用模式生物青鳉概观硬骨鱼性别决定及性分化研究进展

鱼类性别决定和性分化呈多元性,既有雌雄同体也有雌雄异体。性腺的雌雄性分化过程受遗传和环境因素(如温度、光照、激素和pH值等)影响,具有可逆可塑性。随着生物技术和基因组学的迅速发展,近年来脊椎动物性别决定和性分化的研究有了重大进展和显著突破。本文通过聚焦青鳉及其他硬骨鱼纲保守存在的dmrt1、gsdf、amh等雄性因子,探讨硬骨鱼普遍存在的雌雄性别可塑可逆信号通路,并介绍了新的基因组编辑和性控育种技术,为单性选育等水产养殖技术的研发提供参考。     

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.     

Expression pattern of anti-Müllerian hormone (amh) in the hybrid fish complex of Squalius alburnoides

In fish of the Squalius alburnoides complex, hybridisation and polyploidy have affected sex ratios, resulting in strong correlations between sex and genotype. The preponderance of females among triploids and the occurrence of an all male lineage among diploids seem to imply that sex ratio deviations should have a strong genetic basis. Until now, no information has been gathered regarding the molecular basis of sex determination in this intricate hybrid system. Thus, putative regulatory elements of the cascade that potentially are involved in sex determination in S. alburnoides have to be investigated. Being reported to have an important role in teleost sex determination, and more particularly in male gonad development, the anti-Müllerian hormone, amh was a good initial candidate. Here we report the isolation, cloning and characterization of the amh ortholog in S. alburnoides and the ancestral species S. pyrenaicus. In adult S. alburnoides and S. pyrenaicus of both sexes, amh shows a gonad specific expression pattern, restricted to the Sertoli cell lineage in testis and to granulosa cells in ovaries. During development, it plays an early role in male gonad differentiation in S. alburnoides. Overall the observed patterns are similar to what has been reported in other teleost species. This suggests a conserved role of amh and implies that its expression dynamics cannot be directly responsible for the sex ratio deviations reported in S. alburnoides. It is possible that a conjunction of other factors could be contributing for sex ratio imbalance. The present results constitute the starting point in the characterization of the S. alburnoides sex determination cascade, a process that we expect to shed some light on the molecular basis of sex distribution, within the context of hybrid system evolution.     

Cross talk between germ cells and gonadal somatic cells is critical for sex differentiation of the gonads in the teleost fish, medaka (Oryzias latipes)

To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka ( Oryzias latipes ), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei , with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.     

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.     

Female‐specific increase in primordial germ cells marks sex differentiation in threespine stickleback (Gasterosteus aculeatus)

Gonadal sex differentiation is increasingly recognized as a remarkably plastic process driven by species‐specific genetic or environmental determinants. Among aquatic vertebrates, gonadal sex differentiation is a frequent endpoint in studies of endocrine disruption with little appreciation of underlying developmental mechanisms. Work in model organisms has highlighted the diversity of master sex‐determining genes rather than uncovering any broad similarities prompting the highly conserved developmental decision of testes versus ovaries. Here we use molecular genetic markers of chromosomal sex combined with traditional histology to examine the transition of the bipotential gonads to ovaries or testes in threespine stickleback (Gasterosteus aculeatus). Serially‐sectioned threespine stickleback fry were analyzed for qualitative and quantitative indications of sexual differentiation, including changes in gonadal morphology, number of germ cells and the incidence of gonadal apoptosis. We show that threespine stickleback sampled from anadromous and lacustrine populations are differentiated gonochorists. The earliest sex‐specific event is a premeiotic increase in primordial germ cell number followed by a female‐specific spike in apoptosis in the undifferentiated gonad of genetic females. The data suggest that an increase in PGC number may direct the undifferentiated gonad toward ovarian differentiation. J. Morphol., 2008. © 2007 Wiley‐Liss, Inc.     

Sexual determination and differentiation in teleost fish

The present work reviews the latest information on the cellular, molecular and physiological aspects of sexual determination and differentiation in teleost fish. The group exhibits a large variety of mechanisms of sexual determination. These may be genetic, or depend on environmental conditions such as temperature, pH, and social factors, all of which can influence the proportion of the sexes. Additionally, sex steroids play an important role in the regulation of sexual differentiation. The patterns of gonadal sexual differentiation are diverse, and species may be hermaphroditic or gonochoristic, some of the latter displaying juvenile hermaphroditism. In recent years, several genes involved in the sexual determination and differentiation pathways in vertebrates, particularly in mammals, have also been characterized in teleosts. Conserved as well as diversified functions have been proposed.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.     

