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. 2016 Jun 20:6:28347.
doi: 10.1038/srep28347.

The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model

Juan Lin  1 , Fei Chen  2 , Ming-Ju Sun  2 , Jiang Zhu  2 , You-Wei Li  2 , Liu-Zhu Pan  1 , Jie Zhang  1 , Jing-He Tan  1   2
Affiliations

The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model

Juan Lin et al. Sci Rep. .

Abstract

The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration.

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Figures

Figure 1
Figure 1. GV chromatin configurations of mouse oocytes before (0 h) or following OH for 1 or 2 h at 37 or 39 °C.
Photographs (A) and A’, (B) and B’, (C) and C’, (D) and D’, (E) and E’, and (F) and F’ are the same oocytes observed with phase contrast and fluorescence, respectively, after Hoechst 33342 staining. A’, B’ and C’ show, respectively, oocytes with NSN, intermediate (IN) and SN configurations before OH. D’ shows an oocyte with condensed NSN (c-NSN) configuration, and E’ and F’ show oocytes with condensed IN (c-IN) configuration. Original magnification ×1300. Scale bar is 10 μm. The table shows percentages of oocytes with different chromatin configurations. a-d: Values without a common letter in their superscripts differ significantly (P < 0.05) in the same column. Each treatment was repeated 5 times with each replicate containing 50–60 oocytes.
Figure 2
Figure 2. Laser confocal photomicrographs show acetylation (ace) of histone H3K14, H4K12 and H4K16, dimethylation (met) of H3K9 and H3K4, and phosphorylation (pho) of H3S10 in the SN oocytes before or after OH of prepubertal mouse ovaries at different temperatures for different times.
Acetylated, dimethylated or phosphorylated histone was pseudo-colored red. Original magnification ×200. Scale bar is 10 μm.
Figure 3
Figure 3. Apoptosis of MGCs and cumulus cells following OH of prepubertal mouse ovaries at different temperatures for different times.
Micrographs (A–E) show MGCs smears and micrographs (H,I) show cumulus cells smears stained with Hoechst 33342 and observed under a fluorescence microscope. The heterochromatin was heavily stained with Hoechst and gave bright fluorescence. Whereas the apoptotic cells showed pyknotic nuclei full of heterochromatin (Arrows), healthy cells showed normal nuclei with sparse heterochromatin spots. Smears (A–E) show MGCs from ovaries before (A) or after OH at 37 °C for 1 h (B), 39 °C for 1 h (C), 37 °C for 2 h (D) and 39 °C for 2 h (E), respectively, while smears (H,I) show cumulus cells from ovaries before (H) and after OH at 37 °C for 1 h (I), respectively. Original magnification ×400. Scale bar is 20 μm. Graphs (F,G) show percentages of apoptotic cells and the level of active caspase-3 (Casp-3) in MGCs, respectively, and graph (J) shows percentages of apoptotic cumulus cells. In graph (G), the molecular weights of active caspase-3 and β-tubulin are 17 and 55 kDa, respectively. To measure percentages of apoptotic cells, each treatment was repeated 3–5 times with each replicate containing 10 smears, and to quantify the level of active caspase-3 by western blotting, each treatment was repeated 3 times with each replicate containing MGCs from 4 ovaries. a–e: Values without a common letter above their bars differ significantly (P < 0.05).
Figure 4
Figure 4. Annexin-positive oocytes with different chromatin configurations after OH of prepubertal mouse ovaries at different temperatures for different times.
Photographs in the same column show the same oocytes observed under phase contrast (original magnification ×1300, scale bar is 10 μm) and fluorescence after Hoechst (×1300) and annexin (×400, scale bar is 15 μm) staining, respectively. Photographs in different columns show oocytes with NSN, SN, c-NSN and c-IN configurations, respectively. The table shows percentages of annexin-positive oocytes with various chromatin configurations after OH at 37 or 39 °C for 1 or 2 h. a–d: Values without a common letter in their superscripts differ (P < 0.05) in the same column. Each treatment was repeated 4-5 times with each replicate containing about 30 oocytes.
Figure 5
Figure 5. Levels of FasL in MGCs and Fas receptors in oocytes and cumulus cells after OH of prepubertal mouse ovaries.
The OH conditions include before OH (0 h) and after OH at 37 °C for 1 h (37C1h), 39 °C for 1 h (39C1h), 37 °C for 2 h (37C2h) and 39 °C for 2 h (39C2h). Panel A shows FasL expression in MGCs revealed by western blotting. The molecular weights of FasL and β-tubulin (Tublin) are 31 and 55 kDa, respectively. Panel B shows Fas quantification by immunocytochemistry in oocytes. Each treatment was repeated 3–4 times with each replicate containing about 30 oocytes. Panel C shows confocal micrographs showing Fas localization in oocytes. Original magnification ×400. Scale bar is 30 μm. Panel D shows Fas quantification in cumulus cells by immunocytochemistry. Each treatment was repeated 3–4 times with each replicate containing about 30 cells from 15 images. Panel E shows confocal micrographs showing Fas localization in cumulus cells. Original magnification ×650. Scale bar is 5 μm. In the confocal images, DNA and Fas were pseudo colored blue and red, respectively. In the graphs, a–e indicates values without a common letter above their bars differ significantly (P < 0.05).
Figure 6
Figure 6. Percentages of annexin-positive oocytes and levels of H4K12 acetylation in oocytes with different chromatin configurations after OH under different conditions in wild-type and gld prepubertal mice.
The micrographs are laser confocal images of SN oocytes in which Hoechst 33342 (upper row) and H4K12 (lower row) were pseudo-colored blue and red, respectively. Original magnification × 200. Scale bar is 10 μm. Images in the same column are from the same oocyte observed at different optical wavelengths. The SN oocytes shown in different columns include oocytes before OH (0 h) or after OH at 39 °C for 1 h (39C1h) from wild-type (W) or gld (G) mice. The two tables show % annexin-positive oocytes and relative levels of H4K12 acetylation among different oocytes, respectively. Each treatment was repeated 4 times with each replicate containing about 30 oocytes. a–c: Values in the same column without a common letter in their superscripts differ (P < 0.05).
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