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1.  CDKN2D-WDFY2 Is a Cancer-Specific Fusion Gene Recurrent in High-Grade Serous Ovarian Carcinoma 
PLoS Genetics  2014;10(3):e1004216.
Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.
Author Summary
High-grade serous carcinoma (HG-SC) is the most common subtype of ovarian cancer observed in women. This subtype of ovarian cancer is typically detected at advanced stages due to lack of effective early screening tools. Recurrent cancer-specific gene fusions resulting from chromosomal translocations have the potential to serve as effective screening tools as well as therapeutic targets. Here we identified CDKN2D-WDFY2 as a cancer-specific fusion gene present in 20% of HG-SC tumors, by far the most frequent gene recombinant event found in this highly heterogeneous disease. We also presented evidence that the expression of this fusion may affect the PI3K/AKT pathway that is important for cancer progression. Thus CDKN2D-WDFY2 could very well represent a major cellular lineage important for detecting and classifying heterogeneous ovarian carcinomas, and could provide insight into the underlying mechanism of this deadly disease. This is critical, given that ovarian cancer kills 140,200 women worldwide each year, and few ovarian cancer-specific molecular alterations are currently available for targeting and screening.
doi:10.1371/journal.pgen.1004216
PMCID: PMC3967933  PMID: 24675677
2.  The biology of infertility: research advances and clinical challenges 
Nature medicine  2008;14(11):1197-1213.
Reproduction is required for the survival of all mammalian species, and thousands of essential ‘sex’ genes are conserved through evolution. Basic research helps to define these genes and the mechanisms responsible for the development, function and regulation of the male and female reproductive systems. However, many infertile couples continue to be labeled with the diagnosis of idiopathic infertility or given descriptive diagnoses that do not provide a cause for their defect. For other individuals with a known etiology, effective cures are lacking, although their infertility is often bypassed with assisted reproductive technologies (ART), some accompanied by safety or ethical concerns. Certainly, progress in the field of reproduction has been realized in the twenty-first century with advances in the understanding of the regulation of fertility, with the production of over 400 mutant mouse models with a reproductive phenotype and with the promise of regenerative gonadal stem cells. Indeed, the past six years have witnessed a virtual explosion in the identification of gene mutations or polymorphisms that cause or are linked to human infertility. Translation of these findings to the clinic remains slow, however, as do new methods to diagnose and treat infertile couples. Additionally, new approaches to contraception remain elusive. Nevertheless, the basic and clinical advances in the understanding of the molecular controls of reproduction are impressive and will ultimately improve patient care.
doi:10.1038/nm.f.1895
PMCID: PMC3786590  PMID: 18989307
3.  H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts 
PLoS ONE  2013;8(8):e72689.
The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.
doi:10.1371/journal.pone.0072689
PMCID: PMC3744460  PMID: 23967333
4.  Germ Cell Intercellular Bridges 
Stable intercellular bridges are a conserved feature of gametogenesis in multicellular animals observed more than 100 years ago, but their function was unknown. Many of the components necessary for this structure have been identified through the study of cytokinesis in Drosophila; however, mammalian intercellular bridges have distinct properties from those of insects. Mammalian germ cell intercellular bridges are composed of general cytokinesis components with additional germ cell–specific factors including TEX14. TEX14 is an inactive kinase essential for the maintenance of stable intercellular bridges in gametes of both sexes but whose loss specifically impairs male meiosis. TEX14 acts to impede the terminal steps of abscission by competing for essential component CEP55, blocking its interaction in nongerm cells with ALIX and TSG101. Additionally, TEX14-interacting protein RBM44, whose localization in stabile intercellular bridges is limited to pachytene and secondary spermatocytes, may participate in processes such as RNA transport but is nonessential to the maintenance of intercellular bridge stability.
Mammalian cytoplasmic bridges are composed of cytokinesis components and germ cell–specific factors (e.g., TEX14). Ring canals in Drosophila are also formed during cytokinesis but by different mechanisms.
doi:10.1101/cshperspect.a005850
PMCID: PMC3140685  PMID: 21669984
5.  Endothelial pentraxin 3 contributes to murine ischemic acute kidney injury 
Kidney international  2012;82(11):1195-1207.
