| Generally, in teleost fish, production of mono-sex population and investigation of sex-determining mechanism supplement each other. The results during the mono-sex production will be beneficial for the investigation of sex-determining mechanism, meanwhile clear sex-determining mechanism will provide theoretical guide for sex control. The diversity of sex-determining mechanism and lability of sex differentiation of fish are theoretical basis and prerequisite for mono-sex production in the fish species which display sexual specificity, e.g. different in growth, ornamental value, gonadal value, and reproductive value. Applying three mating strains (normal XY male, sex reversed XY female, and YY super-male) produce all-male population is the best example of using the knowledge of sex-determining mechanism to produce mono-sex population. Primitiveness (heteromorphic and homomorphic sex chromosomes), diversity (genetic sex determination and temperature-dependent sex determination, gynogenesis, hermaphrodite and gonochorism), and lability (affected by temperature, exogenous hormones, and other environmental factors) of sex-determining mechanism in fish highlight the importance of fish in the research field of sex-determining mechanism evolution, sexual selection, population sex ratio adaptability in vertebrate. In the other hand, sex control is one of the most important methods to study sex-determining mechanism. For example, sex reversal induction by exogenous hormones, hormonogenic enzyme inhibitor (aromatase inhibitor), antagonist of sex hormone receptor, and temperature etc., are unexceptionable approach to investigate the function of sex hormones or temperature conditions in the processes of sex determination and sex differentiation, and are important method to explore the sex-determining genes, sex differentiation related genes, and molecular pathway in sex determination. Meanwhile, offspring sex ratio result by mating sex reversed individual (e.g. phenotypic female) and normally developed, opposite phenotypic sex individual (e.g. male) is important approach to preliminarily distinguish the type of sex chromosome (e.g. XX/XY, ZW/ZZ). Sex inversion induced by temperature treatment is the single way to differentiate temperature-dependent sex determination, genetic sex determination, and genetic sex determination plus temperature effects.The present dissertation are mainly focus on the sex control and sex determination mechanism in yellow catfish and bluegill sunfish. Yellow catfish is a popular and economically important fish species in China, but in other Asian countries, e.g. North Korea, Japan, and Vietnam as well. Bluegill sunfish is one of the most popular sport fish in North America, as well as one of the five aquaculture species in northern Unite State; as important food fish, it has been introduced into China. The growth pattern of yellow catfish and bluegill shows sexual dimorphism, that the males grow faster and reach bigger ultimate size than females. Both of them reach sexual maturation early and could propagate in rearing conditions. Bluegill, especially, could reproduce multiple times during spawning season in a single year. Plasticity or lability of phenotypic sex allow us to produce mono-sex population of fish. A series of problems in large-scale industry of yellow catfish and bluegill, e.g. early sexual maturation, high reproductive ability, high density, slow growth rate, and high size variation could be solved theoretically and practically by mono-sex production. Bluegill sunfish display extraordinary sex determination mechanism and reproductive biology, e.g. coexist of genetic sex determination and temperature-dependent sex determination, displaying alternative mating tactics (two types of male:parental males and cuckolder males).In the present study, we investigated the effects of different concentrations of two masculinizing chemicals on sex reversion of yellow catfish; base on previous results of sex control in bluegill, and in the background of complicated sex-determining mechanism in bluegill, we clarified temperature-dependent sex determination in bluegill, and studied sex differentiation related genes prior to and during sex differentiation in different rearing temperatures. Main results are shown as follows.(1) Effects of17a-methyltestosterone or aromatase inhibitor Letrozole on masculinization in yellow catfishThe masculinization of yellow catfish (Pelteobagrus fulvidraco) by oral administration of various doses of17a-methyltestosterone (MT) and aromatase inhibitor letrozole (LZ) was investigated and their effects on the survival, growth performance, sex ratio and changes of gonadal structure were evaluated. Three doses of MT treatments from10days post-hatching (DPH) to59DPH failed to alter the male percentage; however, a certain proportion of intersex were detected in all MT treatments. Growth performance in MT treatments was depressed significantly compared with control group in a dose-dependent manner at the end of the masculinization experiment, and the suppression effect discontinued45days after the termination of treatments. LZ treated groups produced75.0%,83.3%, and75.0%male respectively, which were significantly higher than the control (37.5%). Low dose LZ treatment significantly advanced growth performance compared with the control, with no advancement in two high-dose treatments. Enlarged lobule lumens and a large amount of spermatozoa in male histological sections suggested the promoting potential of testicular development in early gonad development stage following the treatments of LZ in male yellow catfish. The changes of sex ratios after the termination of LZ treatments indicated that female germ cells possess some degree of bipotentiality. The present study demonstrated that LZ possesses the ability of masculinization in yellow catfish, and LZ treatment at low levels could promote growth performance and advance testicular development. Oral MT administration (from20to100mg kg-1) did not reverse the sex from phenotypic female to male, but repressed growth during treatments.