• Clinical and Experimental Reproductive Medicine
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• v.15 no.1
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• pp.25-34
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• 1988

The intrafo1licular echoes of cumulus oophoruses within ovarian follicles were assessed with the use of ultrasound in 86 women taking part in an in vitro fertilization(IVF) or gamete intrafallopian transfer(GIFT) program, stimulated with pure follicle-stimulating hormone(FSH)/human menopausal gonadotropin(hMG)/human chorionic gonadotropin (hCG). When intrafo1licular echoes were clearly separated from the follicular wall or relatively dispersed within the follicle, they were considered to be a dissociated cumulus, and when they were only slightly prominent from the follicular wall, they were suspected to be a nondissociated cumulus. A cumulus was seen in 62.1% of the follicles larger than 10 mm diameter and 75.1% of them were dissociated. The larger the follicles in size, the more the cumuluses in number and dissociation. The number of follicles and intrafollicular echoes per woman was not different whether or not she would be pregnant, but the number of dissociated cumuluses was significantly more in pregnant women. The number of observed dissociated cumuluses correlated significantly with the number of recovered mature oocytes. When an intrafollicular echo is seen, it can be taken as evidence of a sign of maturity of that particular follicle and oocyte. Ultrasonographic monitoring of intrafollicular echoes and follicular size is very helpful to predict follicular maturation in ovulation stimulation cycles.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.2
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• pp.83-89
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• 2013

Objective: To investigate outcomes of stimulated IVF cycles in which GnRH antagonist was omitted on the ovulation triggering day. Methods: A total of 86 women who underwent controlled ovarian hyperstimulation with recombinant FSH and GnRH antagonist flexible multiple-dose protocols were recruited and prospectively randomized into the conventional group (group A) or cessation group (group B). The GnRH antagonist, 0.25 mg/day of cetrorelix, was started when the leading follicle reached 14 mm in diameter and was continuously administered until the hCG triggering day (group A, 43 cycles) or until the day before hCG administration (group B, 43 cycles). The maturity of oocytes, fertilization rate, embryo quality, and implantation and clinical pregnancy rates were evaluated. Results: The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in group B than group A ($2.5{\pm}0.9$ vs. $3.2{\pm}0.8$ ampoules, p<0.05). There was no premature luteinization in any of the subjects. The proportion of mature oocytes and fertilization rate were not significantly different in group B than group A (70.7% vs. 66.7%; 71.1% vs. 66.4%, respectively). There were no significant differences in the implantation or clinical pregnancy rates. Conclusion: Our prospective randomized study suggested that cessation of GnRH antagonist on the hCG administration day during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising its effects on pregnancy rates.

• Journal of Aquaculture
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• v.8 no.1
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• pp.31-46
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• 1995

Gonadal maturation and annual reproductive cycle in ovoviviparous oblong rockfish, Sebastes oblangus on the basis of monthly gonadosomatic indices (GSI), hepatosomatic indices (HSI) and histological observations of gonadal tissues. GSI values of female were in a wide range from $0.l5\pm0.0l\;(July)\;to\;58.54\pm3.86$ (December) and began to increase in August and reached the maxium in December, then decreased rapidly thereafter. Male GSI values were in a range from $0.08\pm0.03$ (July) to $1.55\pm0.27$ (September) and began to increase rapidly in July and reached the maximum in September, then decreased gradually, thereafter. Female HSI was in a range from $0.89\pm0.12$ (December) to $3.73\pm0.15$ (October), and male's was from $2.09\pm0.76$ (October) to $3.62\pm0.48$ (August). HSI reached the maximum values one or two months before GSI reached their maxium values in both sex, and then decreased rapidly thereafter. Mature oocytes began to appear in late October as being oocytes began to mature in August, and the type of oocyte development is categorized in the roup-synchronous oocyte development'. Ovulation and fertilization of ripe oocytes occurred in November, and hatched larvae were born from December to January. Maturation of testis was progressed in short term from August to October and spermatozoa were released in October. Sperm balls consisted of many spermatozoa were preserved in ovarian cavity of female after copulation. These results may suggest that the annual reproductive cycle of oblong rockfish could be divided into the following successive stages: growing (August and September), mature (September and October), gestation (November and December), parturition (December and January) and resting (February to July) in female, and growing (August and September), mature (September and October), copulation (October) and resting (November to July) in male.

