Junji Masaki

Endocrine profiles and embryo quality in superovulated Japanese Black cattle

The relationships between plasma concentrations of progesterone (P4), estradiol-17beta (E2) and lutenizing hormone (LH) and embryo yield and quality were examined in 40 superovulated Japanese Black cattle. The results indicated that the ovarian function, especially the function of corpus luteum on the first treatment day, is an important factor for reliable superovulation in cattle. The levels of plasma E2 and LH at estrus were related to embryo yield and quality.

Serum inhibin, FSH, LH and testosterone levels and testicular inhibin content in beef bulls from birth to puberty

Developmental changes in testicular inhibin content and serum inhibin levels were observed with serum levels of gonadotrophins and testosterone in beef bulls up to 8 months of age. Twenty-two beef bulls (8 Japanese Black and 14 Japanese Shorthorn) were assigned to monthly groups from 1 to 8 months old (n=2 for 1 and 8 months, and n=3 for 2–7 months). Inhibin activity in the testicular extracts was determined using an in vitro rat pituitary cell culture system. Serum levels of inhibin and other hormones were measured by radioimmunoassays. The concentrations of inhibin in the testis (U/g) were high during the first 3 months (P < 0.05). They decreased temporarily between 4 and 6 months and then increased (P < 0.05). Serum inhibin levels were high during the first 4 months and decreased after 5 months (P < 0.05). Serum FSH levels were higher at 2 months than at any other month of age (P < 0.05). Serum testosterone levels remained low during the first 3 months, increased at 4 months and continued to rise thereafter. A significant positive relationship was found between serum FSH and serum inhibin levels (r=0.068; P < 0.001), whereas a negative relationship was found between serum testosterone and serum inhibin levels (r = −0.58; P < 0.01). During the first 5 months of age, the concentrations of inhibin in the testis (U/g) correlated positively with serum FSH levels (r=0.69; P < 0.01), while they correlated negatively with serum testosterone levels (r=−0.85; P <0.001). These results indicate that the immature bovine testis produces and secretes high levels of inhibin, and that serum inhibin levels reflect this high inhibin production in the testis before the onset of puberty. After the onset of puberty, inhibin production and inhibin release into the circulation are no longer parallel, which is probably due to the formation of the blood-testis barrier. It is suggested that FSH is the principal regulator of inhibin production in the testis during the first 5 months of age in bulls. After the onset of puberty, testosterone might play a dominant role in suppression of FSH from the pituitary.

Plasma endocrine profiles and total cholesterol levels in superovulated cows

Abstract Changes in plasma follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β (E2) and progesterone levels between superovulatory hormone injection and flushing were monitored. FSH level at gonadotropin injection was significantly correlated with the peak level (r = 0.73) and the total level between 30 h after cloprostenol injection and first service (r = 0.76). There was a significant relationship between the parameters of the FSH level and the number of transferable embryos. Early or delayed preovulatory LH surge was observed in 33.3% of the cows ( 6 18 ; Group I). Three cows (16.7%) had low basal FSH levels of less than 5 ng/ml between the gonadotropin injection and the preovulatory LH surge (Group II). The remaining nine cows (50.0%) made up Group III. The numbers of recovered and transferable embryos in each group were 4.5 and 0.5 (Group I); 4 and 3 (Group II); and 8 and 4 (Group III), respectively. A significant difference in the number of transferable embryos was observed between the groups (P The number of recovered embryos in the cows with a total cholesterol level of less than 130 mg/dl was significantly less than those in the cows with a total cholesterol level of 130 mg/dl and higher (4:11, P Our study indicated that early or delayed preovula tory LH surge, low FSH, and low total cholesterol levels adversely affect superovulation results.

