Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation.

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.

Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo.

Chicken embryonic stem (CES) cells are pluripotent cells derived from chicken early blastoderm. In order to identify new genes specifically expressed in these pluripotent cells, we have used a gene trap strategy and cloned a novel gene family called cENS for chicken Embryonic Normal Stem cell gene. The cENS genes expression decreases after induction of CES cells differentiation in culture and is restricted in vivo to the very early embryo. We have characterized three different cENS genes. One, cENS-1, is composed of an open reading frame inserted between two terminal direct repeats which are the common point of the cENS genes. cENS-1 encodes a protein identical to cERNI, a recently described protein. cENS-2 is a truncated form of cENS-1. cENS-3 presents two adjacent open reading frames coding respectively for env and pol related proteins. The presence of conserved direct repeats, of retrovirus related genes and the absence of introns argue in favor of a retroviral origin of the cENS genes. In the cENS we identified a promoter region whose activity is strong in CES cells and decreases after induced differentiation showing a highly specific transcriptional activity specific of undifferentiated chicken embryonic stem cells.

Predominant expression of a Z-chromosome-linked immunoglobulin superfamily gene, ZOV3, in steroidogenic cells of ovarian follicles and in embryonic gonads of chickens.

A cDNA clone, pZOV3, was isolated from the cDNA library of immature chicken ovaries and its gene was mapped to the middle of the short arm of the Z chromosome. The cDNA sequence suggests that ZOV3 is a novel member of the immunoglobulin superfamily. cDNA clones of homologues of chicken ZOV3 were also obtained from Japanese quail and pigeon. Northern blot hybridization suggests that the high-level expression of the ZOV3 gene is restricted to the gonads: embryonic, immature and mature ovaries, and embryonic and immature testes. Western blot analysis and immunocytological detection using specific polyclonal antibodies against amino- and carboxyl-terminal regions of ZOV3 demonstrate that ZOV3 is a plasma membrane-bound glycoprotein that exists in granulosa cells and islets of cells in the theca externa layer of ovarian follicles. The latter islets coincide with those producing estradiol-17 beta. In male and female embryos, production of ZOV3 is first prominent in medullary and seminiferous codes, respectively, of developing gonads. Then, after hatching, it is shifted to the cortex surrounding the primitive follicles in the ovary or is continued weakly in the primary seminiferous tubules in the testis. Expression of the ZOV3 gene and production of ZOV3 are no longer detectable in the mature testis. ZOV3 is unique among the immunoglobulin superfamily proteins in that it is produced predominantly in gonads. Its possible role in differentiation or maintenance of steroidogenic cells in an ovarian follicle is discussed.

Molecular cloning of acid alpha-glucosidase cDNA of Japanese quail (Coturnix coturnix japonica) and the lack of its mRNA in acid maltase deficient quails.

Acid alpha-glucosidase (GAA) hydrolyzes alpha-1, 4 and alpha-1, 6 glucosidic linkages of oligosaccharides and degrades glycogen in the lysosomes. The full-length GAA I cDNA, pQAM8, was isolated from a cDNA library derived from Japanese quail liver. The cDNA is 3569 base pairs long and has an open reading frame capable of coding 932 amino acids. The deduced amino acid sequence shares 52% identity with human GAA. Transfection of expression vector pETAM8 into COS-7 cells or acid maltase deficient (AMD) quail embryonic fibroblasts increased the level of GAA 20-50-fold. Compared to normal quail, the levels of GAA I mRNA were significantly reduced in the muscle, liver, heart, and brain of AMD quails, suggesting the GAA deficiency in AMD quail is due to a lack of GAA I mRNA. A second GAA II cDNA was identified after probing the cDNA library from the ovarian large follicles of quails with a PCR product derived from cultured quail skin fibroblasts. This clone having 3.1 kb insert, has GAA activity as well (3 to 10 fold increase). This cDNA, designated GAA II, predicted an 873 amino acid polypeptide showing 63% identity to human GAA and 51% identity to the GAA I. The RT-PCR analysis demonstrated that GAA II mRNAs were barely detectable in normal tissues, while they were enhanced to higher levels in AMD tissues. These results suggest that GAA II expression is up-regulated at the transcription levels, and quail GAA gene redundancy performs the same function of satisfying GAA demand at the two different phases represented by normal and AMD.

Identification and localization of two genes on the chicken Z chromosome: implication of evolutionary conservation of the Z chromosome among avian species.

A cDNA clone containing an insert of about 3.4 kb, pCIREBP, was isolated from the chicken liver cDNA library and identified as a clone for the chicken homologue of iron-responsive element-binding protein (IREBP). The deduced amino acid sequence showed 88% identity with that of the mouse IREBP and 17 out of the 20 active site residues of the pig heart mitochondrial aconitase were conserved. Another cDNA clone, pZOV3, containing an insert of about 4.5 kb was isolated from the chicken ovary cDNA library. This cDNA contained an open reading frame for 327 amino acid residues, whose sequence had partial similarity to two immunoglobulin superfamily proteins; mouse GP-70 and chicken HT7. Fluorescence in situ hybridization using corresponding genomic clones revealed that both genes are localized on the Z chromosome; the ZOV3 gene at the middle of the short arm and the IREBP gene at the boundary of heterochromatin on the long arm. Southern blot hybridization to male and female genomic DNA preparations from six species representing five avian genera suggested that these two genes are Z-linked in all the species tested.