Effects and Mechanism of the Leontopodium alpinum Callus Culture Extract on Blue Light Damage in Human Foreskin Fibroblasts.

Leontopodium alpinum is an important source of raw material for food, medicine, and modern cosmetics. The purpose of this study was to develop a new application for protection against blue light damage. To investigate the effects and mechanism of action of Leontopodium alpinum callus culture extract (LACCE) on blue light damage, a blue-light-induced human foreskin fibroblast damage model was established. The contents of collagen (COL-I), matrix metalloproteinase 1 (MMP-1), and opsin 3 (OPN3) were detected using enzyme-linked immunosorbent assays and Western blotting. The calcium influx and reactive oxygen species (ROS) levels were measured via flow cytometry and the results showed that the LACCE (10-15 mg/mL) promoted the production of COL-I, inhibited the secretion of MMP-1, OPN3, ROS and calcium influx, and may play a role in inhibiting the activation of blue light on the OPN3-calcium pathway. Thereafter, high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry were used to quantitatively analyze the contents of nine active ingredients in the LACCE. The results indicated that LACCE has an anti-blue-light-damage effect and provides theoretical support for the development of new raw materials in the natural food, medicine, and skin care industries.

Role of epigenetics in the etiology of hypospadias through penile foreskin DNA methylation alterations.

Abnormal penile foreskin development in hypospadias is the most frequent genital malformation in male children, which has increased dramatically in recent decades. A number of environmental factors have been shown to be associated with hypospadias development. The current study investigated the role of epigenetics in the etiology of hypospadias and compared mild (distal), moderate (mid shaft), and severe (proximal) hypospadias. Penile foreskin samples were collected from hypospadias and non-hypospadias individuals to identify alterations in DNA methylation associated with hypospadias. Dramatic numbers of differential DNA methylation regions (DMRs) were observed in the mild hypospadias, with reduced numbers in moderate and low numbers in severe hypospadias. Atresia (cell loss) of the principal foreskin fibroblast is suspected to be a component of the disease etiology. A genome-wide (> 95%) epigenetic analysis was used and the genomic features of the DMRs identified. The DMR associated genes identified a number of novel hypospadias associated genes and pathways, as well as genes and networks known to be involved in hypospadias etiology. Observations demonstrate altered DNA methylation sites in penile foreskin is a component of hypospadias etiology. In addition, a potential role of environmental epigenetics and epigenetic inheritance in hypospadias disease etiology is suggested.

Downregulated miR-181a alleviates H2O2-induced oxidative stress and cellular senescence by targeting PDIA6 in human foreskin fibroblasts.

BACKGROUND: Oxidative stress is strongly associated with cellular senescence. Numerous studies have indicated that microRNAs (miRNAs) play a critical part in cellular senescence. MiR-181a was reported to induce cellular senescence, however, the potential mechanism of miR-181a in hydrogen peroxide (H2O2)-induced cellular senescence remains obscure. OBJECTIVE: The aim of this study is to investigate the role and regulatory mechanism of miR-181a in H2O2-induced cellular senescence. METHODS: Human foreskin fibroblasts (HFF) transfected with miR-181a inhibitor/miR-NC with or without H2O2 treatment were divided into four groups: control + miR-NC/miR-181a inhibitor, H2O2 + miR-NC/miR-181a inhibitor. CCK-8 assay was utilized to evaluate the viability of HFF. RT-qPCR was used to measure the expression of miR-181a and its target genes. Protein levels of protein disulfide isomerase family A member 6 (PDIA6) and senescence markers were assessed by western blotting. Senescence-associated ß-galactosidase (SA-ß-gal) staining was applied for detecting SA-ß-gal activity. The activities of SOD, GPx, and CAT were detected by corresponding assay kits. The binding relation between PDIA6 and miR-181a was identified by luciferase reporter assay. RESULTS: MiR-181a inhibition suppressed H2O2-induced oxidative stress and cellular senescence in HFF. PDIA6 was targeted by miR-181a and lowly expressed in H2O2-treated HFF. Knocking down PDIA6 reversed miR-181a inhibition-mediated suppressive impact on H2O2-induced oxidative stress and cellular senescence in HFF. STUDY LIMITATIONS: Signaling pathways that might be mediated by miR-181a/PDIA6 axis were not investigated. CONCLUSION: Downregulated miR-181a attenuates H2O2-induced oxidative stress and cellular senescence in HFF by targeting PDIA6.

