Unusually Giant Solitary Osteochondroma of the Ilium: A Case Report with Review of Literature.

Introduction: Osteochondroma is the most common benign bone tumor where a chondrogenic lesion is derived from aberrant cartilage from the perichondral ring. Although it commonly arises from the growing ends of long bones, less commonly, it may arise from the scapula, pelvis, or vertebra. Case Report: We encountered a 16-year-old male patient with a painless left pelvic solid mass for 3 years, which was suggestive of osteochondroma on X-ray and magnetic resonance imaging findings. Besides cosmetic issues, the main indication for surgery was the constant discomfort in wearing pants/shorts/belts. He underwent en bloc excision followed by a biopsy of the surgical specimen by two independent histopathologists confirming the tumor to be osteochondroma. He was followed up for 2 years with no signs of post-operative complications or recurrence. This case represents one of the very few reported so affecting the iliac wing, where the excision was performed before skeletal maturation. We also performed a review of the current literature on iliac wing osteochondroma to understand the tumor better, identify gaps in current knowledge, and suggest areas for future research. Conclusion: Since one of the differential diagnoses includes secondary chondrosarcoma, which could be a rare progression of osteochondroma, early recognition and comprehensive evaluation of such unusual cases needs to be dealt with a high index of suspicion to avoid misdiagnosis and to provide effective treatment.

Evaluation of mixture toxicity literature and chemical space: a data centric approach.

Using the Abstract Sifter tool to analyse PubMed, we reveal published mixture related research most commonly relates to water pollutants, pesticides, environmental pollutants, insecticides, soil pollutants, and chemicals described as persistent, bioaccumulative, and toxic. Furthermore, we discern individual chemicals that also identify as priority chemicals in biomonitoring initiatives and using an ontology-based chemical classification, at the level of the chemical subclass, found these priority chemicals overlap with just 9% of the REACH chemical space.

Identifying the landscape of developmental toxicity new approach methodologies.

BACKGROUND: The dynamics and complexities of in utero fetal development create significant challenges in transitioning from lab animal-centric developmental toxicity testing methods to assessment strategies based on new approach methodologies (NAMs). Nevertheless, considerable progress is being made, stimulated by increased research investments and scientific advances, such as induced pluripotent stem cell-derived models. To help identify developmental toxicity NAMs for toxicity screening and potential funding through the American Chemistry Council's Long-Range Research Initiative, a systematic literature review was conducted to better understand the current landscape of developmental toxicity NAMs. METHODS: Scoping review tools were used to systematically survey the literature (2010-2021; ~18,000 references identified), results and metadata were then extracted, and a user-friendly interactive dashboard was created. RESULTS: The data visualization dashboard, developed using Tableau® software, is provided as a free, open-access web tool. This dashboard enables straightforward interactive queries and visualizations to identify trends and to distinguish and understand areas or NAMs where research has been most, or least focused. CONCLUSIONS: Herein, we describe the approach and methods used, summarize the benefits and challenges of applying the systematic-review techniques, and highlight the types of questions and answers for which the dashboard can be used to explore the many different facets of developmental toxicity NAMs.

Expression patterns of chemokine (C-C motif) ligand 2, prostaglandin F2A receptor and immediate early genes at mRNA level in the bovine corpus luteum after intrauterine treatment with a low dose of prostaglandin F2A.

