EFFECTS OF PHOTOPERIOD ON MORPHOLOGY, SEX HORMONE LEVELS AND GENE EXPRESSION OF GONADAL DIFFERENTIATION OF BLACK ROCKFISH (SEBASTES SCHLEGELII)
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摘要: 研究以35日龄(dpb)许氏平鲉(Sebastes schlegelii)仔稚鱼为对象, 研究不同光周期(短光照组L﹕D=8﹕16、长光照组L﹕D=16﹕8和对照组L﹕D=12﹕12)对性别分化、相关激素水平及基因表达水平的影响。结果显示非自然的光周期尤其是较短的光照, 会不同程度地影响性腺分化时期性腺发育程度, 并且短光照会导致部分性腺雄性化; 雌激素(E2)在短光照组中更早出现峰值, 而雄激素(T)在3个处理组中均在实验第9天时达到峰值; 4个卵巢分化相关基因cyp19a1a、ERα、ERβ2和foxl2中, ERα、ERβ2和foxl2受短光照影响显著, 实验中后期出现明显的抑制(P<0.05); 4个精巢发育相关基因sox3、sox9、amh和dmrt1相对表达水平未见明显规律, 可能与精巢分化时间较晚有关。综合而言, 较短的光照会影响性腺的发育以及性腺的分化, 抑制卵巢分化基因的表达, 诱导原始性腺雄性化。Abstract: Genetic sex determination (GSD) along with environmental sex determination (ESD) are two crucial sex determination mechanisms. This study investigated gonadal differentiation mechanisms under 3 different photoperiod conditions (long light photoperiod: L﹕D=8﹕16; short light photoperiod: L﹕D=16﹕8; control: L﹕D=12﹕12) of black rockfish (Sebastes schlegelii) larvae, which are demersal at coast in the mid and high latitudes, at 35 dpb (day post birth). The result showed that the unnatural photoperiod conditions impacted the gonadal differentiation and development with the short light photoperiod condition to induce gonad masculinization. Estrogen level (E2) first reached the peak value in the short light photoperiod condition, while androgen (T) level reached the peak value at 9d after treatment in all three conditions. The short light photoperiod condition significantly reduced the expression of the ovary differentiation related genes such as ERα, ERβ2 and foxl2 (P<0.05). Consider that the testis differentiation time was relatively late, the testis differentiation related genes of sox3, sox9, amh, and dmrt1 did not impact by photoperiod. In conclusion, short photoperiod could affect the gonad differentiation and development by restrain the ovary differentiation related genes expression and induce primitive gonad masculinization.
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Keywords:
- Gonad differentiation /
- Photoperiod /
- Sex hormone /
- Gene expression /
- Black rockfish
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图 1 不同光周期下第0天许氏平鲉的性腺组织切片
A. 短光照(L﹕D=8﹕16)组卵巢; B. 短光照组精巢; C. 对照(L﹕D=12﹕12)组卵巢; D. 对照组精巢; E. 长光照(L﹕D=16﹕8)组卵巢; F. 长光照组精巢; 比例尺=50 μm; MD. 中肾管; BV. 血管; OC. 卵巢腔; O. 卵巢; T. 精巢; I. 肠
Figure 1. The gonads histologic section of Sebastes schlegelii at day 0 under the different photoperiod conditions
A. Ovarian under the short light photoperiod; B. Testis under the short light photoperiod; C. Ovarian under the control condition; D. Testis under the control condition; E. Ovarian under the long light photoperiod; F. Testis under the long light photoperiod. Scale=50 μm. MD. mesonephric ducts; BV. blood vessel; OC. ovarian cavity; O. ovarian; T. testis; I. intestines
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图 2 不同光周期下第21天许氏平鲉的性腺组织切片
A. 短光照组卵巢; B. 短光照组精巢; C. 对照组卵巢; D. 对照组精巢; E. 长光照组卵巢; F. 长光照组精巢; 比例尺=50 μm; MD. 中肾管; BV. 血管; SG. 精原细胞; OC. 卵巢腔; OG. 卵原细胞; O. 卵巢; SD. 输精管; T. 精巢; I. 肠; GC. 生殖细胞
Figure 2. The gonads histologic section of Sebastes schlegelii at day 21 under the different photoperiod conditions
A. Ovarian under the short light photoperiod; B. Testis under the short light photoperiod; C. Ovarian under the control condition; D. Testis under the control condition; E. Ovarian under the long light photoperiod; F. Testis under the long light photoperiod. Scale=50 μm. MD. mesonephric ducts; BV. blood vessel; SG. spermatogonia; OC. ovarian cavity; OG. Oogonia; O. ovarian; SD. sperm ductus; T. testis; I. intestines; GC. germ cell
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图 3 不同光周期对许氏平鲉仔稚鱼E2(A)、T(B)水平的影响
图中标注的小写字母表示同一时间点不同光周期间存在显著性差异(P<0.05), 大写字母表示同一光周期不同时间点间存在显著性差异(P<0.05); 下同
Figure 3. Effects of the E2 (A) and T (B) level of Sebastes schlegelii larvae under different photoperiod condition
Lowercase letters mean significant difference between different photoperiod at the same time (P<0.05), and capital letters means significant difference between different time at the same photoperiod (P<0.05); the same applies below
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图 4 不同光周期下许氏平鲉卵巢分化相关基因mRNA相对表达水平(A、B、C和D分别为cyp19a1a、 ERα、ERβ2和foxl2 mRNA相对表达水平)
Figure 4. The relative mRNA expression level of ovary differentiation related genes in Sebastes schlegelii under different photoperiod condition (A, B, C and D showed the relative mRNA expression level of cyp19a1a, ERα, ERβ2 and foxl2, respectively)
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图 5 不同光周期下许氏平鲉精巢分化相关基因mRNA相对表达水平(A、B、C和D分别为sox3、sox9、amh和dmrt1 mRNA的相对表达水平)
Figure 5. The relative mRNA expression level of testis differentiation related genes in Sebastes schlegelii under different photoperiod condition (A, B, C and D showed the relative mRNA expression level of sox3, sox9, amh and dmrt1, respectively)
表 1 基因表达分析的特异性引物
Table 1 Primer sequences for mRNA expression analysis
引物名称Primers 核苷酸序列Sequence (5′—3′) cyp19a1a F: ACCTGGACAGTTTGGCTCA R: GAGGCAGTCCAAGGTAAGG ERβ2 F: GCCACGGACCTCTAACCT R: GACTTCCTCCTCTGACCTCTT foxl2 F: CAAGAAAGGTTGGCAGAA R: GTAGGACATCGGAGTGGG ERα F: CCTGACGCAGAGCAAGCA R: GAGTCCAGAGGAGCAGAGTA sox9 F: AGAGGCTCCTTCCATCCG R: AGCACGCCAGACTCAGAC amh F: AGCCTCACCGTGTCCCTA R: TCCCGTGTTCGTTCAAGTC sox3 F: CTGCTGCGTGGTGGTGTA R: CCTCATCAGCACCATAGCA dmrt1 F: TGGAGGCTATCATCACGG R: CAGGCAACTCTTCATTCTTA β-actin F: TGGAGGCTATCATCACGG R: CAGGCAACTCTTCATTCTTA 
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