绵羊卵母细胞小群培养条件优化

doi:10.11843/j.issn.0366-6964.2025.12.023

摘要/Abstract

摘要： 旨在优化绵羊卵母细胞小群(5枚/组)体外培养体系，改善其成熟质量及后续发育潜能。本研究通过设置不同培养体积(50 COCs/500 μL、5 COCs/500 μL、5 COCs/50 μL)及添加外源BMP15与GDF9蛋白(200 ng·mL^-1)，评估颗粒细胞扩展指数及其相关HAS2基因表达、卵丘和卵母细胞凋亡相关基因(BAX、BCL2、C-Myc)表达、卵母细胞氧化还原稳态(GSH、ROS、GPX-1、GSR)及胚胎发育指标(卵裂率、囊胚率、囊胚平均细胞数)，并进一步优化体外成熟培养时长(18 h、21 h、24 h)，确立优化方案。结果表明，添加BMP15和GDF9显著提升卵丘扩展指数(P<0.01)，上调HAS2基因表达，改善氧化还原稳态(GSH水平升高，ROS降低)；500 μL培养液结合21 h成熟方案使小群培养卵母细胞体外受精后的48 h卵裂率达到70.23%±2.78%及囊胚率35.33%±1.53%，而21 hIVM组较24 h IVM组BAX表达量和BAX/BCL2比值显著降低。5 COCs /500 μL小群培养效果优于5 COCs/50 μL，联合添加BMP15和GDF9蛋白可降低细胞凋亡并提升胞内氧化还原能力，体外成熟时长由24 h缩短至21 h可通过降低细胞凋亡并促进囊胚OCT4、CCNB1基因表达并且提高囊胚平均细胞数。

关键词: 绵羊, 卵母细胞, 小群培养

Abstract: This investigation aimed to optimize the in vitro culture system for small cohorts of ovine COCs(5 per group), and enhance in vitro maturation quality and subsequent developmental potential. Experimental variables included culture volume (50 COCs/500 μL,5 COCs/500 μL, 5 COCs/50 μL,) supplemented with exogenous BMP15 and GDF9 proteins (200 ng·mL^-1), and maturation durations tested (18, 21, 24 h). Comprehensive evaluations encompassed cumulus expansion index, HAS2 gene expression profiles, apoptosis-related gene expression (BAX, BCL2, C-Myc) in cumulus-oocyte complexes, oocyte redox homeostasis parameters (GSH, ROS, GPX-1, GSR), and embryonic developmental indexes (cleavage rate, blastocyst rate, mean cell numbers/blastocyst). Addition of the combination of BMP15 and GDF9 into maturation medium significantly enhanced cumulus expansion indexes (P<0.01), upregulating HAS2 expression and improving redox balance through elevated GSH levels and reduced ROS accumulation. The optimized protocol employing 500 μL culture medium with 21 h maturation achieved superior outcomes, yielding cleavage rate of 70.23%±2.78% and blastocyst rate of 35.33%±1.53% at 48 h after in vitro fertilization of oocytes cultured in small group. Notably, the 21-hour maturation group exhibited significantly lower BAX expression and BAX/BCL2 ratios compared to the 24-hour group. Culturing 5 COCs in 500 μL microdrops yielded better results than culturing 5 COCs in 50 μL microdrops. Co-supplementation with BMP15 and GDF9 proteins reduced apoptosis and enhanced intracellular redox capacity. Shortening the in vitro maturation (IVM) duration from 24 h to 21 h reduced apoptosis, promoted OCT4 and CCNB1 gene expression in blastocysts, and increased the average blastocyst cell count.

Key words: ovine, oocyte, small group cultivation

中图分类号:

S826.3

胡伯欣, 高程远, 刘从, 陈茹曼, 朱捷, 田树军. 绵羊卵母细胞小群培养条件优化[J]. 畜牧兽医学报, 2025, 56(12): 6204-6218.

HU Boxin, GAO Chengyuan, LIU Cong, CHEN Ruman, ZHU Jie, TIAN Shujun. Optimization of Culture Conditions for Small Groups of Ovine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6204-6218.

