Post doctoral reasearcher INRAE, PRC, 37380, Nouzilly, France Nouzilly, France
Abstract Authors: Coline Mahé1; Aleksandra Maria Zlotkowska2; Karine Reynaud1; Régis Lavigne3,4; Emmanuelle Com3,4 ; Charles Pineau3,4; Elke Albrecht5 ; Pascal Mermillod1; Jennifer Schoen2, 6; Marie Saint-Dizier1,7
1. CNRS, IFCE, INRAE, Université de Tours, PRC, 37380, Nouzilly, France
2. Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, Germany
3. Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR-S 1085, F-35000 Rennes, France
4. Protim, Univ Rennes, Biosit – UMS 3480 CNRS, US 018 Inserm, F-35000 Rennes, France
5. Institute of Muscle Biology & Growth, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
6. Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany
7. Tours University, Faculty of Sciences and Techniques, Tours, France
Abstract Text: The caudal isthmus part of the oviduct serves as a sperm reservoir before ovulation in many mammalian species. Sperm cells bind to the epithelium and its apical structures, especially to cilia, which prolongs their survival and ensures the availability of sperm able to fertilize until the time of ovulation. However, very few sperm receptors on oviduct cilia have been identified so far. Therefore, our aim was to compare the apical epithelial proteome of bovine oviducts between anatomical regions (ampulla vs. isthmus) before and after ovulation to identify new receptor candidates for sperm binding.
Oviducts from cyclic adult cows were collected at a local slaughterhouse. Only pre- and post-ovulatory oviducts ipsilateral to the ovulation site were used (4 cows/stage = 16 samples). After 6 h fixation and paraffin embedding, 6 µm serial sections of the isthmus and ampulla were used for laser capture microdissection of the apical epithelium. Proteins were digested and analyzed by nano liquid chromatography coupled with tandem mass spectrometry. Proteins were identified using the Uniprot Bos taurus database and considering minimum two unique peptides. Proteins were then quantified by precursor intensities-based label-free. Ciliary origin of proteins was assessed with the Human Protein Atlas and the list of proteins previously identified in human airway cilia. Prediction of subcellular localizations and molecular interactions were performed using TMHMM, MBPPred and ProteinPrompt tools. Protein abundance were compared between stages and anatomical regions by ANOVA and t-tests considering a p-value ≤ 0.05. Functional gene ontology enrichment analysis was performed using Metascape online tool.
In total, 505 proteins were identified in the microdissected samples, of which 106 were previously identified in cilia. Among those 106 ciliary proteins, 5 were predicted to be transmembrane and 9 predicted to interact with membrane phospholipids, including the annexins (ANXA1, A2, A4, A5) and the voltage-dependent anion-selective channel proteins 1 and 2 (VDAC1, VDAC2). After ANOVA and hierarchical clustering, 66 proteins were differentially abundant and clustered according to the region: 32 proteins were overabundant in the isthmus (group 1) and 34 were overabundant in the ampulla (group 2). Proteins from group 1 were mainly involved in antioxidant processes while the ones from group 2 were mainly involved in protein binding.
Among the proteins overabundant in the pre-ovulatory isthmus (the time and place of the sperm reservoir) compared to the pre-ovulatory ampulla, 6 were previously identified in cilia including the peroxiredoxin 6 (PRDX6) and dynein light chain roadblock-type 2 (DYNLRB2). Among the proteins overabundant in the pre-ovulatory isthmus compared to the post-ovulatory isthmus, 5 were previously identified in cilia including the tubulin beta-2B chain (TUBB2B) and peroxiredoxin 2 (PRDX2). Furthermore, 108 identified proteins were predicted to interact with the sperm-coating Bovine Seminal Plasma proteins (BSPs), including ANXA2, ANXA5, DYNLRB2, TUBB2B, VDAC1 and VDAC2 and 32 predicted to interact with the sperm-coating β-defensin 126 protein including ANXA2, PRDX6, VDAC1 and VDAC2.
This is the first proteome of the apical oviduct epithelium in mammals. This study highlights changes in apical proteins along the oviduct during the periconception period and reports new candidates potentially involved in the oviduct sperm reservoir.
