Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.
Cinnamon clownfish, Amphiprion melanopus, typically live as part of a mated adult pair or an adult pair and an immature individual and social ranking in the group controls the sexes of the fish. In general, the female is the larger and dominant individual. If a dominant female dies or is absent, the male partner undergoes a sex change to become a female. This special reproductive characteristic provides a good model for which to study the mechanism of sex change. Sexual development and gonadal maturation in fish are regulated by hormones of the hypothalamus-pituitary-gonad (HPG) axis including gonadotropin-releasing hormone (GnRH) and other neurohormones, gonadotropins, and gonadal steroid hormones and peptides. Specially, light is among the most important natural environmental factors that regulate reproduction in fish. Light-emitting diodes (LEDs), which is a new form of lighting technology can be designed to output specific wavelengths such as narrow bandwidth light which is important because it can be tuned to the environmental sensitivity of a target species. In the experiment, the white fluorescent bulb and different LED (red, peak at 630 nm; green, 530 nm) were used. The effects of different illuminations were assessed by measuring the mRNA and protein expressions of HPG axis genes and sex steroid hormone level. For green LED light, significantly higher levels of HPG axis genes expressions, GSI, and plasma sex steroid hormone were obtained, compared to the red LED light spectra. Histological analysis also revealed the presence of vitellogenic oocytes in fish exposed to green LED light. The results indicate that exposure to short wavelength lighting accelerates gonadal maturation and sex change, and is likely to facilitate development of more energy-efficient aquaculture procedures. Also, have investigated that light reaction in marine organism's mechanism using LED lights, and high potential of application of Bio-photonics for the aquaculture.
The advent of Next Generation Sequencing (NGS) technology has changed the research paradigm and become an essential tool for recent biological and medical study. In today’s market, there are several sequencing platforms which have specific sequencing principle, and the result of each sequencing platform has different characteristics among them. Hence, each sequencing method became more specialized for specific research purpose, and researchers who consider NGS analysis have to understand the very basic characteristics of each NGS platform. NGS is used in various studies and they are usually classified into 5 categories (Re-sequencing, RNA-seq, de novo assembly, Metagenomics and Epigenomics) of analysis. In this session, we will introduce the characteristics of sequencing platforms and examples of recent research on each of the 5 analysis categories. In addition, we will talk about the benefit of NGS study compared to the traditional study and how these NGS technologies can be applied in developmental biology research.
Ovarian cancer is the most fetal gynecological malignancy leading cause of cancer-related deaths in women worldwide. Diagnosis of ovarian cancer is hard at an early stage when 90% of patients can be cure due to lack of symptom and early detection markers. Therefore, most of patients with this disease are detected at advanced stage (Stage Ⅲ-Ⅳ) occurring low survival rate (< 30%). More than 90% of ovarian carcinomas are originated from ovarian surface epithelium and it is called as epithelial-derived ovarian cancer (EOC). Recently, previous studies have been showed ovarian cancer could arise from oviduct and oviduct-related genes are up-regulated in hen EOC, the most relevant animal model. Therefore, the objectives of this study were to determine: 1) the distribution and localization of oviduct developmental-regulatory genes including A2M, GAL11, SERPINB3, SERPINB11 and SPP1 in normal and cancerous ovaries of laying hens; 2) the expression pattern of target genes among normal and cancerous ovarian cells of hens and human ovarian cancer cell lines; and 3) the functional role of target gene in human EOC. Results of the present study showed five genes were abundant only in the glandular epithelium of cancerous ovaries of hens. And SERPINB3 was abundant in the nucleus of both chicken and human ovarian cancer cells whereas SERPINB11 was abundant in the cytoplasm. Further, several microRNAs were discovered to influence SERPINB3, GAL11 and SPP1 expression via their 3’-UTR which suggest that post-transcriptional regulation influences target gene expression in chickens. Moreover, in 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) patients showed weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance, and for poor progression-free survival. Therefore, oviduct developmental-regulatory genes, especially SERPINB3, may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC.
The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin- like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy; 2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cells; and 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA- transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1; by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processes; however, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.
