Ikuo Domeki

Epigenetic Modifications Necessary for Normal Development Are Established During Oocyte Growth in Mice

Abstract The ability of maternal chromatin to support full-term development is attained during oocyte growth. The aim of this study was to identify when during the growth phase the maternal chromatin developed the capacity to support term development. Mature metaphase II-arrested oocytes that contained chromatin from oocytes at different stages of oocyte growth were constructed by micromanipulation. The oocytes were fertilized in vitro, developed to the blastocyst stage in vitro, and transferred to recipients to assay developmental potential. The results demonstrate, firstly, that the origin of the maternal chromatin has no effect on the rate of oocyte maturation, fertilization, or development to the blastocyst in vitro. Secondly we demonstrate that maternal chromatin is first competent to support development to term during the latter half of oocyte growth when oocytes are 60–69 μm in diameter in juvenile mice or 50–59 μm in diameter in adult mice. These data show that epigenetic modifications necessary for postimplantation development occur during a specific phase of oocyte growth.

Effect of fetal mass, number and stage of gestation on pregnancy-specific protein B concentrations in the bovine

In this study we characterized the peripheral plasma pregnancy-specific protein-B (PSPB) profile throughout gestation and examined the effect of stage of gestation, fetal mass and number on this profile in Holstein cows after non surgical embryo transfer. Cows (n = 12) were divided into 2 groups: Group 1 = single embryo recipient cows (n = 5), Group 2 = twin-embryo recipient cows (n = 7). Blood was collected approximately every third day from Day 0 (Day 0 = first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d post partum. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from the group. The time trend concentrations of plasma PSPB were significantly affected by the stage of gestation (P < 0.001) and fetal number (P < 0.001). In both groups PSPB increased gradually, with the mean levels being significantly higher (P < 0.01) in the twin-bearing group from Day 50 onwards (0.7 +/- 0.2 vs 9.2 +/- 4.5 ng/ml, singleton and twin-bearing cows, respectively) except for Day 10 pre-partum. By mid-gestation (Day 140), mean PSPB levels increased in the singleton (P < 0.001) cows by thirty-fold (21.2 +/- 3.2 ng/ml) as opposed to a ten-fold (98.4 +/- 13.2 ng/ml) increase in the twin-bearing (P < 0.001) group. The mean PSPB concentrations between Days 30 to 20 prepartum dramatically increased by about 700 to 200% in singleton (128.8 +/- 46.3 to 745.6 +/- 66.7 ng/ml) and twin-bearing cows (375.6 +/- 130.4 to 861.5 +/- 127.9 ng/ml), respectively. The PSPB levels between Day 10 prepartum to parturition were significantly higher (P < 0.001) in the twin-bearing group than in the singleton group (745.6 +/- 66.7 to 1627.4 +/- 238.9 ng/ml vs 861.5 +/- 127.9 to 3103.0 +/- 643.0 ng/ml in singleton and twin-bearing groups, respectively). Calf birthweight was correlated (P < 0.01) to peripheral PSPB concentration in singleton cows; however, this relationship decreased with the subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited abnormal PSPB profiles. These results indicate that peripheral PSPB levels are correlated to the stage of gestation and fetal number. In addition, the peripheral pattern of PSPB is a valuable guage for predicting fetoplacental viability.

Plasma bovine placental lactogen concentration throughout pregnancy in the cow; relationship to stage of pregnancy, fetal mass, number and postpartum milk yield

This study characterized the peripheral plasma placental lactogen (bPL) profile throughout gestation and examined the relationship between the stage of gestation, fetal mass, number, and postpartum lactation with circulating levels of bPL in Holstein cows after nonsurgical embryo transfer. Cows (n = 12) were divided into two groups: Group 1 = single embryo recipient cows (n = 5); Group 2 = twin embryo recipient cows (n = 7). Blood was collected about every third day from Day 0 (Day 0 was defined as the first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d postpartum. The cows were milked twice daily at 0800 and 1800 hr. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from that of the group and reported separately. The time trend concentrations of plasma bPL were significantly affected by the stage of gestation (P < 0.01) but not fetal number (P < 0.21). In both groups bPL levels remained low during the first two trimesters, then increased rapidly (P < 0.01) to peak concentrations between Days 200 and 220, and stabilized at this elevated level until parturition. Postpartum milk yields were indistinguishable between the singleton and twin bearing cows. Calf birth weight and postpartum lactation were both correlated (P < 0.01) to peripheral bPL concentration in singleton cows, however, this relationship decreased with a subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited a deviating pBL profile. These results indicate that peripheral bPL levels are positively associated with the stage of gestation but not with fetal number. Otherwise, the peripheral pattern of bPL is a valuable index for predicting feto-placental viability.