The present study evaluated expression patterns of chemokine (C-C motif) ligand 2 gene/Monocyte chemoattractant protein-1 gene (CCL2/MCP-1), prostaglandin F2 alpha receptor gene (PTGFR) and immediate early genes including nuclear receptor subfamily 4, group A, member 1 (NR4A1), early growth response 1 (EGR1) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in cells of the bovine corpus luteum after intrauterine infusion of a low dose of prostaglandin F2α (PGF2A) aimed at enhancing our understanding of the mechanisms of luteolysis. Holstein dairy cows were superovulated (>6 corpora lutea [CL]) and on day 9 of the estrous cycle were infused with a low dose of PGF2A (0.5 mg PGF2A in 0.25 ml phosphate buffered saline) into the greater curvature of the uterine horn ipsilateral to the CL. Ultrasound-guided biopsy samples of different CL were collected at 0 min, 15 min, 30 min, 1h, 2h and 6h after PGF2A infusion. Expression profiles and localization of mRNA for PTGFR, CCL2/MCP-1, and immediate early genes (NR4A1, EGR1 and FOS), were investigated by using qPCR and in situ hybridization. The concentrations of early response genes including FOS, NR4A1, and EGR1 exhibited the greatest increase at 30 min after PGF2A, compared to other time points. Expression profile of CCL2 mRNA increased gradually after intrauterine infusion of PGF2A with maximal up-regulation for CCL2 at 6h. Abundance of PTGFR mRNA only increased at 15 min and significantly decreased at 6h, compared to 0 min. Cellular localizations of all studied genes except CCL2 (primarily localized to apparent immune cells) were predominantly visualized in large luteal cells. Interestingly, early response genes demonstrated a changing profile in cellular localization with initial responses appearing to be in both large luteal cells and endothelial cells, although no staining for PTGFR mRNA was observed in endothelial cells. Later, sustained responses, were only observed in large luteal cells, although PTGFR mRNA was decreasing in large luteal cells over time after PGF2A. The involvement of the immune system was also highlighted by the immediate increases in CCL2 mRNA that became much greater over time as there was an apparent influx of CCL2-positive immune cells. Thus, the temporal and cell-specific localization patterns for the studied mRNA demonstrate the complex pathways that are responsible for initiation of luteolysis in the bovine CL.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Diarrhea in kidney transplant recipients: Etiology and outcome.

BACKGROUND AND AIM: Diarrhea in kidney transplant recipients influences outcome of transplantation. Data from India in this regard are sparse and do not address the differential outcome of infective and non-infective diarrhea. We studied the demographic data, laboratory findings, treatment response, disease duration, and outcome of diarrhea in kidney transplant recipients, and the differential outcome between infective and non-infective diarrhea, if any. METHODS: All kidney transplant recipients who were referred to the Division of Gastroenterology with diarrhea between June 2015 and February 2017 were prospectively included. Demographic, clinical and laboratory data, graft function, treatment administered, and outcome were noted, and the patients were followed up for 3 months. RESULTS: Forty-seven patients (median age 45 years, range 16-78; 34 men) with 64 episodes of diarrhea were studied. Thirty-three (51.5%) episodes were attributed to infections. Eleven (17%) were immunosuppressant-induced (mycophenolate 8, tacrolimus 2, cyclosporine 1). Twenty (31%) were due to other causes (antibiotics 6, laxatives 3, irritable bowel syndrome 3, sepsis 8). Fifty-three episodes (82%) had graft dysfunction during the diarrheal episodes. Mean increase in serum creatinine was 45% in the infectious diarrhea group and 95% in the non-infectious diarrhea group (p < 0.05). Median time to resolution of diarrhea was 3 days. With improvement in diarrhea, return to pre-diarrhea creatinine levels occurred in 87% of episodes at 3 months. CONCLUSION: One-half of episodes of diarrhea in kidney transplant recipients were non-infectious in origin. Seventeen percent were attributed to immunosuppressants, requiring dose modification. More than 80% were associated with worsening of graft function. Recovery of graft function to baseline was seen in a majority of cases with the resolution of diarrhea.

Making a Positive Diagnosis of Intestinal Tuberculosis with the Aid of New Biologic and Histologic Features: How Far Have We Reached?

BACKGROUND: The diagnosis of intestinal tuberculosis (TB) and its differentiation from Crohn's disease (CD) remain a challenge. We review here in detail the various methods for the diagnosis of intestinal TB. SUMMARY: Colonoscopy findings in intestinal TB are useful and suggestive; histopathology of colonoscopic biopsies is contributory but rarely confirmatory. Increasing the number of colonoscopic biopsies increases the histological yield. Recent culture methods that have improved the yield for TB offer hope. Mycobacteria Growth Indicator Tube (MGIT) culture is now the standard of care as its yield is superior to that of the traditional Lowenstein-Jensen medium. Increasing the number of colonoscopic biopsy samples for MGIT culture can increase the yield. The culture and histology are complimentary. Even then a significant proportion of patients do not have a positive diagnosis of intestinal TB. Scoring systems have been developed with a sensitivity and specificity of 90 and 60%, respectively, but their utility in routine practice is yet to be established. Similarly, the ratio of visceral fat to total fat is helpful in differentiating CD from intestinal TB. Polymerase chain reaction has been used but its value seems uncertain. Gene Xpert® in an emerging technique that has been found to be useful in the diagnosis of pulmonary TB, and its utility in intestinal TB needs to be looked at. Newer technologies like TB-LAMP (loop-mediated isothermal amplification) need to be assessed in clinical studies. KEY MESSAGE: Optimization of the present diagnostic tools (taking an adequate number of biopsies for histology and culture) and study of newer techniques to learn their actual utility seems to be the way forward.

