Seasonal Changes in Plasma Hormones, Sex-Related Genes Transcription in Brain, Liver and Ovary during Gonadal Development in Female Rainbow Trout ( Oncorhynchus mykiss )

: The purpose of this study was to investigate the periodic seasonal changes in endocrine activity and gonadal development of female rainbow trout ( Oncorhynchus mykiss ) in a high-altitude cold-water environment. The ﬁsh were sampled monthly from January to November and the levels of plasma hormones (estradiol (E 2 ), cortisol and thyroid hormones (TH S )) and vitellogenin (VTG) were measured by ELISA. Moreover, the transcriptions of sex-related genes in the ovary, brain, and liver were detected by qRT-PCR. The results showed a seasonal ﬂuctuation of plasma hormones and VTG together with the development of the ovary, which reached a peak from August to October. Similarly, the transcription of hypothalamic gonadotropin-releasing hormone-2 ( cgnrh-2 ), hypothalamic gonadotropin-releasing hormone receptors ( gnrhr ) and follicle-stimulating hormone ( fsh ) in the brain varied from January to September, but the highest level was detected in September to November. In addition, the transcription of sex-related genes located in the ovary and liver increased signiﬁcantly during August to October, accompanied by a continuous increase in the gonadosomatic index (GSI) and a decrease in the hepatosomatic index (HSI). Therefore, plasma hormones and sex-related genes regulate the development and maturation of O. mykiss oocytes with the change in seasons and peaked in November. The results of this study provide a reference for improving the efﬁciency of the artiﬁcial reproduction of O. mykiss .


Introduction
Reproduction is the basis of the whole ontogeny, which requires energy, ecology, anatomy, biochemistry and endocrine adaptations [1]. The obtainability of a high-quality fry and the capability to control fish breeding are the confining factors for fish culture [2]. Therefore, sufficient information related to reproductive constant, developmental biochemistry, and molecular regulation mechanism of gonadal development are very important in aquaculture.
Throughout the reproductive cycle, seasonal changes in tissue biochemical composition are related to gonadal weight, especially in females, in which the hepatic metabolism are stimulated during vitellogenesis [3]. Hence, monthly variations in the gonadosomatic index (GSI) helps to determine the breeding season of the fish, and study of the hepatosomatic index (HSI) is also important because the liver is a key organ in fish for production of vitellogenin which plays a significant role in the development of eggs [4,5]. Moreover, several studies have reported the seasonal endocrine and aromatase changes associated of the circulatory system for a month in Yunnan Agricultural University and fed twice a day to satiety. Thirty-three female O. mykiss were sampled monthly from January to November 2018 (Table 1). After a fast for 2 d, three fish were anesthetized by 100 mg/L MS-222 (tricaine methane sulfonate, Sigma, St. Louis, MO, USA) before sampling every month. Plasma was separated from blood samples after centrifuging at 3000 rpms for 15 min, and then stored at −80 • C until assayed. The liver, brain and ovary were separated from the abdominal cavity and ventricle, then collected in an RNAase-free tube and stored at −80 • C for further testing. The experimental animals used in this experiment are strictly in accordance with the requirements of the guidelines for the use of Experimental Animals of Yunnan Agricultural University, and were approved by the Experimental Ethics Committee of Yunnan Agricultural University (YNAU2017llwyh131). Table 1. Body weight, liver weight and ovary weight of female O. mykiss sampled monthly (n = 3).

Determination of HSI and GSI
In order to calculate the values of HSI and GSI, the liver and ovary were weighed immediately after decapitation. HSI was calculated using the equation: HSI = [liver weight (g)/body weight (g)] × 100%. GSI was calculated as [ovary weight (g)/body weight (g)] × 100%.

