Pituitary LH Stores Research Articles

27 Neurokinin B, Kisspeptin, and Gnrh Hormone Analogs Fail to Stimulate Secretion of Luteinizing Hormone (LH) in KISS1 Knockout Boars

Abstract We generated kisspeptin knockout (KISS1-/- ) pigs exhibiting hypogonadotropic hypogonadism and pubertal failure. Humans and rodents with inactivating KISS1 mutations exhibit increased gonadotropin secretion in response to GnRH and kisspeptin analogs, but it is unknown if KISS1-/- pigs respond similarly. Our goal was to define endocrine responses of KISS1-/- boars to hormone agonists that target different levels of the hypothalamic-pituitary-gonadal axis. Boars (302.6 ± 0.9 d of age) were fitted with indwelling jugular catheters to collect serial blood samples. Serum concentrations of LH and testosterone were quantified with radioimmunoassay. Data were analyzed as repeated measures with genotype (KISS1+/+ ; KISS1+/- ; KISS1-/- ) and time as fixed effects. Blood samples were collected beginning 60 to 120 min before a bolus injection (i.v.) of the agonist and continued for 5 to 48 h after treatment, depending on experiment, with at least 1 wk between studies. Boars received an agonist of either: 1) neurokinin B (senktide; 10 mg/kg BW; Exp. 1); 2) kisspeptin (C6; 15 nMol/kg BW; Exp. 2); 3) GnRH (cystorelin, 150 ng/kg BW; Exp. 3); or 4) LH (hCG; 20 IU/kg BW; Exp. 4). There was a genotype x time interaction for all experiments (P < 0.09). In the 60 min following senktide treatment (Exp. 1), mean LH increased 2.5-fold in KISS1+/+ and KISS1+/- boars, but was unchanged in KISS1-/- boars (P < 0.04). In Exp. 2, C6 increased LH concentrations in KISS1+/+ and KISS1+/- boars within 10 min (P < 0.05) and LH did not return to baseline until 28 h after treatment. However, secretion of LH in KISS1-/- boars was unaffected by C6. Next, cystorelin treatment (Exp. 3) increased circulating LH (P < 0.05) in KISS1+/+ and KISS1+/- males (2.1 ± 0.2 ng/mL), but not in KISS1-/- boars (0.5 ± 0.2 ng/mL). In Exp. 4, hCG increased mean testosterone concentrations for 48 h in KISS1+/+ (32.9 ± 6.7 ng/mL) and KISS1+/- (26.7 ± 4.7 ng/mL) boars, but testosterone in KISS1-/- animals remained at near undetectable levels after hCG administration. Therefore, we have shown for the first time that both neurokinin B and kisspeptin agonists (senktide and C6, respectively) stimulate robust secretion of LH in pigs. In addition, these agonists stimulate similar secretory patterns of LH in KISS1+/+ and KISS1+/- boars, confirming that a single functional copy of KISS1 is sufficient to confer normal reproductive endocrine function in pigs. Failure of a GnRH agonist to stimulate LH secretion in KISS1-/- boars likely reflects insufficient pituitary stores of LH, resulting from a lack of GnRH priming, and underlies the inability of either senktide or C6 to stimulate LH secretion in these animals. Lack of hCG-stimulated testosterone secretion in KISS1-/- boars may indicate a gonadotropin-independent deficit in testicular Leydig cells.

Effect of copper status, supplementation, and source on pituitary responsiveness to exogenous gonadotropin-releasing hormone in ovariectomized beef cows1,2,3

