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.

• Keywords
• apoptosis, caspases, early blastula embryos, fish, in-vitro-aged oocytes, spontaneous activation,
• MeSH
• Apoptosis * physiology   MeSH
• Embryo, Nonmammalian physiology   MeSH
• Embryonic Development * physiology   MeSH
• Carps * embryology   physiology   MeSH
• Caspases metabolism   genetics   MeSH
• Oocytes * physiology   MeSH
• Aging * physiology   MeSH
• Gene Expression Regulation, Developmental physiology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Caspases MeSH

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

• Keywords
• Ctenopharyngodon Idella, Epigenetics, Fertilization, HAT activity, Histone acetylation, Ovulation,
• MeSH
• Acetylation MeSH
• Histone Acetyltransferases * metabolism   genetics   MeSH
• Histones * metabolism   MeSH
• Carps * metabolism   MeSH
• Oocytes * MeSH
• Protein Processing, Post-Translational MeSH
• Cellular Senescence MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Histone Acetyltransferases * MeSH
• Histones * MeSH

INTRODUCTION: Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes. METHODOLOGY: We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates. RESULTS: The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2-associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and pro-apoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO. DISCUSSION: Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish.

• Keywords
• apoptosis, cell death, fertilization, membrane integrity, trypan blue, zebrafish,
• Publication type
• Journal Article MeSH