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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Sex Determination in Birds from the Perspective of Studies on Triploid IntersexesB.L. Sheldon1, M.H. Thorne1,2 1CSIRO Division of Animal Production, Locked Bag 1, Delivery
Centre, Blacktown, NSW 2148 Australia; 2Current Address: 41 Roma
Rd, St. Ives, NSW 2075 Australia Recent advances in knowledge of sex determination in mammals have depended largely on analysis of individuals with aberrant sex chromosome constitution, such as XO or XY females and XX or XXY males. Analogous individuals, ZZ females and ZW males, have not been detected in chickens. The development of a unique "triploidy" line of chickens has enabled the study of large numbers of triploid female intersex chickens (3A.ZZW), and a few associated chimeric individuals. These studies are reviewed in this paper. They demonstrate that (i) masculinization of the external phenotype of 3A.ZZW apparent females becomes obvious only from about sexual maturity, (ii) initial determination and differentiation of 3A.ZZW embryos as female is normal and complete, (iii) masculinization of the left ovary and development of a right testis in place of a regressed right ovary already starts before hatching, (iv) this masculinization of the gonads is reversible (at least temporarily) by injection of estrogen at 96 h of incubation, (v) the sex phenotype of the chimeras is dependent primarily on the presence of a W chromosome, but affected by the proportions of diploid and triploid cells and the dosage of Z and W chromosomes. The results support the hypothesis that there is a female (ovary) determining gene on the W chromosome, the effect of which is able to be inhibited or partly reversed by more than one dose of male (testis) determining gene(s) on the Z chromosome. Expression of Genes Involved in Estrogen Synthesis and Expression of the Estrogen Receptor Gene during Early Gonadal Development of ChickensS. Mizuno, O. Nakabayashi, H. Kikuchi1, T. Kikuchi1 Laboratory of Molecular Biology, Department of Applied
Biological Chemistry, Faculty of Agriculture, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 Japan;
1Department of Animal Model of Human Disease, National Institute
of Neuroscience, NCNP, Kodaira, Tokyo, Japan Administration of an aromatase inhibitor during an early period of chicken embryogenesis causes the development of testis in a genetic female (Elbrecht and Smith, 1992), suggesting that exposure to estrogen during the early phase of gonadal development is crucial for the normal development of the ovary. We examined the expression of five genes involved in the synthesis of estradiol-17_ and the expression of the estrogen receptor (ER) gene during the development of the gonads. Changes in the expression of mRNA for these genes were determined by in situ hybridization with cDNA probes with embryonic tissue sections. The expression of P-450scc, 3_-HSD and P-450c17 genes was already detectable at the 7th day of incubation in the right and left gonads in both sexes. Expression of 17_-HSD and P-450arom genes was also detectable in right and left gonads but only in female embryos. ER mRNA was detected after 7th day of incubation in the left gonad of both sexes but was undetectable in the right gonad. Signals for ER mRNA in the left gonad of males declined in conjunction with the growth arrest of germinal epithelium and became undetectable by the 14th day of incubation. These observations are discussed in relation to the onset and asymmetric differentiation of ovaries in chickens. Genotypic and Phenotypic Sex ReversalR.J. Etches, H. Kagami Department of Animal and Poultry Science, University of
Guelph, Guelph, ON N1G 2W1 Canada Sexual differentiation was altered by in ovo exposure to estradiol, by in ovo exposure to CGS, an inhibitor of estrogen synthesis, by surgical removal of the left ovary within 10 days after hatching, by combining in ovo exposure of CGS with sinistral gonadectomy, and by producing mixed-sex blastodermal cell chimeras. At hatching, all of the chicks exposed to estradiol had female external genitalia but sexual dimorphism in growth rate and 2° sexual characteristics at 18 weeks corresponded to the genetic sex of each bird. Spermatogenesis in males exposed to estradiol was abolished, but ovarian development was unaffected. Development of the shell gland was impaired in hens exposed to estrogens and in males, partial development of the Müllerian ducts was evident. Exposure to CGS had no effect on genetic males but masculinized genetic females. At hatch, during growth, and after sexual maturity, genetic females possessed male 2° sexual characteristics; at 30 weeks of age, these birds had an ovotestis and a partially developed oviduct. Left ovariectomy produced birds with a male phenotype and a right ovotestis at 30 weeks. Females that were treated in ovo with CGS and sinistrally gonadectomized developed male secondary sexual characteristics; at 30 weeks of age they possessed a right ovotestis and a partially developed oviduct. Transfer of male blastodermal cells into female recipient embryos produced female chimeras containing ZZ "oogonia" which differentiated