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Abstract.

In order to clarify the mechanism of hyperprolactinaemic anovulation, the medial basal hypothalamic (MBH) catecholamine (CA) turnover and LRH concentration, and the serum levels and pituitary contents of gonadotrophins and prolactin (Prl) in hyperprolactinaemic female rats were examined. Hyperprolactinaemia (HPrl) was produced by oral administration of sulpiride for 10 consecutive days; each measurement made on the sulpiride-treated rats was compared with that of control dioestrus rats. Prl, LH, FSH and LRH were determined by radioimmunoassay; CA turnover, as assessed by the accumulation of CA following monoamine oxidase inhibition, was assayed by high performance liquid chromatography with electrochemical detection. Sulpiride treatment induced (1) an increase in the serum Prl and a decrease in the serum LH, (2) an increase in the pituitary FSH and LH contents, (3) an increase in the MBH LRH concentration, and (4) an increase in the MBH dopamine (DA) turnover. These results suggest that HPrl may induce anovulation by impaired LH secretion which was caused by the suppression of LRH release due to an increase in DA turnover in the MBH.

Abstract. The effect of prolactin (Prl) on oestrogeninduced gonadotrophin secretion was examined in vitro in a sequential double chamber perifusion system. As control groups, mediobasal hypothalamus (MBH)-pituitary pairs or pituitaries without the MBHs were perifused with Medium 199. As an experimental group, MBH-pituitary pairs were perifused with Medium 199 containing 1 μg/ml of rat Prl. These groups were stimulated with 10⁻⁷ m oestradiol-17β (E₂) for 30 min, and luteinizing hormone (LH) in the serial fractions of effluent was measured.

In the control group of MBH-pituitary pairs perifused with medium without Prl, secretion of LH began to rise within 30 min after the beginning of stimulation, reached a peak 30 min after the end of stimulation and then remained at a plateau for the rest of the experimental period, whereas in the control group of pituitaries alone no significant response was observed. In the experimental group perifused with medium containing Prl, LH-secretion showed peaks 20 and 80 min after the end of E₂-stimulation, respectively, and the first peak was significantly (P < 0.01) less than the level in the control group.

These data demonstrate that Prl at this concentration suppressed the rapid LH release induced by E₂. Its site of action is suggested to be at the hypothalamic level, and its possible mechanism of action is discussed.

Abstract. The effect of hydrocortisone on the release of luteinizing hormone (LH) and LH-releasing hormone (LRH) in response to clomiphene citrate (clomiphene) were examined in a sequential double chamber perifusion system by perifusing the mediobasal hypothalami (MBH) and/or pituitaries excised from normal female rats in dioestrus. When the MBH and the pituitary were perifused in sequence with medium containing 5 × 10⁻⁶ m hydrocortisone, a significant release in LH (100– 150% increase, P < 0.01–P < 0.05) was observed 40 min after the administration of 3 × 10⁻⁸ mol clomiphene. Clomiphene had no effect on LH release from the pituitary when perifused in series with the MBH without basal hydrocortisone infusion. Administration of clomiphene did not cause a significant increase in LH from the pituitary perifused alone, with or without medium containing hydrocortisone. The concentration of LRH in the efflux was significantly increased 40 min after clomiphene administration when MBH was perifused with medium containing hydrocortisone, whereas clomiphene had no effect when perifused with medium only. These data indicate that hydrocortisone stimulates the effect of clomiphene on LRH release from the hypothalamus, which in turn induces LH release from the pituitary.

Abstract. The effect of prostaglandin D₂ on the release of luteinizing hormone was studied in a superfusion system by superfusing human pituitary gland. Perfusion with 30 μg of prostaglandin D₂ induced a significant increase of luteinizing hormone secretion. This is the first evidence of a direct effect of prostaglandin D₂ on the secretion of luteinizing hormone from the human pituitary gland. This finding suggests the possible role of prostaglandin D₂ in human reproductive function

Abstract. The effects of vasoactive intestinal peptide (VIP) on the releases of LH and GnRH were examined in a sequential double chamber perifusion system by perifusing the medio-basal hypothalamus and/or pituitary excised from normal female rats in diestrus or ovariectomized rats. When the medio-basal hypothalamus and pituitary from normal rats in series were perifused with VIP (10⁻⁶ mol/l), the concentration of LH in the efflux was increased by 59–181% above that before the injection (P < 0.05), VIP having a dosedependent effect. VIP had no effect on LH release from the pituitary perifused alone. Infusion of VIP at 10⁻⁶ mol/l induced a significant release (84–159% increase, P < 0.05) of GnRH from the medio-basal hypothalamus. Infusion of 10⁻⁶ mol/l VIP induced a significant release (41–99% increase, P < 0.05) of LH in ovariectomized rats. These findings suggest that VIP induces GnRH release from the medio-basal hypothalamus, resulting in LH release from the pituitary, and that this process does not require ovarian estrogen.

Abstract. The effects of epidermal growth factor (EGF) on pituitary luteinizing hormone (LH) release and on the releases induced by oestradiol (E₂) and LH-releasing hormone (LRH) were examined in a sequential double chamber perifusion system. In this system the mediobasal hypothalami (MBH) and/or pituitaries excised from normally cycling female rats in dioestrus were perifused with test media.

