Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (8): 1543-1550.doi: 10.3864/j.issn.0578-1752.2017.08.018

• RESEARCH NOTES • Previous Articles    

A Modified Paraffin-Section Technique for Ovine Cumulus-Oocyte Complexes

LI HaiJun1, YU BoYang2, DUAN YunJiao1, LIU XingYu1, WENG YaZheng1, DU ChenGuang1, WANG XiuMei1   

  1. 1College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018；2 Psychosomatic Medicine Laboratory , Inner Mongolia Medical University, Hohhot 010110
  • Received:2016-11-28 Online:2017-04-16 Published:2017-04-16

RichHTML

3

PDF

668

Abstract: 【Objective】 During the ovine antral follicle development, the multilayer cumulus cells closely surround the oocyte to form a special structure named cumulus-oocyte complex (COC), existing in the follicular cavity. The ovine COC will be discharged tube from the mature follicle into the fallopian to be fertilized. The existing technologies for protein identification in COC choose the ovarian tissue containing follicles to be sliced up or to immunostain the whole COC after the COC isolated from the follicle. However, there are significant defects when using the two methods to detect protein expression in ovine COC from the antral follicle. In the present study, the conventional paraffin section technology was improved so as to meet the demand of ovine COC protein identification. 【Method】 The COCs were obtained from the antral follicles by aspiration, and the special structure containing multiple ovine COCs was created by paraffin embedding. Together with the healthy ovarian tissue, the target protein expression, urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), were identified in the COCs structure and the immunostaining effects and procedures were compared between the modified and conventional paraffin immunohistochemical technologies. 【Result】 The samples of ovine COCs and ovaries were obtained by paraffin embedding. The single COC sheets distributed in glass slides and the thin ovarian layer (5µm) was formed following sectioning, plastering and dewaxing. The indirect immunostaining results showed that, ? The target protein expression was consistent in the two types of COCs, which revealed that uPAR was expressed in both bovine cumulus and oocyte, while uPA was only expressed in cumulus. ? In the COC section, a well-preserved bovine COC structure with well-defined layers and easily identified protein location in either the oocyte or cumulus was observed. In the ovarian section, the follicle remained intact, and the clear protein localization in oocyte could be observed; however, cumulus layers and its protein localization were unclear. ?A comparison of the staining procedures between the both methods described herein showed that ovine COCs paraffin-sectioning technique provided a more simplified process and significantly shorter handling for fixation, dehydration, transparent processing, waxing and dewaxing. For example, fixation was shortened from 24 h to 2 h; dehydration was converted from the granual steps of 10 h to the two-step of 1 h; transparent processing was shortened from 30 mins to 10mins; waxing and dewaxing were simplified from 6 h to 1 h, and from the granual steps of 25 mins to the two-step of 10 mins, respectively. 【Conclusion】 The modified COCs paraffin sectioning technique showed a better immunostaining effect and featured the higher generation rate for COCs, the clearer outline of oocyte and cumulus cells, and the more simplified operation process. Based on the complete COCs structure, this new technology effectively detected the target protein expression in ovine COCs, with great methodological significance to reveal the developing mechanism of ovine follicles.

Key words: sheep, cumulus-oocyte complexes-like tissue, ovarian tissue, paraffin section, protein expression

