• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1652-1656
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• 2004

The aim of this study was to obtain mature ova or embryos at a single cell stage, which can be used in avian transgenesis and nuclear transfer through multiple ovulations, in vitro fertilization and culture. Chicken anterior pituitary extract (CAPE) or acetone-dried chicken anterior pituitary extract (ACAPE) was used to induce multiple ovulations in hens pretreated with pregnant mare' serum gonadotrophin (PMSG). In vitro fertilization of the multiple ovulated ova was performed by inseminating sperm onto the germinal disks in m-Ringer' solution and incubating the ova at 41$^{\circ}C$, 5% $CO_2$ for 10 h in DME-F12 medium containing 20% liquid albumen. The in vitro fertilization process was observed using an environmental scanning electron microscope. When normal laying hens (white Leghorn) were administered daily with PMSG (100 IU), egg laying ceased in most hens within 3 to 8 days. Ovulation began to occur about 7.5 h after injection of CAPE and ACAPE. The number of ovulated ova was 1.00${\pm}$0.00, 2.33${\pm}$0.52 and 2.20${\pm}$0.45, respectively, after receiving 100, 200 and 300 mg CAPE. The number of ovulated ova was 2.00${\pm}$0.00, 2.86${\pm}$0.69 and 3.00${\pm}$1.22, respectively, after receiving 10, 15 and 20 mg ACAPE. The fertilized and cultured ova were able to develop into embryos up to the 32 cell stage. The present experiments demonstrated that multiple ovulations can be induced by CAPE and ACAPE successfully, and the ova resulted from the treatment retained the capability for further fertilization and embryonic development. These data provide new information to support the establishment of an in vitro culture system for future avian transgenesis studies.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.274.2-275
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• 2003

The aim of this study was designed to determine the induction of rat growth hormone(rGH) by extracts of a popular herb, Glycyrrhizae radix(GR), roots of Glycyrrhiza glabra $\sub$Linne/, and Glycyrrhiza uralensis $\sub$Fischer/. In vitro study was carried out using primary rat pituitary cell culture for 3 days and then was treated with methanol extract corresponding to 1 mg of dried weight of herb per 1 $m\ell$ of culture solution. (omitted)

• The Korean Journal of Pharmacology
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• v.30 no.1
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• pp.19-28
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• 1994

The effects of gonadoropin-releasing hormone (GnRH) and ovarian steroid hormones on the release of luteinizing hormone (LH) and its subunit mRNA levels were investigated in anterior pituitary cells in culture. LH concentration was measured by a specific radioimmunoassay and mRNA levels of u and $LH{\beta}$ subunits by RNA slot blot hybridization assay. GnRH stimulated LH release in a dose-dependent manner from cultured pituitary cells. However, the basal LH release in the absence of GnRH was not changed during the course of 24h culture, strongly suggesting that release of LH is directly controlled by GnRH. The treatment of the pituitary cells with GnRH increased $LH{\beta}$ subunit mRNA levels in a dose-dependent manner, reaching the maximum with $2\;{\times}\;10^{-10}M$ GnRH while no significant increase in ${\alpha}$ subunit mRNA levels was observed after GnRH treatment. Estradiol did not augment GnRH-induced LH release while progesterone augmented GnRH-induced LH release in a dose-dependent manner at the level of pituitary. However, estradiol and progesterone increased basal and GnRH-induced $LH{\beta}$ subunit mRNA levels in a dose-dependent manner. The treatment of estrogen antagonist, LYI17018 blocked the effect of estradiol on GnRH-induced $LH{\beta}$ subunit mRNA levels in a dose-dependent manner while progesterone antagonist, Ru486 tended to block the effect of progesterone on GnRH-induced $LH{\beta}$ subunit mRNA levels. It is therefore suggested that GnRH Playa a major role in LH release and subunit biosynthesis by influencing the steady state $LH{\beta}$ subunit mRNA loves and ovarian steroid hormones modulate subunit biosynthesis via directly acting on pituitary gonadotropes.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.2
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• pp.282-290
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• 1997

Hepatocytes of Anguilla japonica have been prepared using a collagenase perfusion technique. The isolated cells attached efficiently to fibronectin-coated culture dishes and subsequently formed monolayers in serum-free medium. These cultures maintained in appropriate medium at least for 10 days with minimal cell loss. The effects of estradiol and pituitary hormones on vitellogenin (Vg) synthesis were examined in primary hepatocyte culture of the immature eels. In fish, as in other oviparous vertebrates, estrogen is a major inducer of Vg synthesis. However, $estradiol-17\beta(E_2)$ alone was insufficient to induce Vg synthesis in cultures of eel hepatocytes. Combination of $E_2$ with growth hormone (GH) and/or prolactin (PRL) markedly stimulated Vg synthesis. Even in cultures exposed to $E_2$ or precultured without hormones for 8 days, $E_2$ alone could not fully induce Vg synthesis. The synthesis of Vg was dramatically increased when hepatocytes were cultured in medium supplemented with $E_{2}+GH+PRL$ for 6 days. At this point, even though GH and/or PRL were eliminated from the medium, Vg synthesis was not influenced by these factors during culture of further 3 days. These results indicate that pituitary hormones, in particular GH and PRL, play important roles in the regulation of Vg synthesis in primary cultures of eel hepatocytes.

