Coxsackievirus and adenovirus receptor (CAR) is a member of the Ig-type superfamily of cell adhesion molecules. In polarized epithelial cells CAR is expressed at the tight junction. The mouse CAR gene is composed of at least eight exons, and CAR splice variants that differ at the end of the cytoplasmic tail have been identified in a number of tissues. The present study aimed to examine the expression of (CAR), a TJ protein sealing the muticellular contact point during preimplantation embryos and role of CAR in the formation and integrity of the blastocoel. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG in oviducts and uterus, and we also obtained gestation day 5 and 6.5 embryos in uterus. All samples were subjected to RT-PCR, immunofluorescence and immunohistochemistry analysis. To analyze epithelial permeability of CAR, we examined permeability of FITC-labeled dextran (MW 40 kDa) following functional blocking of CAR in blastocysts. Long isoform of CAR mRNA was expressed from throughout the preimplantation development and markedly increased at morulae stage onward. Small amount of short isoform CAR mRNA was expressed at blastocyst stage. On Western blot, 64 kDa protein was detected together with 43 kDa protein corresponding to short and long forms CAR, respectively in blastocysts. CAR immunoreactivity was found in cell contacts between blastomeres from 4-cell stage onward. Under Ca2+ switching condition blocking antibodies for CAR increased the permeability of blastocysts to FITC-dextran, a permeability tracer. At 5 dpc, trophoblasts of the implanting embryos were immunoreactive with anti-CAR IgG. At 6.5 dpc, the egg cylinder stage in mouse, the visceral and parietal endoderm were immunoreactive with anti-CAR IgG. Our results suggest that alternative splicing of CAR transcript is highly dependent on the development of expanding blastocyst. CAR may play a crucial role as a barrier to adenovirus infection and adhesion molecule for epithelial permeability during peri-implantation period.

Amnionless (AMN) is a plasma membrane protein that binds to cubilin and megalin in various epithelia and mediates endocytosis of extracellular ligands. This function has been studied in the kidney where it plays a key role in vitamin B12 and vitamin D homeostasis. Present study aimed to elucidate developmental pattern of expression of AMN during the peri-implantation period in mouse embryos. In an effort to understand functional role of AMN in the histiotropic nutrition in blastocyst, endocytotic function of AMN for apoplipoprotein was examined in blastocyst. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. To obtain embryos, females were mated with males. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG by flushing oviducts and uterus, and we also obtained gestation day 6.5, 7.5 and 8.5 embryos in uterus. All samples were subjected to quantitative RT-PCR, whole-mount immunofluorescence and immunohistochemistry analysis. To analyze endocytotic function of AMN, we examined uptake experiment of FICT labeled apolipoprotein A-I (ApoA-I-FITC) following functional blocking of AMN in blastocysts. AMN and cubilin mRNA was expressed in all developmental stages of mouse embryos. Megalin was the first detected at morula stage. AMN protein was expressed in trophectoderm (TE) and inner cell mass (ICM). AMN and cubilin were expressed in visceral endoderm of GD 6.5 and 7.5 embryos and visceral yolk sec of GD 8.5 embryos. In normal IgG treated embryos, ApoA-I-FITC was detected in intracellular vesicles of TE and ICM. However, in the presence of anti-AMN antibody, ApoA-I-FITC was weakly detected in apical surface of plasma membrane of TE. To date, AMN has been believed to be expressed in visceral endoderm of post implantation embryos. Our results demonstrated that AMN is the important molecular partner of cubilin and megalin in the preimplantation embryos and that AMN mediates endocytosis of apoplipoprotein, which may play a crucial role in embryonic development and normal growth via supporting histiotropic nutrition during peri-implantation period.