Damage prevention effect of milk-derived peptides on UVB irradiated human foreskin fibroblasts and regulation of photoaging related indicators.

Long-term exposure to UVB can trigger acute inflammation of the skin and lead to skin photoaging. To scrutinize the anti-photoaging functions of peptides obtained from milk, the physicochemical including molecular weight and amino acid compositions were first analyzed. Totally 267 peptides were screened out and identified by PEAKS X software, and then evaluated through Peptide Ranker and BIOPEP-UMW. Six peptides with the highest antioxidant ability and relative abundance were selected. This study was then conducted in UVB-damaged human foreskin fibroblasts with proadministration of peptides. The results indicated that at concentrations of 0.08-0.10 mg/mL, milk-derived peptides could realize a damage prevention effect through inhibiting the generation of reactive oxygen species (ROS) and lipid peroxidation malondialdehyde (MDA). Also, these peptides were found to promote the photoaging related enzyme activities of superoxide dismutase (SOD) and catalase (CAT), while to block the production of matrix metalloproteinases-1. Through this study, we found that milk-derived peptide mixture is effective in preventing photoaging damage. Milk-derived peptides found in this study could serve as raw materials for future development of antioxidant functional foods.

Repression of Mafb promotes foreskin fibroblast proliferation through upregulation of CDK2, cyclin E and PCNA.

Mafb plays a significant role in the development and differentiation of various organs, tissues and cells. Nevertheless, its role in the control of external genital cell proliferation and function in the mechanism of hypospadias remains unknown. In this study, the expression of Mafb in foreskin fibroblasts was inhibited by siRNA. The Cell Count Kit-8 (CCK-8) assay showed cell proliferation increased after transfection, and the number of cells entered the S phase significantly increased via flow cytometry. Both mRNA and protein levels of cyclin E, cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) were significantly upregulated in the siRNA group. Meanwhile, twenty-five prepuce tissue samples were collected from hypospadias repair surgery. These samples were divided into two groups: the severe and mild groups. Normal prepuce tissue specimens were obtained during circumcision as the normal control. The upregulated expression of cyclin E, CDK2 and PCNA and downregulated Mafb expression were observed in the hypospadias group. This study reveals for the first time that the reduction in Mafb promotes the foreskin fibroblast proliferation. Thus, downregulated Mafb expression may cause hypospadias by upregulating CDK2, cyclin E and PCNA. These findings can shed new light on the embryonic development of the urethra.

Why human prepuce is a valuable resource in science? Histological, ultrastructural, immunohistochemical and statistical analysis / ¿Por qué el prepucio humano es un recurso valioso en la ciencia? Análisis histológicos, ultraestructurales, inmunohistoquímicos y estadísticos

SUMMARY: This research was to examine the histological and ultrastructural characteristics of prepuce samples, as well as vimentin and S100 protein localization and statistical analysis. Urologists have long struggled with the prepuce, which is used to treat a variety of urethral problems. Skin biopsies were collected from the prepuce at the moment of circumcision and processed for light microscopy, electron microscope examination, immunohistochemical techniques, and statistical analysis in a total of six boys. Histologically, the prepuce epidermis displayed focal spiky ridges, which are saw-toothed interspersed with sulci, slight hyperpigmentation, looser connective tissue and plentiful vascular components. Immunohistochemically, the existence of melanocytes and Langerhans cells in the epidermis, as well as smooth muscles in the dermis, was stained positively for vimentin. Also, there was a positive reactivity of the Langerhans cells in the epidermis and around Meissner's corpuscles in the dermis for S100 protein staining. Ultrastructurally, the prepuce's intercellular gaps were widened, melanocytes rested on a folded basement membrane, and desmosomal content was reduced, with a prominent active euchromatic nucleus. Cytoplasmic projections were distended and elongated, and the interstitial blood vessels were surrounded by endothelial cells and rested on a basement membrane. There were also minimal collagen fibers in the interstitium. The prepuce's histological and ultrastructural features, as well as immunohistological studies using vimentin and S100 protein as intermediate filaments and statistical analysis, all demonstrated that it is a useful scientific resource.

