Genetic Manipulation of Sex Ratio for the Large-Scale Breeding of YY Super-Male and XY All-Male Yellow Catfish (Pelteobagrus fulvidraco (Richardson))

Yellow catfish has become one of the most important freshwater aquaculture species in China. The mono-sex male yellow catfish has important application value in aquaculture because the male grows generally faster than the sibling females under the same conditions. This study has screened YY super-male and YY physiological female yellow catfish by sex reversal, gynogenesis, and progeny testing, which can help to achieve the large-scale production of YY super-male and XY all-male. From 2008 to 2010, about 123,000 YY super-male were produced, and about 81 million XY all-male fry were produced with 100 % male rate by random sampling. Therefore, these results indicate that YY super-male and YY physiological female yellow catfish can be viable and fertile. We conclude that the mono-sex breeding technique by YY super-male yellow catfish is stable and reliable, which has great potential for application in yellow catfish aquaculture.     

利用鱼类性逆转技术创制黄颡鱼XX雄鱼的方法

目前黄颡鱼(Pelteobagrus fulvidraco Richardson)生产上使用的母本非常混杂, 成为影响黄颡鱼产业发展的一个突出问题。建立一个优良性状稳定的全雌家系对于全雄黄颡鱼和杂交黄颡鱼的生产尤为重要, 而将XX雌性黄颡鱼逆转为XX雄性黄颡鱼是创制全雌家系中的关键一步。黄颡鱼性染色体连锁分子标记的开发为鉴定XX雄鱼提供了技术支撑。研究使用不同浓度的17α-甲基睾酮(MT)和芳香化酶抑制剂来曲唑(LZ)处理黄颡鱼鱼苗54d (7—60日龄)。61日龄测量并统计各组鱼的存活率、体长及体重。解剖观察性腺结构, 结合性腺组织切片和性别分子标记分析各组实验鱼中XX鱼性腺发育情况。结果显示: 2种药物对黄颡鱼的存活率影响较小, 与对照组没有显著差异; MT处理的XX性腺为空腔状精小囊结构, 不能逆转为功能性精巢; 适当剂量的LZ可以将XX雌性黄颡鱼性逆转成正常的XX雄鱼, 诱导效果随着剂量的增加而增强。用完全逆转的XX生理雄鱼分别与XX雌鱼和YY雌鱼交配, 能够正常繁殖并具备较好的繁殖能力。研究建立了一种使用芳香化酶抑制剂来曲唑创制黄颡鱼XX雄鱼的方法, 为全雄黄颡鱼的品种改良及新品种的培育奠定了基础。     

Genetic Differentiation and Efficient Sex-specific Marker Development of a Pair of Y- and X-linked Markers in Yellow Catfish

Pf62-Y and Pf62-X is a pair of allelic Y chromosome-linked and X chromosome-linked markers, and have been used to identify YY super-males, XY males and XX females for commercial production of all-male populations in yellow catfish (Pelteobagrus fulvidraco). However, the SCAR primers used previously have only two nucleotide difference, which restricts the wide utility because of nucleotide polymorphism. In this study, a continuous 8102 bp Pf62-Y sequence and a 5362 bp Pf62-X sequence have been cloned by genome walking, and significant genetic differentiation has been revealed between the corresponding X and Y chromosome allele sequences. Moreover, three pairs of primers were designed to efficiently identify YY super-males, XY males and XX females in an artificial breeding population, and to distinguish XY males and XX females in various wild populations. Together, the three new sex-specific genetic markers develop a highly stable and efficient method for genetic sex identification and sex control application in sustainable aquaculture of all-male yellow catfish.     