Water channel proteins, aquaporins (AQPs) contribute to transepithelial water movement in many tissues. To date, 13 mammalian AQPs have been identified. Of these, AQP5 plays an important role in the fluid homeostasis and cell volume control in epithelial cells. In an effort to understand the role of AQP5 in testis, we investigated the expression of AQP5 in developing mouse testis, its regulation by estrogen and LH, and the change of steroidogenesis by AQP5 knockdown. Testes and Leydig cells were isolated from male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and estrogen receptor alpha knockout (ERαKO) mice. In mouse testis, AQP5 immunoreactivity was negligible by PND 14. From PND 28 onward, AQP5 immunoreactivity was found in Leydig cells. In ERαKO mouse Leydig cells, AQP5 mRNA level was significantly lower than wild type. In primary adult Leydig cell culture, the expression of AQP5 mRNA was increased by 17β-estradiol (E2) and human chorionic gonadotropin (hCG), but was not changed in ERαKO Leydig cells. Moreover, the expression of AQP5 mRNA was increased by E2 and ERα-selective agonist PPT, but was not changed by ERβ-selective agonist DPN in primary Leydig cells and mLTC-1. In silico analysis and chromatin immunoprecipitation (ChIP) assay revealed that there are putative estrogen response elements (EREs) and cAMP response elements (CRE) in AQP5 promoter region. Testosterone secretion and steroidogenic pathway genes (StAR, Cyp11a1, Cyp17a1, and 3β-HSD6) expression were decreased by AQP5 siRNA in primary Leydig cells. In conclusion, AQP5 expression was coupled with functional differentiation of adult Leydig cells. AQP5 may play an important role in the fluid homeostasis and cell volume control during development of adult Leydig cells. The expression of AQP5 in Leydig cells could be regulated by ERα and LH signaling and AQP5 may be involved in steroidogenesis.

설치류에서 약 200여 가지의 homeobox 유전자가 밝혀져 있고, 그 중 1/3은 생식소에서 발현하는 것으로 알려져 있다. Homeobox는 전사인자를 암호화 하는 서열로 이 중 Rhox cluster는 개체 발생과 생식에 중요한 역할을 한다. 총 13가지로 Rhox 유전자 중 Rhox5는 포유류에서만 발견되는 homeobox 유전자로 주로 정소, 부정소, 난소, 자궁과 같은 생식과 관련된 조직에서 선택적으로 발현되며, 다른 종류의 Rhox5 발현에 영향을 미치는 master Rhox이다. 생쥐 부정소에서 Rhox5는 두부에서 주로 발현되는 반면 쥐에서는 미부부정소에서 발현되는데 이는 차등적 promoter(proximal, distal) 사용에 따른 것으로 알려져 있다. 본 연구에서는 생쥐의 출생 후 성숙과정에서 부정소 부위별 Rhox5 발현을 분석하는 한편, 성체 생쥐 부정소에서 남성호르몬에 의한 Rhox5 발현 변동을 분석하였다. 신생 생쥐, 생후 1, 2, 4, 8주령의 수컷 생쥐의 부정소를 대상으로 정량적 RT-PCR 및 면역조직화학 법으로 Rhox5 발현을 확인하였다. 성체 부정소에서는 caput, corpus, cauda 부위별로 Rhox5 발현을 확인하였다. 성체 생쥐 부정소에서 남성호르몬에 의한 Rhox5 발현 변동을 분석하기 위해 성체를 거세시킨 후 2주 후 5a-dihydrotestosterone(5aDHT)을 7일간 투여하고 마지막 투여 6시간 후 분석에 공시하였다. 주령별 생쥐 부정소의 RT-PCR 결과, Rhox5 mRNA는 출생 직후, 출생 후 1, 2주령의 생쥐에서는 발현되지 않았고, 출생 후 4, 8주령의 생쥐에서는 발현이 유의적으로 증가하였다. 성체 부정소의 caput, corpus, cauda에서 caput에서 Rhox5 mRNA 발현이 유의적으로 높았다. 면역조직화학 염색결과 수출관, 두부의 상피조직의 핵에서 다량 발현되었고 체부에서 가장 낮고 미부에서 소량 발현하였다. 거세 생쥐 부정소에서는 두부에서 Rhox5 mRNA 및 protein이 급격히 감소하였고 5aDHT에 의해 부분적으로 회복되었다. 거세 생쥐 부정소 체부와 미부에서는 Rhox5 mRNA 발현에는 큰 변화가 없는 반면 protein은 증가 하였고 5aDHT에 의해 다시 감소하였다. 결론적으로 생쥐 부정소에서 Rhox5 발현은 사춘기 직전 아직 남성호르몬이 충분치 않은 조건에서 이미 개시되고 사춘기에 추가적으로 증가하므로 부정소 내재적 인자에 의해 Rhox5 유전자 전사가 조절되는 것으로 사료된다. 성체 부정소에서 Rhox5 발현은 두부에서 매우 높으며 부분적으로 남성호르몬의 조절을 받으며, 체부와 미부에서는 억제되던 Rhox5가 남성호르몬 결핍 시 비정상적으로 유도되는 것으로 사료된다. 특히 체부와 미부에서는 Rhox5 mRNA와 단백질 발현량이 일치하지 않는 것으로 보아 Rhox5는 번역후 조절을 받는 것으로 사료된다.

