Relaxin과 insulin이 돼지 난포 과립막세포의 스테로이드 호르몬 분비에 미치는 영향을 연구하기위하여 체외에서 황체화된 과립막세포에서 prosesterone과 의 생산을 조사하였다. 돼지난포 과립막세포를 혈청 존재하에 배양접시에 부착 후 48시간 동안 체외배양하고 무혈청 배지에서 24시간 배양하였다. Relaxin과 insulin의 용량의존성을 확인하기 위하여 다양한 농도 (10, 100, 1,000 ng/ml)를 각각 무혈청 배지에 첨가하였다.

Adipose tissue is one of the major endocrine gland. More recently, local production of steroids in adipocytes differentiated from mouse 3T3-L1 cell-line was reported. We hypothesized that rat adipocytes have steroidogenic machinery and the expression patterns of the components might be differentially regulated, depending on the distribution and sex. To verify this hypothesis, we collected the adipose tissues depot- and sex-specifically at postnatal day (PND) 30, and performed quantitative RT-PCRs. In overall aspects, the abundances of the transcripts were lower in the brown adipose of both sexes. 3β-HSD transcript levels in female abdominal and reproductive adipose, CYP17 transcript levels in female reproductive adipose, 17β-HSD transcript levels in female abdominal and reproductive adipose, and CYP19 transcript levels in female abdominal adipose were significantly lower than those of male counterparts. Similar to steroidogenic factors, the abundance of the ER-α transcripts were generally lower in the brown adipose of both sexes. ER-β transcripts were more abundant in male white adipose depots than their female counterparts. The levels of LHR transcripts in female reproductive adipose were significantly higher than those of male counterpart. In conclusion, our study demonstrated that the expressions of steroidogenesis-related genes were depot- and sex-specifically occurred in the immature male and female rat adipose tissues. Our study suggested that the adipose tissues are not only targets but de novo synthesizing sites of sex steroid(s), though the synthesizing activities could be much less than in gonads. Further researches in this field will be helpful for understanding the adipose physiology and for medical application such as sex-specific steroid supplement therapies for older populations.

Estrogen is an important regulator of reproduction in both male and female. The two forms of estrogen receptor (ER) are known, ERα and ERβ. To understand the role of ERα in the testis, we investigated the expression of ERα in the mouse Leydig cells during postnatal development and the effects of estrogen on steroidogenesis and proliferation in progenitor Leydig cells (PLCs). In the testis, ERα mRNA and protein levels were markedly increased from postnatal day (PND) 1 to 14 and decreased thereafter until PND 56. During postnatal development ERα immunoreactivity was strong in the nucleus of Leydig cells at PND 14 when PLCs were abundant in the interstitium and low in the mature adult Leydig cells (ALCs). In fetal Leydig cells (FLCs), ERα immunoreactivity was negligible at birth and became increased at PND 14. This suggests an important role of ERα in Leydig cells during neonatal period. In isolated PLCs, 17β-estradiol (E2) and ERα-selective agonist, PPT suppressed the hCG-induced progesterone production and steroidogenic pathway genes expression. The hCG-induced PLCs proliferation was significantly inhibited by E2 and PPT. In conclusion, estrogen - ERα signaling may negatively regulate functional differentiation and proliferation of PLCs.

