• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.29 no.4
• /
• pp.279-288
• /
• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Development and Reproduction
• /
• v.8 no.1
• /
• pp.57-64
• /
• 2004

This study was carried out to examine the expression of the circadian clock genes in the mouse ovary and testis at different developmental stages. Expression of Period1(Per 1), Period2(Per2), Period3(Per3), Cryptochrome1(Cry1), Cyptochrome2(Cry2), Clock Small and Prokineticin1 and Prokineticin2 receptor(Prok1r, Prok2r) genes in mouse ovary was explored by semiquantitative reverse transcription Polymerase chain reaction(RT-PCR) according to the developmental stage(post partum day; ppd 1, 7, 10, 21 and 35). Immunohistochemistry using PER1 antibody was also analyzed. The differential expression pattern of clock genes was presented according to stages of the mouse ovarian development (ppd 1, 7, 10, 21 and 35). In the cases of ovaries, at the starting point of follicle growth at ppd 7 and 10, the clock gene expression patterns were changed vastly. According to the developmental stages, the clock genes were highly expressed at ppd 7 and 10 in mouse testis also. Receptors for Prok2, the circadian output molecule of SCN, were also expressed in ovary at ppd 7 and in testis at ppd 1 and 7, respectively. Immnunohistochemical analysis of PER1 showed positive signals in the cytoplasm of oocytes and granulosa cells. The level or PER1 expression was increased in cells at the spermatogonia and the condensing spermatids. The expression pattern of Perl and localization of PER1 were showed similar patterns according to the developmental stages in ovary and testis. Taken together, it could be observed that the expression of clock genes was highly correlated with gonadal development and germ cell differentiation in mice. Therefore, in this study, circadian programming of the genes in the ovary and testis is strongly imposed across a wide range of core reproductive cycles and normal development of gametes. Although the existence of circadian genes is clearly investigated, further studies on the direct evidence is required for the understanding of the relationship between circadian genes and regulation of gonadal differentiation and germ cell development.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.26 no.4
• /
• pp.337-344
• /
• 2000

Germ-line mutations at DNA repair loci confer susceptibility to colon cancer in hereditary non-polypopsis colorectal cancer. Somatic loss of DNA mismatch repair gene has been reported in a large variety of other tumor types. Replication errors(RERs) judged by microsatellite instability(MSI) and its associated mutations have been recognized as an important mechanism in various tumor types. To investigate associations between MSI and oral squamous cell carcinoma, the frequency of MSI using 12 microsatellite markers were analyzed for the series of oral tumors. Of 17 tumors, 8 cases(47%) did not show instability at any of the 12 loci; 5(29%) showed instability at $2{\sim}3$ loci; and 4(24%) showed instability above 4 loci. The 4 cases showing widespread MSI did not differ from those without evidence of instability in terms of age at diagnosis, degree of differentiation, metastasis to lymph node, tumor location or the presence of mutations in the p53 tumor suppressor gene. DCC and D17S 796 were the most frequently detected in MSI analysis. There were no correlation between smoking and MSI frequency, instead, smoking was suggested to increase the mutation rate of p53 and development of oral carcinomas.

• Journal of Aquaculture
• /
• v.20 no.1
• /
• pp.47-50
• /
• 2007

The primordial germ cells of the oblong rockfish, Sebastes oblongus were buried under fibrous mesenchymal tissues between gut and mesonephric duct of pre-larva with 4.0 mm total length (TL) at 1 day after the parturition. In the juvenile of 22.0 mm TL at 71 days after the parturition, the gonad composed of a large number of gonial cell and formed of cavity along the lateral side of the gonad, differentiated to the ovary. At this time, the gonad formed seminiferous tubules by somatic cells, was differentiated to the testis. The smallest oblong rockfish that possessed primary oocytes was about 42.1 mm TL at 141 days. Spermatogonia remained quiescent until most fish were over 42.1 mm TL at 141 days. The oblong rockfish the differentiated directly into male or female without an intermediate female phase at the early indifferentiated stage. Therefore, the oblong rockfish belongs to the differentiated type of gonochoristic teleosts.

• Journal of the Korea Organic Resources Recycling Association
• /
• v.23 no.1
• /
• pp.70-81
• /
• 2015