Sirt1 belongs to class III histone/protein deacetylase. Sirt1 KO mice have spermatogenic defects. In this study, expression of Sirt1 and acetylated histone 3k9 (H3k9ac) was examined in developing mouse testes. Among two splicing variants of sirt1 mRNA long form was dominant in developing testis. Testicular sirt1 mRNA levels were low at birth, increased until 14 days post partum (pp) and remained constant thereafter. Sirt1 immunoreactivity was weak to negligible in gonocytes, moderate in spermatogonia, absent in preleptotene spermatocytes, moderate in zygotene spermatocytes, strong in pachytene spermatocytes, and weak in diplotene spermatocytes. Round and elongating spermatids were negative for Sirt1. Acetylated histone 3k9 (H3k9ac) immunoreactivity was moderate in spermatogonia and weak to negligible in preleptotene to pachytene spermatocytes but strong in round and elongating spermatids. In Sertoli cells, nuclear Sirt1 immunoreactivity was absent at birth, increased at 14 days pp and markedly increased at 28 days pp onwards. In immature Sertoli cells culture, FSH and testosterone increased sirt1 mRNA, suggesting that Sirt1 participates in protein deacetylation events during the differentiation of Sertoli cells by gonadotropin. In the Leydig cells, nuclear Sirt1 immunoreactivity was weak until 2 weeks pp and decreased in 4 weeks pp onward. suggesting the protein deacetylation by Sirt1 in Leydig cell precursor/progenitor cells. Mutually exclusive expression between Sirt1 and H3k9ac in pachytene spermatocytes in testis suggests that deacetylation of H3K9ac by Sirt1 participates in the gene silencing and/or chromosome behavior in pachytene sspermatocytes.

Expression of claudin-11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testes. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the non-breeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonular occludens-1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those of non-breeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the BTB where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJ’s expressing CLDN11 at the BTB in amniotes.

