RFRP-3对猪卵母细胞体外成熟的影响

doi:10.11843/j.issn.0366-6964.2022.04.013

Abstract

Abstract: The aim of this study was to investigate the effect of RFRP-3 on in vitro maturation of porcine oocyte. In this study, GV stage oocytes from the ovaries of healthy sows collected from slaughterhouses were randomly divided into 3 groups. The maturation rate of porcine oocytes in each group was calculated after incubating with 0, 10^-6 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. In the subsequent experiment, the collected oocytes were randomly divided into 2 groups. Porcine oocytes were cultured in medium with 0 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. The cumulus expansion of porcine oocytes was observed and the cumulus expansion index and maturation rate of oocytes in each group were calculated. The expression of cumulus expansion factors (PTGS2, HAS2, PTX3), oocyte secretory factors (GDF9, BMP15) and cyclin related genes (CCNB1 and CDK1) were detected by qRT-PCR. The contents of MPF and cAMP in oocytes were detected by ELISA kit. The concentrations of progesterone and estrogen were measured by radioimmunoassay. Oocytes in each group were no less than 100, and each test was repeated 3 times. Compared with the control group, the maturation rate of porcine oocytes cultured with 10^-8mol·L^-1 RFRP-3 was extremely significantly reduced (P<0.01). No significant change in cumulus expansion rate and cumulus expansion index were observed(P>0.05), but the expression of cumulus expansion factors (PTGS2, HAS2, PTX3) was extremely significantly decreased (P<0.01). RFRP-3 could extremely significantly reduce MPF content in porcine oocytes (P<0.01), and had no significant effect on cAMP content (P>0.05). After the addition of RFRP-3, the expression levels of GDF9 (P<0.01) and BMP15 (P<0.05) were promoted, while the expression levels of CCNB1 (P<0.05) and CDK1(P<0.05) were inhibited; meanwhile, the concentrations of progesterone and estrogen in medium of experimental groups were extremely significantly decreased (P<0.01). In conclusion, RFRP-3 inhibits in vitro maturation of porcine oocytes by regulating the expression of maturation related factors and cumulus expansion factors and the secretion of steroid hormones. This study will lay a theoretical foundation for elucidating the regulatory effect of RFRP-3 on mammalian oocytes.

Key words: RFRP-3, porcine, oocyte, IVM

CLC Number:

S828.3

JIA Jinmei, FENG Jianhao, YANG Meixia, ZHANG Yufang, ZHANG Hongliang, WANG Shuilian. Effect of RFRP-3 on in Vitro Maturation of Porcine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1133-1142.

