@misc{oai:az.repo.nii.ac.jp:00003178,
 author = {百目鬼, 郁男 and Domeki, Ikuo},
 month = {2013-03-21, 2014-08-08, 2013-02-08, 2013-02-08},
 note = {For the purpose of studying the correlation between the fertilization failure or early embryonic death and the anomaly of secretion of sex steroids in a cow, concerning the chemical measuring method and the radioimmunoassay method for measuring estrogen and gestagen in the blood of cow, the methods were established at first, and using these methods in combination with the hitherto used biological assay method, the aspects of estrogen and gestagen in the peripheral plasma in a cow during the estrous cycle and in early pregnancy were clarified. Secondly, an repeat breeding cow was autopsied in 15-28 days after artificial insemination, and the presence of embryo in the uterus or its growing state and the anomaly of sexual organs were investigated, and at the same time the aspects of estrogen and gestagen in the peripheral plasma before the artificial insemination up to the time of autopsy were pursued, and the fact that there is a remarkable difference in the secretion type of these sex steroids between an infertile cow and a fertile cow was made clear.
 In the following is described of an outline of the results obtained.

1. Fluorometric determination of free plasma estrogens during the estrous cycle in the cow

 The fluorescence method of Ittrich was applied to the determination of free estrogens in the peripheral blood of two Japanese beef cows during the estrous cycle.
 The patterns of blood levels of estrone and estradiol were almost the same in tendency in the two cows during the estrous cycle. Estradiol was generally higher in level than estrone. Estriol was not detected at all. In the two cows, total estrogen increased in the proestrous phase, presenting a sharp peak at a level of 35.3 and 99.8 ng/liter (estrone, 5.9 and 16.0 ng/liter ; estradiol, 29.4 and 83.8 ng/liter), respectively, prior to ovulation, and decreased rapidly to a minimum level, or 3.8 and 5.3 ng/liter (estrone, 1.6 and 1.9 ng/liter; estradiol, 2.2 and 3.4 ng/liter) after ovulation. In the luteal phase (6-8 days after ovulation), it reached a peak again at a level of 10.1 and 27.0 ng/liter (estrone, 2.4 and 3.4 ng/liter ; estradiol, 7.7 and 23.6 ng/liter). In short, two peaks appeared during one estrous cycle.

2. Fluorometric determination of plasma progesterone during the estrous cycle in the cow

 The method of Heap for the determination of progesterone was modified by introducing thin layer chromatography (TLC) and improving some conditions for coloration.
 TLC was applied to isolation and purification of progesterone and 20β-hydroxyprogesterone. Satisfactory results were obtained by developing a sample twice with n-hexane : ethylacetate (5:2) and once with benzene : ethylacetate (2:1).
 The most intense and stable fluorescence was obtained when the color was developed with concentrate sulfuric acid : ethanol (3:2) at 60℃ for 10 minutes. When determined by the spectrofluorometer, the maximum wavelength of this fluorescence presented peaks at 468 nm for excitation and 525 nm for emission. Under the same conditions as these the minimum amount of detection of 20β-hydroxyprogesterone was 2.5 ng.
 The blood level of progesterone was determined by the modified method during the estrous cycle in four Japanese beef cows. Its minimum value was 0.2-0.8 ng/ml during the estrous period, and its maximum value 2.6-6.2 ng/m1 during the luteal phase (8 to 20 days after ovulation).

3. Radioimmunoassay of blood plasma estrogen and progesterone during estrous cycle and early pregnancy in the cow

 Peripheral plasma estrogen and progesterone levels in the cow during the estrous cycle and early pregnancy were determined by radioimmunoassay.
 In six cows showing normal estrous cycle, blood level of estrone and estradiol fluctuated with almost same patterns, but the concentration of estradiol was generally higher than that of estrone in three of six cows. Level of total estrogen formed a medium peak (9.0-10.5 ng/ml) during luteal stage of 3-12 days after ovulation, and a sharp peak (10.6-19.5 pg/ml) in proestrus, 3-1 days before next ovulation. In three other cows, no increase in total estrogen was found during luteal stage, but, they showed its sharp and high peak (12.7-20.0 pg/ml) 2-1 days before next ovulation. Plasma progesterone showed its minimum level (0.2-0.3 ng/ml) at estrus and maximum level (1.8-4.2 ng/ml) during luteal stage of 11-17 days after ovulation. It began to decrease rapidly 4 to 5 days before next ovulation.
 In five cows of early pregnancy, within 32 days after insemination, plasma estradiol showed higher level than that of estrone. In two of them, concentration of total estrogen increaded (12.4 and 7.2 pg/ml) temporarily 5 days after insemination, but it maintained of low level (2.3-6.0 pg/ml) until about 12 days after insemination in three others. Thereafter, the level of total estrogen rose gradually in all of them and showed considerable high level (6.2-11.6 pg/ml) 27-28 days after insemination. Concentration of plasma progesterone increased rapidly after the ovulation and showed higher level of 6.0 ± 1.7 ng/ml at 31-32 days of pregnancy than that in luteal stage of the estrous cycle.