黄颡鱼和杂交黄颡鱼“黄优1号”形态及性腺发育的比较

选取相同养殖条件的10月、22月和34月龄的黄颡鱼和新品种杂交黄颡鱼(黄颡鱼P. fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)“黄优1号”进行形态及性腺发育的比较研究。通过形体指标测量发现“黄优1号”生长性能显著优于黄颡鱼; 在22月和34月龄黄颡鱼中, 雄性的体重是雌性的2倍左右, 雄性生长速度显著高于雌性; 而在“黄优1号”中, 两性生长异形现象被显著减弱。基于性腺解剖形态分析发现雌性“黄优1号”卵巢完全退化, 呈细线状结构且没有卵子产生, 故“黄优1号”雌鱼完全不育; 雄性“黄优1号”精巢组织呈现透明状和退化状态。精巢组织切片HE染色分析发现10月龄“黄优1号”的精小囊为空腔状几乎没有精子产生, 22月和34月龄“黄优1号”的精小囊内出现极少量精子。计算机辅助精子分析系统(CASA)分析发现, 相比于黄颡鱼, “黄优1号”精巢中精子量非常少, 有效活力低下; 经过繁殖能力测试, 22月龄“黄优1号”雄鱼不具备繁殖能力。新品种杂交黄颡鱼“黄优1号”在生长性能提高上显现了杂交优势, 具有推动黄颡鱼产业发展的潜力。     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

高温和皮质醇对黄颡鱼性别分化的影响

研究以性染色体类型已确定且已有性别特异分子标记的黄颡鱼为研究对象,开展高温与皮质醇诱导黄颡鱼(Tachysurus fulvidraco Richardson)XX个体雄性化组织学进程研究,以期为环境应激诱导鱼类雄性化提供研究基础。通过对每尾鱼采用性别特异性标记鉴定遗传性别(XX或XY)及组织学鉴定生理型性别,仅经过24d的处理(12—35日龄),高温或皮质醇便能诱导XX遗传型个体雄性化。在此过程中,部分XX遗传型个体卵母细胞受到抑制,之后发育成带有卵巢腔的精巢结构。62日龄时, XX伪雄鱼性腺较正常XY雄鱼大, XX伪雄鱼体重与正常XY雄鱼相近,而显著大于未发生性逆转的XX雌鱼。122日龄时, XX伪雄鱼从62日龄带有卵巢腔的精巢结构发育成具有典型的精小叶结构样精巢,且都具有生理性雄鱼特有的生殖突,推测这些雄鱼可能具有与正常雄鱼类似的生殖能力。部分XX个体对高温处理不敏感,没有发生性逆转,温度处理反而加快了卵巢发育的进程,这些个体对高温的耐受性和另外一些发生性逆转的个体对温度的敏感性值得进一步研究。     

黄颡鱼XX伪雄鱼诱导与全雌种群规模化繁育

研究利用3种雄性化因素, 包括17α-甲基睾丸酮(MT, 5 mg/kg)、来曲唑(LZ, 300 mg/kg)和高温(33.5℃) 联合处理12—65日龄黄颡鱼幼鱼, 并将性成熟的XX伪雄鱼与正常XX雌鱼进行人工繁殖, 开展了全雌黄颡鱼(Tachysurus fulvidraco)规模化繁殖与苗种培育工作。研究发现, MT、LZ和高温共同作用可诱导XX黄颡鱼逆转为生理型雄性, 完全性逆转个体运动型精子比例与XY雄鱼无显著性差异, 组织学切片也显示其精巢中存在大量精子细胞, 推测XX伪雄鱼具有正常的繁殖功能。随后, 以XX伪雄鱼为父本, 正常XX雌鱼为母本开展了规模化人工繁殖, 获得了57万尾基因型全部为XX的黄颡鱼苗种, 并将其成功培育成大规格鱼种。在幼鱼60日龄和120日龄时取样发现, 分别有2.8%和12.0%的个体发生了不同程度的雄性化, 推测其可能受到池塘自然高温的影响而发生了性逆转。其余XX雌鱼卵巢发育良好, 来年繁殖季节可作为规模化人工繁殖的雌性亲本。研究成功开展了全雌黄颡鱼规模化繁育工作, 为全雌黄颡鱼规模化繁育体系的建立提供了基础, 也为黄颡鱼新品种选育中雌性选育提供了保障。     

Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.     

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.     

Heteromorphic sex chromosome system with an exceptionally large Y chromosome in a catfish Steindachneridion sp. (Pimelodidae)

The chromosomes and banding patterns of Steindachneridion sp., a large catfish (Pimelodidae), endemic to the Igua?u River, Brazil, were analyzed using conventional (C-, G-banding) and restriction enzyme banding methods. The same diploid number (2n = 56) as in other members of the genus and the family was found but the karyotype displayed an XX/XY sex chromosome system. The X chromosome was the smallest submetacentric, while the Y was the largest chromosome in the karyotype. Meiotic analysis showed 27 autosomal bivalents plus one heteromorphic XY bivalent during spermatogenesis. Sex chromosomes had no particular pattern after C-banding but G- and restriction enzyme bandings showed specific banding characteristics. The present finding represents the first report of a well-differentiated and uncommon sex chromosome system in the catfish family Pimelodidae.     

