Aquacult. Sci. 64（1），29－42（2016） Quantitative comparisons of maternal transcripts related to cell division between good and poor quality eggs from artificially matured Japanese eel Anguilla japonica 1 2 3 1 Hikari IZUMI , Koichiro GEN , Moemi HORIUCHI , Noriaki MATSUYA , 1 1,＊ Shigeho IJIRI and Shinji ADACHI Abstract: The current method of artificial egg production in Japanese eel does not always yield good quality eggs, but the molecular basis for the decrease in egg quality remains unclear. In this study, we compared the levels of 74 maternal transcripts related to cell division among good quality eggs, developing poor quality eggs (i.e., those producing a viable morula embryo without hatch- ing), and non-developing poor quality eggs (i.e., developmentally incompetent eggs) to determine the molecular characteristics of poor quality eggs produced by artificially matured Japanese eel. Only one transcript (lsm7) had a lower level in developing poor quality than in good quality eggs, with the others being present at similar levels between the two types of egg. The levels of 11 tran- scripts (e.g., cdc26, psma7) were lower in non-developing poor quality than in good quality eggs. These results suggest that insufficient or reduced levels of maternal mRNA related to cell division are associated with the loss of egg quality in artificially matured Japanese eel, and that maternal characteristics other than quantity may affect the developmental competence of poor quality eggs. Key words: Japanese eel; Egg quality; Maternal mRNA; Cell division The Japanese eel Anguilla japonica is a major until much later (Tanaka et al. 2003), with aquaculture species in Japan. The seedling second-generation larvae finally obtained by stock for aquaculture is derived from glass eels artificial production in 2010 (Tanaka 2011). caught in the wild. Recently, natural glass eel However, there remain many challenges in the stocks have drastically diminished (Tanaka current method of artificial eel maturation. One et al. 2006) and in 2014, the species was listed problem is that many artificially matured eels as endangered on the International Union for produce poor quality eggs, which decreases Conservation of Nature Red List (Jacoby and larvae production efficiency, although this has Gollock 2014). Therefore, artificial production recently seen some improvement owing to of glass eels is necessary in order to ensure methodological advances (Kagawa et al. 2005; a seedling supply and preserve this natural Unuma et al. 2011; Okamura et al. 2014; Tanaka resource. Fertilizable eggs and larvae were et al. 2015). first produced by artificial hormone treatment Egg quality is assessed by the percentage of in the 1970s (Yamamoto and Yamauchi 1974), eggs that are successfully fertilized and hatch, but glass eels were not generated in captivity as well as the rate of deformity during early Received 16 September 2015; Accepted 14 December 2015. 1 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan 2 Seikai National Fisheries Research Institute, Fisheries Research Agency, Taira-cho, Nagasaki 851-2213, Japan 3 School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan ＊Corresponding author: Tel & Fax, (+81) 138-40-5545; Email, [email protected] (S. Adachi). 30 H. Izumi, K. Gen, M. Horiuchi, N. Matsuya, S. Ijiri and S. Adachi development. Good quality eggs are those that fertilization, maternal gene products drive early exhibit normal developmental competence and embryonic development in a transcriptionally have sufficient amounts of substrate required silent embryo (Vasudevan et al. 2006). In fish, for embryogenesis including yolk proteins, the onset of zygotic transcription̶referred to cytoskeleton and membrane proteins, lipids, as zygotic genome activation (ZGA)̶occurs RNAs, hormones, and vitamins (Brook et al. at the mid-blastula transition (MBT), which is 1997). In contrast, poor quality eggs are those characterized by the lengthening of the cell that show reduced developmental competence cycle and an asynchronous appearance after the and might have abnormal composition or first 10 synchronous cleavage divisions (Kane abundance of these substances. In aquacul- and Kimmel 1993; Lee et al. 2014). Maternal ture, multiple factors can influence egg quality transcripts begin to degrade shortly before (see review Brook et al. 1997; Bobe and Labbé ZGA, and developmental control is transferred 2010). Environmental factors such as photo- from maternal to zygotic gene products in a period, temperature, salinity, pH of the water, process known as the maternal-zygotic transi- and diet affect oogenesis by altering the phys- tion (MZT), which is a critical step for the pro- iological condition of broodstock, ultimately gression