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1.  Amino Acid 72 of Mouse and Human GDF9 Mature Domain Is Responsible for Altered Homodimer Bioactivities but Has Subtle Effects on GDF9:BMP15 Heterodimer Activities1 
Biology of Reproduction  2014;91(6):142.
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted paralogs of the transforming growth factor beta (TGFbeta) superfamily. In mammals, these two growth factors play critical roles in folliculogenesis. As previously reported, an arginine in the pre-helix loop of GDF5 defines the high binding specificity to its type 1 receptor. Interestingly, bioactive mouse GDF9 and human BMP15 share the conserved arginine in the pre-helix loop, but their low-activity counterparts (mouse BMP15 and human GDF9) have a glycine or a proline instead. To address the question of whether the arginine residue defines the different activities of GDF9 and BMP15 homodimers and their heterodimers in human and mouse, we used site-directed mutagenesis to change the species-specific residues in human and mouse proteins, and examined their activities in our in vitro assays. Although amino acid 72 of mature GDF9 is responsible for altered homodimer bioactivities, neither the corresponding BMP15 amino acid 62 nor the intact pre-helix loop is indispensable for BMP15 homodimer activity. However, amino acid 72 in GDF9 only has only subtle effects on GDF9:BMP15 heterodimer activity. Based on previous studies and our recent findings, we provide hypothetical models to understand the molecular mechanism to define activities of the homodimeric and heterodimeric ligands. The arginine residue in the pre-helix loop of GDF9 homodimer may prevent the inhibition from its pro-domain or directly alter receptor binding, but this residue in GDF9 does not significantly affect the heterodimer activity, because of suggested conformational changes during heterodimer formation.
PMCID: PMC4434983  PMID: 25253739
follicular development; growth factors; ovary
2.  Lineage Specification of Ovarian Theca Cells Requires Multi-Cellular Interactions via Oocyte and Granulosa Cells 
Nature communications  2015;6:6934.
Organogenesis of the ovary is a highly orchestrated process involving multiple lineage determinations of ovarian surface epithelium, granulosa cells, and theca cells. While the sources of ovarian surface epithelium and granulosa cells are known, the origin(s) of theca progenitor cells have not been definitively identified. Here we show that theca cells derive from two sources: Wt1+ cells indigenous to the ovary and Gli1+ mesenchymal cells migrated from the mesonephros. These progenitors acquire theca lineage marker Gli1 in response to paracrine signals Desert hedgehog (Dhh) and Indian hedgehog (Ihh) from granulosa cells. Ovaries lacking Dhh/Ihh exhibit theca layer loss, blunted steroid production, arrested folliculogenesis, and failure to form corpora lutea. Production of Dhh/Ihh in granulosa cells requires Growth differentiation factor 9 (GDF9) from the oocyte. Our studies provide the first genetic evidence for the origins of theca cells and reveal a multicellular interaction critical for the formation of a functional theca.
PMCID: PMC4413950  PMID: 25917826
3.  Epigenetic Activation of Neuronal Gene Expression by JMJD3 is Required for Postnatal and Adult Brain Neurogenesis 
Cell reports  2014;8(5):1290-1299.
The epigenetic mechanisms that enable lifelong neurogenesis from neural stem cells (NSCs) in the adult mammalian brain are poorly understood. Here we show that JMJD3, a histone H3-lysine 27 (H3K27) demethylase, acts as a critical activator of neurogenesis from adult subventricular zone (SVZ) NSCs. JMJD3 is upregulated in neuroblasts, and Jmjd3-deletion targeted to SVZ NSCs in both developing and adult mice impairs neuronal differentiation. JMJD3 regulates neurogenic gene expression via interaction at not only promoter regions, but also neurogenic enhancer elements. JMJD3 localizes at neural enhancers genome-wide in embryonic brain, and in SVZ NSCs, JMJD3 regulates the I12b enhancer of Dlx2. In Jmjd3-deleted SVZ cells, I12b remains enriched with H3K27me3, and Dlx2-dependent neurogenesis fails. These findings support a model in which JMJD3 and the poised state of key transcriptional regulatory elements comprise an epigenetic mechanism that enables the activation of neurogenic gene expression in adult NSCs throughout life.
PMCID: PMC4201382  PMID: 25176653
4.  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.
PMCID: PMC3668851  PMID: 23676498
5.  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.
PMCID: PMC3099586  PMID: 21293030
epigenetics; gamete biology; histone demethylase; spermatogenesis; testis
6.  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.
PMCID: PMC3056269  PMID: 20456819
male infertility; Sertoli cell; spermatogenesis; knockout mouse; testis signaling
7.  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.
PMCID: PMC2831102  PMID: 20179198
microRNA; serous ovarian carcinoma; cancer therapy; miR31; TP53
8.  Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair 
Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound.
PMCID: PMC4087116  PMID: 19079322
activin; dermis; epidermis; follistatin; wound healing
9.  Follistatin Operates Downstream of Wnt4 in Mammalian Ovary Organogenesis 
Wnt4−/− XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFβ superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary. Developmental Dynamics 230:210–215, 2004.
PMCID: PMC4046253  PMID: 15162500
sex determination; ovary; coelomic vessel; germ cells; follistatin; Wnt4
10.  UBE2B mRNA alterations are associated with severe oligozoospermia in infertile men 
Molecular Human Reproduction  2013;19(6):388-394.
