• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.5
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• pp.423-435
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• 1984

The reproductive cycle of the small filefish, Rudarius ercodes was investigated based on the annual variations of gonadosomatic index(GSI) and hepatosomatic index(HSI) by electronic and photic microscophy. The specimens used were collected at the coastal area of Benden island, Sizuokagen, Japan, from September 1982 to August 1983. GSI began to increase from March, starting season of longer daylength and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between November and February without any evident variation. The annual variations of HSI were not distinct in male filefish and were negatively related to GSI in female : HSI decreased in the summer season when the ovary was getting mature and reached the maximum in the winter season when the ovary was getting retrogressive. The ovary consisted of a pair of saccular structure with numerous ovarian sacs branched toward the median cavity. Oogonia divided and proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophile cytoplasm showed several scattering nucleoli along the nuclear membrane. when the oocytes growing to about 300 ${\mu}m$, nuclear membrane to disappear with nucleus migrating toward the animal pole. The regions of protoplasm were extremely confined within the animal hemisphere in which most of cytoplasms were filled with yolk materials and oil drops. After ovulation, residual follicles and growing oocytes remaining in the ovarian sacs degenerated. But perinucleatic young oocytes without follicles formed were not degenerated, and growing continuously still in the next year. Mitochondria and endoplasmic reticula in the cytoplasm remarkably increased with oocytes maturing and yolk accumulating. Those were considered to be functionally related to the yolk accumulation. Five or six layers of possible vitellogenin, oval-shaped disc structures with high electron density, appeared in the apex of follicular processes stretching to the microvilli pits of mature oocytes. Testis consisting of a pair of lobular structures in the right and left were united in the posterior seminal vesicle, Cortex of testis was composed of several seminiferous tubules, and medulla consisting of many sperm ducts connected with tubules. Steroid hormone-secreting cells with numerous endoplasmic reticula and large mitochondria of well developed cristae were recognized in the interstitial cells of the growing testis. Axial filament of spermatozoon invaginated deeply in the central cavity of the nucleus and the head formed U-shape with acrosome severely lacking, mitochondria formed large globular paranuclei at the posterior head, and microtubular axoneme of the tail represented 9+9+2 type. The annual reproductive cycles could be divided into five successive stages : growth(March to July), maturation(May to September), Spawning(mid May to early October) and resting stages(October to February). The spawning peak occurred from June to August.

• Journal of Aquaculture
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• v.11 no.4
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• pp.475-485
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• 1998

To clarify annual reproductive cycle of Koran dark sleeper, odontobutis platycephala, we examined the seasonal changes of gonadosomatic index(GSI), testicular development stages and sex steroid hormones in blood from December 1995 to November 1997. Testis was podlike shape from July to October, and tadpole-like shape from November because of its expanded posterior part. GSI was 0.14~0.18 from July to September and increased to $0.43{\pm}0.04$ in October and then was not changed significantly until February. GSI was reincreased to $0.52{\pm}0.09$ from March and then was kept at similer levels until May, but fell down to $0.28{\pm}0.05$ in June. As results of histological observation, testis was divided into 3 parts(anterior, boundary, posterior) in the development progress of germ cells. In July, the testis was composed of only spermatogonia without seminiferous tubules in most fishes. In the anterior part of testis, the ferquency of spermatogenesis stage seminiferous tubules appearing in August was more than 80% from September to December. decreased gradually from January to March and drastically in April, and then disappeared in June. The frequency of spermiogenesis stage seminiferous tubules appearing in December, increased gradually from January to March and drastically to 80% in April, and reached to 90% the highest levels of the year in June. Post-spawning stage seminiferous tubules did not appear throughout the year. The frequency of spermatogonia was 100% and 65% in July and August, and less than 20% in the rest period of the year. In the boundary part, the frequency of spermatogenesis stage seminiferous tubules appearing in August increased from September and reached to 82% in November, decreased from December, adn disappeared in March. The frequency of spermiogenesis stage seminiferous tubules appearing in November was less than 18% until February, and increased to 29%~57% from March to June. The frequency of post-spawning stage seminiferous tubules appeared 12%~25% only from March to June. The frequency of spermatogonia was 100% in July, decreased to 85% in August and 10% in November, and increased gradually from December to 50% in April, and decreased again from May to June. In the posterior part, seminiferous tubules with some seminiferous tubules increased drastically 80%~85% in August and September, decreased drastically from October to November and remained below 10% until February, and disappeared after March. The frequency of spermiogenesis stage seminiferous tubules appearing in August increased sharply from October and reached to 75% in November. decreased to 15% in December and no significant changes until March, and disappeared after April. The frequency of post-spawning stage seminiferous tubules appearing very early in November increased to 82% in December and 85%~95% until June. The frequency of spermatogonia was 100% in July, decreased drastically to 15% in August, disappeared from October to Mrch, but reappeared from April and kept at less than 10% until June. The blood level of testosterone (T) increrased gradually from August was $0.61{\pm}0.09 ng/m\ell$ in November, increrased drastically to $3.99{\pm}1.22 ng/m\ell$ in December and maintained at in similar level until March, and decreased to $0.25{\pm}0.14 ng/m{\ell} ~ 0.17{\pm}0.13ng/m{\ell}$ in April and May and no significant changes until July (P<0.05). The blood level of 17, 20 -dihydroxy-4-pregnen-3-one $ng/m{\ell}$in the rest of year without significant changes(P<0.05). Taken together these results, the germ cell development of testis progressed in the order of posterior, boundary, anterior part during annual reproductive cycle in Korean dark sleeper. The testicular cycle of Korean dark sleeper was as follows. The anterior part of testis : i.e. spermatogonial proliferation period (July), early maturation period (from August to November), mid maturation period (from December to March), late maturation period (from April to May) and functional maturation period (June) were elucidated. The boundary of testis, i.e. spermatogonial proliferation period (July), early maturation period (from August to October), mid maturation period (from November to February) and the coexistence period of late maturation, functional maturation and post-spawn (from March to June) were elucidated. The posterior of testis, i.e. spermatogonial proliferation period (July), mid maturation period (from August ot September), late maturation period (October), functional maturation period (November) and post-spawn period (from December to June) were elucidated. It was showed that the changes of sex steroid hormone in blood played a important roles in the annual reproductive cycle of Korean dark sleeper.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.292-301
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• 2010