Water channel proteins, aquaporins (AQPs) contribute to transepithelial water movement in many tissues. To date, 13 mammalian AQPs have been identified. Of these, AQP5 plays an important role in the fluid homeostasis and cell volume control in epithelial cells. In an effort to understand the role of AQP5 in testis, we investigated the expression of AQP5 in developing mouse testis, its regulation by estrogen and LH, and the change of steroidogenesis by AQP5 knockdown. Testes and Leydig cells were isolated from male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and estrogen receptor alpha knockout (ERαKO) mice. In mouse testis, AQP5 immunoreactivity was negligible by PND 14. From PND 28 onward, AQP5 immunoreactivity was found in Leydig cells. In ERαKO mouse Leydig cells, AQP5 mRNA level was significantly lower than wild type. In primary adult Leydig cell culture, the expression of AQP5 mRNA was increased by 17β-estradiol (E2) and human chorionic gonadotropin (hCG), but was not changed in ERαKO Leydig cells. Moreover, the expression of AQP5 mRNA was increased by E2 and ERα-selective agonist PPT, but was not changed by ERβ-selective agonist DPN in primary Leydig cells and mLTC-1. In silico analysis and chromatin immunoprecipitation (ChIP) assay revealed that there are putative estrogen response elements (EREs) and cAMP response elements (CRE) in AQP5 promoter region. Testosterone secretion and steroidogenic pathway genes (StAR, Cyp11a1, Cyp17a1, and 3β-HSD6) expression were decreased by AQP5 siRNA in primary Leydig cells. In conclusion, AQP5 expression was coupled with functional differentiation of adult Leydig cells. AQP5 may play an important role in the fluid homeostasis and cell volume control during development of adult Leydig cells. The expression of AQP5 in Leydig cells could be regulated by ERα and LH signaling and AQP5 may be involved in steroidogenesis.

In this study, oocyte steroidogenesis are investigated in relation to oocyte development in the starry flounder, Platichthys stellatus, a marine multiple spawner. Vitellogenic (0.52 and 0.55 mm oocyte diameter) and mature oocytes (0.63, 0.66 and 0.71 mm oocyte diameter) were incubated in vitro in the presence of [3H]17α-hydroxyprogesterone ([3H]17α- OHP) as a precursor. Steroid metabolites were extracted from the incubated media and oocytes, the extracts were separated and identified by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatographymass spectrometry (GC-MS). The major metabolites produced from [3H]17α-OHP were androgens [androstenedione (A4) and testosterone (T)] and estrogens [17β-estradiol (E2) and estrone (E1)] and progestins [17α,20α-dihydroxy-4-pregnen- 3-one (17α20αP) and 17α,20β-dihydroxy-4-pregnen-3-one (17α20βP)] in vitellogenic and mature oocytes. The results from this study suggest the potential roles of E1 in the oocytes with diameter 0.52-0.71 mm, 17α20αP and 17α20βP at the oocytes of 0.63, 0.66 and 0.71 mm.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

스테로이드 호르몬은 다양한 생리작용의 조절자로서 잘 알려져 있으며, 생리적 활성에 따라 5그룹 (mineralocorticoids, glucocorticoids, estrogens, progestagens, androgens)으로 나뉜다. 그 중 estrogen류는 특히 암컷 포유류의 이차성징을 유도함이 잘 알려져 있다. 본 연구는 사춘기 전후 암컷 흰쥐난소에서의 steroidogenesis 조절 효소의 유전자 발현에 대해 정밀 조사한 것이다. 미성숙한 암컷 흰쥐를 생후 29일에서 43일까지 2일 간격으로 희생한 뒤 total RNA를 추출하여 semi-quantitative RT-PCR을 시행하여 steroidogenesis 관련유전자인 StAR(steroidogenic acute regulatory protein), CYP11A1(side-chain cleavage enzyme), CYP17(17α-hydroxylase), 3β-HSD(3β-hydroxysteroid dehydrogenase), 17β-HSD(17β-hydroxysteroid dehydrogenase), CYP19(Aromatase)의 발현 양상을 조사하였다. 연구에 사용된 암컷 흰쥐는 대체로 37일에 질구개방이 일어났다. 유전자 발현 조사에서 StAR는 생후 41일, 43일에 유의하게 증가(p<0.05, p<0.01)하였다. CYP11A1은 43일에 유의하게 증가(p<0.01)하였다. CYP17의 경우, 35일과 39일에서 유의한 감소(각 p<0.05)를 보였다. 3β-HSD, 17β-HSD, CYP19의 경우는 유의한 변화를 보이지 않았다. 전반적으로 스테로이드 합성 관련 유전자 모두 생후 35일에 감소하였다가 암컷의 성 성숙 지표인 질구개방이 대체로 일어난 37일을 기준으로 유전자 발현이 급증됨을 확인하였다. 이는 사춘기 개시를 위한 다량의 sex steroid 합성을 위한 것으로 추정된다. 이러한 결과는 사춘기 전후시기 난소의 성 성숙에 관한 기초자료로 사용될 수 있을 것으로 사료된다.

