PROCEEDING The 7th AIC-ICMR on Health and Life Sciences The Annual International Conference 2017 Syiah Kuala University “Advancing Knowledge, Research, and Technology for Humanity” ISSN: 2089-208X Banda Aceh, Aceh, Indonesia October 18-20, 2017 Proceedings of the 7th AIC-ICMR on Health and Life Sciences Comparing Oviductal Expression During Oestrous Cycle of Epithelial Oviductin (OVGP1) in Peranakan Ettawah (PE) Goat Herawati1*, Yudit Oktanella2, Aulia Firmawati2, Nurul Isnaini3 1Departement of Veterinary Public Health; 2Departement of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang 65145, Indonesia 3Departement of Animal Husbandry, Animal Husbandry Faculty, Brawijaya University, Malang 65145, Indonesia *Correspondence e-mail: [email protected] / [email protected] Abstract Over hundreds types of proteins are present in the oviductal tract, where fertilization occurs and zygotes begin to develop. Oviductin, also called Oviductal glycoprotein 1 (OVGP1), has been shown to be expressed by the oviductal epithelial cells and is suggested to play a role in fertilization and sperm–egg interaction. The expression pattern of oviductin become important since the number of studies suggested that its transcription is regulated via estrogen-dependent during oestrous cycle. The aim of this study was to identify the characteristics of oviductin expression in oviduct during oestrous cycle, especially on folicullar and luteal phase. Twenty-four fresh reproduction organ of Peranakan Ettawah (PE) goat were collected from abattoir, then divided into two groups (Group1: Follicular phase; Group2: luteal phase) based on the ovary performance macroscopically then we performed immunohistochemistry to identify the oviductin expression in the oviduct. Our results showed that the percentage of oviductin expression vary throughout the estrous cycle, which is higher in follicular phase. In both, the ampulla and infundibulum, the expression of OVGP1 was found to be higher at folicullar phase than luteal phase; but there was no significant difference of oviductin expression during luteal phase in isthmus. In conclusion, this finding suggests that the difference expression‘s patterns in both folicullar and luteal phase is important in regulating fertilization and embryogenesis on PE goat. Key words: Oviductin, oviduct, PE goat, protein expression INTRODUCTION Although fertilization can be achieved in vitro without any contributions from the oviduct or oviductal proteins, some of these proteins play an important roles or yet remain unknown roles in fertilization and embryo development. In the pig, sheep, goat, cow, hamster, and rabbit, oviductal fluid is responsible for prefertilization ZP hardening (understanding ZP hardening as ZP changes that increase the ZP‘s resistance to proteolysis and reduce sperm binding. Oviductin that binds into ZP makes it more resistant to sperm attachment and decreases the risk of polyspermy in the pig and the cow (Coy et al. 2010). It undergoes numerous sequential morphological changes over the course of oestrous cycle, driven by cyclic fluctuations in several reproductive hormones. 191 The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia Oviductal fluid contains amino acids, proteins, simple carbohydrates and complex ions, fats and phospholipids. These components include metabolic subtract such lactic, pyruvic acid, amino acids and glucose that the level is different in uterine liquids and serum (Hugentobler et al. 2010). Volume and components of some proteins in oviductal liquids is changing during estrous cycles. Part of the complex of proteins in oviductal liquids is produced from transudate serum, however there is also a specific protein synthesis and secretion of oviduct epithelium, and the secretion of these proteins is regulated by hormonal changes, followed by an increase in biosynthesis of periovulatory period (Buhi, 2002). During the oestrous cycles, the main function of cervical and oviduct on the reproduction process involves interaction with gametes, the estrus phase. Therefore, it can be hypothesized that the regulation of proteins secretion in cervical fluid and oviduct when the oestrous- oriented interacting with gametes. On the other hand, when the luteal phase, the amount of differential protein in the uterus can changes in the concentration and type. Oviductin (OVGP1) is estrogen-dependent glycoprotein secreted on epithelial oviduct by almost all mammals (Coy and Yanagimachi, 2015). Oviductin in swine, sheep, goats, cows and rabbits