Gonadal Development and Associated Changes in Estradiol, Thyroid Hormones, and Sex-Related Genes During Different Growth Stages in Cultured Female Rainbow Trout (Oncorhynchus Mykiss) in Yunnan

Background: Rainbow trout (Oncorhynchus mykiss) is an important shing resource in Yunnan. However, the oocyte quality and fecundity are particularly affected by environmental factors when sh are undergoing sexual maturation, which hinders the development of the O. mykiss industry. Results: Estradiol (E 2 ), thyroid hormones (THs), and sex-related genes transcription associated with gonadal development were analyzed in different growth stages of O. mykiss cultured in Yunnan. Females were classied into four growth stages: growth stage (cid:0) , growth stage (cid:0) , growth stage (cid:0) , and growth stage (cid:0) . Histological analysis found a correlated relationship between body growth and ovary development. Serum levels of E 2 and THs increased during ovarian development and reached the highest level at growth stage (cid:0) indicated that E 2 and THs play important roles in the development and maturation of oocytes in O. mykiss. Furthermore, real-time PCR analysis showed that the vitellogenin mRNA (vtg) transcription level increased signicantly both in ovaries and liver during ovarian development, then reaching its peak at the post-vitellogenetic (growth stage (cid:0) ) period. The same transcription trend was detected in the levels of ovarian estrogen receptors (ers). Conclusions: These results indicated that the high level of E 2 promotes the transcription of the vtg mediated by different types of ers, and then accelerates the maturation of oocytes, and THs also participate in the regulation of maturation of oocytes in cultured O. mykiss during the individual development in Yunnan.


Background
Yunnan located in the Yunnan-Guizhou Plateau are topographically and climatically diverse. Yunnan is in a subtropical humid monsoon climate zone that includes tropical, subtropical, temperate and boreal climates [1]. Environmental conditions such as water temperature, water quality, lighting, and dietary nutrition, which in turn affect production traits including gonads development, reproduction, and growth in sh [2][3][4]. Rainbow trout (Oncorhynchus mykiss) is an important shing resource in Yunnan, and it is also an expanding industry in several regions. Because of the oocyte quality and fecundity are particularly affected by environmental factors when sh are undergoing sexual maturation, it is important to attain knowledge about the oocyte development and how factors such as serum hormones and sexrelated genes involved in growth changed in cultured O. mykiss in Yunnan.
In teleost, it is well known that the hypothalamic-pituitary-gonadal (HPG) axis regulates the vitellogenesis and the nal oocyte maturation during individual growing that mediate gonadotropic hormones (GtHs) actions via steroids secreted by the follicular cells surrounding the oocyte [5]. Correlation between changes in levels of sexual steroids (testosterone and 17β-estradiol (E 2 )) and oocyte development have been well documented in sh [6][7][8]. Moreover, studies showed that the hypothalamus-pituitary-thyroid (HPT) axis is also involved in gonad development [9]. For instance, thyroid hormones (THs) ampli ed the effects of GtH on E 2 secretion by isolated ovarian follicles in rainbow trout (Salmo gairdneri) [10]. In Atlantic salmon (Salmo salar), THs were elevated before vitellogenesis and then decreased around the time when E 2 and vitellogenin (VTG) levels were known to rise [11]. However, in female brown trout (Salmo trutta), the discrepancies of plasma sex steroids observe between wild and cultured females may be due to differences in stress susceptibility, environmental conditions, life cycles, or to genetic divergence between the strains [12]. Therefore, changes in serum hormones during individual growth are important to be characterized and it will broaden the understanding of the relationship with the oocyte development and serum hormones in cultured O. mykiss in Yunnan.
VTG is a glycolipophosphoprotein produced in all oviparous species, which is biosynthesized in the liver under estrogen regulation and transported to the growing oocytes participating in the process of yolk formation [13,14]. In the liver of mature females teleost, vtg transcription is stimulated via estrogen receptors (ers) signaling pathways due to the simultaneous increase of estrogen concentration [15].
Matured VTG produced in the liver was then transported to the ovary through the circulatory system.
Thus, the amounts of vtg transcription and translation have been widely used as biomarkers during oocyte development in certain teleost. In addition, ers play a crucial role in the mediation of estrogen activities. Four er isoforms (erα1, erα2, erβ1, and erβ2) have been detected and dominatingly expressed in the liver of O. mykiss [16]. These receptors may be important for estrogen signal conduction that initiates the next cycle of germ cell development in the ovary [17]. Therefore, understanding the function of ers in oocyte development during growing processes will be bene cial for improving the quality of cultured O. mykiss oocytes in the future. Although the endocrine system of Oncorhynchus species has been relatively well studied, limited information is available about the relationship among oocyte development, serum hormones and sexrelated genes related to different growth stages. In the present study, we described the characteristics of oocyte development and analyzed the regulatory relationship among E 2 , THs, and sex-related genes during different growth stages of O. mykiss. This study aims to clarify the regulation of oocyte development of O. mykiss cultured in plateau of Yunnan.

