Preparation of an enriched fraction of luteinizing hormone-secreting cells from a mixed population of rat anterior pituitary cells.

To determine whether female rat gonadotropes differentially secrete only LH, only FSH, or both LH and FSH, a new method of preparing an enriched fraction of LH-secreting cells from a mixed population of cells was developed. Dispersed female rat pituitary cells were incubated for 4 h in glass tubes with magnetic beads coated with anti-LH antibody. The tubes were then placed in a magnetic particle concentrator to separate cells bound to antibody-coated beads (518b7-bound) from the rest of the cell population (518b7-unbound). Several controls were used in this experiment: 1) mixed cells immediately plated without any preincubation (control); 2) mixed cells incubated without magnetic beads (incubated-only); 3) mixed cells incubated with magnetic beads but not separated (nonseparated); and 4) mixed cells first incubated with magnetic beads coated with normal mouse serum (NMS) and then separated into NMS-bound and NMS-unbound populations. After removal of magnetic beads from cell populations, the cells were plated at 200,000 cells/well. The 518b7-bound cells secreted 1.5-, 2-, 2-, and 5-fold more LH than control, incubated-only, nonseparated, or 518b7-unbound cells, respectively (p < 0.05); 518b7-bound cells secreted 2.5-, 3.8-, 3.8-, and 11-fold more FSH than control, incubated-only, nonseparated, and 518b7-unbound cells. The ratio of LH:FSH secreted did not vary between the different treatment groups, but the ratio of LH:FSH contained in 518b7-selected cells was greater than that for unbound cells. Our data indicate that we have prepared an enriched fraction of LH-secreting pituitary cells that also secrete FSH but have a greater capacity for producing LH than FSH in culture. These results are supportive of the view that LH and FSH are secreted by the same cells.

Cortisol regulates secretion and pituitary content of the two gonadotropins differentially in female rats: effects of gonadotropin-releasing hormone antagonist.

To determine if LH and FSH respond to cortisol exposure the same way in females as they do in males, metestrous females were implanted with cholesterol or cortisol (F) subcutaneously, and either ovariectomized or left intact 4 days later. Tail vein injections of 1000 ng of GnRH in saline, or saline alone, were given 4.5, 23.5, or 47.5 h after the time of ovariectomy. Animals were killed 30 min after the injections at 5, 24, and 48 h after surgery. F attenuated the postovariectomy increase in serum LH at 48 h. F also suppressed GnRH-stimulated LH release 24 and 48 h after surgery in ovariectomized animals and in intact animals at 48 h. Pituitary content of LH was increased moderately by F at 5 h. These effects of F are similar to those seen in males. In contrast to LH, F increased serum FSH in intact females and suppressed levels in ovariectomized animals at 24 and 48 h, while inducing a remarkable increase in pituitary FSH content at all three times. These divergent effects of F on serum FSH (suppression in gonadectomized and stimulation in intact groups) were not seen in males, and the increase in pituitary FSH as a result of exposure to F was much more profound and reliable in females than in males. To determine if the F-induced increase in pituitary FSH was dependent on endogenous secretion of GnRH, intact metestrous females were implanted with either cholesterol or F pellets. Each implant group received sc injections of 100 micrograms GnRH antagonist or control injections every 48 h beginning at the time of steroid implantation. Animals were killed 5 days after implantation. The antagonist suppressed both serum and pituitary LH. F also suppressed serum LH levels, but had no effect on pituitary content of LH. Neither the antagonist nor F affected serum FSH. F greatly increased pituitary content of FSH in the presence or absence of GnRH antagonist. These data suggest that 1) LH responds to F treatment in a similar way in females and males; 2) pituitary FSH content is more sensitive to the enhancing effect of F in females than in males; 3) the ability of F to increase pituitary FSH in females is not dependent on GnRH.

Effect of treatment with cortisol in vivo on secretion of gonadotropins in vitro.

To investigate the site of action of glucocorticoids in modulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitaries of male rats, we implanted intact male rats with 250-mg pellets of cortisol (F) or cholesterol (C). Four days later, we collected and enzymatically dispersed the pituitaries. After the dispersed pituitaries had been in culture for 2 days, we treated the cells with gonadotropin-releasing hormone (GnRH) (0-150 nM) and determined the concentrations of LH and FSH in the medium after 6 h of incubation. Cells from donor animals pretreated with F secreted 30-60% more LH approximately 75% more FSH than cells from donor animals pretreated with C. This increase occurred regardless of the presence of F or C in the incubation medium in vitro. The slopes and ED50s of the GnRH dose-response curves were not altered. These data show that glucocorticoids have stimulatory effects on both LH and FSH. The inhibitory effects observed in vivo must be exerted by some mechanism that is not carried over to the in vitro model, and perhaps involve sites of action in addition to the pituitary.

Dual role of glucocorticoids in regulation of pituitary content and secretion of gonadotropins.