Edar is a downstream target of beta-catenin and drives collagen accumulation in the mouse prostate.

Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.

Do additional colonoscopic biopsies increase the yield of Mycobacterium tuberculosis culture in suspected ileo-colonic tuberculosis?

BACKGROUND: Isolation of Mycobacterium tuberculosis on culture is vital for differentiating intestinal tuberculosis (ITB) from Crohn's disease (when histology is not diagnostic) and for diagnosis of multidrug-resistant tuberculosis. The current yield of TB culture (< 50%) from colonoscopic biopsy tissue is not satisfactory. AIM: To determine whether more colonoscopic biopsies can increase the yield of TB culture in patients with ITB. METHODS: In this prospective study, in patients who underwent colonoscopy for suspected ITB, four biopsies were taken (container 1) followed by an additional four biopsies (container 2) for TB culture, from involved regions. The culture was done using Mycobacterium Growth Indicator Tube (MGIT) 960. A final diagnosis of ITB was made if TB culture was positive, there was unequivocal histological evidence of TB, or there was unequivocal evidence of TB elsewhere in the body, in the absence of another diagnosis. RESULTS: Of 182 patients enrolled (mean age 37.5 [SD 17.2] years; 93 [51.5%] women), 70 (38.4%) were finally diagnosed to have ITB. MGIT culture was positive in 29 (41.4%), 27 (38.5%), and 37 (52.8%) of 70 patients from containers 1, container 2, and combined eight biopsies, respectively. The incremental yield of eight biopsies was 11.4% (95% confidence interval [CI] 5.1 to 21.3%) as compared to container 1 and 14.3% (95% CI 7.1 to 24.7%) as compared to container 2. CONCLUSION: Additional four (total eight) colonoscopic biopsies improved the yield of TB culture positivity over four biopsies by 11.4% to 14.3%, to 52.8%; this increase is clinically useful.

Drug resistance in intestinal tuberculosis: A reason to worry?

BACKGROUND AND AIM: Emergence of drug resistance in intestinal tuberculosis (ITB) makes the treatment of this condition challenging. While there is growing evidence of multiple and extensive drug resistance in pulmonary and glandular tuberculosis (TB), literature regarding susceptibility and resistance patterns in ITB is scarce. The aim of the current paper was to study the prevalence of drug resistance in patients with ITB. METHODS: Among patients presenting to a tertiary care hospital in Mumbai between 2008 and 2016, records of all patients with ITB, whose mucosal biopsy (obtained at ileocolonoscopy) tissue culture was positive for Mycobacterium tuberculosis and in whom drug sensitivity testing was performed, were retrospectively analyzed. Sensitivity and resistance to single or multiple anti-TB drugs were noted. RESULTS: A total of 43 patients were included, of whom 10 (23.2%) patients were diagnosed to have resistance to at least one first-line anti-TB drug. Resistance to isoniazid was the most common (nine patients), followed by rifampicin (six), pyrazinamide (five), streptomycin and ethionamide (four each), ethambutol, moxifloxacin and ofloxacin (three each), and p-amino salicylic acid (one). Six patients (13.9%) had multidrug-resistant TB and needed second-line anti-TB therapy as per drug sensitivity pattern. There was no patient with extensive drug-resistant TB. CONCLUSION: Twenty-three percent of our patients with ITB tested for drug resistance had drug resistance, 13.9% being multidrug resistant and needing second-line anti-TB therapy.

Beta-catenin and estrogen signaling collaborate to drive cyclin D1 expression in developing mouse prostate.

