Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

BACKGROUND: DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. OBJECTIVES: In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. METHODS: We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. RESULTS: We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. CONCLUSIONS: The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 µg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 µg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Clinical, ultrasonography and haematology of aglepristone-induced mid-gestation pregnancy terminations in rabbits.

Aglepristone is a safe abortifacient in cats, dogs and rabbits. Although no serious side effects have been reported, there is no information available about the effects of the medicine on haematological parameters. For the first time clinical and ultrasonographic features and haematological profiles were evaluated in rabbits treated with aglepristone 15 and 16 days after mating. Ten healthy 10-14 month-old New Zealand White female rabbits were mated with fertile bucks and pregnancies were confirmed by ultrasound 15 days later. Of these, 5 does were treated with aglepristone (test group, n = 5) whilst the remaining five (control group, n = 5) were treated with a saline solution (0.9% NaCl). The treatment dose was 10 mg/kg body weight, administered subcutaneously once daily on two consecutive days (day 15 and 16 post mating). Ultrasonographic, clinical and haematological assessments were performed daily. Aglepristone treatment induced embryonic fluid resorptions without foetal death in mid-gestation terminations. Following ultrasonographic and haematological examinations, it was established that aglepristone is a safe abortifacient in rabbits.

Investigations into the mechanisms controlling parturition in cattle.

A pronounced increase in fetal cortisol concentrations stimulating an increase in estrogen production at the expense of progesterone precursors in the placenta, luteolysis, and progesterone withdrawal is considered as a key event during the complex signal cascade leading to the initiation of parturition in cattle. However, there are many questions concerning the exact functional and/or temporal relationships between these individual processes which finally result in the expulsion of the calf and the timely release of the placenta. Thus, parturition was induced in 270-day pregnant cows using the progesterone receptor blocker aglepristone (group AG, n=3), the prostaglandin F(2α) analog cloprostenol (group PG, n=4), and the glucocorticoid dexamethasone (group GC, n=4) to characterize the effect on maternal steroid and prostaglandin levels and to identify immediate subsequent changes in placental morphology and gene expression as compared with untreated controls sampled on day 272 (group D272, n=3) and cows during normal parturition (group NT, n=4). All calves of the treatment groups were born on days 271-272, whereas gestational length in NT cows was 280.5±1.3 days. However, none of the treatments significantly induced the prepartal remodeling of placentomes characterized by a decline in trophoblast giant cells and reduction of the caruncular epithelium. Data on placental CYP17 and COX2 expression confirm that these key enzymes are upregulated by GC, whereas placental aromatase expression was not affected by any treatment. Maternal progesterone and prostaglandin profiles suggest differential effects of the treatments on luteal function and placental or uterine prostaglandin production. The results provide new information on the initiation of parturition in cattle but raise many new questions.

Use of the progesterone (P4) receptor antagonist aglepristone to characterize the role of P4 withdrawal for parturition and placental release in cows.

In late pregnant cows, progesterone (P(4)) is mainly of luteal origin. However, the trophoblast may provide high local P(4) concentrations in the uterus. To test for the importance of a complete P(4) withdrawal for parturition-related processes and placental release, the P(4) receptor (PGR) blocker aglepristone (Ap) was administered to three cows on days 270 and 271 of pregnancy. A complete opening of the cervix was observed 46.5±7.3 h after the start of treatment. However, expulsion of the calves was impaired obviously because of insufficient myometrial activity, and placental membranes were retained for at least 10 days. Measurement of P(4) concentrations indicated that PGR blockage induced luteolysis. To investigate the role of P(4) withdrawal for the prepartal tissue remodeling of the placentomes, the caruncular epithelium was evaluated by morphometry, and the percentage of trophoblast giant cells (TGCs) relative to the total number of trophoblast cells were assessed. Caruncular epithelium in Ap-treated cows (D272+Ap) was immature (30.5±3.3%) and not different from untreated controls (elected cesarean section (CS) on day 272; D272-CS; 31.5±1.4%), whereas it was significantly reduced at normal term (D280.5±1.3; 21.0±6.1%; P=0.011). Correspondingly, the percentage of TGCs were 20.1±1.4 in D272+Ap, 22.1±4.8 in D272-CS, and 9.8±3.9 at term (P=0.001). No effect was detected on placental estrogen synthesis. The results showed that in late pregnant cows, P(4) withdrawal only induces a limited spectrum of the processes related to normal parturition and is not a crucial factor for the prepartal tissue remodeling in placentomes and the timely release of the placenta.