Plasma Hormones and Vitellogenin (VTG) Analyses
Cortisol, E 2, and VTG were determined by the ELISA kit, which was produced by Shanghai Enzymatic Biotechnology Co., Ltd. (Shanghai, China), according to the instructions of the kit. The Intra-assay CV(%) is less than 10% and Inter-assay CV(%) is less than 15%. The minimum detectable dose of cortisol, E 2, and VTG are typically less than 10 pg/mL, 0.1 pmol/L and 1.0 ng/mL, respectively. TH S were also determined by ELISA kit, which was produced by Beijing North Institute of Biotechnology Co., Ltd., Beijing, China, according to the instructions of the kit. The Intra-assay CV(%) and Inter-assay CV(%) is less than 15%. The minimum detection concentration is less than 10 ng/mL, 0.4 ng/mL, 4.0 pmol/L, and 2.0 pmol/L for T 4 , T 3 , FT 4 , and FT 3 , respectively. All samples were measured at 450 nm wavelength using a 96 Flat Bottom Transparent Polystyrol (Greiner BioOne, Kremsmünster, Austria). The standard curve was established to calculate the concentration of cortisol, E 2 , VTG, and TH S .

RNA Extraction and cDNA Synthesis
The total RNA of the brain, liver, and ovary of three female O. mykiss in each month were extracted using the TRIpure reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) following the manufacturer's instruction. The quality and concentration of the total RNA were detected by NanoDrop 2000c apparatus (Thermo Fisher Scientific, Waltham, MA, USA), with an A260 nm/A280 nm ratio from 1.8 to 2.1. Then 1 µg total RNA was used for reverse transcription according to the manufacturer's instruction of the TRUEscript 1st Strand cDNA Synthesis Kit user manual with gDNA Eraser (Aidlab Biotechnologies Co., Ltd., Beijing, China).

Gene Transcription
The transcription of sex-related genes during the oocyte development was detected using the quantitative real-time PCR. PCR reactions (20 µL) contained 1 µL of cDNA, 0.5 µL of each primer (10 µM), and 10 µL of 2 X TSINGKE ® Master qPCR Mix (SYBR Green I, Beijing TsingKe Biotech Co., Ltd., Beijing, China). The amplification procedure of these genes was: pre-heating at 95 • C for 3 min, followed by 40 cycles of 95 • C for 20 s, 55 • C for 20 s, and a final extension step at 72 • C for 20 s. The samples were analyzed in triplicate with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The elongation factor 1-alpha (ef1α) was used as the internal control to calculate the relative transcription of target gene by the 2 − Ct method [31]. The primers of genes were shown in Table 2.

Statistical Analysis
The Tukey method of a one-way analysis of variance (ANOVA) was used to evaluate the differences of value among the months through the SPSS 16.0 software (IBM Inc., Armonk, NY, USA). A value of p < 0.05 was considered statistically significant. All data were expressed as mean ± standard deviation (SD).

Seasonal Changes in HSI and GSI
From January to July, no monthly significant differences in the HSI were observed. However, in August, HSI significantly reduced. After that, HSI returned to the normal levels from September to November ( Figure 1A). Moreover, from January to October, no significant differences of GSI values were observed. The highest value of GSI of 4.11% was observed in November ( Figure 1B).

Statistical Analysis
The Tukey method of a one-way analysis of variance (ANOVA) was used to evaluate the differences of value among the months through the SPSS 16.0 software (IBM Inc., Armonk, NY, USA). A value of p < 0.05 was considered statistically significant. All data were expressed as mean ± standard deviation (SD).

Seasonal Changes in HSI and GSI
From January to July, no monthly significant differences in the HSI were observed. However, in August, HSI significantly reduced. After that, HSI returned to the normal levels from September to November ( Figure 1A). Moreover, from January to October, no significant differences of GSI values were observed. The highest value of GSI of 4.11% was observed in November ( Figure 1B).

