Roles of AFAP1-AS1 in Gynecology and Urogenital System.

BACKGROUND: Human disease onset and progression are strongly associated with aberrant long noncoding RNA (lncRNA) expression, highlighting the functional regulatory role of lncRNA. Actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1), a member of lncRNAs, is located on the antisense strand of Actin filament-associated protein 1 (AFAP1). METHODS: We conducted a comprehensive review of AFAP1-AS1's functions in gynecology and urogenital systems using the "PubMed" database. RESULTS: Our analysis reveals that AFAP1-AS1 is overexpressed and engages in the initiation and process of gynecological and urogenital diseases. The regulatory mechanisms employed by AFAP1-AS1 involve four major strategies: gene-level effects, competition for microRNA (miRNA) repression, protein binding, participation in signaling networks that influence cellular processes such as proliferative phenotype, migration, invasiveness, epithelial-mesenchymal transition (EMT), cycle regulation, drug resistance, and more. Furthermore, AFAP1-AS1 is implicated in guiding clinicopathological characteristics. CONCLUSION: AFAP1-AS1 holds promise as a potent diagnostics and treatment option for gynecological and genitourinary systems in the future.

In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo: Part II: Pathology: Part II: Pathology.

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change-size, number, and physicochemical composition-in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.

Primary Amyloidosis of the Genitourinary Tract.

CONTEXT.­: Amyloidosis is caused by the deposition of misfolded proteins as insoluble eosinophilic material in the extracellular tissues of the body, leading to impairment of organ function. It can be systemic or localized. Localized genitourinary tract amyloidosis is rare and can be incidentally seen; however, in some cases, it can be the only presenting disease. OBJECTIVE.­: To review the clinical presentation and pathologic findings related to primary amyloidosis of the urogenital system and highlight some of the associated pathologic findings based on our personal experience. DATA SOURCES.­: Published peer-reviewed literature and personal experience of the senior author. CONCLUSIONS.­: Primary localized amyloidosis within the urogenital tract can present as a neoplastic process and may be clinically and radiologically considered as a mass. Awareness of primary amyloidosis by pathologists and clinicians is required for accurate diagnosis and proper patient management.

Ectopic subcutaneous transplantation of fetal rat urogenital sinus and seminal vesicle promotes the organ growth and formation.

PURPOSE: The fate of subcutaneously transplanted urogenital sinus (UGS) and seminal vesicle (SV) was investigated in the present study. MATERIALS AND METHODS: Fetal UGS and SV extracted from 20-embryonic-day-old male normal and GFP transgenic rats were subcutaneously transplanted into 7-week-old male immunologically inhibited rats. The transplants were then examined at 2, 4, 8, and 16 weeks after transplantation. We analyzed the survival ratio, weight, and histopathology as well as the immunohistochemical characteristics of the transplanted tissues. For control experiments, 2-, 4-, 8-, and 16-week-old normal male rats were used. RESULTS: Almost all of the transplanted tissues survived under the skin, and the tissue weights increased over time after transplantation. The histopathological characteristics and immunohistochemical staining pattern with certain antibodies of the transplanted tissues were similar to those of normal adult rat prostate and seminal vesicle. The transplanted GFP transgenic tissues demonstrated spontaneous growth and organ formation under the skin, showing distribution and movement of transplanted cells and tissues. CONCLUSION: Subcutaneously transplanted fetal UGS and SV were able to develop into mature adult organs.

Practical Molecular Testing in a Clinical Genitourinary Service.

CONTEXT.­: Molecular testing is increasingly playing a key role in the diagnosis, prognosis, and treatment of neoplasms of the genitourinary system. OBJECTIVE.­: To provide a general overview of the clinically relevant molecular tests available for neoplasms of the genitourinary tract. DATA SOURCES.­: Relevant medical literature indexed on PubMed. CONCLUSIONS.­: Understanding of the molecular oncology of genitourinary neoplasms is rapidly advancing, and the pathologist must be aware of the practical implications of molecular testing. While many genomic abnormalities are not yet clinically relevant, there is an increasing library of ancillary tests that may guide diagnosis, prognosis, and/or treatment of many neoplasms. Recurrent genomic abnormalities have been identified in many types of renal cell carcinoma, and some types of renal cell carcinoma are specifically defined by the molecular abnormality. Two major routes of developing urothelial carcinoma have been molecularly described. Recurrent translocations involving ETS family genes are found in approximately half of prostate cancer cases. Testicular germ cell tumors typically harbor i(12p). Penile neoplasms are often high-risk human papillomavirus-driven cancers. Nonetheless, even as genitourinary neoplasms are increasingly better understood at the molecular level, further research with eventual clinical validation is needed for optimal diagnosis, prognosis, and treatment of aggressive malignancies in the genitourinary tract.