Where does gonadal sex differentiation begin? Gradient of histological sex differentiation in the gonads of pejerrey,Odontesthes bonariensis (Pisces,Atherinidae)

This study investigated the possibility that the histological process of gonadal sex differentiation in pejerrey (Odontesthes bonariensis), a fish with marked temperature-dependent sex determination (TSD), occurs through a predictable gradient of differentiation as opposed to simultaneous or random differentiation throughout the gonad. For this purpose, fish reared at 17 degrees, 24 degrees, and 29 degrees C from hatching were sampled weekly for 11 weeks, fixed, and prepared for histological observation of serial cross-sections of the gonads. The thermal manipulation and sampling procedure ensured the availability of males and females at various degrees of gonadal sex differentiation. The location of the differentiated area(s) was estimated in the right and left gonads of 17 females and 14 males selected among the available specimens so as to represent increasing degrees of differentiation. The analysis revealed that sex differentiation followed a gradient from the anterior to posterior areas of the gonads regardless of sex. Furthermore, plotting of the degree of sex differentiation in the right gonad as a function of the degree of differentiation of the left gonad clearly showed that sex differentiation only begins in the right gonad when 10-30% of the length of the left gonad has already differentiated. The mean rostral edge of the differentiated areas in females was 9% and 10.8% for the left and right gonads, respectively, while for males these values were 7.3% and 7.0%, respectively. Thus, it was established that ovarian and testicular differentiation in pejerrey follow both a cephalocaudal and a left-to-right gradient. Possible explanations for this gradient and its relevance for TSD in pejerrey, that is, as a mechanism to prevent discrepant differentiation of male and female features within the same gonad, are discussed.     

Sex-specific and left-right asymmetric expression pattern of Bmp7 in the gonad of normal and sex-reversed chicken embryos

A genetic switch determines whether the indifferent gonad develops into an ovary or a testis. In adult females of many avian species, the left ovary is functional while the right one regresses. In the embryo, bone morphogenetic proteins (BMP) mediate biological effects in many organ developments but their roles in avian sex determination and gonadal differentiation remains largely unknown. Here, we report the sex-specific and left-right (L-R) asymmetric expression pattern of Bmp7 in the chicken gonadogenesis. Bmp7 was L-R asymmetrically expressed at the beginning of genital ridge formation. After sexual differentiation occurred, sex-specific expression pattern of Bmp7 was observed in the ovary mesenchyme. In addition, ovary-specific Bmp7 expression was reduced in experimentally induced female-to-male reversal using the aromatase inhibitor (AI). These dynamic changes of expression pattern of Bmp7 in the gonad with or without AI treatment suggest that BMP may play roles in determination of L-R asymmetric development and sex-dependent differentiation in the avian gonadogenesis.     

Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis

Sex determination in mammals and birds is chromosomal, while in many reptiles sex determination is temperature dependent. Morphological development of the gonads in these systems is conserved, suggesting that many of the genes involved in gonad development are also conserved. The genes SF1, WT1 and DAX1 play various roles in the mammalian testis-determining pathway. SF1 and WT1 are thought to interact to cause male-specific gene expression during testis development, while DAX1 is believed to inhibit this male-specific gene expression. We have cloned SF1 and DAX1 from the American alligator, a species with temperature-dependent sex determination (TSD). SF1, DAX1 and WT1 are expressed in the urogenital system/gonad throughout the period of alligator gonadogenesis which is temperature sensitive. SF1 appears to be expressed at a higher level in females than in males. This SF1 expression pattern is concordant with the observed pattern during chicken gonadogenesis, but opposite to that observed during mouse gonadogenesis. Although the observed sexual dimorphism of gonadal SF1 expression in alligators and chickens is opposite that observed in the mouse, it is probable that SF1 is involved in control of gonadal steroidogenesis in all these vertebrates. DAX1 and WT1 are both expressed during stages 22-25 of both males and females. However, there appear to be no sex differences in the expression patterns of these genes. We conclude that DAX1, WT1 and SF1 may be involved in gonadal development of the alligator. These genes may form part of a gonadal-development pathway which has been conserved through vertebrate evolution.