RESUMEN: El presente trabajo de investigación se realizó para examinar las características histológicas y ultraestructurales de las muestras de prepucio, así como la localización y el análisis estadístico de la vimentina y la proteína S100. Los urólogos han intentado trabajar durante mucho tiempo con el prepucio, que se usa para tratar una variedad de problemas uretrales. Se recolectaron biopsias de piel del prepucio de seis niños en el momento de la circuncisión y se procesaron para microscopía óptica, examen con microscopio electrónico, técnicas inmunohistoquímicas y análisis estadístico. Histológicamente, la epidermis del prepucio mostraba crestas puntiagudas focales, intercaladas con surcos, hiperpigmentación leve, tejido conectivo más laxo y abundantes componentes vasculares. Inmunohistoquímicamente, la existencia de melanocitos y células dendríticas epidérmicas (células de Langerhans), así como músculo liso en la dermis, se tiñeron positivamente para vimentina. Además, hubo una reactividad positiva de las células dendríticas epidérmicas en la epidermis y alrededor de los corpúsculos del tacto (de Meissner) en la dermis para la tinción de la proteína S100. Ultraestructuralmente, los espacios intercelulares del prepucio se ensancharon, los melanocitos descansaban sobre una membrana basal plegada y el contenido desmosómico se redujo, con un núcleo eucromático activo prominente. Las proyecciones citoplasmáticas estaban distendidas y alargadas, y los vasos sanguíneos intersticiales estaban rodeados por células endoteliales y descansaban sobre una membrana basal. También había fibras de colágeno mínimas en el intersticio. Las características histológicas y ultraestructurales del prepucio, así como los estudios inmunohistológicos utilizando vimentina y proteína S100 como filamentos intermedios y el análisis estadístico, demostraron que es un recurso científico útil.

Transcriptional Differences in Lipid-Metabolizing Enzymes in Murine Sebocytes Derived from Sebaceous Glands of the Skin and Preputial Glands.

Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.

ß2-adrenoreceptors control human skin microvascular reactivity.

Topical α1- and α2-adrenoreceptor (ADRA1 and 2) agonists are effective in alleviating permanent vasodilation and facial erythema associated with rosacea by inducing skin vasoconstriction. Although ß-adrenoreceptor (ADRB) antagonists are used off-label for rosacea, pharmacological and pharmacodynamic data pertaining to these receptors in skin micro-vessels are lacking. Objectives: To analyse the expression of different adrenergic receptors and their contribution to vasoreactivity in skin micro-vessels. Small arteries (500-800 µm) and arterioles (<200 µm) were studied in human foreskin tissue. Specifically, ADR-A1, -A2, -B1 and -B2 expression was assayed by immunofluorescence, polymerase chain reaction (PCR), and western blotting. Small skin artery reactivity was evaluated using ex vivo myography (500-800 µm) or a visible microscope perfusion system with precision-cut skin slices (<200 µm). ADRB2 was the most highly expressed receptor in small skin arteries and arterioles, followed by ADRA2. ADRA2 activation via brimonidine-induced vasoconstriction was greater in skin arterioles than in small skin arteries, and more potent than that with norepinephrine (NE). The use of prazosin (ADRA1 inhibitor) partially attenuated brimonidine-induced vasoconstriction, indicating some activation of ADRA1 by brimonidine, at least at 10-µM concentrations. Small skin arteries and arterioles, pre-treated with prazosin and stimulated with NE, exhibited ADRB2-mediated vasodilation, which was inhibited by the beta blockers, propranolol or timolol. This study shows that ADRB2 is predominantly expressed in small skin arteries and arterioles, and that ADRBs plays a functional role in vasodilation. The data presented here indicate that ADRBs can be a therapeutic target for the treatment of rosacea.

