Müllerian inhibiting substance (MIS) is a protein that encoded by MIS gene. It has also been called Müllerian inhibiting factor (MIF) and anti-Müllerian hormone (AMH). Mis expression occurs in ovarian granulose cells of females postpartum, and serves as a molecular biomarker for relative size of the ovarian reserve. In humans, the number of cells in the follicular reserve can be used to evaluate the reproductive function and fertility of female. Pagrus major is typical cultured fish in Korea but there is no clear evidence for their gene identification. However, in many teleost, MIS genes were demonstrated already. Present study aimed to identify the Mis gene in Pagrus major and seasonal difference of its expression. Using conserved sequence of the other known teleost Mis genes, we make conserved primers. Pagrus major’s ovary samples were obtained from the sea rim farm (Geoje, Korea) and kept in RNAlater®Solution or fixed in 4% paraformaldehyde containing 0.16% picric acid. RNA was isolated from kept sample and cDNA was synthesized. The PCR products were performed ligation with TOPO vector and transformation in TOP10 cell and sequenced the Mis mRNA fragment. MIS was localized in the follicle cells. Its mRNA levels were higher in summer than spring or fall. Based on them, it is suggested that MIS can be used to estimate the fertility of this fish.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Telomeres at the end of the eukaryotic chromosomes consist of tandem repeats of (TTAGGG)n DNA sequence and shelter in protein complex. Telomeres have the essential functions in chromosome stability and genome integrity and are hence related to cell senescence and cancer. Stripped, Black and White Cattle (Endangered Korean Native Cattle) characterized by their coat color, live in the Korean peninsula. However, they are endangered, with very small populations remaining. To investigate the karyotypic pattern of chromosome and also to quantify the amount of telomeric DNA was carried out from the traditional Korean beef cattle species, HanWoo and endangered cattle bull. We quantified the amount of telomeric DNA by the Quantitative-Fluorescence in situ Hybridization (Q-FISH) technique using the telomeric DNA probe and chromosome analysis of lymphocytes was carried out using GTG-banding in 9 bull at age of 18 months. In results, we found that the normal (60, XY) male karyotype were detected in metaphase chromosomes from korean native cattle including Hanwoo, Stripped, Black and White cattle, respectively. In addition, there were no significant differences in the relative amount of telomeric DNA among the korean cattle bull. However, the relative amount of telomeric DNA of Hanwoo was slightly higher than that of White cattle. In conclusion, this study reported karytype and the amount of telomeric DNA which could serve as baseline information for comparison in conditions of physiological and health status of endangered Korean native cattle. Although we have no definitive explanations as to why this occurs, further investigations are needed to continue investigation of these animals throughout their life spans.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

As an endocrine disruptor, bisphenol-A (BPA) causes several functional and behavioral abnormalities related to reproduction. The current study was design to evaluate the effect of perinatal exposure of female mice to BPA on sperm function of adult F(1) offspring. Pregnant female mice F(0) were gavaged with three different concentration of BPA, such as 50 μg/kg/day (tolerable daily intake value by the European Food Safety Authority), 5 mg/kg/day (no-observed-adverse-effect level; NOAEL), and 50 mg/kg/day (lowest-observed-adverse-effect level; LOAEL) and corn oil (7 mg/kg/day; vehicle control). The functional parameters of F(1) spermatozoa were studied both before and after capacitation, whereas the fertility assessment was evaluated by in vitro and in vivo assay using unexposed females. Our results showed that spermatozoa hyperactivated motility, capacitation, intracellular ATP, Ca2+, and ROS levels after capacitation were significantly affected using NOAEL and LOAEL concentration of BPA. However, the sperm motility was only affected by LOAEL dose after capacitation. All of the tested parameters were potentially unaffected by BPA before capacitation, except intracellular ATP that decreased by all concentrations. Although both NOAEL and LOAEL concentration were effectively reduced the rate of fertilization and embryonic development in vitro, however the average litter size was only affected by LOAEL dose. Our finding suggested that perinatal exposure of 50 μg/kg/day did not produce significant effects; however both NOAEL and LOAEL affects overall sperm function after capacitation, leading to impairments in the fertility of F(1) male offspring.

