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.

The delay in fertilization after ovulation or retention of ovulated oocytes in the fish body causes postovulatory aging. Postovulatory aging leads to time-dependent deterioration of oocyte quality and loss of fertilization capacity. The mechanisms behind losing oocyte quality and developmental capacity due to postovulatory oocyte aging remain elusive. The emerging climate change issues in nature and unfavorable spawning conditions have caused the retention of ovulated oocytes in the female body. Analyzing the apoptotic parameters to understand the fate of these aged oocytes and the consequences of this aging on embryo development was the main objective of this study. The results obtained from this study indicate that aged oocytes die by apoptosis. The embryos from aged oocytes show more apoptosis, stating that oocyte aging affects embryo development by affecting the intensity of apoptosis.

Department of Endocrinology and Metabolism Charité Universitätsmedizin Berlin Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin 10115 Berlin Germany

DZHK Partner Site Berlin 10115 Berlin Germany

Research Institute of Fish Culture and Hydrobiology South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske Budejovice 389 01 Vodňany Czech Republic

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