Plasma Cortisol, E 2 and VTG Levels of Female O. mykiss during Oocyte Development
Plasma cortisol levels of females gradually increased from January to July (354.02 ± 19.60~485.85 ± 10.65 pg/mL). Then, cortisol levels decreased rapidly in August (423.58 ± 22.31 pg/mL) and increased again in September (594.65 ± 7.05 pg/mL) and remained at a high level until November (Figure 2A). Plasma E 2 levels of females fluctuated from January to July (5.80 ± 0.07~12.00 ± 0.45 pmol/L) and peaked in May. E 2 levels then increased rapidly in September (13.05 ± 0.77 pmol/L) and decreased again from October and November ( Figure 2B). Plasma VTG levels of females fluctuated and were low in January to July (65.56 ± 3.95~97.96 ± 5.70 ng/mL), increased in September and reached the peak (126.09 ± 1.66 ng/mL). VTG levels then decreased rapidly in October and remained at a low level until November ( Figure 2C). January to July (5.80 ± 0.07~12.00 ± 0.45 pmol/L) and peaked in May. E2 levels then increased rapidly in September (13.05 ± 0.77 pmol/L) and decreased again from October and November ( Figure 2B). Plasma VTG levels of females fluctuated and were low in January to July (65.56 ± 3.95~97.96 ± 5.70 ng/mL), increased in September and reached the peak (126.09 ± 1.66 ng/mL). VTG levels then decreased rapidly in October and remained at a low level until November ( Figure 2C).

Plasma THS Levels of Female O. mykiss during Oocyte Development
Plasma concentrations of both T4, FT4, T3, and FT3 were determined in female O. mykiss during the oocyte development ( Figure 3). Plasma T4 levels remained at around 30 ng/mL from January to May, and then significantly increased in June. The highest plasma T4 levels were observed in August (122.59 ± 15.08 ng/mL). After that, T4 levels gradually declined from September to November ( Figure 3A). Plasma T3 levels of females fluctuated throughout the year. From January to April, T3 levels increased first and then decreased (1.01 ± 0.05~1.74 ± 0.23 ng/mL). After that, plasma T3 remained elevated from May and reached the peak in June (2.24 ± 0.10 ng/mL). However, T3 concentrations continued to decrease from July to November ( Figure 3B). The changes in FT4 levels in the plasma were similar to T4. Plasma FT4 increased in June and reached peak levels by the beginning of July (8.69 ± 0.21 pmol/L), and then maintained a high level until October (10.80 ± 0.22~10.97 ± 1.00 pmol/L) ( Figure 3C). Then, a significant decrease was observed in November. Similar to T3, FT3 levels fluctuated irregularly throughout the year. The highest level of FT3 appeared in October (9.48 ± 0.48 pmol/L) ( Figure 3D). Different letters denote statistically significant differences among the groups (Tukey method of the one-way ANOVA test, p < 0.05).

Plasma TH S Levels of Female O. mykiss during Oocyte Development
Plasma concentrations of both T 4 , FT 4 , T 3, and FT 3 were determined in female O. mykiss during the oocyte development ( Figure 3). Plasma T 4 levels remained at around 30 ng/mL from January to May, and then significantly increased in June. The highest plasma T 4 levels were observed in August (122.59 ± 15.08 ng/mL). After that, T 4 levels gradually declined from September to November ( Figure 3A). Plasma T 3 levels of females fluctuated throughout the year. From January to April, T 3 levels increased first and then decreased (1.01 ± 0.05~1.74 ± 0.23 ng/mL). After that, plasma T 3 remained elevated from May and reached the peak in June (2.24 ± 0.10 ng/mL). However, T 3 concentrations continued to decrease from July to November ( Figure 3B). The changes in FT 4 levels in the plasma were similar to T 4 . Plasma FT 4 increased in June and reached peak levels by the beginning of July (8.69 ± 0.21 pmol/L), and then maintained a high level until October (10.80 ± 0.22~10.97 ± 1.00 pmol/L) ( Figure 3C). Then, a significant decrease was observed in November. Similar to T 3 , FT 3 levels fluctuated irregularly throughout the year. The highest level of FT 3 appeared in October (9.48 ± 0.48 pmol/L) ( Figure 3D).

