개체 사이의 사회적 상호작용은 인간을 포함한 대부분의 척추동물의 적절한 성성숙 및 번식활동을 위하여 중요한 과정이므로, 행동양식을 결정하는 조절인자들은 뇌를 포함한 중추신경계에 분포하고 있으며, 이 신호들을 조절하는 신경내분비계는 척추동물 내에서 잘 보존되어 있다. 척추동물 경골어류의 번식현상은 다양한 환경적, 계절적, 사회적 요소들에 의해 많은 영향을 받지만, 궁극적으로는 이러한 요소들의 자극이 뇌에서 통합되며, 뇌-뇌하수체-생식소 축에 의해서 번식활동이 제어된다. 이러한 신호 전달은 뇌의 시상하부 preoptic 지역에 위치한 생식선자극호르몬방출호르몬(GnRH1) 함유 뉴런에 의해 시작되며, 축삭이 뇌하수체 생식선자극호르몬 생산세포로 투사되어 1차적으로 생식선의 성장과 스테로이드 호르몬 생산을 조절한다. 경골어류의 일부 종은 성적 가소성이 현저하며, 사회적 계급 사회를 형성하는 흥미로운 지견을 제공한다. 아프리칸 시크리드 Astatotilapia burtoni 수컷 성어들의 소수 지배계급층(Territorial dominant, T)은 번식능력이 있으며, 공격적이고 고유 수역에 대한 보호본능이 있지만, 나머지 피지배계급층(Non-territorial subordinate, NT) 수컷들은 번식능력도 없으며, 보호본능을 상실한다. 그러나, NT와 T는 수 시간~수 일 이내에 변화되며, 충분히 가역적이다. 가장 현저한 변화는 4일 이내에 GnRH1 뉴런 세포체의 체적이 크게 변화하는 것이다. 그러나, 이 세포크기의 변화를 유도하며, 결과적으로 GnRH1 뉴런의 신경내분비학적 활성을 조절하는 분자는 현재 알려져 있지 않지만, GnRH1 세포 크기를 조절하는 후보의 하나로서 세포체 크기, 수상돌기의 국소적 단백질 합성, 축삭의 성장, 시냅스 가소성을 결정하는 역할을 담당한다고 알려진 serine-threonine protein kinase에 속하는 TOR(target of rapamycin)가 추정된다. 포유동물의 mTOR는 세포 성장과 시냅스 가소성을 통제하는 기능이 시사되었지만, 포유동물 이외에는 그 기능이 잘 알려져 있지 않다. 또한, GnRH 뉴런과 같은 신경내분비세포의 세포크기를 조절하는지는 불명확하다. 본 연구에서는 GnRH1 뉴런의 세포의 크기에 영향을 미치는 분자로 추정되는 mTOR 억제자로 알려진 rapamycin을 T 수컷의 제3뇌실에 주사하여 9개의 대표적인 번식관련 행동, GnRH1 뉴런의 세포형태, 정소의 생식세포에 미치는 영향을 분석하였다. Rapamycin 주사 14시간 후에는 대조군에 비해 상대적으로 번식관련 행동의 빈도수가 낮게 관찰되는 경향을 보였다. 한편, 36시간 후에는 번식행동의 패턴은 주사하기 전의 패턴과 유사한 상태로 회복되는 현상이 관찰되었다. H/E염색과 광학현미경으로 정소를 관찰한 결과, 정소세포 및 정자의 분포와 발달상에는 큰 차이가 없었다. 현재, Rapamycin 주입에 의한 GnRH1 뉴런의 변화를 관찰하고 있으며, T 수컷의 번식행동 패턴의 변화와의 상관관계를 조사하고 있다.

