It is well known that bitches present a peculiar oocyte maturation, characterized by ovulation of immature oocytes and a long period of viability in the oviduct. Thus, the oviduct in this species plays an essential role in oocyte maturation, in addition to fertilization and early stage embryo development. Different approaches have been used in order to identify the factors involved in each step (from resumption of meiosis through development of embryos) and recently a new technique was used for spatial identification of oviductal proteins in the domestic cat (1). With the purpose to contribute to the understanding of these factors, we applied MALDI imaging mass spectrometry (MALDI-IMS) to obtain protein profiling and imaging of canine oviducts. Reproductive tracts were collected from 4 bitches in estrus (cross-breed, 2 to 6 years old) undergoing routine ovariohysterectomy. Post-ovulatory period was confirmed by blood serum progesterone concentrations (P4 mean±SD: 15.5±2.5) and ovarian morphology. The oviducts were carefully dissected, divided into three segments (distal to the ovary-isthmus, proximal to the ovary-infundibulum and the mid-section between the two-ampulla; further confirmed by histology), snap-frozen in liquid nitrogen and stored at -80ºC until use. Then, they were sectioned (11 µm) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides, while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (α-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 µm. Mass spectra were characterized by abundant ions of m/z 2279, 2401, 3458 and 4976; which have been tentatively attributed to actin cytoplasmic 2, keratin type 1, neutrophil defensin 1 and thymosin β4, respectively. Actin has been detected in oviduct fluid of alpaca (2) and is related to epithelial cell renewal or secretory activity (3). As previously described in queens (1), defensin, keratin and thymosins are defense proteins that integrate the innate immune systems and are involved in the biological response to cellular damage. These data might contribute to piecing together the puzzle of factors that are involved in the peculiar aspects of the domestic dog reproductive physiology that might hamper in vitro embryo production. 1) Apparicio M, Santos VG, Rocha DFO et al. Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins. Reprod Domest Anim. 2017;52 (Suppl. 2):88–92. (2) Apichela SA, Argañaraz ME, Zampini R et al. Biochemical composition and protein profile of alpaca (Vicugna pacus) oviductal fluid. Anim Reprod Sci. 2015,154:79-85 (3) Steffl M, Schweiger M, Sugiyama T et al. Review of apoptotic and non-apoptotic events in non-ciliated cells of the mammalian oviduct. Ann Anat. 2008,190: 46-52.
|Titolo:||Chemical imaging of canine oviducts during the post-ovulatory period|
|Data di pubblicazione:||2018|
|Parole Chiave:||dog; MALDI; reproduction|
|Settore Scientifico Disciplinare:||Settore VET/10 - Clinica Ostetrica e Ginecologia Veterinaria|
|Citazione:||Chemical imaging of canine oviducts during the post-ovulatory period / M. Apparicio, D.F.O. Rocha, F. Negrão, D.G. Borges, C.B.M.L. Felippe, B.I. Macente, M.R. Tavares, G.C. Luvoni, M.N. Eberlin, W.R.R. Vicente. ((Intervento presentato al 21. convegno International Congress EVSSAR tenutosi a Venezia nel 2018.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|