The present study was designed to investigate whether or not prostaglandins (PGs) were produced by human luteal cells (HLC) and their effects on the luteal cells by monolayer culture. The following results were obtained.
Cultured HLC secreted progestrone (P), prostaglandin F (PGF) and prostaglandin E (PGE) into a medium at concentrations of 276.6±38.6, 1.95±0.36, 2.44±0.45ng/ml/1×10⁵ cells/day (mean±SE), respectively. Cultured HLC was able to convert ¹⁴C-arachidonic acid to ¹⁴C-PGF_2α, ¹⁴C-PGE₂.
These two results indicated that HLC had the ability to produce PGF and PGE.
Cultures were carried out in the presence of indomethacin (Ind), PGF_2α and PGE₂ alone as well as in a combination.
P production by HLC was reduced in the presence of Ind. P production in the presence of Ind+PGE₂ was more than that in the presence of Ind alone. There was no significant difference in P production between the presence of Ind and Ind+PGF_2α.
It was concluded that HLC had the ability to produce PGs and that PGE₂ significantly stimulated P production in as low concentrations as HLC could produce physiologically while PGF_2α did not.

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Extensive studies have recently been conducted on the expression of hormone actions, especially on its mechanisms. Sutherland, et al. identified the function of C-AMP as an intracellular messenger of peptide hormones. Recent studies by several investigators including ours have concluded that the role of C-GMP and calcium are as important as that of C-AMP. In this report we present our findings on the role of Ca ions in the acceleration of steroid production in isolated porcine cells of the corpora lutea. (1) The incubation time needed to compare the production of progesterone and that of C-AMP is at least 120 mins. (2) Production rates of both C-AMP and progesterone in the presence of HCG were maximum at 10^-7g/ml HCG. Inclusion of Ca ions in the medium increases this production rate. (3) When Ca ions are included in the medium, the accelerated rate of progesterone production takes on its highest value at 1.2mM Ca. (4) Verapamil and cycloheximide inhibit the production of progesterone. (5) The binding capacity of ¹²⁵I-HCG combined with corpora lutea cells is independent of the Ca concentration in the medium. (6) Ca uptake into corpora lutea cells increases according to the concentration of HCG until it reaches a plateau at 10^-7g/ml HCG. This acceleration by HCG is affected by Ca and is remarkably inhibited by verapamil.
These results indicate the important role of Ca ions in the acceleration of progesterone production compared to that of C-AMP.

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【目的】哺乳類において排卵後に形成される黄体は，妊娠が成立しなかった際に機能と構造を急速に失う (黄体退行)。Matrix metalloproteinases (MMPs) の主な作用は細胞外基質 (ECM) の分解であり退行期に黄体組織中の MMPs 発現が増加することが知られているが，MMPs の産生源，発現調節機構，さらに MMPs の生理的役割は不明である。我々は黄体退行時にがリンパ管を介して卵巣外へ流出することで黄体が急速に消失することを報告したが (PLoS One, 2014)，その機構は明らかでない。がリンパ管へ流出するためにはが黄体組織から分離される必要があることから，本研究ではが MMPs を産生分泌し，ECM を分解することでリンパ管へ流出するという仮説を立て，における MMPs 発現ならびに黄体退行因子の及ぼす MMPs mRNA 発現への影響を検討した。【方法】黄体退行因子として prostaglandin F2α (PGF)， 腫瘍壊死因子 (TNF) および interferon-γ (IFNG) に着目した。中期黄体より酵素的に単離した (2.0×10⁵ cell/ml) に PGF (0.01, 0.1, 1 μM)，TNF (0.05, 0.5, 5 nM) および IFNG (0.05, 0.5, 5 nM) を添加し，24 時間後の MMP-1，MMP-2，MMP-9 および MMP-14 mRNA 発現を real-time RT-PCR 法を用いて調べた。【結果】における MMP-1 mRNA 発現は PGF ならびに IFNG によって有意に刺激された。MMP-9 mRNA 発現は PGF (0.01 μM) 添加区において濃度依存的に有意な上昇がみられた。PGF，TNF ならびに IFNG はMMP-2 および MMP-14 mRNA 発現に影響しなかった。本研究によりに MMPs mRNA 発現が確認され，その発現は PGF ならびに IFNG によって刺激されることが明らかとなったことから，由来の MMPs が周囲の ECM を分解することでのリンパ管への流出を促進し，急速な黄体消失に貢献している可能性が示された。

