To establish the gene expression profile and cascade subsequently on avian sex-determining candidate genes, seven avian sex-determining candidate genes including DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1 and SOX9 were analyzed at early embryogenesis. Quantitative reverse transcription PCR (Quantitative RT-PCR) technology was used to establish the gene expression profiles among these genes at four, five, six and seven days of embryos. The results of quantitative RT-PCR reveal that the DMRT1 was expressed in chicken embryos of both sexes. DMRT1 gene expressions were up-regulated at four, five and six days of chicken embryos. DMRT1 expression increased at 5-Dpc. of male embryos, however, expression was not signification different in females embryos. Gene expression of FET1, FOXL2, LHX9 and HINT1 were higher in females than in males. The SMC2L1 and SOX9 were expressed in both sexes. Also, to identify the novel sex-determination genes in early chicken subtractive embryos, cDNA libraries from male-minus-female and female-minus-male 3.5 Dpc. embryos cDNA were established. Gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA) and TIGR gene indices (The Institute for Genome Research, USA). A total 548 of colonies in male-minus-female library and 79 sequences were annotated. However, a total of 589 of colonies in female-minus-male library and 16 sequences were annotated. Sequences were homologous to the steroid 5£\-reductase protein (SRD5A1) using BLASTx in male-minus-female subtractive library. The SRD5A1 may play a sex-differentiation role in male chicken. We need more study to know function of steroid 5£\-reductase protein in future.

Two hypotheses currently exist regarding to the determining factors for sexual development and differentiation in birds. One is based on the unbalanced sex chromosome, meaning that avian sex determination is dominated by ¡§Z-chromosome dosage¡¨. The other brings up (reconsider this) the key factor of ¡§W chromosome¡¨ which is a particular sex chromosome in female birds (ZW). In the previous studies, we constructed a female-subtract-male cDNA library before morphological gonad differentiation. After sequencing and annotation, a total of 279 expression sequence taqs (ESTs) were identified, with potentially higher expression levels in females. By utilizing quantitative RT-PCR, 16 potential ESTs and three marker transcripts (HINT1, FET1 and WDR36), which identified to be involved in sexual development at 3, 5, 7, 9 days post-coitum (dpc) was analyzed in chicken embryos. Results indicated that AGR2, CPT2, DUSP19, HINTW, LOC771368 and EY53070791 had higher expression levels in female than in male embryos at 3 and 5 dpc; FET1 expression level in female embryos gradually increased from 3 to 9 dpc. Moreover, both HINT1 and WDR36 were higher expressed in male than in female embryos across 3 to 9 dpc. However, HINT1 exhibited higher expression levels starting at early stage, whereas WDR36 at later stage. Next, we constructed HINT1-GFP fusion protein and overexpressed this protein in chicken B-cell line (DT40), resulting in upregulation of WDR36 expression. On the contrary, overexpressed HINTW-GFP fusion protein in DT40 cells had decreased WDR36 expression level. Moreover, we designed a small hairpin RNA by utilizing RNA interference technique to knockdown expression of HINTW, which resulted in WDR36 upregulation. Finally, we then estimated the regulation of WDR36 promoter activity through analyzing HINT1-GFP overexpression. Results had shown that HINT1-GFP can improve WDR36 promoter activity. Therefore, we suppose that HINT1 can regulate WDR36 transcription via WDR36 proximal promoter region. Ongoing HINT1 responsive element(s) must be identified to characterize whether HINT1 or HINTW regulates WDR36.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

From the sexual preselection point of view, understanding sex determination/differentiation mechanisms in the bird is critical in both evolutionary and industrial applications. The chicken embryo provides an unique vertebrate model in the field of development biology. Morphological sex development in the chick gonad starts at 6.5 embryonic day (E6.5), however, genetic sex determination and development should occur earlier. In order to comprehend genes and their underlying mechanisms being involved in sex-determination/development during early embryogenesis, we not only made a male-subtract-female and a female-subtract-male cDNA library as early as embryonic day 3 (E3; Hamburger and Hamilton Stage 20), but also examined early transcripts related to male development in chicken embryo and their expression profiles in this study. A total of 89 and 127 candidates of male-development transcripts represented respectively for 83 known and 119 unknown non-redundant sequences, which were characterized in an E3 male- subtract-female complementary DNA library. In this study, thirty-five selected transcripts being validated by quantitative reverse transcription-polymerase chain reaction that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Notably, twelve of these transcripts mapped to the Z chromosome. At 72 weeks of age, twenty transcripts were expressed at higher levels in testes than in ovaries. Meanwhile, four transcripts were expressed at higher levels in brains of male than in brains of female chickens (P < 0.05). By using of methods of whole mount and frozen cross-section in situ hybridization, the expressions of riboflavin kinase (RFK), WD repeat domain 36 (WDR36) and EY505808 transcripts on E7 chicken male gonads were corroborated to be better than female gonads. This result was confirmed by using of western blotting analysis which also showed the expressions were specifically on gonads than other tissues. Treatment with an aromatase inhibitor formestane at E4 depicted the effect of the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), which was similar to the observed gene expressions for both doublesex and mab-3 related transcription factor 1 gene. Additionally, the interaction effects of sex with formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). This study demonstrated a panel of potentially candidate male development transcripts being identified during early chicken embryogenesis; some might be regulated by sex hormones.