• Journal of Microbiology and Biotechnology
• /
• v.15 no.2
• /
• pp.395-402
• /
• 2005

Biologically active recombinant human follicle stimulating hormone (rhFSH) was produced in Chinese hamster ovary cells and purified by a series of chromatographic steps. The chromatographic steps included anion-exchange chromatography (DEAE Sepharose F/F, Q Sepharose F/F), hydrophobic interaction chromatography (Source 15 PHE), and hydroxyapatite chromatography (Macro-Prep ceramic hydroxyapatite type I). A distinctive step of the purification process developed was the use of ZnCl$_2$ for the removal of non-glycosylated or lowly-glycosylated FSH and impurities through co-precipitation with Zn$^{2+}$. Purified rhFSH was identified and characterized by several physicochemical and biological methods such as gel electrophoresis, high-performance liquid chromatography, amino acid analysis, carbohydrate analysis, and biological activity. The overall yield of the purification was ~$30\%$. The rhFSH preparation obtained showed high purity (>$99\%$) and high in vivo potency (>16,000 IU/mg). Carbohydrate analysis suggested that the purified rhFSH contained approximately $40\%$ (w/w) carbohydrate with di­or tri-antennary structure on average, which is somewhat more heavily sialylated than commercially available rhFSH. In conclusion, the results of these analyses established an identity of the purified rhFSH with natural FSH from human pituitary glands, and furthermore, the purified rhFSH preparation showed higher in vivo potency and was slightly more heavily sialylated than commercially available rhFSH.

• Animal cells and systems
• /
• v.1 no.1
• /
• pp.99-105
• /
• 1997

Using three species of Korean frogs (Rana dybowskii, R. rugosa and R. nigromaculata), the annual spermatogenic pattern, the seasonal changes in the steroidogenic competence, and responsiveness of testis to gonadotropins in terms of testosterone secretion in vitro were examined. The spermatogenic pattern of R. dybowskii was classified as a discontinuous type since spermatogenesis stops completely after spawning in late winter (February) until mid-summer (July). In contrast, the pattern of R. nigromaculata and R. rugosa was classified as a potent continuous type since sperm was always present in the seminiferous tubules all year round. In all three species, the levels of testicular testosterone and that of testosterone secreted by testis following in vitro culture were very low in late summer (August), but increased thereafter until winter (hibernation period). Interestingly, responsiveness of testis in vitro to gonadotropins in terms of testosterone secretion increased markedly in November (early hibernation period). Specifically, bullfrog LH was more effective than FSH in stimulating the secretion of testosterone by frog testis in vitro during hibernation period. This fact suggests that testosterone secretion by testis during hibernation is at least regulated by the pituitary gonadotropin rather than environmental factors. Taken together, the data presented here suggest that testicular cycles of three species of Korean frogs are closely linked to their females breeding cycles, and are eventually controlled by various environmental cues.

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

• Animal cells and systems
• /
• v.10 no.4
• /
• pp.211-217
• /
• 2006

Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01-100 ${\mu}M$), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH-induced pregnenolone ($P_5$) production by the follicles ($EC_{50},\;4.0{\mu}M,\;25.6{\mu}M\;and\;5.7{\mu}M$, respectively ), and also suppressed the conversion of testosterone (T) to estradiol $17{beta}\;(E_2)\;(EC_{50},\;4.2{\mu}M,\;7.5{\mu}M\;and\;80.0{\mu}M) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as $C_{17-20}$ lyase and $17{\beta}$-hydroxysteroid dehydrogenase ($17{\beta}$-HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.3
• /
• pp.383-387
• /
• 1999

Objective: There is increasing evidence for the expression of rat in gene in several extrapituitary sites including testis and ovary. We also have demonstrated that the local LH expression in the rat epididymis and uterus, the major accessory sex organs in male and female reproductive system, respectively. Design: The present study was undertaken to elucidate whether the gene for LH receptor is expressed in rat uterus and whether the expressions of uterine LH and its receptor are differentially regulated during estrous cycle. Presence of the transcripts for rat LH receptor in the rat uterine tissue were confirmed by touchdown reverse transcription-polymerase chain reaction (RT-PCR). Results: In $LH{\beta}$ semi-quantitative RT-PCR, the highest expression level was shown in estrus stage. The level of ill receptor transcripts was also fluctuated during estrous cycle. In ovariectomized rats (OVX + Oil), the expressions of both uterine LH and LH-R were markedly reduced when compared to those from normal rats. Supplement with estradiol $17{\beta}$ to the ovariectomized rats (OVX + $E_2$) restored the expression levels of LH and its receptor to the levels in uteri from normal rats. Conclusion: Our findings indicated that 1) LH and its receptor gene are expressed in the rat uterus from cycling rats, 2) the expression of uterine LH and its receptor is mainly, if not all, under the control of ovarian sex steroid(s). These results suggested that the uterine LH may act as a local regulator with auto and/or paracrine manner, though the posibility that the pituitary LH may act directly on the regulation of uterine functions could not be discarded.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.35 no.6
• /
• pp.676-681
• /
• 2002