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[目的]Insulin-like growth factor(IGF)とその結合蛋白であるIGF binding protein(IGFBP)は諸臓器の機能調節に関わり，卵胞発育や黄体形成にもIGF-IGFBP系は重要な役割を果たしていることが知られている．本研究では，ヒト顆粒膜培養系を用いて，顆粒膜によるIGFBP産生分泌と黄体機能の関係を明らかにすることを目的とした．[方法]体外受精胚移植の採卵時に卵胞液，卵胞洗浄液より顆粒膜を採取して培養し，培養液を24時間毎に交換して培養液中estradiol(E2)，progesterone(P4)濃度を測定した．また，培養開始48時間後にhuman chorionic gonadotropin(hCG)を0，30，300，3000 mIU/ml添加して48時間培養し，無血清培養液に交換して，さらに48時間培養(培養開始後96～144時間)を続けた．この培養液中E2，P4濃度を測定するとともに，¹²⁵I-IGF-IIを用いたWestern ligand blotにてIGFBP解析を行い，bioimage analyserを用いてIGFBP濃度を定量化した．[結果]顆粒膜培養液中E2濃度 は時間経過に伴う変化を示さずほぼ一定であったが，P4濃度は培養開始後上昇し，培養開始後96時間でピークに達した後，120～144時間に有意な低下を示した．顆粒膜細胞培養開始後48～96時間にhCGを添加し，96～144時間に無血清培養した培養液中P4濃度は，hCG非添加群に比べてhCG 30 mIU/ml添加群で1.8倍，300 mIU/mlで3.7倍，3000mIU/mlで3.6倍に上昇した．この培養液のIGFBP解析ではIGFBP-2とIGFBP-3が検出され，IGFBP-2，IGFBP-3量はhCG添加濃度の増加に伴い減少した．とくにIGFBP-2はhCG 300，3000 mIU/ml添加群で非添加群に比較して有意な低下を示した．[結論]培養ヒト顆粒膜からのIGFBP-2，IGFBP-3産生分泌が機能の低下時期に一致して認められ，とくにIGFBP-2産生分泌はhCG添加による機能促進と逆相関を示して減少したことより，活性の制御に関与していると考えられた．〔産婦の進歩55(3)：281-287，2003(平成15年8月)〕

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【目的】一酸化窒素 (NO) は黄体退行因子であり、ウシ黄体において NO は主に血管内皮細胞 (LEC) が分泌すること、また、LEC の NO 分泌を prostaglandin F2α (PGF) が刺激することが知られている。ウシ顆粒層細胞において NO 合成酵素 (NOS) の発現することから、においても NOS の発現する可能性があるものの詳細は分かっていない。そこでウシ黄体退行機構に関する研究の一環として、ウシにおける NO 分泌調節機構およびが分泌する NO が自身に及ぼす影響を調べた。【材料および方法】1) ウシにおける血管内皮型NOS (eNOS) および誘導型 NOS (iNOS) mRNA 発現を RT-PCR 法により調べた。2) に PGF (1 µM) を添加し、培養上清中 NO 濃度を griess 法により調べた。3) に PGF ならびに NO 合成阻害剤 L-NAME (100 µM) を単独または組み合わせて添加し、培養上清中プロジェステロン (P4) 濃度を EIA により調べた。また、に PGF, L-NAME, 腫瘍壊死因子 (TNF; 0.5 nM) およびインターフェロン γ (IFNG; 0.5 nM) を単独または組み合わせて添加し、細胞生存率を WST-1 法により調べた。【結果】1) において eNOS および iNOS mRNA発現が認められた。2) PGF 添加区において control 区と比較し培養上清中 NO 濃度が高かった。3) PGF および L-NAME を組み合わせて添加した区において control 区と比較し P4 濃度は有意に高かった。PGF 添加区において control 区と比較し細胞生存率は有意に高かった。PGF および L-NAME を組み合わせて添加した区では PGF 添加区と比較し細胞生存率は有意に低かった。また、サイトカインのみを添加した区と比較し、サイトカインと L-NAME を組み合わせて添加した区において細胞生存率が有意に低かった。以上の結果より、NO はからも分泌されており、由来の NO は自身の P4 分泌を抑制する一方で細胞生存率を高める 2 つの相反する作用を有することが示唆された。