To verify the sex steroids which are involved in oocyte maturation of the blacktip grouper, , we incubated vitellogenic oocytes (0.41 and 0.50 mm in average diameter) in the presence of exogenous steroid precursor (-hydroxyprogesterone). Steroids were extracted, separated and identified by thin layer chromatography. The major metabolites produced were androstenedione, estradiol-, estrone and progestogens. Progestogen metabolites in the oocytes of 0.50 mm were more abundant than those of 0.41 mm. Also, we investigated the effects of human chorionic gonadotropin (HCG; 5, 50 and 500 ), -dihydroxy-4-pregnen-3-one () and -trihydroxy-4-pregnen-3-one (; 5, 50 and 500 , respectively) on oocyte maturation. In the oocytes of 0.41 mm, treatment with 50 IU HCG stimulated GVBD () compared with controls (, <0.05). In the oocytes of 0.50 mm, treatment of (50 and 500 ) stimulated GVBD ( and , respectively) compared with controls (, <0.05). Treatment with 500 IU HCG also stimulated GVBD () compared with controls (<0.05). Taken together, these results suggested that both HCG and were effective on in vitro oocyte maturation and may act as a maturation inducing hormone in blacktip grouper.

Simazine은 triazine계 제초제로서, 잡초와 일년생 풀들을 통제하는데 우리나라를 포함하여 세계적으로 널리 사용되고 있으며, 미국, 유럽, 오스트레일리아의 경우, 물에서 두 번째로 많이 검출되는 살충제로 알려져 있다. Simazine에오염된 토양과 물을 통하여 사람에게 노출되어 이후 생체 내에 수년간 잔존하며 특히, 생물체의 지방 및 조직에 농축되는특징을 갖는 내분비계 장애물질로 분류되어졌다. 하지만 simazine이 정소세포의 사멸 또는 생존

6-hydroxydopamine (6-OHDA)은 dopamine 뉴런과 norepinephrine 뉴런을 사멸시키는 신경독소로서, 실험동물의 뇌에 직접 미세주입하여 Parkinson's disease (PD)와 유사한 증세를 야기하는 모델에 널리 사용된다. 그런데, 6-OHDA 주사후 뇌내 생식내분비 관련 유전자들의 활성을 조사한 연구는 매우 제한적이며, 더욱이 시상하부-뇌하수체-표적기관 조절 개념에서 하부 표적기관 (말초조직)에서의 활성조사는 이루어진 바가 거의 없다. 따라서 본 연구는 6-OHDA에 의한 PD 유도 흰쥐 모델을 사용하여 정소와 부신의 활성 변화를 조사하고 비교하였다. 수컷 흰쥐 (3 month)에 6-OHDA (200 ㎍ in 10 ㎕ of saline/animal)을 좌측 제3뇌실에 주입하고 2주 후 희생시켰다. 시상하부-뇌하수체-정소 호르몬 축 (생식)과 시상하부-뇌하수체-부신 호르몬 축 (스트레스)의 유전자 활성과 혈중 testosterone과 coticosterone 수준을 측정하였다. 생식관련 유전자 발현의 경우, 6-OHDA 주사에 의해 시상하부에서의 KiSS-1, GPR 54, GnRH, 뇌하수체에서의 common α, LHβ, FSHβ subunit, 정소에서의 StAR, CYP11A1, CYP17A1 mRNA 수준이 모두 유의하게 감소하였으며, 혈중 testosterone 수준도 유의하게 감소하여 생식기능의 저하를 시사하였다. 스트레스 관련 유전자 발현의 경우, 6-OHDA 주사에 의해 시상하부에서의 TH, CRH, 뇌하수체에서의 ACTH mRNA 수준은 유의하게 낮았으나, 부신 수질의 catecholamine 합성 효소들인 TH와 PNMT mRNA 수준은 오히려 유의하게 증가하였고, 부신 피질의 스테로이드 합성 관련 유전자들인 StAR, CYP11A1, 3β-HSD, CYP11B1 mRNA 수준 역시 유의하게 증가하였다. 혈중 corticosterone 수준은 증가하는 경향을 보였으나 유의성은 없었다. 이 결과는 정소와 부신 (피질)의 스테로이드 합성 기구들이 중추신경에서의 catecholamine 결핍에 상반된 반응을 나타냄을 보여준 것으로, 6-OHDA 주사 모델이 PD의 진행과 말초조직들의 기능 변화에 대한 이해를 증진시키는데 도움이 될 것으로 사료된다.