Different types of transcripts encoding growth hormone (GH) were identified from cDNA libraries constructed with pituitaries of a marine fish species, greenling (Hexagrammos otakii). GH-homologous cDNA clones were isolated using the high-density filter hybridization and the expressed sequence tag techniques. Of 39 full-length positive cDNA clones, 31 clones ($79\%$) displayed an identical sequence, however, remaining 8 clones exhibited several polymorphisms in their sequences including (1) the length and sequence variability in the 5' upstream region, (2) insertional sequences in open reading frame, and (3) deletion and/or single nucleotide polymorphism in the untranslated 3' region. Based on RT-PCT and RNA dot blot analyses, these transcripts were proven to be expressed in a pituitary-specific manner.

• Journal of Korean Biological Nursing Science
• /
• v.1 no.1
• /
• pp.1-24
• /
• 1999

The purpose of this study was to define the content of requisite human structure and function knowledge needed for clinical knowledge of nursing practice. Subjects of human structure and function were divided into 10 units, and each unit was further divided into 21 subunits, resulting in a total of 90 items. Contents of knowledge of human structure and function were constructed from syllabus of basic nursing subjects in 4 college of nursing, and textbooks published by nurse scholars prepared with basic nursing sciences. The degree of need of 90 items was measured with a 4 point scale. The subjects of this study were college graduated 136 nurses from seven university hospitals in Seoul and three university hospitals located in Chonnam Province, Kyungbook Province, and Inchon. They have been working at internal medicine ward, surgical ward, intensive care unit, obstetrics and gynecology ward, pediatrics ward, opthalmology ward, ear, nose, and throat ward, emergency room, rehabilitation ward, cancer ward, hospice ward, and their working period was mostly under 5 years. The results were as follows: 1. The highest scored items of human structure and function knowledge necessary for nursing practice were electrolyte balance, blood clotting mechanism and anticoagulation mechanism, hematopoietic function, body fluid balance, function of plasma, and anatomical terminology in the order of importance. The lowest scored items of human structure and function knowledge necessary for nursing practice was sexual factors of genetic mutation. 2. The highest order of need according to unit was membrane transport in the living unit, anatomical terminology in movement and exercise unit, mechanism of hormone function in regulation and integration unit, component and function of blood in oxygenation function unit, structure and function of digestive system in digestive and energy metabolism unit, temperature regulation in temperature regulation unit electrolyte balance in body fluid and electrolyte unit, concept of immunity in body resistance unit, and genetics terminology in genetics unit. The highest order of importance according to subunit was membrane transportation in cell subunit, classification of tissues in tissue unit, function of skin and skin in skin subunit, anatomical derivatives of the skeleton subunit, classification of joints in joint subunit, an effect of exercise on muscles in muscle subunit, function of brain in nervous system subunit, special sense in sensory subunit mechanism of hormone function in endocrine subunit, structure and function of female reproductive system in reproductive system unit, structure and function of blood in blood unit, structure of heart, electrical and mechanical function in cardiovascular system unit, structure of respiratory system in respiratory system subunit, structure and function of digestive system in digestive system subunit, hormonal regulation of metabolism in nutrition and metabolism subunit, function of kidney in urologic system subunit, electolyte balance in body fluid, electolyte and acid-base balance subunit. 3. The common content of human structure and function knowledge need for all clinical areas in nursing was structure and function of blood, hematopoietic function, function of plasm, coagulation mechanism and anticoagulation mechanism, body fluid, electrolyte balance, and acid-base balance. However, the degree of need of each human structure and function knowledge was different depending on clinical areas. 4. Significant differences in human structure and function knowledge necessary for nursing practice such as skin and derivatives of the skin, growth and development of bone, classification of joint, classification of muscle, structure of muscle, function of muscle, function of spinal cord, peripheral nerve, structure and function of pancrease, component and function of blood, function of plasma, structure and function of blood, hemodynamics, respiratory dynamics, gas transport, regulation of respiration, chemical digestion of foods, absorption of foods, characteristics of nutrients, metabolism and hormonal regulation, body energy balance were demonstrated according to the duration of work. 5. Significant differences in human structure and function knowledge necessary for nursing practice such as classification of tissue, classification of muscles, function of muscles, muscle metabolism, classification of skeletal muscles, classification of nervous system, neurotransmitters, mechanism of hormone function, pituitary and pituitary hormone, structure and function of male reproductive organ, structure and function of female reproductive organ, component and function of blood, function of plasma, coagulation mechanism and anticoagulation mechanism, gas exchange, gas transport, regulation of respiration, characteristics of nutrients, energy balance, function of kidney, concept of immunity, classification and function of immunity were shown according to the work area. Based on these findings, all the 90 items constructed by Korean Academic Society of Basic Nursing Science should be included as contents of human structure and function knowledge.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.12
• /
• pp.1659-1664
• /
• 2001