The effect of Cu status, supplementation, and source on pituitary responsiveness to exogenous GnRH was evaluated using nine multiparous, nonpregnant, nonsuckling, ovariectomized Angus cows (7.1 +/- 3.3 yr; 622.9 +/- 49.8 kg; BCS = 6.0 +/- 0.5). Cows were considered Cu-deficient based on liver Cu concentrations (< 30 mg of Cu/kg of DM) after receiving a low-Cu, forage-based diet supplemented (DM basis) with 5 mg of Mo/kg and 0.3% S for 216 d. Copper-deficient cows were stratified based on age, BW, BCS, and liver Cu concentration and assigned randomly to repletion-phase treatments. Treatments included 1) control (no supplemental Cu); 2) organic (ORG; 100% organic Cu); and 3) inorganic (ING; 100% inorganic CuSO4). Treatments were formulated to meet all NRC recommendations, except for Cu, which was supplemented to ORG and ING cows at 10 mg of Cu/kg of dietary DM. During the 159-d repletion phase, Cu status was monitored via liver biopsy samples, and all cows received exogenous progesterone. A controlled intravaginal drug-release device (replaced every 14 d) was used to maintain luteal phase progesterone as a means to provide negative feedback on the hypothalamic-pituitary axis. During the repletion phase, liver Cu concentrations did not differ between ORG and ING cows at any time. By d 77 of the repletion phase, all supplemented cows were considered adequate in Cu, and liver Cu concentrations were greater in supplemented than in nonsupplemented control cows on d 77 (P < 0.05) and throughout (P < 0.01) the repletion phase. Beginning on d 99, exogenous GnRH was administered to all cows at low (0, 3, and 9 microg; Exp. 1) and high doses (0, 27, and 81 microg; Exp. 2) at six different times. Cows were catheterized every fifth day, and blood samples were collected every 15 min for 1 h before and 4 h after GnRH administration and analyzed for LH concentration. In Exp. 1, Cu status and supplementation did not affect basal or peak LH concentrations, but total LH released tended (P < 0.07) to be greater in Cu-supplemented vs. control cows when 3 microg of GnRH was administered. In Exp. 2, there was no effect of Cu supplementation or source on basal, peak, or total LH released, regardless of GnRH dose. Pituitary LH concentrations did not differ across treatments. In conclusion, Cu status, supplementation, and source did not affect GnRH-induced LH secretion or pituitary LH stores in ovariectomized, progesterone-supplemented cows in this experiment.

Gonadotrope responsiveness in orchidectomized sheep: effect of duration of a simulated follicular phase.

The effect of duration of a simulated follicular phase on gonadotrope responsiveness was assessed in orchidectomized sheep (wethers). The oestrogenic and hypothalamic inputs characteristic of the ovine follicular phase were simulated by continuous infusion of oestradiol (5 micrograms h-1 in 10% (v/v) ethanol saline) and circhoral delivery of GnRH (200 ng per hourly pulse) for 0, 6, 12, 24, 48 or 96 h (n = 6 wethers per group). Responsiveness increased (P < 0.05) with increasing duration of simulated follicular phase. In a second experiment, responsiveness was assessed 96 h after initiation of infusion of oestradiol in wethers receiving hourly pulses of GnRH or saline. Concurrent administration of GnRH reduced (P < 0.05) the magnitude of the oestradiol-induced increase in gonadotrope responsiveness. In a companion study, anterior pituitary tissue was collected 96 h after the start of infusion of oestradiol and circhoral delivery of GnRH or saline. Pituitary stores of LH and tissue concentrations of GnRH receptor and mRNA encoding the GnRH receptor were increased (P < 0.05) by oestradiol infusion. The magnitude of these oestradiol-induced responses was not affected (P > 0.05) by concurrent GnRH treatment. Tissue concentrations of FSH and mRNA encoding the FSH beta subunit were decreased (P < 0.05) by oestradiol infusion. This suppressive effect of oestradiol was not reversed by GnRH. These results indicate that oestradiol stimulation, but not concurrent delivery of GnRH, is essential for full expression of surge-like secretion of LH. In addition, the oestradiol-induced increase in gonadotrope responsiveness during the simulated follicular phase is sustained throughout the period of oestradiol delivery.

Pituitary function is altered during the same cycle in women with polycystic ovary syndrome treated with continuous or cyclic oral contraceptives or a gonadotropin-releasing hormone agonist