[1]    QUIRK S M, COWAN R G, HARMAN R M, HU C L, PORTER D A. Ovarian follicular growth and atresia: the relationship between cell proliferation and survival. Journal of Animal Science, 2004,82 (E-Suppl.): E40 -52.
[2]    沙红英，陈建泉，钱旻，成国祥. 哺乳动物卵泡发育的调控机制. 生殖与避孕, 2004,24(5): 295-299.
SHA H Y, CHEN J Q, QIAN M, CHENG G X. Regulation of mammalian follicle development. Reproduction and Contraception, 2004, 24(5): 295-299. (in Chinese)
[3]    ARMSTRONG D T, KOTARAS P J, EARL C R. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reproduction Fertility and Development, 1997, 9(3): 333-339.
[4]    LI H J, SUTTON-MCDOWALL M L, WANG X, SUGIMURA S, THOMPSON J G, GILCHRIST R B. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions. Human Reproduction, 2016, 31(4): 810-821.
[5]    KAWASHIMA I, LIU Z, MULLANY LK, MIHARA T, RICHARDS JS, SHIMADA M. EGF-like factors induce expansion of the cumulus cell-oocyte complexes by activating calpain-mediated cell movement. Endocrinology, 2012, 53(8):3949-3959.
[6]    FABBRI R, MACCIOCCA M, VICENTI R, PASQUINELLI G, CAPRARA G, VALENTE S, SERACCHIOLI R, PARADISI R. Long-term storage does not impact the quality of cryopreserved human ovarian tissue. Ovarian Research, 2016, 9(1):50.
[7]    SHIKINA S, CHIU Y L, CHUNG Y J, CHEN C J, LEE Y H, CHANG C F. Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis. Scientific Reports, 2016, 6: 258-268.
[8]    GRIFFIN D, LIU X, PRU C, PRU J K, PELUSO J J. Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells. Biology of Reproduction, 2014, 91(2):36.
[9]    FRANK L A, SUTTON-MCDOWALL M L, BROWN H M, RUSSELL D L, GILCHRIST R B, THOMPSON J G. Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90. Human Reproduction, 2014, 29(6):1292-1303.
[10]   PELUFFO M C, TING A Y, ZAMAH A M, CONTI M, STOUFFER R L, ZELINSKI M B, HENNEBOLD J D. Amphiregulin promotes the maturation of oocytes isolated from the small antral follicles of the rhesus macaque. Human Reproduction, 2012, 27(8):2430-2437.
[11]   XU J, LAWSON M S, YEOMAN R R, PAU K Y, BARRETT S L, ZELINSKI M B, STOUFFER R L. Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Human Reproduction, 2011,26:1061-1072.
[12]   DU C, XILINGAOWA, CAO G, WANG C, LI H,vZHAO Y, SIQINGAOWA, vCAO J. Expression of the orexigenic peptide ghrelin in the sheep ovary. Domestic Animal Endocrinology, 2009, 36(2): 89-98.
[13]   SILVA C C, GROOME N P, KNIGHT P G. Immunohistochemical localization of inhibin/activin alpha, betaA and betaB subunits and follistatin in bovine ooc ytes during in vitro maturation and fertilization. Reproduction, 2003, 125 (1):33-42.
[14]   ALBANO R M, GROOME N, SMITH J C. Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development, 1993, 117(2): 711-723.
[15]   SIMÓN C, FRANCES A, PIQUETTE G, POLAN M L. Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biology of Reproduction, 1994, 50(2):449-457.
[16]   YUAN S, WEN J, CHENG J, SHEN W, ZHOU S, YAN W, SHEN L, LUO A, WANG S. Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway. Cell Cycle, 2016, 15(21):2895-2905.
[17]   FENWICK M A, HURST P R. Immunohistochemical localization of active caspase-3 in the mouse ovary: growth and atresia of small follicles. Reproduction, 2002, 124(5):659-665.
[18]   陈华絮, 杨素娇. 兔卵巢组织石蜡切片技术的改良. 生物学杂志, 2007, 24(5):59-60.
