Inhibition of ovine in vitro fertilization by anti-Prt antibody: hypothetical model for Prt/ZP interaction

BACKGROUND: The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood. Likewise, the role of prnt gene, encoding the prion-like protein testis specific (Prt), in ram reproductive physiology remains largely unknown. In this study, we assessed the effect of Prt in ovine fertilization by using an anti-Prt antibody (APPA) in fertilization medium incubated with spermatozoa and oocytes. Moreover, a computational model was constructed to infer how the results obtained could be related to a hypothetical role for Prt in sperm-zona pellucida (ZP) binding. METHODS: Mature ovine oocytes were transferred to fertilization medium alone (control) or supplemented with APPA, or pre-immune serum (CSerum). Oocytes were inseminated with ovine spermatozoa and after 18 h, presumptive zygotes (n = 142) were fixed to evaluate fertilization rates or transferred (n = 374) for embryo culture until D6-7. Predicted ovine Prt tertiary structure was compared with data obtained by circular dichroism spectroscopy (CD) and a protein-protein computational docking model was estimated for a hypothetical Prt/ZP interaction. RESULTS: The fertilizing rate was lower (P = 0.006) in APPA group (46.0+/−6.79%) when compared to control (78.5+/−7.47%) and CSerum (64.5+/−6.65%) groups. In addition, the cleavage rate was higher (P < 0.0001) in control (44.1+/−4.15%) than in APPA group (19.7+/−4.22%). Prt CD spectroscopy showed a 22% alpha-helical structure in 30% (m/v) aqueous trifluoroethanol (TFE) and 17% alpha in 0.6% (m/v) TFE. The predominant alpha-helical secondary structure detected correlates with the predicted three dimensional structure for ovine Prt, which was subsequently used to test Prt/ZP docking. Computational analyses predicted a favorable Prt-binding activity towards ZP domains. CONCLUSIONS: Our data indicates that the presence of APPA reduces the number of fertilized oocytes and of cleaved embryos. Moreover, the CD analysis data reinforces the predicted ovine Prt trend towards an alpha-helical structure. Predicted protein-protein docking suggests a possible interaction between Prt and ZP, thus supporting an important role for Prt in ovine fertilization

Morphologycal Changes Of Islet Of Langerhans In An Animal Model Of Type 2 Diabetes

A deficiência da secreção de insulina na diabetes tipo 2 deve-se à menor massa de células ß e à disfunção destas células. A redução da massa celular na doença humana é variável, mas não superior a 50%. Não há alterações específicas da morfologia do ilhéu diabético humano; pode surgir deposição amilóide e, ocasionalmente, fibrose insular. Com este estudo cronológico comparativo entre ratos Wistar normais (W) e ratos Goto-Kakizaki (GK), um modelo animal de diabetes tipo 2 não obesa, fizemos a avaliação da morfologia do pâncreas endócrino; a aplicação de técnicas histológicas e imuno-histoquímicas permitiu caracterizar a estrutura insular e com o estudo morfométrico computorizado de secções imunomarcadas para insulina foi possível avaliar as áreas insulino-positivas. Foi também feita uma avaliação clínica, igualmente de forma cronológica: peso corporal, glicémia (jejum e após glicose oral) e outros parâmetros bioquímicos do sangue. Os ratos GK foram sempre intolerantes à glicose e a hiperglicémia em jejum surgiu após as quatro semanas de vida; os ratos W foram primeiras semanas. A área de células insulinopositivas foi sempre menor nos pâncreas diabéticos, mas superior a 40%. Às 12 semanas e atingindo progressivamente mais ilhéus com o avançar da idade surgiram, numa subpopulação destes, lesões da normal arquitectura insular, não relacionáveis com processos inflamatórios ou imunes nem com deposição amilóide, e caracterizadas por irregularidade de contornos, perda da normal distribuição relativa das células endócrinas e aumento da matriz extracelular com deposição aumentada de material PAS positivo e fibrose. Os nossos resultados demonstram que, nos ratos GK da colónia de Coimbra, a redução da massa de células ß constitui um dos factores primários da síndrome diabética. As lesões estruturais dos ilhéus, capazes de reduzir ainda mais a massa celular e de comprometer a função insular, surgem mais tarde e serão, pelo menos em parte, causadas pela hiperglicémi

Medical grade honey as a promising treatment to improve ovarian tissue transplantation

Ovarian tissue cryopreservation is a female fertility preservation technique that presents major challenges for the maintenance of follicular viability after transplantation. The aim of this study was to evaluate and compare the application of L-Mesitran Soft®, a product containing 40% medical grade honey (MGH), with other strategies to improve ovarian grafts’ viability. For this purpose, bovine ovarian tissue was vitrified, warmed, and randomly assigned to culture groups: (1) control, (2) MGH 0.2% in vitro, (3) MGH in vivo (direct application in the xenotransplantation), (4) vascular endothelial growth factor (VEGF 50 ng/mL) and (5) vitamin D (100 Nm), during a 48 h period. A sixth group (6) of fragments was thawed on transplantation day and was not cultured. The tissue was xenotransplanted into immunodeficient (Rowett nude homozygous) ovariectomized rats. Grafts were analyzed 48 h after culture, and 7 and 28 days after transplantation. The tissue was subjected to histological and immunohistochemical analysis. Treatments using MGH showed the highest angiogenic and cell proliferation stimulation, with cellular apoptosis, within a healthy cellular turnover pathway. In conclusion, MGH should be considered a potentially effective and less expensive strategy to improve ovarian tissue transplantation.info:eu-repo/semantics/publishedVersio

Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors—FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares’ oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns

Microvascularization and Expression of Fibroblast Growth Factor and Vascular Endothelial Growth Factor and Their Receptors in the Mare Oviduct.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors-FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns