Role of nuclear receptors in blastocyst implantation.

The regulation of blastocyst implantation in the uterus is orchestrated by the ovarian hormones estrogen and progesterone. These hormones act via their nuclear receptors to direct the transcriptional activity of the endometrial compartments and create a defined period in which the uterus is permissive to embryo implantation termed the "window of receptivity". Additional members of the nuclear receptor family have also been described to have a potential role in endometrial function. Much of what we know about the function of these nuclear receptors during implantation we have learned from the use of mouse models. Transgenic murine models with targeted gene ablation have allowed us to identify a complex network of paracrine signaling between the endometrial epithelium and stroma. While some of the critical molecules have been identified, the mechanism underlying the intricate communication between endometrial compartments during the implantation window has not been fully elucidated. Defining this mechanism will help identify markers of a receptive uterine environment, ultimately providing a useful tool to help improve the fertility outlook for reproductively challenged couples. The aim of this review is to outline our current understanding of how nuclear receptors and their effector molecules regulate blastocyst implantation in the endometrium.

Fertilization of squirrel monkey and hamster ova in the rabbit oviduct (xenogenous fertilization).

Homologous sperm and ova of either squirrel monkeys or hamsters were placed in the oviducts of pseudopregnant rabbits. Xenogenous fertilization rates of 36 and 60 percent were obtained for squirrel monkey and hamster gametes, respectively.

Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform.

Progesterone regulates reproductive function through two intracellular receptors, progesterone receptor-A (PR-A) and progesterone receptor-B (PR-B), that arise from a single gene and function as transcriptional regulators of progesterone-responsive genes. Although in vitro studies show that PR isoforms can display different transcriptional regulatory activities, their physiological significance is unknown. By selective ablation of PR-A in mice, we show that the PR-B isoform modulates a subset of reproductive functions of progesterone by regulation of a subset of progesterone-responsive target genes. Thus, PR-A and PR-B are functionally distinct mediators of progesterone action in vivo and should provide suitable targets for generation of tissue-selective progestins.

Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene.

The in vivo biological function of a steroid receptor coactivator was assessed in mice in which the SRC-1 gene was inactivated by gene targeting. Although in both sexes the homozygous mutants were viable and fertile, target organs such as uterus, prostate, testis, and mammary gland exhibited decreased growth and development in response to steroid hormones. Expression of RNA encoding TIF2, a member of the SRC-1 family, was increased in the SRC-1 null mutant, perhaps compensating partially for the loss of SRC-1 function in target tissues. The results indicate that SRC-1 mediates steroid hormone responses in vivo and that loss of its coactivator function results in partial resistance to hormone.

Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression.

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.

Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.

Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.

Episomal maintenance of a bovine papilloma virus vector in transgenic mice.

We have used a bovine papillomavirus-based vector to generate transgenic mice. Transgenic mice result from either pronuclear or cytoplasmic injections of the vector into fertilized eggs. Of 30 mice generated by microinjection, 27 (90%) contained the vector in its episomal form, at less than one copy per cell. This represents a highly efficient means of gene transfer in which the transgene is in a controlled genetic environment.

Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX).

This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.

Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation.

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF(-/-) embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF(-/-) embryos suffer significant defects in posterior development. Unlike GCNF(+/+) embryos, GCNF(-/-) embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF(-/-) embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF(-/-) embryos is 13 instead of 25 as in the GCNF(+/+) embryos. Interestingly, the tailbud of GCNF(-/-) embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF(-/-) embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

Ligand-inducible and liver-specific target gene expression in transgenic mice.

Transgenic mice have been used as models for tissue-specific gene regulation and to examine the molecular and cellular effects of altered expression of specific gene in disease processes such as tumorigenesis. Because of the deleterious effects of constitutive expression of transgenes, which frequently result in prenatal or postnatal death, only a limited number of disease models have been established in transgenic mice. We report an inducible binary transactivation system that permits the control of transgene expression in a tissue-specific and inducible fashion in mice. In this system, transcription of the target transgene is kept silent until turned on by the administration of an exogenous compound. We also demonstrate that expression level of the target gene can be induced three to four orders of magnitude and can be controlled by the administrated compound in a dose-dependent manner.

Steroid hormone regulation of Clca3 expression in the murine uterus.

Progesterone (P4) and its cognate receptor, the progesterone receptor (PGR), have important roles in the establishment and maintenance of pregnancy in the murine uterus. In previous studies, using high-density DNA microarray analysis, we identified a subset of genes whose expression is repressed by chronic P4-PGR activation in the uterus. The Clca3 gene is one of the genes whose expression is the most significantly downregulated by P4 and PGR. In the present study, we performed real-time RT-PCR and in situ hybridization to investigate the regulation of Clca3 by P4 and determine the pattern of expression of Clca3 in the uterus during early pregnancy. This analysis shows that Clca3 mRNA transcripts were detected in the luminal and glandular epithelium of the pseudopregnant uterus at day 0.5 and that the expression of Clca3 was not detected after day 3.5. P4 represses Clca3 mRNA synthesis in the luminal epithelial and glandular epithelial cells of the uterus in ovariectomized wild-type mice, but not in Pgr knockout (PRKO) mice. Conversely, estrogen (E2) induces Clca3 expression in the luminal epithelium and glandular epithelium, and this induction was repressed by P4 in the murine uterus. Analysis of the promoter region of Clca3 by in silico and transient transfection analysis in HEC-1A cells identified the regulation of Clca3 by estrogen receptor-alpha (ESR1) within the first 528 bp of 5'-flanking region of the Clca3 gene. Our studies identified Clca3 as a novel downregulated gene of PGR that is a direct target of E2 regulation.

