CRISPR/dCas9-mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig-like cells.

Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig-like cells. We first constructed the stable Hsd3b-dCas9-MPH-HFF cell line using the Hsd3b-EGFP, dCas9-VP64 and MS2-P65-HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real-time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig-like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig-like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.

Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation.

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

Biological characters of human dermal fibroblasts derived from foreskin of male infertile patients.

Dermal fibroblasts play a vital role in maintaining skin function. They not only synthesize and secrete extracellular matrix molecules, but also produce a complex mixture of bioactive factors, which both contribute to immune regulation and wound healing. Fibroblasts isolated from skin tissue exhibit wide range of potentials, especially in regenerative medicine. The use of fibroblast cultures for medical purposes requires standardization of cell preparations. To achieve this, we isolated and characterized dermal fibroblasts from human foreskin with a standardized method. The obtained cells grew as typical morphology of fibroblasts, and expressed intermediate filament protein vimentin and nestin. Immunophenotypic analysis indicated that the isolated fibroblasts expressed mesenchymal surface markers CD73, CD90, CD44 and CD105, and were negative for haematopoietic markers CD45 and CD34. Growth kinetics analysis of the cells showed high proliferative properties. Furthermore, cryopreservation had no influence on cell morphology and growth properties. Here, we describe a standardized, repeatable method for isolation of fibroblasts from human foreskin tissues and identify their biological characters according to morphologic, immunohistologic and proliferative criteria, which would be meaningful for future clinical trials and regenerative medicine purposes.

2D:4D indicates phimosis risk: A study on digit ratio and early foreskin development.

BACKGROUND: Many researchers have adopted 2D:4D (second to fourth finger length ratio) as a noninvasive retrospective biomarker for prenatal androgen exposure in recent years. It is thought to be related to diverse traits including behavioral phenotypes, disease susceptibility, and development of urogenital system. OBJECTIVE: To examine the relationship between 2D:4D and early foreskin development. METHODS: We analyzed the digit ratio and foreskin condition in 176 cases (range 0-6years). The boys were divided into four groups according to their ages: group 1, neonates (below 28days, n=13); group 2, infants (1-12months, n=45); group 3, toddlers (1-2years old, n=42); group 4, preschool children (3-6years old, n=76). We measured the lengths of the second and fourth digits of the left and right hands. The foreskin status was classified into 4 types. Type I (phimosis), type II (partial phimosis), type III (adhesion of prepuce), type IV (normal). RESULTS: The phimosis rate was 92.3%, 82.2%, 45.2%, and 38.7% in group 1 to group 4. In contrast, the proportion of normal foreskin increased from 0% in neonates to 13.2% in preschool children. The percentage of higher level of foreskin development shows a downward trend with the increase of digits ratio, and as the age grows, the percentage of normal foreskin cases also increases. CONCLUSIONS: These results suggest that a higher R2D:4D (right hand 2D:4D) is a risk factor for phimosis in the early human development. Age is also a significant influence factor of foreskin conditions. Additional research is required to identify pathophysiologic mechanisms and to determine clinical significance.

Neonatal skin maturation--vernix caseosa and free amino acids.

Neonatal skin hydration decreases rapidly postnatally and then increases, indicating adaptive changes in stratum corneum water handling properties. Transition from high to low humidity at birth may initiate filaggrin proteolysis to free amino acids. Neonatal skin with vernix caseosa retained is more hydrated than skin with vernix removed. This study examines the potential roles of free amino acids and vernix in postnatal adaptation of infant stratum corneum in vivo. Specifically, the ontogeny of free amino acid generation in neonatal stratum corneum and the role of vernix caseosa in postnatal adaptation were examined using high performance liquid chromatography. Free amino acids were quantified for infant skin samples collected at (i) birth and 1 month and (ii) birth and 24 hours after vernix caseosa retention or removal and compared to neonatal foreskin, vernix caseosa, and adult stratum corneum using t-tests, analysis of variance, or univariate procedures. Free amino acids were extremely low at birth, significantly higher 1 month later but lower than in adults. Vernix caseosa retention led to significantly higher free amino acids 24 hours after birth compared to infants with vernix caseosa removed, and it paralleled the higher stratum corneum hydration of vernix caseosa-retained skin. Vernix caseosa contained free amino acids, with glutamic acid and histidine levels higher than in infants. Free amino acids in vernix caseosa-retained skin appear to originate from vernix caseosa. Free amino acids were lower in neonatal foreskin than adult forearm stratum corneum. Arginine was higher than citrulline at birth, but levels were comparable in older infants. The free amino acid increase at 1 month may be initiated by the humidity transition at birth and supports results in animals. The findings have implications for infant skin care practices.