Oligozoospermia (low sperm count) is a common semen deficiency. However, to date, few genetic defects have been identified to cause this condition. Moreover, even fewer molecular genetic diagnostic tests are available for patients with oligozoospermia in the andrology clinic. Based on animal and gene expression studies of oligozoospermia, several molecular pathways may be disrupted in post-meiotic spermatozoa. One of the disrupted pathways is protein ubiquitination and cell apoptosis. A critical protein involved in this pathway is the ubiquitin-conjugating enzyme 2B, UBE2B. Absence of Ube2b in male mice causes spermatogenic meiotic disruption with increased apoptosis, leading to infertility. To examine the association between messenger RNA defects in UBE2B and severe oligozoospermia (0.1–10 × 106 cells/ml), sequencing of sperm cDNA in 326 oligozoospermic patients and 421 normozoospermic men was performed. mRNA alterations in UBE2B were identified in sperm in 4.6% (15 out of 326) of the oligozoospermic patients, but not found in control men, suggesting strong association between mRNA defects and oligozoospermia (χ2 = 19, P = 0.0001). Identified UBE2B alterations include nine splicing, four missense and two nonsense alterations. The follow-up screen of corresponding DNA regions did not reveal causative DNA mutations, suggesting a post-transcriptional nature of identified defects. None of these variants were reported in the dbSNP database, although other splicing abnormalities with low level of expression were present in 11 out of 421 (2.6%) controls. Our findings suggest that two distinct molecular mechanisms, mRNA editing and splicing processing, are disrupted in oligozoospermia. We speculate that the contribution of post-transcriptional mRNA defects to oligozoospermia could be greater than previously anticipated.
PMCID: PMC3655768  PMID: 23378580
male infertility; oligozoospermia; UBE2B alterations; abnormal splicing; post-transcriptional errors
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.
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.
PMCID: PMC2805395  PMID: 19020301
follicle; gamete biology; gametogenesis; oocyte development; ovary
13.  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.
PMCID: PMC3967933  PMID: 24675677
14.  Luteinizing hormone promotes gonadal tumorigenesis in inhibin-deficient mice 
The inhibins are secreted α:β heterodimers of the TGF-β superfamily that are mainly synthesized in Sertoli cells and granulosa cells, and are critical regulators of testicular and ovarian development and function. Mice homozygous for a targeted deletion of the inhibin α subunit gene (Inha-/-) develop sex cord-stromal tumors in a gonadotropin-dependent manner. Here, we determine the contribution of LH to gonadal tumorigenesis by generating mice deficient in both inhibins and LH. Inha-/-Lhb-/- mice have increased survival and delayed tumor progression, and these observations correlate with lower serum FSH and estradiol levels compared to Inha-/- controls. Double mutant testicular tumors demonstrate decreased expression of cyclin D2, while double mutant ovarian tumors have elevated expression of p15INK4b and trend toward higher levels of p27Kip1. We conclude that LH is not required for tumor formation in the absence of inhibins but promotes tumor progression, likely through alterations in serum hormone levels and cell cycle regulators.
PMCID: PMC2743404  PMID: 18657590
inhibin; luteinizing hormone; ovarian cancer; testicular cancer
15.  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.
PMCID: PMC2717030  PMID: 17383626
TEX14; intercellular bridge; cytoplasmic bridge; ring canal; knockout; centralspindlin; septins; midbody
16.  Conditional Deletion of Smad1 and Smad5 in Somatic Cells of Male and Female Gonads Leads to Metastatic Tumor Development in Mice▿ †  
Molecular and Cellular Biology  2007;28(1):248-257.
The transforming growth factor β (TGFβ) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFβ function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFβ pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.
PMCID: PMC2223289  PMID: 17967875
17.  Activin-Like Kinase 2 Functions in Peri-implantation Uterine Signaling in Mice and Humans 
PLoS Genetics  2013;9(11):e1003863.
Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3′ UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.
Author Summary
A couple is defined as infertile when failing to become pregnant after one year of regular, unprotected intercourse. Infertility affects more than 10% of couples. The implantation of the embryo in the uterus is one of the most critical steps of pregnancy, and it has been estimated that 75% of pregnancy fails because of peri-implantation defects. An intricate network of molecular pathways regulates the peri-implantation process. It is known that the bone morphogenetic protein (BMP) pathways are part of this network, and herein we investigated how one of the BMP signaling receptors interacts with other factors in the uterus. Our results show an essential and conserved role of this BMP receptor during the implantation of the embryo in mice and humans. Furthermore, we discovered that BMPs act in a linear pathway upstream of two other key regulators of implantation, CEBPB and PGR.
PMCID: PMC3828128  PMID: 24244176
18.  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.
PMCID: PMC3786590  PMID: 18989307
19.  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.
PMCID: PMC1626139  PMID: 17080191
20.  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.
PMCID: PMC3744460  PMID: 23967333
21.  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.
PMCID: PMC3140685  PMID: 21669984
22.  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.
PMCID: PMC3499641  PMID: 22895517
23.  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.
PMCID: PMC3654696  PMID: 21382200
mendelian genetics; male infertility; asthenozoospermia; oligospermia
24.  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.
PMCID: PMC3244884  PMID: 21911476
male infertility; teratozoospermia; abnormal head morphology; ZPBP1 cDNA mutations; sp38
25.  Small-Molecule Inhibition of BRDT for Male Contraception 
Cell  2012;150(4):673-684.
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.
Graphical Abstract
► 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.
PMCID: PMC3420011  PMID: 22901802

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