Canine sperm motility is associated with telomere shortening and changes in expression of shelterin genes.

BACKGROUND: Motion quality is a critical property for essential functions. Several endogenous and exogenous factors are involved in sperm motility. Here, we measured the relative telomere length and evaluated the gene expression of its binding-proteins, shelterin complex (TRF1, TRF2, RAP1, POT1, TIN2, and TPP1) in sperm of dogs using relative quantitative real-time PCR. We compared them between two sperm subpopulations with poor and good motion qualities (separated by swim-up method). Telomere shortening and alterations of shelterin gene expression result from ROS, genotoxic insults, and genetic predisposition. RESULTS: Sperm kinematic parameters were measured in two subpopulations and then telomeric index of each parameter was calculated. Telomeric index for linearity, VSL, VCL, STR, BCF, and ALH were significantly higher in sperms with good motion quality than in sperms with poor quality. We demonstrated that poor motion quality is associated with shorter telomere, higher expression of TRF2, POT1, and TIN2 genes, and lower expression of the RAP1 gene in dog sperm. The levels of TRF1 and TPP1 gene expression remained consistent despite variations in sperm quality and telomere length. CONCLUSION: Data provided evidence that there are considerable changes in gene expression of many shelterin components (TRF2, TIN2, POT1and RAP1) associated with shortening telomere in the spermatozoa with poor motion quality. Possibly, the poor motion quality is the result of defects in the shelterin complex and telomere length. Our data suggests a new approach in the semen assessment and etiologic investigations of subfertility or infertility in male animals.

How to maintain and transport equine adipose tissue for isolating mesenchymal stem cells?

BACKGROUND: Adipose tissue (AT) is one of the most important mesenchymal stem cell (MSC) sources because of its high quantities, availability and ease of collection. After being collected samples, they should be transported to a laboratory for stem cell (SC) isolation, culture and expansion for future clinical application. Usually, laboratories are distant from animal husbandry centers; therefore, it is necessary to provide suitable conditions for adipose tissue transportation, such that adipose-derived MSCs are minimally affected. In the current study, the impact of tissue maintenance under different conditions on MSCs derived from these tissues was evaluated. We aimed at finding suitable and practical transportation methods in which ASCs go through the slightest changes. RESULTS: In the current study, after being collected, equine AT was randomized into eight groups: four samples were maintained in stem cell culture media at 25 οC and 4 οC for 6 and 12 hrs. as transportation via SC media groups. Three samples were frozen at three different temperatures (- 20, - 75 and - 196 οC) as cryopreserved groups; these samples were defrosted 1 week after cryopreservation. Fresh and unfrozen AT was evaluated as a control group. The tissue samples were then initiated into enzymatic digestion, isolation and the culturing of SCs. Cells at passage three were used to evaluate the ability to form colonies, proliferation rate, plotting of the cell growth curve, and viability rate. All experiments were performed in triplicate. Stem cell isolation was successful in all groups, although purification of SCs from the first series of cryopreservation at - 196 οC and two series of - 20 οC was unsuccessful. There was no significant difference between the surface area of colonies in all groups except for - 20 οC. The growth rate of transportation via stem cell media at 25 οC for 6 hrs. was similar to that of the control group. MTT analysis revealed a significant difference between 25 οC 12 hrs. Group and other experimental groups except for control, 4 οC 12 hrs. and - 196 οC group. CONCLUSION: Data have shown freezing at - 75 οC, transportation via stem cell media at 4 οC for 12 hrs. and 25 οC for 6 hrs. are acceptable tissue preservation and transportation methods due to minor effects on MSCs features.

Characterization of Iranian canarypox and pigeonpox virus strains.

Avipoxviruses (APVs) are large DNA viruses that are detected widely in many species of birds. Little information is available regarding genetic variations in these host-specific viruses. In the present study, nine canarypox virus and five pigeonpox virus isolates were collected from northeastern Iran and isolated via the chorioallantoic membrane of chicken embryos. Further investigations were conducted using analysis of virus growth in chicken embryo fibroblasts, histopathology, electron microscopy, and molecular techniques such as polymerase chain reaction (PCR) combined with sequencing and phylogenetic analysis to investigate variations in the highly conserved P4b gene of poxviruses. Virus replication and pock lesions were evident, and microscopic examination revealed eosinophilic intracytoplasmic inclusion bodies and biconcave enveloped virus particles with randomly arranged surface filaments, which are characteristic features of poxviruses. PCR results confirmed the presence of an APV-specific 578-bp fragment in all of the samples. Sequence analysis and phylogenetic analysis of 578-bp P4b fragments of eight isolates confirmed that our canary and pigeon isolates clustered with previously reported isolates. The similarity between the nucleotide sequences of most of our isolates and those isolated previously in other countries could be due to the high degree of conservation of these fragments. However, the FZRC6V isolate from a canary in this study did not have a canarypox virus origin according to the sequence analysis, and might have originated from cross-infection with different strains of avipoxviruses.