단백질의 기능을 다양화하기 위한 하나의 방법으로 전사 후 단백질 변형(post-translational modification)을 통해 단백질 활성이 조절된다. 또한, 최근 단백질의 sumoylation에 의한 활성 변화와 조절기전에 관해 많은 연구가 진행되어 여러 종류의 small ubiquitin-like protein(Ubl)이 밝혀졌다. 그 중 SUMO-1(small ubiquitin-related modifier 1)에 의한 sumoylation을 통해 androgen receptor(AR)가 modification 되어진다. Sumoylation은 protein을 targeting하고, 단백질을 안정화시키며, 전사를 활성화시키는 다양한 역할을 가지고 있다. 본 연구에서는 부정소에서 sumo-1 발현에 미치는 남성호르몬의 기능을 규명하고자 하였다. 본 연구에서는 생후 8주인 수컷 생쥐를 이용하여 정소가 제거 되지 않은 생쥐(Sham)와 정소를 제거한 생쥐(ORX), 정소를 제거한 후 1주간 Dihydrotestosterone(DHT)를 subcutaneous에 주사한 생쥐(ORX+DHT) 등으로부터 적출한 부정소를 이용하였다. 면역조직화학법을 이용하여 부정소 내 SUMO-1의 발현 부위와 정량적 RT-PCR를 통해 SUMO-1 mRNA 발현을 두부, 체부, 미부로 나누어 분석하였다. 또한, Western blot을 이용하여 부정소 내 sumo-1에 의한 단백질 패턴을 확인하였다. 생쥐 부정소에서 SUMO-1 immunoreactivity는 Sham군보다 ORX군에서 강하게 발현되었으며, ORX+DHT군에서 다시 감소하는 것으로 확인되었다. 특히, ORX군에서 두부보다는 체부와 미부에서 강하게 발현된 것을 확인하였다. 정량적 RT-PCR에 의하면 두부와 체부 부정소에서는 Sham군에 비해 ORX군에서 SUMO-1 mRNA 발현이 유의적으로 감소하였다. 하지만 미부에서는 변화가 나타나지 않았다. 또한, sham군보다 ORX+DHT군 미부에서 SUMO-1 mRNA 발현 유의성이 증가하였지만 두부와 체부에서는 변화를 나타내지 않았다. ORX+DHT군에서 SUMO-1 mRNA 발현이 ORX군에 비해 모든 부위에서 유의적으로 증가하였다. Western blot에서는 각 부정소에서 단백질 발현 패턴이 다른 것을 확인하였고 특히 sham군에서 발현된 단백질은 ORX군에서는 발현되지 않았으며 ORX군에서 발현된 단백질은 sham군에서는 발현되지 않았다. 부정소에서 sumo-1 전사와 단백질량은 음의 상관성을 보였으며 sumo-1의 전사는 두부, 체부가 유사하고 미부는 이들과 다른 양상을 보였다. 각 부정소에서 단백질 발현 패턴이 상이한 것을 알 수 있었고 남성호르몬에 의해서 ORX군에서 감소하고 증가한 단백질들을 전반적으로 회복시키는 것으로 사료된다. ORX군 상피조직의 핵에서 sumo-1 증가는 AR의 감소를 수반하는 것으로 보아 상피세포에서 AR등과 같은 전사인자의 분해를 촉진하는 것으로 추측된다. 반대로 AR이 감소한 경우 남성호르몬에 의한 AR의 전사활성 감수성을 증가시키기 위한 기작으로 추측할 수 있는 것으로 사료된다.

Aromatase is an enzyme that converts testosterone to estrogen. This enzyme, present in the sperm as well as various tissue and cells, has been considered to be related to the fertility of human and mouse sperm. Therefore, we examined effect of aromatase inhibitor on viability and fertility of sperm, and quantity of aromatase in sperm groups with different density in pig. To analyze the effect of aromatase on sperm viability, we treated aromatase inhibitor to the sperm with different concentrations (0, 10, 20, 50, 100, 200, 500 μM) at different time (0.5, 1, 2, 4, 8 hours). After the treatment, the sperm viability was calculated by hypo-osmotic swelling test. We selected 0, 50, 100 μM concentration during 0.5 hour as inhibitor treatment condition before in vitro fertilization. Next, we examined fertility and quantified aromatase protein in sperms with different density. In the first experiment, viability of sperm was decreased following the increasement of inhibitor concentration. The aromatase inhibited sperm showed lower penetration rate and cleavage rate than those of non-treated sperm. Concentration of 50 μM inhibitor had no significant effect on the sperm viability, but it significantly reduced sperm fertility. Second, sperms with low density showed higher penetration rate, but no significant difference between sperms with high density. In conclusion, aromatase is responsible for viability and fertility of porcine sperm similar to mouse and human, however, density of sperm has no correlation with quantity of aromatase protein.

The present study aims to investigate the effect of BPA on sperm functions, fertilization and to evaluate their association with the activity of fertility related proteins in spermatozoa. We used a comprehensive in vitro test system to evaluate the effect of various concentrations of BPA (0.0001, 0.01, 1, and 100 μM) on mouse spermatozoa following 6 h of incubation. Our results showed that high concentration of BPA inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa. Simultaneously, exposure of spermatozoa to high concentrations of BPA increased the tyrosine phosphorylation of sperm proteins involved in PKA-dependent regulation and induced a robust AR, ultimately results in poor fertilization and compromised embryonic development. Finally, BPA effects on selected group of fertility related proteins in spermatozoa, such as it degraded the β-actin, whereas the levels of peroxiredoxin-5, glutathione peroxidase, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase were increased. Based on these results, we propose that high concentration of BPA may alter overall sperm functions, fertilization and embryonic development, in association with degradation and/or phosphorylation of fertility related proteins in spermatozoa.