Although the toxicological impacts of the xenoestrogen bisphenol-A (BPA) have been studied extensively, but its mechanism of action is poorly understood. Eventually, no standard method exists for evaluating the possible health hazard of BPA. Considering mice spermatozoa as a potential in vitro model, here we demonstrated the effects of BPA exposure (0.0001, 0.01, 1, and 100 μM for 6 h) on spermatozoa and the related mechanisms of action. Our results demonstrated that high concentrations of BPA negatively affect sperm motility, viability, intracellular ATP, and mitochondrial functions by activating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A pathways. The same doses were also employed to identify the differential expressed proteins of exposure and screen their functional affiliation to diseases using sperm proteomics and informatics, respectively. Our results demonstrated that a high concentration of BPA (100 μM) induced differential expression (> 2-fold) of 24 proteins in spermatozoa (16 down- and 9 up-regulated), that are putatively involved in the pathogenesis of several diseases. To the best of our knowledge, this is the first study to demonstrate the mechanisms of BPA action in spermatozoa and to identify the possible biomarkers of exposure. Moreover, we anticipated that current strategy might apply for the hazard assessment of other toxicological agents.

Prognosis and diagnosis of male fertility is a most important for animal breeding system and human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Thus, new methods for diagnosis and prognosis of male fertility will need to be developed to ensure more accurate assessments. Proteomics have used to find candidate biomarkers for male fertility, but the relationship between the proteome and fertility was not fully understood. Therefore, we performed a comprehensive proteomic approach to investigate small and large litter size boar spermatozoa and identify proteins related to negative male fertility. In present study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins were abundantly expressed in the small litter group. Interestingly, only one protein was highly expressed in the large litter size spermatozoa. We then identified signaling pathways associated with the differentially expressed protein markers. Glutathione S-transferase Mu3 and glutathione peroxidase 4 were related to the glutathione metabolic pathway and arginine vasopressin receptor 2 was linked to vasopressin R2/STAT. Taken together, our results suggest that identified negative fertility-related biomarkers may be used as negative biomarkers for the detection of inferior male fertility such as sub-fertility or infertility.

Cryopreservation allows for the advances of the reproductive technique and livestock industry. However, cryopreservation inevitably causes various types of stress, such as cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. Although cryoprotectant agent (CPA) is added to protect spermatozoa from freezing damage during cryopreservation, it has intrinsic toxicity that can affect components of the sperm membrane. Moreover, the addition of CPA induces osmotic stress and excessive reactive oxygen species (ROS) generation, resulting in disruption of mitochondrial membrane potential, alteration of membrane permeability, and damage of sperm surface proteins. To identify the effects of CPA to spermatozoa, we analyzed the sperm movement, capacitation status, and viability using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we performed two-dimensional electrophoresis to find protein markers related CPA addition in cryo processes. CPA addition reduced sperm motility (%), viability (%), and non-capacitated spermatozoa, whereas acrosome-reacted spermatozoa increased significantly (p<0.05). Following addition of CPA, a total of ten proteins were altered their expression (eight increased, two decreased) (>3 fold, p<0.05). Among these, four differentially expressed proteins were related to several canonical pathways, such as the ephrinR-actin, ROS metabolism, actin cytoskeleton assembly, actin cytoskeleton regulation, and respiratory chain and oxidative phosphorylation pathway (p<0.05). The present study suggests that CPA significantly alters the functions and proteome content of spermatozoa. Additionally, we anticipated that the differentially expressed proteins might consider as biomarker of CPA-induced stress.

Autophagy is a self-degradative process which accompanies the formation of double-membraned vesicles inside the cell. In the mouse uterus, autophagy is enhanced during steroid hormone deprivation and associated with acute inflammation. There are 17 major Autophagy related genes (Atg). Herein we investigated the role for Atg7 by using uterine cell-specific deletion model of this gene. We crossed Atg7flox/flox (Atg7f/f) mouse and Anti-Mullerian hormone type 2 receptor (Amhr2)-Cre mice (Amhr2-Cre; Atg7f/f). Amhr2 is mainly expressed in stroma and myometrium in the uterus, ovary, and oviduct, during 30 to 60 days. To confirm the region of Cre expression and to monitor whether conditional deletion of Atg7 was by Cre recombinase, we isolated uterine epithelial and stromal cells from 8 and 16 weeks mice by enzymatic digestion and performed RT-PCR. We confirmed that Amhr2-Cre is expressed in stoma and myomotrium, but not in epithelium. Then we examined the uterine histology and embryonic development of day 3 pregnant Amhr2-Cre; Atg7f/f mice. However, there was no specific difference between Atg7f/f (control) and Amhr2-Cre; Atg7f/f mice. To examine the effect of hormone deprivation, we performed western blotting and immunofluorescence staining of p62 (SQSTM1), an indicator of autophagic flux, and LC3B, a marker of autophagic activation, in Amhr2-Cre; Atg7f/f mice ovariectomized (OVX) for 2 weeks. p62 increased dramatically in OVX Amhr2-Cre; Atg7f/f uteri but not in control mice, suggesting that autophagic activation did not occur in the absence of Atg7 in the uterine stroma and that this led to massive accumulation of p62 in this cell type. p62 marks to-be-degraded proteins and target them for autophagic-lysosomal degradation. Thus it is predictable that Atg7-driven uterine autophagy is responsible for degradation of macromolecules during hormone deprivation.