Epigenetic change is dynamic during germ cell development. DNA methylation and histone modification are the most important epigenetic process to regulate the gene expressions. They are very close reciprocal relationship on the specific genomic regions called CpG islands (CGI). The CGIs are located on the promoter regions and recruit various epigenetic regulators including, CFP1, KDM2A, KDM2B, TET1 and MLL. They contain a common domain which is the zinc finger CxxC domain. The CxxC domain reads non-methylated CpG and recruits other regulatory elements such as SET1, PRC, COMPASS and SIN3A to modify Histone proteins. CFP1 contains a CxxC domain. CFP1 protein therefore imposes an ability to distinguish its important regulatory element, “non-methylated CpG” from the genome. After binding the CpG, CFP1 recruits SET1 complex, which is involved in the histone H3 lysin 4 (H3K4) methylation. However, the functional consequence of CFP1 in the germ cell development remains unknown. In this study, we demonstrated that CFP1 is critical for the both spermatogenesis and oogenesis using conditional knockout system.
Epigenetic regulations including DNA methylation, long noncoding RNAs and histone modification are considered to be involved in many biological processes. Such regulations in general begin with change of covalent bonds on the substrates. Moieties involving the covalent bond include methyl- and acetyl-group, glucose, SUMO and etc. Among them, methyl group-mediated modulation is commonly observed in all three substrates. Mouse primordial germ cells (PGCs) first appear at embryonic day (E)7.25 on the base of the allantois, and then migrate through the hindgut to the genital ridge. Once PGCs reach genital ridge, they become dimorphic, in that female PGCs undergo meiosis whereas male PGCs are mitotically arrested. Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA. I will discuss role of Ten-to-Eleven translocation (Tet) in DNA demethylation in the process of PGC reprogramming.
The NGS technologies of genome DNA structure, expression profiling and epigenome elements have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454, Illumina/Solexa and PacBio systems along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. One of the advantages of the NGS systems is the cost-effectiveness to obtain the result of high-throughput DNA sequencing for genome, RNAnome, and miRNAnome studies. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data and for resequencing the samples with a reference genome DNA sequence. To construct high-quality contig consensus sequences, each DNA fragment read length is important to obtain de novo assembly with long reading sequences of the Roche/454 and PacBio systems. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly, as hybrid assembly for novel genome sequencing would be cost-effective. In some cases, Illumina/Solexa data are used to construct scaffolds through de novo assembly with high coverage depth and large diverse fragment mate paired-end information, even though they are already participating in assembly and have made many contigs. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. MAQ and CLC software are useful to both SNP discovery and genotyping through a comparison of resequencing data to a reference genome. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples, such as control and exposed tissue. The long read sequence data of PacBio are more powerful to find full length cDNA sequence through de novo assembly in any whole-genome sequenced species. An average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference EST sequence. In an in silico method, conserved miRNA and novel miRNA discovery is available on massive miRNAnome data in any species. Particularly, the discovered target genes of miRNA could be robust to approach genome biology study.
Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing LIF and bFGF. After establishment, this cells were cultured and stabilized in LIF or bFGF contained media. This cell lines displayed a dome-shaped colony morphology in both culture condition. The cell lines were maintained in both condition over an extended time period and were able to differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or bFGF expressed different pluripotency markers. LIF-dependent pEGCs expressed naive-pluripotency markers such as OCT4, SOX2, NANOG and SSEA1, while bFGF-dependent pEGCs expressed primed-pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. However, as a result of analysis of XCI, two cell lines showed hemi-methylated pattern similarly in XIST promoter regions. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Pluripotent state of pEGCs were regulated by modulation of culture condition. In LIF supplement, pEGCs showed naive-pluripotency expressing SSEA1, while pEGCs show primed-pluripotency expressing SSEA4 in bFGF condition. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.