Seasonal changes in inhibin activity in seminal plasma and serum concentrations of FSH, LH and testosterone in the male goat ( Capra hircus )

Inhibin activity in goat seminal plasma was measured by in vitro assay throughout successive 9 months and its relationship with the serum FSH, LH and testosterone concentrations was investigated. Total inhibin activity (TIA) in seminal plasma gradually increased from spring to summer, reduced in autumn (P<0.05) and recovered toward winter (P<0.05). Serum FSH and LH reached a peak in mid-summer (P<0.01) and returned to the low levels in autumn. Serum testosterone also increased in mid-summer and kept the high levels until the early winter (P<0.05). Some positive correlation was found in monthly levels between seminal TIA and serum FSH (r=0.305; P<0.05). Results suggest that the summer increase of inhibin activity in seminal plasma relates with the mid-summer rise of serum FSH levels in the male goat.

Subcellular localization of the antigenic sites of relaxin in the luteal cells of the pregnant rat using an improved immunocytochemical technique

Abstract Using an improved immunocytochemical technique, the present work has demonstrated the antigenic sites of relaxin in the luteal cells of the pregnant rat with preservation of the ultrastructural details. Tissues collected were fixed with either 4% paraformaldehyde (PFA), 4% PFA containing 0.3% glutaraldehyde (GLA) or Zamboni solution followed or not by 1% osmium. Ultrathin sections of the tissues embedded in epoxy resin were incubated with rat relaxin antiserum and antibody binding sites were visualized with protein A-gold. Among these fixatives, the use of 4% PFA-0.3% GLA, followed by osmium, allowed excellent preservation of fine cellular structure with maintenance of antigenic activity and immobilization of the antigen. The majority of the gold particles indicative of relaxin immunoreactivity were localized on the dense core of the membrane-limited granules, while a minor fraction was localized on the Golgi apparatus. The results suggest that the subcellular localization of relaxin can be detected after post-fixation with osmium and that the antigenic sites of relaxin exist mostly in the dense granules with a small fraction in the Golgi apparatus.

Subcellular location of the maturation process of relaxin in rat luteal cells during pregnancy as revealed by immunogold labeling

The protein A-gold immunocytochemical technique and rat relaxin antiserum were used to study the subcellular location of the maturation process of relaxin in rat luteal cells on Days 15–19 of pregnancy. We investigated both the distribution of intracellular organelles with immunoreactivity for relaxin and differences in their density of labeling by the gold particles, which revealed immunoreactive sites. On Days 15–19 of pregnancy, relaxin immunoreactive sites were located in the cisternae and vacuoles of the Golgi apparatus and in the Golgi-associated small granules as well as in the secretory granules. However, no gold particles were detected in rough endoplasmic reticulum at this stage of pregnancy. Quantitative analysis revealed that the density of labeling increased from the Golgi apparatus through the Golgi-associated small granules to the secretory granules, which showed significantly more labeling than the background (P<0.01). This finding indicates that the Golgi complex and Golgi-associated granules are both immunoreactive sites for relaxin, and suggests their involvement in the maturation of this peptide in rat luteal cells during pregnancy.

Correlation of the mating behaviour of bulls with patterns of gonadotrophins and oestrogens in oestrous cows

Abstract The correlation between mating behaviour of bulls and patterns of gonadotrophins (GTH, i.e. FSH and LH) and oestradiol-17β (E 2 ) was examined in oestrous cows, using two bulls. Four cows were placed with each bull in a free stall (10 m × 15 m) at 44 h after the second injection of PGF 2α analogue (cloprostenol). Mating (mounting and ejaculation) behaviour of the bull was observed and serum levels of FSH, LH and E 2 were measured in cows during the next 33 h. The bull ejaculated only with cows in which elevated E 2 levels and a GTH surge occurred. When the time of the GTH peak was designated as 0, and the patterns of hormones in cows with mating behaviour were combined, it was found that 92.3% of ejaculating and 79.1% of mounting behaviour occurred within the range of the GTH surge, indicating that the former behaviour was coincident with the GTH surge.

