@misc{oai:az.repo.nii.ac.jp:00003237,
 author = {紫野, 正雄 and Shino, Masao},
 month = {2013-02-19, 2014-08-18, 2013-02-19},
 note = {Sows and gilts of reproductive age repeat the 2～3 days of estrus in 21-day cycle throughout the year; they are so called the polyestrus animal.
 Each animal species exhibits the different signs during estrus. One of the characteristic changes displayed by sows and gilts during estrus is the neutrophils' appearance to mucus secretion of uterine cervix (cervical mucus). This phenomenon has been known, clinically; however, its biological significance and the mechanism were hardly elucidated. Generally, neutrophilic migration to local tissue is considered the sign of inflammation. It is interesting that similar phenomenon occurs in estrus, which is a transient physiological phenomenon, and furthermore, it is only seen in cervical mucus of sows and gilts. Therefore, we conducted the series of experiments for the purpose of clarifying the endocrinological mechanism of this neutrophilic migration.

Chapter 1
The Neutrophilic Count in Cervical Mucus and External Signs in the Reproductive Cycle

 First, the fluctuations of neutrophilic count in cervical mucus (NCCM) and external signs in reproductive cycle were analyzed. Neutrophilic counts from cervial mucus were taken twice, in morning and afternoon, every day throughout the period of estrus, pregnancy, farrowing, and lactation, on two sows with normal cycle of estrus. Cervical mucus was collected by cotton swab inserted in 1cm-diameter vinyl chloride tubing; the measured size cotton swab was pressed against the mucus wall area of second ruga in cervix. Cotton swab was washed in 1 mL phosphate buffer solution, and the resulting cell-suspension fluid was subjected to cell count. On the other hand, as the external signs of estrus, observation were made and recorded on the appearance of male receptivity, and hyperemia and swelling of vulva. As the result, except for the several days immediately after farrowing, the period when numerous neutrophils appear in cervical mucus was identified to be within one week prior to and within one week from the estrus, the day of the external signs were conspicuous. Evidently during this period, the number of neutrophil count in cervical mucus was 2.5 to 15 times the level of other period. The possibility, which the neutrophilic count increase by the effect of estrogen, is suggested from this finding.

Chapter 2
The Neutrophilic Count in Cervical Mucus and External Signs and Their Relationship to Ovulation Time

 While in the estrus, external signs become conspicuous, and at the same time, follicles are maturing and ovulation occurs in ovary. I further analyzed which part of these stages is the most closely related to the increase of NCCM. For this investigation, 7 sows without any clinical abnormality in reproductive organs and displaying normal estrus cycle were used. Daily records of external signs and neutrophils counts were entered for the 4 sows among the above, for 9 days before and after the starts of estrus, which include two starting days, or 14 days. For remaining 3 sows, their ovulation was identified, and in order to estimate the ovulation timing, they were bred and subjected to laparotomy operation. After the observation was made on ovary, ova were collected by perfusion of physiological saline solution in oviduct and uterus area. Simultaneously, these 3 sows' blood progesterone and estrogen levels and white blood cell count in peripheral blood were measured.
 The total of 8 estrus was observed in aforementioned 4 sows. As the result, the increase in NCCM occur from the day before the start of estrus to the next day of the start, when the external signs are the most notable. Duration of time the increase was observed was within 24 to 36 hours. The finding within the individual had shown that there is a case (1 sow in 4) shows the increase of neutrophils at the same timing and the same level in first and the second estrus, and a case (1 sow in 4) shows at the different timing and the different level. Though in the latter case, the difference in timing remained within 24 hours.
 The three sows subjected for identification of ovulation were mated either once or twice within 36 hours after standing heat. One sow was laparotomically incised after 28 hours, and 5 embryos at 3 to 4-cell stage were identified. No ovulation was identified in one saw by exploratory incision after 15 hours and 25 hours from mating; however, when sacrificed after approximately 30 hours, 8 embryos at 2-cell stage were collected. In the remaining sow, one ovum was collected from the exploratory incision after 38 hours; however, the fertilization was not identifiable about this ovum. In general, fertilized swine ova need 14～19 hours to reach 2-cell stage embryo, and 24 hours, to 4-cell stage. Presumably, from this information, the ovulation timing of subject sows, identified with fertilized ova, were speculated 28 and 38 hours each after the start of estrus. These time period almost overlap or extremely close to the peak hours of neutrophils count in cervical mucus.
 The blood estrogen level of the above three sows began to increase 2～3 days before the start of estrus, and decreased as the sow approaches to the start of estrus or estimated ovulation date. On the other hand, progesterone level displayed the low-grade increase in the day before the start of estrus towards estimated ovulation date. When the time period the neutrophils count in cervical mucus was at the peak, estrogen level was decreased, and progesterone level begins slight increase. Clear correlation was not identified between the neutrophils count in cervical mucus and white blood cell count in circulation.
 From above results, it is concluded that the increase of the neutrophils count in cervical mucus is limited to the short period of time in estrus, and at the same time, the increase is closely related to ovulation and blood estrogen/progesterone level.

