• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.4
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• pp.383-391
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• 1995

In order to find out the distribution of neuropeptide Y (NPY) recently known as the gonadotropin (GtH) stimulation neurohormone in the brain tissues of marine teleost, detection and localization of NPY like substance in brain of two rockfish species, Sebastes oblongus and S. schlekeli were done by immunohistochemisty. Distribution of GtH cells in hypophysis were also observed by aldehyde fuchsin (AF)-fast green-orange G stain to compare with gonadal phases of the rockfish species. NPY immunoreactive cells were detected in olfactory bulb, telencephalon and mesencephalon of the brain, and NPY immunoreactive fibers were distributed not only in olfactory bulb, telencephalon and mesencephalon but also in optic nerve, hypothalamus and optic tectum. Regardless of ovarian maturation in two rockfish species, NPY immunoreactive fibers were observed in the neurohypophysis adjacent to the AF negative cells in the rostral pars distalis of hypophysis in both species. Moreover, the fibers were distributed in the rostral and proximal pars distalis near to the GtH cells of the hypophysis in both species possessing the growing or mature oocytes. Slight AF stainable GtH cells were detected in hypophysis of two species before parturition (S. oblongus) and in mature stage (S. schlegeli), but AF stainability of the cells in the proximal pars distalis after parturition was more increased than that of the cells Tn mature stage or before parturition. The size and nucleus diameter of GtH cells in S. oblongus and S. schlegeli before parturition were significantly bigger than those of GtH cells in individuals after parturiton (S. oblongus) or with resting ovary (S. schlegeli) (P<0.01).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.18 no.3
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• pp.253-261
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• 1985

The reproduction of marbled sole Limanda yokohamae, caught near around the southeastern coast of Korea, from December 1983 to November 1984, was investigated based on such annual variations as gonadosomatic index(GSL), gametogenesis, reproductive cycle, spawning number, hepatosomatic index (HSI), and fatness. GSI began to increase in the autumn season with the onset of shorter day length and colder water temperature, and reached the maximum value in December with the shortest day length and the lowest temperature over the year. The gonad activated the proliferation of oogonia and spermatogonia in June, reached the mature stage in October, ripe in December, and spawning from the end of December to January. After spawning, it showed the resting stage which gonad remained regressive and suppressive from February to May. In addition, the adult individuals observed discharged eggs only once during their spawning period. At yolk globular stage, the substance of vitellogenin synthesized from the liver was considered to participate in the active yolk accumulation of oocytes. Marbled sole was concluded to be a typical winter spawning species in that such environmental factors as short day length and low water temperature were closely related with the gametogenesis, the stimulation of oocyte maturation, and were also affecting the ovulation.

• Fisheries and Aquatic Sciences
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• v.1 no.1
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• pp.82-93
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• 1998

Monthly changes of the gonad follicle index (GFI), reproductive cycle, egg-diameter composition, first sexual maturity of the Pacific oyster, Crassostrea gigas, were studied based on the samples which have been collected from the intertidal zone of Poryong west coast of Korea, from January to December, 1996. C. gigas, is dioecious, while a few individuals are alternatively hermaphroditic. Monthly variation of gonad follicle index (GFI) used for determination of spawning period, coincided with the reproductive cycle. GFI increased from April when seawater temperatures gradually increased and reached the maximum in May. And then, GFI sharply decreased from June to September due to spawning. Reproductive cycle of this species can be divided into five successive stages: in females, early active stage (March to April), late active stage (April to May), ripe stage (May to August), partially spawned stage (June to September) and spent/inactive stage (September to February); in males, early active stage (February to March), late active stage (April to May), ripe stage (May to September), partially spawned stage (June to September) and spent/ inactive stage (September to February). The diameter of fully mature eggs are approximately 50um. Spawning occurred from June to September, and two spawning peaks were observed in June and August when the seawater temperature was above $20^{\circ}C$. Percentages of the first sexual maturity of males of 20.1-25.0 mm in shell height were over $50\%$, while those of females of 25.1-30.0 mm in shell height were over $50\%$. All the males of > 30.1 mm and all the females of ^gt; 35.1 mm completed their first sexual maturity. The results suggest that C. gigas has a protandry phenomenon. Sex ratios of 919 oysters observed were 453 females $(49.29\%)$, 429 males $(46.68\%)$, 16 hermaphrodites $(1.74\%)$, and 21 indeterminate individuals $(2.29\%)$. In age class I, sex ratio of males were $64.00\%$, thus, a higher percentage than that of females. It was noted that $64.00\%$ of the young males (age class I) were more functional than females in age class I, but 2-3 year-old oysters showed higher percentage of females. Percentages of hemaphrodites in 2-3 year classes were relatively higher than those in other year classes. Histological pattern of hermaphrodites can be divided into two types: Type I (hermaphrodite having a number of newly formed developing oocytes on the oogenic tissues within a degenerating spermatogenic follicle after discharge of numerous spermatozoa) and Type II (hermaphrodite having two separate follicles in the same gonad).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.45 no.3
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• pp.253-261
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• 2012