난자에서 감수분열이 일어나는 동안 centromere와 microtuble의 부착이 일어난다. 이때 잘못된 부착이 일어나면, 염색체 비분리로 인해, 배아의 염색체 이수성, 착상실패 및 자연유산을 야기시킨다. 그러나, 이러한 중요성에도 불구하고, 난자의 감수분열에서의 동원체와 미세소관의 부착에 대한 연구가 미미한 실정이다. 이에 본 연구에서는 난자의 감수분열동안 염색체와 미세소관 부착의 연결고리 역할을 하는 단백질 Zwint-1에 관하여 분석하였다. Zwint-1은 바깥 표면의 동원체 단백질로써 RZZ복합체(Rod/Zwilch/Zw10), KMN(KNL-1/Mis12/ Ndc80)복합체와 상호작용을 한다고 알려져 있다. 본 연구에서는 미세주입법을 통한 RNAi 방법을 사용하여 난자에서 Zwint-1의 발현을 Knock down 시켰다. 그 후 RT-PCR법을 이용하여 난자내의 Zwint-1의 발현감소를 확인하였으며, 면역형광법을 이용하여 Zwint-1의 위치와 단백질 발현 등을 관찰하였다. 또한 Zwint-1과 상호작용하는 단백질인 Zw10의 위치를 확인하였으며, Spindle assembly checkpoint(SAC)의 활성을 조사하였다. 이를 통해, 난자의 감수분열 동안, Zwint-1이 kinetochore와 microtubule의 부착을 매개함으로써, SAC의 활성을 조절하는 것을 확인하였다. 따라서, 본 연구결과는 Zwint-1이 체세포 분열뿐 아니라, 난자의 감수분열에서도 kinetochore와 microtubule의 부착을 조절하는 중요한 매개인자임을 보여준다.

The histological changes in the Ussurian bullhead, Leiocassis ussuriensis, and the Korean bullhead, Pseudobagrus fulvidraco, were observed during the early period of growth. The retinas size of both species increased in the 9 days post-hatching (DPH) (P<0.05). In the just-hatched Ussurian bullhead, the retina already consisted of six layers: the epithelial layer, ganglion cell layer, inner nuclear layer, inner plexiform layer, outer limiting membrane layer, and rod and cone layer. The Korean bullhead had the same components. At 50 DPH, the thickness of the retina was 538.0±7.19 μm in the Ussurian bullhead and 558.9±9.44 μm in the Korean bullhead. The relative thickness of each layer of the retina did not differ significantly in the two species. Although the growth of the Korean bullhead’s retina was faster, the relative thickness of each layer in the retina did not change during early development. After hatching, some parts of the tissue gradually became denser. Immediately after hatching, the kidney and midgut epithelium of the Ussurian bullhead and Korean bullhead were already formed and grew gradually thereafter. From 0 DPH to 30 DPH, the nuclear height in the midgut epithelium did not differ significantly between the two species, but at 50 DPH, it was 11.4±2.45 μm in the Korean bullhead and 9.9±2.13 μm in the Ussurian bullhead. During the experimental period, the major axes, minor axes, surface areas, and volumes of the proximal tubule cells in the kidney did not differ significantly between the two species. Thus, the early histological development of the Ussurian bullhead is similar to that of the Korean bullhead.

Measurements of closely related sets of classical and truss dimensions were analyzed to discriminate species of scorpaenidae including the dark banded rockfish, Sebastes inermis, the black rockfish, S. schlegeli, and gobioninae including the striped shiner, Pungtungia herzi, and the slender shiner, Pseudopungtungia tenuicorpa. The measurements of the dimensions were arc sin square root transformed, and compared as a function of the standard length of each species for statistical analysis. For values of the classical dimensions of the rockfish, 6 were greater for the dark banded rockfish than for the black rockfish, 1 value was smaller for the former, and for 2 values there was no statistically significant difference (P>0.05). For values of the classical dimensions of the shiners, 9 values were greater for the striped shiner than for the slender shiner, 2 values were smaller for the former, and for 1 value there was no statistically significant difference (P>0.01). For values of the truss dimensions of the rockfish, 6 were greater for the dark banded rockfish than for the black rockfish, 1 was smaller for the former, and for 4 values there was no statistically significant difference (P>0.05). For values of the truss dimensions of the shiners, 13 values were greater for the striped shiner than for the slender shiner, 3 values were smaller for the former, and for 6 values there was no statistically significant difference (P>0.01). The dimension sets used in this study may be useful as taxonomic indicators for discriminating among fish species in Korea.