Inhibitory Effects of Bilirubin on Colonization and Migration of A431 and SK-MEL-3 Skin Cancer Cells Compared with Human Dermal Fibroblasts (HDF).

This study evaluated the inhibitory effects of bilirubin on colony formation and cell migration of melanoma and non-melanoma skin cancer cell lines SK-MEL-3 and A431, compared with normal human dermal fibroblasts (HDF). The IC50 obtained from the MTT assay was 125, 100, and 75 µM bilirubin for HDF, A431, and SK-MEL-3 cells, respectively. The colony formation and cell migration of cancer cells, treated with 100 µM bilirubin, were reduced significantly (p < 0.05). Bilirubin decreased cell adhesion and inhibited cell colonization via inducing apoptosis and cell death. Also by interaction with migration main factors, bilirubin caused inhibition the cell migration.

Human Induced Pluripotent Stem Cell-Based Skin for Assessing Transdermal Drug Permeability and Irritancy.

To establish a system for assessing drug permeation and irritation of the skin, the permeation of benzoic acid and isosorbide dinitrate, which are listed in the Pharmacopoeia, and the chemical irritation were evaluated using skin generated from human induced pluripotent stem cells (iPSCs). Multilayer structures and cellular markers (keratin 14 and 10, which are in basal and suprabasal epidermal layers) were clearly detected in our iPSC-based skin. Transepidermal water loss (TEWL) decreased after iPSC-derived keratinocytes were cultured on collagen gels from human primary fibroblasts. These results indicate that the barrier function was partly increased by formation of the living epidermis. The cumulative amount of benzoic acid and isosorbide dinitrate across human iPSC-based skin gradually increased after an initial lag time. Moreover, the irritancy of various chemicals (non-irritants: ultrapure water, allyl phenoxy-acetate, isopropanol, and hexyl salicylate and irritants: 5% sodium dodecyl sulfate (SDS), heptanal, potassium hydroxide (5% aq.) and cyclamen aldehyde) to iPSC-based skin was almost met the irritation criteria of the Organisation for Economic Co-operation and Development (OECD) guideline. The results of our iPSC-based skin evaluation provide useful basic information for developing an assessment system to predict the permeation and safety of new transdermal drugs in human skin.

Evaluation of the effects of IL­22 on the proliferation and differentiation of keratinocytes in vitro.

Psoriasis is one of the most common chronic inflammatory skin diseases, it is characterized by hyperproliferation of keratinocytes and infiltration of inflammatory cells. Several in vitro studies have reported that interleukin (IL)­22 is involved in excessive proliferation and abnormal differentiation of human keratinocytes. However, the association between IL­22 and CCAAT enhancer binding protein α (C/EBPα) in the pathogenesis of psoriasis remains unclear. Therefore, the present study aimed to investigate the association between IL­22 and C/EBPα, and the effects of IL­22 on the proliferation and differentiation of keratinocytes. Keratinocytes were treated with different concentrations of IL­22 (30, 60 and 90 ng/ml) and subsequently cells were collected at different time intervals. The expression levels of the key molecules of the mitogen­activated protein kinase (MAPK) signaling pathway were detected using western blot analysis. In addition, the effect of IL­22 on the proliferation rate of keratinocytes and the mRNA expression levels of C/EBPα were determined using a Cell Counting Kit­8 assay and reverse transcription­quantitative PCR, respectively. Furthermore, keratinocytes were transfected with C/EBPα small interfering (si)RNA or control using Lipofectamine® 2000. The results revealed that IL­22 significantly induced the proliferation of keratinocytes and the expression of phosphorylated (p)­JNK, p­ERK and p­p38 (P<0.05). Additionally, IL­22 significantly inhibited the differentiation of keratinocytes, and the mRNA and protein expression of C/EBPα (P<0.05). Furthermore, downregulation of C/EBPα increased the proliferation rate of keratinocytes and reduced the expression levels of cytokeratin 10 and involucrin. Therefore, these results suggested that the effect of IL­22 on the proliferation and differentiation of keratinocytes may be mediated via the regulation of the MAPK signaling pathway and the expression of C/EBPα.