Histology
Different growth stages of oocytes observed in the ovaries of O. mykiss were shown in Fig. 1. On growth stage and growth stage , oocytes at primary yolk stage appeared and showed a higher percentage among other oocyte groups. On growth stage , ovaries were mostly occupied by lling yolk stage oocytes. On growth stage , mature oocytes lled the whole ovary (Fig. 1). Serum hormones pro le in female O. mykiss E 2 levels in female O. mykiss ranged from 5.958 pg/mL to 1496.072 pg/mL in Fig. 2. E 2 content was positively correlated with individual growth and oocyte development. There was no signi cant difference between growth stage and growth stage . However, as the growth of O. mykiss, signi cantly elevated of E 2 level has been detected in growth stage (p < 0.05). Moreover, the highest value was recorded in growth stage when the oocytes were matured (p < 0.05).
The levels of THs (T 4 , FT 4 , T 3 , FT 3 ) peaked in growth stage (p < 0.05) and were correlated with O. mykiss growth and oocyte development. No signi cant difference in T 4 was detected among growth stage , growth stage , and growth stage . But the highest level of T 4 was showed in growth stage . T 3 showed an increasing trend during O. mykiss growing and reached the highest level in growth stage . The trend of FT 4 during O. mykiss growing is similar to that of T 4 , while a higher level was also found in growth stage . FT 3 started to increase from growth stage and maintain high levels during the mature stage   The THs in each growth stage is presented as mean ± SD (n = 3). Different superscript letters indicate signi cant differences among different growth stages (p < 0.05).
Transcription pro les of sex-related genes during different growth stages in O. mykiss Variations of the ers transcription during the different growth stages were analyzed in this study (Fig. 4a). The ers showed an irregular trend throughout the different growth stages, and on signi cant differences have been detected in the liver. In contrast, signi cant increases of erα1 and erβ1 transcription have been detected in the gonad and the highest level was shown in growth stage (p < 0.05), while erβ2 did not uctuate signi cantly during the different growth stages.
vtg transcription was also continuously detected in all the analyzed individuals from 80.0 to 1200.0 g. The relative vtg transcription in the liver was low of all detected individuals at growth stage . However, when the individuals continue to grow, vtg transcription is gradually upregulated, and the highest level was detected in growth stage . Similarly, the transcription of vtg in gonads was low of all detected individuals at growth stage , growth stage , and growth stage . However, when the individuals growing enter into stage , vtg transcription showed a big uctuation and signi cantly increased (Fig. 4b).