into ova and were fertilized. Transfer of male blastodermal cells into female recipient embryos also produced male chimeras, indicating that the presence of male cells in a female embryo can masculinize development. Transfer of female blastodermal cells into male recipient embryos produced only male chimeras indicating that the presence of female cells does not femininize development of males. Both ZW and ZZ spermatogonia in mixed-sex, male chimeras produced fertile Z-bearing spermatozoa. The absence of albino offspring from matings of sex-linked albino hens to mixed-sex chimeras indicated that W-bearing spermatozoa were incapable of fertilization. These data demonstrate that sexual differentiation originates as a genetic signal that is difficult to override by hormonal manipulation. In the correct environment, however, genetically male cells can become oogonia and genetically female cells can become spermatogonia. W-bearing spermatozoa are, however, non-functional. Are NonGonadal Factors Involved in Sexual Differentiation of the Neural Song Circuit in the Zebra Finches?A.P. Arnold Department of Physiological Science and Laboratory of
Neuroendocrinology of the Brain Research Institute, UCLA, 621
Circle Drive South, Los Angeles, CA 900951527, USA The brain regions controlling song are much larger in male songbirds than in females, and the song system is one of the only systems available for study of the mechanisms of anatomical sexual differentiation of the avian brain. One theory proposes that the brain is masculinized in males by estrogens and androgens, which derive from testicular secretions. Another theory suggests that the ovary may secrete an antimasculine factor, which itself may be estrogen. Although it is easy to promote masculine patterns of neural development by treating hatchling female zebra finches with estrogen, it has been much more difficult to block masculine patterns of development in males by blocking estrogen synthesis or action. Recently, we have created genetic females that develop large amounts of testicular tissue by treating embryos with the aromatase inhibitor fadrozole (Wade and Arnold, in press). These females possess a right testis and left ovotestis. The testes contain sperm and secrete androgen, as evidenced by the hypertrophy of the androgendependent syrinx. When adult, these females have feminine neural song circuits, which are masculinized little or not at all. Testicular secretions may thus not be solely responsible for masculine patterns of neural development. Ovarian antimasculine factors, or direct (i.e., nonhormonal) genetic factors may also be casual factors in the sexual differentiation of the neural song circuit. (Supported by NIH grants DC00217 and HD32050) Novel Approaches to Study Avian Sexual Differentiation using Mixed-Sex Chimeric ChickensH. Kagami, T. Tagami, Y. Matsubara, H. Hanada, M. Naito1 National Institute of Animal Industry; 1National Institute of
Animal Health, Tsukuba, Ibaraki, Japan Mixedsex chimeras were produced to investigate the interaction of sex between donor and recipient. A portion of recipient blastoderm was biopsied and dissociated donor blastodermal cells were injected into the recipient. The donors and recipients were sexed by PCR reaction. The embryos were cultured for 5 days and wholemount in situ hybridization using Wchromosome specific DNA probe was performed. When a female donor and a female recipient was combined, the entire body was stained dark violet, while embryos produced by a male and a male were not colored. Embryos produced by a male and a female and viceversa were stained about 60% or 10% of the body, respectively. A series of chimeras were produced and donor derived feather pigmentation was observed. The production of somatic chimeras was greatly increased when the recipient tissue was biopsied prior to injection of donor cells. The donor contribution to the germline will be assessed by mating these chimeras with donor chicken strains. From these studies, it will be determined how mixedsex chimeras develop and how the germ cells and somatic cells contribute in the process of sexual differentiation. Protein Kinase Genes Expressed in Chicken Primordial Germ CellsE. Sasaki, Y. Kaku, H. Hikono, T. Kuwana1, M. Naito, M. Sakurai1 National Institute of Animal Health, Tsukuba, Ibaraki 305,
Japan; 1National Institute for Minamata Disease, Minamata,
Kumamoto 867, Japan Chicken primordial germ cells (PGCs) are known to circulate temporarily via the blood vascular system and colonize into the germinal ridges. Then finally they differentiate into eggs or spermatozoa. Germline chimeric chickens have been produced by transferring PGCs isolated from embryonic blood or germinal crescents. However, biological or biochemical features of chicken PGCs and mechanisms of proliferation of chicken PGCs are largely unknown. Protein kinases (PKs) play important roles in cellular proliferation and differentiation. To identify PKs expressed in chicken PGCs, mRNA was prepared from completely purified PGCs taken from stage 1315 embryos and PKrelated sequences were amplified by polymerase chain reaction (PCR) using the mRNA as a template. PCR primers were designed from the conservative regions VIB and IX of PKs. The amplified DNA fragments were subcloned into pUC118 and their sequences compared with the published sequences of chicken and mammalian PKs.Twenty distinct kinaselike sequences were obtained. Six and nine of these sequences represented cDNAs for receptortype protein tyrosine kinases (PTKs) and for non receptortype PTKs, respectively. One clone corresponded to a human serine/threonine kinase. The other 4 sequences seemed to represent novel serine/threonine kinase genes. An Analysis of the Divisional Ability of Chicken Primordial Germ Cells Before and After Settling in the Germinal RidgeT. Maeda Faculty of Applied Biological Science, Hiroshima University,