To determine if continuous oral contraceptive (OC) therapy was superior to a cyclic regimen in achieving persistent pituitary suppression of LH in patients with polycystic ovary syndrome (PCOS). Fourteen women (ages 16 to 41 years) with PCOS received one of three treatment groups: continuous OC therapy (30 micrograms ethinyl E2 plus 150 micrograms desogestrel), cyclic OC therapy, or monthly injections of a GnRH agonist (GnRH-a, leuprolide acetate depot 3.75 mg) for 3 months. Basal hormone levels were obtained before initiating therapy, on days 15 to 17 of the 3rd month of treatment (study 1) and again on days 26 to 28 of the 3rd month (study 2). A GnRH stimulation test was also performed during study 1 and study 2. After 3 months of treatment, LH levels were decreased significantly in all groups with less effective suppression observed in the cyclic OC group compared with the continuous OC or GnRH-a groups. A significant rise in LH was found only in the cyclic OC group after 5 to 7 days of placebo treatment (study 1 versus study 2). An increase in T was also observed in the cyclic OC group during study 2, whereas the continuous OC and GnRH-a groups showed continued inhibition of T levels. Although there was no significant difference in LH area under the curve (AUC) measurements after GnRH stimulation in study 1 versus study 2, the LH AUC was significantly greater in both studies in the cyclic OC group compared with the continuous OC or GnRH-a groups. Increased LH secretion during the week of placebo in the cyclic OC group was associated with a concomitant increase in T. The striking rise in LH secretion after GnRH stimulation in the cyclic OC group may represent increased pituitary sensitivity in patients receiving cyclic OCs regardless of the phase of the treatment cycle, perhaps secondary to increased pituitary stores of LH in these women.

Continuous gonadotropin-releasing hormone infusion stimulates dramatic gonadal development in hypogonadal female mice.

Adult hypogonadal (hpg) mice, lacking GnRH, have infantile reproductive systems and levels of pituitary gonadotropins that are lower than normal. The mutant mice respond to brain grafts containing GnRH neurons with gonadal development and increased production of gonadotropins. In view of the substantial literature regarding the nature and necessity of pulsatile GnRH stimulation of gonadotropins, we were not surprised in earlier studies to find that the majority of hpg mice with successful grafts have pulsatile LH secretion. It is not known, however, why LH pulsatility was undetectable in some animals with significant gonadal development. The present experiment was intended to determine the degree to which hpg mice respond to continuous infusion of GnRH via osmotic minipumps. Unexpectedly, female hpg mice exhibited dramatic ovarian and uterine growth after 15 or 30 days of continuous exposure to GnRH, with five- and eightfold increases in ovarian and uterine weights, respectively. Despite evidence of increased gonadotropin secretion in the treated hpg mice, pituitary stores of FSH and LH remained low. Similar treatment of normal female mice for 15 days also depleted pituitary concentrations of LH and FSH without significantly altering gonadal weights or plasma gonadotropin levels. It is clear from the present that inferences of pulsatile GnRH secretion based on stimulation of gonadal development in hpg mice should be made with caution.

Use of gonadotropin-releasing hormone, estrogen, or a combination to increase releasable pituitary luteinizing hormone in early transitional mares.

A lack of pituitary LH stores has been implicated as the cause of seasonal anestrus and failure to ovulate during the spring transition period in mares. In this experiment, 40 mares were used to study the effects of GnRH, estrogen, and an estrogen-GnRH combination on increasing releasable pituitary LH. Mares were stratified based on their ability to secrete LH in response to a 950-micrograms challenge of GnRH (n = 10 per group) and then assigned to one of four treatment groups: 1) controls, given no treatment; 2) 1 mg of estradiol-17 beta in oil i.m. daily for 8 d; 3) 200 micrograms of GnRH analogue des-Gly10, [D-ala6]-LHRH ethylamide in saline i.m. twice daily for 8 d; or 4) estradiol for 4 d then estradiol plus GnRH for four subsequent days. Blood was collected on d 1, 3, 5, and 7 of treatment, and serum was assayed for LH. On d 10 after initiation of treatment, mares were again challenged with GnRH (950 micrograms), and blood was collected for 4 h. Concentrations of serum LH did not vary significantly in control, estradiol-treated, or estradiol plus GnRH-treated mares among treatment days. In contrast, administration of GnRH alone increased (P < .05) concentrations of LH on d 5 and 7. Response to GnRH challenge, as measured by area under the LH curve (AUC) and peak LH, was greater (P < .05) for mares administered GnRH (7,307.1, 67.6 ng/mL, respectively) and GnRH plus estradiol (5,691.4, 60.3 ng/mL) than for mares given estradiol alone (1,519.4, 22.1 ng/mL) or no treatment (1,213.8, 19.4 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrope Responsiveness in Orchidectomized Sheep: III. Effect of Estradiol Withdrawal after Continuous Infusion1