Discussion
In this paper, we illustrated the relationship among the oocyte development, E 2 , 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 lled 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 [18]. 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) [19]. 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 sh, the serum concentrations of E 2 signi cantly 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 E 2 were observed at 30 d before ovulation in the wild strain [12]. In cultured strain of S. trutta, the E 2 levels were the highest at the beginning and declined throughout two weeks before ovulation [12]. In female S. salar, E 2 were low from December to July and increased to peak levels in September and October during reproductively maturing period, and then decreased signi cantly during the period of ovulation in November [11]. In contrast, the levels of E 2 were not signi cantly different among different gonadal development stages in Sterlet (Acipenser ruthenus) [20]. These results indicated that E 2 has different in uences on different sh species and strains. Therefore, high level of E 2 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 [21]. 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 [22]. Similarly, a recent study showed that the treatment to female gold sh (Carassius auratus) with T 3 resulted in an increase of VTG [9]. In contrast, rabbit sh (Siganus guttatus) fed T 4 during vitellogenesis did not advance oocyte maturation, but could induce spawning [23]. Moreover, in female S. trutta from a wild strain, the increase in plasma THs around the time of ovulation had an in uence on nal maturation of oocytes, while the absence of any appreciable change in pro le of T 4 indicated that the increase of THs are not necessary for ovarian maturation in cultured strain [12]. In our study, THs (T 3 , T 4 , FT 3 , FT 4 ) all peaked when the O. mykiss growing entered into stage , which was correlated with ovarian development. Similar to E 2 , elevated levels of THs contribute to the growing and nal maturation of oocytes in cultured O.
mykiss. Comparison of these results are complicated by several factors including different species and stock. E 2 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 [24]. 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 [25]. Estrogen receptor genes, such as erα and erβ2, are involved in E 2 -induced liver vitellogenesis in medaka (Oryzias latipes), especially erβ2 [26]. In female zebra sh (Danio rerio), higher transcription levels of erα and erβ2 were detected in the liver than erβ1 [27]. Similarly, as an important estrogen receptor gene subtype, erα is also participated in the synthesis of liver vitellogenin induced by E 2 in several teleost sh [28]. Moreover, the up-regulation of erα mediated by normal E 2 is regulated by erβ1 and erβ2 [29]. In this study, as the growth of O. mykiss, there was no signi cant 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 indicated that signi cantly increasing of vtg transcription in the liver is stimulated by high levels of E 2 instead of ers in the later stage of vitellogenesis. Furthermore, the study of female Korean rock sh (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 [30]. 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 [31]. The highest levels of erα mRNA were found in late vitellogenetic ovaries of O. mykiss [32]. 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 [33]. The previous study reported that vtg is mainly expressed in the liver, but also various extrahepatic vtg transcription was observed in shes [34]. 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 [35]. 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 ). 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 [36]. 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 E 2 , indicated that E 2

Fish sampling
The cultured O. mykiss was obtained from Tanghao Aquaculture Company located in Kunming, China. Sampling permissions were also obtained for the Tanghao Aquaculture Company. Sampling was carried out in July when sh were in the peak of growth and development. The bodyweight of O. mykiss ranged from 80.0 g to 1200.0 g (n = 12). They were divided into four different growth stages, which are growth stage (80-120 g), growth stage (160-180 g), growth stage (280-400 g), growth stage (700-1200 g), respectively. Fish were fasted for 24 h and then anesthetized by 100 mg/L tricaine methanesulfonate (MS-222, Sigma, St. Louis, MO) till the cessation of opercular movements for about 5 min before sampling. Blood samples were withdrawn from the sh caudal vein with a syringe and centrifuged at 1,500 g for 30 min. The serum of shes in each growth stage was pooled and then quickly frozen at -80 °C for sequential detect of E 2 , and THs (T 3 , T 4 , FT 3 , FT 4 ). Then, the ovaries were rapidly excised and xed in Bouin's uid for determined the histological characteristics. Furthermore, the ovaries and liver of each sh were frozen at -80 °C for the determination of gene transcription. The usage of sh was in strict accordance with the recommendations of the Guidelines for the Use of Experimental Animals of Yunnan Agricultural University. The protocol for animal care and handling used in this study was approved by the Committee on the Ethics of Animal Experiments of Yunnan Agricultural University.

Histology
For histological studies, ovaries of each growth stage were embedded in para n and sectioned at 5-6 µm. Sections were stained with hematoxylin and eosin. After staining, the slides were allowed to dry and then examined with a light microscope (Olympus BX51, Japan).

E 2 analyses
The levels of E 2 were measured by using commercially available 125 I-RIAs assay (Beijing North Institute of Biotechnology Co., Ltd., Beijing, China) in serum according to the manufacturer's instruction. The kit uses competitive radioimmunoassay to simultaneously compete for binding of radiolabeled antigens and non-labeled antigens to a limited amount of speci c antibodies. The radioactive count of the labeled antigen-antibody complex is determined by separating the unbound labeled antigen.