INVESTIGATION OF GENE EXPRESSION PATTERN OF ZONA PELLUCIDA DOMAIN 4 (ZP4) IN PACIFIC ABALONE Haliotis discus hannai AND ITS EXPRESSION IN EARLY STAGE OF THE GONAD DIFFERENTIATION.  

Several genes were shown to be exclusively expressed in female or male gonads have been reported in teleosts. To study the mechanism of sex differentiation in marine invertebrate, especially in economically valuable marine invertebrate, pacific abalone Haliotis discus hannai, molecular markers for determining the sex determination are need to be developed. From transcriptome analysis of H. discus hannai, zona pellucida domain 4 (ZP4) was shown to be expressed in a female gonad and to be related with a growth rate of abalone. In order to understand further the sexual maturation of abalone and its relationship to the growth, RT-PCR was carried out with RNAs isolated from egg, and gonads collected from abalones of 50, 100, 150, and 200 days post fertilization (dpf). ZP4 transcripts were shown to be detected from gonads of female abalones from 200 dpf. The frequencies of abalone expressing ZP4 transcript in their gonad will be analyzed with different size abalones of various dpfs.

To examine the developmental stage-dependent expression of ZP4 together with sex differentiation, RNAs were isolated from eggs as a positive control and individual abalones collected at 50, 100, 150, and 200 dpf. Amplification of cDNA was carried out by using ZP4 specific primers and ribosomal protein L3 (RPL3) primer as an internal control. Developmental stage-dependent expression of ZP4 in abalone was shown by the detection of ZP4 in abalones from 200 dpf (Figure 1). The result indicates that the sex differentiation process to female abalone seems to start from 200 dpf.

Differential expressions of ZP4 were examined in different (small and large) size   abalones collected from 200 (Figure 2-a) and 300 (Figure 2-b) dpfs. The result indicating a higher frequency detection of ZP4 in a large size abalone as compared to that of small size abalone suggest a faster growth rate of female abalone. This, together with its female specific expression, indicated that ZP4 may be used as a marker for identification of fast growth and female-specific sex determination in H. discus hannai.