The magnitude of GnRH-induced (1600 ng/hourly pulse for 24 h) preovulatory surge-like secretion of LH was assessed in orchidectomized sheep (wethers) during infusion of estradiol (E2, 5 micrograms/h in 10% ethanol-saline [vehicle]) or at 0, 12, 24, or 48 h after E2 withdrawal (n = 6 wethers/group). In one group, infusion of E2 was continued for 48 h, with concurrent circhoral delivery of GnRH during the final 24 h of the E2 delivery period. In other treatment groups, infusion of E2 or vehicle was continued for 24 h. Circhoral delivery of GnRH was initiated at the conclusion of E2 delivery or 12, 24, or 48 h thereafter, or at 48 h after cessation of vehicle infusion. Total GnRH-induced LH secretion in wethers receiving concurrent E2 was significantly augmented relative to the GnRH-induced secretory response in wethers receiving vehicle alone. The magnitude of GnRH-induced LH release was significantly reduced in animals in which E2 delivery was halted at the beginning of the GnRH challenge period. Further reductions in gonadotrope responsiveness were noted 12, 24, and 48 h after cessation of E2 delivery. Responsiveness 48 h after halting the E2 infusion did not differ (p > 0.05) from the responsiveness of wethers that had not been treated with E2. In a companion study, anterior pituitary tissue was collected at the end of E2 infusion (5 micrograms/h for 24 h) or at 12, 24, or 48 h thereafter (n = 6 wethers/group). Anterior pituitary tissue of control animals (n = 6) was collected after halting the infusion of vehicle alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin stimulates LH production by the anterior pituitary gland of the rat.

Gonadotrophin-releasing activity of oxytocin has previously been demonstrated in vitro and in vivo. This study investigated whether oxytocin is also able to induce LH accumulation in pituitary cells. Following trypsin digestion and mechanical dispersion, pituitary cells from female rats were incubated with oxytocin (100 nmol/l) for 24 h. LH release stimulated by oxytocin increased (P less than 0.001) progressively during the incubation indicating a different secretory pattern from the more rapid but less sustained secretion stimulated by gonadotrophin-releasing hormone. Oxytocin also enhanced (P less than 0.01) total LH accumulation in the incubation system (released plus cell contents) which was apparent after 7-11 h of stimulation. The release of LH stimulated by oxytocin was reduced by the protein synthesis inhibitor cycloheximide (10 mumol/l). However, cycloheximide did not completely block oxytocin-stimulated LH release; there remained some LH release above that seen in nonstimulated controls (P less than 0.01) revealing the presence of a cycloheximide-resistant component in the release mechanism. Furthermore, accumulation of total LH in 24 h incubations was suppressed (P less than 0.01) by cycloheximide. The advancement in LH release which oxytocin has been shown to induce in vivo in pro-oestrous rats was accompanied by an early reduction of pituitary LH stores. However, the fall normally observed in LH content during the surge was markedly attenuated by the oxytocin treatment. Thus, loss of pituitary LH stores was less in oxytocin-treated rats than in saline-treated controls, even though net LH release into plasma was increased. Therefore, oxytocin stimulated the replenishment of LH stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a Direct Negative Effect of Estradiol at the Level of the Pituitary Gland in Sheep1

The purpose of this experiment was to determine if pituitary stores of LH could be replenished by administration of GnRH when circulating concentrations of both progesterone and estradiol-17 beta (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days. One group of 5 steroid-treated OVX ewes was given GnRH for an additional 42 days (250 ng once every 4 h). Steroid treatment alone reduced (p less than 0.01) the amount of LH in the anterior pituitary gland by 77%. Pulsatile administration of GnRH to steroid-treated ewes resulted in a further decrease (p less than 0.01) in pituitary content of LH. Compared to the OVX ewes, concentrations of mRNAs for alpha- and LH beta-subunits were depressed (p less than 0.01) in all steroid-treated ewes, whether or not they received GnRH. The ability of the dosage of GnRH used to induce release of LH was examined by collecting blood samples for analysis of LH at 15 days and 42 days after GnRH treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of GnRH responded with increased serum concentrations of LH after GnRH administration during the first and second bleedings, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Suckling as a regulator of postpartum rebreeding in cattle: a review.