외과적으로 정소를 제거하는 물리적 거세와 약물을 이용한 화학적 거세는 의학적 치료나 연구의 목적으로 다양하게 사용되고 있다. 최근 흰쥐 정소에 20%의 고장성 식염수를 직접 주사할 경우 혈중 테스토스테론 수준이 외과적 거세와 유사한 수준까지 저하되어 화학적 거세 상태에 들어감이 보고되었다. 그러나 고장성 식염수를 정소에 직접 주사함으로서 나타나는 염증반응으로 인해, 안전성에 대한 우려가 제기되고 있다. 본 연구는 기존 연구에서 보다 낮은 농도의 식염수를 주사하여 농도에 따른 효과와 효용성을 조사하고자, 조직학적 변화를 확인하였다. 성체 수컷 흰쥐에 멸균한 5%, 10%, 20% 생리 식염수를 정소 당 500 ㎕씩 직접 주사하여 농도별 식염수 주사군(saline-injection)과 대조군(control)로 분류하고, 실험군의 체중을 측정하여 기록하였다. 주사 후 1주 뒤에 모든 동물들의 체중을 측정한 후 희생하였다. 각 실험군의 체중 변화와 조직 무게 변화를 비교하였고, 생식기관인 정소, 부정소, 저정낭, 전립선은 Hematoxylin & Eosin 염색법을 사용하여 조직학적 변화를 관찰하였다. 정소 무게는 5% 주사군과 20% 주사군에서 유의하게 감소하였고, 10% 주사군에서는 대조군에 비해 감소하였으나 유의하지는 않았다. 부정소의 경우 20% 주사군에서 매우 유의하게 감소하는 양상을 보였고, 저정낭의 무게는 10% 주사군과 20% 주사군에서 유의하게 감소하였다. 전립선의 경우 20% 주사군에서만 유의하게 감소하였다. 기타 조직에서는 면역기능을 담당하는 비장의 무게가 10% 주사군에서 유의하게 증가하는 양상을 보였고, 신장은 20% 주사군에서 유의하게 감소하였다. 조직학적 관찰 결과, 10% 주사군과 20% 주사군의 정소에서 전체적인 형태 변화와 세정관의 손상을 확인할 수 있었고, 부정소는 20% 주사군의 경우 도관 내 정자가 거의 관찰되지 않았다. 저정낭의 경우 농도의존적으로 내강이 감소하며 위축되는 양상을 보였다. 전립선은 20% 주사군에서 확연한 내강의 감소와 도관의 격리됨을 확인하였다. 본 연구는 기존에 보고된 정소 내 식염수 주사모델의 독성 문제가 제기됨에 따라 보다 낮은 농도에서의 효용성을 조사하고자 한 것이다. 생식 기관의 조직학적 조사를 통해 비교적 낮은 농도의 식염수 주사모델이 기존의 20% 식염수 주사 모델과 유사한 효과를 나타냄을 확인하였다. 그러나 물리적, 화학적 거세모델과 유사한 거세 효과를 유발할 수 있는지에 대해서는 보다 심층적인 연구가 필요할 것으로 사료된다.