Expression of endogenous retroviruses in pre-implantation stages of bovine embryo.

Endogenous retroviruses (ERVs) are involved in cellular proliferation, pluripotency, tissue-specific remodelling and regulation of developmental processes. These elements are transcriptionally active in mouse and human pre-implantation embryos. Empirical evidence indicates that regulatory networks involved with ERV transcripts are responsible for pluripotency and totipotency at certain stages of mouse and human pre-implantation development. Yet, the expression in pre-implantation bovine embryo remains unidentified. To determine whether two members of bovine endogenous retroviruses, BERV-K1 and BERV-K2, are expressed in the pre-implantation bovine embryo, each embryonic stage developed in vitro and was subjected to RNA release, reverse transcription and quantitative PCR. We found that BERV-K1 and BERV-K2 are expressed throughout different stages of pre-implantation development. The higher level of expression was detected in embryonic blastomeres with totipotent/pluripotent status (two-cell to 16-cell stages), while the more differentiated blastocyst stage showed significantly lower levels of ERVs expression. These findings suggest a possible role for endogenous retroviruses in the establishment of totipotent and pluripotent states in pre-implantation bovine embryo, similar to functions which have been suggested for these elements in human and mouse embryos.

In vitro study of matrix metalloproteinases 1, 2, 9, 13 and serum amyloid A mRNAs expression in equine fibroblast-like synoviocytes treated with doxycycline.

Application of synthetic matrix metalloproteinases (MMPs) inhibitors, such as doxycycline is one of the possible therapeutic options for osteoarthritis. However, little is known about the protective mechanism of doxycycline in equine models on MMPs inhibitors as well as on serum amyloid A (SAA) gene expression. This study investigated the effects of doxycycline on mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA of equine fibroblast-like synoviocytes (FLSs). The FLSs were established from synovial fluids of clinically normal metacarpophalangeal joints of 6 skeletally mature horses. The cells were treated with either 10 or 100 µg/mL of doxycycline for 48 h. The mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA were assessed using real-time polymerase chain reaction (PCR). Treatment with doxycycline resulted in significantly decreased mRNA expression of MMP-1 in FLSs at both concentrations (P = 0.001). No significant differences were detected among groups for MMP-2, MMP-9, and MMP-13 (P > 0.05). Only a tendency towards a decrease in mRNA expression level of SAA in the presence of doxycycline could be detected. Doxycycline inhibits MMP-1 gene expression at the transcript level. These findings indicate that doxycycline can protect the articular environment through inhibition of MMP-1 at transcript level.

L'application d'inhibiteurs synthétiques des métalloprotéinases de la matrice (MMP), telle que la doxycycline, est une des options thérapeutiques possibles pour l'ostéoarthrite. Toutefois, peu de choses sont connues sur le mécanisme protecteur de la doxycycline dans les modèles équins des inhibiteurs des MMP, de même que sur l'expression génique de l'amyloïde sérique A (SAA). La présente étude visait à déterminer les effets de la doxycycline sur l'expression de l'ARNm de MMP-1, MMP-2, MMP-9, MMP-13, et SAA des synoviocytes équins apparentés aux fibroblastes (FLS). Les FLS ont été établis à partir du liquide synovial provenant d'articulations métacarpo-phalangiennes cliniquement normales de six chevaux squelettiquement matures. Les cellules ont été traitées avec 10 ou 100 µg/mL de doxycycline pendant 48 h. L'expression d'ARNm de MMP-1, MMP-2, MMP-9, MMP-13, et SAA a été évaluée par réaction d'amplification en chaîne par la polymérase en temps réel. Le traitement avec la doxycycline a causé une diminution significative de l'expression de MMP-1 par les FLS et ce pour les deux concentrations (P = 0,001). Aucune différence significative ne fut détectée parmi les groupes MMP-2, MMP-9, et MMP-13 (P > 0,05). Seulement une tendance à la diminution de l'expression d'ARNm de SAA en présence de doxycycline pouvait être notée. La doxycycline inhibe l'expression génique de MMP-1 à l'étape de la transcription. Ces informations indiquent que la doxycycline peut protéger l'environnement articulaire en inhibant MMP-1 à l'étape de la transcription.(Traduit par Docteur Serge Messier).