An extended and variable period of anovulation and anestrus occurs in suckled beef cows following parturition. This imposes both biological and economic constraints on the efficiency of beef production. A large body of knowledge has linked these phenomena to an attenuation of neuroendocrine signals that subserve gonadal function. During late gestation, high concentrations of placental estrogen inhibit the synthesis of LH, and pituitary stores of LH are depleted at parturition. Maximum pituitary stores of LH, releasable pools of LH and the ability of hypothalamic centers to respond to positive feedback effects of estradiol occur within 3 to 4 wk after calving in suckled cows. However, the requisite pattern of pulsatile LH secretion, which accompanies similar changes in nonsuckled cows within 2 to 3 wk after calving, occurs in only 30 to 50% of suckled animals. The period of acyclicity that continues in the remainder is exacerbated by poor body condition and may persist in some females for periods exceeding 100 d. Utilizing data from the cow, as well as other species, a model for suckling-induced inhibition of pulsatile LH release is proposed. The biological and economic impact of current and future techniques for controlling suckling-induced anestrus also is considered, including a conceptual analysis of nonbiological limitations.

Studies on the modulation of the desensitization of the pituitary gland by luteinizing hormone-releasing hormone in the ovariectomized rat.

In ovariectomized rats the desensitization of the LH cells in vivo, which develops during constant rate infusion of LHRH, 1) does not depend on a concomitant depletion of the pituitary LH stores, 2) proceeds normally when the hypothalamo-pituitary connection has been severed and 3) is a process in which LH itself is not involved.

Inhibition of gonadotropin-releasing hormone release as the basis of pituitary-gonadal dysfunction during treatment with 4-aminopyrazolo-(3,4-d)-pyrimidine.

The inhibitor of hepatic lipoprotein release, 4-aminopyrazolo-(3,4-d)-pyrimidine (4-APP), has been shown to reduce testosterone production via impairment of pituitary gonadotropin secretion rather than through decreased cholesterol availability. It was previously shown that serum LH levels were reduced by more than 75% in male rats treated with 4-APP, but pituitary stores of LH and the gonadotropin response to exogenous GnRH were maintained. Also, there was a reduction in pituitary GnRH receptors which was consistent with hypothalamic GnRH deficiency. The present studies were undertaken to examine the mechanism by which 4-APP inhibits GnRH synthesis and/or release. Intact, adult male or 2-week ovariectomized female rats were treated daily with 25 mg/kg 4-APP for 3 days. Both sexes showed lowered basal serum levels of LH and absence of the elevations in serum LH normally elicited by the opiate antagonist, naloxone. In pituitary portal plasma collected from normal male rats, GnRH was significantly elevated by naloxone treatment, confirming that naloxone acted at the level of hypothalamic GnRH release. However, naloxone stimulation of GnRH secretion into portal blood was absent in rats treated with 4-APP. In vitro, the potassium-induced release of GnRH from perifused medial hypothalami was reduced by 60% in 4-APP-treated male rats while hypothalamic GnRH content remained unchanged. These data indicate that 4-APP has an inhibitory effect on the mechanism of GnRH release, and that analysis of its actions should clarify the processes involved in neurohormone secretion.

Desensitization of the pituitary gland induced in vivo by luteinizing hormone-releasing hormone (LRH) or by the LRH-analogue buserelin does not affect the autonomous secretion of luteinizing hormone and follicle stimulating hormone as observed in vitro.

Three-weeks ovariectomized rats were sc implanted with Alzet osmotic minipumps which released either LRH or the LRH-analogue buserelin at the rate of 250 ng/h. Control rats were implanted with a silastic 'shampump'. After explantation, 6 days later, the pituitary glands of part of these rats were exposed to the maximally active LRH concentration of 1 micrograms/ml for a period of 6 h, using a perifusion system. In a second group of rats explantation and perifusion was done not directly, but 5 days after cessation of the LRH pre-treatment. After 6 days in vivo pre-treatment with LRH or with buserelin the pituitary LH and FSH stores were partially depleted, the depletion after buserelin being stronger than after LRH. The pituitary glands of the first group of rats showed rates of both maximally LRH-stimulated and unstimulated (autonomous) LH- and FSH-secretion which were strongly impaired, the impairment after buserelin being stronger than after LRH. In the group with a 5 days interval between in vivo LRH/buserelin pre-treatment and explantation the pituitary LH and FSH stores were restored to the range of pre-treatment levels. Of these pituitaries the autonomous secretion of LH and FSH as well as the maximally LRH-stimulated secretion of FSH was restored to the normal level; the maximally LRH-stimulated secretion of LH, however, remained depressed, indicating that 5 days after cessation of exposure to LRH or to buserelin, and in spite of restored pituitary LH/FSH contents, the sensitivity of the LH releasing system to LRH was still subnormal.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate causes long-lasting suppression of the autonomous and the LRH-stimulated secretion of luteinizing hormone and follicle stimulating hormone. An in vitro study.