Toll-like receptor 4 (TLR4), a receptor forDamage Associated Molecular Pattern Molecules and also the lipopolysaccharide receptor, is required for early endothelial activation leading to maximal inflammation and injury during murine ischemic acute kidney injury. DNA microarray analysis of ischemic kidneys from TLR4-sufficient and deficient mice showed that pentraxin 3 (PTX3) was upregulated only on the former while transgenic knockout of PTX3 ameliorated acute kidney injury. PTX3 was expressed predominantly on peritubular endothelia of the outer medulla of the kidney in control mice. Acute kidney injury increased PTX3 protein in the kidney and the plasma where it may be a biomarker of the injury. Stimulation by hydrogen peroxide, or the TLR4 ligands recombinant human High-Mobility Group protein B1 or lipopolysaccharide, induced PTX3 expression in the Mile Sven 1 endothelial cell line and in primary renal endothelial cells suggesting that endothelial PTX3 was induced by pathways involving TLR4 and reactive oxygen species. This increase was inhibited by conditional endothelial knockout of Myeloid differentiation primary response gene 88, a mediator of a TLR4 intracellular signaling pathway. Compared to wild type mice, PTX3 knockout mice had decreased endothelial expression of cell adhesion molecules at 4 hours of reperfusion possibly contributing to a decreased early maladaptive inflammation in the kidneys of knockout mice. At 24 hours of reperfusion, PTX3 knockout increased expression of endothelial adhesion molecules when regulatory and reparative leukocytes enter the kidney. Thus, endothelial PTX3 plays a pivotal role in the pathogenesis of ischemic acute kidney injury.
doi:10.1038/ki.2012.268
PMCID: PMC3499641  PMID: 22895517
6.  Mendelian genetics of male infertility 
Infertility is defined as the inability of a couple to conceive despite trying for a year, and it affects approximately 15% of the reproductive-age population. It is considered a genetically lethal factor, as the family lineage stops at that individual with no progeny produced. A genetic defect associated with an infertile individual cannot be transmitted to the offspring, ensuring the maintenance of reproductive fitness of the species. However, with the advent of assisted reproductive techniques (ART), we are now able to overcome sterility and bypass nature’s protective mechanisms that developed through evolution to prevent fertilization by defective or deficient sperm.
doi:10.1111/j.1749-6632.2010.05917.x
PMCID: PMC3654696  PMID: 21382200
mendelian genetics; male infertility; asthenozoospermia; oligospermia
7.  BMPR2 is required for postimplantation uterine function and pregnancy maintenance 
The Journal of Clinical Investigation  2013;123(6):2539-2550.
Abnormalities in cell-cell communication and growth factor signaling pathways can lead to defects in maternal-fetal interactions during pregnancy, including immunologic rejection of the fetal/placental unit. In this study, we discovered that bone morphogenetic protein receptor type 2 (BMPR2) is essential for postimplantation physiology and fertility. Despite normal implantation and early placental/fetal development, deletion of Bmpr2 in the uterine deciduae of mice triggered midgestation abnormalities in decidualization that resulted in abnormal vascular development, trophoblast defects, and a deficiency of uterine natural killer cells. Absence of BMPR2 signaling in the uterine decidua consequently suppressed IL-15, VEGF, angiopoietin, and corin signaling. Disruption of these pathways collectively lead to placental abruption, fetal demise, and female sterility, thereby placing BMPR2 at a central point in the regulation of several physiologic signaling pathways and events at the maternal-fetal interface. Since trophoblast invasion and uterine vascular modification are implicated in normal placentation and fetal growth in humans, our findings suggest that abnormalities in uterine BMPR2-mediated signaling pathways can have catastrophic consequences in women for the maintenance of pregnancy.
doi:10.1172/JCI65710
PMCID: PMC3668851  PMID: 23676498
8.  Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse 
PLoS Genetics  2013;9(3):e1003377.
Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1−/+/Hdac2−/− or Hdac2−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2−/− oocytes. The infertility of mice harboring Hdac1−/+/Hdac2−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.
Author Summary
Oocyte development is becoming of increasing interest not only in the broad research community but also within the general public due, in part, to the ever increasing demand for and use of assisted reproductive technologies (ART) to treat human infertility, and because the oocyte-to-embryo transition encompasses a natural reprogramming of gene expression, a process central to forming iPS cells. Dramatic changes in chromatin structure and gene expression occur during oocyte development, but the role of such changes in generating oocytes that are capable of maturing, being fertilized, and giving rise to offspring is very poorly understood. Histone deacetylases (HDACs) are critically involved in modulating chromatin structure. Here, we describe the effect of specifically deleting the gene encoding Hdac2 in mouse oocytes and find the fertility of female mice harboring such oocytes is compromised. Although such mutant oocytes can grow they fail to mature properly to become an egg. The primary defect is that histone H4 acetylated on lysine 16 fails to become deacetylated as the oocyte matures to become an egg, with the consequence that the ability of chromosomes to interact with spindle microtubules is compromised, which in turn leads to improper chromosome segregation.