Ovarian tissue cryopreservation and transplantation causes follicle depletion. To overcome this problem, we investigate the effect of Anti-Müllerian hormone (AMH), a follicle recruitment control hormone, supplementation before and/or after mouse ovarian transplantation. A total of 120 5-week-aged BD F-1 female mice were used. The mice were randomly divided into four groups according to AMH doses (0, 5, 25, 125 μg/mL, respectively). AMH was injected intraperitoneally on every other day for a week before, after, or before and after transplantation of ovaries under kidney capsules was performed. One week after transplantation, follicular normality was evaluated by histological analysis and TUNEL assay. In Group A and C, morphologically intact follicle (G1) ratios of AMH treated groups showed no statistically significant difference. In Group B, G1 ratios of 25 and 125 μg/mL of AMH treated groups were higher than those of 5 μg/mL treated group, but there was no improvement in G1 ratio after AMH treatment. In every group, apoptotic follicle ratios did not show any trend according to AMH treatment. Proportions of primordial follicle were not significantly different according to AMH treatment in all groups. The result of the present study demonstrated that AMH treatment during on transplantation of cryopreserved ovaries has no significant effect on follicle survival and prevention of follicle depletion.

Polyethyleneglycol-adsorbed–superoxide dismutase (PEG-SOD), has been proposed as an effective agent for reducing free radical-mediated injury. The objective of this study was to investigate a protective effect of PEG-SOD supplementation on ovarian tissue during transplantation. Ovaries from F-1 mice were collected and vitrified. After warming, ovaries were autotransplanted under kidney capsule. Mice were randomly divided into four groups according to dose of PEG-SOD, (0 U/ml, 100 U/ml, 1,000 U/ml and 10,000 U/ml respectively). Grafted ovaries were retrieved 2, 7 and 21 days later. PEG-SOD was treated by intraperitoneal injection once every 48 hours and especially for 21 days group, after first week treatment, PEG-SOD was treated once every 4 days. Morphology of ovaries was assessed histological analysis and ELISA for FSH was performed to evaluate restoration of ovarian function. In 2 days groups, morphologically intact follicle ratio of 10,000 U/ml group was significantly higher than other groups. In 7 days groups, morphologically intact follicle ratio was significantly higher in all treatment groups. In 21 days groups, there was no significant difference of intact follicle ratio in total follicles in all groups but intact primordial, primary and secondary follicles ratio was higher in 10,000 U/ml group. FSH levels in blood serum were decrease as time goes on, but there is no statistical difference in each groups. In conclusion, the data of the present study show that PEG-SOD has a beneficial effect on preservation of the morphologically intact follicle.

The follicle loss of transplanted ovarian tissue (OT) is caused by ischemia and slow revascularization. To shorten the ischemic period and promote angiogenesis, some angiogenic factors have been treated for transplanted tissues. Angiopoietin-2 (ANG-2) is one of the major angiogenic factors and has been reported to promote blood vessels and increase vascular permeability in the ischemic and/or hypoxic environment. So, this study was designed to assess the impact of ANG-2 on follicle integrity and revascularization of mouse OT grafts. The 5-week-old B6D2F1 female mice were divided into 3 groups (a control and 2 ANG-2 groups) followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without ANG-2 injection (50 or 500 ng/kg), and then killed at day(D)2, 7, 21 and 42 after transplantation. Total 2,437 follicles in OT grafts were assessed for the follicular density, integrity and classification by H&E staining. Apoptosis, revascularization, and serum FSH levels were evaluated by TUNEL assay, CD31 immunohistochemistry, and ELISA respectively. All the ANG-2 groups showed remarkable increase of morphologically intact follicle ratio across all the grafting duration except D21 (no statistical difference). The numbers of CD31(+) vessels (the sum of 3 fields at ×400 magnification) were significantly increased in both ANG-2 groups compared with the control group at all the grafting duration. Especially at D42, the 500ng ANG-2 group showed significantly more vessels than the 50 ng ANG-2 group as well as the control group. However the mean follicle numbers of grafts, apoptosis ratio and serum FSH levels showed no significant difference among the groups. In this study, remarkably well preserved follicles and larger amount of vessels were appeared in ANG-2 treated groups. So we thought that ANG-2 treatment is effective for OT transplantation and improve transplantation outcomes.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

