Sakaguchi Kenichiro, Huang Weiping, Yang Yinghua, Yanagawa Yojiro, Nagano Masashi
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan.
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan; Anhui Provincial Laboratory of Animal Genetic Resources Protection and Breeding, College of Animal Sciences and Technology, Anhui Agricultural University, Hefei, Anhui, China.
Theriogenology. 2017 Jul 15;97:113-123. doi: 10.1016/j.theriogenology.2017.04.030. Epub 2017 Apr 23.
Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5-1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E), but less progesterone (P). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E production decreased from days 8-12, and P production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E production were observed between groups from days 4-8, regardless of whether BMP-4 was added without FSH; however, E production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E production from days 8-12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4 without FSH showed the lowest P production among all groups for all culture periods. OCGCs that produced mature oocytes tended to secrete more E and less P than OCGCs that produced immature oocytes. In conclusion, until day 8 of the IVG culture, P production by OCGCs was suppressed by the addition of 10 ng/mL BMP-4 in the absence of FSH, without inhibiting E production. These conditions appear to mimic growing follicles until day 8 and mimic degenerating follicles from days 8-12 of culture.
骨形态发生蛋白-4(BMP-4)和促卵泡激素(FSH)在体内卵泡生长和类固醇生成过程中发挥着重要的调节作用。本研究旨在探讨BMP-4和FSH对牛卵母细胞-卵丘-颗粒细胞复合体(OCGCs)体外生长(IVG)和类固醇生成的影响。我们将从早期窦卵泡(0.5 - 1毫米)收集的OCGCs在不含BMP-4和FSH的培养基中培养4天,并观察OCGCs的外观及其类固醇生成情况。在IVG的前4天,形态正常的OCGCs产生的雌二醇-17β(E)较多,但孕酮(P)较少。将形态正常的OCGCs在补充有BMP-4(0、10和50纳克/毫升)和FSH(0和0.5纳克/毫升)的培养基中继续培养至第12天。我们在培养8天和12天后检测了OCGCs的活力和类固醇生成情况。同时还研究了卵母细胞生长、颗粒细胞特征以及卵母细胞的成熟能力。在第8天,未添加FSH培养的OCGCs中,10纳克/毫升BMP-4组的活力高于50纳克/毫升BMP-4组(P < 0.05)。添加FSH培养的各组之间,无论是否添加BMP-4,活力均无显著差异,且FSH提高了50纳克/毫升BMP-4组的活力,使其与10纳克/毫升BMP-4组相似。在第8天,添加FSH培养时,10纳克/毫升BMP-4组的颗粒细胞总数多于50纳克/毫升BMP-4组(P < 0.05)。无论是否添加BMP-4和FSH,从第8天到第12天E的生成量均下降,而在整个IVG培养过程中P的生成量增加(P < 0.05)。在第4天至第8天期间,无论是否添加BMP-而不添加FSH,各组之间E的生成量均无显著差异;然而,FSH抑制了添加50纳克/毫升BMP-4组的E生成。无论是否添加FSH,BMP-4在第8天至第12天均抑制E的生成。在所有培养阶段,未添加FSH的10纳克/毫升BMP-4组的P生成量在所有组中最低。产生成熟卵母细胞的OCGCs比产生未成熟卵母细胞的OCGCs倾向于分泌更多的E和更少的P。总之,在IVG培养至第8天时,在无FSH的情况下添加10纳克/毫升BMP-4可抑制OCGCs的P生成,而不抑制E生成。这些条件在培养至第8天时似乎模拟了生长中的卵泡,而在培养第8天至第12天时模拟了退化中的卵泡。