References 38

[1]	张羽芳,阳美霞,汤亚茹,等.哺乳动物卵母细胞成熟相关因子研究[J].医学信息,2020,33(14):17-20.ZHANG Y F,YANG M X,TANG Y R,et al.Study on factors related to maturation of mammalian oocytes[J].Medical Information, 2020,33(14):17-20.(in Chinese)
[2]	WIGGLESWORTH K,LEE K B,O’BRIEN M J,et al.Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes[J].Proc Natl Acad Sci U S A, 2013,110(39):E3723-E3729.
[3]	RUSSELL D L,GILCHRIST R B,BROWN H M,et al.Bidirectional communication between cumulus cells and the oocyte:old hands and new players?[J].Theriogenology, 2016,86(1):62-68.
[4]	GE L,SUI H S,LAN G C,et al.Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus:observations of nuclear and cytoplasmic events[J].Fertil Steril, 2008,90(6):2376-2388.
[5]	田长永,杨景晁,马泽芳,等.卵丘细胞对辽宁绒山羊卵母细胞体外成熟与孤雌发育的影响[J].畜牧兽医学报,2010,41(5):543-548.TIAN C Y,YANG J C,MA Z F,et al.Effects of cumulus cells on in vitro maturation and parthenogenetic development of Liaoning cashmere goats oocytes[J].Acta Veterinaria et Zootechnica Sinica, 2010,41(5):543-548.(in Chinese)
[6]	MAREI W F,ABAYASEKARA D R E,WATHES D C,et al.Role of PTGS2-generated PGE2 during gonadotrophin-induced bovine oocyte maturation and cumulus cell expansion[J].Reprod Biomed Online, 2014,28(3):388-400.
[7]	CHAUBEY G K,KUMAR S,KUMAR M,et al.Induced cumulus expansion of poor quality buffalo cumulus oocyte complexes by Interleukin-1beta improves their developmental ability[J].J Cell Biochem, 2018,119(7):5750-5760.
[8]	LI Q L,RAJANAHALLY S,EDSON M A,et al.Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15[J].Mol Hum Reprod, 2009,15(12):779-788.
[9]	PANGAS S A,MATZUK M M.The art and artifact of GDF9 activity:cumulus expansion and the cumulus expansion-enabling factor[J].Biol Reprod, 2005,73(4):582-585.
[10]	王兆琛,赵勇超,杜炜,等.AREG对绵羊小腔卵泡卵母细胞体外成熟的影响[J].畜牧兽医学报,2020,51(6):1238-1247.WANG Z C,ZHAO Y C,DU W,et al.Effect of AREG on in vitro maturation of small antral follicular oocytes of sheep[J].Acta Veterinaria et Zootechnica Sinica, 2020,51(6):1238-1247.(in Chinese)
[11]	EDRY I,SELA-ABRAMOVICH S,DEKEL N.Meiotic arrest of oocytes depends on cell-to-cell communication in the ovarian follicle[J].Mol Cell Endocrinol, 2006,252(1-2):102-106.
[12]	LEAL G R,MONTEIRO C A S,SOUZA-FABJAN J M G,et al.Role of cAMP modulator supplementations during oocyte in vitro maturation in domestic animals[J].Anim Reprod Sci, 2018,199:1-14.
[13]	VALENCIA C,PÉREZ F A,MATUS C,et al.Activation of bovine oocytes by protein synthesis inhibitors:new findings on the role of MPF/MAPKs[J].Biol Reprod, 2021,104(5):1126-1138.
[14]	TSUTSUI K,SAIGOH E,UKENA K,et al.A novel avian hypothalamic peptide inhibiting gonadotropin release[J].Biochem Biophys Res Commun, 2000,275(2):661-667.
[15]	UBUKA T,INOUE K,FUKUDA Y,et al.Identification,expression,and physiological functions of Siberian hamster gonadotropin- inhibitory hormone[J].Endocrinology, 2012,153(1):373-385.
[16]	LI X,SU J,LEI Z H,et al.Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig:cDNA cloning,expression in tissues and expression pattern in the reproductive axis during the estrous cycle[J].Peptides, 2012,36(2):176-185.
[17]	LI X,SU J,FANG R,et al.The effects of RFRP-3,the mammalian ortholog of GnIH,on the female pig reproductive axis in vitro [J].Mol Cell Endocrinol, 2013,372(1-2):65-72.
[18]	汪瑶,李珣,李敏婕,等.GnIH对母猪生殖调控的研究[J].中国农业科学,2014,47(18):3716-3724.WANG Y,LI X,LI M J,et al.Regulation of GnIH on reproduction of female pig[J].Scientia Agricultura Sinica, 2014, 47(18): 3716-3724.(in Chinese)
[19]	ZMIJEWSKA A,CZELEJEWSKA W,DZIEKONSKI M,et al.Effect of kisspeptin and RFamide-related peptide-3 on the synthesis and secretion of LH by pituitary cells of pigs during the estrous cycle[J].Anim Reprod Sci, 2020,214:106275.
[20]	PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Res, 2001,29(9):e45.