Several studies have been done regarding carcass traits and growth in pigs. Recently, these have progressed to examine increases in economic traits, including meat quality and meat quantity, by using candidate genes. One of them is the pituitary-specific protein PIT-1, a member of the POU (Pit-Oct-Unc) family of transcription factors playing an important regulatory role in developmental processes. In addition, muscle development is known to be regulated in part by growth factors and cytokines locally produced. Therefore, studies were performed to analyze PIT-1 genotypes and serum cytokines (IGF-I, IGF-II, TGF-${\beta}1$, EGF, cortisol, DHEA-S, IL-2, and IL-6) in castrated male pigs for their possible involvement in the development of carcass traits. The genotypes of PIT-1 gene were analyzed by PCR-RFLP with MspI restriction enzyme. But, only CD and DD genotypes, not CC genotype, have been detected. Based on PIT-1 genotyping, a significant difference in EGF expression beween CD type (78.8 ng/ml) and DD type (46.0 ng/ml) was detected (p<0.05), whereas other cytokines did not show any statistical significance depending on PIT-1 genotypes. Collectively, these results suggest the possibility that EGF could affect the formation of carcass traits.

• Asian-Australasian Journal of Animal Sciences
• /
• v.5 no.1
• /
• pp.173-181
• /
• 1992

Duck is an important domestic animal, especially in Asia. Eighty five percent of ducks in the world are kept in Asia, especially in the East and South Asia regions. The ancestor of domesticated ducks was mallard (Anas platylhynchos), which are still migrating between north and southern parts in Eurasia. Ducks have been domesticated in China for at least 3000 years ago. Phylogenetic studies on ducks, employing electrophoresis of blood proteins, indicate a marked difference of genetic constitution between duck breeds in southeast Asia and those in northeast Asia. Duck embryonic ovary is much more active in secretion of sex steroid hormones especially estradiol than the embryonic testes. Estradiol secreted by the embryonic left ovary has an important role in female sexual differentiation in ducks. In the female ducks, plasma LH, estradiol and testosterone levels increase and reach peaks shortly before the first egg, while progesterone level reach a peak shortly after the first egg. In laying ducks oviposition mostly occurs in the last 3 hr of darkness and first hr of light ranging 02:00-06:00 under 14 hr light (05:00-19:00) and 10 hr darkness photoperiodic condition. Measurements of plasma hormone levels reveal that onset of darkness is a major signal for LH release from the pituitary and the subsequent release of progesterone from ovary, and for induction of ovulation in the female duck.

• The Korean Journal of Zoology
• /
• v.36 no.4
• /
• pp.487-495
• /
• 1993

TRH (Thvrotropin-Releasing Hormone) known to regulate the transcription of the TSH (Thyroid-Stimulating Hormones gene in pituitary cells, but little is understood about the mechanism(sl involved. re present study was attempted to elucidate the role of Ca2+ movement through the voltage-gated channels in the regulation of TSH gene transcription. The c-fos is one of immediate early genes and used as model system for the investigation of signaling pathwavs involved in various stimuli. The changes of c-fos mRNA levels were determined after treatment of various agents using Northern and slot hybridization analysis. The c-fos mRNA was rapidly and transiently induced by TRH (about 3-fold) in GH3 cells and this induction was repressed by calcium chelating agent (EGTA), calcium channel blocker (verapamil) anti protein kinase C inhibitor (aminoacridine). The abilities of forskolin (adenvlate cvclase activators, PMA (protein kinase C activator), and A23187 (calcium ionophore) to affect c-ios gene transcription, either alone or in combination with TRH were tested in the same cells. All of them significantly increased the level of c-fos mRUA. However, no additive relationship was observed in all combined treatments except forskolin. These results suggest that TRH action on the c-fos gene activation is mediated by calcium influx as well as through protein kinase C.