Three eel species such as Anguilla japonica (AJ), Muraenesox cinereus (MC) and Conger myriaster (CM), belonging to the order Anguilliformes, are the most popular marine products in Korea because of their taste and nutritional value, and Koreans consume them in large quantities. Eel, ecologically important warm water fish species widely distributed on the coast of the Yellow Sea, southern sea and the several sea areas under the natural ecosystem. However, in spite of their economic and scientific consequences, a little information currently exists regarding the genetic levels only of eel species in Korea. In this study, to explicate the genetic distances and differences among geographical eel species, the author accomplished a clustering analysis of three eel species collected from the Yellow Sea. PCR analysis was performed on DNA samples extracted from a total of 21 individuals using seven oligonucleotides primers. Muscle tissues were obtained separately from individuals from Anguilla japonica, Muraenesox cinereus and Conger myriaster, respectively. Eel muscle was collected in sterile tubes, instantaneously placed in liquid nitrogen, and stored at －40℃ until the genomic DNA extraction. Genomic DNA was extracted and purified under the conditions described previously (Yoon, 2008). After several washings, lysis bufferⅠ (155 mM NH4Cl; 10 mM KHCO3; 1 mM EDTA) was added to the samples, and the mixture tubes were gently inverted. The concentration of the extracted genomic DNA was measured by optical density at 260 nm by a spectrophotometer (Beckman Coulter, Buckinghamshire, UK). PCR was performed using two Programmable DNA Thermal Cyclers (MJ Research Inc., Waltham, MA, USA). Euclidean genetic distances within- and between-species were also calculated using the hierarchical dendrogram program Systatver.10 (SPSSInc., Chicago, IL, USA). Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers generated 191 specific loci in the AJ species, 226 in the MC species and 181 in the CM species, respectively. The specific loci generated by oligonucleotides primers exhibited inter-individual-specific characteristics, thus revealing DNA polymorphisms. The gDNA isolated from three eel species were amplified by PCR. Here, the seven oligonucleotide primers were used to generate the unique shared loci to each species and shared loci by the three eel species. With regard to average bandsharing value (BS) results, individuals from Conger myriaster species (0.808) exhibited higher bandsharing values than did individuals from Muraenesox cinereus species (0.729) (P<0.05). The dendrogram resulted from reliable seven oligonucleotides primers, indicating three genetic clusters composed of group I, group II and group III. The longest genetic distance (0.430) displaying significant molecular difference was also between individual no. 01 within Anguilla japonica eel species and individual no. 04 within Anguilla japonica species. From what has been said above, the potential of this analysis to ascertain diagnostic markers for the identification of three eel species has also been verified (McCormack et al., 2000; Yoon, 2008).

Red spotted grouper (Epinephelus akaara) is one of the most popular and important grouper species for aquaculture in South-East and East Asia thanks to its fast growth and high market value. This species is known as a protogynous hermaphrodite which first differentiates into females and changes to males later. Natural sex change in this species occurs at 2 or 3 years of age. Many studies have been conducted, so far, to develop standardized methods for artificial sex reversal by treatment with sex steroid hormones (Bhandari et al., 2005; Lee et al., 2014). However, sex-changed male groupers showed testes with ovarian cavity, and their sperm production was very low. Induction of primary sex differentiation directly into males would be an alternative approach. Identification of the exact primary sex differentiation period is the prerequisite for this approach. Red spotted grouper were reared in indoor tanks and acclimatized at 25±2℃ under natural photoperiod. Fish were sampled at 60 days after hatching. The samples were processed for histological analysis using standard techniques in an automatic tissue processor, sectioned at 8 μm in thickness and stained with haematoxylin and eosin. The result of this study shows the morphological characteristics of gonadal primordium, and suggests that the timing of gonadal differentiation in red spotted grouper is at least 10 days earlier than the previous study of Lee et al. (2014) which observed gonadal primordium at 70 days after hatching.

Light characteristics are very specific in the aquatic environment. Fish vision and different light spectra perception are related to each species’ natural habit. Light is one of the main environmental conditions and can be easily manipulated in artificial rearing settings. Mucus-secreting goblet cells are the main regulators of digestion. In this study, we established whether the light spectrum (natural condition, full spectrum: green, 520 nm; red, 590 nm, and blue, 480 nm) influences growth performance and digestive activity related to mucus-secreting goblet cell activity in order to develop a good management protocol and optimal rearing system for nursery stage of Epinephelus akaara. For each light spectrum, fish (11.5 ± 0.2 g in mean initial body weight, 9.0 ± 0.1 cm mean initial total length) were reared 16 weeks under a flow-through system and fed commercial pellet diets twice daily. At the end of the experiment, the final body weights differed among the fish reared under different light spectra. The highest growth performance value and feed efficiency were observed in fish reared under the green light condition. Mucus-secreting goblet cell activity was significantly higher in the fish under green light condition than in the fish under the natural, red, and blue light conditions. Rearing of E. akaara under the green light condition had positive effects on fish growth performance and digestion. We recommend that the appropriate light spectrum for nursery stage of E. akaara is the green light condition from the perspective of growth performance and the synergistic effects of mucus-secreting goblet cells. However, longer light treatment periods are needed in future investigations to clarify the effects of light spectrum on each growing stage of E. akaara.