hamsters known to efect in increasing the resistance of the zona pellucida, when pre-fertilization against proteolysis and decrease the possibility of polyspermy (Mondejar et al., 2013). Oviductin is located in perivitelline space and the membrane of the embryo each species before implatation. Their densely glycosylated mucin-type domains would act as a protective shield around the oocyte and the early embryo and would be important for early stages of embryo development (Buhi 2002, Wolf et al. 2003, Gon alves et al. 2008). Oviductin in pigs known to increase the post-cleavage embryo development to the blastocyst is formed (Kouba et al. 2000, McCauley et al. 2003). In goats, oviductin increase the level of cleavage in the cleavage stage, as well as the morula and blastocyst formation (Pradeep et al., 2011). Detection and analysis of proteins, synthesized and secreted from the oviduct epithelial find their distribution temporal and spatial of macro molecules and the differences in the distribution of proteins in various species. Moreover, this analysis has allowed the identification of the major secretory products oviduct specific glycoprotein, whose secretion is dependent estrogen, and has a high molecular weight. Knowledge of the oviductin expression derived from normal histological appearance of the reproductive tract at each stage of the oestrous cycle is essential when evaluating female reproductive tissues for evidence of its regulation during fertilization and embryogenesis. This paper is mainly focus 192 Proceedings of the 7th AIC-ICMR on Health and Life Sciences on oviductal tract since these two processes mainly occur in isthmus, ampulla, and infundibulum. MATERIALS AND METHODS Female reproductive organs derived from waste of Slaughterhouse Animal Gandang, Malang, collected a maximum of 2 hours after the slaughtering process. For the preparation of the organ, oviduct washed twice using physiological NaCl media and placed in a petri dish, then dissection. Determination of the follicular and luteal phases was seen by observing the dominant form in ovarian macroscpicly. The follicular phase marked by the formation of the dominant follicle in the ovary, meanwhile the luteal phase marked by the formation of the corpus luteum with minimum diameter of 0.3 mm. Result of grouping ovarian then placed in the organ pot containing 10% formalin for immunohistochemistry (IHC) staining. The IHC specimen made by goat-monoclonal antibody anti-oviductin (Abcam®), then the IHC specimen was observed under a microscope with magnification 400X and 1000X. The identification procedure oviductin expression by immunohistochemical methods, the oviduct specimen was made in object glass dipped in xylol twice, alcohol-rise (100%, 90%, 80%, 70% and 30%) consecutively. Then washed in PBS at pH 7.4 for 3 times each for 5 minutes. Then soaked in hydrogen peroxidase (H2O2) 3% for 5-10 minutes. Then soaked in 1% BSA in PBS for 10-30 minutes at room temperature. Added oviductin primary antibody for 1 hour at room temperature. Washed in PBS pH 7.4 for 3 times 5 minutes. SA-HRP was added for 30-60 minutes at room temperature. Washed in PBS pH 7,4 for 3x5 minutes. Added Cromogen DAB (3,3-diaminbenzidine tetrahydrochloride) for 10-20 minutes. Washed 3 times in PBS for 5 minutes at room temperature and then added with a counterstain for 3 minutes and then mounting with entellan. Observations were carried out by using a microscope at a magnification of 400x and 1000x times. Determination of the amount of oviductin expression can be seen from the percentage extensive DAB (brown color change in the lamina propria / oviduct epithelial cells) were compared among groups using software Immunoratio Oviductin expression data analysis. RESULTS AND DISCUSSION Identification of oviductin expression in the oviductal tract were analyzed by IHC staining by comparing the level of expression in the follicular and luteal phase. Based on it consideration that oviductin is a glycoprotein that is secreted by the oviduct epithelial cells 193 The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia are estrogen-dependent, although after fertilization also reported a significant increase, so that secretion is directly associated with changes in each estrus cycle. Figure 5.1 Oviductin expression in the cell nucleus that is contained at the lamina propria region in channel oviduct during the follicular phase (A) and luteal phase (B). Image 1. Oviductin expression in the oviduct PE goats with IHC staining. Description: