• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.20 no.4
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• pp.308-316
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• 1987

The flying fish, Prognichthys agoo, is widely distributed in the coastal waters of south-eastern Korea. On July 14, 1986, mature adults of flying fish were captured from U-do, Cheju-do. The eggs were stripped and fertilized by the wet method on the ship. The mature eggs are demersal and adhesive with 30-40 filaments. The egg diameter varied from 1.42 to 1.58 mm. The water temperature throughout incubation ranged from 23.70 to $27.82^{\circ}C$, and salinity was maintained at $30.75-33.76\%_{\circ}$. The hatching took place in 174 hours after fertilization. The newly hatched larvae measured 4.75-5.25 mm in total length possessing yolk sac and about 45-46 myotomes. The larvae cultured for ten days after hatching reached 11.45-12.60 mm in total length and entered the juvenile period of life. Twenty days after hatching, the juveniles measured 20.01 mm in mean total length, and the scales were formed behind the pectoral fin.

• Journal of Sericultural and Entomological Science
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• v.5
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• pp.25-38
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• 1965

I. Breeding of tussah silkworm(preliminary report). The preliminary examination for bleeding has been carried out since 1963 in tussah silkworms. 1) The strain(l-MG-B)of the heaviest silk quantity was the green silkworm and brown cocoon in univoltine, and the strains(2-G-B, 2-MG-B) of the heaviest silk quantity were also the green silkwom and brown cocoon in bivoltine in both spring and fall in 1965. 2) It looks like the voltinism, the body color and the cocoon color have reached to pure line up to 1965. II. Best place for the winter of tussah pupa. This work was aimed to find out good ways for the winter of tussah pupa. 1) The hatch of bivoltine was better than that of univoltine. 2) The cocoons covered with the leaves were good in the emergence of moth. 3) The cocoons which were kept at natural temperature till the first emergence of moths would show bad in both hatch and emergence. 4) If some of the pupae kept under natural condition were controled at proper temperature for a few days, hatch and laying eggs were best. 5) The best places for the winter were the egg storage and the rearing room. III. Relation between incubation temperature and voltinism. 1) When the tussah pupa are kept at natural temperature during winter, the moths do not come out of the pupa. 2) There is no difference between about 18$^{\circ}C$ and about 25$^{\circ}C$ during incubation in hatching ratio. 3) The tussah silkworms of univoltine in mortality are stronger than that of bivoltine. 4) There is not any relation between voltinism and high or low temperature for pupa and eggs. IV. Induced mutation by gamma-ray and neutron in tussah silkworm. This work was carried out in order to induce the mutation by treating the pupa or the eggs of tussah silkworm with gamma my and neutron. The results obtained are as follows. 1. Though the whole pupa treated with neutron become moths, the moths have no ability to copulate each other. The only moths emerged from pupa treated with neutron, 4000${\gamma}$ are able to lay all un-fertilized eggs, some of which have a hole on the surface and nothing of contents. 2. The non-diapause eggs are treated with neutron in spring, but the hatching ratio is 50∼60 percent, but the whole eggs treated with gamma ray are never hatched. 3. The sensitivity of the pupa to neutron is weaker than that of the eggs. 4. The hatching ratio is in direct proportion to the gamma ray dose. 5. Author find out a new mutant which is excellent in the cocoon quality, so he will do the progeny test next hear.

• Applied Microscopy
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• v.34 no.1
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• pp.71-81
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• 2004

When U. unicinctus mature oocytes were fertilized in vitro, germinal vesicle breakdown (GVBD) and meioses occurred and the zygotes entered cleavage stage. A modified pattern of spiral cleavages, suggestively based on behavior of mitotic spindles, have been observed in this work. The first and second cleavages were meridional and the third was equatorial, and then followed by repetitions of meridional-equatorial cleavage. The cleavage of the isolecithal egg were equal and holoblastic and its patterns were spiral. The anti-${\alpha}-,-{\beta}$- tubulin reactions and confocal microscopy revealed mitotic apparates tilted obliquely at each mitosis causing oblique displacements of the blastomeres. Despite isolecithal distribution of yolk, this observations implicated that tilting of mitotic apparates induced spiral cleavage and the displacements of blastomeres. However, these features would not be the typical spiral cleavage, but represented a modified pattern of known Spiralian s in the sense of the equal cleavage. During the first cleavage, heart-shaped eggs have been produced. Electron microscopies exhibited the first cleavage furrow extended with its membranous structure deeply into the cytoplasm. Contractile ring has not been observed.