Seasonal Changes in the Levels of Female O. mykiss Brain Genes during Oocyte Development
The transcription levels of genes in the brain were examined through an areal-time PCR assay (Figure 4). The transcription of cyp19b did not fluctuate significantly from January to July. However, a significant decrease was detected in August and September. From October to November, cyp19b levels gradually raised and then peaked in November ( Figure 4A). The levels of cgnrh-2 were low in January to August, and increased significantly in September and October in association with active vitellogenesis. In November, the cgnrh-2 level decreased to the similar levels of January to August ( Figure 4B). Moreover, similar to cgnrh-2, the levels of gnrhr were low from January to August. After that, the levels of gnrhr substantially increased in September and October, and the level in October was significantly higher than those in other months. The transcription of gnrhr significantly decreased in November ( Figure 4C). The transcription levels of fsh were low from January to August. However, the levels of fsh fluctuated irregularly from September to November, showing a transcription trend that decreased first and then increased. The transcription peaks of fsh were observed in September and November ( Figure 4D).  Different letters denote statistically significant differences among the groups (Tukey method of the one-way ANOVA test, p < 0.05).

Seasonal Changes in the Levels of Female O. mykiss Brain Genes during Oocyte Development
The transcription levels of genes in the brain were examined through an areal-time PCR assay (Figure 4). The transcription of cyp19b did not fluctuate significantly from January to July. However, a significant decrease was detected in August and September. From October to November, cyp19b levels gradually raised and then peaked in November ( Figure 4A). The levels of cgnrh-2 were low in January to August, and increased significantly in September and October in association with active vitellogenesis. In November, the cgnrh-2 level decreased to the similar levels of January to August ( Figure  4B). Moreover, similar to cgnrh-2, the levels of gnrhr were low from January to August. After that, the levels of gnrhr substantially increased in September and October, and the level in October was significantly higher than those in other months. The transcription of gnrhr significantly decreased in November ( Figure 4C). The transcription levels of fsh were low from January to August. However, the levels of fsh fluctuated irregularly from September to November, showing a transcription trend that decreased first and then increased. The transcription peaks of fsh were observed in September and November ( Figure 4D).

Seasonal Changes in the Levels of Female O. mykiss Gonadal Genes during Oocyte Development
The real-time PCR was further used to validate the differentially expressed gonadal genes during seasonal changes ( Figure 5). The transcription levels of fshr did not change significantly during the period from January to May, followed by a gradually increase from June to September, which reached the highest levels in September. In October and November, the transcription of fshr also remained at a high level ( Figure 5A). Different from fshr, the levels of lhr transcription were low from January to September, and then

Seasonal Changes in the Levels of Female O. mykiss Gonadal Genes during Oocyte Development
The real-time PCR was further used to validate the differentially expressed gonadal genes during seasonal changes ( Figure 5). The transcription levels of fshr did not change significantly during the period from January to May, followed by a gradually increase from June to September, which reached the highest levels in September. In October and November, the transcription of fshr also remained at a high level ( Figure 5A). Different from fshr, the levels of lhr transcription were low from January to September, and then gradually increased and reached its peak in November ( Figure 5B). Transcripts of cyp19a1a remained at the same levels from January to July, and then significantly increased from August to November. The transcription peak of cyp19a1a was observed in November ( Figure 5C). The levels of foxl2 transcription were low from January to July, and significantly increased from August and reached the maximum in September. After that, the transcription of foxl2 gradually decreased to the same level in August ( Figure 5D).
According to the real-time PCR data, erα1 and erα2 were expressed at low levels from January to August and then became significantly high in September, followed by a gradually decrease from October to November ( Figure 5E,F). However, irregular fluctuations of the erβ1 transcription were observed from January to August, which showed transcription peaks in September ( Figure 5G). Similar to erα1 and erα2, the erβ2 transcription remained at the same levels from January to August, and then significantly increased in September. After that, the transcription of erβ2 gradually decreased to the same level in January to August ( Figure 5H).