We investigated the reproductive capacity of starry flounder Platichthys stellatus from the Uljin (UJ) marine ranching area and compared it to that of P. stellatus from the coastal waters of Pohang (PH). In UJ, female gonadosomatic index (GSI) peaked in October ($3.14{\pm}0.87$) and male GSI was high in October and December. In PH, female GSI peaked in January ($18.64{\pm}2.15$) while male GSI began to increase in October and remained high until March. Most ovaries of UJ females were immature with perinucleus oocytes, although the testes of UJ males were ripe in January. Both the ovaries and testes of PH starry flounders were ripe from January to March. The plasma estradiol-$17{\beta}$ ($E_2$) levels of UJ females were highest in October ($4.09{\pm}1.90$ ng/mL) although the testosterone (T) levels of UJ males were highest in December ($3.81{\pm}0.78$ ng/mL) and decreased gradually until April. The $17{\alpha}$,$20{\beta}$-dihydroxy-4-pregnen-3-one ($17{\alpha}20{\beta}P$) levels of UJ females were not detected. The E2 levels of PH females were highest in December ($36.25{\pm}33.07$ ng/mL) and $17{\alpha}20{\beta}P$ levels were highest in March ($5.51{\pm}0.95$5 ng/mL). The T levels of PH males were highest in December ($4.03{\pm}1.34$ ng/mL) and decreased gradually until October. Taken together, these results suggest that most females from UJ did not reach maturation with a spawning period that was considered to be between December and January.

• Development and Reproduction
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• v.7 no.2
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• pp.113-118
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• 2003

Follicular fluid(FF) of mammalian Graafian follicles contains various kinds of proteins and proteinases that are believed to play important roles during follicular growth oocyte maturation and ovulation of mature oocytes. Previous studies of human FF(hFF) demonstrated the presence of many serine/threonine proteinases and matrix metalloproteinases such as gelatinases, however, little is known about the caseinases. Present study was aimed to examine the presence and the property of caseinolytic enzyme in hFF. Using casein zymographic method, it was found that hFF, human adult serum and cord serum exhibited one intense 80 kDa and another weak 78 kDa bands having caseinolytic activity. When inhibitors were added to the zymographic substrate buffer, caseinolytic activity of both 80 kDa and 78 kDa proteins were inhibited by othylenediarnine tetraacetic acid(EDTA) or soybean trypsin inhibitor(SBTI), but not by E-64, phenylmethylsulfonyl fluoride(PMSF) or 1,10-phenanthroline. Thus both enzymes appear to belong to a family of trypsin-like enzyme. Addition of EDTA to the zymographic substrate buffer almost abolished the caseinolytic activity of both enzymes. However, further addition of a divalent metal ion such as CaC $l_2$, MgC $l_2$, MnC $l_2$ or ZnC $l_2$ to the same buffer fully restored the enzyme activity at 5 mM concentration despite the presence of EDTA. Based upon these observations, 80 kDa and 78 kDa caseinolytic enzymes are present in human follicular fluid and they appear to be trypsin-like enzymes of which caseinolytic activity needs the presence of $Ca^{++}$, aM $g^{++}$, M $n^{++}$ or Z $n^{++}$././././.