doi:10.1371/journal.pgen.1003377
PMCID: PMC3597510  PMID: 23516383
9.  Local Signaling Environments and Human Male Infertility: What Can Be Learned from Mouse Models 
Infertility is one of the most prevalent public health problems facing young adult males in today’s society. A clear, treatable cause of infertility cannot be determined in a large number of these patients, and a growing body of evidence suggests that infertility in many of these men may be due to genetic causes. Studies utilizing animal models, and most importantly, mouse knockout technology, have been integral not only for the study of normal spermatogenesis but also for identifying proteins essential for this process, which in turn are candidate genes for causing human male infertility. Successful spermatogenesis depends on a delicate balance of local signaling factors, and this review focuses specifically on the genes that encode these factors. Normal functioning of all testicular cell types is not only essential for normal fertility but, as recently hypothesized, may also be crucial to prevent germ cell oncogenesis. Analysis of these processes using mouse models in vivo has provided investigators with an invaluable tool to effectively translate basic science research to the research of human disease and infertility.
doi:10.1017/S1462399410001468
PMCID: PMC3056269  PMID: 20456819
male infertility; Sertoli cell; spermatogenesis; knockout mouse; testis signaling
10.  Association of mutations in the zona pellucida binding protein 1 (ZPBP1) gene with abnormal sperm head morphology in infertile men 
Molecular Human Reproduction  2011;18(1):14-21.
Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ2 = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.
doi:10.1093/molehr/gar057
PMCID: PMC3244884  PMID: 21911476
male infertility; teratozoospermia; abnormal head morphology; ZPBP1 cDNA mutations; sp38
11.  Tektin 3 is required for progressive sperm motility in mice 
Tektins are evolutionarily-conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial non-redundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for non-syndromic asthenozoospermia in humans.
doi:10.1002/mrd.20957
PMCID: PMC2657187  PMID: 18951373
coiled-coil; axoneme; knockout; motility
12.  Mouse TEX14 Is Required for Embryonic Germ Cell Intercellular Bridges but Not Female Fertility1 
Biology of Reproduction  2009;80(3):449-457.
A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to have roles in communication of large numbers of germ cells. In testis expressed gene 14 (Tex14) knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, demonstrating an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to have a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. In addition, we demonstrate that mitotic kinesin-like protein 1 (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14-null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but are absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14-null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14-null neonatal ovaries, folliculogenesis was still active at 1 yr of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo.
TEX14 is present in embryonic germ cells, but it is not required for female fertility.
doi:10.1095/biolreprod.108.070649
PMCID: PMC2805395  PMID: 19020301
follicle; gamete biology; gametogenesis; oocyte development; ovary
13.  Conversion of midbodies into germ cell intercellular bridges 
Developmental biology  2007;305(2):389-396.
Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP), and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7, and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.
doi:10.1016/j.ydbio.2007.02.025
PMCID: PMC2717030  PMID: 17383626
TEX14; intercellular bridge; cytoplasmic bridge; ring canal; knockout; centralspindlin; septins; midbody
14.  Small-Molecule Inhibition of BRDT for Male Contraception 
Cell  2012;150(4):673-684.
Summary
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.
PaperClip
Graphical Abstract
Highlights
► Bromodomain, testis-specific (BRDT) is a contraceptive target ► JQ1 is a BRDT inhibitor that causes a reversible contraceptive effect in male mice ► JQ1 alters spermatogenesis at the spermatocyte and round spermatid stages ► JQ1 treatment targets the male germline and reduces spermatozoa number and motility
Inhibition of the chromatin reader protein BRDT with the small molecule JQ1 provides an approach for reversible male contraception.
doi:10.1016/j.cell.2012.06.045
PMCID: PMC3420011  PMID: 22901802
15.  Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells 
PLoS ONE  2012;7(6):e38914.
Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.
doi:10.1371/journal.pone.0038914
PMCID: PMC3374785  PMID: 22719986
16.  The Testis-Enriched Histone Demethylase, KDM4D, Regulates Methylation of Histone H3 Lysine 9 During Spermatogenesis in the Mouse but Is Dispensable for Fertility1 
Biology of Reproduction  2011;84(6):1225-1234.
