Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

Deducing the stage of origin of Wilms' tumours from a developmental series of Wt1-mutant mice.

Wilms' tumours, paediatric kidney cancers, are the archetypal example of tumours caused through the disruption of normal development. The genetically best-defined subgroup of Wilms' tumours is the group caused by biallelic loss of the WT1 tumour suppressor gene. Here, we describe a developmental series of mouse models with conditional loss of Wt1 in different stages of nephron development before and after the mesenchymal-to-epithelial transition (MET). We demonstrate that Wt1 is essential for normal development at all kidney developmental stages under study. Comparison of genome-wide expression data from the mutant mouse models with human tumour material of mutant or wild-type WT1 datasets identified the stage of origin of human WT1-mutant tumours, and emphasizes fundamental differences between the two human tumour groups due to different developmental stages of origin.

11ß-Hydroxysteroid dehydrogenase type 1, but not type 2, deficiency worsens acute inflammation and experimental arthritis in mice.

Glucocorticoids profoundly influence immune responses, and synthetic glucocorticoids are widely used clinically for their potent antiinflammatory effects. Endogenous glucocorticoid action is modulated by the two isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). In vivo, 11ß-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11ß-HSD2 catalyzes the reverse reaction, inactivating intracellular glucocorticoids. Both enzymes have been postulated to modulate inflammatory responses. In the K/BxN serum transfer model of arthritis, 11ß-HSD1-deficient mice showed earlier onset and slower resolution of inflammation than wild-type controls, with greater exostoses in periarticular bone and, uniquely, ganglion cysts, consistent with greater inflammation. In contrast, K/BxN serum arthritis was unaffected by 11ß-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11ß-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11ß-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11ß-HSD1-deficient mice showed greater numbers of inflammatory cells in pleural lavages in carrageenan-induced pleurisy with lung pathology consistent with slower resolution. These data suggest that 11ß-HSD1 limits acute inflammation. In contrast, 11ß-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11ß-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

Smooth muscle cell-specific knockout of androgen receptor: a new model for prostatic disease.

Androgen-driven stromal-epithelial interactions play a key role in normal prostate development and function as well as in the progression of common prostatic diseases such as benign prostatic hyperplasia and prostate cancer. However, exactly how, and via which cell type, androgens mediate their effects in the adult prostate remains unclear. This study investigated the role for smooth muscle (SM) androgen signaling in normal adult prostate homeostasis and function using mice in which androgen receptor was selectively ablated from prostatic SM cells. In adulthood the knockout (KO) mice displayed a 44% reduction in prostate weight and exhibited histological abnormalities such as hyperplasia, inflammation, fibrosis, and reduced expression of epithelial, SM, and stem cell identify markers (e.g. p63 reduced by 27% and Pten by 31%). These changes emerged beyond puberty and were not explained by changes in serum hormones. Furthermore, in response to exogenous estradiol, adult KO mice displayed an 8.5-fold greater increase in prostate weight than controls and developed urinary retention. KO mice also demonstrated a reduced response to castration compared with controls. Together these results demonstrate that prostate SM cells are vital in mediating androgen-driven stromal-epithelial interactions in adult mouse prostates, determining cell identity and function and limiting hormone-dependent epithelial cell proliferation. This novel mouse model provides new insight into the possible role for SM androgen action in prostate disease.

Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5-16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg(-/-)) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

Persistence of functional hepatocyte-like cells in immune-compromised mice.

BACKGROUND: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. AIMS: In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. METHODS: hESC-derived HLCs were injected into the spleen of acutely injured NODscid(IL-2Rγ) null mice and analysed at various time points post-transplantation up to 3 months. RESULTS: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. CONCLUSIONS: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1.

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal-epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover.

Deletion of androgen receptor in the smooth muscle of the seminal vesicles impairs secretory function and alters its responsiveness to exogenous testosterone and estradiol.

The seminal vesicles (SVs), like much of the male reproductive tract, depend on androgen-driven stromal-epithelial interactions for normal development, structure, and function. The primary function of the SVs is to synthesize proteins that contribute to the seminal plasma and this is androgen dependent. However, the cell-specific role for androgen action in adult SVs remains unclear. This study analyzed the SV in mice with targeted ablation of androgen receptors specifically in smooth muscle cells (PTM-ARKO) to determine in vivo whether it is androgen action in a subset of the SV stroma, the smooth muscle cells, that drives epithelial function and identity. These mice have significantly smaller SVs in adulthood with less smooth muscle and reduced epithelial cell height. Less epithelial cell proliferation was observed in adult PTM-ARKO SVs, compared with controls, and production of seminal proteins was reduced, indicating global impairment of epithelial cell function in PTM-ARKO SVs. None of these changes could be explained by altered serum testosterone or estradiol concentrations. We also demonstrate altered SV responsiveness to exogenous testosterone and estradiol in PTM-ARKO mice, indicating that smooth muscle androgen receptors may limit the SV epithelial proliferative response to exogenous estrogens. These results therefore demonstrate that the smooth muscle cells play a vital role in androgen-driven stromal-epithelial interactions in the SV, determining epithelial cell structure and function as well as limiting the SV epithelial proliferative response to exogenous estrogens.