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The present investigation was designed to study the effect of synthetic ACTH on the synthesis of progesterone, 20α-OH-progesterone and adenosine-3', 5'-monophosphate (cAMP) in isolated luteal cells. Experiments were conducted on rats from three groups. Group I : PMS primed immature rats. Group II : PMS plus hCG primed immature rats. Group III : pregnant rats. Luteal cells were isolated by the Kumai Method (7) using a sucrose density gradient after digestion with trypsin and collagenase. Luteal cells were divided into three fractions; S-1, S-2 and S-3.
Following an 18 hr-incubation of S-1 cells of day 7 after PMS injection, a significant increase in progesterone release by ACTH lasted from 6 to 18 hr, whereas 20α-OH-progesterone decreased significantly by ACTH from 8 to 18 hr. There were no differences in progesterone and 20α-OH-progesterone releases from S-2 cells of day 7 between media with and without ACTH.
Progesterone release without ACTH reached the peak on day 7 in Group I and on day 6 in Group II. ACTH stimulated progesterone and inhibited 20α-OH-progesterone releases in Groups I and II. The maximum effect of ACTH in both Groups was noted on days 6 and 7. There were no differences in the Effective Dose (ED₅₀) and the Inhibitory Dose (ID₅₀) of ACTH between Group I and Group II.
In Group III, releases of progesterone and 20α-OH-progesterone from S-1 cells to media with or without ACTH decreased remarkably after 11 days of gestation. On days, 3, 5 and 8 of gestation, a significant increase in progesterone release from S-1 cells was noted by the ACTH addition. 20α-OH-progesterone release from S-1 cells to media wiLth or without ACTH decreased as the pregnancy advanced.
Total cAMP of intra- and extra-cellular S-1 cells (Group II) on day 5 after PMS injection was assayed. ACTH induced an increase in intracellular cAMP.
The present results indicate the following : (1) In Group I and Group II, progesterone release increased with the age of the luteal cells and ACTH dose. In Group III, ACTH stimulated progesterone release in a dose-dependent manner in early gestation. (2) 20α-OH-progesterone release decreased by ACTH dose in Group I and Group II, and decreased in Group III as the gestation advanced. (3) Intracellular cAMP increased by ACTH in Group II.

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卵巣の着床周辺期における性格に関してウイスター系ラット卵巣を使用して, 脂質, 3β-HSD活性, G-6-PDH活性, 20α-OH-HSD活性の推移と各卵巣要素における3H-thy-midine, ³H-uridine, ³H-methionineの取込み状態とについて, 系統的な組織化学的検討を行なった.
その結果, この時期を通じ, 最大の変化を生ずる場は新生黄体であり, 一方, 卵胞ではL3に軽度ながら卵胞発育が認められ, 問質腺はほとんど変化しないことが判別した. また, 新生黄体および内莢膜の機能的分化は着床周辺期に完成する. などの興味ある知見を得た.

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妊娠5ヵ月のウシの黄体の微細構造を調べたところ,大型および小型についてそれぞれ次のような結果が得られた。(1)大型(直径30~45μm),細胞表面には多数の微絨毛がみられた。細胞質には断片状で発達の悪い粗面小胞体,中等度に発達した小胞状の滑面小胞体および少量の脂肪滴が観察された。(2)小型(直径15~25μm)。細胞表面には微絨毛はあまりみられず,比較的平坦であった。細胞質には層板状の発達した粗面小胞体,中等度に発達した管状の滑面小胞体および多量の脂肪滴が認められた。核,管状クリステを持つミトコンドリア,ゴルジ装置,ライソゾーム様小体および遊離リボゾームは両種細胞の間に形態上,分布上の差異はみられなかった。以上のことから,妊娠のそれと異なる主な点は,大型,小型とも,ミトコンドリアが著しく多く,滑面小胞体の発達が良くないことであった。

