Shuji Sasamoto

Sertoli cell function declines earlier than Leydig cell function in aging Japanese men

In order to evaluate the age-related changes in Leydig cell and Sertoli cell function, we measured serum levels of total testosterone (TT), free testosterone (FT), inhibin, LH and FSH in 116 healthy Japanese men, aged 24-92 years. Serum TT remained constant up to the age of 80 years and decreased thereafter. Serum FT declined linearly with aging and was significantly lower in men in their forties than in younger men (24-39 years old). Serum inhibin levels also declined with aging, with serum concentrations significantly lower in men older than 40 and markedly lower in men over 80 years old. LH and FSH were elevated in men over 60 and 40 years old, respectively. We also examined relationships between gonadotropins and gonadal hormones in these men divided into three age groups, young (24-39 years old), middle aged (40-59 years old) and aged (60-92 years old) men. Although there was a significant inverse correlation between LH and TT or FT for the entire population, subset analysis demonstrated that this inverse correlation was confined to men over 60 years old. In fact, in young men, TT and FT were positively correlated with LH. Overall, there was also an inverse correlation between FSH and inhibin. In subset analysis this relationship was present in both middle aged and aged men. These findings suggest that in Japanese men testicular endocrine functions decline after the fourth decade of life, and that Sertoli cell function declines earlier than Leydig cell function.

Effects of aluminum adjuvant on systemic reactions of lipopolysaccharides in swine.

In vivo effects of aluminum adjuvant on systemic reaction of bacterial lipopolysaccharide (LPS) in piglets were investigated. Intramuscular injection of 0.1 mg kg-1 of LPS added to aluminum hydroxide gel (LPS(+)AL) mitigated the leukopenia, trembling and serum levels of TNF-alpha and cortisol compared with the injection of LPS suspended in LPS-free saline (LPS(+)SALINE). The serum endotoxin levels were reduced remarkably but relatively long-lasting in the LPS(+)AL. The lethality in mice injected with LPS added to aluminum hydroxide gel was significantly reduced. Likewise, the Limulus activity of a test LPS was reduced by the addition of aluminum hydroxide gel or aluminum chloride.

Superovulation of holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animals own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.

Changes in the secretion of inhibin and steroid hormones during induced follicular atresia after hypophysectomy in the rat.

Sequential changes in the function of antral follicles during the period of follicular atresia were investigated after hypophysectomy (Hypox) at 1100 hr on proestrus. Within 6 hours after Hypox, concentrations of progesterone (P), testosterone (T) and estradiol-17 beta (E) decreased abruptly in ovarian venous plasma (OVP) and follicles showed a reduced ability to ovulate. Six hours after Hypox, ovulation was still induced by human chorionic gonadotropin (hCG) in all animals but with significantly fewer number of oocytes compared to the group given hCG at 1100 hr on the day of proestrus. Nine hours after Hypox, several granulosa cells of all large follicles (greater than 400 microns in diameter) exhibited morphological signs of atresia. Twelve hours after Hypox, all large and medium sized (200-400 microns in diameter) follicles showed advanced stages of atresia and almost all follicles failed to ovulate in response to hCG. Inhibin activity in OVP declined more slowly compared to the profiles of steroid hormones and 53% of the initial inhibin activity was still maintained at 18 hours after the operation. Inhibin activity further decreased to 7% of the initial level at 24 hours and was undetectable by 48 hours after Hypox. These results suggest that fully developed Graafian follicles gradually lose their ability to secrete inhibin in contrast to the rapid decrease in secretion of steroid hormone during the process of atresia.

Relationship between characteristics of immunoreactive LH/FSH cells and the levels of gonadotropin in the female rat.

Morphological and functional changes of pituitary LH/FSH cells in the female rat were investigated using the parameters on the radioimmunoassay, immunohistochemistry and ultrastructure. Changes in immunostainability, populations of intensely immunostained LH and FSH cells and total volume of secretory granules were correlated with the changes in pituitary LH and FSH contents during the estrous cycle. The immunohistochemical feature of gonadotropin release is the transformation of intensely immunostained gonadotrophs into the weakly stained ones. Secretory granules of small diameter (less than 150 nm) were numerous just before LH and FSH surges then sharply declined along with LH and FSH surges. The number of secretory granules of large diameter (larger than 150 nm) also decreased when LH and FSH surges took place. Then the number increased progressively until 17.00 h on the day of diestrus, corresponding to the increase in pituitary LH and FSH contents. It is suggested that small secretory granules are a release pool while large ones are a reserve pool.