• Korean Journal of Veterinary Research
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• v.34 no.4
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• pp.735-744
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• 1994

Catheters were placed into the external jugular veins of immature female rats. On the following day (day 28 of age), the animals were injected subcutaneously with pregnant mare serm gonadotropin(PMSG): 4IU(control) or 20IU(superovulation). Each animal was sequentially bled at Ohr and 12hr and subsequently at 6hr intervals until sacrifice at 72hr after PMSG. The superovulatory dose of PMSG significantly(P<0.05) increased the ovulatory response by 4.0 fold above controls. On the other hand, superovulated oocytes displayed considerably different stages of meiotic maturation: prophase I (14.7%), anaphase I (36.2%), telophase I (10.3%), metaphase I/II (32.4%), while in control rats a majority of the oocytes examined(94.0%) consistently showed a metaphase II configuration. Serum luteinizing hormone(LH) levels were determined by RIA. Both groups exhibited a similar time relationship with two distinct peaks: an initial slight rise at 0-18hr and a second sharp rise at 54-60hr. However, there was a marked change in the magnitude of LH levels between the two groups. In superovulated animals, prior to the second peak, overall LH levels were significantly(P<0.001) higher than controls. In contrast, at the peak occurring at 60hr, LH concentrations were significantly(P<0.001) reduced by 54% below that of control. Additionally, a maximum increase of mean ${\Delta}LH$ between two peaks was much less in superovulated as compared to control rats. The initial prolonged elevation of serum LH before 54hr in superovulated rats was found to result from actual cross-reaction of the injected PMSG with LH antibody in the assay, while a precipitous second elevation between 54hr and 60hr resulted primarily from an endogenous LH surge. This study clearly defines time-course features of serum LH in PMSG-treated rats. The overall results indicate that, following superovulatory treatment with PMSG, the increased ovulatory response is primarily associated with PMSG-derived intrinsic gonadotropin, and that the recovery of immature or asynchronously mature oocytes at ovulation may reult from the circulatory alteration of LH activity characterized by an initial prolonged elevation of serum LH and its subsequent attenuation.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.265-274
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• 2009

Objective: MicroRNAs (miR) are known to repress target genes at post-transcriptional level and play important roles in development and maturation of cell. However, the expression profiles of miR during ovarian follicle maturation have not been fully elucidated. Here, we designed this study to investigate the expression profiles of miR in oocytes and granulose cells (G-cells) after in vitro culture according to gonadotropins and adding hCG. Methods: Ovaries from 12-day-old mice (C57BL6) were removed and preantral follicles were isolated and cultured in $20\;{\mu}L$-drop of culture media with supplementation of either rFSH, rLH, or rFSH+rLH. After their full maturation, follicles were incubated with rhCG and rEGF. RNA was isolated from oocytes and G-cells, and real-time PCR were performed with primers of miR known to be expressed in the mouse ovary (mmu-miR-16, -miR-27a, -miR-126, -miR-721). Results: FSH+LH group showed the highest ovulation and MII rates among gonadotropin groups. The profiles of miRs in oocytes and G-cells differed according to gonadotropin groups and adding hCG. The profiles of miRs showed divergent changes between oocytes and G-cells. Conclusion: miR expression profiles are altered by gonadotropins and supplementation of hCG during in vitro maturation of murine follicles. Target gene study must be necessary to validate these findings.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.79-86
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• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.249-254
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• 2009

Objective: The aim of this study was to compare the fertilization and cleavage rates of human in vitro matured oocytes after fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Methods: A total of 135 GV stage oocytes were obtained from 59 women who received ovarian stimulation and IVF during Jan 2007 to Oct 2008. Ovarian hyperstimulation was performed using hMG or recombinant FSH with GnRH antagonist and then ovulation triggered by recombinant hCG. The immature oocytes obtained from stimulation cycles were cultured in IVM medium up to 48 hrs; commercial medium supplemented with rFSH 75 mIU/mL, rhCG 0.5 IU/mL and rEGF 10 ng/mL. The in vitro matured oocytes were fertilized by conventional IVF (41 GV oocytes) or ICSI method (94 GV oocytes). Results: Maturation rate were 51.2% and 59.6% in conventional IVF group and ICSI group, respectively. There was no significant difference in fertilization rates between two groups; 71.4% and 80.4%, respectively. The cleavage rate was also similar in two groups. Conclusion: The presented data suggest that conventional IVF has comparable fertilization and cleavage potential compared with ICSI as the insemination method of immature human oocytes obtained from stimulated cycle.

• 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.

• 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 $n^{++}$././././.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.8-8
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• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.