Single cell transcriptomics of human epidermis identifies basal stem cell transition states.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

Fluorinated Chitosan Microgels to Overcome Internal Oxygen Transport Deficiencies in Microtissue Culture Systems.

Poor oxygen transport is a major obstacle currently for 3D microtissue culture platforms, which at this time cannot be grown large enough to be truly physiologically relevant and replicate adult human organ functions. To overcome internal oxygen transport deficiencies, oxygenating microgels are formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water-in-oil miniemulsion method. Microgels that are on the order of a cell diameter (≈10 µm) are formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it is demonstrated that incorporating oxygenating microgels at ratios ranging from 50:1 to 400:1 (# of cells:# of microgels) into dense human fibroblast-based spheroids facilitated the growth of larger human cell-based spheroids, especially at the highest incorporation percentages (50:1), which lacked defined hypoxic cores. Quantification of total double-stranded (ds)-DNA, a measure of number of live cells, demonstrated similar results to hypoxia quantification, showing more ds-DNA due incorporation of oxygenating microgels. Finally, oxygen concentrations are measured at different depths within spheroids directly and confirmed higher oxygen partial pressures due to chitosan-PFC microspheres.

Foreskin surface area is not associated with sub-preputial microbiome composition or penile cytokines.

OBJECTIVE: Male circumcision (MC) reduces acquisition of HIV-1 in heterosexual men by at least 60%, but the biological mechanism for this protection is incompletely understood. Previous studies have shown that a larger foreskin size, increased abundance of anaerobic bacteria in the sub-preputial space, and higher levels of pro-inflammatory cytokines on the penis are all prospectively associated with risk of HIV-1 acquisition. Since coverage of the glans on the non-erect penis is dependent on foreskin size, a larger foreskin could result in a less aerobic environment that might preferentially support anaerobic bacterial growth and induce inflammation. We therefore assessed the relationship between foreskin size, penile microbiome composition and local inflammation. METHODS: This is a retrospective, cross-sectional analysis of 82 HIV-uninfected men who participated in a randomized trial of MC for HIV-1 prevention in Rakai, Uganda between 2003-2006. Sub-preputial swabs were collected prior to MC and assessed for cytokines (multiplexed immunosorbent assay) and bacterial load (qPCR) and taxon abundance (sequencing). Foreskin size was measured immediately after MC. RESULTS: Foreskin surface area did not correlate with total bacterial load (rho = 0.05) nor the abundance of key taxa of bacteria previously associated with HIV-1 risk (rho = 0.04-0.25). Foreskin surface area also did not correlate with sub-preputial cytokine concentrations previously associated with HIV-1 risk (IL-8 rho = 0.05). CONCLUSIONS: Larger foreskin size is not associated with either increased penile anaerobes or pro-inflammatory cytokines. These data suggest that foreskin size does not increase HIV-1 risk through changes in penile microbiome composition or penile inflammation.

Phenolic and Non-Polar Fractions of the Extracts from Fruits, Leaves, and Twigs of Elaeagnus rhamnoides (L.) A. Nelson-The Implications for Human Barrier Cells.