lon-1 regulates Caenorhabditis elegans body size downstream of the dbl-1 TGF beta signaling pathway

Although the primitive vasculature is identical in XX and XY genital ridges until 11.5 days postcoitum (dpc), by 12.5 dpc the XY gonad develops a distinct vasculature. This male-specific vasculature, which includes the development of a large coelomic vessel, develops coincident with expression of Sry and formation of testis cords. We show that similar levels of proliferation and vasculogenesis expand the primary vasculature in XX and XY gonads. However, soon after Sry expression begins, the XY gonad recruits a large number of endothelial cells from the adjacent mesonephros, a mechanism totally absent in XX gonads. These migrating cells do not contribute to venous or lymphatic development. Instead, these cells contribute to the arterial system, as indicated by expression of ephrinB2 and by elements of the Notch signaling pathway. This newly formed arterial system establishes a new pattern of blood flow in the XY gonad, which we speculate may have an important role in export of testosterone to masculinize the XY embryo.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Sex differentiation in mammals and tempo of growth: probabilities vs. switches

In the conventional model of sex differentiation in placental mammals, a switch is envisaged to steer the indifferent gonad into the path of either testicular or ovarian development. The immediate cause of the switch is thought to be the presence or absence of Sertoli cells, which in turn is controlled by the presence or absence of the testis-determining factor on the Y chromosome (TDF in humans, Tdy in mice). Quantitative investigations indicate, however, that the rate of growth of XY gonads is faster than that of XX gonads before the formation of Sertoli cells, and furthermore, that XY embryos develop faster than XX embryos long before the formation of gonadal ridges. Since the genetic constitution of the sex chromosomes appears to manifest itself from the earliest embryonic stages onwards, the concept of indifferent gonads being switched into alternate pathways becomes inappropriate. A model is proposed in which gonadal differentiation depends on developmental thresholds: the formation of Sertoli cells needs to occur by a particular stage in time in a sufficiently developed gonad, failing which the gonad will enter the ovarian pathway. While TDF is the principal factor enhancing the rate of gonadal growth, other factors which influence development rates can modulate the probability of a gonad becoming either a testis or an ovary.     

Sex reversal of pejerrey (Odontesthes bonariensis), a species with temperature-dependent sex determination,in a seminatural environment

This study examined the changes in sex ratios and sex reversal rates in pejerrey Odontesthes bonariensis that occur with the progression of the spawning season in a seminatural setting. Four groups of hatchery-produced pejerrey larvae were stocked in floating cages in La Salada de Monasterio lake (Pampas region), a natural habitat of this species, and reared from hatching beyond gonadal sex determination with minimum human interference. Cage 1 was stocked at the beginning of the spring spawning season and the other cages were stocked with monthly delays until cage 4 in early summer. The genotypic (amhy+, XY/YY; amhy−, XX) and phenotypic (testis, male; ovary, female) sex ratios and proportions of genotype/phenotype mismatched individuals were estimated and their relation to water temperature and daylength during the experiment was analysed by generalized linear modelling. Water temperature varied between 11 and 30.5°C, and daylength duration between 11 h 22 min and 14 h 35 min. Sex genotyping revealed nearly balanced sex ratios of XY/YY (46%–49.1%) and XX (50.9%–54%) fish in cages 2–4 whereas the genotypic sex ratio in cage 1 was clearly biased towards XY/YY fish (60.6%). Phenotypic males ranged from 42% to 54.4% in cages 1–3. Cage 4, in turn, had significantly more phenotypic males (66%). The percentage of XX males (phenotypic male/genotypic female) was 23.1% in cage 1, decreased to a minimum of 5.4% in cage 2 and gradually increased in cages 3 and 4 to a maximum of 40.7% in the latter. The percentages of XY/YY females (phenotypic female/genotypic male) were highest in cage 1 (30%) and decreased progressively in the other cages to a significantly lower value (4.3%) in cage 4. These results generally support the findings of laboratory studies on the effect of temperature on the sex determination of this species and also provide novel evidence of a XX genotype-specific masculinizing effect of short daylength.     

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.