of embryonic development (Tadros dictating egg quality. Stress and reproductive and Lipshitz 2009; Lee et al. 2014). Maternal age can also play a role. In some aquaculture factors are required for early stages of embry- species including Japanese eel in which ovula- onic development as well as for MBT and tion must be artificially induced even if oogen- ZGA (Shimuta et al. 2002; Dosch et al. 2004; esis is induced normally, eggs ovulated at the Lindeman and Pelegri 2010; Lee et al. 2013). inappropriate time show low developmental Additionally, maternal factors also influence competence (Adachi 2000; Unuma et al. 2011; subsequent development after MZT (Wagner et Ishihara et al. 2014). This is the main problem al. 2004). Therefore, abnormal composition or in artificially matured fish. Even in eggs ovu- abundance of maternal mRNA in eggs is likely lated at the optimum timing, postovulatory to result in reduced egg quality. aging, and post-stripping handling can directly Several previous studies have investigated the impact eggs and undermine egg quality (Ohta relationship between egg quality and maternal et al. 1996a; Bobe and Labbé 2010; Nomura et mRNA abundance in aquaculture species (see al. 2013). Additionally, genetic factors such as review Sullivan et al. 2015). One transcriptome breeding between genetically close individu- analysis of fully grown ovarian follicles from als are known to have a major impact on egg the striped bass Morone saxatilis identified 95 quality (Brook et al. 1997). It is very likely that maternal transcripts related to cell division the above-described factors negatively influ- among the 133 informative genes that were cor- ence egg components required for normal related with egg quality (Chapman et al. 2014), embryogenesis, resulting in poor quality eggs. leading the authors to propose that dysfunction However, the molecular basis for the loss of egg of the cell cycle contributes to embryo mortal- quality as a result of the above-described factors ity prior to MBT. This suggests that abnormal has not been widely investigated in aquaculture levels of maternal mRNA related to cell division species, including Japanese eel. are a molecular characteristic of poor qual- Comparing the molecular characteristics of ity eggs. However, quantitative PCR (qPCR) poor vs. good quality eggs can provide insight analysis of mRNA levels was not carried out into the molecular basis for the loss of egg in this report. In artificial maturation of aqua- quality. One type of substance required for culture species, it is often observed that poor embryogenesis is maternally inherited tran- quality eggs develop into viable embryos at the script (maternal mRNA), which accumulates in synchronous cleavage stage but fail to hatch; eggs during oogenesis and is translated during however, maternal mRNA abundance in these final oocyte maturation and egg activation. After eggs has not been investigated. Additionally, Maternal mRNA and egg quality in Japanese eel 31 there have been no studies to date examining Artificial fertilization the relationship between maternal mRNA in Fertilization and hatching rates were Japanese eel eggs and egg quality. determined using an individual rearing method The present study investigated the molecular (Unuma et al. 2004) in which 2 g of ovulated characteristics of poor quality eggs resulting eggs were inseminated with 1 ml of pre-diluted from multiple factors, including inappropri- milt (sperm mortality >50%) and transferred ate timing of ovulation induction in artificially to 100 ml filtered seawater. A 3-ml volume of matured Japanese eel. Egg quality was esti- seawater containing about 120 eggs was trans- mated by fertilization and hatching rates; eggs ferred to a glass dish, and the eggs were trans- with high hatching rates were classified as good ferred to 48-well culture plates (one egg per quality eggs; those with high fertilization and well). Each well was filled with 1 ml filtered sea- low hatching rates were classified as developing water containing 100,000 IU/l penicillin G potas- poor quality eggs; and those with low fertiliza- sium, 0.1 g/l streptomycin sulfate, and 1μg/ml tion rate were classified as non-developing poor polyethylene glycol, and the eggs were incu- quality eggs. We compared the maternal mRNA bated at 23°C. Normal morula embryos and levels of cell division-related genes identified hatched larvae were counted 4 h after insemina- in the striped bass study among good quality, tion and 3 days after fertilization, respectively. developing poor quality, and non-developing Fertilization and hatching rates were calculated poor quality eggs by qPCR. using the following formulae: Materials and Methods Fertilization rate (%)=100×number