• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.315-324
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• 2004

Cytochrome P450 aromatase is the enzyme responsible for biosynthesis of female sex hormone(estrogen) and 19-nortestosterone(nandrolone), a unique steroid hormone endogenously synthesized in the pig. By use of RT-PCR coupled with DHPLC technique (WAVE analysis), expression pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. The result from the DHPLC demonstrated that PCR amplified DNA fragments of ovary and testis tissues. using unique PCR primers for all three types of aromatase genes, were different from those of type II and ill genes. Further nucleotide sequence analyses of the plasmid clones containing the PCR products revealed that nucleotide sequences of all clones were identical to type I aromatase gene(ovary type). Thus, the result from the present study indicates that the ovary and testis express the same type of aromatase gene. Therefore, the efficacy of DHPLC techniques used for this study helped us to analyze tissue-specific expression of isoform of genes containing the nucleotide sequences with high homology.

• Environmental Mutagens and Carcinogens
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• v.26 no.2
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• pp.53-58
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• 2006

Normally, environmental toxicants are classified as endocrine disruptors if they interfere with regulation of cellular function by endogeneous steroids through inhibition of receptor binding and/or transcriptional activation. So, many studies have been performed about agonist/antagonist of hormone receptor to study mechanisms of endocrine disruptors. If toxicants affect steroid biosynthesis and/or degradation and alter hormone homeostasis, these also are classified as endocrine disruptors. But there are not many studies of the mechanisms of endocrine disruptors on the basis of alteration of steroid biosynthesis and/or degradation. Isolation and culture of Leydig cells from testis is one of methods for the steroidogenesis screening assays to evaluate a substance for altering steroidogenesis. Leydig cells were harvested using the method described by Klinefelter with modifications. Leydig cells were purified by perfusion of testis and incubation ($34^{\circ}C$, 80cycles/minute, 20 minutes) with collagenase (0.25 mg/kg), centrifugal elutriation, percoll gradient centrifugation and BSA multidensity gradient centrifugation. To confirm if this method is one of appropriate tools to evaluate a substance for altering steroidogenesis, ketoconazole, positive control was administered to purified Leydig cells. Ketoconazole ($10^{-8}M$ and above) significantly reduced testosterone production in purified Leydig cells. From above results, we suggest that this method for steroidogenesis screening assay appears to be a appropriate tool to detect suspected compounds for altering steroidogenesis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.5
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• pp.309-315
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• 2005

Changes of sex steroid hormones in the plasma of yellowfin goby, Acanthogobius flavimanus were investigated in relation to the gonadosomatic index (GSI), the hepatosomatic index (HSI) and gonadal development. The GSI in females rose rapidly in November and remained high from December to May $(7.26\pm0.89­6.62\pm0.02)$. The Male's GSI also increased gradually from November and was highest in May $(0.16\pm0.08)$. The HSI in both sexes was in reverse correlation with the GSI, and the HSI was low during the spawning season (February-May). In females, the $estradiol-17{\beta}\;(E_2)$ level increased during vitellogenesis (November and December) and reached its maximum $(8.13\pm2.87 ng/mL)$ at the maturing period, in January. $17{\alpha},\;20{\beta}$-dihydroxy-4-pregnen-3-one$(17{\alpha}20{\beta}OHP)$ gradually increased from October $(0.063{\pm}0.02ng/mL)$ to March $(0.16{\pm}0.02ng/mL)$ and increased rapidly in May. The level of testosterone (T) showed a similar tendency of $E_2$. In males, T increased gradually during spermatogenesis from September to December $(0.14{\pm}0.06­0.26{\pm}0.10ng/mL)$ and peaked in January $(0.36{\pm}0.29 ng/mL)$ when the spermatozoa filled the testis. 11-KT also rose markedly in January and then decreased. On the other hand, $17{\alpha}29{\beta}OHP$ in males did not show any clear tendencies.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.93-102
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• 1998

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptides, which may mediate or potentiate gonadotropin action. Inhibin also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia. Therefore, we studied the differences of serum inhibin, gonadotropin and androgen levels in the women with only the ultrasound findings and no disorder, and polycystic ovary (PCO) with ovulatory disturbance. We prospectively analysed forty-three women with PCO. The diagnosis of PCO was based on typical appearance of the ovaries on TVS. Twelve women with regular menstrual cycle and normal ovarian morphology were selected as control. Basal levels of inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol $(E_2)$, testosterone (T), androstenedione (ADD), dehydroepiandrosterone-sulfate (DS), prolactin and TSH in serum were determined. There were significant differences in basal LH levels and LH/FSH ratio between the control and the women with PCO. The basal levels of inhibin and $E_2$ in the oligo-amenorrheic PCO (N=34) were significantly higher than those in the control. There was higher negative correlation between the inhibin and T levels in the oligo-amenorrheic PCO, but, not in the regular cycling PCO. Also, there was higher positive correlation between the LH and T levels in the oligo-amenorrheic PCO, but not in the regular cycling PCO. These data presume that the initial event of PCO is elevated pituitary LH secretion. Elevated levels of LH may down-regulate LH receptors on granulosa cells and also cause hypertrophy of the thecal layer. High level of androgen secreted by the hypertrophied thecal layer may stimulate inhibin secretion from granulosa cells and can be converted to estrogen by extraovarian tissues and could serve to augment pituitary sensitivity to GnRH with a resultant secretion of more LH than FSH. Inhibin may inhibit FSH action on granulosa cell in the PCO follicle, impairing follicular development and dominant follicle selection resulted in ovulatory disturbance.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.293-299
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• 2000

