Differential expression of the MHM region and of sex-determining-related genes during gonadal development in chicken embryos.

The chicken (Gallus gallus) embryo has been used as a classic model system for developmental studies because of its easy accessibility for surgical manipulation during embryonic development. Sex determination in birds is chromosomally based (ZZ for males and ZW for females); however, the basic mechanism of sex determination is still unknown. Here, the dynamics of expression of candidate genes implicated in vertebrate sex determination and differentiation were studied during embryonic chicken gonadal development. Gene expression profiles were obtained before, during, and after gonadal sex differentiation in females and males for DMRT1, SOX3, SOX9, DAX1, SCII, HINTZ, HINTW, and the male hypermethylated (MHM) region. Transcripts for the HINTZ, DMRT1, DAX1, SCII, and SOX9 genes were observed in both sexes, but expression was higher in male gonads and may be correlated with testicular differentiation. The expression patterns of HINTW, SOX3, and MHM suggest that they may act in ovary development and may be involved in meiosis entry. MHM was upregulated and DMRT1 was downregulated in females at the same developmental stage. This may indicate a regulation of DMRT1 by MHM ncRNA. Similar dynamics were observed between HINTW and HINTZ. This study reports on the MHM expression profile during gonadal development and its correlation with the expression of genes involved in vertebrate sex determination.

Sexing single bovine blastomeres using TSPY gene amplification.

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.

Identification of a DNA methylation point in the promoter region of the bovine CYP21 gene.

The CYP21 (steroid 21-hydroxylase) gene is involved in the synthesis of steroid hormones. Bov-A2 is a retroposon that is common in ruminant genomes. The promoter region of bovine CYP21 contains a short interspersed nucleotide element of Bov-A2, which overlaps a putative Sp1 binding site. We looked for RFLP/HpaII polymorphism in the Bov-A2 element in bovine Zebu breeds by PCR-RFLP, and examined whether polymorphism in this element is associated with methylation. Among DNA samples from 135 Brazilian Zebu breed cattle, we identified an RFLP/HpaII polymorphism (T/C), which, based on a restriction methylation-sensitive assay employing HpaII and isoschizomer MspI enzymes (methylation-sensitive and -non-sensitive enzymes, respectively), appears to be a DNA methylation point. This is the first report of this polymorphism and on DNA methylation in the bovine CYP21 promoter region in Brazilian Zebu cattle.