• Development and Reproduction
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• v.5 no.2
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• pp.123-129
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• 2001

When mammalian oocytes undergo maturation, cumulus cells surrounding the oocyte exhibit remodeling of their structure known as cumulus expansion. Many molecules including hyaluronic acid participate in this remodeling. The present study aimed to investigate a possible existence of matrix metalloproteinases(MMPs) in the extracellular matrix(ECM) of human oocyte-cumulus complex. ECM was extracted from the human oocyte-cumulus complex. Gelatin gel zymogram of ECM exhibited 7 gelatinases having molecular weight of 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, and 84kDa. This gelatinase profile was very different from that of ovarian mural granulosa cell extract or white blood cell extract, indicating that the oocyte-cumulus complex donating ECM was free from other than cumulus cells. When ethylenediaminetetraacetic acid or 1', 10'-phenanthroline was added to the reaction buffer during zymographic development, almost gelatinase activities were abolished, suggesting that they were MMPs. Following incubation of ECM in the presence of aminophenylmercuric acetate, an activator of proMMPs, 4 gelatinases of 240kDa, 180kDa, 97kDa, and 84kDa disappeared with the concomitant appearance of 80kDa, 65kDa, and 60kDa gelatinases. Based upon these observation, it is suggested that ECM of the human oocyte-cumulus complex consists of gelatinases, presumed to be MMP-2 and MMP-9 isoforms.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.21-26
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• 2007

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 $100{\mu}1$와 $10{\mu}1$ 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건($S;\;10{\mu}1$ 소적) 및 그룹 배양 조건 ($G;\;100{\mu}1$ 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 $1{\mu}g/ml$ $estradiol-17{\beta}$를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene glycol로 직접 이식법에 의한 동결 방법으로 동결을 실시하였고, 세포수는 융해 후 생존 수정란에 대해 조사하였다. 체외 배양 시간이 $16{\sim}17$시간 배양 조건에서 난자의 성숙율은 그룹 배양 조건$(27.1{\pm}16.8%)$보다는 단독 배양조건$(57.1{\pm}15.0%)$에서 성숙율이 높았다(p<0.05). 그러나 체외 배양 시간이 $18{\sim}19$ 시간과 $20{\sim}21$시간 배양시는 유사한 성숙율을 보였다. 난자의 체외 배양율은 체외 배양 시간의 증가에 의해 성숙도가 $86.3{\pm}9.9%$로 증가하였다. 접합체(zygote)의 분할율은 단독이나 그룹 배양 조건에서도 유사한 결과를 얻었다. 배반포 발달율은 배양 $7{\sim}8$일째에 조사한 결과 단독 배양 방법보다는 그룹 배양 방법에서 발달율이 높았으나, 분할된 접합체를 기준으로 한 경우 배반포 발달율$(S;\; 21.4{\pm}10.6%,\;G;\;39.0{\pm}13.1%)$에서는 유의적인 차이가 없었다. 단독 배양과 그룹 배양에서 $6.5{\pm}8$일 사이에 배반포로 발달된 수정란의 세포수 조사에서는 유사한 결과를 얻었다. 동결 융해 후 24시간 배양 후 배반포 생존율(5; 24.2%, G; 30.2%), 부화율(S: 20.9%, G: 12.7%) 및 생존 수정란수(S; 45.2%, G: 42.8%)에서도 배양 조건에 따른 유의적인 차는 없었다. 결론적으로 mSOF배양액을 이용하는 경우 미성숙 난자의 체외 성숙 유도 배양 시 단독이나 그룹 배양 시 배반포 발달율에서 그룹간에 유의적인 차가 인정되었다(p<0.01).

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.5
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• pp.583-590
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• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.1
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• pp.83-91
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• 1986

The activity of the liver cells of greenling, Agrammus agrammus were histologically investigated under photo-and electron microscopy, and studied by comparing seasonal changes of hepatosomatic index (HSI). The materials were monthly collected at the costal area of Tongbaeksom, Pusan, Korea, from September 1983 to August 1984. The annual variations of HSI of male were not distinct, but those of HSI in female began to increase in autumn, and reached the maximum in winter when the ovary was getting mature. During the period of yolk accumulation in the oocytes, the female liver and its hepatic cells were seen to large and nuclei and nucleoli were hypertrophic also. At this time the amounts of glycogen and lipid in the cells gradually decreased, while basophilic substance (RNA) increased. And well-developed granular endoplasmic reticula binding ribosomes were supposed to play the leading role in protein synthesis and deposition for vitellogenin in the cystoplasm. Just prior to spawning, glycogen and lipid droplets were decreased, but basophilic substances(RNA) were found in a high concentration especially at the peripheral region of the liver cells of females. In the liver cells of males, were hardly altered by gonadal maturation, basophilic substances gradually increased, glycogen particles and lipid droplets were still observed in large quantities. After spawning, basophilic subtances decreased in the liver cells of female and male.