In this paper, we illustrated the relationship among the oocyte development, E2, THs, and sex-related genes in O. mykiss during the different growth stages. The results showed that these indicators have an increasing trend among different growth stages associated with the oocyte development of cultured O. mykiss in Yunnan.
Investigating the developmental period of the oocytes during growing can reduce the time and cost to determine whether the cultured O. mykiss has reached sexual maturity. The result showed that oocytes at the primary yolk stage appeared and showed a higher percentage among other oocyte groups during 80–180 g of body weight. When the body weight above 700–1200 g, mature oocytes filled the whole ovary and can be ready for spawning. Study in sequential hermaphroditism species marbled swamp eel (Synbranchus marmoratus) showed that the females were sexually active with the mean total body length of 43.5 cm while the secondary males with about 58.5 cm . Moreover, year 2–3 ovaries of 0.85–1.63 kg body weight were at stage I and year 4–5 ovaries of 3.67–6.30 kg body weight were at stage II in different ages of Amur sturgeon (Acipenser schrenckii) . Hence, there is a correlated relationship between body growth and ovary development. On the other hand, as a key hormone to promote the development of gonad in fish, the serum concentrations of E2 significantly increased during the body growth of O. mykiss in this study, which corresponded with the development of ovarian histology. This result is consistent with the study of S. trutta in which the peak level of plasma E2 were observed at 30 d before ovulation in the wild strain . In cultured strain of S. trutta, the E2 levels were the highest at the beginning and declined throughout two weeks before ovulation . In female S. salar, E2 were low from December to July and increased to peak levels in September and October during reproductively maturing period, and then decreased significantly during the period of ovulation in November . In contrast, the levels of E2 were not significantly different among different gonadal development stages in Sterlet (Acipenser ruthenus) . These results indicated that E2 has different influences on different fish species and strains. Therefore, high level of E2 contribute to the development and maturation of oocytes of cultured O. mykiss in this study.
THs also play important roles in the regulation of many biological processes in vertebrates, such as growth, metabolism, morphogenesis, and reproduction . An early study demonstrated that THs can promote all stages of reproduction including the early stages gonadal (oocyte) development, vitellogenesis, and maturation leading to successful ovulation and spawning success . Similarly, a recent study showed that the treatment to female goldfish (Carassius auratus) with T3 resulted in an increase of VTG . In contrast, rabbitfish (Siganus guttatus) fed T4 during vitellogenesis did not advance oocyte maturation, but could induce spawning . Moreover, in female S. trutta from a wild strain, the increase in plasma THs around the time of ovulation had an influence on final maturation of oocytes, while the absence of any appreciable change in profile of T4 indicated that the increase of THs are not necessary for ovarian maturation in cultured strain . In our study, THs (T3, T4, FT3, FT4) all peaked when the O. mykiss growing entered into stage Ⅳ, which was correlated with ovarian development. Similar to E2, elevated levels of THs contribute to the growing and final maturation of oocytes in cultured O. mykiss. Comparison of these results are complicated by several factors including different species and stock.
E2 is synthesized by the cooperation of the theca and granulosa cell layers surrounding the oocytes, and secreted into the blood subsequently. VTG is produced by the liver in an estrogen-dependent manner by entering through diffusion, binding and activating the ERs . Only three subtypes of ers (erα1, erβ1, and erβ2) can be detected in our study. In teleost, ers expressed early during embryonic development and gonadal differentiation, suggesting the important role for estrogen in sexual differentiation in the early stage . Estrogen receptor genes, such as erα and erβ2, are involved in E2-induced liver vitellogenesis in medaka (Oryzias latipes), especially erβ2 . In female zebrafish (Danio rerio), higher transcription levels of erα and erβ2 were detected in the liver than erβ1 . Similarly, as an important estrogen receptor gene subtype, erα is also participated in the synthesis of liver vitellogenin induced by E2 in several teleost fish . Moreover, the up-regulation of erα mediated by normal E2 is regulated by erβ1 and erβ2 . In this study, as the growth of O. mykiss, there was no significant correlation among the transcriptions of ers in the liver with the ovarian development. However, the transcription of vtg in the liver gradually raised and reached its peak at growth stage Ⅳ indicated that significantly increasing of vtg transcription in the liver is stimulated by high levels of E2 instead of ers in the later stage of vitellogenesis. Furthermore, the study of female Korean rockfish (Sebastes schlegeli) showed erβ1 and erβ2 highly expressed in the ovary at the vitellogenetic stage, which indicated that ers might play an important role in gonadal development . In orange-spotted grouper (Epinephelus coioides), erα highly expressed in mature ovaries, erβ1 mainly expressed in immature ovaries and erβ2 varied greatly during ovarian development. It speculated the potential roles of ers during female maturation . The highest levels of erα mRNA were found in late vitellogenetic ovaries of O. mykiss . Similarly, in the present study, it was shown that ovarian ers (including erα and erβ1) were sharply increased during O. mykiss growing and oocyte maturation. This result revealed an important role of ovarian ers in the maturation of O. mykiss oocyte cultured in Yunnan.
The vtg gene, which encodes VTG, is a precursor of lipo- and phospho-proteins involved in the formation of the yolk during oogenesis in the majority of vertebrates . The previous study reported that vtg is mainly expressed in the liver, but also various extrahepatic vtg transcription was observed in fishes . In female A. ruthenus, vtg transcription has been detected both in the liver and ovary, and they both reached its peak at the late vitellogenetic stage . During the O. mykiss growing and ovary development, vtg transcription in this study exhibited an increasing trend both in the liver and ovaries, and reached its peak at the late growth and vitellogenetic stage (growth stage Ⅳ). In general, the rise in vtg level in both liver and ovaries during vitellogenesis period, which correspond with the VTG and eggshell proteins incorporation into the oocyte, and the accumulation of enough nutrients for developing embryos . We also propose that the liver and ovaries are both the sites of VTG synthesis in O. mykiss. Moreover, the transcription of vtg was in parallel with the level of serum E2, indicated that E2 plays an important role in inducing the transcription of vtg. Summarizing the results of ers transcription, it demonstrated that the high level of E2 promotes the transcription of the vtg mediated by different types of ers, and then accelerates the maturation of oocytes in cultured O. mykiss.