4. Radioimmunoassay of blood plasma progesterone during estrous cycle in the cow

 Radioimmunoassay method was compared to competitive protein assay and fluorometric assay method for the determination of plasma progesterone of the cow. Peripheral blood progesterone of the during estrous cycle was determined by radioimmunoassay.
 High potent antiserum against progesterone-3-oxime-BSA (optimal dilution, 1:40,000) was prepared for radioimmunoassay. Calibratiin curve made by the antiserum showed clear lineality between 0 and 400 pg of progesterone, and percent binding of 3H-progesterone was 70% or higher at 0 pg. The antiserum also showed highly specificity against progesterone, and no significant cross reaction was found against other various steroids except 5α-pregnanedione.
 In radioimmunoassay method, values of progesterone determined on the crude samples which was extracted from blood samples with ether (direct method), were compared to those determined on the purified samples obtained by carring out column chromatography (chromatographic method). There was a good correspondence in the values of progesterone between direct and chromatographic methods.
 No significant differences were found between the direct method of radioimmunoassay, competitive protein binding assay and fluorometric assay method, in values of progesterone, determined on the same plasma samples collected from the cow at various stages of the estrous cycle.
 Concentrations of plasma progesterone, determined by the direct method of radioimmunoassay in 9 cows during estrous cycle, showed minimum level (0.2-0.3 ng/ml) at estrus and maximum level (3.2-5.8 ng/ml) during luteal stage of 11 to 16 days after ovulation. The concentration was rapidly decreased about 4 to 6 days before the next ovulation.

