ER、bcl-2和p53在鸡与鹌鹑属间杂交种早期胚胎中的mRNA表达

通过人工授精获得鸡(♂10只)与鹌鹑(♀100只)属间杂交种蛋并同机入孵, 采用Wpkci引物和多重PCR鉴定66~120 h的鸡(♂)与鹌鹑(♀)属间杂交种活胚的性别后, 选取不同时间点雌、雄胚胎共300枚, 以b-actin为内标, 通过RT-PCR分别测定雌激素受体(ER)和细胞凋亡因子(bcl-2、p53)的mRNA相对丰度; 探讨ER、bcl-2和p53对杂交种早期胚胎发育及性别分化的影响。结果表明: (1) 杂交种胚胎ER mRNA 表达在66～84 h期间雌性极显著高于雄性(P＜0.01), 由此推测杂交种的性分化时间大致在胚胎发育的66~84 h范围内; (2) bcl-2和p53 的mRNA表达在杂交胚胎发育过程中具有明显的时序性, 说明bcl-2和p53基因对早期杂交胚的发育具有重要的影响。     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.     

鸟类性别决定候选基因在性反转鸡胚中的表达

DMRT1、PKCIW和FET1是鸟类性别决定过程中重要的候选基因。以芳香化酶抑制剂处理的鸡胚为实验材料, 对这3个基因的表达变化进行了研究。结果表明, 在整个性别决定关键时期(E4.5 ~ E10.5), DMRT1在雄性的表达量显著高于雌性, 并且在ZW性反转鸡胚中表达大幅上升, 表明DMRT1的上调表达是与睾丸形成相关的。PKCIW基因在雌性特异表达并在性反转鸡胚表达上升, 这可能与其特殊作用模式有关, 即使性反转鸡胚PKCIW代偿性的表达升高, 却也未能阻止睾丸的形成。此外, FET1为雌性特异表达, 但在性反转鸡胚中表达无变化。综上, 实验结果支持了DMRT1是鸟类睾丸发育决定因子的假说。     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Xenogenic oogenesis of chicken (Gallus domesticus) female primordial germ cells in germline chimeric quail (Coturnix japonica) ovary

In present study, chicken primordial germ cells (PGCs) were transferred into quail embryos to investigate the development of these germ cells in quail ovary. Briefly, 2 microl of chicken embryonic blood (stage 14) or about 100 purified circulating PGCs were transferred into quail embryo. Contribution of chicken PGCs were detected in gonads of chimeric quail embryos (stage 28) by immunocytochemical staining of cell surface antigen SSEA-1, and by in situ hybridization (ISH) with female chicken specific DNA probe. As a result, 52.0+/-43.2 (n=18) and 42.7+/-27.3 (n=17) chicken PGCs were found in the gonads of chimeric quail embryo that was injected with chicken embryonic blood (stage 14) and about 100 purified circulating PGCs, respectively. Furthermore, the ovaries of 81.8% (9/11) 12 days post incubation (dpi) chimeric quail embryos were observed with a mean of 457.6+/-237.1 female chicken PGCs-derived oogonia scattered in ovarian cortex area. In 9 out of 12 newly hatched and one week old chimeric quail chicks, on average of 2883.0+/-1924.1 primary oocytes and 3 follicles derived from chicken PGCs were found, respectively. The present results suggest that chicken female PGCs are able to migrate, colonize, proliferate and differentiate into oogonia, primary oocytes in chimeric quail ovary.     

Sex Determination: Two Copies for One Cock

The molecular mechanism of sex determination in birds has long remained mysterious. Genetically male chicken embryos, which have two Z sex chromosomes, develop female gonads when the Z chromosome-linked gene DMRT1 is knocked out. This suggests that sex is determined by Z chromosome dosage.     

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.     

Ultrastructural study of embryonic and early adult germ cells,and their support cells,in both sexes of Xiphophorus (Teleostei:Poeciliidae)

The ultrastructure of the early spermatogonia in mature testes of the platyfish, Xiphophorus maculatus, was compared to that of oogonia in mature ovaries of X. maculatus and the related X. nigrensis. Both cell types were very similar and, characterized as being large, oval to round cells containing large, central nuclei with prominent nucleoli. Abundant mitochondria with sparse transverse cristae were located at one pole or around the nucleus. Annulate lamellae and electron-dense granular material (nuage) were present. Other organelles were not prominent. A female that had received a testis graft had testicular tissue containing mature spermatozoa within the ovary, indicating that cells were present that could develop along the male line. Special crosses were carried out to obtain all-male embryos of X. maculatus and all-female embryos of X. nigrensis. The ultrastructure of the germ cells in all embryonic gonads was similar to that of the adult cells. These results suggest the presence of sexually undifferentiated germ cells in the adult gonads of both sexes. The support cells investing all of these germ cells were also similar structurally and appeared to be undifferentiated.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus)

In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.