본 연구에서는 nonylphenol(NP)과 2,2',4,6,6'-pentachlorobiphenyl(PCB104)이 성숙한 그물베도라치의 난소 스테로이드 대사과정에 미치는 영향을 조사하였다. 그물베도라치의 성숙란을 방사 표지된 스테로이드 전구물질인 -hydroxyprogesterone()와 함께 NP와 PCB104를 각각 100 ng/의 농도로 첨가하여 배양하였다. 배양 후 배양액과 난모세포로부터 스테로이드 호르몬을 추출하여 박막 크로마토그래피를 통해

GnRH는 국부적으로 난소에서 합성되며, 난소내 과립 및 황체세포에 직접적으로 작용하여 난소의 기능을 조절하는 것으로 알려져 있으며, 특히, GnRH는 난소내 과립-황체화 세포의 세포자연사를 유도하는 것으로 보고하고 있다. 그러나 GnRH에 의한 세포자연사가 FSH에 의해 회복될 수 있는지는 명확히 밝혀져 있지 않다. 따라서 본 실험에서 난자 채취시 획득한 사람 과립-황체화 세포를 배양한 후 5, 50, 100 ng/ GnRH와 1 IU/ FSH를 처리

Cortisol은 난소내 다량으로 존재하며, 난소 세포에 그 수용체가 있는 것으로 보고되고 있다. 또한 사람의 과립 및 황체화 세포에서 cortisol은 스테로이드 생성과 세포 대사에 영향을 미치는 것으로 알려지고 있으나, 배란 후 난포액에 높은 농도로 존재하는 cortisol이 과립-황체화 세포에 어떤 영향을 미치는 지는 정확히 밝혀져 있지 않다. 따라서 본 실험에서 과배란 유도후 획득한 사람 과립-황체화 세포를 배양하면서 5, 50, cortisol

Some organotin compounds such as butyltins and phenyltins are known to induce impo-sex in various marine animals and are considered to be endocrine disruptors. In this study, the effect of organotins on follicular steroidogenesis in amphibians was examined using ovarian follicles of Rana dybowskii and Rana catesbeiana. Isolated follicles were cultured for 6 or 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the levels of product steroids in the culture media oassay. Among the butyltin compounds, tributyltin (TBT) strongly and dose-dependently inhibited the FPH-induced synthesis of pregnenolone () and progesterone () by the follicles. TBT also strongly suppressed the conversion of cholesterol to and partially suppressed the conversion of to . A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin and tetrabutyltin had negligible effects. The toxic effect of TBT or DBT was irreversible and a short time of exposure (30 min) was enough to suppress steroidogenesis. All the phenyltin compounds significantly inhibited FPH-induced synthesis by the follicles. The effective dose of 50% inhibition by diphenyltin was and those of monophenyltin and triphenyltin were and , respectively. However, none of the phenyltin compounds significantly suppressed the conversion of to -hydroxyprogesterone (-OHP) (by -hydroxylase), -OHP to androstenedione (AD) (by lyase), or AD to testosterone by the follicles. Taken together, the data show that among the steroidogenic enzymes, P450scc in the follicles is the most sensitive to organotin compounds and that an amphibian follicle culture system can be a useful screening model for endocrine disruptors.