17ß-estradiol improves the efficacy of exploited autologous bone marrow-derived mesenchymal stem cells in non-union radial defect healing: A rabbit model.

Exploiting mesenchymal stem cells (MSCs) appears to be an appealing alternative to the traditional clinical approach in the treatment of non-union bone defects. It has been shown that 17ß-estradiol improves the osteogenesis and proliferation potential of the MSCs via estrogen receptors. We investigated the effect of 17ß-estradiol on exploiting autologous BMSCs (bone marrow-derived MSCs) for the purpose of healing of radial non-union segmental defect in rabbit. Twenty rabbits were divided into 4 experimental groups: 1. Control group; 2. MSC treatment group; 3. 17ß-estradiol (E2) treatment group; and 4. E2+MSC treatment group. Isolated BMSCs were seeded in a critical-sized defect on radial mid-diaphysis that was filled with autologous fibrin clot differently in 4 groups: 1. intact fibrin clot (control); 2. Fibrin clot containing MSCs; 3. Estradiol; and 4. E2 and MSCs. Defect healing was assessed by radiological (week 0, 2, 4, 6, 8 and 10) and histopathological evaluation (week 10). Radiological evaluation data demonstrated that quantities for the E2+MSC group were significantly the greatest in comparison with the other groups at week 4 to 10 inclusive. Moreover, Histopathological evaluation indicated that the E2+MSC group had the highest score which was significantly greater than the E2 group and the control group (P<0.05). In-vivo application of in situ 17ß-estradiol provides the seeded BMSCs with improved osteogenic capacity in tandem with an accelerated rate of bone healing. This obviously more qualified approach that yields in a shorter time appears to be promising for the future cell-based clinical treatments of the non-union bone fractures. Exploiting mesenchymal stem cells (MSCs) appears to be an appealing alternative to the traditional clinical approach in the treatment of non-union bone defects. It has been shown that 17ß-estradiol improves the osteogenesis and proliferation potential of the MSCs via estrogen receptors. We investigated the effect of 17ß-estradiol on exploiting autologous BMSCs (bone marrow-derived MSCs) for the purpose of healing of radial non-union segmental defect in rabbit. Twenty rabbits were divided into 4 experimental groups: 1. Control group; 2. MSC treatment group; 3. 17ß-estradiol (E2) treatment group; and 4. E2+MSC treatment group. Isolated BMSCs were seeded in a critical-sized defect on the radial mid-diaphysis that was filled with autologous fibrin clot differently in 4 groups: 1. intact fibrin clot (control); 2. Fibrin clot containing MSCs; 3. Estradiol; and 4. E2 and MSCs. Defect healing was assessed by radiological (week 0, 2, 4, 6, 8 and 10) and histopathological evaluation (week 10). Radiological evaluation data demonstrated that quantities for the E2+MSC group were significantly the greatest in comparison with the other groups at week 4 to 10 inclusive. Moreover, Histopathological evaluation indicated that the E2+MSC group had the highest score which was significantly greater than the E2 group and the control group (P<0.05). In-vivo application of in situ 17ß-estradiol provides the seeded BMSCs with improved osteogenic capacity in tandem with an accelerated rate of bone healing. This obviously more efficient approach that yields in a shorter time appears to be promising for future cell-based clinical treatments of the non-union bone fractures.

Wrapped omentum with periosteum concurrent with adipose derived adult stem cells for bone tissue engineering in dog model.

Adipose derived adult stem cells (ASCs) are multipotent cells that are able to differentiate into osteoblasts in presence of certain factors. The histological characteristics of periosteum makes it a specific tissue with a unique capacity to be engineered. Higher flexibility of the greater omentum is useful for reconstructive surgery. These criteria make it suitable for tissue engineering. The present study was designed to evaluate bone tissue engineering with periosteal free graft concurrent with ASCs and pedicle omentum in dog model. Twelve young female indigenous dogs were used in this experiment. In omental group (n = 4), end of omentum was wrapped by periosteum of the radial bone in abdomen of each dog. In omental-autogenously ASCs group (n = 4), 1 ml of ASCs was injected into the wrapped omentum with periosteum while in omental-allogenously ASCs group (n = 4), 1 ml of allogenous ASCs was injected. Lateral view radiographs were taken from the abdominal cavity postoperatively at the 2nd, 4th, 6th and 8th weeks post-surgery. Eight weeks after operation the dogs were re-anesthetized and the wrapped omenum by periosteum in all groups was found and removed for histopathological evaluation. Our results showed that omentum-periosteum, omental-periosteum-autogenous ASCs and omental-periosteum-allogenous ASCs groups demonstrated bone tissue formation in the abdominal cavity in dog model. The radiological, macroscopical and histological findings of the present study by the end of 8 weeks post-surgery indicate bone tissue engineering in all three groups in an equal level. The present study has shown that the wrapped omentum with periosteum concurrent with ASCs (autogenous or allogenous ASCs) lead to a favorable bone tissue formation. We suggested that it may be useful when pedicle graft omentum used concurrent with periosteum in the bone defect reconstruction, and this phenomenon should be studied in future.

