Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration.

AIM: (i) To assess the expression profiles of stem cell-associated markers including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and Cd9, (ii) analyze the nanotopography of the MIC-1 stem cells and (iii) evaluate the efficiency of live stem cell implants and stem cell culture derivatives on the regeneration of bone deficiencies in rabbit mandibles. MATERIALS AND METHODS: The expression profiles of stem cell-associated genes, including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and CD9 were assessed using reverse transcription polymerase chain reaction and flow cytometry. Nanotopography of the antlerogenic MIC-1 cell lineage was analyzed using atomic force microscopy. The effect of MIC-1 stem cells, their homogenate and supernatant on the regeneration of bone deficiencies in rabbit mandibles was evaluated using histological analysis. The effect of MIC-1 stem cells and stem cell-based derivatives on the immune responses of the animals was assessed by analyses of acute phase protein levels (haptoglobin and fibrinogen). RESULTS: We found that the MIC-1 cells isolated from the apical regions of growing antlers exhibited molecular features that were characteristics of pluripotent stem cells. Using atomic force microscopy, we determined the details of the cell surface morphologies with a particular emphasis on the patterns of formation of plasma extensions for interlinking adjacent cells. We also demonstrated that not only implanted stem cells but also cell homogenates and cell post-culture supernatants have potential in the regeneration of bone deficiencies in the rabbit mandible. CONCLUSIONS: Our findings indicate that the use of both antlerogenic stem cell implants and the preparations derived from the cells offer alternative approaches to those based on autologous stem cells in the biological stimulation of osteogenesis and in bone regeneration.

Influence of the intramedullary nail locking method on the stiffness of the bone-implant system.

PURPOSE: Bone fractures are one of the most commonly seen problems in veterinary traumatology. The authors of this study strive to find a new intramedullary nail, which is intended for treating femoral bone fractures for canine patients. The purpose of this study was to analyze biomechanical parameters of the intramedullary nails, which use a new bolt system concept. METHODS: Dissected femoral bones of a large breed dog were cut in order to simulate interfragmentary gap, and then the bones were stabilized using intramedullary nail with locking bolts. Bone-nail systems were subjected to cyclic loading using force which corresponds to the load on the femoral bone in the first few days after surgery. Micro-CT scans were taken of the bone samples around implant in order to determine deformation and structural parameters of bone tissue. RESULTS: The calculation of the bone-nail system stiffness was done through analysis of the force- displacement curves recorded during experimental studies. Using monocortical locking bolts resulted in smaller stiffness of the bone-nail system than using bicortical locking bolts. CONCLUSIONS: The results obtained in this study can indicate that the intramedullary nail could work well when used for treatment of bone fractures in dogs. The authors focused on using monocortical bolts which provides good stability and adequate biomechanical environment. Described fixation method is easily adjustable to a particular patient individual pa- rameters.

Chromosome abnormalities in dogs with disorders of sex development (DSD).

Disorders of sex development (DSD) caused by chromosome abnormalities are rarely diagnosed in dogs. In this report, there is a focus on five DSD cases in which the dogs had abnormal karyotypes. All animals were recognized by owners as females, however, these dogs had a large number of reproductive defects. Among these were abnormal external genitalia such as an enlarged clitoris, abnormal development of the labia, abnormal location of the vulva and urethral orifice, and other abnormalities were observed in four dogs. Gonadal histology assessments were conducted on three dogs and there were diagnoses of the presence of an ovary, inactive testes, and ovotestis with calcification in ovarian follicles. Results from cytogenetic analysis indicated there were the following karyotypes: (a) X trisomy in a mosaic form (79,XXX/78,XX); (b) Robertsonian translocation in a mosaic form (77,XX,rob/78,XX); (c) nonmosaic X/autosome translocation (78,X,t(X;A)); (d) X/autosome translocation in a mosaic form (78,X,t(X;A)/78,XX); and (e) leukocyte chimerism (78,XX/78,XY). The findings in the present study, emphasize that cytogenetic analysis is essential for elucidating the pathogenesis of DSD in dogs.

Parameters of Hemostasis in Sheep Implanted with Composite Scaffold Settled by Stimulated Mesenchymal Stem Cells-Evaluation of the Animal Model.

Implantation of composite scaffolds could be potentially associated with the risk of hemostatic disturbances in a recipient. However, there is a lack of information on possible alterations in clotting mechanisms resulting from such a procedure. The aim of the present work was to investigate changes in hemostatic parameters in sheep implanted with a scaffold composed of poly(ε-caprolactone) and hydroxyapatite and tricalcium phosphate (9:4.5:4.5), settled previously with mesenchymal stem cells stimulated by fibroblast growth factor-2 and bone morphogenetic protein-2. Nine Merino sheep were examined for 7 days, and measurements of clotting times (PT, aPTT), activities of antithrombin, protein C and clotting factors II-XII, and concentrations of fibrinogen and D-dimer were carried out before and 1 h, 24 h, 3 days and 7 days after scaffold implantation. The introduction of scaffold initially resulted in a slowdown of the clotting processes (most evident 24 h after surgery); PT and aPTT increased to 14.8 s and 33.9 s, respectively. From the third day onwards, most of these alterations began to return to normal values. The concentration of fibrinogen rose throughout the observation period (up to 8.4 g/L), mirroring the ongoing inflammatory reaction. However, no signals of significant disturbances in hemostatic processes were detected in the sheep tested.

Screening for structural variants of four candidate genes in dogs with disorders of sex development revealed the first case of a large deletion in NR5A1.

Disorders of sex development (DSD) are important causes of infertility and sterility, and are risk factors for gonadal carcinogenesis. Many DSDs are caused by genetic factors, mainly sex chromosome abnormalities or mutations of genes involved in sexual development, as well as structural variants (SVs) - large deletions, duplications, and insertions, if these overlap genes involved in sex development. The aim of this study was to determine if there were SVs in four candidate genes - NR0B1 (DAX1), NR5A1, RSPO1, and SOX3 - using droplet digital PCR (ddPCR). There was study of two cohorts of dogs with DSD, including 55 animals with XX DSD and 15 with XY DSD. In addition, 40 control females and 10 control males were included in the study. Among cases, for which there were evaluations, a large deletion consisting of four exons of the NR5A1 gene was identified in a Yorkshire Terrier with a rudimentary penis, hypospadias, bilateral cryptorchidism, and spermatogenesis inactive testes. This is the first mutation in the NR5A1 gene leading to XY DSD phenotype to be reported in domestic animals. There were no SVs in the genes evaluated in the present study in the cohort of dogs with XX DSD. The results from this study provide evidence that the large structural variants of these genes are rarely associated with the DSD phenotype in dogs.