Biological potential of plant extracts are widely described. Because their oral or topical administration is usually recommended, intestinal mucous and skin are the first surfaces exposed to such preparations. Therefore, we asked the question whether phenolic and non-polar fractions of the extracts from fruits, twigs, and leaves of sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) would be able to modulate the functions of human physiological barrier. The study was carried on caucasian colon epithelial-like Caco-2 cells and human foreskin fibroblasts HFF-1 line. Cell secretory activity (ELISA), the expression of cell surface molecules (flow cytometry), cell migration during wound healing in vitro (scratch assay) were assessed. It was demonstrated for the first time, that sea buckthorn extracts can improve intestinal and skin barrier by increasing of ICAM-1 expression on colon epithelial cells and intensification of IL-8 production by fibroblasts. On the other hand, an inhibition of fibroblasts migration in the presence of those preparations was noted. Therefore, greater attention should be paid on precise description of plant extracts effect depended on target cells and their role to give adequate recommendations for such preparations use.

Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases.

The protozoan parasite Toxoplasma gondii lives inside a vacuole in the host cytosol where it is protected from host cytoplasmic innate immune responses. However, IFNγ-dependent cell-autonomous immunity can destroy the vacuole and the parasite inside. Toxoplasma strain differences in susceptibility to human IFNγ exist, but the Toxoplasma effector(s) that determine these differences are unknown. We show that in human primary fibroblasts, the polymorphic Toxoplasma-secreted effector GRA15 mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the vacuole membrane, which enhances recruitment of ubiquitin receptors (p62/NDP52) and ubiquitin-like molecules (LC3B, GABARAP). This ultimately leads to lysosomal degradation of the vacuole. In murine fibroblasts, GRA15-mediated TRAF6 recruitment mediates the recruitment of immunity-related GTPases and destruction of the vacuole. Thus, we have identified how the Toxoplasma effector GRA15 affects cell-autonomous immunity in human and murine cells.

Leydig-like cells derived from reprogrammed human foreskin fibroblasts by CRISPR/dCas9 increase the level of serum testosterone in castrated male rats.

In the past few years, Leydig cell (LC) transplantation has been regarded as an effective strategy for providing physiological patterns of testosterone in vivo. Recently, we have successfully converted human foreskin fibroblasts (HFFs) into functional Leydig-like cells (iLCs) in vitro by using the CRISPR/dCas9 system, which shows promising potential for seed cells. However, it is not known whether the reprogrammed iLCs can survive or restore serum testosterone levels in vivo. Therefore, in this study, we evaluate whether reprogrammed iLCs can restore the serum testosterone levels of castrated rats when they are transplanted into the fibrous capsule. We first developed the castrated Sprague Dawley rat model through bilateral orchiectomy and subsequently injected extracellular matrix gel containing transplanted cells into the fibrous capsule of castrated rats. Finally, we evaluated dynamic serum levels of testosterone and luteinizing hormone (LH) in castrated rats, the survival of implanted iLCs, and the expression levels of Leydig steroidogenic enzymes by immunofluorescence staining and Western blotting. Our results demonstrated that implanted iLCs could partially restore the serum testosterone level of castrated rats, weakly mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, through 6 weeks after transplantation. Therefore, this study may be valuable for treating male hypogonadism in the future.

Expression of androgen receptor and estrogen receptor-beta in foreskin tissue of hypospadiac and healthy boys. / Expresión de receptores androgénicos y estrogénicos beta en la piel prepucial de hipospadias y niños sanos.