Effects of bull seminal plasma extract on release of FSH and LH by GnRH injection in pubertal beef bulls

The effects of bull seminal plasma (BSP) injection on gonadotrophins release by GnRH were examined in pubertal intact and castrated bulls (n=4). An ethanolic extract of BSP (0.18 g dissolved in 1 ml of saline) or heated-BSP (h-BSP) was injected repetitively 10 times with a total dose of 1.8 g (equivalent to 30 ml of BSP) via jugular cannula at intervals of 20 min for 3 h. Just after the last BSP injection, GnRH (20 μg) was challenged intravenously. Blood samples were collected via cannula at 30 min intervals for 14 h starting 3 h prior to commencement of BSP injections. BSP had no distinct effects on basal FSH and LH concentrations during 3 h-injection. A GnRH challenge led to a gonadotrophin peak within 30 min in all animals. In the intact group, the peak height showed no significant differences between injections (mean ± s.e.m.) of FSH (78.6 ± 15.6 ng/ml for h-BSP vs. 61.3 ± 10.4 ng/ml for BSP) and LH (125.9 ± 11.3 ng/ml for h-BSP vs. 115.6 ± 17.0 ng/ml for BSP). In the castrated group, an evident suppression of FSH peak by BSP injection was observed compared with h-BSP injection (261.4 ± 9.1 ng/ml for h-BSP vs. 188.8 ± 7.7 ng/ml for BSP; P < 0.001), whereas the peak height of LH was not different between injections (141.2 ± 41.0 ng/ml for h-BSP vs. 139.0 ± 44.5 ng/ml for BSP). The inhibiting effect of BSP on FSH release was greater in the peak height than in the secretion rate (209.2 ± 14.5 ng/ml/min for h-BSP vs. 163.3 ± 9.1 ng/ml/min; P <0.05), which means a particular inhibiting effect of BSP in the period of the initial part of the peak. Actually, a marked FSH suppression was observed within the first 1 h from GnRH challenge in each animal. The results of this study show that a heat-labile ethanolic extract of bull seminal plasma can suppress FSH release by GnRH in castrated pubertal bulls, and suggest that endogenous feedback of the testis is involved in the different effect of BSP between intact and castrated bulls.

Scanning eletron microscopical observations on the rat sperm head trapped by ovum microvilli during the penetration into ovum in vivo.

ラット受精過程のうち,精子頭部が卵細胞に接触後,卵細胞質突起で覆われるまでの過程を走査型電顕で観察した。精子頭部は卵細胞に接触後,線維状に伸びた卵子微絨毛によって巻き込まれていた。微絨毛は規則正しく糸巻き状を示し,特に赤道域中央付近で多く認められた。その後,微絨毛によって捕獲された精子頭部は卵細胞質突起によって覆われる。

Phospholipase A Activity in Bull and Boar Seminal Plasma

ウシ精漿には, 赤血球, 卵子および精子に対して細胞膜の融解またはリン脂質の減少をひきおこす作用のあることが知られている. このような反応には, リン脂質分解酵素が関与している可能性があるため, 本研究ではウシ精漿のボスホリパーゼA活性の測定を試みた. また, 上記の作用がみられないブタ精漿およびボスホリパーゼA2活性の存在が知られているヒト精漿も比較のため供試した. アッセイにはヒドロキサメート法を適用し, インキュベーションはCaを20μmol含む50mMトリスー塩酸緩衝液 (pH7.5) を用い3時間行った。ホスファチジルコリンを基質とした場合, ウシ精漿は他の精漿に比べて高い活性を示した (加水分解エステルとして, ウシ8.66±1.08, ブタ1.09±0.51, ヒト1, 22±0.48μmol/ml/3hrs, 平均値±標準誤差). ヒト精漿はホスファチジルコリンよりもホスファチジルエタノールアミンをより多く加水分解した. ブタ精漿の活性はいずれの基質に対しても低かった. ウシ精漿に高いホスホリパーゼA活性が認められたことから, 本酵素が細胞膜融解を伴う変化に関与している可能性のあることが示唆された.