• Toxicological Research
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• v.22 no.4
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• pp.307-313
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• 2006

The development of in vitro assays has been recommended to screening and testing the potential endocrine disruptors (EDs). These assay systems focus only on identifying the estrogenic or antiestrogenic activity of EDs, whereas a few studies have been carried out to screen the thyroid hormone (TH) disruptors. The aim of this study was to evaluate a test system to detect TH disruptors using rat pituitary tumor $GH_3$ cells. The test system is based on the TH-dependent increase in growth rate. As expected, L-3,5,3-triiodothyronine ($(T_3)$ markedly induced a morphological change in $GH_3$ cells from flattened fibroblastic types to rounded or spindle-shaped types. $T_3$ stimulated $GH_3$ cell growth in a dose-dependent manner with the maximum growth-stimulating effect being observed at a concentration $1{\times}10^9M$. In addition, $T_3$ increased the release of growth hormone and prolactin into the medium of the $GH_3$ cells culture. Using this assay system, the TH-disrupting activities of bisphenol A (BPA) and its related compounds were examined. BPA, dimethy/bisphenol A (DMBPA), and TCI-EP significantly enhanced the growth of $GH_3$ cells in the range of $1{\times}10^{-5}M\;to\;1{\times}10^{-6}M$ concentrations. In conclusion, this in vitro assay system might be useful for identifying potential TH disruptors. However, this method will require further evaluation and standardization before it can be used as a broad-based screening tool.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.2
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• pp.204-210
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• 1999

The present studies were conducted to evaluate the effects of activin-A on gonadotropins (GTHs) release in testosterone-treated immature rainbow trout Oncorhpynchus mykiss. The administration of testosterone elevated pituitary level of GTH II but not of GTH I. In this study using primary cultures of dispersed pituitary cells in static incubation, dose-dependent increases in GTH II release was observed in the activin-treated group at day 3 of incubation (long-term incubation), but not at day 1 of incubation (short-term incubation). Dopamine, a potent inhibitor of gonadotropin-releasing hormone (GnRH)-stimulated GTH II release in rainbow trout, was only partially effective in decreasing actvin-induced GTH II release. Furthermore, salmon GnRH (sGnRH)-stimulated GTH II release was not potentiated by the pretreatment with activin. However, the control mechanisms of GTH I release by activin and other hormones were not observed in the all tested experiments. The results of these studies support the contention that in contrast with the usual stimulatory effects of activin on GTH release in mammals, activin exerts long-term stimulatory actions on GTH II release in rainbow trout. The control mechanism of GTH I release, however, is a question that remains to be elucidated.

• Animal cells and systems
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• v.3 no.4
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• pp.385-391
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• 1999

Changes in the levels of prostaglandian F$_{2a}$ (PGF$_{2a}$) and E$_2$ (PGE$_2$) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12-O-tetradecanoylphorbol-13-acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF$_{2a}$ to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE$_2$ increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF$_{2a}$ induced ovulation but PGE$_2$ suppressed FPH- or PGF$_{2a}$-induced oocyte ovulation. Basal levels of PGs Increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell-enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOS. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF$_{2a}$ plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) thecal epithelium layer is responsible for the PGs production in Rana.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.430-436
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• 2010

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

• Animal cells and systems
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• v.10 no.4
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• pp.203-209
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• 2006

Azoles are widely used antifungal agents, which inhibit the biosynthesis of fungal cell-membrane ergosterol. In this study, using an amphibian follicle culture system, the effects of azoles on follicular steroidogenesis in frogs were examined. Itraconazole (ICZ), clotrimazole (CTZ) and ketoconazole (KCZ) suppressed pregnenolone ($P_5$) production by the follicles ($ED_{50};\;0.04_{\mu}M,\;0.33_{\mu} M,\;and\;0.91_{\mu}M$, respectively) in response to frog pituitary homogenates (FPH). However, fluconazole (FCZ), miconazole (MCZ) and econazole (ECZ) were not effective in the suppression of $P_5$ production. Not all the azoles examined suppressed the conversion of exogenous $P_5$ to progesterone ($P_4$) (by $3{\beta}$- HSD) or $P_4$ to $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) (by $17{\alpha}$-hydroxylase), or androstenedione (AD) to testosterone (T) (by $17{\beta}$-HSD). In contrast, CTZ, MCZ and ECZ in medium partially suppressed the conversion of $17{\alpha}$-OHP to AD (by C17-20 lyase) ($ED_{50};\;0.25{\mu} M,\;4.5{\mu}M,\;and\;0.7{mu}M$, respectively) and CTZ, KCZ, ECZ and MCZ strongly suppressed the conversion of exogenous T to estradiol ($E_2$) (by aromatase) ($ED_{50};\;0.02{\mu}M,\;8{\mu}M,\;0.07{\mu}M,\;0.8{\mu}M$, respectively). These results demonstrated that some azole agents strongly suppress amphibian follicular steroidogenesis and particularly, P450scc and aromatase are more sensitive to azoles than other steroidogenic enzymes.

• Korean Chemical Engineering Research
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• v.43 no.1
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• pp.103-109
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• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.