Higashi-Hiroshima, 739 Japan To clarify the divisional ability of primordial germ cells (PGCs), the quantity of DNA and frequency of the final stage of metaphase in PGCs at stages 14-15 (before settling in the germinal ridge) and stages 17-20 (after settling in the germinal ridge) were analyzed using an interactive laser cytometer. When the quantity of DNA was graphically presented, two peaks were recognized in all stages. The value of second peak was approximately twice that of the first. The PGCs having two nuclei in one cell were recognized in each stage and were considered to be the final stage of metaphase in cell division. The amount of DNA in metaphase correlated with that in the second peak. The frequency of final stage metaphase in PGCs after settling in the germinal ridge was markedly increased comparing with that before, and significantly increased with advancing embryonic stage. From these results, it seems that intense proliferation of chicken PGCs occurs after settling in the germinal ridge. Expression of Exogenous DNA in Embryonic Gonads by Transferring Primordial Germ Cells Transfected in VitroM. Naito, a. Tajima1, Y. Yasuda2, M. Sakurai, T. Kuwana3 National Institute of Animal Health, Tsukuba 305, 1Tsukuba
University, Tsukuba, 305, 2Kumamoto University, Kumamoto 860,
3National Institute of Minamata Disease, Kumamoto 867, Japan Gene transfer into primordial germ cells (PGCs) and the production of viable offspring from germline chimeric chickens provides a powerful tool for studying avian endocrinology. PGCs collected from the embryonic blood of White Leghorns (WL) were concentrated. Transfection of PGCs was achieved using a cationic lipid (DOTAP, Boehringer Manheim Biochemica). The PGCs were incubated with DOTAP:DNA (pAcZ, lacZ gene under the control of chicken ß-actin gene promoter) mixture for 5 hours at 38°C, washed with culture medium and 300 PGCs were injected into the blood stream of recipient embryos from which blood had been drawn prior to the injection. The recipient embryos were incubated for 3 days and lacZ gene expression was detected in the gonads by X-gal staining. The percentage of embryos which expressed the lacZ gene in the left and/or right gonads was 71.2% (37/52), and the number of positive cells for the lacZ gene in the gonads was variable; a few to more than 50 cells. Thus, the lacZ gene was successfully introduced into the PGCs in vitro and was expressed in the gonads of recipient embryos. This technique provides an experimental system for testing the expression of exogenous DNA in the gonads of developing chick embryos, and also contributes to the production of transgenic chickens. Asymmetrical Germ Cell Commitment in the Avian GonadK. Simkiss School of Animal and Microbial Sciences, The University of
Reading, Reading, RG6 6AJ England The vertebrate gonad consists of two distinct components. The stroma is formed from the coelomic epithelium and mesonephric tissues, while the gametes are derived from primordial germ cells (PGCs). The decision as to whether a PGC will differentiate into a sperm or an oocyte is dependent not upon its genotype (i.e. whether it is ZZ male or ZW female) but by the type of stroma in which it develops. In the normal female bird the left ovary is functional but the right gonad remains as a rudiment. Destroying the left ovary results in the right gonad proliferating but as a testis, producing sperm from female PGCs. Clearly there are two phenomena in this process that relate to basic developmental biology. The first is the initiation of asymmetry while the second depends on cellcell signalling.We are studying the first of these by determining the distribution of aromatase in the germinal stroma while cellcell interactions are being studied by tracing artificially introduced ZW PGCs into male embryos and following the W chromosome into the cockerels sperm. Long Term Culture and Characterization of Avian Embryonic Stem Cells with Multiple Morphogenetic PotentialitiesB. Pain, M.E. Clark1, R.J. Etches1, J. Samarut Ecole Normale Superieure de Lyon, UMR 49-LA-INRA, Laboratoire
de Biologie Moleculaire et Cellulaire, Lyon, France; 1Department
of Animal and Poultry Science, University of Guelph, Guelph, ON
