ProAKAP4 protein marker: Towards a functional approach to male fertility.

Spermatozoa are highly differentiated cells whose ultimate function is fertilization to successfully transfer the male genome. This achievement relies on the expression, localization, organization, and proper functionality of their molecular components. For years, proteomics emerged as a remarkable approach for fertility research to identify specific protein markers related to sperm competency. Such biomarkers, undetected with conventional semen analysis methods, are next-generation tools to assess sperm functionality and predict male fertility. Among them, the proAKAP4 marker is a sperm-specific protein, synthesized as the precursor of AKAP4, highly conserved among mammals and only present in the principal piece of the flagellum. ProAKAP4 entails a disposable stock of AKAP4 to ensure long-lasting sperm motility up to the fecundation site, capacitation, and fertility. By being either converted into mature and active AKAP4 or degraded by proteolysis, proAKAP4 is a stress sensor, acting as a potential gatekeeper to prevent transmission of unfavorable genetic damage to the next generation. Loss or decrease of AKAP4 expression does not affect the number of spermatozoa produced but it impacted sperm motility, viability, and fecundation. A method based on proAKAP4 concentration measurement in a defined number of spermatozoa recently appeared as a simple, reliable, reproducible, robust, and quantitative tool to assess more objectively semen quality and predict male fertility. In this review, we will discuss how fundamental discoveries around the proAKAP4 biomarker provide a yet missing molecular dimension in semen analysis assessments to ensure higher semen quality and reproductive performance in veterinary clinics, zoos, and wild animal reproduction centers.

Synergy between dinotefuran and fipronil against the cat flea (Ctenocephalides felis): improved onset of action and residual speed of kill in adult cats.

BACKGROUND: The cat flea, Ctenocephalides felis felis (C. felis), is a cosmopolitan hematophagous ectoparasite, and is considered to be the most prevalent flea species in both Europe and the USA. Clinical signs frequently associated with flea bites include pruritus, dermatitis and in severe cases even pyodermatitis and alopecia. Ctenocephalides felis is also a vector for several pathogens and is an intermediate host for the cestode Dipylidium caninum. Treatment of cats with a fast-acting pulicide, that is persistently effective in protecting the animal against re-infestation, is therefore imperative to their health. In addition, a rapid onset of activity ("speed of kill") may also reduce the risks of disease transmission and flea allergic dermatitis. The aim of this study was to evaluate the in vitro insecticidal activity and potential synergism between dinotefuran and fipronil against C. felis. A further aim was to evaluate the onset of activity and residual speed of kill of the combination in vivo on cats artificially infested with C. felis. METHODS: In the first study, the insecticidal activity of dinotefuran and fipronil separately and dinotefuran/fipronil (DF) in combination, at a fixed ratio (2:1), was evaluated using an in vitro coated-vial bioassay. In the second study, the onset of activity against existing flea infestations and residual speed of kill of DF against artificial flea infestations on cats was assessed in vivo. Onset of activity against existing flea infestations was assessed in terms of knock-down effect within 2 h post-treatment and onset of speed of kill assessed at 3 h, 6 h and 12 h post-treatment. Residual speed of kill was evaluated 6 h and 48 h after infestation, over a period of six weeks post-treatment. RESULTS: In vitro results revealed that the DF combination was synergistic and more potent against fleas than either compound alone. The combination also proved effective when tested in vivo. Efficacy was > 97% [geometric mean (GM) and arithmetic mean (AM)] at 3 h after treatment, and ≥ 99.8% (GM and AM) at 6 h and 12 h post-treatment. At 6 h after flea re-infestations, the efficacy of DF remained ≥ 90.8% (GM and AM) for up to 28 days, and at 42 days post-treatment persistent efficacy was still ≥ 54.3% (GM and AM). At 48 h after flea re-infestations, DF remained almost fully effective for up to 28 days, with efficacies ≥ 99.4% (GM and AM) and was persistently ≥ 93.0% (GM and AM) effective for up to 42 days post-treatment. CONCLUSIONS: The combination of dinotefuran and fipronil in a single formulation exhibited strong synergistic insecticidal activity against C. felis in vitro, and also proved effective on artificially infested cats. This activity had a rapid onset that persisted for 6 weeks against re-infestations of C. felis on cats. The rapid curative insecticidal effect was observed as early as 3 h after treatment, and as early as 6 h after re-infestations for up to 6 weeks post-treatment. The insecticidal activity profile of DF makes it an optimal candidate for the protection of cats against flea infestations, and possibly also associated diseases.