Alterations in glucose homeostasis in SSTR1 gene-ablated mice.

SSTR1 is found on the majority of human pancreatic beta cells, however, its role in insulin secretion has yet to be elucidated. In this study, we used the SSTR1 knockout mouse model to examine the role of SSTR1 in insulin secretion and glucose homeostasis in mice. Despite the reported effect of SSTR1 in inhibiting growth hormone secretion, SSTR1-/- mice had significantly reduced body weight with growth retardation. Perfusion of isolated mouse pancreata at 3 months of age demonstrated a significant increase in insulin secretion in SSTR1-/- mice compared with that of WT controls. We also found that at 3 months of age, SSTR1-/- mice had significantly decreased levels of systemic insulin secretion and were glucose intolerant. However, SSTR1 gene-ablated mice had a much higher rate of insulin clearance compared to WT mice at the same age. When challenged at 12 months of age, we found SSTR1-/- mice had increased glucose tolerance with exaggerated increase of insulin levels at the end of the experiment. Immunochemical analysis showed that the pancreatic islets of SSTR1-/- mice had significantly decreased levels of somatostatin staining and a significant decrease of SSTR5 expression. These results demonstrate that SSTR1 plays an important role in the regulation of insulin secretion in the endocrine pancreas in mice.

Molecular mechanisms involved in progesterone receptor regulation of uterine function.

The ovarian steroid hormone progesterone is a major regulator of uterine function. The actions of this hormone is mediated through its cognate receptor, the progesterone receptor, Pgr. Ablation of the Pgr has shown that this receptor is critical for all female reproductive functions including the ability of the uterus to support and maintain the development of the implanting mouse embryo. High density DNA microarray analysis has identified direct and indirect targets of Pgr action. One of the targets of Pgr action is a member of the Hedgehog morphogen Indian Hedgehog, Ihh. Ihh and members of the Hh signaling cascade show a coordinate expression pattern in the mouse uterus during the preimplantation period of pregnancy. The expression of Ihh and its receptor Patched-1, Ptc1, as well as, down stream targets of Ihh-Ptch1 signaling, such as the orphan nuclear receptor COUP-TF II show that this morphogen pathway mediates communication between the uterine epithelial and stromal compartments. The members of the Ihh signaling axis may function to coordinate the proliferation, vascularization and differentiation of the uterine stroma during pregnancy. This analysis demonstrates that progesterone regulates uterine function in the mouse by coordinating the signals from the uterine epithelium to stroma in the preimplantation mouse uterus.

Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein.

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice.

The transcription factor achaete-scute homologue-1 (ASH1) is essential for neural differentiation during fetal development and is a cardinal feature of neuroendocrine (NE) tumors such as small cell lung cancer. To explore the potential of ASH1 to promote NE differentiation and tumorigenesis in the lung, we constitutively expressed the factor in nonendocrine airway epithelial cells using transgenic mice. Progressive airway hyperplasia and metaplasia developed beginning at 3 weeks of life. ASH1 potently enhanced the tumorigenic effect of SV40 large T antigen in airway epithelium. These doubly transgenic animals developed massive NE lung tumors, implying that ASH1 may cooperate with defects in p53, pRb, or related pathways in promoting NE lung carcinogenesis.

Metastatic prostate cancer in a transgenic mouse.

We have previously reported the development of a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (transgenic adenocarcinoma mouse prostate) model. We now describe the temporal and spatial consequences of transgene expression and report the identification and characterization of metastatic disease in the TRAMP model. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastases can be detected as early as 12 weeks of age. The common sites of metastases are the periaortic lymph nodes and lungs, with occasional metastases to the kidney, adrenal gland, and bone. By 28 weeks of age, 100% harbor metastatic prostate cancer in the lymph nodes or lungs. We have also demonstrated the loss of normal E-cadherin expression, as observed in human prostate cancer, as primary tumors become less differentiated and metastasize. The TRAMP model provides a consistent source of primary and metastatic tumors for histopathobiological and molecular analysis to further define the earliest molecular events involved in the genesis, progression, and metastasis of prostate cancer.

Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B.

Cdc25 A and B are dual-specificity phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely undefined. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia.

Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor.

BACKGROUND: The mechanisms responsible for tumor necrosis factor (TNF)-induced LV structural remodeling in the adult heart are not known. METHODS AND RESULTS: We generated a line of transgenic mice (MHCsTNF) with cardiac restricted overexpression of TNF that develop progressive LV dilation/remodeling from 4 to 12 weeks of age. During the early phases of LV structural remodeling, there was a significant increase in total matrix metalloproteinase (MMP) activity that corresponded to a decrease in total myocardial fibrillar collagen content. As the MHCsTNF mice aged, there was a significant decrease in total MMP zymographic activity that was accompanied by an increase in total fibrillar collagen content. The changes in total MMP activity and myocardial fibrillar collagen content were related to a time- dependent increase in myocardial tissue inhibitor of metalloproteinases (TIMP)-1 levels, resulting in a significant time-dependent decrease in the MMP activity/TIMP level ratio in the MHCsTNF mice. To determine a possible mechanism for the increase in myocardial fibrosis, we also measured levels of TGF-beta(1) and TGF-beta(2) protein levels, which were shown to be significantly elevated in the hearts of the MHCsTNF mice. CONCLUSIONS: Our results suggest that progressive time-dependent changes in the balance between MMP activity and TIMP activity are responsible, at least in part, for the spectrum of TNF-induced changes in the myofibrillar collagen content that occur during LV structural remodeling in the MHCsTNF mice.

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice.

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.

Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene.

The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.