Throughout their meiotic maturation in most mammals, oocytes are arrested twice, prophase I and metaphase II. Being released from these arrests, transient or oscillation of intracellular Ca2+ concentration is observed in the ooplasm, which is not answered in relation to the specific role in the resumption of meiotic arrest. Recently, Ca2+/calmodulin-dependent protein kinase II (CaM KII) has been known as a Ca2+ oscillation decoder from the in vitro experiment. CaM KII is multifunctional serine/threonine kinase observed in most cells. Present studies were performed to investigate the role of CaM KII during resumption of meiotic arrest and activation in vitro of mouse oocytes. It was questioned whether CaM KII might be involved in the meiotic resumption of mouse oocytes. Compared to the control, both of CaM KII inhibitors, KN-93 and KN-62, significantly inhibited germinal vesicle breakdown (GVBD) of mouse oocytes in a dose-dependent manner. As the concentration of KN-93 increased, concomitant decrease of intracellular Ca2+ concentration ([Ca2+]i) was also observed using confocal laser scanning microscope (CLSM) and an intracellular Ca2+ indicator, fluo 3-AM. When GVBD oocytes were treated with 6% ethanol, small [Ca2+]i transient was observed in oocytes bathed with Ca2+-free medium and large increase was observed in oocytes bathed with Ca2+-containing medium, suggesting that [Ca2+]i transient could happen from intracellular Ca2+ store as well as Ca2+ influx through Ca2+-channel on the oolemma. However, KN-93 inhibited the [Ca2+]i transient of GVBD oocytes in both cases. Using monoclonal antibodies against α-subunit of CaM KII, tubulin and microtubule-assocaited proteins (MAPs), CaM KII has been colocalized on the spindle with tubulin and MAPs. The present study also demonstrated the presence of α-subunit of CaM KII in heart, kidney, testes, ovary as well as in brain of the mouse. In ovarian follicles, CaM KII was expressed in granulosa cells and oocytes. Based on overall the above results, followings are suggested. First, CaM KII might be involved in the regulatory mechanism of meiotic resumption. Second, CaM KII might play a regulatory role in the stabilization of microtubule.
Developing preimplantation embryos require appropriate energy source and express stage specific gene expression for proper development. During early stage embryo development, major energy sources were pyruvate and lactate, after then glucos is used as a main source. Aquaporins (AQPs), also, is suggested as key molecules for blastocoels formation, and energy and meytabolic homeostasis. In this study, we analyzed the expression profiles of AQPs and lactate dehydrogenase (LDH) which convet lacte to pyruvate and back. During development in vivo condition, the expression patterns can be classificed six clusters. AQP2,-3, -5, -8, -9, and -11 were detected at various stages but others were not. Cluster 1 is for an only express at blastocyst stage. Cluster 2 is for an incrase continuosuly from 4-cell stage. Cluster 3 is for a peak at both 4-cell and blastocyst stages. Cluster 4 is for a sharp decrease at morula stage. Cluster 5 is for a sharp decrase at 2-cell and morula stages. Cluster 6 is for continuous decrease after 4-cell stage. Cluster 7 is for no expression AQPs. LDHs expression has three patterns. First is for sharp decrase at both 2-cell stage and morula a stage. Second is for a continuous decrease from 4-cell stage. Third is for an existings until fertilized oocyte, 1-cell stage. Interestingly the expression profiles of AQPs and LDHs were totally changed by in vitro culture. All of the AQPs and LDHs were detected except AQP8. The leves of LDHA and LDHB were significantly decreased in vitro but those of LDHC and LDHD were increased. These results suggest that early stage embryos themselves adaptate to their conditon through modulation of the specific gene expression such as AQPs and LDHs.
Objective : To investigate the effects of Simvastatin and Methylprednisolone on ovarian tissue cryopreservation and transplantation using mouse models. Methods : The mice were randomly distributed into 1 control and 3 experimental groups. The B6D2F1 mice were given oral Simvastatin (5 mg/kg), intravenous Methylprednisolone (15 mg/kg), or a combination of both at 2 hours before ovariectomy. Same volume of normal saline was given perorally in the control group at 2 hours before ovariectomy. The ovarian tissues were vitrified accrording to our protocols. The vitrified ovaries were warmed 1 week later and auto-transplanted under bilateral kidney capsules. The ovaries and blood sera were collected at 2, 7 or 21 days after transplantation. Histological analysis, TUNEL assay, immuno-histochemistry for CD31, serum AMH level and embryonic development after in vitro fertilization were assessed for evaluation. Results : With regard to the total grade 1 follicle rate, both Simvastatin or Methylprednisolone treated groups were significantly increased at 2, 7 or 21 days after transplantation (except Simvastatin treated group at 7 days). A combination of Simvastatin and Methylprednisolone group was significantly improved in terms of the total G1 follicle rate, apoptotic follicle rate, CD31 positive area and serum AMH after ovarian tissue transplantation. However, there were no statistically difference with respect to the oocyte maturation rate, blastulation rate, and the other embryonic development parameters after in vitro fertilization procedure among the four groups. Conclusion : Our results suggest that combined donor Simvastatin and Methylprednisolone have beneficial effects on the quality and function of transplanted ovarian tissues.
