On The Sex Ratio Of Chicken And Sexual Differentially Expressed Genes In Chicken Embryos During Early Sexual Differentiation

Sex control of chicken is signality in modern commercial poultry production, but unfortunately, there is no authorized way yet to realize. Current approaches to control the sex of chicken include manipulating the factors which have effect on chicken sex ratio to make it deflect according to the requirement of production, producing sex reversal chicken by interfere with the sex differentiation etc. In present research, two topics are focused on: firstly, the sex ratios under normal condition of chicken production would be studied by an approach being able to identify the sex of embryo during the early development stage and the causes to lead the deflection of sex ratios would be simply attributed, and secondly, sexual differentially expressed genes in chicken embryos during early sexual differentiation would be isolated by using suppressive subtraction hybridization (SSH), and the developmental expression profiles of the isolated genes and some candidates which involve in the sex determination and differentiation of other species would be studied to form the basis of molecular mechanism of chicken sex determination and sex differentiation. Details as follows:A molecular approach to sex the chicken and the embryo was established by using duplex PCR which simultaneously amplifys a 360 bp EE0.6 fragment on W chromosome and 970 bp OVR fragment on Z chromosome. It has been validated to be accurate completely (100 %) and is used successfully to identify the sexes of chicken embryos at early stage, and the sexes of 92.34 % embryos died during early developmental stage can be identified by using this way. Offspring sex ratios of 140 hens during 30 days under normal condition are studied, it shows that the sex ratios of hatching and all offspring (including embryos died) respectively in "all hens" group and "high fecundity" group, i. e. 76 hens among all hens, do not deviate significantly from expected 1:1, but the number of female embryos died is higher significantly than that of males (P＜0.05) which has validated through the study of a large number of dead embryos. Parental sex ratio of most "high fecundity" hens appear bias in different degree, of which the offspring sex raio of 7 hens (account for 9.21%) deviate significantly from 1:1 (P＜0.05), and 37 hens produce successively more than 4 eggs with the same sex.Forward and reverse subtracted cDNA libraries between female and male are constructed from chicken embryonic gonads at days 3.5-6 post-incubation by SSH. The subtractive efficiency of 2⁵ folds is obtained in the two libraries by using the housekeeping gene encoding for glyceraldehyde-6-phosphate dehydrogenase (GAPDH) as the reference. PCR of 96 recombined clones picked randomly from each library shows that most of them contained inserts of 250-750 bp, and 93.8 % clone in the F-M library, in which female embryos as tester, male embryos as driver, is recombinants and corresponding, 90.6 % in the M-F. Dot Blot hybridization is carried out to screen differentially expressed genes by using forward subtracted cDNA and reverse unsubtracted cDNA as probes, results show that, respectively in F-M and M-F library, there are 31% and 26 % clones having obviously different expression (three times difference at least) among 1200 clones detected in F-M and 900 in M-F. BLAST of sequences of 152 sequenced clones reveals that 86 clones from F-M library represent 41 genes, including 39 being identified and 2 having homologous EST sequence, and 66 clones from M-F library represent 47 genes, 16 being identified, 12 having homologous EST sequence and another 25 having no homolog in GenBank database. Based on the function of the homologs in mammals, all 41 genes identified (31 in F-M, 10 in M-F) are predicted to be functional molecular belonging to DNA association, RNA association, structural protein, enzyme, cancer relation and signing transport etc. It is demonstrated that most of 88 differentially expressed genes locate on macrochromosomes (1-5), one from F-M library locate on W and 2 from F-M library, 3 from M-F library on Z. The cDNA sequences of differentially expressed genes are blasted with EST sequences in two reported chicken gonad related libraries (gonad cDNA library and gPGC cDNA library) from GenBank, it reveals that there are 80.49 % and 34.04 % differentially expressed genes respectively from F-M and M-F library having homologous sequences in two reported libraries, and accordingly, expression feature in chicken embryonic gonad of differentially expressed genes are predicted by comparing the redundancy of differentially expressed cDNA in subtracted library with that in two reported gonad libraries.Expression profiles of seven candidate genes putatively involved in chicken gonadal development and 46 differentially expressed genes newly isolated from subtracted cDNA libraries (34 in F-M, 12 in M-F) during chicken early gonadal development are studied by RT-PCR. Results show that the cBrd3, cVnn1, cPpt1, cLhx9 and cGATA4 are sexually dimorphic expression with the exception of cEki and cFog2 having similar expression pattern in both sexes, and comparative expression analysis between mammals and chickens show both conserved and divergent elements: expression patterns of cVnn1 and the interaction between cGATA4/cFog2 are similar to those in mammals, while others appearing differences. Expression profiles of genes newly isolated reveals that most of them (respectively 85.29 % in F-M and 91.67 % in M-F) present really different expression. There are 13 and 6 genes, respectively in F-M and M-F, presenting the difference of more than 2 fold relative expression and among them, 10 genes in F-M and 1 in M-F showing obvious difference before sex differentiation (i. e. at 4-5 days). In addition, there are 16 and 5 genes respectively in F-M and M-F library presenting difference of less than 2 times relative expression. The expression profile of SMARCE1 in urogenital system of chicken embryos at days of 5.5 and 8.5 are studied by WISH, a method for positional gene expression, it indicates that the expression of SMARCE1 is similar in both sexes in the urogenital system including embryonic mesonephros and gonad at 5.5-day, and positive expression of this gene has been found in the gonad and the lateral of mesonephros of embryo at 8.5-day.Full-length cDNA sequences of a new transcript of chicken HMG-14A (Clone F071) and a non-coding molecular (Clone A041) on Z chromosome are obtained by using SMART technology, and these two sequences are submitted to GenBank with accession number EF422210 and EF422212. The cDNA sequence of F071 is 1026 bp, with largest ORF of 318 bp, encoding 105 amino acids predicted by ORF Finder. BLAST of nucleotide and amino acid sequences of F071 show that the coverage rate of homologous nucleotide sequence of F071 with its homolog HMG-14A (NM₀01079480.1) is 85 %, and for the similarity of amino acid, 100 %. The structure and function of the protein encoded by F071 gene are predicted by using Signal P3.0, ProtParam and Prosite software, results show that this protein is Lysine-rich but non-secretory, having HMG14 and HMG17 signature, a cAMP-and cGMP-dependent protein kinase phosphorylation site, a protein kinase C phosphorylation site, an amidation site, a casein kinaseâ…¡phosphorylation site and a N-glycosylation site. Phylogenetic tree of HMG14 (HMGN1) among several species are constructed by using MEGA3.1 based on CLUSTALW, it shows that chicken HMG-14A (F071) is far away from HMGN1 of other compared species. A041 is found having two transcripts with difference length in 5' UTR and the full length cDNA sequence are 1249 bp and 790 bp respectively, its largest ORF predicted by ORF Finder is 144 bp, encoding possibly 47 amino acids. No any homolog of A041 is found in NCBI and it is tentatively recognized as a non-coding RNA (ncRNA).