Seasonal Changes in the Levels of Female O. mykiss Liver Genes during Oocyte Development
Both the erα1 and erα2 gene transcription profile showed the lowest transcription levels from January to July, then a significant elevation of erα1 transcription was observed in November, but erα2 transcription increased sharply between October and November ( Figure 6A,B). There was no significant change in erβ1 transcription during the annual cycle. The erβ2 transcription was detected in the liver during the annual cycle with low levels of transcription, except for the October, in which the transcription level was significantly higher compared with January to July ( Figure 6C,D).

Seasonal Changes in the Levels of Female O. mykiss Liver Genes during Oocyte Development
Both the erα1 and erα2 gene transcription profile showed the lowest transcription levels from January to July, then a significant elevation of erα1 transcription was observed

Seasonal Changes in the Levels of Female O. mykiss vtg Gene during Oocyte Development
Transcription levels of vtg in the ovary were low from January to August, and its levels increased significantly from September to November when a peak was reached in November ( Figure 7A). The vtg transcription levels in the liver increased significantly from August to November and reached a peak in November ( Figure 7B).
in November, but erα2 transcription increased sharply between October and November ( Figure 6A,B). There was no significant change in erβ1 transcription during the annual cycle. The erβ2 transcription was detected in the liver during the annual cycle with low levels of transcription, except for the October, in which the transcription level was significantly higher compared with January to July ( Figure 6C,D).

Seasonal Changes in the Levels of Female O. mykiss vtg Gene during Oocyte Development
Transcription levels of vtg in the ovary were low from January to August, and its levels increased significantly from September to November when a peak was reached in November ( Figure 7A). The vtg transcription levels in the liver increased significantly from August to November and reached a peak in November ( Figure 7B).

Seasonal Changes in the Levels of Female O. mykiss vtg Gene during Oocyte Development
Transcription levels of vtg in the ovary were low from January to August, and its levels increased significantly from September to November when a peak was reached in November ( Figure 7A). The vtg transcription levels in the liver increased significantly from August to November and reached a peak in November ( Figure 7B).