Epigenetic modifications, and methylation of histones in particular, dynamically change during spermatogenesis. Among various methylations of histone H3, methylation of histone H3 lysine 9 (H3K9) and its regulation are essential for spermatogenesis. Trimethytransferases as well as dimethyltransferase are required for meiotic progression. In addition, didemethylase of H3K9 is also critical for spermatogenesis through transcriptional regulation of spermatid-specific genes. However, the requirement for demethylation of trimethylated H3K9 (H3K9me3) during spermatogenesis remains to be elucidated. Here, we report the targeted disruption of KDM4D, a testis-enriched tridemethylase of H3K9. Kdm4d-null mice are viable and fertile and do not show any obvious phenotype. However, H3K9me3 accumulates significantly in Kdm4d-null round spermatids, and the distribution of methylated H3K9 in germ cells is dramatically changed. Nevertheless, the progression of spermatogenesis and the number of spermatozoa are normal, likely secondary to the earlier nuclear localization of another H3K9 tridemethylase, KDM4B, in Kdm4d-null elongating spermatids. These results suggest that demethylation of H3K9me3 in round spermatids is dispensable for spermatogenesis but that possible defects in Kdm4d-null elongating spermatids could be rescued by functional redundancy of the KDM4B demethylase.
The testis-enriched histone demethylase, KDM4D, regulates global methylation of histone H3 lysine 9 during spermatogenesis but is not essential for completion of spermatogenesis and fertility.
doi:10.1095/biolreprod.110.088955
PMCID: PMC3099586  PMID: 21293030
epigenetics; gamete biology; histone demethylase; spermatogenesis; testis
17.  Shaping the sperm head: an ER enzyme leaves its mark 
Journal of Clinical Investigation  2006;116(11):2860-2863.
Lipid storage diseases are debilitating inherited metabolic disorders that stem from the absence of specific lysosomal enzymes that degrade selected lipids. Most characteristically, these disorders affect the nervous and the reticulo-endothelial systems, with massive organomegaly resulting from the presence of engorged, lipid-laden macrophages. In this issue of the JCI, Yildiz et al. describe the role of the ER-resident enzyme β-glucosidase 2 (GBA2) in mice (see the related article beginning on page 2985). Surprisingly, GBA2 deficiency leaves bile acid and cholesterol metabolism intact, instead causing lipid accumulation in the ER of testicular Sertoli cells, round-headed sperm (globozoospermia), and impaired male fertility.
doi:10.1172/JCI30221
PMCID: PMC1626139  PMID: 17080191
18.  Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium 
PLoS Genetics  2012;8(2):e1002500.
The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. We observed overlapping expression of these factors in uterine epithelial and stromal compartments of pregnant mice prior to embryo implantation. Conditional ablation of both Msx1 and Msx2 in the uterus resulted in female infertility due to a failure in implantation. In these mutant mice (Msx1/2d/d), the uterine epithelium exhibited persistent proliferative activity and failed to attach to the embryos. Gene expression profiling of uterine epithelium and stroma of Msx1/2d/d mice revealed an elevated expression of several members of the Wnt gene family in the preimplantation uterus. Increased canonical Wnt signaling in the stromal cells activated β-catenin, stimulating the production of a subset of fibroblast growth factors (FGFs) in these cells. The secreted FGFs acted in a paracrine manner via the FGF receptors in the epithelium to promote epithelial proliferation, thereby preventing differentiation of this tissue and creating a non-receptive uterus refractory to implantation. Collectively, these findings delineate a unique signaling network, involving Msx1/2, Wnts, and FGFs, which operate in the uterus at the time of implantation to control the mesenchymal-epithelial dialogue critical for successful establishment of pregnancy.
Author Summary
During implantation, various tissue compartments within the uterus, including epithelium and stroma, undergo sequential proliferation and differentiation as the embryo attaches to the uterus and invades into the maternal tissue. There is only limited understanding of the molecular signaling pathways that interconnect these tissue compartments to achieve a functional state of the uterus conducive to implantation. This study reveals that a unique signaling network regulated by the homeobox transcription factors MSX1 and MSX2 in the mouse uterus critically controls female fertility. Targeted mutation of Msx1 and Msx2 genes in female mice, which results in infertility, established that these factors suppress signaling by the morphogenic ligands, WNTS, in the uterus. In the absence of Msx1 and Msx2, the WNT signaling is elevated, leading to the production of a subset of fibroblast growth factors (FGFs) in uterine stroma. These FGFs act in a paracrine manner on the uterine epithelium to promote epithelial proliferation, which results in lack of uterine receptivity and implantation failure. This work, therefore, uncovers an important mechanism in mammalian reproduction and development by identifying key paracrine signals that arise from the uterine stroma to control epithelial function during implantation.
doi:10.1371/journal.pgen.1002500
PMCID: PMC3285581  PMID: 22383889
19.  Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function 
PLoS Genetics  2011;7(10):e1002320.
