전암컷 3배체 넙치 생산

Abstract

본 연구에서는 전 암컷 3배체 넙치를 유도하기 위해서 국립수산과학원 육종연구센터에서 선발육종기법을 통하여 생산된 킹넙치(KingNupchi) 5가계 암컷을 이용하여 자성발생성 2배체를 유도하고, 성호르몬 및 환경 조절을 통해 성전환을 유도함으로써 자성발생성 2배체 수컷을 생산하였다. 생산된 자성발생성 2배체 수컷 5가계 중 우수한 가계를 선정, 이들을 대상으로 3배체를 유도하여 3배체의 세포유전학적 분석, 생식소 성숙 여부, 세포 주기, 혈액학적 특징 및 질병저항성 특징 등에 대해 조사하였다. 강도다리와 넙치의 세포유전학적 분석을 수행한 결과, 적혈구 세포 핵의 표면적 및 부피의 경우, 강도다리는 7.60±0.93 μm2 및 12.80±1.75 μm3 이었고, 넙치는 8.56±0.82 μm2 및 16.76±2.50 μm3 이었고, DNA 함량은 강도다리가 0.66 pg/haploid cell, 넙치가 0.71 ph/haploid cell로 관찰되어 강도다리 DNA 함량은 넙치의 약 93%로 확인되었다. 염색체 수와 핵형은 두 종 모두 2n=24 그리고 차단부 염색체 24쌍으로 구성되었으며, 암·수간, 개체간 및 세포간 염색체 다형현상도 관찰되지 않았다. 넙치는 24쌍의 차단부 염색체 중 가장 큰 1쌍의 차단부 염색체에서 NORs가 확인되었고, 강도다리는 가장 작은 1쌍의 차단부 염색체에서 확인되었다. 반수체 넙치의 특이적인 기형발생 현상을 관찰한 결과, 넙치의 반수체 배아는 이배체에 비하여 배체의 형태가 넓고, 짧으며, 분명하지 않은 상태로 발생하였으며, Kupffer’s vesicle이 제대로 형성되지 않으면서 대부분 꼬리가 형성되지 않거나 짧게 형성되었다. 반수체의 최초의 부화는 수정 후 55시간으로 이배체와 동일하였으나, 반수체의 경우 거의 대부분 부화하지 못하였고, 부화하였을지라도 6시간 이내에 모두 죽는 것으로 관찰되었다. 자성발생성 2배체 수컷을 생산하기 위해서 킹넙치 암컷 5미로부터 확보된 난과 자외선 5,400 erg/mm2의 자외선을 조사한 킹넙치 정자를 수정하였고, 수정 3분 후 2℃에서 45분간 저온처리하여 자성발생성 2배체를 유도하였다. 유도된 자성발생성 2배체에 대한 세포유적학적 분석에서 대조군과 차이가 없는 것이 확인되었다. 수컷으로 성전환 하기 위해서 웅성화 호르몬(17α-methyltestosterone) 첨가 사료 공급법 및 수온 조절법을 사용하여 자성발생성 2배체 수컷(XX)를 생산하였다. 유도된 자성발생성 2배체 수컷 5가계(Z1-1788, Z1-1697, Z1-0335, Z1-0219 및 Z1-1779) 중 우수한 가계를 선정하고자 생존, 성장, 질병저항성 및 생식능력 등에 대하여 가계별 특징을 비교하였고, 전암컷 3배체 생산을 위한 수컷 가계로 Z1-0219를 선정하였다. 전암컷 3배체 집단을 생산하기 위해서 일반 넙치의 난와 자성발생성 2배체 수컷 Z1-0219의 정자를 수정하였고, 수정 3분 후 2℃에서 45분간 저온처리하여 3배체를 유도하였다. 유도된 3배체를 대상으로 세포유적학적 분석을 수행한 결과 적혈구 세포 크기, 염색체 수, DNA 함량 및 NORs 수는 2배체의 약 1.5배 인 것으로 확인되어 성공적으로 3배체가 유도된 것으로 확인되었다. 3배체 넙치의 특징을 확인하기 위해서 난발생, 세포주기, 혈액학적 분석, 질병저항성 및 생식소 발달에 대한 분석을 수행하였다. 3배체의 난발생 형태 및 발생속도는 2배체와 동일한 것으로 확인되었고, 세포 주기는 3배체가 2배체에 비해 G2+M기의 비율이 낮은 것으로 확인되어 2배체가 3배체보다 유사분열이 활발한 것으로 확인되었다. 혈액학적 분석의 경우 3배체가 2배체보다 적혈구 세포의 크기(MCV)는 1.6배 크고, 세포 수(RBC)는 0.55배 적은 것으로 확인되었으며, 평균 적혈구 헤모글로빈 수치(MCH)는 3배체가 2배체보다 2.13배 높았으나 3배체의 hematocrit 수치는 2배체 보다 낮은 것으로 확인되었다. 생식소 발달의 경우, 3배체 넙치 암컷과 수컷에서 생식소의 발달이 제대로 이루어지지 않거나 비정상적인 발달이 관찰되어 불임인 것으로 확인되었다. 그러나 3배체 정소는 난소에 비해 다소 많이 발달된 형태인 정세포 및 정자가 관찰되었으나, 이들은 비정상적인 크기 및 형태의 정세포 및 정자로 확인되어 3배체 암컷과 수컷 모두 불임인 것으로 확인되었다.

The olive flounder Paralichthys olivaceus is one of the most economically important marine fishes in Korea. Sterility protects somatic growth, survival, and flesh quality from the negative effects of sexual maturation. Due to these advantages, all-female triploid of P. olivaceus was induced by sex reversal and chromosomal set manipulation techniques. Cytogenetic analysis was conducted to obtain basic information for the chromosome manipulation of olive flounder and starry flounder, Platichthys stellatus. The nuclear surface area and volume of the etrythrocyte were 7.60±0.93 μm2and12.80±1.75μm3 in starry flounder and 8.56±0.82μm2 and 16.76±2.50μm3 in olive flounder, respectively. The haploid DNA content of starry flounder was 0.66 pg/haploid cell, which corresponds to 93% of olive flounder (0.71 pg/haploid cell). A karyotype analysis was also carried out with the species through conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra- individual