The effect of a combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate (EB) on the autonomous and the 'supra-maximally' LRH-stimulated in vitro release of LH and FSH by pituitary glands of 2 weeks ovariectomized (OVX) rats was studied using a perifusion system. The concentration of LRH in the perifusion medium was 1 microgram/ml. Pre-treatment with LRH during 6 days was effected by means of sc implanted Alzet osmotic minipumps (MP). Control rats received a piece of silastic with the dimensions of a minipump ('sham-pump'; Sh-P). EB, 3 micrograms/injection or solvent (arachis oil) was sc injected on days-3 and -1 (day of perifusion: day 0). Of the pituitary glands of EB-injected, Sh-P-implanted rats both the autonomous and the LRH-stimulated secretion of LH and the LRH-stimulated secretion of FSH were significantly higher than those of the oil-injected, Sh-P-implanted rats without EB administration. Pretreatment with LRH for 6 days had a suppressing effect on the autonomous and the LRH-induced depletion of the pituitary LH and FSH stores. In combination with EB, the suppressing effect of LRH pre-treatment on the LRH-stimulated secretion of LH and FSH was still greater: the pituitary gland appeared to be fixed in a relatively unresponsive state with very low autonomous LH and FSH secretion. It is discussed that increase of pituitary LRH-responsiveness due to EB demands withdrawal of the pituitary gland from the influence of LRH, an effect which is in vivo achieved by the negative feedback of oestrogen on the hypothalamus.

Changes in pituitary LH content and LH secretion in the long-term ovariectomized rat during prolonged infusion of LRH.

LH secretion and the depletion of the pituitary LH stores caused by continuous infusion of LRH, were studied in the 5-weeks ovariectomized rat. After 1 day of infusion at the rate of 416 ng/h, pituitary LH content decreased to about 40% of its initial value. Upon continuation of the infusion the LH content increased slowly until, after 7 days, it increased to about 54% of the original value. The plasma LH concentrations, after having reached peak values of 7122 +/- 291 ng/ml after 2 h of infusion, declined to a plateau at about 300 ng/ml but it took 5 days to reach this value. When after either 2 or 8 days of LRH infusion at the rate of 416 ng/h the infusion was changed to a 'supramaximal' rate of 10,400 ng/h for 24 h, a transient secondary rise of LH secretion was induced. The magnitude of the secondary response, was essentially the same after 2 or 8 days of LRH infusion, but these secondary responses were significantly smaller than the LH-response during a similar LRH-infusion in previously untreated ovariectomized rats.

Effects of estradiol-17beta on the induction of gonadotropin release by electrical stimulation of the hypothalamus in rhesus monkeys.

Serum LH and FSH were measured at 60, 30, and 0 min before, at 5, 15, and 30 min during, and at 10, 45, and 90 min after bilateral electrical stimulation (ES) of various hypothalamic regions in 12 unanesthetized ovariectomized rhesus monkeys. ES of the arcuate-ventromedial nuclei (medial basal hypothalamus; MBH) induced a prompt increase in serum LH that persisted throughout stimulation and returned to basal levels within 90 min thereafter. FSH was also released, but the release was slower and less dramatic than that of LH. Sham stimulation (0muA) caused no change in serum gonadotropins. The amount of LH released after MBH-ES depended upon current strength (1.0 mA greater than 0,5 or 0.7 mA). Three sequential 30-min MBH-ES trials at 90-min intervals induced comparable LH responses and 3 h of continuous MBH-ES maintained elevated serum LH levels throughout the stimulation period, suggesting that these stimulation period, suggesting that these stimulation parameters did not completely deplete pituitary stores of releasable LH. The character of the LH response was similar in individual monkeys through 3 to 24 trials during 4 to 18 months. Comparisons were made of the effects of estradiol-17beta (E2) treatment at different doses and for different intervals of time before MBH-ES. ES-induced LH release was not affected by low levels (25 and 55 pg/ml) ofE2 for 48 h, but was reduced by higher E2 concentrations (100 or 230 pg/ml). E2 concentrations of 100 pg/ml had no effect at 24 h, but reduced MBH-ES-activated LH release at 48 to 96 h; the degree of depression was time-related (48 h less than 72 h less than 96 h). ES of the preoptic-suprachiasmatic region (rostral hypothalamus; RH) in non-E2-treated monkeys also released LH, but this increase was less than after MBH-ES. FSH release was not measurable after RH-ES. In contrast to the depressed LH response to MBH-ES after 48 h of E2 (100 pg/ml), the response to RH-ES was not inhibited by this E2 regimen. These data suggest that ES of an area extending caudally from the rostral hypothalamus to the arcurate-median eminence region will evoke LH release in rhesus monkeys. This electrically induced gonadotropin release was affected by administration of physiological levels of E2 but the nature of effect depended on the specific region stimulated: distinct inhibition of the gonadotropic response to MBH-ES and slight facilitation of the response to RH-ES.