5. Peripheral blood plasma sex steroids before and after insemination in repeat breeding cows

 An experiment was carried out to investigate the relationships between the secretion pattern of sex hormones and infertility in cows.
 Nine repeat breeding cows and two normal cows obtained from individual farms were inseminated experimentally. The animals, except one which was held under observation up to 70 days after insemination, were autopsied 15-28 days later, and their genital organs were examined. The levels of estrogen and gestagen in the peripheral blood plasma were assayed by the method of Sulman et al. and of Hooker & Forbes.
 In one of nine repeat breeding cows, a dead fetus was found in the uterus, but not any other cows had fetus or any other conceptus. There was no evidence of bacterial infections in the uterus and oviduct of any of all these cows. Of two normal cows, one had a fetus developed normally in the uterus, and the other continued the normal pregnancy up to days after insemination. At estrus, estrogen levels in the fertile cows reached a maximum 2-3 days before ovulation. It decreased rapidly to a minimum immediately after ovulation. In the infertile cows, however, the time showing the maximum levels was later 1-2 days than that of the fertile cows. It appeared on the day of ovulation in four of eight cows. In the infertile cows, except one, the time exhibiting the minimum levels was also late, which appeared 1-3 days after ovulation, Estrogen levels observed in the luteal stage following insemination were classified to the two groups ; i.e. a high estrogen group, and a low estrogen group. The normal pregnant cows were belonged to the later group and the cow having a dead fetus in the uterus to the former group. The gestagen levels showed a peak at around estrus in all three fertile and six of eight infertile cows, almost simultaneously with, or a little later than, the time of estrogen peak. In the luteal stage following insemination, the gestagen levels were relatively high in six infertile cows which formed a peak at estrus, but low in other two infertile cows. In two normal pregnant cows, the gestagen levels increased with advance of the pregnancy, and was maintained at a high level. However, it remained at a low level in the cow having a dead fetus in the uterus.
 From these results, it may be postulated that when some abnormal secretions of estrogen and gestagen occurred before and after ovulation, fertlization failure or death of fertilized ova might be induced, and that when it occurred in the luteal stage following insemination, early embryonic death might be induced., 牛の繁殖障害のなかでリピートブリーダーは卵巣疾患，子宮疾患とならぶ主要なものである。リピートブリーダーの不受胎の主な原因は胚の早期死滅であると考えられており，その誘因としては遺伝因子，生殖器の感染，炎症，ホルモンの分泌異常ならびに受精卵の細胞質的欠陥などが推測されているが，なかでもホルモンの分泌異常は最も重視さるべきものである。
　従来，牛体液中の性ステロイドの測定には比較的感度の高い生物学的手法が応用されていた。しかるに，近年アイソトープおよび各種のクロマト技術が進歩するに至り，化学的測定法ならびにcompetitive radioassay法などの近代的手法が生物学的手法に代って応用されるようになってきた。とりわけradioimmunoassay法は，感度，特異性がきわめて高く，かつ操作が簡便で，比較的短時間に大量の検体を処理し得る優れた方法である。しかし，牛の末梢血中性ステロイド濃度は他の哺乳動物のそれに比べて著しく低いために諸種の繁殖状態におけるこの動態を精細に検討した報告は数少ない。
　本研究では牛における受精障害，あるいは胚の早期死滅と性ホルモンの分泌異常との関連性を究明する目的で，先ず牛の血中estrogenおよびgestagenを測定するための化学的測定法ならびにradioimmunoassay法について測定条件を吟味して，手法を確立し，これらの方法ならびに従来の生物学的測定法を併せ用いて，正常性周期および妊娠初期における牛末梢血中estrogenおよびgestagenの動態を明らかにした。ついでリピートブリーダー牛を実験的に授精後15～28日の間に屠殺解剖して，子宮における胚の存在，あるいはその発育状態ならびに生殖器の器質的異常，細菌感染などについて検索するとともに授精前から屠殺時までの末梢血中estrogenおよびgestagenの動態を追求して，不受胎牛ならびに受胎牛の間にはこれら性ステロイドの分泌型に顕著な相違があることを明らかにした。
　以下に得られた成績の概要を記す。
1.性ステロイドの測定法
　1)化学的測定法