The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.
Author Summary
Approximately 20% of infertile couples in the United States have unexplained causes. Many vital aspects of female fertility are regulated by a family of growth factors called the transforming growth factor β (TGFβ) superfamily. These factors exert their functions via specific receptors and downstream signal mediators. Perturbation of components in this pathway can lead to reproductive dysfunction. We identified a novel role for a TGFβ receptor (called TGFBR1) in female fertility. We demonstrated that female mice with disruption of Tgfbr1 in the reproductive tract are unable to successfully conceive, although they can ovulate and produce fertilizable oocytes. Most importantly, these mice have a striking deformity in the oviduct, marked by the formation of oviductal outpouchings (diverticula) that prevent embryos from reaching the uterus. Concomitant aberrations in the uterine smooth muscle layers are additional features of mice lacking TGFBR1. Therefore, TGFBR1 is critical for the structural integrity and function of the female reproductive tract. Our model can be further exploited to study the development of smooth muscle cells of the female reproductive tract. Genetic mutations in TGFBR1 or other TGFβ signaling machinery may lead to fertility defects in women.
doi:10.1371/journal.pgen.1002320
PMCID: PMC3197682  PMID: 22028666
20.  Activin Bioactivity Affects Germ Cell Differentiation in the Postnatal Mouse Testis In Vivo1 
Biology of Reproduction  2010;82(5):980-990.
The transforming growth factor beta superfamily ligand activin A controls juvenile testis growth by stimulating Sertoli cell proliferation. Testicular levels are highest in the first postnatal week, when Sertoli cells are proliferating and spermatogonial stem cells first form. Levels decrease sharply as Sertoli cell proliferation ceases and spermatogenic differentiation begins. We hypothesized that changing activin levels also affect germ cell maturation. We detected an acute and developmentally regulated impact of activin on Kit mRNA in cocultures of Sertoli cells and germ cells from Day 8, but not Day 4, mice. Both stereological and flow cytometry analyses identified an elevated spermatogonium:Sertoli cell ratio in Day 7 testes from InhbaBK/BK mice, which have decreased bioactive activin, and the germ cell markers Sycp3, Dazl, and Ccnd3 were significantly elevated in InhbaBK/BK mice. The flow cytometry measurements demonstrated that surface KIT protein is significantly higher in Day 7 InhbaBK/BK germ cells than in wild-type littermates. By Day 14, the germ cell:Sertoli cell ratio did not differ between genotypes, but the transition of type A spermatogonia into spermatocytes was altered in InhbaBK/BK testes. We conclude that regulated activin signaling not only controls Sertoli cell proliferation, as previously described, but also influences the in vivo progression of germ cell maturation in the juvenile testis at the onset of spermatogenesis.
Activin modulates germ cell and Sertoli cell development in the mouse testis in vivo.
doi:10.1095/biolreprod.109.079855
PMCID: PMC3142881  PMID: 20130270
activin; KIT; KITL; Sertoli cells; spermatogenesis; spermatogonia; testis
21.  Molecular profiling uncovers a p53-associated role for microRNA-31 in inhibiting the proliferation of serous ovarian carcinomas and other cancers 
Cancer research  2010;70(5):1906-1915.
MicroRNAs (miRNAs) regulate complex patterns of gene expression, and the relevance of altered miRNA expression to ovarian cancer remains to be elucidated. By comprehensively profiling expression of miRNAs and mRNAs in serous ovarian tumors and cell lines and normal ovarian surface epithelium, we identified hundreds of potential miRNA-mRNA targeting associations underlying cancer. Functional overexpression of miR-31, the most underexpressed miRNA in serous ovarian cancer, repressed predicted miR-31 gene targets including cell cycle regulator E2F2. MIR31 and CDKN2A, which encodes p14ARF and p16INK4A, are located at 9p21.3, a genomic region commonly deleted in ovarian and other cancers. p14ARF promotes p53 activity, and E2F2 overexpression in p53 wild-type cells normally leads via p14ARF to an induction of p53-dependent apoptosis. In a number of serous cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, and SKOV3), miR-31 overexpression inhibited proliferation and induced apoptosis; however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Additionally, the osteosarcoma cell line U2OS and the prostate cancer cell line PC3 (p14ARF-deficient and p53-deficient, respectively) were also sensitive to miR-31. Furthermore, miR-31 overexpression induced a global gene expression pattern in OVCAR8 associated with better prognosis in tumors from patients with advanced stage serous ovarian cancer, potentially impacting many genes underlying disease progression. Our findings reveal that loss of miR-31 is associated with defects in the p53 pathway and functions in serous ovarian cancer and other cancers, suggesting that patients with cancers deficient in p53 activity might benefit from therapeutic delivery of miR-31.