Many methods have been developed for screening chemicals with hormonal activity. Using recombinant yeasts expressing either human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127 (YER)] or androgen receptor [S. cerevisiae AR + 8320 (YAR)], we evaluated the hormonal activities of several chemicals by induction of ${\beta}-galactosidase$ activity. The chemicals were $17{\beta}-estradiol$ (E2), testosterone (T), ${\rho}-nonylphenol$ (NP), bisphenol A (BPA), genistein (GEN), 2-bromopropane (2-BP), dibutyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and butylparaben (BP). To assess the estrogenicity of NP, the result of the in vitro recombinant yeast assay was compared with an E-screen assay using MCF-7 human breast cancer cells and an uterotrophid assay using ovariectomized mice. In the YER yeast cells, E2, NP, BPA, GEN, and BP exhibited estrogenicity in a doseresponse manner, while TCDD did not. All the chemicals tested, except T, did not show androgenicity in the YAR yeast cell. The sensitivity of the yeast (YER) assay system to the estrogenic effect of NP was similar to that of the E-screen assay. NP was also estrogenic in the uterotrophic assay. However, in terms of convenience and costs, the yeast assay was superior to the E-screen assay or uterotrophic assay. These results suggest that the recombinant yeast assay can be used as a rapid tool for detecting chemicals with hormonal activities.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.1
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• pp.83-98
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• 1985

In order to study the mechanism of follicular atresia, the follicles of the porcine ovary were isolated according to the presence or absence of the corpus luteum and their size, and then classified to the normal? or atretic?follicle on the morphological observation such as the transparency, the vascularization of follicle, the nuclear phase of oocyte, and the homogeneity of the granulosa cell layer. The viability of granulosa cells was examined. The concentrations of progesterone ($P_4$), testosterone (T), and estradiol-17 beta ($E_2$) in each follicular fluid were estimated by the radioimmunoassay. The viability of granulosa cells in the atretic follicle was much lower than that of the normal one. The concentration of each steroid hormone increased as the follicular size was increased, was not different in quantity between the normal- and the atretic follicle of which diameter was below 3mm, and were much higher in the atretic follicle than those in the normal one of which diameter was above 7mm. The ratio of the concentration of E2 to T in the large atretic follicle valued higher than that in the normal one, but smaller in the small and medium atretic follicle than that in the normal one. The present study suggests that the mechanism of atresia of the large follicle may be different from that of the small and the medium follicle and that the amount of steroid hormones regarded as the one of the criteria for the atretic follicles.

• Toxicological Research
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• v.16 no.2
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• pp.163-172
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• 2000

Recently, there is a worldwide concern that a great number of man-made chemicals have a hormone-like action both in humans and in animals. DECD is developing screening programs using validated test systems to determine whether certain substances may have an effect in humans. In the present study. the establishment oj repeated-dose toxicity test method was tried. Flutamide. an anti-androgenic agent. was administered by gavage to Sprague-Dawley rats for 28 days at dose levels of 0. 0.5. 3 and 18 mg/kg body weight (10-15 rats/sex/group) to examine the effects on general findings. especially reproductive and endocrine parameters. Clinical signs. body weights, food consumption, and sexual cycle were checked and measured. For the gross and microscopic examinations. 10 rats/sex/group were sacrificed at the end of dosing period and the remaining animals of control and high dose groups (5 each) were sacrificed after 14 days recovery. Examinations for hematology and clinical chemistry were carried out at necropsy. There were no treatment-related changes in clinical signs. body weights, food consumption. gross necropsy. hematology and clinical chemistry at all doses of both sexes. The period and regularity of sexual cycle were not adversely affected at all doses by the test agent. At 18 mg/kg. both decreased weights of prostate, seminal vesicle and epididymis in males and increased weights of spleen and thymus in females were observed. In addition, decreased number of spermatids and sperms. increased serum testosterone concentration and increased incidence (100%) of interstitial cell hyperplasia were seen in males. At 18 mg/kg of the recovery group. decreased prostate weight. reduced sperm count and increased incidence (20%) of interstitial cell hyperplasia in males and increased thymus weight in females were observed. At 3 mg/kg. reduced sperm count was found. There were no adverse effects on parameters examined at 0.5 mg/kg of both sexes. The results suggested that the potential target organs of flutamide may be accessory sexual glands including testes for males and spleen and thymus for females. Taken together. this test method was found to be a useful screening test system for endocrine disrupting chemicals.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.81-86
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• 1996