Bone tissue engineering with periosteal-free graft and pedicle omentum.

BACKGROUND: The histological characteristics of periosteum make it a specific tissue with a unique capacity to be engineered. Higher flexibility of the greater omentum is useful for reconstructive surgery as it facilitates not only filling of the site of infections such as myelitis, but also is effective in filling complicated defects of the soft and hard tissues, and these criteria make it suitable for tissue engineering. The present study was designed to evaluate bone tissue engineering with periosteal-free graft concurrent with pedicle omentum and compare it with subcuticular periosteal grafting in a dog model. This is the first report in which periosteum-free graft has been used as bone tissue engineering. METHODS: Eight young female indigenous dogs were used in this experiment. In omental group (n = 4), end of omentum was wrapped by periosteum of the radial bone in the abdomen of each dog, while in the subcutaneous group (n = 4), the harvested periosteum was sutured on the subcutaneous layer. Lateral view radiographs were taken from the abdominal cavity post-operatively at 2, 4, 6 and 8 weeks post surgery. Eight weeks after operation, the dogs were re-anaesthetized and the omental or subcutical grafted periosteom was found and removed for histopathological evaluation. RESULTS AND DISCUSSION: Radiological, gross and histopathological evaluations revealed a superior bone formation in the wrapped omentum with periosteum compared with that of the subcuticular periosteal grafting. This is a novel and efficient technique in producing mature trabecular bone and could be used as a potential source of bone tissue engineering for autotransplantation.

Effects of adipose tissue stem cell concurrent with greater omentum on experimental long-bone healing in dog.

Repair of large bone defects resulting from trauma, tumors, and osteitis is a current challenge to surgeons. Adipose-derived adult stem cells (ASCs) are multipotent cells that are able to differentiate into osteoblasts in the presence of certain factors. In this study, the role of greater omentum as a scaffold incorporation of ASCs was evaluated in long-bone defect healing in dog model. Sixteen 3-4-year-old, male adult mongrel dogs, weighing 25.2 ± 3.5 kg, were used in this study. In the control group (n = 4), the defect was left empty. In the omental group (n = 4), the defect was filled with harvested omentum. In the omental-ASCs group (n = 4), the defect was filled with omentum and 1 mL of ASCs was injected into the grafted omentum. In the omental-culture medium group (n = 4), 1 mL of culture medium was injected into the grafted omentum. Finally, the injured radial bones were fixed with plate and screw. Radiographs of each forelimb was taken postoperatively on the first day and at the second, fourth, sixth, and eighth weeks postinjury to evaluate bone formation, union, and remodeling of the defect. The operated radii were removed on the 56th postoperative day and were histopathologically evaluated. In this study, both omental-culture medium and omental-ASCs groups demonstrated superior osteogenic potential in healing the radial bone defect. Compared to those of the omental and control groups, more advanced bone healing criteria were present in the omental-culture medium and omental-ASCs groups at radiological and histopathological levels at 8 weeks postsurgery.

Developmental and lactational exposure to environmentally relevant concentrations of dieldrin does not alter pregnancy outcome and mammary gland morphology in BALB/c mice.

The objectives of this study were to: (1) determine the dosing range necessary to produce serum levels of dieldrin in mice representative of human body burdens; and (2) define the effect of developmental exposure to environmentally relevant concentrations of dieldrin on mammary gland development. Sexually mature female BALB/c mice (n=140) were randomly assigned to receive vehicle, 0.45, 2.25, 4.5, and 22.5 microg dieldrin/g body weight (BW)/day. Serum levels of dieldrin were quantified by gas chromatography in pooled samples (n=4/treatment group). Target levels of 10-30 ng/ml were achieved in 0.45 and 2.25 microg/g dose groups by the end of 2 weeks of treatment. Vehicle or dieldrin (0.45, 2.25, and 4.5 microg/g BW) was administered weekly to sexually mature female BALB/c mice (n=48) throughout mating, pregnancy, and lactation. Treatments had no effect on fertility parameters in dams or mammary gland morphology at sexual maturity. Developmental exposure to dieldrin has no effect on mammary gland development in aged BALB/c mice.