To verify the inhibitory effect of cortisol (F) on secretion of luteinizing hormone (LH) 24 h postorchidectomy, we implanted cholesterol (C) or F subcutaneously into male rats, and 4 days later orchidectomized or sham orchidectomized them under ether anesthesia. We injected gonadotropin-releasing hormone (GnRH) or saline into these rats 24 h postorchidectomy, collected blood 30 min later, and measured LH and follicle-stimulating hormone (FSH) in serum and pituitaries. F inhibited GnRH-induced secretion of LH without affecting secretion of FSH. We then implanted C, corticosterone (B), or F into rats, performed the same surgeries, and collected pituitaries 24 h after surgery for quantitation of receptors for GnRH. Neither F nor B affected the number of receptors for GnRH or their affinity for a GnRH analogue. Suppression of LH in serum occurred without decreased pituitary content of LH. In contrast, F increased pituitary content of FSH. Implantation of progesterone in a similar experiment did not affect circulating concentrations or pituitary contents of FSH or LH. These data suggest that glucocorticoids may inhibit responsiveness to GnRH by some mechanism distal to the receptor for GnRH that affects only LH.

Differential effects of follicle-stimulating and luteinizing hormones on testosterone production by mouse testes.

In adult mice, direct intratesticular injection of ovine follicle-stimulating hormone (o-FSH-13; AFP 2846-C, from NIAMDD, less than 1% LH contamination) at 10, 100 or 1000 ng significantly elevated concentrations of testosterone (T) within the testis. These effects were rapid, with peak values attained by 15 min, and transient, with return to values comparable to that in the contralateral, saline-injected testis within 90 min. Intratesticular injection of FSH (1 microgram) significantly increased testicular T levels in 15- and 60-day old mice. This contrasted with the effects of intratesticular administration of human chorionic gonadotropin (hCG), which stimulated T production significantly at 30 days of age through adulthood. In adult mice, the equivalent LH to the possible contamination in the FSH preparation (1 ng) had no effect. Intratesticular injection of 10 ng LH produced comparable stimulation to that by 100 ng FSH (approximately 7-fold). Systemic pre-treatment with a charcoal-treated porcine follicular fluid (PFF) extract for 2 days reduced plasma FSH levels [86 +/- 17 (5) vs 700 +/- 8 (6); P less than 0.05], but had no effect on plasma LH. Twenty-four hours after the last treatment, the response to intratesticular injection of hCG (2.5 mIU), FSH (100 ng) or LH (10 ng) was also significantly attenuated in these mice. Intratesticular injection of PFF had no direct effect on testicular T levels. In vitro T production in the presence of hCG, LH or FSH were differentially affected by the concentrations of calcium (Ca2+) or magnesium (Mg2+) in the incubation media. The stimulatory effects of FSH were apparent at significantly lower levels of Ca2+ or Mg2+, than were those of LH or hCG. The results of these studies indicate that FSH is capable of stimulating testicular T production. Furthermore, the responsiveness to FSH is qualitatively different than that to LH/hCG in terms of the age pattern, as well as the dependence on Ca2+ or Mg2+. In addition, plasma FSH levels appear to influence testicular responsiveness to direct exogenous administration of gonadotropins. These studies indicate that FSH stimulation of T production can be differentiated from those of LH, and that these effects of FSH can be observed under physiological conditions.

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.

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.

Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: effects of inhibition of protein synthesis.

Studies were undertaken to investigate the possibility that receptors for LH in monolayer cultures of enzymatically dissociated ovine luteal cells are recycled. Cultured cells (3-10 X 10(5) total steroidogenic cells/dish) were incubated with or without cycloheximide (CHX; 10(-4) M) and 2 X 10(6) cpm [125I]iodo-hCG in the presence or absence of 30 micrograms nonradioactively labeled hCG for 0, 12, 24, 36, or 48 h. At each time point, the amounts of radioactivity bound to the cells (bound [125I]iodo-hCG), located intracellularly (intracellular [125I]iodo-hCG), and degraded and returned to the medium as [125I]monoiodotyrosine (degraded [125I]iodo-hCG) were determined. The number of receptors for LH was determined by Scatchard analysis. Cell viability was also monitored by: 1) trypan blue dye exclusion, 2) the ability of the cells to synthesize protein, and 3) basal and hCG-stimulated secretion of progesterone. More than 90% of the cells remained viable after 48 h of culture, and CHX had no effect on cell viability. Protein synthesis in CHX-treated cells was inhibited by more than 90%. Basal and hCG-stimulated secretion of progesterone were also inhibited by CHX. Treatment with CHX increased the amounts of membrane-bound and internalized [125I]iodo-hCG and decreased the amounts of [125I]iodo-hCG that were degraded. When the quantities of radioactivity in these three fractions (plasma membrane-bound, internalized, and degraded) were added together to obtain a value for the total amount of [125I]iodo-hCG that had been bound to receptor during the 48-h time course (total receptor-associated [125I]iodo-hCG), the value for control cells was not significantly different from the value for CHX-treated cells. Furthermore, the total receptor-associated [125I]iodo-hCG was approximately 2-fold greater than the amount of [125I]iodo-hCG required to saturate receptors at time zero. These data indicate that synthesis of new receptors is not required for the continued binding, internalization, and degradation of [125I]iodo-hCG. Further, the data are compatible with the hypothesis that receptors for LH are recycled or that a portion of the total receptor population is in an unavailable form when the cells are intact but are available for binding after homogenization of the cells.