Helicases are known to be a proteins that use the chemical energy of NTP binding and hydrolyze to separate the complementary strands of double-stranded nucleic acids to single-stranded nucleic acids. They participate in various cellular metabolism in many organisms. DEAD-box proteins are ATP-dependent RNA helicase that participate in all biochemical steps involving RNA. DEAD-box3 (DDX3) gene is belonging to the DEAD-box family and plays an important role in germ cell development in many organisms including not only vertebrate, but also invertebrate during asexual and sexual reproduction and participates in stem cell differentiation during regeneration. In this study, in order to identify and characterize DDX3 gene in the earthworm, Perionyx excavatus having a powerful regeneration capacity, total RNA was isolated from adult head containing clitellum. Full length of DDX3 gene from P. excavatus, Pe-DDX3, was identified by RT-PCR using the total RNA from head as a template. Pe-DDX3 encoded a putative protein of 607 amino acids and it also has the nine conserved motifs of DEAD-box family, which is characteristic of DEAD-box protein family. It was confirmed that Pe-DDX3 has the nine conserved motifs by the comparison of entire amino acids sequence of Pe-DDX3 with other species of different taxa. Phylogenetic analysis revealed that Pe-DDX3 belongs to a DDX3 (PL10) subgroup of DEAD-box protein family. And it displayed a high homology with PL10a, b from P. dumerilii.

• Journal of Aquaculture
• /
• v.11 no.4
• /
• pp.529-546
• /
• 1998

Gonadal part that developed by indifferentiation period for 6 months after hatching is made as gonad and fat body. These gonad are thin semi-transparant and undistinguished germ cell. Germinal epithelium is distinguished by development of gonad epithelial tissue from 7 months after hatching. Sex differentiation is begun by oogonia develoment at 8 months after hatching. Primary oocytes grow over germinal epithelium of gonadal cavity, at 9 months after hatching, gonadal cavity become ovarian cavity as they increasing. As soon as oocytes at 13 months after hatching are filled with the whole part of gonad, degeneration of oocyte is begun. And then, gonad has cavity tissue, a small number of oocyte are located in gonadal cavity. At 15 months after hatching, new primary oocyte develop and cavity of ovarian tissue in the central of ovarian cavity. Spermatogonia multiplicate and cavity tissue consist of testicular tissue. These gonad become hermaphrodite and then ditermine the sex of female and male. These results show the red sea bream is juvenile hermaphrodite and undif-ferentiated gonochoristic teleost. Male and female differentiation type of gonad is divided in undifferentiation stage, oogonia-like stage, ovary-like stage, ovary development stage, hermaphroditic testis stage, hermaphroditic ovary stage, and testis development stage. Undifferentiation stage is continued total lenth 18cm at 13 months after hatching. ovary-like stage is continued total length 11~18cm at 13 months after hatching. Ovary-like stage is continued total length 14~26cm at 10~14 months after hatching. Ovary development stage begins from total length 20cm, 14 months after hatching. At 20 months after hatching, 44 percent of total sampled individuals had ovary. Hermaphroditic ovary stage first begins total length 19~20 cm at 15 months after hatching, but it is not observed total length 28~29cm at 20months after hatching. Hermaphroditic testis stage first begins total length 21~22cm at 20months after hatching and is continued for 20months. Testis development stage first begins total length 20~21cm at 20 months after hatching, and is occupied 33 percent total length 28~29cm at 20 months. The beginning of sex differentiation more than 50 percent is from total length 16cm at 11 months after hatching. Sex determination begins total length 20cm, 14months after hatching in female and total length 20cm, 15 months after hatching in male. Sex determination more than 50 percent begins total length 23cm,, 17 months after hatching. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks. This appearence is showed the same tendency in 3-year old red sea bream. 1.9mm larvae after hatching grow about 19mm larvae for 47 days. The relationship between the total length and body weight of larvae and juveniles in $BW=4.45{\times}10^{-6}TL^{3.4718}$ r=0.9820. Fishes in cage culture grow to maximum total length 28.4cm. The relationship between the total length and body weight of these fishes is $BW=2.36{\times}10^{-2}TL^{2.9180}$, r=0.9971. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks.

• Journal of Aquaculture
• /
• v.17 no.1
• /
• pp.30-38
• /
• 2004

To estimate the spawning period the annual change of gonadosomatic index (GSI) were examined from January 2000 to December 2001. Fecundity, spawning frequency and egg diameter were measured by ocular observation. Germ cell differentiation during gametogenesis, the reproductive cycle and the first sexual maturity of greenling Hexagrammos otakii were observed under light microscopy from January to December, 2000. GSI began to increase in August and reached the maximum in November when ovary was getting mature. The reproductive cycle of H. otakii can be divided into five successive stages in females: early growing stage (July), late growing stage (July to August), mature stage (September to October), ripe and spent stage (September to December), and recovery and resting stage (December to June). Males showed four successive stages : growing (June to August), mature (August to October), ripe and spent (September to December), and recovery and resting stage (December to May). According to the frequency distributions of egg diameter in spawning season, H. otakii could be one of polycyclic species spawning 2 times or more during one spawning season. Number of total eggs and mature eggs in the absolute fecundity were related to the standard length and body weight, respectively. Number of total eggs and mature eggs in relative fecundity were also proportional to the standard length, but rather these numbers decreased with body weight. Percentages of first sexual maturity of females and males in greenling were over 50% from 19.1 to 21.1cm in length, and 100% for fish over 25.1cm in length. Therefore, both sexes are ready to reproduce after one year old.