• Korean Journal of Ichthyology
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• v.26 no.2
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• pp.89-98
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• 2014

Eggs development and early life history of the endangered Korean dwarf loach, Kichulchoia brevifasciata (Teleostei: Cobitidae) was investigated to provide basic information regarding biological characteristics and restoration. Adult fish specimens were sampled using a spoon net at Geurnsan-myeon, Goheung-gun, Jeollanam-do, Korea from June to July 2011. Since, spawning characteristics were analyzed, and females were induced to spawn by injecting Ovaprim (0.5 mL/kg) and their eggs were artificially fertilized with sperms by the dry method in the laboratory. Total length of mature female were 46~76 mm with GSI $9.6{\pm}3.77%$, and total length of mature male was 42~52 mm with GSI $3.5{\pm}1.04%$. Sex ratio (♂/♀) was 0.10, and there were no secondary sexual characteristics. The number of mature eggs was averaged $60{\pm}28.7$ per female. The lemon yellow eggs were slightly adhesive $1.46{\pm}0.07mm$ in diameter. The embryo hatched approximately 66 h after fertilization at $25^{\circ}C$, and the hatched larvae were averaged $5.5{\pm}0.07mm$ in total length (TL). At 6 days after hatching, the larvae averaged $9.0{\pm}0.29mm$(TL) and their yolk sac was completely absorbed. At 17 days after hatching, they entered the juvenile stage and reached $12.6{\pm}0.24mm$ (TL). At 80 days after hatching, the band patterns and external form of the juveniles were similar to those of adults, and they averaged $33.0{\pm}2.19mm$(TL).

• Journal of Aquaculture
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• v.13 no.2
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• pp.119-128
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• 2000

Exposure to air and increased temperature induced successful spawning in Mytilus edulis, M. coruscus Crassostrea gigas and Pinctata fucata martensii. Developmental durations required for an egg to attain D-shaped larva and the D-shaped larva to reach pediveliger stage were estimated in these bivalves. Size of fertilized eggs was the largest (70.3 ${\mu}m$) in M. coruscus and the smallest (45.3 ${\mu}m$) in P. fucata martensii. At 17$^{\circ}C$, M. edulis and M. coruscus attained D-shaped larval stage within 48 hours after fertilization but those of C. gigas and B. fucata martensiii within 24 and 22 hours at 21 and 26$^{\circ}C$, respectively. The development duration required for a D-shaped larva to attain pediveliger stage was the longest (27 days) in M. coruscus and ranged between 20 and 22 days for the others. The shell length of the pediveliger was the longest (274.9 ${\mu}m$) in C. gigas and smallest (190.9 ${\mu}m$) in P. fucata martensii Length and height of larval shell was highly correlated with each other in all the 4 species. The shell height of C. gigas was more than the shell length beyond the size of 100 ${\mu}m$ shell length. However, shell length of the others was always longer than shell height at the larval stage.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.9 no.1
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• pp.43-55
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• 1976