　牛血中estrogenの測定に，比色法の高い特異性と螢光法の高い感度をとり入れたIttrich螢光法を応用するために，その測定条件を吟味して，Xenonランプを使用した日立分光螢光光度計MPF-2Aおよび203型によるIttrich colorの最大波長はestrone, estradiolおよびestriolともに共通で，励起光538nm，螢光552.5nmであることを明らかにし，さらに実際の測定においては両波長の接近を避けるために510～520nmで励起して，螢光を552.5nm前後で読む方法を検討して検出感度1ngの微量測定法を確立した。
　つぎにprogesteroneの化学的測定法としてprogesteroneを20β-hydroxy-steroid-dehydrogenaseにより20β-hydroxy-progesteroneに転換して測定するHeapの螢光法において，抽出純化の過程にペーパークロマトグラフィーの代りに操作が簡便で能率的な薄層クロマトグラフィーを導入して，n-hexane:ethyl acetate=5:2で2回, benzene:ethyl acetate=2:1で1回展開して，好結果が得られることを明らかにした。さらに発色性は濃硫酸:ethanol=3:2，10分，60℃で最も強く，かつ安定で日立分光螢光光度計MPF-2Aおよび203型による本螢光の最大波長は励起光468nm，螢光525nmであることを明らかにした。Heapの螢光法を改良した本法の牛血中progesteroneの最少検出量は2.5ngで精度，特異性ともに満足し得るものである。
　2)radioimmunoassay法
　牛血中estrogenのradioimmunoassayにestrone-17-oxime-bovine serum albumin, estradiol-17β-6-oxime-bovine serum albuminならびにestriol-6-oxime-bovine serum albuminに対する家兎抗血清を使用して，検出感度がestroneおよびestradiolでは10pg, estriolでは20pgである高感度のestrogen測定法を確立した。
　また牛血中progesterone，20β-hydroxy-progesterone，17α-hydroxy-progesteroneのradioimmunoassayにそれぞれprogesterone-3-oxime-bovine serum albumin，20β-hydroxy-progesterone-3-oxime-bovine serum albuminならびに17α-hydroxy-progesterone-3-oxime-bovine serum albuminに対する家兎抗血清を使用して，検出感度がそれぞれ10～20pgである高感度のgestagen測定法を確立した。
　さらに，progesteroneの抽出過程で粗抽出物をクロマトにより精製した場合と，この操作を省いた場合のそれぞれの測定値はほぼ等しいことを明らかにして，radioimmunoassay法による牛血中progesteroneの簡易測定法を確立した。
　以上の結果から，牛血中estrogenならびにgestagenの測定にradioimmunoassay法を応用し得ること，また本法は感度，精度，特異性，測定操作の迅速性においてきわめて優れていること，さらにprogesteroneについてはクロマトによる精製操作を省いた直接法を応用し得ることなどが明らかにされた。
2.性周期における末梢血中性ステロイドの動態
　正常性周期を示す牛21頭を用い，頸静脈から経日的に採取した血液材料についてestrogenおよびgestagenの濃度を生物学的測定法，化学的測定法およびradioimmunoassay法で測定して，性周期におけるこれら性ステロイドの消長を比較検討した結果，生物学的測定法による測定値は他のふたつの測定法によるそれに比べて高い値を示すが，いずれの方法によっても性周期の各時期における末梢血中のestrogenおよびgestagenあるいはprogesteroneの測定値はほぼ同様の傾向で増減することを認めた。
　すなわちestrogenは発情期，排卵前に鋭いピークを示し最高値に達し，排卵後に低下した後，黄体期にはふたたび増加する傾向を示した。発情期，排卵後および黄体期における総estrogenのピーク値は生物学的測定法ではそれぞれ3.34～8.90μg/l，0.21～0.84μg/lおよび3.34～8.90μg/l，化学的測定法では13.7～99.8ng/l，3.8～5.3ng/lおよび10.1～27.0ng/l，radioimmunoassay法では10.6～20.0pg/ml，5.4～11.0pg/mlおよび2.4～11.8ng/mlであった。
　gestagenは全般に黄体期に最高値，発情期に最低値を示したが，生物学的測定法では排卵前に一過性にわずかに増加することを認めた。黄体期におけるgestagenあるいはprogesteroneのピーク値および発情期におけるそれらの濃度水準は生物学的測定法では8.00～10.67μg/mlおよび2.67～5.33μg/ml，化学的測定法では2.6～6.2ng/mlおよび0.2～0.8ng/ml，radioimmunoassay法では1.8～5.8ng/mlおよび0.2～0.3ng/mlであった。
　しかし各個体間におけるこれら性ステロイドの濃度水準およびピーク形成の時期にはかなり大きい差異があることが注目された。
　発情開始から排卵までの間の末梢血中progesterone，20β-hydroxy-progesteroneならびに17α-hydroxy-progesteroneの動態を1頭の牛について2時間々隔で採取した血液材料を用いて詳細に検討したところ，progesteroneならびに17α-hydroxy-progesteroneの測定値が排卵前に一過性に上昇する傾向が認められ，排卵にこれらのgestagenが一役を演じている可能性が示唆された。
3.妊娠初期における末梢血中性ステロイドの動態
　正常妊娠牛5頭身ついて発情前期から授精後31～32日までの間に頸静脈から採取した血液材料について，妊娠初期におけるestrogenおよびgestagenの濃度をradioimmunoassay法により測定した結果，estrogenは全般に低値(2.3～6.0pg/ml)で経過し，やや高い場合があっても，その期間はきわめて短く一過性であること，またprogesteroneのピーク値は6.0±1.7ng/mlで性周期における黄体期のそれに比べて増加速度が早く，かつ濃度水準が高い傾向があることを認めた。さらに20β-hydroxy-progesteroneはprogesteroneとほぼ同様に消長するが，17α-hydroxy-progesteroneは排卵後12日まではprogesteroneとほぼ同様に消長し，その後は急減して比較的低値で経過することを認めた。
4.リピートブリーダーにおける末梢血中性ステロイドの動態
　農家から導入したリピートブリーダー9頭に授精を行ない，授精前後から授精後15～28日までの間に頸静脈から採取した血液材料についてestrogenおよびgestagenの濃度を生物学的測定法によって測定し，発情前期から妊娠初期にかけてのこれら性ステロイドの消長を正常妊娠牛におけるそれと比較したところ，リピートブリーダーの発情期におけるestrogen値は，最高値に達する時期および低下を開始する時期が正常妊娠牛のそれにくらべて1～2日遅延する傾向があることを認めた。
　さらに正常妊娠牛における妊娠初期のgestagen値は黄体の発育に伴って増加し，estrogenは低値のまま経過するが，リピートブリーダーにおいて妊娠初期に胚死亡が認められた例ではgestagen値がきわめて低く，estrogenが高値を示したことが注目された。
　以上の結果から，estrogenおよびgestagenの分泌異常が排卵前後に生じた場合には受精障害あるいは受精卵の死滅を招き，これが妊娠初期に生じた場合には胚の早期死滅を招くことが示唆された。},
 title = {性周期ならびに妊娠初期における牛末梢血中性ステロイドの動態に関する研究},
 year = {}
}