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. 2016 Feb 26;22(6):397–409. doi: 10.1093/molehr/gaw019

Figure 4.

Figure 4

Analysis of the association of post-ovulatory aging with deficiencies in a pathway including MAPK3/1 and MLCK. (A) A hypothesized pathway [also based on McGinnis et al. (2015)], noting that MAPK3/1 activity is inhibited with U0126, an inhibitor of the upstream activating kinases MEK1/2 and that MLCK activity is inhibited by ML-7. (B) Data from previous studies (Yanagimachi and Chang, 1961; Whittingham and Siracusa, 1978; Xu et al., 1997; Phillips et al., 2002b; Tong et al., 2003; Larson et al., 2010; McGinnis et al., 2015), and also puts new data presented here in context with these other data; new data are indicated with bold and brighter color font, and the associated figure cited. (C and D) Analysis of parthenogenetic egg activation in freshly collected ovulated eggs (i.e. assessed shortly after isolated from oviducts; C, n = 12 experiments), and in ovulated eggs cultured for 2 h after isolation from oviducts (D; n = 4 experiments). For the studies shown in D, aged eggs were either left untreated and cultured for 2 h, or were treated with zinc pyrithione (ZnPT) for 10 min prior to culture for an additional 2 h. The extent of parthenogenesis in the ZnPT-treated group versus the untreated groups is statistically significantly (P = 0.003). (E) Outcomes from IVF of young zona pellucida-free eggs that were pre-treated with 15 µM ML-7 (or the solvent control DMSO) for 1 h, then inseminated for 1.5 h with 50 000 sperm/ml. The x-axis indicates number of sperm fused and the y-axis shows the percentage of eggs with the indicated number of sperm fused (n = 18 experiments). The overall distributions between the control and the ML-7-treated group are statistically significantly different (P < 0.0001); other differences are noted in the main text. The values for average number of sperm fused per egg (± SEM) were 2.29 ± 0.083 sperm fused per DMSO-treated egg (n = 429 eggs), and 2.61 ± 0.14 sperm fused per ML-7 treated egg (n = 512 eggs) (P = 0.0613). For the subset of fertilized eggs, these values were 2.34 ± 0.083 sperm fused per DMSO-treated fertilized egg (n = 420 eggs), and 3.99 ± 0.16 sperm fused per ML-7-treated egg (n = 335 eggs) (P < 0.0001).