We investigated the change mRNA expression of GtHs subunits (FSHβ, LHβ) in the pituitary, androgen receptor (AR), estrogen receptor (ERα) in gonad and histological observation of gonads in longthooth grouper Epinephelus bruneus by treatment Femara, an aromatase inhibitor (AI). Longtooth grouper (body weight 408±43.1 g; one year) cultured in Future Aquaculture Research Center, NFRDI were used in the experiments. The experiment was conducted for 12 weeks from 21 August 2013. Fish received intramuscular injection of AI at 5 mg/g BW dose in three times every 3 weeks. Fish were sampled pituitary and gonads at 3, 6, 12 weeks post-injection (n=50). The mRNA levels of FSH-β, LH-β in pituitary and AR, ERα mRNA in gonad were evaluated using qRT-PCR and qPCR. The histological change of gonads observed on light microscope. The gonads of control group contained most perinucleolus oocyte. At 3 to 6 weeks post-injection, the gonads of AI-treated group contained a few degenerated oocytes, spermatogonia and spermatocytes. At 12 weeks post-injection, gonads contained spermatids undergoing spermatogenesis. From 6 to 12 weeks post-injection, the expression level of GtHs subunits mRNA in pituitary was significantly higher than control group. The expression level of AR mRNA in gonad was higher than control group from 3 to 12 weeks post-injection. The expression level of ERα mRNA in gonad was lower than control group from 6 to 12 weeks post-injection. These results suggest that immature longtooth grouper with AI treatment induced masculinization via change of GtH subunits in pituitary, AR and ERα mRNA in gonad.
Adipogenesis is critical in development and homeostasis of energy metabolism. However, in these days, the obesity has become prevalent and became a cause of medial complication. Various applications have been suggested to prevent or decrease accumulaiton of energy in fat cells. However, those have little usefulness and have various side effects. Diphlorethohydroxy-carmalol (DPHC) is a phlorotannin compound, with various biological activities in vitro and in vivo. In here, we studied that DPHC could modify the accumulation of fat on integument. The size of adipocytes and thickness of the subcutanous fat tissue was analyzed after treatment of cosmetics contained 0, 0.01, 0.1, 1, or 10 % DPHC using NIS Element D 4.10.00 software (Nikon). The viability and proliferation of cell was analyzed after 0, 0.4, 2, 10, or 50μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay (R&D system, Cat # 48090-025-k) and measurment of doubling time. Accumulation of lipids in differentiating preadipocytes was analyzed with spectrophotometer after Oil Red-O staining. The size of adipocyte and thickness in skin was decreased in DPHC treated mice. The metabolic activity and doubling of 3T3-L1 were suppressed by DPHC in concentration dependent manner. DPHC also inhibit accumulation of lipids in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC has antiobesity effects in integument through suppress accumulation of lipids and suppress the proliferation and differentiation both of adipose stem cells and precursor cells.