polymorphism was not detected inolive flounder and starry flounder. The chromosome nucleolar organizer regions (NORs) of olive flounder and starry flounder appeared on the short arm of the largest acrocentric chromosome pairs and in the terminal position of the short arm of the smallest acrocentric pairs, respectively. This study investigated the characteristics of embryonic and abnormal organ development of gynogenetic haploid in olive flounder by comparing the development of eggs and tissues in haploid and diploid individuals. After the mid-blastula transition, abnormal development was observed in haploid fish, including delayed epiboly and malformation of the germ ring and embryonic body. In haploid flounder, Kupffer’s vesicles were irregularly shaped and of variable size compared to diploids. The embryonic body of haploids was shorter and broader than that of diploids and the tail length and size were variable. Most haploid embryos failed to hatch and the few larvae that did so survived for less than 6 hours. The histological analysis of haploid larvae revealed deformed development in diverse organs, including the eyes, otic vesicles, notochord, and neural tube. These results may relate to an abnormality in the axial system of haploid larvae. This study confirmed that the abnormalities of haploid olive flounder were similar to the reported characteristics of haploid syndrome. The abnormalities are caused by delayed epiboly and involution and deformity of Kupffer’s vesicle during embryonic development. Gynogenetic diploid were induced in P. olivaceus through the application of cold shocks to eggs inseminated with sperm that had been genetically inactivated by ultraviolet rays. Embryos inseminated with sperm were irradiated with 5,400 erg/mm2ultraviolet rays, and showed a 100% incidence of haploid syndrome. Fertilized eggs were treated with cold shock (2℃) for 45 minutes to block the second polar body. In the cytogenetic studies, no difference was found between the induced gynogenetic diploids and the normal diploid controls. To produce gynogenetic diploid males (XX), a feeding treatment containing 17α-methyltestosterone (1 μg/g) and a higher water temperature treatment (25℃) than the one used for the control group were applied for 40 days from the 37th day after hatching. The gonad of a 6-month-old gynogenetic diploid male (XX) revealed fully matured spermatozoa in the testes. Moreover, in the F1 progeny test, matings between normal females and gynogenetic diploid males (XX) produced all-female offspring successfully. All-female triploid in P. olivaceus was induced by cold-shocking eggs fertilized with gynogenetic diploid male P. olivaceus (XX), 3 minutes post-fertilization at 2℃ for 45 minutes. Triploid was confirmed by erythrocyte measurement (nuclear volume, 29.15±2.10 μm3), flow cytometry (2.14±0.03 pg/cell), chromosome count (3N=72), Ag-NOR banding, and silver staining. The present study investigated the characteristics of induced all-female triploidy in P. olivacesus. A comparative analysis