The assessment of acrosomal status is important in evaluating the ability of sperm to fertilize the egg. The acrosomal status of sperm from 47 normal volunteers with proven fertility and 167 subfertile men with not to achieve pregnancy for at least 1 year were evaluated with Acrobeads test(FUSO Pharmaceutical Industries, Ltd, Japan) using immunobeads coated with MH61 monoclonal antibody, which is specific for acrosome-reacted sperm. The mean${\pm}$SD of acrobeads score in 47 volunteer group was $2.8{\pm}0.7$, of which 46(97.9%)cases were ${\geq}$ 2. The mean${\pm}$SD of acrobeads score in 167 subfertile group was $1.7{\pm}0.8$, of which 73(79.3%)cases were ${\leq}$ 1. The aerobe ads score in subfertile group were significantly lower(r=0.294, p<0.05) than those in volunteer group. In subfertile group, acrobeads score were well correlated with the sperm density and motility(r=0.275, r=0.281, p<0.01), but not with semen volume(r=0.16) and serum hormone level(FSH r=0.084, LH r=0.036, testosterone r=0.058, prolactin r=0.006 and estradiol r=0.060)(p>0.05). Of 63 subfertile cases with normozoospermia, 22(34.9%)cases showed 0 or 1 of acrobeads score, which means to accompany with a functional defect in spite of normal morphology. As a results, Acrobeads test is not only a technically simple sensitive procedure with good reproducibility in evaluating the sperm fertilizing capacity but also an useful in the evaluation of effectiveness in the treatment of infertility and the separation of acrosome-reacted sperm in the assisted reproductive technique.

• Development and Reproduction
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• v.15 no.2
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• pp.167-172
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• 2011

To monitor the reproductive patterns of endangered common chimpanzee including annual reproductive cycle, amenorrhea, breeding season, and pregnancy diagnosis, Time-Resolved Fluorescence Immuno Assay (TR-FIA) was used to trace MRH (estradiol, progesterone, testosterone) and human chorionic gonadotropin (HCG). In result of this research, age was not the important factor in determining the reproduction capability in common chimpanzee; it was rather greatly influenced by the combination of various factors such as individual's fertilizing ability, presence of obstetrical diseases, the pattern of mating behavior, and the mental communication under the introduction of a mating partner. This research will play an important role in operating conservation project for common chimpanzees and can be extended also for shedding new light on understanding human menopause and obstetrical diseases.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.119-130
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• 1989

본 연구는 생식주기중 폐쇄여포액내에서 생물학적， 면역학적 특성을 나타내는 GTH 의 변화를 조사하고 steroid hormone과의 상관관계를 조사하며 국부조절인자로서 의 GTH의 역 활을 조사하고자 하였다. 가임기간중 215개의 여포와 IVF과정에서 185개의 여포를 얻어 여포액내 GTH의 생물학적 또는 면역학적 활성을 측정하였다. Bioactive LH(bLH)는 생쥐의 Leydig cell-testosterone production assay, bFSH는 흰쥐의 Sertoli cell aromatase assay로 측정 하였 다. Immunological GTH(iLH , iFSH) 는 MaiaClone RIA , Delfia kits를 사용하였다. 여포액내 iLH, iFSH , ihCG 는 hyperstimulation에 의해 형성된 여포의 크기와는 무관하였다. 또 hMG, huFSH 의 처리와도 상관성이 없었다. T의 농도가 높은 여포액내의 iFSH는 현저히 낮았으며 E, P 가 고농도인 여포의 ihCG 양은 현저히 낮았다. 과배란이 유도된 난소의 여포액내 iLH는 LH specific RIA로 측정시 3mIU/ml 이하이었다. 생식주기중 여포액내 bLH, bFSH는 배란기에 현저히 증가 하였다. 혈청내 GTH B/I ratio는 엘정한 반면 여포액내 LH，FSH의 생물학적， 면역학적 활성은 미수정란을 가지거나 폐쇄된 여포내의 활성보다 현저하게 높았다. 위의 결과로 보아 여포액내 생식소자극호르온은 면역학적활성보다 높은 생물학적 활성을 가지며， 생리적 현상의 지표가 된다고 추론된다. 또한 steroid, bGTH는 여포의 선택， 폐쇄를 구분하는 지표로 사용가능하며， 여포가 폐쇄될때 여포액내 B/I ratio가 현저히 낮아지는 것으로 보아 GTH의 활성이 감소되는 것으로 판단된다.