Chapter 3
The External Signs and Neutrophilic Count in Cervical Mucus of Ovariectomised Sows with Induced Pseudo-Estrus

 According to the result of Chapter 2, the effect or estrogen and progesterone (P) administrations were analyzed. As estrogen, estrone (E_1), estradiol-17β(E_2), and estriol (E_3) were used. Administration program was as in following table.

(table)

 In (1) and (2), external signs appeared, and neutrophilic count in cervical mucus increased as well. However, the levels of increase were extremely low. In (3) and (4), the external signs were retained, and NCCM increased 8～10 folds, compared to the level before E administrations. However, transition pattern as observed in natural estrus was not identified, for observations such as multiple peaks and high value after the disappearance of external signs were made. The external signs also appeared in (5) and (6); however, the transition pattern differed from natural estrus, such as the mild increase of NCCM and biphasic peaks, were observed. In cases of (7), (8), and (9), which the amount of E_2 administration was reduced, external signs were observed in each individual, and the increase of NCCM was observed in dose-dependant manner. Moreover, their levels and transition pattern closely resembles that of natural estrus. The external pattern did not appear in (10) and (11), throughout the observation period, and the characteristic change was not observed in NCCM. In (12), (13), and (14) , mild hyperemia and swelling of vulva were observed, but no standing heat was observed. The level of increase observed in (12)'s NCCM was low compared to (9), which administered E_2 dose was the same; however, the transition pattern closely resembles that of natural estrus. As the amount P's administrations increase, the characteristic transition pattern was lost and the amounts of fluctuations were diminished.
 In case of naturally occurred estrus (in #108), NCCM exhibited the characteristic peak in the next day the estrus started, and it was the 7-fold increase. Accompanying with this rise, blood E_2 level reached the maximum of 109 pg/mL; and on the next day, sharply decreased to approximately 10 pg/mL. In (15), the external signs appeared on 3 days after E_2 administration. Blood E_2 concentration suddenly increased 2 days after the administration (to 317 pg/mL), and this level was maintained for 3 days. NCCM increased 7-fold, with the appearance of external signs. Even afterward, the high level of blood E_2 was maintained, and the cyclical rise and fall was repeated in every other day throughout the period.
 From above results, the increase in NCCM, which is characteristically observed during the estrus, requires estrogen, especially the E_2. And progesterone was shown to have inhibitory effect.

Chapter 4
The Morphological Change of Cervical Mucus Tissue in Sows with Naturally Occurred Estrus and Induced Pseudo-Estrus

 In the previous chapters of Chapter 1 through 3, we have clarified the followings. The time periods when the great numbers of neutrophils appear in cervical mucous membrane closely relates to the ovulation time, and corresponds to the time when the estrogen level decrease and progesterone level began mild increase. And the increase of NCCM similar to the natural estrus occurred in ovariectomised sow with induced pseudo-estrus by administration of estrogen. Progesterone was shown to inhibit this phenomenon. Subsequently, histological analysis was conducted to investigate what type of change in tissue yield the migration of neutrophils.
 One sow with natural estrus and 7 ovariectomised sows were subjected to experiment. For 3 of ovariectomised sow, one dose of 1.0 mg, 0.8 mg, and 1.0 mg was administered to each. The tissue samples from second spirette wall area in cervix were obtained by using endometrial biopsy apparatus, immediately before and immediately after the administration; also on day 2, 3, 5, 7, and 9 from the administration. Other 3 sows were allocated as E_1, E_2, and E_3 administration group, and all of them received three consecutive intramuscular injection of 1.0 mg in the first time, 2.0 mg the second, and 1.0 mg the third. The remaining 1 sow was administered 40 mg of P for three consecutive times. The cervical tissue of the last 4 sows were collected on 4 days prior to administration, on the day of administration, and 2, 3, and 6 days after the administration. The biopsies from the sow with natural estrus were conducted on 4 days prior to the predicted date the estrus start, on the day the estrus started, and 2, 3 and 4 days after the start.
 The external signs and the transition of NCCM were mostly the same as results described in Chapter 3. In the E_2 single administration group, proliferation of mucosal epithelium cell in cervical area, infiltration of deformed neutrophils into epithelial cell layer, lamina propira mucosae, and submucosal layer was identified from 2 days after the administration, and the submucosal vessels were dilated. Epithelial cell proliferation began to decelerate after the peak observed on day 5. However, 2 sows among the 1.0 mg-administered sows continued to display dilation of vessels in lamina propira mucosae and submucosal layer up to day 9, and infiltration of deformed neutrophils and white blood cells in the vessels were clearly identified.
 Proliferation of mucosal epithelium layer in cervical area of sows, administered E_2 for 3 consecutive days, became marked on 2 days after the end of administration, and the level of proliferation exceeded that of sow with natural estrus in same period. In sows administered E_1 or E_3, the maximum proliferation of mucosal epithelium layer in cervical area was observed 1 day later. On the other hand, such proliferation was not observed in sows administered P. Similar tendency has been identified in the cases of vessel dilation in lamina propira mucosae and submucosal layer, and deformed neutrophils infiltration. The cells of interior vessel wall was at first cylindrical or spindle shaped. However, as the estrus or pseudo-estrus progress, such observation as the cells became thin as squamous cells and the dilated vessel lumen became filled with white blood cells were observed.
 As described in Chapter 3, the major cause of estrus in these cases was, again, identified to be the estrogen, and especially, the E_2. Moreover, it is speculated that the neutrophils in cervical mucus migrated from the dilated and thinly changed cell-lined vessels. However, at this moment, whether the estrogen directly causes these histological changes or whether there is any unidentified mediating factor presents are unknown.

Chapter 5
The Effect of Indomethacin and Granulocyte Colony Stimulating Factor on Neutrophilic Migration to Cervical Membrane

