The global Ksuc in mouse ovary increases significantly with advancing age and is distributed in various types of mouse ovarian cells
To investigate whether Ksuc is correlated with ovarian aging, the level of Ksuc was detected by immunoblotting and immunohistochemical (IHC). The global Ksuc in the mouse ovary increased extensively with increasing age for physiological ovarian aging (Fig. 1A and 1B). The results of IHC with the anti-pan-Ksuc antibody showed a significant increase from 3 months old to 17 months old (Fig. 1C and 1D). Additionally, we observed that Ksuc occurs in various types of ovarian cells according to the results of IHC (Fig. 1C) and IF (Figure S4A).
Increased Ksuc in ovaries of POI mice and GCs derived from follicular fluid of women with POI
Increased Ksuc in the ovary of mouse POI model caused by CTX and BU was detected (Fig. 2A and 2B). The level of Ksuc in the ovary of POI mice was also higher than that of NOA mice (Fig. 2C and 2D). The same change also occurred in GCs derived from follicular fluid of women with POI (Fig. 2E and 2F). Thus, Ksuc may have a strong correlation with the development of ovarian aging.
Global increase of mouse ovarian Ksuc promoted cell apoptosis in ovaries cultured in vitro
Given the systemic increase of Ksuc in aged and POI ovaries, we further investigated the effect of elevated Ksuc level on mouse ovarian function. It has been reported that disodium succinate can elevate the level of Ksuc by increasing succinyl-CoA (a cofactor of Ksuc) [18]. Our results revealed that in vitro addition of succinate could elevate the Ksuc level in the mouse ovary, which confirmed that succinyl-CoA also works efficiently in the mouse ovary (Fig. 3A and 3B). Meanwhile, the elevation was specific to Ksuc, since succinate had negligible influence on structurally similar PTMs such as Kac (Figure S4B) and lysine malonylation (Kmal) (Figure S4C). To further analyze the effect of excessive Ksuc on the morphological state of the ovaries, HE staining was conducted. As shown in Fig. 3C, the GCs in the ovaries of the succinate administration group were more sparsely arranged around the follicles compared to the control group. However, 40 mM of NaCl had no obvious effect on the ovaries (data not shown), which excluded the possibility that the effects of succinate were caused by osmotic changes in the bath solution.
In order to explore the effect of Ksuc on ovarian cell apoptosis and proliferation abilities, we performed TUNEL staining in ovaries cultured in vitro and determined the expression of B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), cleaved-caspase 3 and proliferating cell nuclear antigen (PCNA). As shown in Fig. 3D, we observed TUNEL positivity at all time-points in preovulatory follicles and mainly in the GCs. Furthermore, a clear decrease in Bcl-2 mRNA and protein levels was detected in the succinate treatment group when compared to the control group (Fig. 3E–3G). The analysis also revealed that the expression of Cleaved-caspase 3, which serves as the terminal effector molecule in many types of apoptosis [19], increased markedly in the succinate group compared with the control group (Fig. 3E–3G). However, the Bax and PCNA mRNA and protein levels in the ovary cultured with succinate were not significantly different from those in the control group (Fig. 3E–3G). These results suggest that the mechanisms causing increased apoptosis in response to high Ksuc level in mouse ovarian GCs might be mediated by the Bcl-2 and Caspase-3.
High Ksuc level could affect the physiological state of mouse ovary in vivo
Based on the above data, we further confirmed the effect of excessive Ksuc on the mouse ovary in vivo. As shown in Fig. 4A and 4B, the ovarian size and weight of mice (18.74 ± 0.95 mg) markedly decreased in the succinate administration group compared with the control group (25.44 ± 1.66 mg). Meanwhile, the ovary index (ovary weight / body weight) in the succinate administration group was also lower than that in the control group (Fig. 4C). Interestingly, we found that the serum levels of AMH and E2 in the succinate group were significantly decreased (Fig. 4D and 4E), along with increased atretic follicles and reduced secondary, antral and total follicles (Fig. 4F–4L). These results suggest that high Ksuc level likely suppresses the ovarian reproductive and endocrine function.
Excessive Ksuc could promote mouse ovarian aging by regulating follicular development and ovarian cell apoptosis and proliferation in vivo
Importantly, the injection with succinate in situ in the ovaries was able to elevate the global Ksuc level of mouse ovaries in vivo as in vitro (Fig. 5A and 5B). Based on this, we would argue that the above phenotypes and the following molecular level changes associated with ovarian dysfunction are due to the high level of Ksuc.
Increased cell apoptosis rate was detected using the TUNEL and IHC staining assay of Cleaved-caspase 3 in the ovaries of succinate-administered mice in contrast to the control mice (Fig. 5C and 5D). We observed positive TUNEL and Cleaved-caspase 3 staining at ovarian cells and mainly in the GCs, indicating that the increased apoptosis of GCs is associated with ovarian dysfunction. Importantly, the mRNA and protein levels of proliferation- and apoptosis-related genes in the ovary administered with succinate in vivo were detected by qRT-PCR and western blot. As shown in Fig. 5E, the mRNA expression levels of ovarian cell proliferation and apoptosis genes were significantly affected by high Ksuc stimulation, particularly for BCL-2 and CASPASE3. Meanwhile, obvious changes in Bcl-2 and Cleaved-caspase 3 protein levels were detected in the succinate group compared with the control group (Fig. 5F and 5G). The changes of these factors were generally consistent with the increased cell apoptosis of ovary cultured in vitro induced by high level of Ksuc, further confirming the role of Ksuc in cell apoptosis of mouse ovary. Moreover, the expression of P21, the aging marker, increased significantly in mouse ovary after succinate administration in vivo (Fig. 5E-5G).