Early growth response(Egr) family는 전사요소로서 Egr1, Egr2, Egr3, Egr4가 알려져 있다. 전사조절 인자로서의 Egr3는 muscle spindle 형성 및 신경 세포에서의 neurite 생성 등 다양한 target 유전자의 발현을 조절하는 것으로 알려져 있다. 본 연구팀은 Egr3가 생쥐 난자의 방추사에 특이적으로 위치하고, 감수분열 과정 중의 microtubule organizing center(MTOC) 주변에서 관찰되며, 마우스 정소의 spermatocyte에서 발현됨을 보고한 바 있다. 본 연구에서는 생식소의 성숙과 Egr3 발현 간의 상관관계를 조사하기 위해 암컷 및 수컷 생쥐의 난소와 정소를 생후 1주, 2주, 3주, 4주에 적출하여 Egr3 RT-PCR 및 면역형광 염색을 수행하였다. RT-PCR 결과, 생후 3, 4주의 난소에서 Egr3의 발현이 미성숙한 1, 2주의 난소에서 보다 더 높게 발현되는 것을 확인하였으며, 1, 2주의 난소에서는 하나의 transcript가 관찰되고 3, 4주의 난소에서는 2개의 transcript가 나타남을 확인하였다. 정소의 경우에도 Egr3의 발현은 유사한 양상을 보였는데, 생후 1, 2주의 정소보다 생후 3, 4주의 정소에서 더 높게 발현되는 것이 확인되었다. 면역 형광 염색 결과, 난소에서 Egr3의 발현이 난포 내 난자를 둘러싸고 있는 cumulus cell과 granulosa cell에 두드러짐을 확인하였다. RT-PCR 결과와 마찬가지로 미성숙한 1, 2주의 난소보다 3, 4주의 난소에서 Egr3의 발현이 더 강함을 확인하였다. 정소에서 Egr3의 발현 위치는 주령에 따라 변화하는 양상을 보였다. 이러한 결과는 암컷 및 수컷 생쥐의 생식소가 성숙함에 따라 생식소 내 Egr3의 발현이 증가함을 보여주며, Egr3가 성적 성숙과 관련된 생식소자극호르몬의 영향을 받을 가능성을 시사한다.

골 형성 단백질(Bone morphogenetic protein, BMP)은 TGF-β superfamily의 구성원 중에 하나이며, 이 들은 원래 뼈 형성을 유도하는 능력에 의해 발견되었지만, 곧 세포분열, 세포자살, 세포이동 그리고 세포 분화를 조절하는 것으로 밝혀졌다. 매우 다기능적인 골 형성 단백질은 포유동물의 reproduction에도 매우 중요한 역할을 하지만 알려진 바가 적다. 그래서 본 연구에서는 골 형성 단백질이 착상 전 oviductal environment에 어떠한 영향을 미치는지 밝히고자 하였다. 먼저, Estrus 시기 생쥐의 oviduct cDNA를 통해 RT-PCR한 결과, BMP1, 2, 3, 4, 5, 6, 7, 8, 10, 15 mRNA와 BMP receptor인 BMPR1A, BMPR1B, BMPR2 mRNA의 발현을 확인하였고, BMP Inhibitor인 Noggin, Follistatin, Chordin mRNA의 발현 또한 확인하였다. 이들 중 BMP2, 3, 7, 15 mRNA의 level이 BMP5, 6, 10 mRNA의 level보다 많은 발현량을 보였다. BMP receptor 중에서는 BMPR2 mRNA의 level이 제일 높았다. BMP Inhibitor 중에서는 Chordin mRNA의 발현량이 제일 많았다. 이들 가운데 가장 높은 발현 수준을 보인 BMP2의 단백질의 localization은 Immunohistochemistry를 통해 확인하였다. BMP receptor와 BMP Inhibitor에 대한 연구는 진행 중에 있지만, 생식주기별 생쥐의 oviduct 내에서 BMP2 단백질의 발현수준은 proestrous, metestrous, diestrous보다 estrous stage에서 가장 강한 발현을 보였다. 이는 BMP2가 steroidogenesis에 영향을 주어 oviductal cell proliferation에 관여한다는 것을 보여준다.

