Aging oocytes: exploring apoptosis and its impact on embryonic development in common carp (Cyprinus carpio)

Abstract

Ovulation, fertilization, and embryo development are orchestrated and synchronized processes essential for the optimal health of offspring. Postovulatory aging disrupts this synchronization and impairs oocyte quality. In addition, oocyte aging causes fertilization loss and poor embryo development. This investigation aimed to unravel the endpoint of in vitro oocyte aging in common carp (Cyprinus carpio) to understand the involvement of apoptosis in postovulatory oocyte death. It was observed that the fertilization ability significantly declined (P < 0.001) at 8-h poststripping (HPS), subsequently triggering apoptosis in the advanced stage of oocyte aging, i.e., 48 HPS. This process included an increase in proapoptotic transcripts (fas, bax, cathepsin D, caspase 8, caspase 9, and caspase 3a) (P < 0.05), elevated levels of caspase 3 protein (P < 0.05), and activation of caspase 3 enzyme (P < 0.001), a key player in apoptosis, in aging oocytes. Furthermore, the effects of oocyte aging on the embryonic apoptosis machinery were examined in embryos at 5-h postfertilization (HPF) and 24 HPF derived from fresh and aged oocytes. Expression of apoptotic genes and caspase enzyme activity remained at the basal level in 5 HPF (early blastula embryos) from both fresh and aged oocytes. In contrast, the zymogenic and active forms of caspase 3 increased in 24 HPF embryos from 8-h-aged oocytes (P < 0.01) compared with those from fresh oocytes. Thus, apoptosis intensified in 24 HPF embryos from aged oocytes without affecting the apoptotic machinery of early blastula embryos. Our findings demonstrate that apoptosis initiated by the Fas/FasL system is an important physiological process accompanying oocyte aging in common carp.

Keywords: apoptosis; caspases; early blastula embryos; fish; in-vitro-aged oocytes; spontaneous activation.

Figure 1.

Schematic representation of experimental design to study the involvement of apoptosis in oocyte aging in common carp (Cyprinus carpio). (A) represents the sampling scheme and (B) represents the molecular and biochemical assays performed on the oocytes and embryo samples. Fert rate: Fertilization rate, DPF: Days Post Fertilization. Source icons from biorender.

Figure 2.

Evaluation of fertilization (A), hatching (B), and malformation rates (C) during in vitro oocyte aging in common carp (Cyprinus carpio). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test are shown by *: P ≤ 0.05, **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001.

Figure 3.

Abundance of apoptosis-related mRNA transcripts during in vitro oocyte aging in common carp (Cyprinus carpio). Depicted are the pro- and anti-apoptotic genes fas (A), bcl2 (B), bax (C), tp53 (D), cathepsin D (E), caspase 8 (F), caspase 9 (G), caspase 3a (H), and caspase 7 (I). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05, **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001. Source icons from biorender.

Figure 4.

The regulation of zymogenic (A) and active forms of caspase 3 (B) during in vitro aging of oocytes in common carp (Cyprinus carpio). Western blot images of caspase 3a with signal at 35 kDa and αtubulin at 54kDa as control from 0, 8, 12, 28, 48, and 72 HPS (C). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05, **: P < 0.01, ***: P < 0.001. Source icons from biorender.

Figure 5.

Regulation of pro- and anti-apoptotic genes in 5 HPF (early blastula) embryos from fresh and aged oocytes of common carp (Cyprinus carpio). Depicted are the pro- and anti-apoptotic genes fas (A), bcl2 (B), bax (C), tp53 (D), cathepsin D (E), caspase 8 (F), caspase 9 (G), caspase 3a (H), and caspase 7 (I). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05. Source icons from biorender.

Figure 6.

The caspase 3 enzyme activity in 5 HPF (early blastula) embryos from fresh and aged common carp (Cyprinus carpio) oocytes. Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05. Source icons from biorender.

Figure 7.

Regulation of pro- and anti-apoptotic genes in 24HPF embryos from fresh and aged common carp (Cyprinus carpio) oocytes. Depicted are the pro- and anti-apoptotic genes fas (A), bcl2 (B), bax (C), tp53 (D), cathepsin D (E), caspase 8 (F), caspase 9 (G), caspase 3a (H), and caspase 7 (I). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05. Source icons from biorender.

Figure 8.

The regulation of zymogenic (A) and active forms of caspase 3 (B) in 24 HPF embryos from fresh and aged common carp (Cyprinus carpio) oocytes. Western blot images of caspase 3a with signal at 35 kDa and αtubulin at 54 kDa as control in embryos from 0, 4, and 8 HPS (c). Data represent mean ± SD. Statistical significance calculated by one-way ANOVA followed by Tukeys multiple comparison test is shown by *: P ≤ 0.05, **: P < 0.01, ***: P < 0.001. Source icons from biorender.