of cell cycles, hematological characteristics, embryogenesis, and histological characteristics in the gonads were analyzed in diploid and induced-triploid P. olivaceus. No significant differences were found among diploid, all female triploid and triploid in terms of embryonic development and immune ability (scuticocidal activity and bacteriocidal activity). Cell cycles were significantly different in the percentage of each cell cycle fraction between diploid and induced triploid P. olivaceus. The S-phase fraction of the diploid group (4.8±1.4%) was higher than that of induced-triploid group (9.7±3.4 and 9.9±1.7%) and the G2+M-fraction of the diploid group (31.2±2.8%) was lower than that of the induced-triploid group (22.5±1.7% and 23.3±2.1%). The differences were statistically significant in the G1-phase fraction. This study also aimed to compare the hematological features of diploid (2N), all-female triploid (AF 3N), and triploid (3N) P. olivaceus for the assessment of the ability of triploid olive flounder to withstand sub-optimal culture conditions. Triploid olive flounder had lower numbers of red blood cells (RBC: 4.60±0.39 cells/pL in 2n, 2.53±0.37 cells/pL in AF 3N and 2.49±0.44 cells/pL in 3n) but they were of a larger size (Mean corpuscular volume [MCV]: 50.93±6.22 fL in 2n 79.70±1.7 fL in AF3N and 80.41±1.02 fL in 3n). However, the decrease in RBC was not compensated by the increase in MCV, and triploidy therefore decreased the haematocrit (Hct: 23.0±2.7% in 2n, 20.6±1.7% in AF3N and 19.5±2.5% in 3n). In contrast, total blood haemoglobin concentration (Hb: 50.53±7.88 g/L in 2n, 58.93±11.20 g/L in AF3N and 58.72±9.86 g/L in 3n), mean corpuscular hemoglobin (MCH: 11.05±0.98 pg in 2n, 24.12±2.29 pg in AF3N and 24.22±3.44 pg in 3n), and MCH concentration (MCHC: 0.22±0.01 pg/fL in 2n, 0.29±0.06 pg/fL in AF3N and 0.30±0.04 pg/fL in 3n) were higher in the triploid group than in diploid flounder. Triploidy-associated changes in haematological features, as determined in the present study, are essential when evaluating the feasibility of triploid olive flounder for intensive aquaculture systems in which unfavorable situations may occur. Early gonad development in diploid and triploid P. olivaceus were carried out using a histological method. Histological analysis of the gonads showed clear differences between the diploid and triploid groups. The ovaries of diploid females had primary growth oocytes (TL 10-20 cm) or early secondary growth oocytes (TL 20-25 cm). However, all ovaries of the triploid group appeared to be filled with considerable number of oogonia (TL 10-25 cm), and a few chromatin nucleolus oocytes (TL 20-25 cm). Even though diploid and triploid testes at TL 10-15 cm attained a similar degree of development consisting of spermatogonia, small gonad sizes in triploid males from TL 15-25 cm were observed compared to the diploid counterparts. Whereas normal spermatids and sperm were present in the testes of the diploid olive flounder, spermatocytes and abnormal sperm were visible in the testes of triploid males at this stage.