OBJECTIVES: This prospective study aimed to investigate the expression of the androgen receptor(AR) and the estrogen receptor-ß (ER-ß) in foreskint issues in boys with and without distal hypospadias. METHODS: Thirty boys with distal hypospadias were evaluated. Fifteen boys who under went elective circumcision over a period of 18 months served as the control group. The presence of AR and ER-ß in foresk in tissues was investigated immunohistochemically. RESULTS: The percentages of AR in epithelial cells were18.9 ± 27.3% in the hypospadias group and 3.3 ±5.3% in the circumcision group, and the difference betweent he groups was significant (p=0.034). Of the stromal cells, 19.5 ± 26.8% in the hypospadias group and2.6 ± 4.4% in the circumcision group were positive lystained for AR (p=0.004). In the hypospadias group,significantly higher stromal cell percentage of ER-ß was found compared to that in the control group (24± 24.5% and 1.3 ± 1.1%, respectively, p<0.001). Moreover, epithelial cell percentage of ER-ß was higher in the hypospadias group than that in the control group,and the respective values were 6.8 ± 10.1% and 0.9± 1.3% (p<0.0001)CONCLUSION: The percent of AR and ER-ß expression were higher in hypospadias-affected foresk in than in the normal foreskin. Whether the normal function of these receptors reveals, there is a need for more detailed studies.

OBJETIVO: Este estudio prospectivo que pretende investigar la expresión del receptor androgénicoy estrogénico en la piel prepucial en niños con y sin hipospadias distal.MÉTODOS: Treinta niños con hipospadias distal fueron evaluados. 15 niños recibieron una circuncisión electiva en un periodo de 18 meses y sirvieron de grupo control.La presencia de RA y RE-ß en la piel prepucial se investigo por immunohistoquimica. RESULTADOS: El porcentaje de expresión del receptor androgenico en células epiteliales fue de 18,9+/-27,3% en el grupo hipospadias y 3,3+/- 5,3% en el grupo de circuncisión. La diferencia entre ambos grupos fue significativo (p=0,034). En las células estromales,19,5+/- 26,8% en el grupo hipospadias y 2,6+/-4,4% en el grupo circuncisión fueron positivos para el RA (p=0,004). En el grupo de hipospadias, un porcentaje mas elevdo de expresión de RE-b ß se evidencio en comparación al grupo control (24+/-24,5%y 1,3+/-1,1, respectivamente, pel porcentaje de células epiteliales con RE-ß fue superior en el grupo hipospadias que en el grupo control; los valores respectivos fueron 6,8+/-10,1% y 0,9+/-1,3%(p<0,0001).CONCLUSIÓN: En este estudio se sugiere que la expresiónde RA y RE fueron superiores en el grupo conhipospadias que en piel prepucial normal. Se requierenmas estudios para determinar el significado de esta expresión.

Expression of androgen receptor and estrogen receptor-beta in foreskin tissue of hypospadiac and healthy boys / Expresión de receptores androgénicos y estrogénicos beta en la piel prepucial de hipospadias y niños sanos

OBJECTIVES: This prospective study aimed to investigate the expression of the androgen receptor(AR) and the estrogen receptor-β (ER-β) in foreskint issues in boys with and without distal hypospadias. METHODS: Thirty boys with distal hypospadias were evaluated. Fifteen boys who under went elective circumcision over a period of 18 months served as the control group. The presence of AR and ER-Beta in foresk in tissues was investigated immunohistochemically. RESULTS: The percentages of AR in epithelial cells were18.9 ± 27.3% in the hypospadias group and 3.3 ±5.3% in the circumcision group, and the difference betweent he groups was significant (p = 0.034). Of the stromal cells, 19.5 ± 26.8% in the hypospadias group and2.6 ± 4.4% in the circumcision group were positive lystained for AR (p = 0.004). In the hypospadias group,significantly higher stromal cell percentage of ER-β was found compared to that in the control group (24 ± 24.5% and 1.3 ± 1.1%, respectively, p < 0.001). Moreover, epithelial cell percentage of ER-β was higher in the hypospadias group than that in the control group,and the respective values were 6.8 ± 10.1% and 0.9 ± 1.3% (p < 0.0001). CONCLUSION: The percent of AR and ER-Beta expression were higher in hypospadias-affected foresk in than in the normal foreskin. Whether the normal function of these receptors reveals, there is a need for more detailed studies