Harmonized actions of ovarian estrogen (E2) and progesterone (P4) regulate cell proliferation and differentiation in the uterus with a spatiotemporal manner. Imbalance between the actions and levels of two major regulators often lead to infertility and gynecological diseases, such as endometriosis and endometrial cancer. While numerous works have shown that reduced expression and/or deletion of uterine factors associated with P4 signaling could disturb uterine physiology, local factor(s) to mediate E2 actions has not been extensively studied yet. Here we demonstrate that early growth response 1 (Egr1), a transcription factor which is rapidly induced in the uterus by E2, is required to maintain coordinated actions of E2 and P4 for uterine receptivity for embryo implantation. Given exogenous gonadotrophins to overcome LHβ deficiency in the pituitary of Egr1(－/－) mice, ovulation, fertilization and embryo development normally occurred in these mice. However, they showed complete failure of embryo implantation with reduced uterine responses to artificial decidualization stimuli. While serum levels of E2 and P4 in Egr1(－/－) mice were comparable, genes regulated by E2 and/or P4 in uterine epithelial cells (ECs) were aberrantly expressed on day 4 of pregnancy. Impaired P4 signaling along with absence of PR in ECs caused hypersensitive E2 responses shown as enhanced expression of E2-responsive genes such Muc1 and Ltf as well as reduced levels of P4-dependent genes, such as Ihh and Areg, in ECs of Egr1(－/－) mice. This is consistent with persistent proliferation in ECs and severely impaired proliferation in stromal cells (SCs) in Egr1(－/－) mice treated with E2+P4. Furthermore, primary co-culture of Egr1(－/－) ECs with Egr1(+/+) SCs and vice versa supported a notion that Egr1 itself is required for proper responses to two major regulators, E2 and P4, in both uterine cell compartments. Collectively, our results show that E2-induced Egr1 participates in P4-dependent modulation on E2 activities in the uterus by regulating a spectrum of genes essential for uterine receptivity and embryo implantation.

Endocrine disruptors are exogenous chemicals that their endocrine disrupting effects mediated by androgenic signaling plays crucial roles in the control of development and several androgen-related diseases. However, there are no authorized in vitro screening and testing methods to evaluation of (anti-)androgenic activity. To find out a better in vitro cell line model, we have previously reported that 22Rv1 cells, a human prostate cancer cells contained functional Androgen Receptor (AR), might be an appropriate model for the evaluation of (anti-)androgenic endocrine disruptors. Based on this result, we developed a stable 22Rv1/mouse mammary tumor virus (MMTV) cell line to test AR-mediated transcriptional activation (TA). Using 22Rv1/MMTV cells, we established the test protocol and optimized the testing condition for AR-TA assay. In this study, we performed the inter-validation assay by four different laboratories to evaluate the 20 coded chemicals which were selected from the ICCVAM list (ICCVAM, 2003) or academic articles that exhibited exact (anti-) androgenic activity. The statistical analysis of the results of the inter-laboratory validation study revealed that there was reproducibility between the four participating laboratories. In conclusion, 22Rv1/MMTV AR-TA assay might be a quick and relatively inexpensive method, which can be used to screen large numbers of chemicals for their potential to activate or inhibit AR-mediated gene transcription. Furthermore, it will provide mechanistic data relevant to understanding adverse reactions observed in intact organisms.

The aim of this study was to enhance the proliferation efficiency of spermatogonial stem cells (SSCs). In order to improve the proliferation efficiency, we investigated new factors that promote the proliferation of SSCs using in vitro culture method with natural plant extracts. Germ cell populations containing SSCs were collected 6- to 8-days-old from C57BL/6-TG-EGFP (C57GFP) mice and SSCs were isolated from the collected cells via magnetic-activated cell sorting (MACS). Since then, SSCs were cultured for a week with culture medium containing natural plant extracts at concentration of 0.1, 1, and 10 μg/mL. After a week of culture, we looked for an increase, especially a dose-dependent increase, in the number of cells compared to that of the control group. A dose-dependent increase, in the number of cells was observed in the Petasides japonicus-treated groups. Furthermore, we carried out repeated experiment that is process consisting of selection and additional segmentation to explore new factors for activating SSCs at the molecular level. As a results, Petasides japonicus butanol fraction significantly increased the proliferation rate of SSCs in a dose-dependent manner among Petasides japonicus fraction samples. We identified normal expression level of PLZF in SSCs cultured with plant extracts using immunocytochemistry method. Furthermore, we also carried out qRT-PCR and identified normal expression level of Lhx1 and GFRα1. The finding of this study could contribute to improvement of proliferation and activation for SSCs, using culture method with natural plant extracts.