Discussion
O. mykiss is an iterative, oviparous fish with a synchronized set of annual reproductive cycles. In the whole reproductive cycle, GSI and HSI may help to identify the breeding period or reproductive peak of fish, and further reflect the ovarian maturation time and spawning time of O. mykiss. In S. Gairdneri, GSI elevated gradually from June to August, increased significantly from September to December and reached a peak in December, and then decreased again from January to May. Similarly, HSI values increased gradually from March to December, and then decreased again in January and February [35]. Therefore, it showed that the ovarian maturation and spawning period of S. Gairdneri is in December. In this study, GSI increased gradually from August to November and peaked in November, while HSI was the lowest in August. When HSI decreases and GSI increases, it indicates that the liver loses weight during reproduction, and the VTG synthesized by the liver is transported to the gonads to promote gonadal maturation [36]. Therefore, the period of rapid gonadal development of O. mykiss is from August to November, and November is the most suitable breeding season for this species.
Steroid hormones such as testosterone, estradiol, progesterone, and corticosteroids play a key role in the reproductive process of fish. These hormones act on the reproductive process of fish directly or through feedback mechanisms. In female fish, E 2 is necessary for ovarian development and controls egg maturation, ovulation, and egg laying. Meanwhile, circulating E 2 regulates the transcription of vtg in hepatocytes, leading to the synthesis of several closely related VTG proteins [37]. In this study, the plasma levels of cortisol, E 2 and VTG in female O. mykiss changed significantly during oocyte development. Especially in the pre-oviposition stage, the level of E 2 in plasma increased, the yolk generation was active, and then the level of VTG in plasma increased subsequently. In the study of catfish (Silurus asotus), the levels of E 2 and plasma cortisol increased during the period of yolk generation and spawning [38]. Female O. mykiss had a low gonad E 2 synthesis January to August. However, the plasma E 2 content increased significantly in September, indicating that the ovarian development of female O. mykiss was the fastest in September in this study. Similarly, the plasma VTG content of female O. mykiss also has a similar trend with E 2 . The content of VTG has a gradually increasing trend from January to June. In September, the plasma VTG content increased significantly, indicating that the synthesis of VTG is the largest in September, followed by the size of the oocyte, which increases rapidly. In addition, the trend of female O. mykiss plasma cortisol and VTG is consistent, reaching a peak in September, and then decreasing. Therefore, the trend of plasma cortisol is consistent with E 2 and VTG, indicating that cortisol is also involved in the development of female O. mykiss oocytes in this study. During the reproductive cycle, plasma cortisol levels also increase from spawning or spawning periods, such as in the plaice (Pleuronectes platessa) [39], two species of trout, Salmo trutta L. and S. gairdneri [40], O. mykiss [41]. Therefore, in O. mykiss reproduction, the increase in the plasma cortisol level during reproductive periods may play a key role in ovarian growth and vitellogenesis in addition to stress [42,43].
In fish species, gonadal steroids can also affect circulating levels of thyroid hormone and/or thyroid activity [18,19]. TH S include T 3 , T 4 , FT 3, and FT 4 [44]. Thyroids participate in the physiologic stress response envisaged chiefly because of the involvement in TH S in almost all aspects of the physiologic processes [45][46][47]. The capability of the thyroid axis is to respond to the stimuli which arise from the other factors that engage in its cross talk [48]. Therefore, thyroid hormones have a wide range of effects on the development, growth, and reproduction of fish [20,21]. These effects are also passed on from generation, because TH S play a pivotal role in the early development of offspring [21,49]. The plasma TH S levels of many fishes show periodic changes. These hormones are related to sexual maturity [50,51] and reproductive cycles [52,53]. In this study, plasma concentrations of TH S were determined in female O. mykiss during the year of oocyte development. Plasma TH S levels increased in August and decreased in November before spawning. This is similar to the study of two strains of rainbow trout (O. mykiss Shasta and Kamloops), the levels of FT 3 and FT 4 , T 3 and T 4 were the lowest during the spawning period, and the circulating TH S showed similar seasonal changes [54]. These results indicated that TH S participated in the regulation of yolk accumulation in rainbow trout.
With the change in seasons, the HPG axis plays an important role in oocyte development of O. mykiss. GnRH, synthesized and released by the hypothalamus, is a key hormone that regulates reproduction [55]. In salmonid fishes, two forms of GnRH, sgnrh-1 and cgnrh-2, have been detected [15]. GnRH is conveyed to the pituitary via the hypothalamohypophyseal portal vessels, and regulates synthesis and release of GtH [56]. In the process of signal transduction, ligands (cgnrh-2, fsh, lh) bind and interact with their receptors (gnrhr, fshr, lhr) distributed in target cells to achieve their physiological functions [57]. In this study, the transcription level of cgnrh-2 and gnrhr gradually increased from August to October. Moreover, a previous study showed that the transcription of fshr is associated predominantly with vitellogenesis, while the lhr is prevalent during oocyte maturation and ovulation [57]. In female greater amberjack (Seriola dumerili) [58], ovarian fshr and plasma E 2 gradually increased during ovarian development, which suggested that fsh plays a role in ovarian development and during the post-spawning period [58]. In this study, the ovarian fsh, fshr of O. mykiss increased continuously from August to September and decreased from September to October, but fsh increased significantly again in November. Similarly, the ovarian lhr increased continuously from August to November, and the peak was in November. Therefore, the transcription of fsh and lh plays a crucial role in the oocyte development of O. mykiss and has seasonal correlation. In addition, GnRH can act as a neuromodulator, and administration of exogenous GnRH facilitates sexual behavior in many species [59]. Therefore, cgnrh-2 can affect the transcription of fsh and lh through gnrhr, and then participate in the regulation of E 2 synthesis in O. mykiss.