参考文献

[1] VIANA J H M. Development of the world farm animal embryo industry over the past 30 years [J]. Theriogenology, 2024, 230: 151-156.
[2] 孙克佳. 活体采卵与小群培养对牛羊体外胚胎生产的影响 [D].保定：河北农业大学 2022.
SUN K J.The impact of ovum pick-up and small group culture on in vitro embryo production in cattle and sheep[D]. Baoding: Hebei Agricultural University, 2022.(in Chinese)
[3] PRIEGO-GONZÁLEZ A, MUNOZ-MACEDA A, CERDEIRA-LOZANO J, et al. Successful ultrasound-guided ovum pick-up (OPU) and subsequent in vitro embryo production in a domestic cat [J]. Theriogenology, 2024, 229: 47-52.
[4] DU Y, XIA Y, XU J, et al. Effects of donor age and reproductive history on developmental potential of ovum pickup oocytes in Japanese Black cattle (Wagyu) [J]. Theriogenology, 2024, 221: 25-30.
[5] SIMMONS R, TUTT D A, GUVEN-ATES G, et al. Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production [J]. Theriogenology, 2023, 199: 77-85.
[6] WIECZOREK J, KOSENIUK J, SKRZYSZOWSKA M, et al. L-OPU in goat and sheep-different variants of the oocyte recovery method [J]. Animals (Basel), 2020, 10(4)：658.
[7] VEGA D A, NARVÁEZ H J. Oocyte quality in adapted Bos taurus taurus cows [J]. Anim Biotechnol, 2023, 34(9): 4675-4679.
[8] GHAFARI F, SALAVATI M, PIERCY R J, et al. Beneficial effects of melatonin on canine oocyte nuclear maturation via reduction of oxidative stress [J]. Reproduction, 2025, 169(4)：e240388.
[9] MISHRA A, REDDY I J, GUPTA P S, et al. Expression of apoptotic and antioxidant enzyme genes in sheep oocytes and in vitro produced embryos [J]. Anim Biotechnol, 2017, 28(1): 18-25.
[10] HUANG K, LI C, GAO F, et al. Epigallocatechin-3-gallate promotes the in vitro maturation and embryo development following IVF of porcine oocytes [J]. Drug Des Devel Ther, 2021, 15: 1013-1020.
[11] MAHMOODI M, CHERAGHI E, RIAHI A. Correction to: The effect of wharton's jelly-derived conditioned medium on the in vitro maturation of immature oocytes, embryo development, and genes expression involved in apoptosis [J]. Reprod Sci, 2023, 30(11): 3400.
[12] TANG Y, LU S, WEI J, et al. Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells [J]. Theriogenology, 2024, 219: 65-74.
[13] JIAO Y, JIANG T, LIN Q, et al. Molecular characterization of the follicular development of BMP15-edited pigs [J]. Reproduction, 2023, 166(4): 247-261.
[14] NISHIO M, HOSHINO Y, SATO E. Effect of droplet size and number of oocytes examined on mouse oocyte quality in in vitro maturation [J]. J Mamm Ova Res, 2011, 28(1): 53-60.
[15] EBRAHIMI M, MARA L, SUCCU S, et al. The effect of single versus group culture on cumulus-oocyte complexes from early antral follicles [J]. J Assist Reprod Genet, 2025, 42(3): 961-976.
[16] 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.
[17] DU C, DAVIS J S, CHEN C, et al. FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro [J]. Reproduction, 2021, 161(2): 205-214.
[18] MORIKAWA R, LEE J, MIYANO T. Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro [J]. J Reprod Dev, 2021, 67(4): 273-281.
[19] CHENG M, CHEN X, HAN M, et al. miR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity [J]. Theriogenology, 2024, 214: 124-133.
[20] STOCKER W A, WALTON K L, RICHANI D, et al. A variant of human growth differentiation factor-9 that improves oocyte developmental competence [J]. J Biol Chem, 2020, 295(23): 7981-7991.