Effect of synthetic luteinizing hormone-releasing hormone on the release of gonadotropins in hypophysogonadal disorders of children and adolescents: VII. Constitutional delay of puberty in males

Serum gonadotropins (LH and FSH) were measured by radioimmunoassay before and after intravenous injection of 0.1 mg/m2 of synthetic luteinizing hormone-releasing hormone in 20 male patients, aged 15 to 18 years, with constitutional delay of puberty. Basal plasma levels of LH and FSH were in the prepubertal range. After administration of LH-RH, the increase in LH was significantly high than in prepubertal control subjects, aged 1 to 13 years; the difference between test patients and pubertal control subjects was not significant. The increase in FSH was in the prepubertal range, significantly lower than that in pubertal control subjects. This discrepancy between LH and FSH responses to LH-RH is similar to that observed in normal boys at the late prepubertal stage and suggests that an elevation of readily releasable pituitary stores of LH correlates with the first step of pubertal onset in males, even if puberty is delayed.

Induction of LH surges by continuous infusion of LH-RH1.

Preovulatory LH surges were measured in cannulated proestrous rats. Such LH peaks were also obtained by infusing pentobarbital-blocked animals with LH-RH for periods of up to 20 h; this suggested that the pituitary LH stores were exhausted. However, the total amount of LH secreted appeared more or less determined by the LH-RH dose, indicating that the size of the releasable LH pool is not fixed.

Progesterone: sites of action in inhibiting ovulation in hamsters.

A single subcutaneous injection of progesterone (0.5 mg/animal at 17.00 hours) on Day 3 of the cycle (Day 1 = day of vaginal discharge) interfered with ovulation and external vaginal discharge in all the animals. Lower doses were only partly active. Intravenous administration of LH, FSH or their combination at 14.00 hours on Day 4 failed to restore ovulation in such animals, implying a direct action of the administered progesterone on the ovary. A large dose of oestradiol benzoate (400 mug/animal) also proved inactive suggesting that progesterone did not interfere with ovulation solely through antioestrogenic action. Depletion of the pituitary LH stores normally found in association with ovulation and ovulatory peak of LH in plasma seen in control animals were not observed in the progesterone-treated hamsters suggesting a further action of progesterone at the pituitary-hypothalamic level in inhibiting ovulation. Administration of synthetic LRF (100 ng/animal, i.v.) on Day 4 to hamsters treated with progesterone caused an increase in plasma LH 15 min later which was comparable to the rise observed in control animals given a similar injection of LH-RF, implying lack of action of progesterone at the pituitary level. It is concluded that progesterone acts centrally, principally on the hypothalamus, and peripherally on the ovary to interfere with ovulation in hamsters. It is possible that the anovulatory state accompanying pregnancy results from these actions.

Participation of the amygdala and of the cerebellum in the feedback effects of progesterone.

Bilateral implantation of small amounts of progesterone in the amygdala, cerebellum, and median eminence of castrated female rats results in an increase of pituitary stores of LH and in a decrease of hypothalamic stores of LH-RF. Similar implants placed subcutaneously or in the cerebral cortex are ineffective. It is suggested that the amygdala and the cerebellum contain receptors sensitive to progesterone and intervene in modulating the negative, inhibitory effect that this steroid exerts onLH secretion.