Effects of prenatal exposure to a low dose atrazine metabolite mixture on pubertal timing and prostate development of male Long-Evans rats.

The present study examines the postnatal reproductive development of male rats following prenatal exposure to an atrazine metabolite mixture (AMM) consisting of the herbicide atrazine and its environmental metabolites diaminochlorotriazine, hydroxyatrazine, deethylatrazine, and deisopropylatrazine. Pregnant Long-Evans rats were treated by gavage with 0.09, 0.87, or 8.73mg AMM/kg body weight (BW), vehicle, or 100mg ATR/kg BW positive control, on gestation days 15-19. Preputial separation was significantly delayed in 0.87 mg and 8.73mg AMM-exposed males. AMM-exposed males demonstrated a significant treatment-related increase in incidence and severity of inflammation in the prostate on postnatal day (PND) 120. A dose-dependent increase in epididymal fat masses and prostate foci were grossly visible in AMM-exposed offspring. These results indicate that a short, late prenatal exposure to mixture of chlorotriazine metabolites can cause chronic prostatitis in male LE rats. The mode of action for these effects is presently unclear.

Foreskin development in 10 421 Chinese boys aged 0-18 years.

BACKGROUND: Few studies on foreskin development and the practice of circumcision have been done in Chinese boys. This study aimed to determine the natural development process of foreskin in children. METHODS: A total of 10 421 boys aged 0 to 18 years were studied. The condition of foreskin was classified into type I (phimosis), type II (partial phimosis), type III (adhesion of prepuce), type IV (normal), and type V (circumcised). Other abnormalities of the genitalia were also determined. RESULTS: The incidence of a completely retractile foreskin increased from 0% at birth to 42.26% in adolescence; however, the phimosis rate decreased with age from 99.7% to 6.81%. Other abnormalities included web penis, concealed penis, cryptorchidism, hydrocele, micropenis, inguinal hernia, and hypospadias. CONCLUSIONS: Incomplete separation of foreskin is common in children. Since it is a natural phenomenon to approach the adult condition until puberty, circumcision should be performed with cautions in children.

[The need of circumcision or prepuce dilation. A study with 1200 boys]. / Necesidad de circuncisión o dilatación del prepucio. Estudio de 1.200 niños.

OBJECTIVES: To demonstrate the increase of preputial retractability with age. To point out the small usefulness of circumcision and preputial forced dilation during childhood. METHODS: The development of the prepuce and its retractability were evaluated in 1200 boys between 0 and 16 years. The prepuce was classified as type I to V depending on its lower or higher retractability in all cases having been dilated previously or not. RESULTS: Prepuce retractability in boys under one year was type I (not retractile) in 63.4%, whereas it was type V (completely retractile) in only 3.7%. The contrary was observed in adolescents (11 to 16 years), in which type I was 0.9% and type V was observed in 80.9%. It was also observed that 309 boys (43. 1%) among the 717 with previous prepuce forced dilation, had types I to IV prepuces, so, they had acquired new balanopreputial adherences by the time of examination for our study. Seventeen boys (0.4%) required circumcision. No children suffered upper urinary tract infections. CONCLUSIONS: All boys are born with the prepuce covering the glans penis, keeping adherences between both structures, which disappear with age, being the detachment complete at the time of puberty in most boys. So, we consider circumcision or forced dilation of the prepuce unnecessary in most boys.