We previously reported that DNA hypermethylation of SRY promoter is associated with emergence of male-to-female sex reversal. The normality of offspring is achieved by relatively complete and correct nuclear reprogramming during somatic cell nuclear transfer and cloning process. The purpose of this study is to determine whether DNA demethylation of SRY promoter induced by 5-aza-2'-deoxycytidine (AzC) DNA methylation inhibitor may get back phenotypic XY sex reversal female to normal male in SCNT cloning. Canine femoral skin fibroblast cells were established from SCNT-cloned XY sex reversed female (GSF335). Using bisulfite genomic sequencing analysis, DNA methylation levels of SRY promoter in non-treated (normal) and 1uM AzC-treated cells were 88.4% and 55.3% in treatment for 4 days respectively. Seven SCNT-cloned puppies were cloned using the AzC-treated cells as donor cell. Six of those clones showed phenotypically normal male, through one puppy (GSF451) was only observed into male-to-female sex reversal with female genitalia. In umbilical cord tissue, DNA methylation levels on SRY promoter of GSF451 clone and the other clones were 79.2% and 5.7% to 62.2% respectively, which was approximately similar to those of non-treated (normal) and AzC-treated cells. Also, cloned puppies originated from AzC-treated cells implied significantly multiple body weight and height compared to age-matched SCNT-cloned control, which may be underlying in size-effect of AzC-treatment. Our findings suggest that DNA demethylated status of SRY promoter induced by AzC is likely to facilitate normal development including sex differentiation through epigenetic alteration of donor cells.
Glycogen synthase kinase-3 α/β는 Apoptosis나 cell survival, 성장인자들과 상관된 다양한 신호전달 중요한 효소이다. GSK-3는 GSK-3α와 GSK-3β인 두 개의 isoform이 존재한다. GSK-3의 아미노산 서열에서 tyrosine 214서열의 인산화, GSK-3α에서는 Serine 21번, GSK-3β에선 Serine 9번의 인산화를 통해서 신호전달기능이 조절된다. 일반적으로 GSK-3는 항상 활성화된 상태이며, 이 활성화가 억제되었을 때 신호 전달이 매개된다. 본 연구에서는 8주령의 수컷 생쥐의 정자를 대상으로 하였다. Western blot을 이용하여 GSK-3α/β, p-GSK-3α/β(tyr 214), p-GSK-3α(ser21), p-GSK-3β(ser9)의 발현을 확인하였다. 또한 면역조직화학법을 이용하여 정자 내 발현부위를 확인하였다. 정자 내 GSK-3α/β의 발현 패턴을 분석하기 위해 생쥐의 뇌, 신장, 생쥐 정소 gonocytes 세포주(GC1-SPG)를 비교 분석하였다. 뇌와 GC1-SPG 세포주에서는 GSK-3α/β는 모두 존재하는데 반해 생쥐 정자와 신장에서는 GSK-3α가 dominant하게 발현됨을 확인하였다. 정자의 수정능 획득에 의한 GSK-3α/β의 발현과 인산화의 변화를 알아보는 실험을 하였다. 부정소 미부의 정자를 추출해 배양하지 않은 정자, 수정능이 생기지 않는 Non capacitated Media (NCM)에 배양한 정자, 수정능을 얻게 하는 Human tubal fluid (HTF)에 배양한 정자들을 western blot을 통해 확인하였다. 배양을 한 정자에서 GSK-3α의 serine 억제성 인산화가 증가하였다. GSK-3α/β의 tyrosine 인산화 역시 증가하는 결과를 보였다. Capacitation에 관한 연구에선 PKA pathway가 매우 중요하다. Forskolin의 처리에 따른 Adenylic cyclase 활성화에 의해 GSK-3α/β serine 억제성 인산화가 증가하였으며, 배양을 통한 capacitation을 진행할수록 GSK-3α/β tyrosine 인산화가 증가하였다. 뿐만 아니라 cell-permeant cyclic adenosine monophosphate analog인 8-Br-cAMP(8-Bromoadenosine 3',5'-cyclic monophosphate)와 phosphodiesterase inhibitor인 IBMX (3-isobutyl-1-methylxanthine)를 이용해 세포내 cAMP 레벨을 증가시켜 PKA pathway 활성화하였다. 이 실험에서도 GSK-3α/β tyrosine 인산화, GSK-3α/β serine 억제성 인산화가 증가하였다. PKA 특이적으로 억제하는 H-89 dihydrochloride hydrate를 통해서도 역시 GSK-3의 억제성 인산화가 감소된 것을 볼 수 있었고 전체 적인 정자의 tyrosine 인산화 패턴이 감소된 것을 볼 수 있었다. 생쥐 부정소 정자에서는 GSK-3α가 우세한 것으로 나타났다. 생쥐 뇌와 신장에 비해서 발현양은 적게 발현된다. 이에 따라 생쥐의 정자에서는 GSK-3α의 기능이 중요하다고 사료된다. 생쥐 부정소 두부 보다 미부에서 GSK-3α/β tyrosine 인산화와 GSK-3α serine 억제성 인산화가 증가하였으므로 정자의 운동성과 수정능이 획득될수록 GSK-3α/β 신호 전달이 활성화되는 것으로 사료된다. 이번 연구를 통해 GSK-3α/β는 PKA pathway dependent한 요소로서 기능하며 포유류 정자 내부의 운동성과 수정능에 대한 신호조절 체계 이해에 필요한 것으로 사료된다.