Sox9 in mouse urogenital sinus epithelium mediates elongation of prostatic buds and expression of genes involved in epithelial cell migration.

Previous studies identified Sox9 as a critical mediator of prostate development but the precise stage when Sox9 acts had not been determined. A genetic approach was used to delete Sox9 from mouse urogenital sinus epithelium (UGE) prior to prostate specification. All prostatic bud types (anterior, dorsolateral and ventral) were stunted in Sox9 conditional knockouts (cKOs) even though the number of prostatic buds did not differ from that of controls. We concluded that Sox9 is required for prostatic bud elongation and compared control male, control female, Sox9 cKO male and Sox9 cKO female UGE transcriptomes to identify potential molecular mediators. We identified 702 sex-dependent and 95 Sox9-dependent genes. Thirty-one genes were expressed in both a sex- and Sox9-dependent pattern. A comparison of Sox9 cKO female vs control female UGE transcriptomes revealed 74 Sox9-dependent genes, some of which also function in cell migration. SOX9 regulates, directly or indirectly, a largely different profile of genes in male and female UGE. Eighty-three percent of Sox9-dependent genes in male UGE were not Sox9-dependent in female UGE. Only 16 genes were Sox9-dependent in the UGE of both sexes and seven had cell migration functions. These results support the notion that Sox9 promotes cell migration activities needed for prostate ductal elongation.

Effects of secondary biological treatment plant effluent administration, as drinking water, to rats' urogenital system in relation to cadmium and lead accumulation.

The aim of this study was to examine the effect of the secondary biological treatment plant effluent administration on the kidneys, urinary bladder, and testis of Wistar rats in relation to lead (Pb) and cadmium (Cd) accumulation, since such an effluent is used for irrigation of edible plants. Male Wistar rats, randomly assigned into 5 groups, were treated with domestic sewage effluent (DSE) for 24 months. Cadmium and lead concentrations in the DSE, rats' tissues, and urine were estimated by means of atomic spectroscopy. Lead was rapidly accumulated in high amounts in rats' kidney and to a lesser extent in the testis whereas Cd concentration was raised in all tissues examined. Deposition of Cd and Pd in the kidney of the rats resulted in profound damage over time. The results showed that long-term administration to DSE as drinking water exposes living organisms to urogenital stress related to heavy metal concentration and pH of the effluent.

Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions.

Background: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damage and improve women's health worldwide. In this study, we evaluated a novel multiplex real-time PCR melting curve assay method for the simultaneous detection of 9 STD pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and herpes simplex virus. Methods: The analytical performance of the method, including its limit of detection (LOD), specificity, repeatability, and effect on different DNA extraction kits were evaluated. Additionally, we obtained 1,328 clinical specimens from 3 hospitals to detect the 9 STD pathogens using multiplex real-time PCR melting curve and Sanger sequencing, to evaluate the sensitivity, specificity, and consistency of the assay method. Results: The results showed that the analytical sensitivity of the novel multiplex real-time PCR melting curve assay is very excellent, with LOD of DNA corresponding to <200 copies/µL for the DNA of the 9 STDs and 1.00 × 104 color change unit /ml for those of UU and UP. Additionally, this assay demonstrated excellent analytical specificity, excellent repeatability, and its results had no effect of different DNA extraction kits. The performance, in terms of sensitivity (91.06-100%) and specificity (99.14-100%), was remarkable, since the consistency between it and Sanger sequencing was more than 0.85 in the clinic. Conclusion: The novel multiplex real-time PCR melting curve assay method has high sensitivity and specificity, relatively low cost, and simple to use for the simultaneous detection of 9 STD pathogens in genitourinary secretions.

The impact of the sepsis on female urogenital system: the role of pregabalin.