To overcome the risk of the ovarian hyperstimulation syndrome (OHSS) in patients have polycystic ovarian syndrome (PCOS) and to prepare emergency fertility preservation in patients undergoing anticancer treatment, several researchers have reported IVM of oocytes retrieved from ovaries exposed by only hCG priming. However, the maturation rate and the developmental potential of embryos from IVM oocytes are significantly lower than those of oocytes matured in vivo. Here, we investigated the optimal time point for immature oocyte collection at post hCG only injection for in vitro maturation, in vitro fertilization and blastocyst formation. Immature GV oocytes were collected from 25 days old B6D2F1 female mouse at 12 hr, 14 hr, 16 hr or 24 hr post hCG injection. Oocytes were collected from antral or late secondary follicle by puncturing with 26 G needle. Collected oocytes were cultured in G2 medium with 10% FBS, FSH, estradiol, and hCG for 16 hr in vitro and subjected in vitro fertilization and further embryonic development. To examine follicular maturation, we estimated the numbers of primordial, primary, secondary follicle and antral follicle on ovaries of each time point post hCG. To confirm the optimal time point post hCG injection for collecting immature oocytes, we recovered the oocytes from each time point. There is no difference in the number of oocytes per mice. Oocytes collected at 14 hr post hCG injection were shown higher maturation rate to MII stage and blastocyst formation compare to other three groups (p<0.01). However, there is no difference in the maturation rate on the other three groups. Also, apoptotic signal with TUNEL assay or anti-PARP staining was not change in ovaries from all experimental groups. Granulosa cell proliferation test with anti Ki-67 or anti AMH was not show any difference. According to these results, there are no significant differences in four different time points at 12 hr, 14 hr, 16 hr or 24 hr of collection of immature oocytes in hCG primed mouse. However, oocytes from 14 hr post hCG injection showed higher percentages of maturation rate, in vitro fertilization rate, blastocyst formation.

The sex of bivalves is classified into gonochorism and hermaphroditism, and hermaphroditism is further divided into simultaneous, and sequential. Simultaneous hermaphroditism is the simultaneous release of eggs and sperm by one organism during the same season. Sequential hermaphrodites are either male or female for one or several annual cycles (Heller, 1993; Gosling 2004; Collin, 2013). T. granosa is a sequential hermaphroditic bivalve undergoing sex change (Lee et al., 2014). However, definitive conclusion on whether the scale and pattern of sex change of T. granosa is always consistent could not be made. Therefore, the objective of this study was to reconfirm sex change in T. granosa and consider the scale and pattern of sex change compared to the results of Lee et al. (2014). The total number of T. granosa used for sex change identification was 777 with a shell length (SL) between 25.1-35.0 (30.9±2.13) mm. For Tegillarca granosa, the scale of sex change during 2006-2007 was reported to be 15.1% (Lee et al., 2014). In this study, the overall scale of sex change in T. granosa was 37.6% during 2011-2012, which was approximately 2.5 times higher than that reported by Lee et al. (2014). In addition, the difference between the sex change ratio from females to males and that from males to females was 15.3% during the period of 2011-2012, which was similar to the finding of 15.0% during 2006-2007 (Lee et al., 2014). The sex change ratio of female→male : male→female was 1 : 3.42 during 2006-2007 reported by Lee et al. (2014). It was 1 : 1.57 during 2011-2012 in this study.