어류의 소화활성은 다양한 소화호르몬에 영향을 받으며, 특히 cholecystokinin과 점액분비세포는 소화기관에 중요한 생리적 역할을 담당한다. 소화호르몬인 cholecystokinin(CCK)은 어류의 뇌와 소화관에서 합성, 분비되며, 장 운동을 활성화시키고, 담낭 수축, 위산과 췌장에서의 분비를 촉진시키며, 또한 식욕을 억제하는 역할을 하기도 한다. 이 연구에서는 바리과 어류인 붉바리 치어를 대상으로 수온에 따른 붉바리의 성장과 이에 따른 소화관 내의 소화호르몬인 CCK의 발현 양상과 점액분비세포인 배상세포의 활성에 대해 조사하였다. 이를 위해 붉바리 치어를 자연수온, 20℃, 24℃, 28℃로 4개의 그룹으로 나눠 2014년 12월부터 2015년 6월까지 7개월간 사육 실험을 진행하였으며, 4주 간격으로 전장과 체중을 측정하였다. 수온에 따른 붉바리 치어의 소화활성을 분석하기 위해 각 그룹별로 5마리씩 각각 뇌와 위, 유문수, 전장, 중장, 후장을 채취하여, 배상세포 활성을 분석하였다. CCK gene은 붉바리의 뇌 조직을 이용하여 클로닝하였고, CCK gene의 조직발현은 뇌, 뇌하수체, 눈, 아가미, 신장, 간, 심장, 소화관, 생식소, 근육을 샘플링하여 분석하였다. 실험 시작 시 자연수온, 20℃, 24℃, 28℃ 실험구의 전장은 각각 7.5±0.7 cm, 7.1±0.6 cm, 7.2±0.6 cm, 7.2±0.5 cm이었고, 체중은 각각 6.80±1.6 g, 6.05±1.5 g, 6.42±1.6 g, 6.16±1.4 g이었다. 실험 종료 시 자연수온, 20℃, 24℃, 28℃ 실험구의 전장은 각각 10.6±1.3 cm, 13.3±1.3 cm, 16.1±1.6 cm, 18.3±1.5 cm이었고, 체중은 각각 21.1±22.3 g, 40.5±11.8, 81.7±22.3 g, 126.0±36.3 g이었다. 성장률을 확인한 결과, 28℃, 24℃, 20℃, 자연수온 순으로 높게 나타났다. CCK의 조직발현 확인 결과, 뇌와 소화관에서 비교적 높은 발현을 보였고, 조직분석을 통한 배상세포의 활성 차이는 실험구 간의 유의적인 차이가 나타나지 않았다. 이 연구를 통해 붉바리 치어의 적정 사육 수온은 24~28℃일 것으로 추정되며, 소화호르몬인 CCK와 점액분비 세포인 배상세포가 소화활성에 영향을 미친 것으로 생각되나, 수온에 따른 CCK와 배상세포의 소화활성에 대한 연구가 더 필요한 것으로 사료된다.

The four transcription factors Oct4, Sox2, Klf4 and c-Myc have been used for making induced pluripotent stem cells. Many efforts have focused on reducing the number of transcription factors, especially c-Myc and Klf4 known as oncogene, for making induced pluripotent stem cells. Recently it have been demonstrated that Oct4 and Sox2 are able to reprogram human fibroblasts or cord blood cells to induced pluripotent stem cells and Oct4 has the ability to reprogram mouse and human neural stem cell to induced pluripotent stem cells. These researches imply cell types for reprogramming experiments have great influence on selection of reprogramming factors. Here we report that pig kidney cortex fibroblasts need only c-Myc factor when they are used for making induced pluripotent stem cells. We used two vector system including drug-inducible vector system and constitutive expression vector system. The two systems generate induced pluripotent stem cells from pig kidney fibroblasts successfully. These one-factor induced pluripotent stem cells are not only similar but also different to pig embryonic stem-like cells. These two one-factor induced pluripotent stem cell lines can express pluripotency related genes and be differentiated into all three germ layers in vitro. However, these two cell lines can be sub-cultured as a single cell by trypsin. Our results support that single factor, c-Myc, is sufficient to converting pig kidney cortex fibroblasts into induced pluripotent stem cells.