Androgen, beta-catenin (CTNNB1), and estrogen pathways stimulate proliferative growth of developing mouse prostate but how these pathways interact is not fully understood. We previously found that androgens induce CTNNB1 signaling in mouse urogenital sinus (UGS) epithelium from which prostatic ductal epithelium derives. Others have shown that low estradiol concentrations induce UGS epithelial proliferative growth. Here, we found that CTNNB1 signaling overlaps cyclin D1 (CCND1) expression in prostatic buds and we used a genetic approach to test whether CTNNB1 signaling induces CCND1 expression. We observed an unexpected sexually dimorphic response to hyperactive CCNTB1 signaling: in male mouse UGS it increased Ccnd1 mRNA abundance without increasing its protein abundance but in female UGS it increased Ccnd1 mRNA and protein abundance, suggesting a potential role for estrogens in stabilizing CCND1 protein. Treating wild type male UGS explants with androgen and either 17ß-estradiol or a proteasome inhibitor increased CCND1 protein and KI67 labeling in prostatic bud epithelium. Together, our results are consistent with an epithelial proliferative growth mechanism linking CTNNB1-driven Ccnd1 transcription and estrogen-mediated CCND1 protein stabilization.

In utero exposure to TCDD alters Wnt signaling during mouse prostate development: linking ventral prostate agenesis to downregulated ß-catenin signaling.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes ventral prostate agenesis in C57BL/6J mice by preventing ventral prostatic budding in the embryonic urogenital sinus (UGS). TCDD (5 µg/kg, po) administered to pregnant dams on embryonic day 15.5 (E15.5) activates the aryl hydrocarbon receptor in the UGS mesenchyme, disrupting the mesenchymally derived paracrine signaling that instructs epithelial prostatic budding. How TCDD alters the mesenchymal milieu is not well understood. We previously showed that TCDD disrupts some aspects of Wnt signaling in UGSs grown in vitro. Here we provide the first comprehensive, in vivo characterization of Wnt signaling in male E16.5 UGSs during normal development, and after in utero TCDD exposure. Vehicle- and TCDD-exposed UGSs were probed by in situ hybridization to assess relative abundance and localization of RNA from 46 genes that regulate Wnt signaling. TCDD altered the staining pattern of five genes, increasing staining for Wnt10a and Wnt16 and decreasing staining for Ror2, Rspo2, and Wif1. We also used immunohistochemistry to show, for the first time, activation of ß-catenin (CTNNB1) signaling in ventral basal epithelium of control UGSs at E16.5. This onset of CTNNB1 signaling occurred immediately prior to the initiation of ventral prostatic budding and is characterized by a pronounced increase in CTNNB1 nuclear localization and subsequent expression of the CTNNB1 signaling target gene, Lef1. In utero TCDD exposure prevented the onset of CTNNB1 signaling and LEF1 expression in the ventral basal epithelium, thereby elucidating a likely mechanism by which TCDD contributes to failed prostatic budding in the ventral UGS.

DNA methylation of E-cadherin is a priming mechanism for prostate development.

In prostate and other epithelial cancers, E-cadherin (CDH1) is downregulated inappropriately by DNA methylation to promote an invasive phenotype. Though cancer frequently involves a reawakening of developmental signaling pathways, whether DNA methylation of Cdh1 occurs during organogenesis has not been determined. Here we show that DNA methylation of Cdh1 mediates outgrowth of developing prostate ducts. During the three-day gestational window leading up to and including prostate ductal initiation, Cdh1 promoter methylation increases and its mRNA and protein abundance decreases in epithelium giving rise to prostatic buds. DNA methylation is required for prostate specification, ductal outgrowth, and branching morphogenesis. All three endpoints are impaired by a DNA methylation inhibitor, which also decreases Cdh1 promoter methylation and increases Cdh1 mRNA and protein abundance. A CDH1 function-blocking antibody restores prostatic identity, bud outgrowth, and potentiates epithelial differentiation in the presence of the DNA methylation inhibitor. This is the first study to mechanistically link acquired changes in DNA methylation to the normal process of prostate organogenesis. We propose a novel mechanism whereby Cdh1 promoter methylation restricts Cdh1 abundance in developing prostate epithelium to create a permissive environment for prostatic bud outgrowth. Thus, DNA methylation primes the prostate primordium to respond to developmental cues mediating outgrowth, differentiation and maturation of the ductal network.

Prostate angiogenesis in development and inflammation.