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and betasubunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, $FSH{\beta}$ subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the $FSH{\beta}$ subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal specificity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified $FSH{\beta}$ protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p<0.05). These observations suggested that the expressed $FSH{\beta}$ protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

• Journal of Life Science
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• v.25 no.10
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• pp.1184-1195
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• 2015

The zebrafish is an emerging vertebrate model organism in reproductive biology. The oocyte maturation of zebrafish is triggered by maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one). In almost all animals, the oocyte maturation is governed by activation of pre-MPF which consists of cyclinB and inactive Cdk1. In the oocyte of Xenopus and mice, the activity of Cdk1 is regulated in two ways, one is the interaction with cyclinB and the other is phosphorylation/dephosphorylation of T14/Y15 residues on the Cdk1 by Wee1 and Cdc25. Unlike Xenopus and mice that have a sufficient amount of pre-MPF, pre-MPF is absent in GV oocyte of most teleost including zebrafish. Therefore, the activation of MPF during zebrafish oocyte maturation might totally depend on de novo synthesis of cyclinB proteins. It is reported that the translation of maternal mRNA is regulated by combination of several RNA binding proteins such as CPEB, Dazl, Pum1/Pum2, and insulin-like growth factor2 mRNA-binding protein 3 in the zebrafish oocytes. However, the definitive mechanism of these proteins to regulate the translation of stored maternal mRNAs remains to be elucidated. Therefore, the investigation of the maturation process of the zebrafish oocyte will provide new information that can help identify the role of translational control in early vertebrate oocyte maturation.

• Journal of Veterinary Clinics
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• v.27 no.6
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• pp.693-697
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• 2010

This study, conducted with four leopard cats (Prionailurus bengalensis), used time-resolved fluoroimmunoassay (TR-FIA) to analyze estradiol and progesterone concentrations in fecal samples. We measured fecal samples taken during estrus period, diestrus period, pregnancy and non-pregnancy period. During estrus (February), the mean minimum estradiol concentration was $4.02{\pm}1.9$ng/g, and the mean maximum was $86.01{\pm}35.2$ng/g (dry fecal weight). During diestrus (November), the mean minimum estradiol concentration was $4.42{\pm}1.32$ng/g and mean maximum was $15.62{\pm}6.48$ng/g (dry fecal weight). Midgestation (April), the mean minimum progesterone concentration was $427{\pm}24.49$ng/g and the mean maximum was $1490{\pm}265.27$ng/g. During non-pregnancy (November), the mean minimum progesterone concentration was $71.25{\pm}29.61$ng/g and the mean maximum was $291.75{\pm}90.30$ng/g. These results suggest that steroid hormone analysis of feces using TR-FIA is a valid method for noninvasively determining ovarian activity associated with estrus and pregnancy in leopard cats. This study will contribute to building breeding management and reproductive plans for endangered species.

• Development and Reproduction
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• v.13 no.2
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• pp.79-87
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• 2009

Due to its well-known function in parturition and milk ejection, oxytocin (OT) has been considered as a 'female neurohypophyseal hormone'. Recent studies, however, clearly indicate that OT has some local roles in male reproduction via both central and peripheral routes. In experimental rodents, OT is released within distinct brain regions in response to social stimuli, and the brain OT receptor (OTR) mediated actions were strongly involved in the regulation of a variety of male behaviors such as mating-associated behaviors. In particular, OT and/or OTR knockout mice provide important clues about the molecular regulatory mechanism of the socio-sexual behaviors. Several lines of evidence also show that OT is synthesized within rodents testis, epididymis and prostate, and the presence of OTRs in these organs. In rodent testes, OT might have a role in the modulation of steroidogenesis via stimulation of the conversion of testosterone (T) to dihydrotestosterone (DHT) by 5alpha-reductase. Similar effects of OT on this androgen conversion were observed in epididymis and prostate suggesting the OT's modulatory role, such as contractility induction, in these androgen-dependent organs. In this context, further investigations on the OT's role in male CNS and reproductive organs are likely to provide better understanding on the complex socio-sexual behaviors and a platform for development of therapeutics to treat some psychological and/or andrological problems.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.