Estrogen plays an important role both in male and female reproduction. Two estrogen receptor isoforms, Esr1 and Esr2, are expressed in male gonad. In the mouse, Esr1 is expressed in Leydig cells of testis and pituitary. Esr1-/- male mice show enhanced androgen synthesis, spermatogenic defect, and infertility. To evaluate the specific function of Esr1 in Leydig cells, we examined spermatogenesis and steroidogenesis in Esr1f/fCyp17iCre male mice in which Esr1 is deleted specifically in Leydig cells. These mice showed normal spermatogenesis and fertility when compared to wild type from young adulthood to old age. Testosterone synthesis in Esr1f/fCyp17iCre mice at 3-12 months old of age was not different from age-matched wild type mice, while, at 18 months old of age, circulating testosterone concentrations were significantly higher than wild type together with increased levels of Star, Cyp17a1, and Hsd17b3 mRNA and with a hypertropy of Leydig cells. In Esr1f/fCyp17iCre mouse pituitaries, Fshb and Lhb mRNA levels were not different from wild type from young adulthood to old age. Taken together, Esr1 in Leydig cells may be not essential for spermatogenesis and fertility under control of endogenous estrogens and may have a role in aged Leydig cell function.

Sirtuin-1(Sirt1)은 NAD+ dependent deacetylase로써 유전자 침묵, 포도당과 지질대사, 세포자살 등 다양한 cellular process에 연관되어 있다. 최근 이뤄진 여러 연구에서 Sirt1 Knockout mouse가 정자형성 장애와 불임증상을 가진다는 것이 보고되었다. Egr1(Early Growth Response 1)은 Egr family에 속하는 전사인자로서 mitogenesis와 differentiation에 필요한 유전자들의 전사를 활성화시킨다. 또한 이는 Sirt1의 전사를 조절하는 것으로 알려져 있다. 본 연구에서는 생쥐 정소 내에서 Egr1의 유무에 의한 Sirt1의 발현 수준 차이를 밝히고자 Egr1 knockout mouse와 wild type mouse를 이용하여 Sirt1의 발현패턴을 분석하였다. Sirt1의 발현을 분석하기 위하여 Quantitative RT-PCR과 conventional RT-PCR을 이용하여 각각 ICR strain adult male mice와 Egr1 Knockout adult male mice의 testicular cDNA에서 mRNA 수준에서 Sirt1의 발현수준을 분석하였다. 또한 Immunohistochemistry를 이용하여 Sirt1단백질의 localization을 분석하였다. 분석한 결과, Wild type과 Egr1 Knockout mouse의 정소 내에서 Sirt1 mRNA의 발현 수준은 큰 차이를 보이지 않았으나, Egr1 Knockout mouse의 정소 내에서 Sirt1 단백질의 발현수준은 wild type과 비교하여 현저히 떨어졌으며, 이 변화는 특히 pachytene spermatocyte에서 큰 차이를 보였다. 반면, Sertoli cell에서는 Pachytene spermatocyte에서만큼 뚜렷한 차이를 관찰할 수 없었다. 본 연구에서 얻은 결과를 바탕으로 Egr1이 pachytene spermatocyte에서 Sirt1 단백질의 발현에 중요한 역할을 할 것이라는 결론을 얻을 수 있었다. Sirt1 Knockout mouse가 불임 증상을 보이며, 정자형성에 장애가 있는 반면, Egr1 KO mouse는 정상적인 생식능력을 가짐으로 보아 premeiotic 또는 meiotic stage에서의 Sirt1 결핍이 이러한 infertility의 원인이 될 것으로 추측된다.