Perifusion with EGF at 1 ng/ml for 30 min induced significant release (80–100% increase, P <0.05) of LH from hypothalamo-pituitary pairs, but not from the pituitary alone. Perifusion of the pituitary alone with medium containing 1 ng/ml EGF, resulted in significant release of LH (70–140% increase, P < 0.05) after adminnistration of 10⁻⁷ m E₂, but did not significantly influence LH release in response to 20 ng/ml LRH.

These findings suggest that EGF may be involved in the regulation of pituitary gonadotrophin secretion by a direct effect on the hypothalamus and indirectly by increasing the pituitary responsiveness to E₂.

Abstract. For determination of the site of action of oestrogen (E) during the negative and positive feedback phases of gonadotrophin secretions, studies were made on the pituitary response to a small amount of LRH and the pulsatility of gonadotrophins after E administration in normal cycling women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 μg of synthetic LRH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated E (Premarin). In the next cycle, the pituitary responses to a same dose of LRH were also observed 2 h before and 56 h after E injection. The mean levels of serum LH and FSH and the peak responses to LRH were significantly (P < 0.05) decreased 8 h after E injection, but were significantly (P < 0.05) increased 56 h after E administration. In the third cycle, the pulsatility of gonadotrophins was evaluated by measuring serum LH and FSH every 15 min for 180 min before and 8 h and 56 h after E injection. The pulse frequencies of gonadotrophins were not significantly different before and 8 h and 56 h after E injection. The amplitudes of pulses 56 h after Premarin injection were significantly higher than those before the injection. These findings suggest that the negative and positive feedback effects of E on gonadotrophin secretion may be caused, in part, by its direct action on the pituitary response to LRH.

Abstract. The direct action of prolactin (Prl) on pituitary LH secretion was studied by examining its effect on oestradiol (E₂)-induced luteinizing hormone (LH) release from the rat pituitary in a perifusion system and determining the oestrogen receptor (ER) content of the pituitary in hyperprolactinaemic female rats. When the pituitary from rats in pro-oestrus was perifused with medium alone, 10⁻⁷ m E₂ significantly (P < 0.05) increased the LH concentration in the effluent by 60–130% of the basal level, but in medium containing 1 μg/ml Prl it did not cause LH release. In hyperprolactinaemic rats produced by implanting 2 anterior pituitary glands under the kidney capsule, the ER content of the cytosol of the pituitary (53.2 ± 2.9 fmol/pituitary) was significantly increased (P < 0.05), but that of the nuclei (6.8 ± 0.2 fmol/pituitary) was significantly decreased (P < 0.05) compared with the contents in rats in pro-oestrus.

These data suggest that Prl has a direct suppressive effect on LH secretion from the pituitary in the rat, and that decreased translocation of ER into the nucleus might relate to impaired LH release by E₂ from the pituitary of hyperprolactinaemic rats.

Abstract. In studies into the mechanism of anovulation in the diabetic condition, the LRH receptor content of the pituitary gland of rats with diabetes mellitus was determined. Normal female rats weighing 180–200 g were injected iv with either streptozotocin (6 mg/100 g body weight) or vehicle in dioestrus of the oestrous cycle. The rats were sacrificed by decapitation 9 days after treatment, and serum LH concentrations and the LRH content of the medial basal hypothalamus (MBH) were measured by radioimmunoassay (RIA), and the LRH receptor content of the pituitary gland was determined.

The serum concentration of LH and the LRH content of the MBH in diabetic rats were 34.4 ± 4.8 ng/ml (mean ± sem) and 2.05 ± 0.04 ng/MBH, respectively, which were similar to the respective values of normal rats in dioestrus. However, the LRH receptor content of diabetic rats (13.2 ± 3.9 fmol/pituitary) was significantly (P < 0.05) lower than that of normal rats in dioestrus (68.0 ± 7.1 fmol/pituitary) and pro-oestrus (59.4 ± 13.6 fmol/pituitary).

These results suggest that anovulation in diabetic rats is at least partly attributable to a low content of LRH receptors in the pituitary gland.

Abstract. The effects of substance P on the release of LH and GnRH were examined in a sequential double-chamber perifusion system by perifusing the medio-basal hypothalamus and/or pituitary excised from normal female rats in dioestrus or ovariectomized rats. When the medio-basal hypothalamus and pituitary from normal rats were perifused in series with substance P (10⁻⁶ mol/l), the concentration of LH in the efflux was significantly (P < 0.05) increased by 70– 120% compared with that before the injection, but substance P had no effect on LH release from the pituitary perifused alone. This LH release by substance P increased in a dose-dependent manner and was blocked by substance P antagonist. Administration of 10⁻⁶ mol/l substance P induced a significant release (40–80% increase, P < 0.05) of GnRH from the medio-basal hypothalamus. Infusion of 10⁻⁶ mol/l substance P induced significant release (50–100% increase, P < 0.05) of LH and GnRH in ovariectomized rats with an implanted oestradiol capsule, but caused no significant increase in LH release in ovariectomized rats without an oestradiol capsule. Progesterone injection to both ovariectomized rats and ovariectomized rats with an implanted oestradiol capsule had no significant effect on the response of LH to substance P. These findings suggest that substance P induces GnRH release from the medio-basal hypothalamus, resulting in LH release from the pituitary, and that oestrogen may be involved in these processes.