiac function. New cardiomyocyte formation was noted in the infarcted zones as well as cell cycle reentry of periinfarct myocardium with an increase in DNA synthesis and mitotic indices. The enhanced cardiac function was serially assessed over time by MRI. Furthermore, the constitutive expression of cyclin A2 appears to augment endogenous regenerative mechanisms via induction of side population cells with enhanced proliferative capacity. The ability of cultured transgenic cardiomyocytes to undergo cytokinesis provides mechanistic support for the regenerative capacity of cyclin A2.

There are two A-type cyclins in higher vertebrates, cyclin A1 and A2. Targeted mutagenesis has shown that cyclin A2 is essential for early embryonic development while cyclin A1 is required only for male meiosis. The embryonic lethality of cyclin A2 knockout mice has obviated understanding its role in other aspects of mammalian development, including the germ line. We reported previously that cyclin A2 expression in the male germ line is consistent with a role in both mitotic and meiotic cell cycles. Usingin situhybridization and immunohistochemistry, we now observe high levels of cyclin A2 in granulosa cells and less-abundant but readily detectable expression in ovarian and ovulated oocytes. A decrease in cyclin A2 protein was observed in oocytes from embryonic stages to post-natal and adult ovaries. Interestingly, cyclin A2 protein was nuclear in oocytes from embryonic day 13.5 to 15.5, changing to largely cytoplasmic in oocytes from embryonic day 16.5 to post-natal and adults. Readily detectable expression of the cyclin-dependent kinases Cdk1 and Cdk2, two common partners for the A-type cyclins, was observed in granulosa cells and oocytes at all stages of folliculogenesis. Cdk1 was predominantly cytoplasmic, whereas Cdk2 was both cytoplasmic and nuclear in oocytes. No cyclin A1 expression, at either the mRNA level or the protein level was detected in either embryonic or adult ovaries, consistent with the full fertility observed in female cyclin A1-deficient mice. These results suggest that in the female germ line, cyclin A2 but not cyclin A1 has distinct roles in both mitosis and meiosis.

The severe degeneration of the germinal epithelium and subsequent male sterility observed in mice null for the retinoic acid receptor alpha (RARalpha) gene suggested its critical role in spermatogenesis, although the etiology and progression of these abnormalities remain to be determined. Previous studies have revealed that elongated spermatids in RARalpha(-/-) testes were improperly aligned at the tubular lumen and did not undergo spermiation at stage VIII(*). We now report a distinctive failure of step 8-9 spermatids to orient properly with regard to the basal aspect of Sertoli cells, resulting in stage VIII(*)-IX(*) tubules with randomly oriented spermatids. By in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL), we noted that elongating spermatids frequently underwent apoptosis. Immunohistochemical analysis revealed that while activated caspase-3, the primary effector caspase in the apoptotic cell death machinery, was detected in the nuclei of primary spermatocytes in the first wave of spermatogenesis and occasionally in spermatogonia of both normal and mutant testes, it was not involved in the death of elongating spermatids in RARalpha(-/-) testes. Thus, sterility in RARalpha(-/-) males was associated with specific defects in spermiogenesis, which may correlate with a failure in both spermatid release and spermatid orientation to the basal aspect of Sertoli cells at stage VIII(*) in young adult RARalpha(-/-) testis. Further, the resulting apoptosis in elongating spermatids appears to involve pathways other than that mediated by activated caspase-3.

An important role of the cell cycle regulatory protein cyclin A1 in the development of acute myeloid leukemia (AML) was previously demonstrated in a transgenic mouse model. We have now turned our attention to study specific aspects of the activity and subcellular distribution of cyclin A1 using bone marrow samples from normal donors and patients with AML, as well as leukemic cell lines. We show that the localization of cyclin A1 in normal hematopoietic cells is nuclear, whereas in leukemic cells from AML patients and cell lines, it is predominantly cytoplasmic. In leukemic cell lines treated with all-trans retinoic acid (ATRA), cyclin A1 localized to the nucleus. Further, there was a direct interaction between cyclin A1 and cyclin-dependent kinase 1, as well as a major ATRA receptor, RARalpha, in ATRA-treated cells but not in untreated leukemic cells. Our results indicate that the altered intracellular distribution of cyclin A1 in leukemic cells correlates with the status of the leukemic phenotype.