BACKGROUND: Prostatic inflammation is an important factor in development and progression of BPH/LUTS. This study was performed to characterize the normal development and vascular anatomy of the mouse prostate and then examine, for the first time, the effects of prostatic inflammation on the prostate vasculature. METHODS: Adult mice were perfused with India ink to visualize the prostatic vascular anatomy. Immunostaining was performed on the E16.5 UGS and the P5, P20, and adult prostate to characterize vascular development. Uropathogenic E. coli 1677 was instilled transurethrally into adult male mice to induce prostate inflammation. RT-PCR and BrdU labeling was performed to assay anigogenic factor expression and endothelial proliferation, respectively. RESULTS: An artery on the ventral surface of the bladder trifurcates near the bladder neck to supply the prostate lobes and seminal vesicle. Development of the prostatic vascular system is associated with endothelial proliferation and robust expression of pro-angiogenic factors Pecam1, Tie1, Tek, Angpt1, Angpt2, Fgf2, Vegfa, Vegfc, and Figf. Bacterial-induced prostatic inflammation induced endothelial cell proliferation and increased vascular density but surprisingly decreased pro-angiogenic factor expression. CONCLUSIONS: The striking decrease in pro-angiogenic factor mRNA expression associated with endothelial proliferation and increased vascular density during inflammation suggests that endothelial response to injury is not a recapitulation of normal development and may be initiated and regulated by different regulatory mechanisms.

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract.

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.

Beta-catenin (CTNNB1) induces Bmp expression in urogenital sinus epithelium and participates in prostatic bud initiation and patterning.

Fetal prostate development is initiated by androgens and patterned by androgen dependent and independent signals. How these signals integrate to control epithelial cell differentiation and prostatic bud patterning is not fully understood. To test the role of beta-catenin (Ctnnb1) in this process, we used a genetic approach to conditionally delete or stabilize Ctnnb1 in urogenital sinus (UGS) epithelium from which the prostate derives. Two opposing mechanisms of action were revealed. By deleting Ctnnb1, we found it is required for separation of UGS from cloaca, emergence or maintenance of differentiated UGS basal epithelium and formation of prostatic buds. By genetically inducing a patchy subset of UGS epithelial cells to express excess CTNNB1, we found its excess abundance increases Bmp expression and leads to a global impairment of prostatic bud formation. Addition of NOGGIN partially restores prostatic budding in UGS explants with excess Ctnnb1. These results indicate a requirement for Ctnnb1 in UGS basal epithelial cell differentiation, prostatic bud initiation and bud spacing and suggest some of these actions are mediated in part through activation of BMP signaling.

TCDD inhibits heart regeneration in adult zebrafish.

Normal adult zebrafish can completely regenerate lost myocardium following partial amputation of the ventricle apex. We report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly impairs this regeneration. Adult male zebrafish were injected with vehicle (control) or TCDD (70ng/g, ip) 1 day prior to partial amputation of the ventricle apex. Gross observation and histological analysis of the amputated heart at 21 days postamputation revealed that TCDD-exposed fish had not progressed beyond the initial clot formation stage, whereas the vehicle control fish showed substantial recovery and almost complete resolution of the formed clot. In contrast, hearts that were not surgically wounded showed no signs of TCDD toxicity. Striking features in the TCDD-exposed hearts were the absence of the normal sheath of new tissue enveloping the wound and the absence of intense cell proliferation at the site of the wound. In addition, the patterns of collagen deposition at the wound site were different between the TCDD and vehicle groups. Because the receptor for TCDD is the aryl hydrocarbon receptor ligand-activated transcriptional regulator, we examined the effects of TCDD exposure on gene expression in the ventricle using DNA microarrays. Samples were collected just prior to amputation and at 6h and 7 days postamputation. TCDD-pretreated hearts had dysregulated expression of genes involved in heart function, tissue regeneration, cell growth, and extracellular matrix. Because embryonic, but not adult, hearts are major targets for TCDD-induced cardiotoxicity, we speculate that the need for embryonic-like cells in regeneration is connected with the effects of TCDD in inhibiting the response to wounding.

Wnt inhibitory factor 1 (Wif1) is regulated by androgens and enhances androgen-dependent prostate development.

Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/ß-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.

Visualization and quantification of mouse prostate development by in situ hybridization.

The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.

Patterns of gene expression in the bovine corpus luteum following repeated intrauterine infusions of low doses of prostaglandin F2alpha.

Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.