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Only a few observations of ovary by electron microscopy, regardless of animal species, have been reported, and none of rabbit corpus luteum. The rabbit ovary has no corpus luteum of sexual cycle like the other species. This is important as an object of study. It was assumed by Limon that interstitial gland cells of rabbit might carry on an endocrynological function and since then many results have been reported of endocrynological significance of the interstitial gland. In view of clarifying these points, the present study has been carried out.
Corpola lutea from pregnant rabbits and rats (at initial stage, middle stage and terminal stage) and the postpuerperal animals (lactating and non-lactating during 5 days) and interstitial glands only from rabbits in each class were employed. After fixation with 1% osmium tetroxide solution being buffered to pH 7.6 with veronal acetate, they were embedded either in methacrylate mixture or epoxy resin 812. In addition, all the left ovaries were examined with Sudan staining, O₂-Schiff reaction and Schulz reaction etc. for reference.
Cell margin, endoplasmic reticulum, mitochondria, liquid granules, vacuolelike granules and vacuolar granules as we call them, and other organelles turn the feature remarkably in the lutein cells of both species with pregnant and postpuerperal passage. As compared with the fine structure of lutein cells from rabbits and rats, it is generally similar, except for the marked difference of the shape of lipid granules. The smooth surfaced endoplasmic reticulum is well developed in the lutein cells and interstitial gland cells, and there are a great number of vesicular components within the cytoplasm through the whole stage. It forms meshe-like area at the initial stage of pregnancy here and there in lutein cytoplasm, and vesicular compoments are the most developed in the lutein cells at the middle and terminal stage of pregnancy. The formation of a meshe-like structure is more conspicuous in the case of rabbit, and there are less mitochondria near the area. It is interesting that the some variations of electron density is observed among numerous vesicles in the cytoplasm of single cell. It is suggested conclusively as follows ; endoplasmic reticulum and mitochondria might play the greatest role in steroid hormone production, and the matter connected with the discontinuous portion of cell margin and endoplsmic reticulum might be the most important in a point of the release. Golgi complex dose not show any considerable extension and transformation of ultrastructural figure in any case. Therefore, it is difficult to find that Golgi complex has a special significance for endocrinological activity. Polymorphous dence bodies or cytolysomes are observed a little in the interstitial gland cells as well as in the lutein cells a t the initial and postpuerperal stages, which are less in rats than rabbits.

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プロスタグランディン(以下PG)E₂は,20~10000 ng/mlの濃度範囲で培養ブタのプロジェテスロン(以下_P4)産生を促進させた.PGF_2αはのP₄産生に対して明確な影響を示さなかった.一方オキシトシンと血小板活性化因子(PAF)は,のP₄産生に抑制的な影響を示した.以上の結果は,ブタにおいては,PGE₂が黄体機能促進の,またオキシトシンおよびPAFは,黄体機能抑制作用を有している可能性を示唆している.

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Thirty one rabbits which were verified as estrous by the vaginal smear test, and eight estrous, 48 hour-hypophysectomized rabbits were administered intramuscularly a single dose of LH Lot #R377242H and LTH-P (Prolactin) Lot #P10011 ; Armour Co. each alone or both combined, and the chemical ana-lysis of urinary pregnanediol and estrogen (just before, and 24 hours, 48 hours and 96 hours after the administration) in combination with the histological and histochemical examination of the ovary (in the non-hypophysectomized cases 5 hours, 10 hours, 24 hours, 48 hours and 96 hours after the administration, in the hypophysectomized cases 96 hours after the administration) were made.
These results in comparison with the results in pregnancy (the chemical analysis in 7 cases and the histochemical examination in 14 cases) were discussed.
The chemical determination was performed according to a modified Stimmel method (1952), and for the histochemical examination were used the Sudan, Schiff, Al-phosphatase, methylgreen-pyronin, V.C., Bielschowsky staining method, and the birefringence technique.
The summary is as follows :
1. In non-hypophysectomized estrous rabbits urinary pregnanediol excretion increased after the administration of LH 10mg and LTH 60 I.U. each alone or both combined, and this increase taken in conjunction with the lacing of the endometrium of the rabbits injected indicate their progesterone secretion.
2. The administration of LH 10mg, 25mg and LTH 60 I.U. each alone to hypophysectomized rabbits induced the increased progesterone secretion.
3. As to the increased progesterone secretion observed in both the estrous LTH treated rabbits, and hypophysectomized LH or LTH treated rabbits, neither of which had corpora lutea, a probable main origin of this hormone is considered to be the interstitial gland, which showed the marked decrease of its lipids including cholesterol and the activated morphological features synchronously.
4. As to the increased progestorone secretion just after mating, a probable main origin of this hormone is also considered to be the interstitial gland.
5. In estrous rabbits the administration of LH 10mg alone or LH and LTH combined induced the increased urinary excretion of estrogen, but LTH 60 I.U. alone did not induce this increase.
6. In hypophysectomized rabbits the administration of LH 10mg, 25mg alone induced the increased urinary excretion of estrogen, but LTH 60 I.U. alone did not induce this increase.
7. In connection of this increase of urinary estrogen after the LH administration and no increase after the LTH administration, histologically were observed some hypertrophy and engorgement of the theca interna after the LH administration, and a tendency of the granulosa cells to degenerate and atrophy after the LTH administration.
8. In estrous rabbits, ovulation and corpora lutea were observed after the administration of LH 10 mg alone or LH 10mg and LTH 60 I.U. combined, but none of them was observed after LTH 60 I.U. alone.
9. In hypophysectomized rabbits, no ovulation was observed macroscopically after the administration of LH 10mg, 25mg or LTH 60I.U..
10. In this experiment, the Sudan and/or Schiff positive lipids of the ovary varied in a close correlation with its endocrine activity.