CHEN H X, YANG S J. Improvement of paraffin-section technique of rabbit ovarian tissue. Journal of Biology, 2007, 24(5):59-60. (in Chinese)
[19]   AL-SAMERRIA S, AL-ALI I, MCFARLANE J R, ALMAHBOBI G. The impact of passive immunisation against BMPRIB and BMP4 on follicle development and ovulation in mice.Reproduction. 2015, 149(5):403-411.
[20]   WENDL J, EBACH K, RODLER D, KENNGOTT R A. Immunocytochemical localization of cytoplasmic and nuclear intermediate filaments in the bovine ovary during Folliculogenesis. Anatomia Histologia Embryologia, 2012, 41:190-201.
[21]   杜晨光, 曹贵方. 绵羊卵泡内Ghrelin的免疫组化定位. 华北农学报, 2008, 23(3):151-153.
DU C G, CAO G F. An immunohis to chemical study of the localization of ghrelin in the ovine follicle. Acta Agriculturae Boreali-Sinica, 2008, 23(3):151-153. (in Chinese)
[22]   LIU Y X, HSUCH A J. Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during  the periovulatory period. Biology of Reproduction, 1987, 36(4): 1055-1062.
[23]   KIM N H, MENINO A R. Effects of stimulators of protein kinase A and C and modulators of phosphorylation and plasminogen activator activity in porcine oocyte-cumulus cells complexes during in vitro maturation. MolecularReproduction andDevelopment, 1995, 40(3): 364-370.
[24]   HUARTE J, BELIN D, VASSALLI J D. Plasminogen activator in mouse and rat oocytes: induction during meiotic maturation. Cell, 1985, 43(2):551-558.
[25]   PARK K W, CHOI S H, SONG X X, FUNAHASHI H, NIWA K. Production of plasminogen activator in bovine cumulus-oocyte complexes during maturation in vitro: effects of epidermal growth factor on production of PAs in oocytes and cumulus cells. Biology of Reproduction, 1999, 61(1):298-304.
[26]   GARCÍA D C, MICELI D C, RIZO G, GARCÍA E V, VALDECANTOS P A, ROLDÁN-OLARTE M. Expression and localization of urokinase- type plasminogen activator receptor in bovine cumulus-oocytecomplexes. Zygote, 2016, 24(2):230-235.
[27]   CONNOLLY B M, CHOI E Y, GARDSVOLL H, BEY A L, CURRIE B M , CHAVAKIS T, LIU, S H, MOLINOLO A, PLOUG M, LEPPLA S H. Selective abrogation of the uPA-uPAR in vivo reveals a novel role in suppression of fibrin-associated inflammation. Blood, 2010, 116(9): 1593-1603.
[28]   CASANO S, GUIDETTI D, PATRIARCA A, PITTATORE G, GENNARELLI G, REVELLI A. MILD ovarian stimulation with GnRH-antagonist vs. long protocol with low dose FSH for non-PCO high responders undergoing IVF: a prospective, randomized study including thawing cycles. Journal of Assisted Reproduction and Genetics, 2012, 29(12): 1343-1351.
[29]   SINGAVARAPU R, BUCHINSKY N, CHEON D J, ORSULIC S. Whole ovary immunohistochemistry for monitoring cell proliferation and ovulatory wound repair in the mouse. Reproductive Biology and Endocrinology, 2010, 8:98.
[30]   任学军, 江昌新. 煮沸法在石蜡切片抗原修复中应用的探讨. 天津医科大学学报, 2001, 7(1): 108-109.
REN X J, JIANG L X. Boiling method application in the antigen repairing of paraffin section technique. Journal of Tianjin Medical University, 2001, 7(1): 108-109. (in Chinese)
[1] GAO YunJie, GUO RuoNan, CHEN ChunWen, DUAN YiFan, ZHANG ZhenJun, NIE ChanChan, LI MengYu, WANG Hui, FENG TingTing, CUI YingYing, DANG GuangHui, LIU SiGuo. Establishment and Preliminary Application of Indirect ELISA Antibody Detection Method for Mycobacterium avium subsp. paratuberculosis in Sheep [J]. Scientia Agricultura Sinica, 2026, 59(3): 655-667.