 From the series of experimental results described in Chapter 2, 3, and 4, ovulation time, blood level of estrogen (E), and progesterone (P, hereafter) were identified to have close relationship with the change in NCCM. Based on these results, following experiment was planned. Prostaglandin (PG) is recognized as the substance relates to inhibition of ovulation, and PG is strongly related to inflammatory events. The change in neutrophilic count and external signs were observed after administering Indomethacin (Ind), which is PG synthesis inhibitor agent. Moreover, endocrinological and macroscopic confirmation of ovary were conducted. Furthermore, the verification is conducted on the inhibition by concomitant administrations of E_2 and Ind on the promoting effect of E_2 to increase NCCM. Also, verification on the effect of concomitant administration of E_2 and granulocyte colony stimulating factor (G-CSF) on the neutrophils of cervical mucus was conducted.
 For the experiment 1, 6 normal pigs of normally repeating estrus cycle were used. As Ind group, 4 of them were administered Ind (720 mg per day, 3 mg/kg), which has inhibitory effect on ovulation. The 7-day administration started on 6, 3, and 1 day before the start of estrus. Ind non-administration group was fed flour. Ovariectimised sows were subjected to the experiment 2. E_2 was administered by intramuscular injection, and Ind was administered for 16 days starting 2 days before E_2 administration., 繁殖年齢に達した雌豚は、21日周期で2から3日間の発情を年間を通して繰り返す、いわゆる多発情動物である。発情時には、動物種ごとに異なる徴候が示される。発情時における豚の特徴的な変化の一つは頸管粘液中に多数の好中球が出現することである。この現象は臨床的には古くから知られていたが、その生物学的意義ならびに機序はほとんど解明されていない。一般に、局所組織への好中球の遊走は炎症時の特徴とされている。同じような現象が繁殖における一過程の生理的現象である発情期に、しかも豚の頸管粘液中に限ってみられるのは極めて興味深い。この現象の内分泌学的機序を解明する目的で一連の実験を行った。
1. 豚の繁殖周期に伴い頸管粘液中好中球(以下好中球と略す)数は変化する。そこで発情期、妊娠期、分娩期、授乳期に出現する好中球数を調べた。経産豚2頭を用い、発情前4日から交配、妊娠、分娩、授乳における150日間毎日、頸管部第2皺壁部から演者考案の綿棒方式で粘液を採取し、粘液中の好中球数を測定した。好中球数は発情前2日目から発情当日にかけて大きな1峰性ピークで増加した。その後、妊娠が進行するとその数は減少する。分娩5日前からその数は次第に増加傾向を示し、分娩後3から5日目にかけて発情期と同等かその約4倍以上となり、1峰性の特異なピークを形成した。
　結果、好中球数は、発情期の前後と分娩後5日目以内に特異的に増加することを確認した。これらのことは特に内分泌学的な変化と密接に関連している可能性が示唆された。
2. 発情期前後を中心に好中球数の変化と内分泌学的関係を検討した。経産豚4頭を用い、発情期を中心に好中球数と外部徴候の変化を2回繰り返し調べた。外部徴候の検査項目は雄許容反応と充血、腫脹とした。供試豚4頭は、2回の発情周期で測定した結果、好中球数は発情開始前1日から発情開始後1日の間に1峰性ピークで増加した。雄許容反応は好中球数が増加し始める前後から出現し、約3日間前後持続した。
　他の経産豚3頭を、好中球数の変化、血中E_2、P濃度と排卵時間帯との関連性を確認するために用いた。血中E_2、P濃度の測定はRIA法、排卵時間帯の確認は試験的開腹手術法と受精卵の分割所要時間によった。
　3頭には発情開始後36時間以内に2回の交配を行なった。その後、開腹手術によって卵管から受精卵を回収した。3頭中1頭では、交配後28時間目に3～4細胞期胚が確認された。他の1頭では、交配後15時間と25時間目で卵子を確認できなかった。しかし30時間後のと殺によって2細胞期胚が認められた。残りの1頭では、交配後38時間後に未受精卵を確認した。ブタの受精卵が2細胞期胚になるまでには14～19時間、4細胞期胚になるには24時間必要とされている。これらを根拠に排卵時間帯を推定すると、発情開始後26～38時間となった。