Peripheral changes in estrone sulfate concentration during the first trimester of gestation in cattle: comparison with unconjugated estrogens and relationship to fetal number

Estrone sulfate originates mainly in the conceptus during gestation in cattle. Its concentration in maternal body fluids is a useful indicator of placental function. The objective of this study was to determine the profiles of estrone sulfate during early gestation in singleton and twin bearing cows using a newly developed extraction method. One or two blastocysts produced in vitro were nonsurgically transferred to regularly cycling Holstein cows on Day 7 (Day 0 was defined as the first day of standing estrus). Pregnancy was diagnosed on Days 30, 45 and 60 by transrectal ultrasonography and finally confirmed at parturition. Six cows with singleton and six with twin pregnancies were used in the experiment. Blood was collected every other morning by jugular venipuncture from the day after transfer to Day 100. Harvested plasma was applied to reversed-phase C18 cartridges. Estrone sulfate and unconjugated estrogens (estrone and estradiol-17beta) retained in the cartridge were eluted separately by methanol stepwise gradient and each measured by validated radioimmunoassay. On average, estrone sulfate concentrations fluctuated between 2 and 6 pg/ml until Day 50 in both groups and then gradually increased. However, the levels of estrone and estradiol- 17beta remained low (1-5 pg/ml) until Day 80. The concentration of estrone sulfate after Day 50 was significantly affected by the day of gestation (P < 0.0001) and the number of fetuses (P < 0.01). After Day 80. estrone sulfate increased drastically, followed by increases in estrone and estradiol-17beta concentrations. The rate of increase in estrone sulfate during Days 80-100 was the greatest among all estrogens (P < 0.05). The rates of increase in estrone sulfate during Days 50-80 and 80-100 were 1.7 times greater in twin pregnancies than in cows having one fetus. These results suggest that the concentration of estrone sulfate in bovine peripheral blood plasma during early gestation has potential application in monitoring embryonic growth as well as fetoplacental development.

Effect of stage of gestation and foetal number on plasma concentrations of a pregnancy serum protein (PSP-60) in cattle.

This study characterised the peripheral plasma concentration of PSP-60 throughout gestation, and examined the effect of stage of gestation and foetal number on this protein in Holstein cows after non-surgical embryo transfer. Cows (n=12) were divided into two groups; Group 1 contained single embryo recipient cows (n=5), Group 2 contained twin-embryo recipient cows (n=7). Blood was collected approximately every third day from day 0 (first day of standing oestrus), then daily for the last 10 days of gestation and until one day post-partum. Two of the twin-embryo recipient cows had abnormal pregnancies, consequently data from them was considered separately. In both groups PSP-60 increased progressively from about day 20 post-oestrus to 20 days pre-partum (from 0.9 +/- 0.2 to 49.7 +/- 8.7 ng ml(-1), and from 1.3 +/- 0.6 to 115 +/- 34.9 ng ml(-1) (mean +/- SEM), in singleton and twin-bearing groups, respectively). The mean concentrations between 20 and 10 days pre-partum increased dramatically by about six-fold (P<0.001) in singleton-bearing cows (from 49.7 +/- 8.7 ng ml(-1) to 283.8 +/- 73.7 ng ml(-1)) to over two-fold in twin-bearing cows (from 115 +/- 34.9 ng ml(-1) to 284 +/- 98.2 ng ml(-1)). The mean concentrations of the two groups were indistinguishable between 10 days pre-partum and parturition. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited abnormal PSP-60 profiles. Our findings indicate that peripheral plasma PSP-60 concentrations are correlated to the stage of gestation and foetal number, and assist in predicting foeto-placental viability.

Establishment of a reliable extraction method of estrone sulfate from bovine plasma for detection of the peripheral level during the regular estrous cycle by radioimmunoassay

A simple extraction and assay technique of estrone sulfate in bovine blood was developed with the object of detecting the peripheral level of estrone sulfate in a normal estrous cycle or in early pregnancy. Estrone sulfate in bovine plasma was extracted with a small reversed phase cartridge. The steroid conjugate retained in the cartridge was eluted with 40% (v/v) methanol. Estrone sulfate was separately recovered from other steroids by the stepwise increase in methanol concentration in the elution solvent. The recoveries of estrone sulfate eluted with 40% methanol were more than 90%, irrespective of the applied plasma volume. The concentration measured by radioimmunoassay with the eluent of 40% methanol was consistent for plasma extraction volumes of 0.5-2.0 ml. The change of estrone sulfate in bovine peripheral plasma during the regular estrous cycle was determined with a small reversed phase cartridge for extraction and 40% methanol for elution. The change in estrone sulfate was found to be similar to the change of estrone and estradiol-17 beta. The concentration of estrone sulfate was not higher than that of both estrogens in cattle.

Ultrasonography for observation of ovary and early pregnancy diagnosis in miniature pig.

医学領域において前立腺検査用に開発された棒状の超音波プローブを利用して, 2頭のミニチュアブタ (CSK×クラウン系1頭, ゲッチンゲン系1頭) の発情周期中における卵巣の超音波画像診断を実施した。また1頭のゲッチンゲン系ミニチュアブタについて体表からの超音波画像による早期妊娠診断およびその後の胎子の観察も試みた。発情発現日における卵巣の超音波画像は, 直径5～8mmのエコーフリーの部位が多く示され, 卵巣に多数の卵胞が存在することが確認された。しかし発情2日目にはエコーフリーの部位は少なくなり, 排卵が確認された。黄体期には直径5mm程度のエコーフリー部位の周りにややエコーレベルの低い層が認められ, この像は黄体であると考えられた。早期妊娠診断においては, 交配後20日目に直径20mmの胎嚢が確認され, 26日目には胎嚢の内部に胎子の存在も認められた。胎子像には交配後47日目に骨格および心拍が認められ, 体長は53日目に50mm, 100日目に100mmを超えることが観察された。以上のことより, ミニチュアブタにおいて超音波診断を実施することで, 発情周期中の卵巣および胎子像の観察は可能であり, ミニチュアブタの繁殖領域における利用がさらに広がるものと思われた。

Activation of quiescent ovaries by administration of PMSG after LH-RH analogue treatment in heifers.

The effect of pregnant mare serum gonadotrophin (PMSG) treatment on activation of quiescent ovaries was examined in heifers. Groups of thirteen, twenty and twelve heifers which showed ovulation within 2 d and corpus luteum (CL) development after injection with a luteinizing hormone releasing hormone analogue (LH-RH-A) were supplementally injected with 500 IU of PMSG (Group I); 500 IU of PMSG and 500 mug of Prostaglandin F(2alpha) analogue (PGF(2alpha)-A; Group II); and 500 mug of PGF(2alpha)-A (Group III) on Day 6 after the injection of 200 mug of LH-RH-A (Day 0), respectively. Estrus appeared in 33.3 to 45.0% of the heifers of the respective groups after the treatment. Ovulation occurred at a significantly (P<0.01) higher rate in Groups I (100%) and II (90.0%) than in Group III (41.7%). The ovarian cyclic activity was initiated in all the heifers that ovulated. Plasma progesterone levels decreased significantly (P<0.05) to about 1 ng/ml on Day 8 and Day 7 in Group I and Groups II and III, respectively. Plasma estradiol-17beta (E(z)) levels increased significantly (P<0.05), reaching a peak on Days 7 to 7.5 in Groups I and II but not in Group III. It is concluded that PMSG treatment stimulates maturation and E(z) secretion of a follicle, thus promoting ovulation and the onset of ovarian cyclic activity.