(2) Effects of non-steroidal aromatase inhibitor Letrozole on sex inversion in yellow catfishThe effects of Letrozole (LZ), a potent non-steroidal aromatase inhibitor (AI), on growth performance, sex inversion and sex changes after the termination of the treatments were investigated in yellow catfish which displays sexual dimorphic growth. Growth performance was promoted significantly in the low dose LZ treatment compared with the control. Four LZ treatments produced dose-dependent male proportions, which were significantly higher than the control. Histological examination of testes treated by LZ displayed a large amount of spermatozoa and enlarged lobule lumens, indicating that LZ treatments have the stimulation potential in the process of spermatogenesis. Changes of sex proportions45days after the end of the LZ treatment prove that the female germ cells possess a certain extent of bipotentiality. These results suggest that aromatase activity plays a vital role in sex differentiation with inhibition of aromatase activity by AI bringing about sex inversion.(3) Temperature-dependent sex determination in bluegill sunfishIncreasing evidence shows that temperature effects on sex ratio in fish species are ubiquitous. In the present study, effects of genotype-temperature interactions on sex determination in bluegill sunfish (Lepomis macrochirus) were investigated using four geographic strains, Hebron, Jones, Hocking, and Missouri. Two opposite sensitive response patterns of sex ratio to temperature and non-sensitive pattern were found in a single fish species for the first time. In the Hebron strain, higher temperature treatment groups (24℃and32℃) produced more males compared with the low temperature group (17℃) from6days post-hatching (dph) to90dph. On the contrary, low temperature treatment produced more males than that of the other two higher temperature treatments in the Jones strain during the same treatment period as Hebron strain. No significant effects of temperature on sex ratio were detected in the other two strains. Combining sex ratio and survival data, it is strongly suggested that both temperature-dependent sex determination and genetic sex determination exist in this fish species, meanwhile, genotype-temperature interactions influence sex determination in bluegill. Therefore, a consumer-and environment-friendly approach to significantly increase the proportion of males could be achieved through selection of temperature sensitivity in bluegill. The evolutionary implications of our findings on sex determining mechanisms were discussed. Results suggested bluegill could be a unique candidate model species for investigations of sex-determining mechanisms and evolution of sexual selection.(4) Expression of foxl2during early gonadal development in bluegill sunfishMany genes associated to sex differentiation express prior to the first sign of morphological sex differentiation. Expression of some of these genes are sex specific. Investigation of these genes allow us to develop molecular markers for identification of individual sex or population sex ratio in early development stage, and will unveil molecular mechanisms of sex differentiation. We investigated the expression profiles of ovary differentiation related genes foxl2and cyp19a1a, as well as testis differentiation related gene dmrtl during early gonadal development. Results show that these sex differentiation related genes expressed as early as7days post-hatching (dph). The mRNA level of foxl2increased sharply during7to17dph, and kept high during17to27dph, then decreased slightly thereafter, suggesting ovary differentiation of bluegill occurred between7and27dph. The expression of foxl2displayed huge individual difference, indicating this gene could be a molecular marker for identification of sex. The expression of foxl2was modulated by temperature on27dph of bluegill, indicating that this gene may involve in temperature induced sex reverse.We studied the effects of two masculinizing chemicals on sex inversion, and optimized the concentrations, produced all-male population of yellow catfish. Results of the present dissertation will provide guidance for mono-sex production, be beneficial for further clarification of sex-determining mechanisms of yellow catfish, as well as increase content of sex-determining mechanism in teleost fish. We also found that female germ cells of yellow catfish possess a certain degree of bipotentiality after the completion of sex differentiation, which will offer new ideas and guidance for future mono-sex production.We investigated temperature-dependent sex determination mechanism in bluegill sunfish, analyzed expression profiles of sex differentiation related genes. We found different response patterns of sex ratio to temperature in a single fish species. These results will enrich sex-determining mechanism of bluegill, provide materials and new research field for temperature-dependent sex determination of fish, and be a reference for environment-and customer-friendly mono-sex production. |
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