As a preliminary investigation into the effect of environmental factors control for gonadal development, we examined the involvement of photoperiod and water temperature in ovarian development of Epinephelus. akaara. For the induction of sexual maturation, E. akaara reared in recirculating aquaculture system (RAS). During November 2013, the photoperiod and water temperature was adjusted to 12L:12D and 18℃, respectively. In the photo-thermal treatment group, every 3 weeks daylight was increased as follows a 13L:11D and 14L:10D, and control group was maintained under natural condition. After 9 weeks, water temperature was increased 23℃ in photo-thermal treatment group. The sampled fish every 3 weeks revealed increase in gonadosomatic index (GSI; 5.18±1.38), oocyte diameter and vitellogenic oocytes (423.9±36.1 ㎛) were observed in gonads 12 weeks under photo-thermal treatment group. However, ovarian development was maintained immature stage in control group. In this environmental factors manipulation trial, seventy one of the 95 females (578.4 ± 25.4 g in mean body weight, 31.0 ± 0.5 cm mean total length) treated with HCG injection (doses 500 IU/kg BW) were induced ovulation by artificial stripping. The total volume of ovulated eggs were 3,470 ml and the total volume of fertilized eggs was 3,295 ml. The fertilization rate and hatching rate were 95% and 98%, respectively. These results suggest that the photoperiod as well as water temperature are major environmental factors in triggering the gonadal development of E. akaara.

염색체분리를 위해서는 미세소관과 동원체의 부착에 관여하는 단백질들이 정확한 시기와 위치에서 조절이 되어야 한다. 이 과정은 주로 Chromosomal passenger complex(CPC)에 의해 조절되게 되는데, 체세포 분열에서는 Histone H3 Thr 3 자리의 인산화가 CPC를 동원체로 불러오게 함으로써 염색체 분리의 과정이 가능하게 해준다고 알려져 있다. 이러한 기작은 미세소관이 동원체에 비정상적으로 부착되었을 시에 발생하는 오류를 바로 잡을 수 있게 해준다. 그러나 이에 대한 과정은 아직까지 감수분열에서 알려진 바가 없다. 먼저, 감수분열에서의 Histone H3 Thr 3의 인산화 활성을 알아보았다. Histone H3 Thr 3의 인산화는 제 1 감수분열 전기에서 중기까지 염색체 전부에서 점차적으로 증가하는 양상을 보였다. 하지만 제 1 감수분열 후기로 접어들면서 염색체의 팔에서 발현되었던 인산화는 줄어들고, 동원체에서만 발현되는 것을 확인할 수 있었다. Histone H3 Thr 3 인산화 기능을 알아보기 위해서, 이를 인산화 하는 Haspin 카이네즈 저해제를 난자에 처리하였다. 저해제가 처리된 난자에서는 Histone H3 Thr 3의 인산화가 사라지면서 세포주기가 중기에서 멈췄고, CPC가 염색체에서 확산되면서 염색체가 적도판에서 비정상적으로 정렬되었다. 따라서 본 연구의 결과를 토대로 감수분열 동안 Histone H3 Thr 3의 인산화 활성이 염색체의 분리가 적절한 시기와 위치에서 이루어지게 하는 중요한 역할을 한다고 할 수 있다.

Melatonin has several known physiological functions, the main one being synchronization of daily and seasonal rhythms. In addition, melatonin has been reported to influence reproduction and behavioral rhythms with varying results depending on the species. To date, it remains unknown how this rhythm in locomotor activity is controlled endogenously, although there must be coordination of chemical and molecular drivers. However, the species is poorly characterized at molecular level with little sequence information available in public databases. The aim of study was to clarify involvement of endogenous melatonin rhythms and locomotor activity in day-night activity of the eel, Anguilla japonica which is an economically important but endangered species. The levels during daytime (zeitgeber time; ZT 6) were significantly (P<0.05) lower than those during nighttime (ZT 18). A similar pattern was persisted under DD conditions, whereas it disappeared under LL conditions and ocular melatonin levels remained low. Therefore, it is likely that ocular melatonin levels of the nocturnal eel reared under LD and DD conditions fluctuate in a daily/circadian manner and night-related physiological processes are dependent on eel locomotor activities which is a nocturnal species. We found that similar number of genes were differentially expressed between day (ZT6) and nighttime (ZT18), suggesting that during the nighttime also important in differential gene expression with daytime. This work also provides essential information for further studies investigating the molecular basis of daily/circadian system in this species.