Synchronization of estrus and ovulation in graz-ing cows with intramuscular injection of prosta-glandin F2α; in the case of administration of synthetic LH-releasing hormone, or human chori-onic gonadotropin followed

黄体期の牛86頭に対してPGF2α10 mgを筋肉内注射し,黄体退行の認められた65頭のうち51頭に合成LH-RH-1200μg(A群)またはHCG 1,000~2,000 i.u.(B群)をPGF2α投与後42~47時間(A-I群8頭,B-I群11頭),57~60時間(A-II群8頭,B-II群7頭),72~74時間(A-II群9頭,B-III群8頭)に筋肉内注射して,これらの発情発現と排卵状況及び受胎性をPGF2α単一処置(C群14頭)のそれと比較検討して,つぎの成績を得た。1. PGF2α投与後37~84時間における発情発現率は,A, B両群のI群ではそれぞれ25.0%,27.3%,II群では37.5%,42.8%であり,C群の71.5%と比べて低かったが,III群では66.7%,62.5%で大差なかった。2. PGF2α投与後84時間以内に発情発現がみられなかった牛は,すべて120時間以内に無発情排卵した。3. PGF2α投与後排卵までの平均(±S.D.)時間は,A,B両群のI群ではそれぞれ74.5(±6.7),79.5(±6.9)時間,II群ではそれぞれ88.0(±3.7),87.0(±4.6)時間,III群ではそれぞれ98.7(±8.2),94.0(±9.8)時間であり,排卵誘起剤の投与時期が早いほど短かった。なお,C群のそれは89.9(±13.3時間)であった。4. PGF2α投与後3~5日の間の授精による受胎率は,A, B両群のI群ではそれぞれ37.5%(3/8頭),12.5%(1/8頭),II群では37.5%(3/8頭),42.9%(3/7頭)でC群の50.0%(7/14頭)に比べて低かったが,III群では55.6%(5/9頭),50.0%(4/8頭)でC群のそれとほぼ同率であった。5. 以上の成績から,PGF2αと合成LH-RHまたはHCGの併用により排卵を効果的に同期化することが可能であり,PGF2α投与後これら薬剤を投与するまでの時間間隔が長いほど処置後の発情発現率は高く,同期化した発情時の受胎成績が良い傾向が認められた。

Effects of intrauterine injection of PGF2α on progesterone levels in peripheral blood plasma of the cow

牛において,排卵後2日ないし15日の黄体期にPGF2αを連日2回(1回用量0.5~1.0 mg)あるいは1回(用量3~6mg),黄体の存在する卵巣と同側の子宮角に注入して,処置後の黄体の変化にともなう末梢血中progesteroneの消長を追求して,次の成績を得た。1) 排卵後2~4日の黄体初期の早い時期に処置した8頭中4頭は処置後8~14日目に排卵した。処置後の血中progesterone値は,A)正常性周期におけるそれとほぼ同様のもの,B)処置後一時減少するが,ふたたび増加して以後A)と同様に維持されるもの,C)処置後暫時増加の傾向を示すが,結局早期に低下するもの,D)処置後いったん低下したのちふたたび増加するが結局早期に低下するもの,などの消長型を示した。2)排卵後4日および5日の黄体初期に連日処置した2頭の牛は,処置後4日目に排卵した。血中pro-gesteroneは処置後急激に減少して,2回目の処置後24時間には処置前の1/3の値を示した。3)排卵後9~11日の黄体開花期に処置した3頭の牛は,いずれも処置後4日目に排卵した。血中pro-gesteroneは処置後3時間にすでに減少の傾向を示し,24時間後には処置前の値の1/2~1/6に減少した。4) 排卵後15日目の黄体後期に処置した2頭の牛は,処置後4日および6日目に排卵した。処置後24時間の血中progesteroneは処置前の値のそれぞれ1/2および1/8以下に減少した。5)血中progesteroneの消長は触診による黄体の形状変化とほぼ一致した。6)以上の成績から,PGF2αの黄体退行作用は排卵後5日以降の黄体期の牛ではきわめて顕著であるが,排卵後4日までの間の牛では弱いことが明らかにされた。