Mode of action of chlorpromazine (CPZ) blockage on 13,14-dihydroprostaglandin F2-alpha formation in rat ovary

The mode of action of the inhibitory effect of CPZ on 13,14-dihydroprostaglandin F2-alpha (13,14H2-PGF2-alpha) formation in rat ovary was examined. The inhibition of 13,14H2-PGF2-alpha formation and of ovulation induced by a proestrus were completely recovered by an injection of hCG (25 IU/rat) or LH-RH (500 ng/rat) at 15:00 on the same day. 13,14H2-PGF2-alpha formation and ovulation were not inhibited by a single injection of prolactin (PRL:6 IU/rat) at 13:00 on the day of proestrus. Repeated injection of PRL inhibited cyclic ovulation and 13,14H2-PGF2-alpha formation. The estrus cycle of PRL treated animals showed a continuous state of diestrus. Although 13,14H2-PGF2-alpha formation and ovulation were inhibited by the repeated injection of CPZ, the repeated-simultaneous injection of CPZ and bromocriptine at 10:00 once a day for 3 days from the first day of diestrus partly restored both and entirely reversed the suppression of the cyclic-changes in the in the vaginal smear pattern. These results indicate that the inhibition of 13,14H2-PGF2-alpha formation induced by a single injection of CPZ probably occurs via the suppression of LH-RH release from the hypothalamus, whereas PRL secretion may participate in the inhibitory effects of repeated injections of CPZ.

Age-related increase in peptidylarginine deiminase in the male rat pituitary

We have measured the activity of peptidylarginine deiminase (EC 3.5.3.15) in male Wistar rat pituitaries at various ages. Pituitaries obtained from 3- and 9-month-old rats showed negligible activities. The mean enzyme activity increased appreciably by 18 months and markedly by 24 months accompanied with actual increases in the enzyme content. The peptidylarginine deiminase mRNA content showed a similar but more gradual increase appreciable from 9 months. Many enzyme-positive cells were present in the pars distalis of 24-month-old male pituitaries. Most of the enzyme-positive cells coincided with lactotrophs. The pituitary prolactin content showed a gradually increasing profile resembling that of the enzyme mRNA, but the serum prolactin concentration did not increase significantly. Neither the serum 17 beta-estradiol content nor the pituitary estrogen receptor content showed significant variation that could account for the marked increase in the pituitary enzyme content between 18 and 24 months of age. These data suggest possible presence of other factors regulating the enzyme content in old male pituitaries.

Relationship between the production capacity of ovarian 13,14-dihydro-prostaglandin F2-alpha and the process of ovulation in immature female rats pretreated with gonadotropin.

The purpose of this work was to investigate the effects of gonadotropin on the production capacity of ovarian 13,14-dihydro-prostaglandin F2-alpha (13,14H2-PGF2 alpha) and whether or not this capacity had any relation to the process of ovulation in rat. To induce the first ovulation, immature rats were injected subcutaneously with PMSG (5 IU/rat) at 8:00 at 26 days of age and some of these rats were followed by an intraperitoneal injection of hCG (10 IU/rat) at 57 hrs after PMSG treatment. The 13,14H2-PGF2 alpha production capacity was unchanged as compared with vehicle control until 57 hrs after PMSG treatment. However, the capacity showed a striking increase at 60 hrs after PMSG treatment. A maximal increase of about 7 fold was observed at 9 hrs after hCG injection just before ovulation. The production capacity of the Graafian follicle (GF) and the part (WO-GF) of the whole ovary (WO) from which the GF is removed at 2:00 on day 29 and the capacity of early corpus luteum at 8:00 on day 29 was greater than that of GF and WO-GF at 0:00 on day 29. These results suggest that the 13,14H2-PGF2 alpha production capacity in rat ovary is regulated by gonadotropin and is closely associated with the process of ovulation.

A role of prostaglandin in the secretion of inhibin and oestradiol-17β in equine chorionic gonadotrophin-primed rats

The role of prostaglandins in the secretion of inhibin and oestradiol-17 beta was examined in immature female rats treated with equine chorionic gonadotrophin. Administration of prostaglandin (PG) F2 alpha to animals whose serum and ovarian levels of inhibin and oestradiol-17 beta had been suppressed by indomethacin given 24 h after equine chorionic gonadotrophin prevented the inhibition or restored the levels to normal. Initiation of PGF2 alpha replacement immediately after treatment with indomethacin was more effective. The results are consistent with the hypothesis that PGs play a regulatory role in the synthesis and secretion of inhibin and oestradiol-17 beta by the rat ovary.