The blastocyst should initiate the dynamic changes in morphology and gene expression during hatching and implantation. Blastocyst morphogenesis includes two major events as the formation of blastocoel cavity for lineage differentiation into trophectoderm and inner cell mass, and the blastocyst hatching for implantation. However, there is little known about the relation of dynamic morphogenesis in blastocyst with hatching and implantation potential. In this study, we investigated effects of the dynamic morphogenesis in blastocyst on hatching and implantation potential by outgrowth assay. The cumulative time between each stages was calculated and analyzed. The feature of contraction was evaluated as follows: the number of contractions and the period of circumference was measured. The percentage of reduction during contraction was classified as weak when it was less than 20% and as strong when 20% or more. Compared to embryos of hatching group, embryos of non-hatching group were significantly delayed time at the compacted morula stage by 375.3 min (p<0.05) and at the early blastocyst stage by 650.1 min (p<0.01), respectively. Compared to blastocysts of outgrowth group, blastocysts of non-outgrowth group were significantly delayed at the compacted morula by 404.0 min (p<0.01) and at the early blastocyst stage by 535.4 min (p<0.01), respectively. There is no significant difference in the feature of contraction between hatching and non-hatching groups. However, blastocyst of outgrowth group showed more number of weak contraction and less number of strong contractions, compared with blastocysts of non-outgrowth group (p<0.01). Period of circumference was not significantly different in hatching and outgrowth process. These results suggested that time of blastocoel formation and number of weak contraction in blastocysts were closely related to hatching and outgrowth potential. Dynamic changes of blastocyst formation and contraction could be useful markers to select embryos for predicting the success implantation and pregnancy in human ART program.

Cytokinesis is the final event in the cell division. After cytokinesis, one parent cell divided into two symmetric daughter cells. Unlike somatic cell which is symmetrically divided, oocyte meiotic maturation is highly asymmetric division, producing mature ovum and polar body. Class III phosphoinositide 3-kinase (PI3K) has been known as a key molecular component that regulates cell cycle progression, autophagy and endosomal trafficking. However, emerging evidences suggest that class III PI3K and its interactors are involved in midbody abscission during cytokinesis. Here we showed that beclin-1, a key component of PI3K is required to regulate midbody abscission during oocyte asymmetric division. Beclin-1 was widely distributed during meiotic maturation forming small vesicles. However, these vesicles were not colocalized with autophagosomal marker LC3. Instead, beclin-1 was detectable at the midbody ring during cytokinesis. Depletion of beclin-1 showed various defects including the failure of cytokinetic abscission, spindle separation and chromosome decondensation. Similar phenotype was observed when class III PI3K activity was inhibited. Therefore, our results demonstrate that PI3K is essential for cytokinesis but not autophagy during oocyte meiosis.

We sought to identify optimal temperatures for aquaculture of juveniles at a length of 7.2 cm ± 0.1 cm. The impact of four temperatures (20℃, 24℃, 28℃, and 32℃) on feeding rate, growth performance, and stress responses (i.e., blood cortisol, glucose, alanine transaminase [ALT], and aspartate transaminase [AST] content) of juvenile red-spotted grouper was measured over a period of 6 wks. Red-spotted grouper had significantly higher final body weight (g) at 24℃ and 28℃ than at 20℃ or 32℃, and greater percentage body weight increase/day at 24℃ than at 28℃. There were significant differences in feeding rate, depending on the number of weeks. The 6-wk feeding rate was highest at 28℃, followed by 32℃, 24℃, and 20℃, while the lowest feed conversion ratio occurred at 24℃, followed by 28℃, 32℃, and 20℃. After 6 wks, there were no significant differences in plasma cortisol or AST levels. However, there were significant increases in plasma glucose and ALT levels at 32℃ compared to at 20℃. Although red-spotted grouper at 32℃ did not show a significant increase in blood cortisol levels, our results suggested that at 32℃ the red-spotted grouper experiences high energy-demand (i.e., high glucose level) and stress (i.e., high ALT), especially in the liver. Based on body weight, % body weight increase/day, feed conversion ratio, and stress indicators, the optimal temperature for aquaculture of red-spotted grouper was ~24℃. Additional studies at temperatures between 24℃ and 28℃ will be required to identify the exact optimal temperature for juvenile red-spotted grouper culture.