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【目的】ウシにおいて排卵後に形成される黄体は、10 日間でその重量が約 20 倍に成長することが知られているが、この黄体の成長はの増殖ではなく、サイズの増加であると考えられている。近年、霊長類のに細胞増殖の指標である Ki-67 タンパク質の発現することが報告されたことから、ウシの増殖する可能性が示唆されるが、その詳細は明らかではない。そこで本研究では、ウシ黄体の形成機構を明らかにする目的で、の増殖能について検討を行った。 【方法】発情周期を通じた黄体組織における Ki-67 タンパク質の局在を免疫組織化学により調べた。また、局在が認められた周期について、Ki-67 との指標となる 3β-HSD の二重染色を行った。次に中期黄体から単離したを播種し、培養 1、4、7 および 10 日後の DNA 含量を DNA assay により検討すると共に、黄体形成ホルモン (LH) 添加区を設け、の増殖能に及ぼす LH の影響を調べた。さらに、発情周期を通じたウシ黄体組織における Pten、Cyclin D2 および p27^kip1 (p27) mRNA 発現を RT-PCR 法により検討した。 【結果】初期、形成期、中期、妊娠期黄体において、Ki-67 陽性細胞が確認された。これらの周期について、Ki-67 および 3β-HSD の二重染色を行った結果、いずれの周期においてもに Ki-67 陽性細胞が認められた。培養中期は、時間依存的に増殖し、このの増殖能は LH 添加により有意に減少した。Pten mRNA 発現量は初期黄体において低かった。また、Cyclin D2 mRNA 発現量は形成期において有意に高く、p27 mRNA 発現量は初期に低い傾向を示した。さらに、細胞増殖を促進する Cyclin D2 と増殖を抑制する p27 mRNA 発現量の比をとった結果、初期および形成期において有意に高かった。本研究より、ウシ黄体ステロイド合成細胞の増殖する可能性が強く示唆された。また、細胞の増殖は発情周期初期、形成期で活発であることが示された。

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Isolation of progesterone secreting cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotropin (PMS) was conducted by a simple procedure which combined the collagenase digestion with a density gradient method. After digestion of the ovarian tissue slices by the enzyme, the residue was gently pressed and placed on a sucrose density gradient. Three bands appearing in the tube after centrifugation were designated as S-1, S-2 and S-3 from the top to the bottom, respectively. The S-1 cells from the ovaries at 6 days after PMS secreted the greatest amount progesterone, i.e. approximately 430ng per 10⁵ cells during the 18th incubation. Progesterone secretion from the S-2 cells was less than 48% of that from the S-1 cells. A physiological interrelation between the S-1 and S-2 cells remains unexplained by the present experiment. The luteal cells were yellow, spheroidal and 15 to 40μ in diameter. Many vesicle like particles were found on the cell surfaces. Progesterone secretion from the prepared cells was stimulated significantly by hCG in vitro. This result indicates that progesterone secreting cells isolated by the collagenase-sucrose density gradient preserve their native function as luteal cells.
The procedure for preparation of luteal cells in the present report may provide a suitable model for in vitro studies on the corpus luteum.

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