doi:10.1158/0008-5472.CAN-09-3875
PMCID: PMC2831102  PMID: 20179198
microRNA; serous ovarian carcinoma; cancer therapy; miR31; TP53
22.  Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein 
PLoS ONE  2011;6(2):e17066.
Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.
doi:10.1371/journal.pone.0017066
PMCID: PMC3045441  PMID: 21364893
23.  Testicular cell adhesion molecule 1 (TCAM1) is not essential for fertility 
Testicular cell adhesion molecule 1 (Tcam1) is a testis-expressed gene that is evolutionarily conserved in most mammalian species. The putative location of TCAM1 on the cell surface makes it an attractive contraceptive target to study. We found that Tcam1 transcription is enriched in the adult testis, and in situ hybridization revealed that Tcam1 is expressed in pachytene to secondary spermatocytes. Immunofluorescence for TCAM1 protein showed strong expression along cell membranes of spermatocytes and weak localization to round spermatids. In light of this evidence, we hypothesized that TCAM1 interacts with an unknown receptor on the surface of Sertoli cells and that this interaction is important for germ cell-Sertoli cell interactions. However, Tcam1 knockout mice that we generated are fertile, and testis weights and sperm counts were not significantly altered. Therefore, we conclude that TCAM1 is not essential for male fertility or germ cell function in Mus musculus.
doi:10.1016/j.mce.2009.09.010
PMCID: PMC2815265  PMID: 19766163
Spermatocyte; Male Fertility; Integral Membrane Protein; Transgenic mice
24.  Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15 
Molecular Human Reproduction  2009;15(12):779-788.
Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.
doi:10.1093/molehr/gap062
PMCID: PMC2776472  PMID: 19651638
BMP15; recombinant protein; oocyte; granulosa cell
25.  Comprehensive 5-Year Study of Cytogenetic Aberrations in 668 Infertile Men 
The Journal of urology  2010;183(4):1636-1642.
Purpose
The causes of male infertility are heterogeneous but more than 50% of cases have a genetic basis. Specific genetic defects have been identified in less than 20% of infertile males and, thus, most causes remain to be elucidated. The most common cytogenetic defects associated with nonobstructive azoospermia are numerical and structural chromosome abnormalities, including Klinefelter syndrome (47,XXY) and Y chromosome microdeletions. To refine the incidence and nature of chromosomal aberrations in males with infertility we reviewed cytogenetic results in 668 infertile men with oligozoospermia and azoospermia.
Materials and Methods
High resolution Giemsa banding chromosome analysis and/or fluorescence in situ hybridization were done in 668 infertile males referred for routine cytogenetic analysis between January 2004 and March 2009.
Results
The overall incidence of chromosomal abnormalities was about 8.2%. Of the 55 patients with abnormal cytogenetic findings sex chromosome aneuploidies were observed in 29 (53%), including Klinefelter syndrome in 27 (49%). Structural chromosome abnormalities involving autosomes (29%) and sex chromosomes (18%) were detected in 26 infertile men. Abnormal cytogenetic findings were observed in 35 of 264 patients (13.3%) with azoospermia and 19 of 365 (5.2%) with oligozoospermia.
Conclusions
Structural chromosomal defects and low level sex chromosome mosaicism are common in oligozoospermia cases. Extensive cytogenetic assessment and fluorescence in situ hybridization may improve the detection rate in males with oligozoospermia. These findings highlight the need for efficient genetic testing in infertile men so that couples may make informed decisions on assisted reproductive technologies to achieve parenthood.
doi:10.1016/j.juro.2009.12.004
PMCID: PMC2969182  PMID: 20172548
infertility; male; aneuploidy; azoospermia; oligospermia; sex chromosome aberrations

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