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。     

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。     

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.     

The etiology of maleness in XX men

Summary Information relating to the etiology of human XX males is reviewed. The lesser body height and smaller tooth size in comparison with control males and first-degree male relatives could imply that the patients never had any Y chromosome. Neither reports of occasional mitoses with a Y chromosome, nor of the occurrence of Y chromatin in Sertoli cells are convincing enough to support the idea that low-grade or circumscribed mosaicism is a common etiologic factor. Reports of an increase in length of one of the X chromosomes in XX males are few and some are conflicting. Nor is there any evidence to support the idea of loss of material. However, absence of visible cytogenetic alteration does not rule out the possibility of translocations, exchanges or deletions.A few familial cases are known. Mendelian gene mutations may account for a number of instances of XX males, similar genes being well known in several animal species. The existing geographical differences in the prevalence of human XX males could be explained by differences in gene frequency. But if gene mutation were a common cause of XX maleness there would be more familial cases.Any hypothesis explaining the etiology of XX males should take into account the following facts. There are at least 4 examples of XX males who have inherited the Xg allele carried by their fathers, and at least 9 of such males who have not. The frequency of the Xg phenotype among XX males is far closer to that of males than to that of females, while the absence of any color-blind XX males (among 40 tested) resembles the distribution in females. Furthermore, H-Y antigen is present in XX males, often at a strength intermediate between that in normal males and females. Finally, in a pedigree comprising three independently ascertained XX males, the mothers of all three are H-Y antigen-positive, and the pattern of inheritance of the antigen in two of them precludes X-chromosomal transmission.Many of the data are consistent with the hypothesis that XX males arise through interchange of the testic-determining gene on the Y chromosome and a portion of the X chromosome containing the Xg gene. However, actual evidence in favor of this hypothesis is still lacking, and the H-Y antigen data are not easy to explain. In contrast, if recent hypotheses on the mechanisms controlling the expression of H-Y antigen are confirmed, a gene exerting negative control on testis determination would be located near the end of of the short arm of the X chromosome. This putative gene is believed not to be inactivated in normal females, for at least two other genes located in the same region, i.e. Xg and steroid sulfatase, are not. Deletion or inactivation of these loci would explain how XX males arise and would be consistent with most, but not all, the facts.There is yet no single hypothesis that by itself can explain all the facts accumulated about XX males. While mosaicism appears very unlikely in most cases, Mendelian gene mutation, translocation, X-Y interchange, a minute deletion or preferential inactivation of an X chromosome, or part thereof, remain possible. The etiology of XX maleness may well be heterogeneous.     

Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice

The sex-determining region of Chr Y (Sry) gene is sufficient to induce testis formation and the subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males, such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth because of germ cell-autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here, we demonstrate that the testicular somatic environment of XX/Sry males is defective in supporting the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, of entering meiosis and of differentiating to the round-spermatid stage. XY-donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, resulting in severe deficiency of elongated spermatid stages. By contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY-donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed the missing expression of several Y-linked genes and the alterations in the expression profile of genes associated with spermiogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, highlighting the idea that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.     

Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y₁₂ receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.     

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.     

Deletion mapping of the testis determining locus with DNA probes in 46,XX males and in 46,XY and 46,X,dic(Y) females.

Eleven Y-specific DNA probes hybridizing with DNA from one or more 46,XX males were isolated from a recombinant phage DNA library constructed from flow sorted human Y chromosomes. Two probes hybridized with DNA from nine out of eleven, i.e. greater than 80% of these 46,XX males. The relative frequency of hybridization of the probes in the 46,XX males and in a 46,X,dic(Y) female, together with in situ hybridization data, allowed mapping of the probes on Yp in relation to a putative testis determining locus. Several of those probes were also absent in a 46,XY female, further refining a model for ordering the probes on Yp. The DNA of one XX male hybridized both with probes from Yp and probes from proximal Yq (excluding the pericentral region). This suggests that complex translocations may occur into the DNA of 46,XX males that involve not only parts of Yp but also parts of Yq.     