Fertilization and early development of turbo cornutus was studied based on the samples which were collected in Yeosu area. Particular emphasis was paid on induction of artificial spawing, fertilization rate, preembryonic development, the growth of the early larva and larval survival to various salinity. Among the various methods for induction of artificial spawning which have been tested for the present study, drying by exposure to air is the. most efficient, and percentage fertilization rate was $83.8-96.4\%$. The diameter of fertilized eggs was $0.182{\pm}0.0028mm$; and the diameter of egg membrane was $0.245{\pm}0.093mm$. Under the temperature range of $20.6-25.4^{\circ}C$ the larvae hatched out after 11:05-11:15 hours of fertilization. After 3.0-3.5 days of fertilization the planktonic larvae begand to settle, and the settlement terminated within 5 days. During the period of 150 days of early culturing the diameter growth of shell(M) and the diameter of shell aperture(A) was formulated as follows: $$1972\;M=0.33e^{0.02070D}$$ $$A=0.19e^{0.02282D}$$ $$1973\;M=0.32e^{0.02282D}$$ $$A=0.16e^{0.02596D}$$ During the same period of early culturing the relative growth of shell diameter and the diameter of shell aperture was formulated as follows : 1972 A=0.6478 S-0.1575 1973 A=0.5897 S-0.0515 After 11 days of larval hatching $0.02-0.18\%$ of planktonic larvae settled. After 150 days of settlement the survival rate of the early shells was $7.4-21.6\%$. Under the temperature range of $21.0-22.7^{\circ}C$ the optimum salinity range for the development of egg and the planktonic larvae was $30-35\%_{\circ}$.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Korean Journal of Ichthyology
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• v.12 no.2
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• pp.129-136
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• 2000

The aucha perch, Coreoperca kawamebari was collected in Tam-jin river from February to June 1998. It was reared in the laboratory and observed the spawning behavior and early life history. Spawning season was from mid of April to the end of May in the Tam-jin river. The fertilized eggs were demersal of adhesive, transparent and spherical in shape. Egg diameter was 2.21~2.65 mm with several oil globule of 0.058~0.343 mm. Hatching occurred about 194 hours 23 minutes after fertilization at water temperature of $18{\sim}22^{\circ}C$. Newly-hatched larvae were 5.09~5.68 mm in total length(TL, mean: 5.38mm) with 10~11+18=28~29 myotomes and opened mouth and anus. Melanophores were distributed on the eye lens, on the head, around the yolk, on the dorsal part and the abdominal region of the trunk. After hatching 5 days larvae attained 6.12~6.68 mm in TL (mean: 6.47 mm), and the yolk sac was completely absorbed and transformed to postlarva stage. The larvae reached to the juvenile stage with all the fins were formed with complete set of fin rays (D. XII-12~13; A. III-8~10; P. 11~13; V. I-4~5) at the 22 days after hatching and of the larvae was 11.54 mm in total length. In 32 days after hatching, the juvenile was 13.05 mm in TL. This period was similar to adult in body form and the spot.

• Korean Journal of Ichthyology
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• v.35 no.2
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• pp.91-100
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• 2023

The early life history of Silurus microdorsalis living in Jahocheon Stream was studied by observing egg and morphological development. Live fish were captured in June 2018, then reared in a circulating filtration system under a 14L : 10D photoperiod with a water temperature of 18℃. To artificially induce spawning, females were injected with 0.5 mL of Ovaprim (Syndel, Nanaimo, BC, Canada) per kg of body weight, and males were injected with 10,000 IU/kg body weight of human chorionic gonadotropin. Approximately 15 h later, eggs were artificially inseminated by the dry method. Mature eggs were light pale yellow, which separated them from immature eggs. Fertilized eggs were 2.16±0.06 mm (n=8) in diameter and fully hatched at 181 h after fertilization. The fertilization rate was 63.1±2.2%, and 10.0±3.7% of the embryos were malformed at 18℃. The rates of development were 181 h at 18℃, 109 h at 21℃, and 76 h at 24℃. The larval size immediately after hatching was 4.64±0.22 mm (n=8), and the larvae displayed negative phototaxis at 1 day after hatching. The total larval length on 7 days after hatching was 12.47±0.53 mm, with 25~30 basal anal fin rays and 14~16 basal caudal fin rays observed. The total larval length was 14.13±0.51 mm on 9 days after hatching, and approximately 90% of the black endoplasmic reticulum was deposited on the head and body. The dorsal fin had formed, and a single basal body was observed. On 15 days after hatching, the total larval length was 16.69±0.31 mm; the number of basal caudal fin rays (18 poles) was an integer because 2 dorsal fin basal rays and 60~63 anal fin basal rays were observed. The total larval length was 28.96±1.10 mm on 50 days after hatching; the numbers of caudal fins (n=18), dorsal fins (n=3), pectoral fins (n=11), and anal fin basal rays (n=67~73) were integers.