[21] FERRER-RODA M, PARAMIO M T, VILA-BELTRÁN J, et al. Effect of BMP15 and GDF9 in the IVM medium on subsequent oocyte competence and embryo development of prepubertal goats [J]. Theriogenology, 2025, 234: 164-173.
[22] WANG L, GAO J, MA J, et al. Effects of hyperhomocysteinemia on follicular development and oocytes quality [J]. iScience, 2024, 27(11): 111241.
[23] JIAO Y, BEI C, WANG Y, et al. Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function [J]. Int J Biol Macromol, 2024, 267(Pt 1): 131417.
[24] LI W, LIU Z, WANG P, et al. The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-β signaling pathway [J]. Int J Biol Macromol, 2023, 238: 124026.
[25] GIUSTARINI D, MILZANI A, DALLE-DONNE I, et al. How to increase cellular glutathione [J]. Antioxidants (Basel), 2023, 12(5)：1094.
[26] ASHIBE S, MIYAMOTO R, KATO Y, et al. Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI) [J]. Theriogenology, 2019, 133: 71-78.
[27] DAVOODIAN N, KADIVAR A, MEHRBAN H. Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation in vitro [J]. Reprod Domest Anim, 2024, 59(1): e14503.
[28] KIM M J, KANG H G, JEON S B, et al. The antioxidant betulinic acid enhances porcine oocyte maturation through Nrf2/Keap1 signaling pathway modulation [J]. PLoS One, 2024, 19(10): e0311819.
[29] READER K L, STANTON J L, JUENGEL J L. The role of oocyte organelles in determining developmental competence [J]. Biology (Basel), 2017, 6(3):35.
[30] LI J, BALBOULA A Z, ABOELENAIN M, et al. Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes [J]. J Reprod Dev, 2020, 66(1): 83-91.
[31] ROBERT C. Nurturing the egg: the essential connection between cumulus cells and the oocyte [J]. Reprod Fertil Dev, 2021, 34(2): 149-159.
[32] YUAN Y, SPATE L D, REDEL B K, et al. Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation [J]. Proc Natl Acad Sci U S A, 2017, 114(29): E5796-E5804.
[33] ZHANG M, ZHANG J, WANG D, et al. C-X-C motif chemokine ligand 12 improves the developmental potential of bovine oocytes by activating SH2 domain-containing tyrosine phosphatase 2 during maturation [J]. Biol Reprod, 2023, 109(3): 282-298.
[34] MARTINEZ C A, CUELLO C, PARRILLA I, et al. Exogenous melatonin in the culture medium does not affect the development of in vivo-derived pig embryos but substantially improves the quality of in vitro-produced embryos [J]. Antioxidants (Basel), 2022, 11(6):1177.
[35] TONG J, SHENG S, SUN Y, et al. Melatonin levels in follicular fluid as markers for IVF outcomes and predicting ovarian reserve [J]. Reproduction, 2017, 153(4): 443-451.
[36] CADENAS J, PORS S E, KUMAR A, et al. Concentrations of oocyte secreted GDF9 and BMP15 decrease with MII transition during human IVM [J]. Reprod Biol Endocrinol, 2022, 20(1): 126.
[37] TAWEECHAIPAISANKUL A, JIN JX, LEE S, et al. The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer [J]. Reprod Domest Anim, 2016,51(6):870-876.
[38] REN X, YUN X, YANG T, et al. Epifriedelanol delays the aging of porcine oocytes matured in vitro [J]. Toxicon, 2023,233:107256.
[39] HUANG K, LI C, GAO F, et al. Epigallocatechin-3-Gallate promotes the in vitro maturation and embryo development following IVF of porcine oocytes [J]. Drug Des Devel Ther, 2021,15:1013-1020.
[40] MASSOUD G, SPANN M, VAUGHT K C, et al. Biomarkers assessing the role of cumulus cells on IVF outcomes: a systematic review [J]. J Assist Reprod Genet, 2024, 41(2): 253-275.