[2] YAN ChuanBo, GONG Ping, ZHENG WenXin, CHEN XinWen, GUO LeiFeng. Research on Sheep Mounting Behavior Recognition in Complex Scenes Based on an Improved YOLOv11 [J]. Scientia Agricultura Sinica, 2025, 58(18): 3783-3798.
[3] CHI RunQing, HAN HaiYin, WANG Peng, LI KaiYang, CHU MingXing, LIU YuFang. Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep [J]. Scientia Agricultura Sinica, 2024, 57(3): 597-612.
[4] ZHANG YingXin, YANG Min, BAI XueBing, CHEN Chang, WU RuiZhi, YANG Ping, CHEN QiuSheng. Morphological Characteristics of Telocytes at Sheep Acupoints and Its Relationship with Surrounding Structures [J]. Scientia Agricultura Sinica, 2023, 56(7): 1417-1428.
[5] ZHAO WeiHong, HAN WenXiong, YANG Bo, MENG WeiKang, CHAI HaiLiang, MA YiMin, ZHANG ZhanSheng, WANG LiFeng, WANG Yan, WANG MingYuan, ZHANG Shan, DING YuLin, WANG JinLing, JIRINTAI Sulijid, WANG FengLong, ZHAO Li, LIU YongHong. Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia [J]. Scientia Agricultura Sinica, 2023, 56(6): 1204-1214.
[6] GUO ZeYuan, DU ZhangSheng, ZHANG YaQi, CHEN ChunLu, MA XiaoYan, CHENG Ying, WANG Kai, LÜ LiHua. Effects of Smad7-Mediated TGF-β Signaling Pathway on Proliferation of Sheep Granulosa Cells [J]. Scientia Agricultura Sinica, 2023, 56(13): 2597-2608.
[7] AYIMUGULI Abudureyimu, ZHANG Chen, CAI Yong, QIN Sheng, LUO WenXue, ZHAXIYINGPAI. The Micro-Structure of Tibetan Sheep Lung and Its HIF-1α and AQP1 Expression Characteristics [J]. Scientia Agricultura Sinica, 2023, 56(11): 2202-2211.
[8] LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(9): 1868-1876.
[9] CHE DaLu,ZHAO LiChen,CHENG SuCai,LIU AiYu,LI XiaoYu,ZHAO ShouPei,WANG JianCheng,WANG Yuan,GAO YuHong,SUN XinSheng. Effect of Litter Bed on Growth Performance and Odor Emission in Fattening Lamb [J]. Scientia Agricultura Sinica, 2022, 55(24): 4943-4956.
[10] SONG ShuZhen, GAO LiangShuang, LI Hong, GONG XuYin, LIU LiShan, WEI YuBing. Effects of Feeding Levels on Muscle Tissue Structure and Muscle Fiber Composition Related Genes in Sheep [J]. Scientia Agricultura Sinica, 2022, 55(21): 4304-4314.
[11] ChunTao ZHANG,Tao MA,Yan TU,QiYu DIAO. Effects of Circadian Rhythm on Rumen Fermentation and Nutrient Digestion of Mutton Sheep [J]. Scientia Agricultura Sinica, 2022, 55(18): 3664-3674.
[12] LIU WangJing,TANG DeFu,AO ChangJin. Effect of Allium mongolicum Regel and Its Extracts on the Growth Performance, Carcass Characteristics, Meat Quality and Serum Biochemical Indices of Captive Small-Tailed Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(17): 3461-3472.
[13] LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen. Association Analysis of the ADIPOQ Variation with Sheep Growth Traits [J]. Scientia Agricultura Sinica, 2022, 55(11): 2239-2256.
[14] KE Na,HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing. The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1 [J]. Scientia Agricultura Sinica, 2022, 55(10): 2047-2056.
[15] WANG Qian,LI Zheng,ZHAO ShanShan,QIE MengJie,ZHANG JiuKai,WANG MingLin,GUO Jun,ZHAO Yan. Application of Stable Isotope Technology in the Origin Traceability of Sheep [J]. Scientia Agricultura Sinica, 2021, 54(2): 392-399.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd