Effects of testosterone implants and hypothalamic lesions on luteinizing hormone regulation in the castrated male rat.

The effects of intrahypothalamic and subcutaneous implants of testosterone (T) and those of hypothalamic lesions on resting levels of circulating LH and pituitary responsiveness to exogenous LHRH were studied in castrated male rats to elucidate hypothalamic and pituitary regulation of LH secretion. Two hundred mug implants of testosterone propionate (TP) in the median eminence region suppressed plasma LH titers before evidence of direct inhibition of pituitary function (as indicated by testing with LHRH) was found. Such implants release appreciable amounts of T into the peripheral circulation in the immediate post-operative period, and SC Silastic (constant release) capsules containing T have similar effects. The findings suggest that, regardless of the site of implant, the initial negative feedback inhibition of LH by T is not dependent on direct action at the pituitary levels but rather appears to be a hypothalamic effect. In the days following exposure to hypothalamic or peripheral implantation of T, however, a progressively developing decline in the response to exogenous LHRH was observed. In order to determine whether this effect results from suppression of endogenous LHRH release, the median eminence-arcuate region was destroyed to remove the source of LHRH. In these animals, the suppression of plasma LH was evident on the first day after the lesion, but pituitary responsiveness to LHRH was unaffected until after one week. When Sialastic capsules were implanted SC into lesioned animals, a more rapid (less than 1 week) inhibition of pituitary responsivity ensued. Suprachiasmatic lesions did not affect basal LH secretion or pituitary responses to LHRH. The data provide evidence for a dual feedback action of T on LH in castrated male rats: an initial inhibitory effect presumably due to hypothalamic inhibition (commencing at around 6h after hypothalamic of SC implantation of T), and a subsequent suppression of pituitary responisveness (after one day) presumably due to direct action of T on the pituitary. In addition to these phenomena, findings in rats bearing median eminence-arcurate lesions suggest that the removal of endogenous LHRH by itself leads to an eventual decline in pituitary responsiveness (greater than one week postoperatively).     

Effect of gonadal steroids on the luteinizing hormone and follicle-stimulating hormone response to 8-bromo-adenosine 3',5'-monophosphate in anterior pituitary cells in culture.

-8-Bromo-cAMP stimulated the release of LH (approximately 20-fold) and FSH (approximately 2-fold) in control rat anterior pituitary cells in primary culture. 8-Bromo-cAMP-induced LH release was 2-3 times greater in cells preincubated for 48 h in the presence of 10(-8) M 17 beta-estradiol (E2). The LH response to 10(-10) M LHRH was similarly increased by E2 pretreatment whereas progesterone (P), which had no effect on the response to 8-bromo-cAMP, led to a reduction of the stimulatory effect of E2 on the LH response to the neurohormone. Preincubation with 10(-8) M testosterone (T) decreased the LH response to 8-bromo-cAMP by about 40%, whereas the response to LHRH was 80% inhibited. 8-Bromo-cAMP-induced FSH release was slightly increased in cells preincubated with E2 but was greatly augmented in cells treated with E2 + P. Basal FSH release was slightly increased (40%) after preincubation with P or T whereas the response to 8-bromo-cAMP was not significantly affected. The sensitivity of the FSH response to LHRH was increased by E2 whereas P and T led to a stimulation of the maximal FSH response to LHRH with no significant effect on the LHRH ED50 value. The present data indicate that P and E2 exert their effects on LH and FSH release at steps before and after cAMP formation, respectively. T appears to act at both steps on the release of the two gonadotropins.     

Effects of gonadal steroids and cycloheximide on the release of gonadotrophins by rat pituitary cells in culture.

The effects of oestradiol-17beta, testosterone and progesterone alone and together with cycloheximide on the basal and gonadotrophin releasing hormone (Gn-RH)-induced release of gonadotrophins were studied in cultured dispersed rat pituitary cells. In the control group (no steroid treatment), GnRH significantly stimulated the release of LH and FSH; cycloheximide partially inhibited this response, although it had no effect on the basal secretion of gonadotrophins. A dose of 5 ng oestradiol/ml had no significant effect on the response to GnRH; at a dose of 100 ng/ml the GnRH-induced release of LH was significantly augmented whereas the release of FSH was inhibited. Cycloheximide blocked the augmenting effect of oestradiol. The basal release of LH was slightly but significantly inhibited in response to 10 ng testosterone/ml and increased in response to progesterone (200 ng/ml). Testosterone at both dose levels and progesterone significantly inhibited the GnRH-induced release of LH and FSH and in testosterone and progesterone-treated groups, the response to GnRH was inhibited by cycloheximide, but not beyond the levels observed in the control group. It is concluded that steroids can act directly on the pituitary cells, that oestradiol stimulates the GnRH-induced release of LH and that cycloheximide blocks this stimulatory effect. Testosterone and progesterone, on the other hand, partially inhibit the response to GnRH.     