• Reproductive and Developmental Biology
• /
• v.29 no.1
• /
• pp.1-7
• /
• 2005

Telomeres consisting of (TTAGGG)n tandem repeat DNA sequences and associated proteins are essential for chromosome stability and related with cell senescence, apoptosis and cancer. The telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. This study was carried out to identify the distribution of telomeres on mouse chromosomes and also to analyze the amount of telomeres and telomerase activity of mouse embryos at early embryonic stages. Germ cells and early embryos from 1 cell to blastocyst stage were analyzed. The amount of telomeres was analyzed by quantitative fluorescence in situ hybridization technique(Q-FISH) using a human telomeric DNA probe, and telomerase activity was measured by telomeric repeat amplification protocol assay(TRAP). In results, the telomeres on mouse chromosomes were distributed at the ends of all autosomes and sex chromosomes. Although the quantity of telomeres varied among chromosomes, most of chromosomes had higher amount in q-arm telomeres than in p-arm telomeres. The results of Q-FISH indicated that the relative amount of telomeres of mouse embryos in each embryonic stage was approximately the same except the higher amount in blastocysts. Using TRAP assay on mouse embryos, telomerase activity was detected in all preimplantation stages from mature oocytes to blastocysts. Especially the telomerase activity was significantly increased at the morula and blastocyst stage. In conclusion, there may be a close association between the amount of telomeres and telomerase activity in early embryonic stages, and analysis of telomere quantity and telomerase activity on early development will be helpful for the investigation of embryogenesis and embryonic cell differentiation in mice.

• Journal of Aquaculture
• /
• v.21 no.4
• /
• pp.331-338
• /
• 2008

Monthly changes in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of wild river puffer Takifugu obscurus, and water quality environment in spawning area during breeding season were investigated from March 1995 to February 1996. Monthly changes in GSI and HSI of T. obscurus, that was cultured in low salinity, were calculated. The external morphology of the gonads, germ cell differentiation during gametogenesis and the reproductive cycle with the gonad developmental phases were investigated by histological analysis. The optimum water quality environment in Ganggyung, Choongcheongnam-do, where is spawning ground of wild T. obscurus, was $15-20^{\circ}C$ (water temperature) and 0 psu (salinity). Monthly changes in the GSI in females and males reached a maximum in May, and then rapidly decreased. Therefore, it is assumed that in the natural condition the spawning period of wild T. obscurus is May to June. In females and males, it showed a negative correlationship between the GSI and HSI. The external morphology of the gonads in female and male T. obscurus, that was cultured in low salinity, is composed of a pair of saccular structure. Based on monthly changes in the GSI, it is assumed that in female T. obscurus, that was cultured in low salinity, spawn from March through May. Therefore, it showed a negative correlationship between changes in the GSI and HSI. On the whole, in females and males, it showed a similar pattern between wild and cultured T. obscurus. The reproductive cycle with the gonad developmental phases can be classified into successive five stages in females: the early growing stage, late growing stage, mature stage, ripe and spent stage, and recovery and resting stage. In males, that can be divided into successive four stages: the growing stage, mature stage, ripe and spent stage, and recovery and resting stage. In case of wild T. obscurus, the spawning period has once a year, however, those cultured in the high water temperature ($20-27^{\circ}C$) - low salinity (under 3.3 psu) condition have reproductive characteristics having possibilities of discharge of eggs and sperms year-round as a multiple spawner.

• Korean Journal of Poultry Science
• /
• v.41 no.4
• /
• pp.287-296
• /
• 2014

Chicken eggs undergo various physiological changes during egg maturation. To study genes associated with the egg maturation in pre-ovulation (immature) and post-ovulation (mature), we compared gene expression patterns between in the immature egg and mature egg using RNA sequencing data. Mature and immature eggs were obtained from a Heuksaek Jaerae-jong of Korean native chicken. Total RNAs obtained from the eggs were sequenced by Illumina HiSeq 2000 platform, and the generated sequence reads were mapped to Galgal4 reference sequence assembly using Tuxedo Protocol. From the comparison of the RNA sequencing data, 315 genes were differentially expressed between mature and immature eggs, and 46 genes were only detected in immature egg. Further gene ontology (GO) analysis was performed for the differentially expressed genes using DAVID, showing that 29 and 28 GO terms were independently clustered from mature and immature, respectively. From those clustered GO terms, genes related to germ cell development, sex differentiation and defense response to bacterium were mainly expressed in the immature egg, while genes related to regulation of apoptosis, steroid metabolic process and lipid homeostasis were mainly detected in the mature egg. Our results could contribute to understand egg maturation before and after ovulation, and develop genetic markers for improving egg quality and productivity.