Pharmacokinetics and efficacy of an ivermectin implant for long-term prevention of Dirofilaria immitis infection in dogs.

An exploratory study was carried out to assess the in vivo efficacy of different prototypes of subcutaneous implants containing ivermectin (IVM) for the prevention of canine Dirofilaria immitis infection. The implants consisted of an ethylcellulose matrix containing IVM (3.0, 4.5, and 6.0 mg/implant; from 0.29 to 0.63 mg/kg bw) as active ingredient designed to release approximately 0.1 ng of IVM/mL in the plasma for at least 12 months. Six dogs were implanted on day -365. On day -12, three heartworm-free dogs were included in the study as a control group. All nine dogs were examined on day -7 and day 0 for circulating D. immitis microfilariae and by an antigen ELISA kit to confirm that the dogs were heartworm-free. The dogs were artificially infected with 75 D. immitis infective larvae (L3) each on day 0. Dogs in the control group were humanely euthanized on day 153 to verify the infectivity of the larvae, while implanted dogs were further examined for circulating D. immitis microfilariae and antigen on days 153, 195, and 246. The treated dogs were not necropsied. The pharmacokinetic profile of the IVM implant was assessed in plasma samples taken on day -364, then at different times until the infection day, and again on days, 15, 30, 60, 90, 120, and 153. Throughout the study, body weights were measured during clinical examination on days 0, 30, 60, 90, 120, and 153. At necropsy, all control dogs were found infected, each with 10-11 adult heartworms. Implanted dogs were negative at both microfilaria and D. immitis antigen examinations until day 246 (8 months from the infection). IVM plasma levels ranged 0.06-0.16 ng/mL on day 0 and remained stable until day 60, afterward they gradually decreased under the limit of quantification of the method. Throughout the study, no side effect was observed. On the basis of these results, it was possible to conclude that the different prototypes of implants were able to protect the dogs from D. immitis artificial infection for at least 12 months.

Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation.

BACKGROUND: Cryopreservation of cortex ovarian tissue before anticancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ovarian graft causes massive follicle loss by apoptosis. VEGF111 is a recently described vascular endothelial growth factor (VEGF) isoform that does not bind to the extracellular matrix, diffuses extensively, and is resistant to proteolysis. These properties confer a significantly higher angiogenic potential to VEGF111 in comparison with the other VEGF isoforms. METHODS: We evaluated the morphology of cryopreserved sheep ovarian cortex grafted in the presence or absence of VEGF111. Ovarian cortex biopsies were embedded in type I collagen with or without VEGF111 addition before transplantation to severe combined immunodeficient mice ovaries. Transplants were retrieved 3 days or 3 weeks later. Follicular density, vasculature network, hemoglobin content, and cell proliferation were analyzed. RESULTS: Addition of VEGF111 increased density of functional capillaries (P=0.01) 3 days after grafting. By double immunostaining of Ki-67 and von Willebrand factor, we demonstrated that proliferating endothelial cells were found in 83% of the VEGF111 group compared with 33% in the control group (P=0.001). This angiostimulation was associated with a significant enhancement of hemoglobin content (P=0.03). Three weeks after transplantation, the number of primary follicles was significantly higher in VEGF111 grafts (P=0.02). CONCLUSION: VEGF111 accelerates blood vessel recruitment and functional angiogenesis and improves the viability of ovarian cortex by limiting ischemia and ovarian cortex damage.

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.