PURPOSE: The aim of the study was to investigate the oxidative damage and inflammatory effects of sepsis on the urogenital system in the Lipopolysaccharide (LPS)-induced sepsis model and ameliorating role of Pregabalin (PGB). METHODS: Twenty-four female Wistar Albino rats (12 months old) were divided into 3 groups as follows: Sepsis group (Group S) (5 mg/kg LPS, i.p, single dose); Sepsis+ PGB group (Group SP) (5 mg/kg LPS, i.p, single dose and 30 mg/kg PGB); Control group (Group C) (0.1 ml/oral and i.p. saline, single dose), 6 h after LPS administration, the animals were killed. Subsequently, analyses of urogenital tissue oxidant/antioxidant status, histopathological and immunohistochemical analyses were performed. RESULTS: Total oxidative status (TOS) and oxidative stress index (OSI) values in the urogenital tissues were increased in Group S (Total anti-oxidative status (TAS) decreased) compared to the Control group (p < 0.05). PGB improved these values (p < 0.05). The immunohistochemical markers [Caspase-3, granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), Serum Amyloid A (SAA) and inducible nitric oxide synthase (iNOS)] were significantly increased in Group S except for bladder (p < 0.001). Statistically significant immunohistochemical positiveness was found only for IL-6 in urinary bladder, though all the others values were negative. With the administration of PGB (Group SP), the expressions of these immunoreactions were markedly decreased (p < 0.001). CONCLUSIONS: These findings demonstrated that sepsis caused oxidative stress and inflammation in the urogenital tissues. We have revealed that PGB ameliorated tissue damage caused by sepsis.

Widespread Cell-Specific Prolactin Receptor Expression in Multiple Murine Organs.

The prolactin receptor (Prlr) mediates not only the multiple effects of prolactin, but also those of the placental lactogens and, in humans, some actions of growth hormone. Although Prlr expression has been reported to be widespread in the body, specific cellular expression patterns within tissues are undefined for many organs. One persisting problem in investigating Prlr function is that the protein is difficult to detect using conventional methods. To allow investigation of Prlr expression with a single cell resolution, we have recently developed a knock-in mouse strain in which Cre recombinase is expressed together with the long isoform of the Prlr using an internal ribosome entry site. When crossed to a Cre-dependent reporter mouse strain, Cre-mediated recombination will genetically label cells that acutely express the Prlr as well as cells that have transiently expressed the Prlr during development. We report here the anatomical distribution of cells which express the fluorescent reporter τ green fluorescent protein in a total of 38 organs prepared from young adult male and female Prlr reporter mice. Our results establish a resource for dissecting the functional role of Prlr in multiple murine tissues.

Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats.

Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralen-treated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.

An extended regulatory landscape drives Tbx18 activity in a variety of prostate-associated cell lineages.

The evolutionarily conserved transcription factor, Tbx18, is expressed in a dynamic pattern throughout embryonic and early postnatal life and plays crucial roles in the development of multiple organ systems. Previous studies have indicated that this dynamic function is controlled by an expansive regulatory structure, extending far upstream and downstream of the gene. With the goal of identifying elements that interact with the Tbx18 promoter in developing prostate, we coupled chromatin conformation capture (4C) and ATAC-seq from embryonic day 18.5 (E18.5) mouse urogenital sinus (UGS), where Tbx18 is highly expressed. The data revealed dozens of active chromatin elements distributed throughout a 1.5 million base pair topologically associating domain (TAD). To identify cell types contributing to this chromatin signal, we used lineage tracing methods with a Tbx18 Cre "knock-in" allele; these data show clearly that Tbx18-expressing precursors differentiate into wide array of cell types in multiple tissue compartments, most of which have not been previously reported. We also used a 209 kb Cre-expressing Tbx18 transgene, to partition enhancers for specific precursor types into two rough spatial domains. Within this central 209 kb compartment, we identified ECR1, previously described to regulate Tbx18 expression in ureter, as an active regulator of UGS expression. Together these data define the diverse fates of Tbx18+ precursors in prostate-associated tissues for the first time, and identify a highly active TAD controlling the gene's essential function in this tissue.

Immunohistochemical expression analysis of the human fetal lower urogenital tract.