OBJETIVO: Este estudio prospectivo que pretende investigar la expresión del receptor androgénicoy estrogénico en la piel prepucial en niños con y sin hipospadias distal. MÉTODOS: Treinta niños con hipospadias distal fueron evaluados. 15 niños recibieron una circuncisión electiva en un periodo de 18 meses y sirvieron de grupo control.La presencia de RA y RE-Beta en la piel prepucial se investigó por immunohistoquimica. RESULTADOS: El porcentaje de expresión del receptor androgenico en células epiteliales fue de 18,9 +/- 27,3% en el grupo hipospadias y 3,3 +/- 5,3% en el grupo de circuncisión. La diferencia entre ambos grupos fue significativo (p = 0,034). En las células estromales,19,5 +/- 26,8% en el grupo hipospadias y 2,6+/-4,4% en el grupo circuncisión fueron positivos para el RA (p = 0,004). En el grupo de hipospadias, un porcentaje más elevdo de expresión de RE-b β se evidencio en comparación al grupo control (24 +/- 24,5%y 1,3 +/- 1,1, respectivamente, pel porcentaje de células epiteliales con RE-Beta fue superior en el grupo hipospadias que en el grupo control; los valores respectivos fueron 6,8 +/- 10,1% y 0,9 +/- 1,3%(p < 0,0001). CONCLUSIÓN: En este estudio se sugiere que la expresiónde RA y RE fueron superiores en el grupo conhipospadias que en piel prepucial normal. Se requierenmas estudios para determinar el significado de esta expresión

FGF8, FGF10 and FGF receptor 2 in foreskin of children with hypospadias: an analysis of immunohistochemical expression patterns and gene transcription.

INTRODUCTION: Fibroblast growth factors (FGFs) play a crucial role in early embryogenesis of the genital tubercle and are involved in the development of hypospadias, affecting both endo- and ectodermally derived tissues. It was hypothesized that expression of FGFs could be qualitatively or quantitatively altered in skin of children with hypospadias. OBJECTIVE: The objective of the study was to investigate expression patterns and transcription levels of FGF8, FGF10, and FGF Receptor 2 (FGFR2) in patients with hypospadias compared to normal controls. PATIENTS AND METHODS: Skin samples from the ventro-lateral aspect of the foreskin of 32 patients with hypospadias (17 distal and 15 proximal, mean age 25 months) and 10 normal foreskin samples (mean age 77 months) were analyzed by immunohistochemistry. Staining, localization, and distribution of positive cells in epidermis and dermis were categorized independently by two researchers. Complementary DNA (cDNA) samples prepared from messenger RNA (mRNA) isolates of the same samples were analyzed by quantitative polymerase chain reaction (qPCR), comparing expressions of FGF8, FGF10, and FGFR2 with loading controls. RESULTS: Patients with hypospadias consistently showed aberrant immunohistochemical staining patterns for FGF8/FGF10/FGFR2 in epidermis and dermis compared to patients without penile malformation (p < 0.01 for all markers). qPCR displayed no difference in expression levels on mRNA level (FGFR2 p = 0.44, FGF8 p = 0.77, and FGF10 p = 0.17) comparing normal foreskin with foreskin from patients with hypospadias. Figure. DISCUSSION: The results point at an impact of FGF signaling during embryological development of hypospadias on skin, as an ectodermally derived tissue. Similar to the urethral development, this might be a result of mesothelial-epithelial interactions. The differing expression patterns in immunohistochemistry are not matched by a quantitative difference in marker expression on the mRNA level, putatively caused by post-translational modifications or alterations of the downstream pathway. FGFs, particularly FGF10 and FGFR2, are critically involved in wound healing. CONCLUSIONS: There are significant differences in localization and distribution of FGF8, FGF10, and FGFR2 in comparisons of normal foreskin to foreskin of patients with hypospadias, whereas there is no difference in the quantitative expression of these markers on the mRNA level. This confirms the notion that penile skin is affected as well by the embryological aberrations during the embryogenesis of hypospadias.