Aromatase is a key enzyme in the synthesis of estrogen, also known as estrogen synthetase. It catalyzes the conversion of testosterone and androstendione to estrone and estradiol in animals, and converts androgen to estrogen. Therefore, aromatase shows its regulatory role in the early differentiation of female gonadal gland in nonmammalian vertebrates [60]. The cyp19 encodes cytochrome P450 aromatase. Two cyp19 genes, cyp19a and cyp19b, which belong to two independent CYP19 subfamilies, were identified. The cyp19a is transcribed in the ovary, while the cyp19b gene is transcribed in the brain [61]. The transcription of the O. mykiss cyp19b is consistent with the gonadal development period. From January to July, the transcription of the O. mykiss cyp19b is at a high level. The cyp19b promotes the release of estrogen and gonadotropin. Then, the transcription of the O. mykiss cyp19b decreased from August, and significantly increased until the gonad maturation process. These results indicate that cyp19b is based on the transcription of the brain, which regulates the final maturation and ovulation of O. mykiss in November.
The foxl2 is the fork transcription factor in the process of the ovarian development [62]. As shown in previous studies, the foxl2 transcription is a genetic factor that activates the transcription of aromatase [63]. In the O. mykiss gonadal development of this study, the transcription of foxl2 reached its peak in September, decreased gradually from October to November, and decreased the lowest in November. Therefore, foxl2 may have a regulatory relationship with cyp19a1a, and the transcription of cyp19a1a gradually increases from August to November and peaked in November. A previous study also revealed that cyp19a1a and cyp19a1b were co-expressed with foxl2, except in the early yolk stage of the ovary [24]. In summary, during fish gonadal development, the highest transcription level of foxl2 was earlier than cyp19a1a, suggesting that foxl2 is involved in fish gonadal differentiation and the maintenance of ovarian function [64].
In addition, estrogen regulates ovarian development, differentiation, and maintenance, as well as oogenesis, and stimulates liver synthesis of vtg and choriogenin [29,30]. These effects are principally mediated through ers which belong to the nuclear hormone receptor superfamily. After binding of a ligand to the ligand-binding domain (LBD) of ERS, this complex binds as homo dimer to estrogen response elements (EREs) in the promoter regions of estrogen responsive target genes and regulates their transcription [65]. Four nuclear estrogen receptor genes were identified in O. mykiss: erα1, erα2, erβ1, and erβ2 [28]. Recent knockout studies in goldfish (Carassius auratus Linnaeus) and zebrafish (Danio rerio) have shown that erβ1 and erβ2 are necessary for the estrogen-mediated up-regulation of erα and vtg transcription [66,67]. The vtg was also positively correlated with the ers subtype [29,30]. In this study, under the effect of estrogen in female O. mykiss, the transcription of ers in the ovary increased significantly in September, and decreased gradually from September to November. Similarly, the transcription of vtg in the ovary increased gradually in August to November and peaked in the November. As an important exogenous organ, the transcription of ers and vtg in the liver also increased with the change in season. These results showed that the increasing of estrogen level and its receptor genes transcription, as well as vtg, participated in the accumulation of vitelloprotein during female O. mykiss ovarian development with seasonal changes. Therefore, cortisol, E 2 , TH S can interfere with the transcription of sex-related genes on the HPG axis, thereby promoting the transcription of vtg genes in the ovary and liver, increasing the plasma VTG levels, and finally leading to the reproduction of O. mykiss in November.

Conclusions
In conclusion, this experiment studied the periodic seasonal changes in plasma hormones, and sex-related genes transcription in the brain, liver, and ovary during the gonadal development of female O. mykiss under the regulation of HPG axis. The plasma hormones gradually increased from January to August, and peaked from August to October, which promotes the synthesis of VTG, and then accelerated the development and maturation of oocytes in O. mykiss. Similarly, the transcription level of sex-related genes in the HPG axis increased significantly since August and maintained a high level until November, which affected the synthesis of estrogen, and then participated in the regulation of gonadal development. In addition, estrogen can affect the transcription of vtg by increasing the ers transcription, thereby regulating the synthesis and accumulation of yolk in O. mykiss oocytes, and ultimately affects the value of GSI in November. Therefore, these sex-related hormones and genes can accelerate the synthesis and accumulation of yolk by affecting the HPG axis from August, and reach a peak in November to promote the spawning and reproduction of O. mykiss. These results strongly proved that November is the most suitable season for O. mykiss breeding in Yunnan.