Familial (9;11)(p22;p15.5)pat translocation and XX sex reversal in a phenotypic boy with cryptorchidism and delayed development

We describe a patient with the co-occurrence of a familial 9;11 reciprocal translocation and an XX sex reversal. The patient had cryptorchidism, delayed development, dysmorphic features and attention deficiency hyperactive disorder (ADHD). The proband's karyotype was 46,XX,t(9;11)(p22;p15.5) and he was positive for SRY gene. The father was found to be the carrier of the similar translocation. The co-occurrence of XX sex reversal and autosomal reciprocal translocation has not been described previously. The possible reasons for the manifestation of features other than those found in XX sex reversal is described.     

Absence of Y-specific DNA sequences in human 46,XX true hermaphrodites and in 45,X mixed gonadal dysgenesis

Summary A search for Y-specific DNA sequences has been performed in a sample of seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case and compared with a sample of 11 XX males. Using six Y-specific DNA probes no hybridization signal was obtained in the hermaphrodite group; in contrast, all XX males gave a positive signal with at least one probe. This difference is statistically highly significant. We conclude that the aetiology of true hermaphroditism is different from that of the XX male syndrome. As all cases of the hermaphrodite group are positive for the serological sex-specific antigen (Sxs) it is concluded that this antigen can be present even in the absence of Y-specific DNA.     

Further cytologic evidence for Xp-Yp translocation in XX males using in situ hybridization with Y-derived probe

Summary Chromosome preparations from seven subjects with aberrations of sex chromosomes were utilized for in situ hybridization studies with the tritium-labeled Y-derived probe p50f. Two subjects had a pseudodicentric chromosome consisting of two copies of Yp and a portion of Y long arm; two were XX males [46,XX,t(Xp;Yp)], one was missing part of the Y short arm, and another had t(5p;Yq); in addition cells from an XYY male as well as a normal 46,XY male, and a 46,XX female, were hybridized with the same probe. The hybridization technique of Harper and Saunders (1981) was used. There was excess labeling of the Yp/paracentromeric regions in the cases with the normal Y, the XYY, the pseudodicentric Y, and the 5/Y translocation. No significant label was seen on metaphases from the normal 46,XX female or the female with the partially missing Y short arm. Excess label was present on the X short arm in the cases of the XX males; there were 8% and 9.5% of cells with label. The combined cytogenetic and hybridization data indicate that one X short arm in these XX males has undergone a translocation with Yp, and that genes for sex determination probably reside on the distal half of the Y short arm.     

The parental origin of X chromosomes in XX males determined using restriction fragment length polymorphisms.

The inheritance of several X-linked restriction fragment length polymorphisms ( RFLPs ) is examined in seven 46,XX males and their immediate relatives. The XX males are shown to have inherited a paternal and a maternal RFLP allele in each of the five (of seven) families in which these X-linked markers are informative. In the other two families, a maternal X-chromosomal contribution is demonstrated, but a paternal contribution cannot be determined. We conclude that most, if not all, XX males inherit one paternal and one maternal X chromosome. A segment of single-copy DNA specific to the short arm of the Y chromosome is found to be absent from the genomes of eight XX males. In one of these XX males, an Xp-Yp translocation had previously been inferred from chromosome-banding studies. Our findings argue against mosaicism involving a Y-containing cell line in the XX males examined here, but they do not exclude an X-Y (or Y-autosome) translocation during paternal meiosis. If such a translocation has occurred, the translocation product received by the XX males does not include the Yp-specific sequence tested here.     

Balanced and unbalanced pericentric inversion of a chromosome 14

Summary Three males with a 46,XX karyotype are described. In two of them, evidence of a Y-containing line was found. In the first case, 1 of 500 lymphocyte metaphases was 47,XXY. In the second, 1 of 400 oral mucosa cells contained a Y body. The proportion of low-grade XX/XXY mosaics found among XX males now stands at about 17%.     

Heteromorphic X chromosomes in 46,XX males: Evidence for the involvement of X-Y interchange