　血中E_2濃度は、発情開始前3日目から上昇しはじめ、発情開始前日から推定排卵日に向けて低下した。一方、P濃度は、発情開始前日から推定排卵日に向けてゆるやかに上昇し始めた。好中球数のピークは、血中E_2濃度のピークから遅れること2日～3日目に認められた。丁度、血中E_2濃度が低下し、P濃度がやや上昇し始める時期にあたっていた。また、好中球数のピークと排卵時間帯との関連は、推定排卵開始1日前から排卵時間帯と丁度重なることが確認できた。外部徴候は、血中E_2、P濃度と密接に関連して変化していることが認められた。
　以上の結果から、好中球数の増加は発情期の短時間内に限定されていて、排卵前または排卵時間の早期に認められ、血中エストロゲン濃度の変化と密接に関連しているかとが確認できた。
3. 発情期における好中球数の増加は、血中エストロゲンが主体となって起こる変化と推定された。そのことを確認するため、卵巣摘出豚を用いて、エストロゲンおよびプロゲステロンを単独および併用投与し疑似発情を誘起した。この実験を通して好中球数の増加が正常な発情と同じように再現されることを期待した。
　卵巣摘出豚7頭を用いた。投与したのはestrone(E_1)、estradiol-17β(E_2)、estriol(E_3)とprogestrone(P)である。これら試験のうちから、副生殖器に対して最も生理的反応の強いとされているE_2を投与した成績を報告する。E_2(投与量0.2、0.5および1.0mg)は、1回投与し、好中球数の特異的な変化が起こるかどうか観察した。また、この試験を通して自然発情と同等の再現がどのE_2投与量でみられるかも観察した。さらにE_2投与量をほぼ一定にしてP量(投与総量300、500、700mg)を投与したとき、P単独投与(総量120mg)したとき、好中球数の特異的な変化にどのような影響生じるかを調べた。
結果、E_2単独投与後4日目前後から好中球数の増加する特異的変化が認められた。好中球数はE_2投与量を増やすと量依存性に増加した。自然発情に最も近似した特徴が再現できたのは、E_2 0.5～1.0mgを1回投与したときであった。E_2とP併用投与では、自然発情様の徴候は全く認められなかった。Pを併用投与すると、量依存的にE_2投与による好中球の特有のパターンが崩れ、変動幅も小さくなった。P単独投与は、正常発情様徴候もなく、好中球数の増加と特異的変化も全く認められなかった。以上の結果から、発情時に認められる好中球の増数にはエストロゲン、特にE_2の存在が不可欠で、プロゲステロンは投与量に依存して抑制的に作用することが示された。
4. 好中球の遊走をもたらす状況がどのような組織の変化であるかを明らかにするために、頸管部粘膜組織の組織学的観察を行なった。供試豚は卵巣摘出豚7頭で、そのうち3頭には1.0、0.8および1.0mgのE_2をそれぞれ1回投与した。投与直前、投与後2、3、5、7および9日目の頸管第2皺壁部組織を子宮内膜バイオプシー器で採取した。エストロゲン投与による影響が最も強く、また、発情の再現性の高いE_2投与による頸管部組織の経時的変化を説明する。投与後2日目から頸管部粘膜上皮細胞の増殖と、変形細胞(変形好中球)の粘膜固有層、粘膜下織への侵入が認められた。粘膜下織の血管は拡張していた。粘膜上皮細胞の増殖は5日目をピークに減弱しはじめたが、E_2 1.0mg投与の2頭では9日目まで粘膜下織や固有層の血管の拡張と間質の疎造化が続き、変形細胞の浸潤や血管内の白血球も明瞭に認められた。以上の結果から、好中球遊走の主因はエストロゲン、特にE_2であることが確認された。
5. indomethacin製剤(以下indと略す)投与による無発情・無排卵豚の好中球数の変化を観察した。排卵期の豚にindを投与すると無発情・無排卵状態が出現する。この機構は排卵にProstagladinが関与し、その合成が阻害されることに起因しているからだとされている。この時に内分泌的変化も合わせ出現することと頸管粘液中好中球数の変化とその関連性について検討した。
　発情を繰り返している経産豚6頭を用いた。そのうち4頭にind(1日690mg)を発情前6日、3日、1日からそれぞれ7日間投与した。投与は小麦粉に混ぜ経口投与とした。この時の排卵確認は直腸検査によった。ind投与豚とind無投与豚の好中球数の変化を比較した。ind無投与豚ではind投与豚の約4倍から40倍の増数が認められた。ind投与豚の血中E_2濃度は発情2日前から急激に減少するか、殆ど変化しなかった。その時の血中P濃度は低値であった。ind投与豚は発情豚と同様な内分泌学的変化が認められなかった。ind投与豚のうち、1頭では、発情開始日に好中球数が僅かに増加したが、発情徴候は全く認められなかった。ind投与豚の卵巣には無排卵性閉鎖卵胞と卵胞嚢腫が認められた。一方、ind無投与豚は正常に排卵し、その後に正常な大きさの黄体形成を認めた。
以上、一連の実験は、豚の発情期に生起する頸管粘液好中球数の増加現象の内分泌学的機序を明らかにすることを目的とした。その結果、頸管粘液中への好中球数の増加と血中E_2濃度との関係が明確に示された。また、血中E_2値が高値に達すると、多数の好中球が頸管部組織中を上皮層表面へ向けて移動を開始することを示唆する組織像を確認することができた。この研究は、特に繁殖学領域での応用として発情期の確認と人工受精時の交配適期を確認するための新たな技術となる。血中エストロゲンによる好中球の子宮頸管部組織への選択的移動を誘起するケミカルメデエーターの解明は今後の研究課題である。},
 title = {発情豚の子宮頸管部粘液への好中球遊走現象に関する研究},
 year = {}
}