Xiangyuan Wang

Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation.

The POU transcription factor Oct-4 is expressed in early mouse embryogenesis and in pluripotent ES and EC stem cell lines. After gastrulation in the embryo, Oct-4 expression is confined to the germline. The present study provides evidence that Oct-4 undergoes downregulation during oogenesis and spermatogenesis, coincident with entry into meiosis. Furthermore, analysis of maturation stages of oocytes showed that Oct-4 is upregulated de novo in the final stages of meiotic prophase I in female germ cells. These data suggest that Oct-4 downregulation in germ cells in both sexes might represent one of the molecular triggers involved in the commitment to meiosis. The upregulation of Oct-4 in oocytes at the completion of the prophase I of meiotic division further suggests a specific involvement of this transcription factor in oocyte growth or the acquisition of meiotic competence.

The first bromodomain of Brdt, a testis-specific member of the BET sub-family of double-bromodomain-containing proteins, is essential for male germ cell differentiation

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, BrdtΔBD1, which lacks only the first of the two bromodomains that uniquely characterize BET proteins. Homozygous BrdtΔBD1/ΔBD1 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 BrdtΔBD1/ΔBD1 testes and chromatin immunoprecipitation revealed that Brdt protein, but not BrdtΔBD1 protein, was associated with the promoter of H1t. Intracytoplasmic sperm injection suggested that the DNA in the BrdtΔBD1 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.

Cyclin A2 Mediates Cardiomyocyte Mitosis in the Postmitotic Myocardium

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 cardiomyoctye 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.

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. Mol. Reprod. Dev. 56:6–11, 2000.

Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin.

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 maleDrosophila 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 G1 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.

Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse

The BET subfamily of bromodomain‐containing genes is characterized by the presence of two bromodomains and a unique ET domain at their carboxyl termini. Here, we show that the founding member of this subfamily, Brd2, is an essential gene by generating a mutant mouse line lacking Brd2 function. Homozygous Brd2 mutants are embryonic lethal, with most Brd2−/− embryos dying by embryonic day 11.5. Before death, the homozygous embryos were notably smaller and exhibited abnormalities in the neural tube where the gene is highly expressed. Brd2‐deficient embryonic fibroblast cells were observed to proliferate more slowly than controls. Experiments to explore whether placental insufficiency could be a cause of the embryonic lethality showed that injecting diploid mutant embryonic stem cells into tetraploid wild‐type blastocysts did not rescue the lethality; that is Brd2‐deficient embryos could not be rescued by wild‐type extraembryonic tissues. Furthermore, there were enhanced levels of cell death in Brd2‐deficient embryos. Developmental Dynamics 238:908–917, 2009.

Oral administration of a retinoic Acid receptor antagonist reversibly inhibits spermatogenesis in mice.

Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.

Retinoic acid receptor α is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process

Targeted mutagenesis of the retinoic acid receptor α (RARα) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RARα‐/‐ 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 RARα‐/‐ testes and confirmed the arrest at step 8–9 spermatids. Retinoid signaling through RARα, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations. Developmental Dynamics 230:754–766, 2004.

Elevated levels and distinct patterns of expression of A-type cyclins and their associated cyclin-dependent kinases in male germ cell tumors

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. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.© 2003 Wiley‐Liss, Inc.

Expression of retinoic acid receptor alpha in the germline is essential for proper cellular association and spermiogenesis during spermatogenesis

Signaling through vitamin A metabolites is indispensable for spermatogenesis, and disruption of retinoic acid receptor alpha (RARα) function resulted in male sterility and aberrant spermatogenesis, which resembled vitamin A deficiency. Here we investigated the lineage- and cell-specific role of RARα-mediated signaling during spermatogenesis using germ-cell transplantation and genetically manipulated mouse models. We demonstrated that RARα-deficient germ-cell stem cells were able to repopulate germ-cell-depleted wild-type testes and initiate spermatogenesis; however, improper cellular associations and abnormal sperm formation were observed. We further generated RARα-deficient mice that expressed RARα-EGFP fusion protein uniquely in haploid germ cells. Strikingly, spermatid orientation, alignment and release, as well as sperm morphology, were normal and there was a partial rescue of sterility. These data provide the first direct evidence for a distinct requirement of RARα-mediated retinoid signaling specifically in germ cells.