Early growth response 1(Egr1)은 zinc finger transcription factor로 다양한 성장인자, 물리적 자극에 따른 세포분화, 생장 및 사멸조절에 관여한다. Egr1 KO 생쥐 정소에서 maturation arrest, Sertoli cell only 등의 비정상 세정관이 증가한다. Glial Cell Derived Neurotrophic Factor(GDNF) 는 정원줄기세포(spermatogonial stem cell)의 자가재생 및 분화를 조절한다. 본 연구에서는 정원줄기세포에서 GDNF 수용체 하위 전사조절네트웍 규명의 일환으로 생쥐 정원줄기세포에서 Egr1 발현 특성을 분석하였다. 출생 0~56일 사이의 생쥐 정소에서 Egr1 발현을 면역조직화학적으로 분석하였다. 생후 6일 생쥐 정소의 세포들을 분산시킨 후 THY1 항체를 이용한 MACS법으로 정원줄기세포를 분리하여 GDNF (100 ng/ml)을 처리하였다. GDNF(100 ng/ml)을 처리 후 0, 2, 12시간에 Egr1, Egr2, Egr3, Egr4, GDNF 수용체인 RET와 GFRA1, 자가재생에 관련된 Etv5, Bcl6b mRNA 발현과 Egr1 단백질 발현량을 분석하였다. 한편 MAPK inhibitor(U0126, 10 μM) 처리 후 GDNF를 각각 2, 12시간 처리하여 mRNA 발현을 분석하였다. 그리고 Egr1 knock out mouse 정원줄기세포에서 GDNF를 12시간 처리하여 mRNA 발현을 분석하였다. 면역조직화학적 분석결과 Egr1은 gonocytes, spermatogonia, peritubular cells, Leydig cell에서 발현하였으나 분화된 세포에서는 발현이 저조하였다. 정원줄기세포에 GDNF 처리 후 2시간에 Egr1 mRNA가 유도되는 반면 Egr2, Egr3 mRNA는 12시간에 유도되었고, Egr4 mRNA는 변화하지 않았다. GDNF 수용체인 RET과 GFRA1 mRNA는 GDNF 처리군에서 증가하였다. Self-renewal에 관련된 Etv5와 Bcl6b mRNA는 12시간에 유도되었다. Egr1 단백질은 2시간에서 GDNF에 의해 증가하였다. U0126과 GDNF를 동시에 처리한 경우 2시간에 Egr1 mRNA가 유도되지 않았으며, 12시간에 Etv5와 Bcl6b mRNA발현도 변화가 없었다. Egr1 knock out mouse 정원줄기세포에 GDNF처리 시 Etv5, Egr2, Egr4 mRNA는 증가하는 반면 Bcl6b, Egr3 mRNA는 감소하였다. Egr1은 미성숙정소에서 gonocyte, spermatogonial stem cell, peritubular cell, Leydig cell에서 주로 발현된다. GDNF는 정원줄기세포에서 MAPK pathway에 의존적으로 Egr1을 유도하며, Egr1은 GDNF 수용체 및 자가재생 유전자발현을 통해 정원줄기세포의 stemness 유지에 중요한 기능을 담당하는 상위 전사인자로 사료된다. 또한 Egr1이 결손되어지면 세포자가재생에 관련된 Bcl6b의 전사가 억제되는 것으로 보아 세포생존, 증식에 중요한 역할을 할 것으로 사료된다.