[21]	WANG X Y,LI X,HU C H.RFRP-3,the mammalian ortholog of GnIH,induces cell cycle arrest at G2/M in porcine ovarian granulosa cells[J].Peptides, 2018,101:106-111.
[22]	SINGH P,KRISHNA A,TSUTSUI K.Effects of gonadotropin-inhibitory hormone on folliculogenesis and steroidogenesis of cyclic mice[J].Fertil Steril, 2011,95(4):1397-1404.
[23]	JOSEFSBERG L B Y,GALIANI D,LAZAR S,et al.Maturation-promoting factor governs mitogen-activated protein kinase activation and interphase suppression during meiosis of rat oocytes[J].Biol Reprod, 2003,68(4):1282-1290.
[24]	PAN B,LI J L.The art of oocyte meiotic arrest regulation[J].Reprod Biol Endocrinol, 2019,17(1):8.
[25]	郭振伟,黄永军,范威宏,等.褪黑素对水牛卵母细胞体外成熟的影响及其受体介导机制的探究[J].畜牧兽医学报,2019,50(2):314-322.GUO Z W,HUANG Y J,FAN W H,et al.The effect of melatonin on the maturation of oocyte in vitro and its receptor-mediated mechanism in buffalo[J].Acta Veterinaria et Zootechnica Sinica, 2019,50(2):314-322.(in Chinese)
[26]	李赞.AMH对小鼠卵母细胞体外成熟及卵丘扩展的调控作用研究[D].武汉:华中农业大学,2018.LI Z.Regulation effect of AMH on in-vitro maturation of oocytes and cumulus expansion in mouse[D].Wuhan:Huazhong Agricultural University, 2018.(in Chinese)
[27]	GILCHRIST R B,LANE M,THOMPSON J G.Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality[J].Hum Reprod Update, 2008,14(2):159-177.
[28]	魏莉娜,方丛,黄睿,等.GDF9和BMP15在PCOS患者刺激周期卵母细胞不同成熟阶段的表达变化及意义[J].中华妇产科杂志,2012,47(11):818-822.WEI L N,FANG C,HUANG R,et al.Change and significance of growth differentiation factor 9 and bone morphogenetic protein expression during oocyte maturation in polycystic ovary syndrome patients with ovarian stimulation[J].Chinese Journal of Obstetrics and Gynecology, 2012,47(11):818-822.(in Chinese)
[29]	彭中友,孙俊铭,李燕,等.GDF9和FST调控猪卵母细胞成熟和胚胎早期发育[J].江苏农业学报,2015,31(3):583-589.PENG Z Y,SUN J M,LI Y,et al.Growth differentiation factor 9 and follistatin regulating porcine oocyte maturation and early embryo development[J].Jiangsu Journal of Agricultural Sciences, 2015,31(3):583-589.(in Chinese)
[30]	CLELLAND E S,TAN Q,BALOFSKY A,et al.Inhibition of premature oocyte maturation:a role for bone morphogenetic protein 15 in zebrafish ovarian follicles[J].Endocrinology, 2007,148(11):5451-5458.
[31]	HSIEH M,ZAMAH A M,CONTI M.Epidermal growth factor-like growth factors in the follicular fluid:role in oocyte development and maturation[J].Semin Reprod Med, 2009,27(1):52-61.
[32]	赵子墨,王庆凯,郑梓,等.卵丘细胞对猪卵母细胞体外成熟发育及GPX3、GPX4基因表达的影响[J].黑龙江畜牧兽医,2020(12):51-55,59,160.ZHAO Z M,WANG Q K,ZHENG Z,et al.Effects of cumulus cells on porcine oocyte maturation-development and the expression GPX3 and GPX4 genes in vitro [J].Heilongjiang Animal Science and Veterinary Medicine, 2020(12):51-55,59,160.(in Chinese)
[33]	NIKOLOFF N,CAMPAGNA A,LUCHETTI C,et al.Effects of EPA on bovine oocytes matured in vitro with antioxidants:impact on the lipid content of oocytes and early embryo development[J].Theriogenology, 2020,146:152-161.
[34]	CAMAIONI A,HASCALL V C,YANAGISHITA M,et al.Effects of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse cumulus cell-oocyte complex[J].J Biol Chem, 1993,268(27):20473-20481.
[35]	PAN Y Y,WANG M,WANG L B,et al.Estrogen improves the development of yak (Bos grunniens ) oocytes by targeting cumulus expansion and levels of oocyte-secreted factors during in vitro maturation[J].PLoS One, 2020,15(9):e0239151.
[36]	NAGYOVA E,NEMCOVA L,MLYNARCIKOVA A,et al.Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium[J].Fertil Steril, 2013,99(6):1739-1748.
[37]	LIU W,XIN Q L,WANG X,et al.Estrogen receptors in granulosa cells govern meiotic resumption of pre-ovulatory oocytes in mammals[J].Cell Death Dis, 2017,8(3):e2662.
[38]	KUROWSKA P,MLYCZY AŃU SKA E,ESTIENNE A,et al.Expression and impact of vaspin on in vitro oocyte maturation through MAP3/1 and PRKAA1 signalling pathways[J].Int J Mol Sci, 2020,21(24):9342.

Effect of RFRP-3 on in Vitro Maturation of Porcine Oocytes

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