We have studied the ontogeny of the developing human male and female urogenital tracts from 9 weeks (indifferent stage) to 16 weeks (advanced sex differentiation) of gestation by immunohistochemistry on mid-sagittal sections. Sixteen human fetal pelvises were serial sectioned in the sagittal plane and stained with antibodies to epithelial, muscle, nerve, proliferation and hormone receptor markers. Key findings are: (1) The corpus cavernosum in males and females extends into the glans penis and clitoris, respectively, during the ambisexual stage (9 weeks) and thus appears to be an androgen-independent event. (2) The entire human male (and female) urethra is endodermal in origin based on the presence of FOXA1, KRT 7, uroplakin, and the absence of KRT10 staining. The endoderm of the urethra interfaces with ectodermal epidermis at the site of the urethral meatus. (3) The surface epithelium of the verumontanum is endodermal in origin (FOXA1-positive) with a possible contribution of Pax2-positive epithelial cells implying additional input from the Wolffian duct epithelium. (4) Prostatic ducts arise from the endodermal (FOXA1-positive) urogenital sinus epithelium near the verumontanum. (5) Immunohistochemical staining of mid-sagittal and para-sagittal sections revealed the external anal sphincter, levator ani, bulbospongiosus muscle and the anatomic relationships between these developing skeletal muscles and organs of the male and female reproductive tracts. Future studies of normal human developmental anatomy will lay the foundation for understanding congenital anomalies of the lower urogenital tract.

Development of the human prostate.

This paper provides a detailed compilation of human prostatic development that includes human fetal prostatic gross anatomy, histology, and ontogeny of selected epithelial and mesenchymal differentiation markers and signaling molecules throughout the stages of human prostatic development: (a) pre-bud urogenital sinus (UGS), (b) emergence of solid prostatic epithelial buds from urogenital sinus epithelium (UGE), (c) bud elongation and branching, (d) canalization of the solid epithelial cords, (e) differentiation of luminal and basal epithelial cells, and (f) secretory cytodifferentiation. Additionally, we describe the use of xenografts to assess the actions of androgens and estrogens on human fetal prostatic development. In this regard, we report a new model of de novo DHT-induction of prostatic development from xenografts of human fetal female urethras, which emphasizes the utility of the xenograft approach for investigation of initiation of human prostatic development. These studies raise the possibility of molecular mechanistic studies on human prostatic development through the use of tissue recombinants composed of mutant mouse UGM combined with human fetal prostatic epithelium. Our compilation of human prostatic developmental processes is likely to advance our understanding of the pathogenesis of benign prostatic hyperplasia and prostate cancer as the neoformation of ductal-acinar architecture during normal development is shared during the pathogenesis of benign prostatic hyperplasia and prostate cancer.

Role of Androgens in Female Genitourinary Tissue Structure and Function: Implications in the Genitourinary Syndrome of Menopause.

INTRODUCTION: Genitourinary conditions in women increase in prevalence with age. Androgens are prerequisite hormones of estrogen biosynthesis, are produced in larger amounts than estrogens in women, and decrease throughout adulthood. However, research and treatment for genitourinary complaints have traditionally focused on estrogens to the exclusion of other potential hormonal influences. AIM: To summarize and evaluate the evidence that androgens are important for maintaining genitourinary health in women and that lack of androgenic activity can contribute to the development of symptoms of the genitourinary syndrome of menopause. METHODS: The role of androgens in the pathophysiology, diagnosis, and treatment of genitourinary syndrome of menopause was discussed by an international and multidisciplinary panel during a consensus conference organized by the International Society for the Study of Women's Sexual Health. A subgroup further examined publications from the PubMed database, giving preference to clinical studies or to basic science studies in human tissues. MAIN OUTCOME MEASURES: Expert opinion evaluating trophic and functional effects of androgens, their differences from estrogenic effects, and regulation of androgen and estrogen receptor expression in female genitourinary tissues. RESULTS: Androgen receptors have been detected throughout the genitourinary system using immunohistochemical, western blot, ligand binding, and gene expression analyses. Lower circulating testosterone and estradiol concentrations and various genitourinary conditions have been associated with differential expression of androgen and estrogen receptors. Supplementation of androgen and/or estrogen in postmenopausal women (local administration) or in ovariectomized animals (systemic administration) induces tissue-specific responses that include changes in androgen and estrogen receptor expression, cell growth, mucin production, collagen turnover, increased perfusion, and neurotransmitter synthesis. CONCLUSION: Androgens contribute to the maintenance of genitourinary tissue structure and function. The effects of androgens can be distinct from those of estrogens or can complement estrogenic action. Androgen-mediated processes might be involved in the full or partial resolution of genitourinary syndrome of menopause symptoms in women. Traish AM, Vignozzi L, Simon JA, et al. Role of Androgens in Female Genitourinary Tissue Structure and Function: Implications in the Genitourinary Syndrome of Menopause. Sex Med Rev 2018;6:558-571.

Incorporation of the δ-subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis.

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, ß, and γ), but a fourth δ-subunit may replace α and form atypical δßγ-ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus αßγ- or δßγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δßγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.

A homozygous missense variant in VWA2, encoding an interactor of the Fraser-complex, in a patient with vesicoureteral reflux.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause (40-50%) of chronic kidney disease (CKD) in children. About 40 monogenic causes of CAKUT have so far been discovered. To date less than 20% of CAKUT cases can be explained by mutations in these 40 genes. To identify additional monogenic causes of CAKUT, we performed whole exome sequencing (WES) and homozygosity mapping (HM) in a patient with CAKUT from Indian origin and consanguineous descent. We identified a homozygous missense mutation (c.1336C>T, p.Arg446Cys) in the gene Von Willebrand factor A domain containing 2 (VWA2). With immunohistochemistry studies on kidneys of newborn (P1) mice, we show that Vwa2 and Fraser extracellular matrix complex subunit 1 (Fras1) co-localize in the nephrogenic zone of the renal cortex. We identified a pronounced expression of Vwa2 in the basement membrane of the ureteric bud (UB) and derivatives of the metanephric mesenchyme (MM). By applying in vitro assays, we demonstrate that the Arg446Cys mutation decreases translocation of monomeric VWA2 protein and increases translocation of aggregated VWA2 protein into the extracellular space. This is potentially due to the additional, unpaired cysteine residue in the mutated protein that is used for intermolecular disulfide bond formation. VWA2 is a known, direct interactor of FRAS1 of the Fraser-Complex (FC). FC-encoding genes and interacting proteins have previously been implicated in the pathogenesis of syndromic and/or isolated CAKUT phenotypes in humans. VWA2 therefore constitutes a very strong candidate in the search for novel CAKUT-causing genes. Our results from in vitro experiments indicate a dose-dependent neomorphic effect of the Arg446Cys homozygous mutation in VWA2.

The Role of the Hydrogen Sulfide Pathway in Male and Female Urogenital System in Health and Disease.

SIGNIFICANCE: The endogenous hydrogen sulfide (H2S) pathway produces an array of biological effects that vary depending on the bodily region. In addition, the H2S pathway's relevance often changes depending on a healthy or disease state. There is abundant evidence pointing to a key role for this pathway in male and female genito-urinary diseases, suggesting it as a possible target for new therapeutic approaches. Recent Advances: The tissue-specific localization of the H2S enzymes in the genito-urinary tract has allowed for a better understanding of its role in the body's pathophysiology. Indeed, in humans, cystathionine-γ-lyase (CSE) plays a major role in corpus cavernosum whereas cystathionine-ß-synthase (CBS) plays a role in bladder functioning. The prostate epithelium expresses CBS and CSE, but stromal CSE only. In the uterus, up- or downregulation of CBS and CSE varies strongly depending on the female's hormonal cycle or pregnancy. CRITICAL ISSUES: There is still the need to better define the male and female's sexual hormonal roles in regulating the H2S pathway, particularly in human pathological conditions. The lack of a correlation between human and animal data should be carefully considered when planning preclinical studies. The unmet need for selective enzymatic inhibitors and the different methodologies for H2S measurements still represent a critical issue in this research field. FUTURE DIRECTIONS: It is feasible that the L-cysteine/H2S pathway can represent an alternative therapeutic target in genito-urinary tract disorders. The research should focus on erectile dysfunction and preeclampsia, characterized by vascular defect, as well as on bladder disorders where the urothelium is compromised. Antioxid. Redox Signal. 27, 654-668.