Mice with DNA repair gene Ercc1 deficiency in a neural crest lineage are a model for late-onset Hirschsprung disease.

The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4-6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.

Topical thymidine dinucleotide application protects against UVB-induced skin cancer in mice with DNA repair gene (Ercc1)-deficient skin.

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.

Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.

Mutationally activated K-ras 4A and 4B both mediate lung carcinogenesis.

To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-ras(tmDelta4A/-) mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras(+/-) mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-ras(tmDelta4A/tmDelta4A) mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-ras(tmDelta4A/tmDelta4A) mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras(+/-) and K-ras(tmDelta4A/-) mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.

Effects on kidney disease, fertility and development in mice inheriting a protein-truncating Denys-Drash syndrome allele (Wt1tmT396).

Denys-Drash syndrome (DDS) is caused by heterozygous mutations of the Wilms' tumour suppressor gene, WT1, characterised by early-onset diffuse mesangial sclerosis often associated with male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that the Wt1tmT396 allele induces DDS kidney disease in mice. In the present study heterozygotes (Wt1tmT396/+) were generated on inbred (129/Ola), crossbred (B6/129) and MF1 second backcross (MF1-N2) backgrounds. Whereas male heterozygotes on each background were fertile, inbred heterozygous females were infertile. Kidney disease (proteinuria and sclerosis) was not congenital and developed significantly earlier in inbred mice, although with variable onset. Disease onset in MF1-N2 stocks occurred later in Wt1tmT396/+ mice than reported previously for Wt1R394W/+ mice, and while no kidney disease has been reported in B6/129 Wt1+/- mice, B6/129 Wt1tmT396/+ mice were affected. Offspring of both male and female B6/129 and MF1-N2 Wt1tmT396/+ mice developed kidney disease, but its incidence was significantly higher in offspring of female heterozygotes. Wt1tmT396/tmT396 embryos exhibited identical developmental abnormalities to those reported for Wt1-/- embryos. The results indicate that the Wt1 (tmT396) allele does not predispose to Wilms' tumourigenesis or male pseudohermaphroditism, its effect on kidney disease and female fertility depends on genetic background, stochastic factors may affect disease onset, and disease transmission is subject to a partial parent-of-origin effect. Since the Wt1tmT396 allele has no detectable intrinsic functional activity in vivo, and kidney disease progression is affected by the type of Wt1 mutation, the data support the view that DDS nephropathy results from a dominant-negative action rather than WT1 haploinsufficiency or gain-of-function.

Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations.

PC4- and SF2-interacting protein 1 (Psip1)-also known as lens epithelium-derived growth factor (Ledgf)-is a chromatin-associated protein that has been implicated in transcriptional regulation, mRNA splicing, and cell survival in vitro, but its biological function in vivo is unknown. We identified an embryonic stem cell clone with disrupted Psip1 in a gene trap screen. The resulting Psip1-betageo fusion protein retains chromatin-binding activity and the PWWP and AT hook domains of the wild-type protein but is missing the highly conserved C terminus. The majority of mice homozygous for the disrupted Psip1 gene died perinatally, but some survived to adulthood and displayed a range of phenotypic abnormalities, including low fertility, an absence of epididymal fat pads, and a tendency to develop blepharitis. However, contrary to expectations, the lens epithelium was normal. The mutant mice also exhibited motor and/or behavioral defects such as hind limb clenching, reduced grip strength, and reduced locomotor activity. Finally, both Psip1(-/-) neonates and surviving adults had craniofacial and skeletal abnormalities. They had brachycephaly, small rib cages, and homeotic skeletal transformations with incomplete penetrance. The latter phenotypes suggest a role for Psip1 in the control of Hox expression and may also explain why PSIP1 (LEDGF) is found as a fusion partner with NUP98 in myeloid leukemias.

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice.

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.

A novel transmembrane MSP-containing protein that plays a role in right ventricle development.

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.

Alopecia areata with lymphocytic mural folliculitis affecting the isthmus in a thoroughbred mare.

A 13-year-old, thoroughbred mare was presented with an 8-year history of multifocal, generalized, noninflammatory alopecia and a 3-month history of alopecia, erythema and scaling of the white star on the forehead and muzzle. Histopathological examination of biopsy samples from multiple sites on the body (mane, neck, shoulder, flank and gluteal region) showed a subtle lymphocytic inflammatory infiltrate affecting and surrounding the anagen hair bulbs, consistent with a diagnosis of alopecia areata. The biopsy sample from the star on the forehead showed atrophic hair follicles with perifollicular and mural mononuclear folliculitis affecting the isthmus. Immunohistochemical staining with a CD3 marker confirmed the T-lymphocytic origin of the inflammatory infiltrate in all the samples. The concurrent presence of lymphocytic infiltration at the bulbar and isthmic level of the hair follicles in the same horse is unusual. This finding may represent a variation of the histological appearance of alopecia areata.