Makoto Tsuji

Roles of pulsatile release of LH in the development and maintenance of corpus luteum function in the goat

The roles of the pulsatile release of LH in the functional development and maintenance of the corpus luteum (CL) during the estrus cycle in the goat were examined using a potent GnRH antagonist. In Experiment 1, to assess the inhibitory effects of the GnRH antagonist on the release of LH during the estrus cycle, 9 goats were divided into 3 groups. Goats in Group I received only saline on Days 0 (day of ovulation), 5, 10 and 15. Goats in Group II received the GnRH antagonist (50 microg/kg, s.c.) on the days mentioned for Group I to inhibit endogenous LH during the periods of luteal development and maintenance. Goats in Group III received saline on Days 0 and 5 and then the GnRH antagonist on Days 10 and 15 to inhibit LH during the period of luteal maintenance. Serial blood sampling took place on Days 1, 3, 5, 8, 13 and 18 to characterize the LH pulses. The LH pulses were observed throughout the estrus cycle in Group I but were completely abolished in Group II. In Group III, the pulsatile release of LH was observed from Day 1 to 8, but the LH pulses were completely abolished on Days 13 and 18. In Experiment 2, 16 goats were divided into the same 3 groups as in Experiment 1 to examine the effects of the GnRH antagonist on the luteal function. The concentration of progesterone in the plasma in Group I increased after ovulation, reached a maximum level around Day 12, and subsequently returned to the basal level on Day 17. The concentrations of progesterone in Group II rose after ovulation, but reached a plateau around Day 6 and maintained the level up to Day 9, then rapidly decreased from Day 9 to 10 to the basal level. The concentrations of progesterone in Group II were lower on Days 7 to 15 than those in Group I (P<0.01). The concentrations of progesterone in Group III increased after ovulation, reached a maximum level around Day 8, then dropped from Day 10 to 13 to the basal level. The concentrations of progesterone in Group III on Days 11 to 15 were lower than those in Group I (P<0.05 on Day 11, P<0.01 on Days 12 to 15). These results demonstrate that endogenous LH is essential for normal development and maintenance of the CL function during the estrus cycle in the goat. Further, this study suggests that while the functional maintenance of the caprine CL depends entirely on LH support, such functional dependence during early CL development is only partial.

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: Changes with age and effects of cryptorchidism

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.

Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs.

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbeccos Modified Eagle Medium with Hams nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.

Anovulation to a luteinising hormone surge in an aged goat with follicular cysts.

local thickening of the area, and probing is mostly inadequate in this area. Stocker and others (1989) and Medl and others (1994) reported that radiography and endoscopy were inefficient in these cases, respectively. Teat stenosis usually appears hyperechoic (Fig la) or sometimes hypoechoic. Foreign bodies, for example, haematoma (Fig 2c) and pus particles, have similar echogenity but these may be seen to move. The presence of milk affects the results of ultrasonographic examinations. These examinations are performed to full advantage when a teat is full of milk; if the teat is not full of milk misleading results can be obtained. If there is not enough milk in the teat despite the oxytocin injection (due, largely, to an obstruction in the annular area or to the absence of milk in mammary lobe), physiological saline solution should be administered to the sinus papillaris, except where there is an obstruction in the ductus papillaris or around the rosette of Furstenberg. When no liquid is given intrammamanly, the quality of the image is reduced. There are advantages and disadvantages of ultrasonographic examinations performed directly or in a water bath. Direct contact can be easily applied; however, the shape of the teat may change because the teat wall near the probe and the rosette of Furstenberg area cannot be seen completely. Nevertheless, direct contact has advantages in diagnosing stenosis at the base of the teats, because the water cup cannot easily be placed at the teat base and its dorsal levels. More detailed information can be obtained by water bath examination in other parts of the teat. As Stocker and others ( 1989) and Saratsis and Grunert (1993) reported, a 5 MHz frequency should be adequate in the diagnosis of teat stenosis. Stocker and others (1989) also reported that 3 5 MHz could be inadequate for the same purpose; however, Cartee and others (1986) reported that the blood vessels and mucosal folds of the teat became clearly visible with 10 MHz. In conclusion, it is suggested that ultrasonography should be used in the diagnosis of teat stenosis because of the better diagnostic characteristics compared with the classic clinical examination methods. Ultrasonography supports and complements the other methods.