Summary G- and R-banded chromosome preparations from eight of twelve 46,XX males, with no evidence of mosaicism or a free Y chromosome, were distinguished in blind trials from preparations from normal 46,XX females by virtue of heteromorphism of the short arm of one X chromosome. Photographic measurements on X chromosomes and on chromosome pair 7 in cells from twelve 46,XX males, eight 46,XX females, and four 46,XY males revealed a significant increase in the size of the p arm of one X chromosome in the group of XX males, independently characterised as being heteromorphic for Xp. No such differences were observed between X chromosomes of normal males and females or between homologues of chromosome pair 7 in all groups. The heteromorphism in XX males is a consequence of an alteration in shape (banding profile) and length of the tip of the short arm of one X chromosome, and the difference in size of the two Xp arms in these 46,XXp+ males ranged from 0.4% to 22.9%. From various considerations, including the demonstration of a Y-specific DNA fragment in DNA digests from nuclei of one of three XX males tested, it is concluded that the Xp+ chromosome is a product of Xp-Yp exchange. These exchanges are assumed to originate at meiosis in the male parent and may involve an exchange of different amounts of material. The consequences of such unequal exchange are considered in terms of the inheritance of genes located on Yp and distal Xp. No obvious phenotypic difference was associated with the presence or absence of Xp+. Thus, some males diagnosed as 46,XX are mosaic for a cryptic Y-containing cell line, and there is now excellent evidence that maleness in others may be a consequence of an autosomal recessive gene. The present data imply that in around 70% of 46,XX males, maleness is a consequence of the inheritance of a paternal X-Y interchange product.     

Successive spontaneous abortions including one with whole-arm translocation between chromosomes 2

Summary A family with five induced and seven spontaneous abortions and no live births is described. Four of the seven spontaneous abortuses were available for cytogenetic examination and three were successfully karyotyped. Their karyotypes were 46,XX; 46,XX/46,XX,t(2;2)(2p2p;2q2q); and 46,XY. The karyotypes of the parents were normal. The origin of the 2p/2p and 2p/2p translocation in one of the abortuses was assigned to an interhomologous whole-arm translocation in an early mitotic division in a conceptus with a 46,XX karyotype.     

XX sex reversal in the American cocker spaniel dog: phenotypic expression and inheritance

Summary This study was conducted to define the range of phenotypic expression and mode of inheritance of XX sex reversal in the cocker spaniel dog. Breeding experiments produced F1, F1BC, and F2 generations in which 29 XX true hermaphrodites and 3 XX males were defined by chromosome constitution, serial histologic sections of the gonads, and examination of the internal and external genitalia. In XX true hermaphrodites, the most common combination of gonads was bilateral ovotestes, followed by ovotestis and ovary, then ovotestis and testis. The amount of testicular tissue in the two gonads was closely correlated within each true hermaphrodite. The distribution of testicular tissue within ovotestes of true hermaphrodites was consistent with the hypothesis that testicular differentiation is initiated in the center of the gonad and spreads outward. XX males had bilateral aspermatogenic testes and the internal ducts and external genitalia were more masculinized than in true hermaphrodites. Results of breeding experiments are consistent with autosomal recessive inheritance, the affected phenotype being expressed only in dogs with an XX chromosome constitution. The phenotypic expression and mode of inheritance of this disorder is compared to XX sex reversal in humans and other animals.     

Fast-developing preimplantation embryo progeny from heterogametic females in mammals.

Karyotyping and cell number estimates in preimplantation embryos from heterogametic (XY*) and homogametic (XX) females of the field mouse Akodon azarae were studied to determine whether XX-XY-XY* differences exist in the rate of preimplantation development. At the morula stage, XY embryos from heterogametic mothers had twice the mean number of cells compared with XX embryos. However, this difference in cell numbers was not seen between XX and XY embryos from homogametic mothers. In this case, mean cell numbers were similar despite embryos being XX or XY. Furthermore, the mean cell number for XX and XY morulae from homogametic females was comparable to that for XX embryos from heterogametic females. It is concluded that XY* embryos (which will develop into heterogametic females) show an accelerated rate of preimplantation development.     

Molecular cytogenetic characterization of ring chromosome 4 in a female having a chromosomally normal child

We present the clinical and molecular findings of mosaic ring chromosome 4. The patient was referred to us for infertility and short stature. Results of three repeated cytogenetic analyses from lymphocytes showed a similar mosaic karyotype with multiple cell-lines [46,XX,r(4)/45,XX,-4/46,XX,dic r(4)/47,XX,r(4),+r(4)/46,XX]. FISH showed deletion of the 4p subtelomeric region and the 4q telomeric region from the ring chromosome 4. The breakpoints were mapped using molecular analysis. Parental karyotypes were normal. During the course of this study, the patient became pregnant without assisted reproductive technology. The result of amniocentesis performed at 16 weeks gestation showed a normal karyotype. Delivery was uncomplicated. This is the first report, to our knowledge, of the presence of ring chromosome 4 having various mosaic conditions in a female having a chromosomally normal fetus.