Mutation Of ZNF230 Gene And Its Association With Azoospermia And Expression Of Its Homolog Rnf141 In Zebrafish Embryo

Azoospermia is a complex disorder caused by both environmental and genetical factors. Some zinc finger genes were reported to be correlated with spermatogenesis failure. The human gene ZNF230 is a newly identified zinc finger gene through mRNA differential display between the normal and the patient testicular tissues, which might be correlated with azoospermia. To test this concept, mutation screening and association analysis of ZNF230 was performed. Recently, the homolog of ZNF230, rnf141, was discovered in zebrafish which is an excellent vertebrate model for research on development, pathogenesis, genetic mechanism and human disease. So, we got the chance to perform analysis of its embryonic expression pattern.At first, we proliferated all 6 exons of ZNF230 from 99 azoospermia patients and 115 normal control. Then, high throughput mutation screening was performed by denaturing high performance liquid chromatography (DHPLC) technology. Finally, the data were confirmed by DNA sequencing and estimated by statistic softwares,such as HWE and SPSS12. 0, to obtain the association information between mutation site and azoospermia.For another purpose, we constructed a recombinant plasmid which could be transcribed into sense or anti-sense mRNA probes for zebrafish homolog rnfl41 with T7 or SP6 RNA polymerase after linearization. The two probes were Dig-labeled and then used for whole mount in situ hybridization (WMISH).We discovered the variation of 316A>G in exon 6. The allele frequencies in patient group were 21.2% (316G) and 78. 8%(316A) respectively, in comparison with those of 13.0% (316G) and 87.0 % (316A) in control group. The genotype and allele frequencies were significantly (K0. 05, K0. 01) higher in the patient group than in the control group, which indicated that this variation may be associated with azoospermia. Further more, this variation was also found to be significantly (fKO. 05) associated with the higher serum follicle stimulating hormone (FSH) level in patients subgroup with 316G. Thus, this variation may be related to the increase of serum FSH level, too. We also discovered a sense mutation of 164OT in exon 2 whose frequency was less than 1%.We exactly obtained the purpose fragment from rnfl41 cDNA and correctly constructed the recombinant plasmid. Sense and anti-sense Dig-labeled RNA probes were successfully prepared as comparison. In accordance with the high similarities of amnio acid and domain sequence of vertebrate RNF141, the expression of rnfl41 is ubiquitous in zebrafish embryo besides the different abundance of rnfl41 mRNA in telencephalon, cerebel and hindbrain. These results indicated that vertebrate RNF141 is conserved in evolution and must play an important role during embryogenesis. In a word,zebraf ish is an interesting and useful model for further researchof rnfl41 gene.