Effects of glucocorticoids on responsiveness of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone by male rat pituitary cells in vitro

To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.     

Effects of castration and gonadal steroids on serum luteinizing hormone and prolactin in old and young rats.

Changes in serum LH and prolactin concentrations in response to bilateral gonadectomy and gonadal steroid replacement were measured in mature young (4-6 months) and old (23-30 months) female and male Long-Evans rats. On day 13 after gonadectomy, female rats were injected with oestradiol benzoate (OB) and male rats with testosterone propionate (TP) for a period of 12 days. They were then permitted a recovery period of 6 weeks. Serum prolactin and LH concentrations were measured by radioimmunassay in single blood samples taken at various intervals before and after gonadectomy and during and after steroid treatment. Serum LH levels were about the same in intact young old female rats, but after ovariectomy LH rose several fold higher in young than in old remale rats. In male rats, after orchidetomy the increase in serum LH was greater in young than in old rats. Oestradiol benzoate and TP injections into female and male young and old rats produced variable effects on LH release. Serum prolactin concentrations were approximately six times higher in old intact than in young intact female rats, and after ovariectomy showed a much greater percentage reduction in old than in young female rats. Administration OB produced a greater absolute increase in serum prolactin in old than in young female rats. Serum prolactin values were about the same in old and young male rats, and the effects of castration and TP administration on serum prolactin were not markedly different in the two age groups. These results indicate that old female and male rats are less capable of releasing LH than young rats of both sexes, but old females release more prolactin than young females.     

Gonadal steroids modulate pulsatile luteinizing hormone secretion by perifused rat anterior pituitary cells

Recent studies have shown that LH secretion is pulsatile and that LH pulse characteristics are affected by the prevailing steroid environment in both male and female rats. In the present study, a cell perifusion system was used to examine the effects of testosterone (T) and 17 beta-estradiol (E) on LHRH-stimulated pulsatile LH secretion. T inhibited LH secretion, increasing the EC50 for LHRH, while E stimulated secretion, lowering the EC50. Steroid effects were independent of both LHRH pulse amplitude and frequency. E also affected the pattern of LH secretion by facilitating both LHRH self-priming and desensitization to LHRH. These results show that steroids can affect pulsatile LH secretion by actions exerted at the pituitary level and that steroids can induce both quantitative and qualitative changes in LH secretion in the presence of an invariant LHRH stimulus. These results help to elucidate the mechanisms underlying steroid feedback in vivo, since reduction in pituitary responsiveness to LHRH may play an important role in T feedback, while facilitation by E of both self-priming and desensitization may serve to increase the magnitude and shorten the duration of the proestrous LH surge.     

Effects of photoperiod on hypothalamic luteinizing hormone releasing hormone in the male hamster.

Male hamsters were maintained on long (14 h light : 10 h darkness; 14L : 10D) or short (6L : 18D) photoperiods. Animals on short-days had reduced levels of LH in the serum and anterior pituitary gland, decreased androgen in the circulation and regressed testes and accessory sex organs. These same hamsters had significantly raised concentrations of hypothalamic luteinizing hormone releasing hormone (LH-RH). There was no significant difference in the response to exogenous LH-RH between groups maintained on long- and short-days. Castration significantly reduced levels of LH-RH in the hypothalamus in the long-day animals but had little effect on this parameter in short-day animals which had already undergone testicular regression. The increased levels of LH-RH in the hyothalami of both intact and castrated hamsters on non-stimulatory photoperiods is interpreted as a decreased release of the neurohormone which subsequently results in a decreased release of LH.     

Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro

To determine if the divergent effects of glucocorticoids on the circulating levels of LH and FSH in female rats are exerted directly on the pituitary, adult female pituitary cells were treated either with no glucocorticoids or with 60 or 600 ng/ml cortisol or corticosterone during one or two 48-h incubations. During the second 48 h, some cells from each group were treated with GnRH (1.7 X 10(-12) - 4.6 X 10(-9) M). Concentrations of LH and FSH in media and cells were measured by RIA. Basal secretion of LH was inhibited 38-43% by different glucocorticoid treatment during the first 48 h and 21% by 600 ng/ml corticosterone during the second 48 h. In contrast, basal secretion of FSH was enhanced 22-64% during the first 48 h and 25-124% during the second 48 h. Secretion of LH in response to maximal stimulation with GnRH was unaffected by glucocorticoids, but maximal secretion of FSH was increased 68%. The responsiveness of the cells to GnRH, as determined from the slope of the GnRH dose-response curve for LH, was increased 43-50% by cortisol. The slope of the dose-response curve for FSH was unaffected, but the mean concentration of FSH as a function of the log dose of GnRH was increased 45-79%. Glucocorticoids had no effect on cell content of LH or total LH per dish, either under basal or maximal GnRH-stimulated conditions. Glucocorticoids increased basal cell content of FSH 41-82%, basal total FSH 35-93%, and maximal GnRH-stimulated total FSH 40-84%. These results suggest that the only negative effect of glucocorticoids on reproduction exerted at the level of the pituitary is a slight suppression of basal LH secretion, that glucocorticoids affect the pituitary directly by increasing FSH synthesis, and that the divergent effects of glucocorticoids on LH and FSH provide a novel model for differential regulation of the gonadotropins.     

In vitro action of hypothalamic luteinizing hormone releasing factor (IRF) on lamb anterior pituitary. I. Kinetics of excretion of luteinizing hormone (LH)

Partially and highly purified sheep LH-releasing factor (LRF) induced secretion of LH during incubations of anterior pituitary slices from ovariectomized ewes treated vaginally or by subcutaneous injection with fluorogestone acetate. The incubation medium contained Krebs-Ringer bicarbonate buffer with glucose, amino acids, mannose, galactose, fucose, glucosamine, galactosamine, sialic acid, calf serum, penicillin, and streptomycin. First, the optimum effect of a dose of 45 mg fluorogestone daily for 12 days before slaughter was demonstrated. These tissues yielded about 2-6 mcg LH per mg tissue in 2 hours. A dose curve of LRF showed 4.7 mcg per mg tissue was optimal, and this dose was adjusted for the purity of the LRF used in subsequent experiments. Added LH, 15.5 mcg highly purified, did not lessen LH production. LH production was fairly constant for 4-5 hours, if the medium were not changed, but increased for up to 7 hours if the medium were renewed. After incubation with labeled amino acids or proline, LH was purified 26-fold over other proteins by ammonium sulfate fractionation DEAE-cellulose chromatography, and Sephadex gel filtration. This LH was biologically active by radioimmunoassay and by the ovary ascorbic acid depletion assay, and of high specific activity, 2.2 Ci per mole LH.     

Plasma and pituitary luteinizing hormone in intact and castrated tree sparrows (Spizella arborea) during a photoinduced gonadal cycle

Plasma and pituitary levels of immunoreactive luteinizing hormone (LH) were measured in female, male, and castrated male tree sparrows during a photoinduced gonadal cycle. Our aims were to compare patterns of LH secretion, particularly during spontaneous gonadal regression, and to determine the testes' influence on the secretory pattern of males.Seven days after females were transferred from 8- to 20-hr daily photoperiods, the concentration of plasma LH had increased 11-fold. Two weeks later, it had declined to a level only 3-fold greater than that in short-day controls, where it remained until day 56, when ovarian growth culminated. The onset of ovarian regression between days 56 and 77 was accompanied by a reduction in plasma LH to the prestimulation level (ca. 0.5 ng/ml) and by a similar reduction in pituitary LH content, which, by contrast, had increased steadily through day 28. The plasma cycle of males was quantitatively similar, but it reflected the transition from photosensitivity to photorefractoriness less incisively. Pituitary cycles of males and females were qualitatively similar. The concentration of plasma LH in castrates, compared with that in intact males, was elevated through day 56. A 4-fold increase in pituitary LH preceded a reduction in the circulating level that paralleled the onset of testicular and ovarian regression.Qualitative similarity among plasma cycles plus the eventual reduction of plasma LH in castrates to the low level characteristic of chronically photostimulated birds of both sexes argues that the LH-release mechanism becomes refractory in castrates as in males and females. We, therefore, conclude that testicular steroids are not obligatory determinants of the temporal pattern of circulating LH in photostimulated males, yet they apparently inhibit the photoresponsive hypothalamohypophysial axis. An elevated plasma level in short-day castrates suggests that a functional relationship between the hypothalamohypophysial axis and the testes exists also in nonphotostimulated males.