The gene encoding mouse cyclin A1, Ccna1, is expressed at highest levels in late pachytene-diplotene spermatocytes, where it is required for meiotic cell division. To begin to understand the mechanisms responsible for its highly restricted pattern of expression, transgenic mouse lines carrying constructs consisting of the cyclin A1 regulatory region fused with the reporter gene lacZ were generated. Analysis of tissue-specific and testicular cell-type-specific transgene expression indicated that sequences within -1.3 kilobases (kb) of the cyclin A1 putative transcriptional start site were sufficient to direct transgene expression uniquely to late spermatocytes while maintaining repression in other tissues. However, sequences located between -4.8 kb and -1.3 kb of the putative transcriptional start site were apparently required to transcribe the reporter at levels needed for consistent X-gal staining. Comparison of the mouse, rat, and human proximal promoters revealed regions of high sequence conservation and consensus sequences both for known transcription factors, some of which are coexpressed with Ccna1, such as A-myb and Hsf2, and for elements that control expression of genes in somatic cell cycles, such as CDE, CHR, and CCAAT elements. Thus, the promoter region within 1.3 kb upstream of the putative Ccna1 transcriptional start can direct expression of lacZ to spermatocytes, while sequences located between -4.8 kb and -1.3 kb of the putative transcriptional start site may enhance expression of lacZ.

The bromodomain, an evolutionarily conserved motif that binds acetyl-lysine on histones, is found in many chromatin-associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET subclass of bromodomain-containing proteins contains two bromodomains and one ET domain and consists of at least four members in mouse and human, Brd2, Brd3, Brd4, and Brdt. We isolated mouse cDNAs for these genes and studied their expression patterns with particular focus on the testis. Northern hybridization revealed that Brd3 is most abundant in testis, ovary, placenta, uterus, and brain; that Brd4 is rather ubiquitously expressed but is most abundant in mid-gestation embryo, testis, ovary, and brain; and that Brdt is specifically expressed in testis. In situ hybridization and immunostaining on histological sections of mouse testes revealed a strikingly specific and dynamic change of cellular specificity in the germ line during the progression of spermatogenesis. Brd4 is expressed in spermatogonia, Brdt is only expressed in mid- to late-spermatocytes, Brd2 is expressed in diplotene spermatocytes and round spermatids and at low levels in spermatogonia, and Brd3 is expressed in round spermatids. This unique expression pattern suggests that genes in this subclass are not simply redundant. Rather, their expression is tightly regulated in the male germ cell lineage, suggesting that they likely have specific roles in different developmental stages and/or cell types.

The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls. Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation. Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.

Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes. Therefore, the concept of stimulating myocyte mitotic divisions has dramatic implications for cardiomyocyte regeneration and hence, cardiovascular disease. Previous reports describing manipulation of cell cycle proteins have not shown induction of cardiomyocyte mitosis after birth. We now report that cyclin A2, normally silenced in the postnatal heart, induces cardiac enlargement because of cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8 into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an increase in cardiomyocyte mitosis, noted in transgenic hearts at all time points examined, particularly during postnatal development. Several stages of mitosis were observed within cardiomyocytes and correlated with the nuclear localization of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These results reveal a previously unrecognized critical role for cyclin A2 in mediating cardiomyocyte mitosis, a role that may significantly impact upon clinical treatment of damaged myocardium.

Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm. Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W(v) mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogonia-specific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

We previously observed high levels of Brd2 (also known as female sterile homeotic related gene-1, Fsrg1) expression in several hormonally responsive tissues, including the ovary. Here, we report distinct localization patterns of Brd2 transcripts throughout ovarian folliculogenesis in normal mice as well as in two strains of mice with aberrant folliculogenesis: mice with mutated growth differentiation factor 9 (Gdf9) and follicle stimulating hormone β (Fshb) genes. The highest level of expression was seen in granulosa cells of growing follicles. Within the oocyte, three patterns of Brd2 RNA localization were observed: diffuse distribution in both the cytoplasm and nucleus, then intense nuclear expression, followed by an absence of Brd2 transcripts from the nucleus. The transition from intense nuclear localization to nuclear exclusion was found to correlate with oocyte maturation and meiotic competence, as determined by nuclear chromatin patterns. These same expression patterns were also seen in oocytes from Gdf9−/− and Fshb−/− mice. Thus, Brd2 expression appears to correlate with stages of oocyte maturation, independent of FSH or GDF9 action and the subsequent disruption in normal follicle development in these models. The distinct patterns of Brd2 localization within the adult ovary supports a role for Brd2 in mitotic and possibly meiotic cell cycle regulation. Mol. Reprod. Dev. 68: 261–268, 2004. © 2004 Wiley-Liss, Inc.

