Cumulus cells accelerate oocyte aging by releasing soluble Fas ligand in mice

Abstract

Although previous studies have suggested that cumulus cells (CCs) accelerate oocyte aging by secreting soluble and heat-sensitive paracrine factors, the factors involved are not well characterized. Because Fas-mediated apoptosis represents a major pathway in induction of apoptosis in various cells, we proposed that CCs facilitate oocyte aging by releasing soluble Fas ligand (sFasL). In this study, we reported that when the aging of freshly ovulated mouse oocytes were studied in vitro, both the apoptotic rates of CCs and the amount of CCs produced sFasL increased significantly with the culture time. We found that oocytes expressed stable levels of Fas receptors up to 24 h of in vitro aging. Moreover, culture of cumulus-denuded oocytes in CCs-conditioned CZB medium (CM), in CZB supplemented with recombinant sFasL, or in CM containing sFasL neutralizing antibodies all showed that sFasL impaired the developmental potential of the oocytes whereas facilitating activation and fragmentation of aging oocytes. Furthermore, CCs from the FasL-defective gld mice did not accelerate oocyte aging due to the lack of functional FasL. In conclusion, we propose that CCs surrounding aging oocytes released sFasL in an apoptosis-related manner, and the released sFasL accelerated oocyte aging by binding to Fas receptors.

Figure 1. Effects of culture time and H₂O₂ on the apoptosis and sFasL release of CCs.

CCs were cultured in regular CZB medium in the presence (+) or absence (−) of H₂O₂. At different times of the culture, the apoptotic rates in the CCs and the concentrations of sFasL in the CM were measured. Micrographs A, B and C show CCs 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, healthy cells showed normal nuclei with sparse heterochromatin spots. Smears A, B and C show CCs collected at 0 h and 24 h of aging culture without or with H₂O₂, displaying approximately 20%, 75%, and 95% apoptotic cells, respectively. Original magnification ×400. Graphs D and E show the percentages of apoptotic CCs and levels of sFasL released by CCs after different treatments, respectively. Each treatment was repeated more than 3 times with each replicate containing 30 oocytes. a–e: Values without a common letter above their bars differ significantly (P < 0.05).

Figure 2. Levels of Fas receptor in aging oocytes.

COCs were cultured in CZB medium for different times before examination for Fas localiztion and quantification by immunocytochemistry or Western blot analysis. A, B, C and D are confocal micrographs (original magnification ×400) showing Fas localization in oocytes that have aged for 0, 12, 24 and 36 h, respectively. E is a graph showing Fas quantification by immunocytochemistry in oocytes aged in vitro for different times. Each treatment was repeated 3–4 times with each replicate containing about 30 oocytes. F is a graph showing Fas levels by Western blot in oocytes aged for different times. β-tub: β-tubulin. a,b: Values with different letters above their bars differ significantly (P < 0.05).

Figure 3. Oocyte activation following ethanol or SrCl₂ treatment in DOs or COCs that had been aged in regular CZB medium for 0, 6 or 12 h.

Each treatment was repeated 3–4 times with each replicate containing about 30 oocytes. a–e: Values without a common letter above their bars differ significantly (P < 0.05).

Figure 4. Development of Sr²⁺ activated embryos after mouse DOs collected 13 h post hCG were cultured for 12 h or 24 h in regular CZB or in different CM.

To prepare CM, CZB was conditioned for 24 h with CCs that had been treated with (CMO) or without H₂O₂ (CM), and some of the CM collected was heated to 56°C to test the heat sensitivity of the aging-facilitating factor (CMH and CMOH). Each treatment was repeated more than 3 times with each replicate containing about 30 oocytes. a–f: Values without a common letter above their bars differ significantly (P < 0.05).

Figure 5. Development of Sr²⁺-activated embryos after mouse DOs collected 13 h post hCG were cultured for 12 h or 24 h in CZB medium containing different concentrations of sFasL.

Each treatment was repeated more than 3 times with each replicate containing 30 oocytes. a–d: Values without a common letter above their bars differ significantly (P < 0.05).

Figure 6. Effects of FasL antibodies containing CM on the development of Sr²⁺-activated oocytes.

CM conditioned for 24 h with H₂O₂-treated CCs was neutralized for 6 h at 37°C with different concentrations of FasL antibodies. DOs collected 13 h post hCG were aged for 12 h or 24 h in the neutralized CM before Sr²⁺ activation was applied. Each treatment was repeated 3–4 times with each replicate containing about 30 oocytes. a–d: Values without a common letter above their bars differ significantly (P < 0.05).

Figure 7. Fragmentation of aging oocytes.

DOs collected 13 h post hCG were cultured for 24 h in CZB alone, CZB with 10 ng/ml sFasL, CM conditioned for 24 h with CCs (CM) or H₂O₂-treated CCs (CMO) before post-treatment aging in CZB alone. At different times of the post-treatment aging, oocytes were observed for fragmentation. Photographs A–E show aged oocytes with different fragmenting patterns. F is graph showing percentages of fragmented oocytes at different times of post-treatment aging. Each treatment was repeated 3–4 times with each replicate containing about 30 oocytes. a–c: Values without a common letter above their bars differ significantly (P < 0.05) within time points of post-treatment aging.

Figure 8. Ethanol-activation rates of oocytes collected from wild-type or gld mice.

The oocytes were collected at 13 h post hCG injection and were cultured for 6 h as COCs in CZB medium, or for 12 h as DOs in CM prepared with wild-type or gld CCs. Each treatment was repeated 3 times with each replicate containing about 25 oocytes. a–b: Values with different letters in their bars differ significantly (P < 0.05).