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. 2017 Apr 6;7:702. doi: 10.1038/s41598-017-00771-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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GLYT1 activity in oocytes, COCs and intact follicles. (a) GLYT1 activity (rate of [³H]-glycine transport) was measured as a function of time after isolation of GV oocytes, COCs, or intact antral follicles. For oocytes and COCs, meiotic arrest was maintained with dbcAMP or NPPC/E₂, respectively. Points represent the mean ± s.e.m. of GLYT1 activity (left) or GVBD (right) for three independent repeats at each time. Data were analysed by 2-way ANOVA with Bonferroni post-test, which indicated significant difference between follicles vs. COCs and GV oocytes at each time from 1–6 h (overall effect between preparations, P < 0.0001; between preparations at each time point: ***P < 0.001), but not between COCs and oocytes (NS, P > 0.05). At 20 h, activity was measured only for oocytes from cultured antral follicles (not included in analysis). There was no significant difference between preparations for GVBD (NS; P = 0.096 by 2-way ANOVA). (b) To assess reversibility of GLYT1 suppression in vitro, antral follicles were cultured for 4 or 24 h as indicated and then the oocytes removed and GLYT1 activity measured immediately (0 h) or after a further 4 h of culture of isolated oocytes. GLYT1 remained suppressed in oocytes within antral follicles for 4 or 24 h, but became activated 4 h after removal. Each bar represents the mean ± s.e.m. of five independent repeats. The effect was significant (overall P < 0.0001 by ANOVA). Bars that do not share the same letter are significantly different (Tukey test; P < 0.001 except b vs c, P < 0.01). (c) Antral follicles were isolated from P21 neonates and cultured overnight with FSH to stimulate LH receptor expression, and then cultured for up to 5 h in the continued absence (−LH) or presence (+LH) of LH, after which the oocytes were isolated and GLYT1 activity measured (left) and GV status determined (right). Each point represents the mean ± s.e.m. of three independent repeats at each time. There was a significant difference between the presence vs. absence of LH at 3 and 5 h for both GLYT1 activity and GVBD (2-way ANOVA with Bonferroni post-test; overall effect of LH: P < 0.0001; between individual time points: a**P < 0.01; ***P < 0.001).