Aberrant expression of several key regulators controlling the G1/S phase of the cell cycle has been implicated in human male germ cell tumorigenesis. Given the critical role of cyclin A2 at both the G1/S and G2/M transitions and the essential role for cyclin A1 in male germ cell development, our present study focused on the involvement of the A-type cyclins in the transformation and progression of male germ cell tumors (GCTs). The expression of the A-type cyclins and their catalytic partners Cdk1 and Cdk2 was examined in all types and stages of human male GCTs, including carcinoma in situ(CIS), seminoma and non-seminoma GCTs, along with normal testis samples. Elevated levels of cyclin A2, Cdk1 and Cdk2 were detected in the majority of GCTs and were correlated with the invasiveness of the tumors (p < 0.05). Cyclin A1 expression was virtually undetectable in CIS and seminoma, but was aberrantly expressed in all non-seminomatous GCTs. Cyclin A2 expression was strongly correlated with that of its catalytic partners Cdk1 and Cdk2 in all types of testicular tumors examined (p < 0.05), whereas a strong correlation between cyclin A1 and Cdk1 or Cdk2 was only seen in non-seminomatous GCTs (p < 0.05). Histone kinase activities of cyclin A1/Cdks and cyclin A2/Cdks were found to be elevated in tumors. Our data suggest that aberrant expression of A-type cyclins and their Cdks is a significant factor in male germ cell tumorigenesis. The abundant ectopic expression of cyclin A1 in non-seminomatous GCTs and its absence in CIS and seminomas is likely linked to the tumor transformation and progression and may be relevant to clinical prognosis.

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.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.

Male mice homozygous for a mutated allele of the cyclin A1 gene (Ccna1) are sterile due to a block in cell cycle progression before the first meiotic division. Meiosis arrest in Ccna1(-/-) spermatocytes is associated with desynapsis abnormalities, lowered MPF activity, and apoptosis as evidenced by TUNEL-positive staining. With time, adult testicular tubules exhibit severe degeneration: some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis. The mechanisms by which the cells sense the cell cycle arrest and the regulation of the decision to undergo cell death are under investigation.

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G(1) and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.

Fsrg1 (female sterile homeotic-related gene 1) is the mouse homolog of the human RING3 protein, which has been shown to associate with the E2 promoter binding factor (E2F) transcription factor and to have a possible role in cell cycle-linked transcriptional regulation. The Fsrg1 protein is 60% identical in sequence to the RNA polymerase II mediator subunit Fsrg4, another member of this subfamily of double bromodomain-containing proteins that are homologs of Drosophila female sterile homeotic. Antibodies against murine Fsrg1 were generated and used in immunoblot and immunoprecipitation experiments to identify proteins interacting with Fsrg1 and RING3. In the presence of acetylated but not nonacetylated histone H3 and H4 peptides, RING3 was shown to interact with E2F, mediator components cyclin-dependent kinase 8 and thyroid receptor-associated protein 220, and the RNA polymerase II large subunit. Fsrg1 mRNA had been previously shown to be expressed at high levels in the epithelium of the adult mouse mammary gland. To determine the physiological relevance of these potential associations, we examined the patterns of expression of Fsrg1 mRNA and protein in the adult mammary epithelia during the reproductive cycle as the tissue is responding to estrogen, progesterone, and prolactin. Changes in the nuclear vs. cytoplasmic localization of Fsrg1 were observed and correlated with physiological changes in mammary gland function. The observations suggested that Fsrg1 may be involved in the transcriptional activities of genes involved in proliferation of the mammary epithelia during pregnancy and in orchestrating postlactation involution and apoptosis. Localization of Fsrg1 on euchromatin, the transcribed portion of the chromosomes, is consistent with its hypothesized function as a transcription regulator.

Mammalian gametogenesis provides a unique system in which to study cell-cycle regulation. Furthermore, understanding the genetic program controlling the mitotic and meiotic divisions of the germ line will provide insight into understanding infertility and new directions for contraception. Male and female germ cells have stages of cell-cycle regulation in common, including a mitotic proliferative stage, entry into meiosis, completion of a reductive division, and entry into a quiescent state awaiting signals at fertilization. However, the timing of these events - and, indeed, even the stage of development at which these events occurs - differs in the two sexes. The genes involved in controlling these specialized mitotic and meiotic cycles of mammalian germ cell differentiation are only now being identified. They include a complex array of kinases, phosphatases, regulatory proteins (e.g., cyclins), and an equally complex array of substrates, including components of the nuclear and cytoplasmic structures involved in cell division. This chapter provides an overview of our current understanding of cell-cycle regulation in mammalian mitotic cells and the importance of restriction points. A summary of observations regarding the expression of various cell-cycle regulatory genes in mouse gametes is provided, along with comments on interesting differences between mitotic and meiotic cells. Finally, the role of the novel A-type cyclin, cyclin A1, during male meiosis is discussed in depth.

Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system.

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.

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. Mol. Reprod. Dev. 56:6–11, 2000. © 2000 Wiley-Liss, Inc.