N1G 2W1 Canada Blastodermal cells that can colonize somatic tissues and the germline when injected into recipient embryos have been identified in early chick embryos. In order to characterize this population of cells, we have used a medium containing the growth factors bFGF, SCF and IGF-1, and the cytokines LIF and IL-11. The morphology of cultured blastodermal cells is round and small; they have a strong alkaline phosphatase activity and they are recognized by the antibodies ECMA-7, SSEA-1, SSEA-3 and EMA-1. All of these features are shared with murine embryonic stem cells. After LIF deprivation, the cells become flat, their growth rate is reduced, and the cells lose epitopes recognized by ECMA-7, SSEA-1, SSEA-3 and EMA-1. Following injection into recipient embryos, cultured cells contributed to the development of phenotypic chimeras that were recognized by their donor-derived pigmented plumage and their recipient-derived non-pigmented plumage. The proportion of injected recipients that are somatic chimeras appears to be independent of the duration of the culture. To date, donor-derived offspring have not been obtained from chimeras made with cultured cells, indicating that cultured cells have not contributed to the germline. Blastodermal cells can be maintained for 20 passages without a loss of ES-like characteristics and can produce embryoid bodies throughout this period. The embryoid bodies can be induced to differentiate into hemopoetic cells, muscle cells, and nerve cells indicating that they contain stem cells for at least the ectodermal and mesodermal lineages. Sexual Differentiation of the Vocal Control System in European StarlingsJ.M. Casto, G.F. Ball Department of Psychology, Johns Hopkins University, Baltimore,
MD 21218, USA We have been studying the timing of the development of the song system and the possible effects of hormones on the development of the vocal control system in European starlings (Sturnus vulgaris). Experiments conducted on zebra finches (Taeniopygia guttata) suggest that exposure of nestling finches to 17ß-estradiol (E2) masculinizes various aspects of the female song system but does not hypermasculinize males. Attempts to block song system masculinization in male zebra finches by administering antiestrogens or aromatase inhibitors during the period when exogenous E2 is effective in females have failed. The action of E2 in relation to sexual differentiation has therefore been determined in the European starling (Sturnus vulgaris), in which we have recently described the ontogeny of sexual differences in the vocal control system. On day 3 after hatching, free-living nestling starlings in nest boxes, were implanted with silastic ropes containing 500 µg of E2. Controls received either blank implants or no implants. Birds were brought into the laboratory at day 11 and hand-reared until independence. Birds were killed at 210 days of age, prior to the onset of measurable gonadal development and marked increases in plasma levels of sex steroid hormones. Volumetric re-construction of 5 song control nuclei, the High Vocal Center (HVC), area X, the robust nucleus of the archistriatum (RA), the lateral and medial parts of the magnocellular nucleus of the anterior neostriatum (1MAN and mMAN) revealed prominent and significant sex differences in all nuclei. Treatment with E2 resulted in significant increases in the volume of HVC (29% increase compared to controls) and area X (27% increase) in males but no significant changes were detected in E2-treated females. These data indicate that E2 exposure alone, at least during this developmental period, is insufficient to masculinize the song system of female starlings. Introduction of an Exogenous Gene into Peking Duck EmbryosZ.D. Li, H.C. Li, D.F. Zhao, M. Du, H. Deng Department of Animal Biochemistry, China Agricultural
University, Beijing 100094, China The ex ova culture of embryos from the chicken and quail has already been achieved. Peking ducks, however, have a quite different incubation period. We examined, therefore, the ex ova culture and introduction of an exogenous gene into the embryos of Peking ducks. Egg yolks with developing embryos after 82 hours of incubation, (stage 18 to 20; Hamberger and Hamilton, 1951), were transferred to other recipient egg shells and incubated with a specially devised incubator at 38 C, RH 60-70%, with rotations at an angle of 30° every 15 min. Survival rates of the transferred embryos decreased drastically, compared with normally developing embryos, especially after 20 days of incubation, resulting in low (ca 16%) hatchability. However, this result shows the ex ova culture of Peking duck embryos is possible, as in chickens and quail. Plasmids containing a andB-actin-lac Z hybrid gene (pmiwZ) were miroinjected by lipofection into the blastoderm of the ex ova cultured embryos. The embryos were cultured for at least one week, and the expression of lac Z activity in the embryonic tissues was determined at 24 h, 62 h and 5 days after incubation. The expression of pmiwZ DNA was detected by a histochemical staining method of andB-galactosidase, X-gal (5-br omo-4-chloro-3-indolyl-andB-D-galactopyranoside) staining. The rates of DNA expression in the embryos, determined by stainability of the tissues, were 57% for 24 h, 96% for 62 h and 37% for 5 days of the incubation, respectively. These results indicate that the technique described here will be useful for the production of transgenic Peking ducks. |