동물의 성은 유전학적 성과 형태학적 성으로 구분되는데, 성의 표현은 일반적으로 형태학적 성을 기준으로 한다. 본 연구는 북방전복의 생식생물학적 정보를 제공하고, 아울러 성의 인위적인 조절에 필요한 정보를 제공하기 위하여 수행하였다. 평균 수온 22℃에서 6개월간 사육한 북방전복의 성분화 과정을 조직학적으로 분석한 결과, 북방전복의 성분화율은 각장 24.2 mm 그룹에서 90.0%였으며, 성비(암:수)는 1:0.64로 나타났다. 북방전복 성체의 생식소는 발과 내장낭 사이에서 긴 원뿔형으로 발달 된 간췌장을 둘러싸고 있으며, 외형의 색깔에 따라 난소는 진녹색, 정소는 옅은 노란색을 나타낸다. 성체의 난소와 정소는 각각 생식소외막에서부터 간췌장까지 생식소 내강 쪽으로 발달된 격벽에 의해 여러 개의 내강으로 구분되며, 각각의 내강에서 생식세포형성소낭(gametogenic follicle)을 따라 생식세포들이 발달한다. 북방전복의 형태학적 성분화 과정은 크게 다음과 같이 5단계로 구분할 수 있었다. 1) 생식소 외막 형성, 2) 장과 간췌장 사이의 결체조직에 시원생식세포(PGC: primordial germ cell) 출현 및 생식소 내강 형성, 3) 생식소 내강 상피층에 PGCs 출현, 5) 생식세포형성소낭(gametogenic follicle) 형성 및 PGCs 출현, 5) 성분화. 북방전복의 각장 5.0~10.0mm 시기에 돌기모양의 간췌장이 형성되어 육안으로 구분이 가능하며, 간췌장의 외막은 이중막으로 형성되어 있다. 이 시기에 안쪽상피층은 편평형 상피세포들로 구성되며, 바깥쪽 상피층은 편평형 상피세포들과 점액세포들로 구성된다. 각장 15.0 mm 전후에는 장과 간췌장 사이의 결체조직에서 처음으로 PGCs의 확인이 가능하다. PGCs의 크기는 약 7.4 μm였으며, 핵의 크기는 약 4.7 μm였다. H-E 염색결과, PGCs는 강한 호염기성의 세포질과 인을 가지고 있었다. 또한, 이 시기에 간췌장의 이중막 가운데 외막이 내막으로부터 분리되면서 생식소 내강이 형성된다. 각장 18.0 mm 전후에는 생식소 내강이 뚜렷해지며, 생식소 내강의 기저부에는 PGCs가 일렬로 배열된다. 이때, PGCs의 크기는 약 10.8 μm였으며, 핵의 크기는 약 7.4 μm였다. 이후, 각장 21.0 mm 전후에는 생식소 내강에 격벽이 형성되고 각각의 격벽 사이에 생식세포 형성소낭이 형성되며, 각각의 소낭 기저부 결체조직에는 PGCs가 증가하며 배열된다. 각장 23.0 mm 전후에 PGCs는 정원세포와 난원세포로 분화되면서 초기 성분화과정이 진행된다. 정원세포들은 크기 감소와 응축과정을 통해 정모세포로 발달하고, 난원세포들은 세포질의 증가와 호염기성의 세포질이 뚜렷한 초기 난모세포로 발달한다.

In animal development, the mechanisms by which localized factors and organelles in egg cytoplasm were exactly distributed into each daughter cell are essential for formation of various cell types. During ascidian Halocynthia roretzi embryogenesis, ooplasmic mitochondria were mainly segregated into muscle and neural precursor cells. At the 32-cell stage, localized mitochondria in the B6.2 blastomeres were preferentially distributed into the B7.4 muscle precursors compared with the B7.3 mesenchyme/ notochord precursors. When the B6.2 blastomeres were isolated from the early 32-cell stage embryos and then allowed to divide 2 times of cell division, the resultant partial embryos showed symmetric distribution of mitochondria, and the partial embryos were composed of equal size cells. In normal development, cell fates of the B7.3 blastomere were correlated with the unequal cleavage of B6.2 lineage cells that normally occurs in the next two-cell division stages to produce a large B8.5 mesenchyme and a small B8.6 notochord cell. Mitochondria are distributed asymmetrically in both cells. When embryos were treated with FGF receptor inhibitor SU5402 and MEK inhibitor U0126 between the 32-cell and the early 64-cell stages, the resultant embryos showed equal cleavage pattern and symmetric distribution of mitochondria in daughter cells of the B6.2 blastomeres. However, blocking of Nodal and Notch signaling did not affect the cell division pattern and mitochondrial distribution in the B6.2 lineage blastomeres between the 32-cell and 110-cell stages. Therefore, it is likely that FGF/MEK signaling is involved in asymmetric distribution of mitochondria and unequal cleavage of the B6.2 lineage blastomeres in ascidian embryo.