Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.     

The use of mouse pituitary fragments to study LH secretion

This work evaluated a perifusion system for studying LH secretion from the anterior pituitary (AP) of female mice. Pituitary fragments were challenged with LHRH, and the effluents assayed for LH. In general, the tissue exhibited augmented release to repeated stimulation. In the dose-response study, the amount of LHRH required to produce maximum and half maximum responses dropped almost 10 fold by the 3rd stimulus. In response to various pulse frequencies LH release increased with the frequency of the 100 nM LHRH dose, but the tissue became refractive to constant nonpulsatile stimulation. Other preparations, subjected to high-frequency 10 nM LHRH pulses, released LH in two distinct episodes. All but the first hour of the response was blocked by cycloheximide, confirming the role of protein synthesis in the sustained release of LH. By varying both the pulse frequency and amplitude, a LHRH protocol was found that produced a proestrous-like surge. Lastly, rat and mouse tissues responded similarly to pulsatile LHRH, verifying their similar LH function during the preovulatory period. These studies demonstrate that the perifusion technique can be used for studying LH secretion in the mouse. Its application to other mouse-oriented studies is planned.     

Differential regulation of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone secretion in ovariectomized rats disclosed by treatment with a LH-releasing hormone antagonist and phenobarbital

Although pulsatile LH release in ovariectomized (OVX) rats appears to be controlled by pulsatile discharges of LHRH, the neuroendocrine regulation of episodic FSH release remains to be explored. The main objective of the present study is to compare and contrast the effects of a potent LHRH antagonist (ALHRH) and a central nervous system depressant, phenobarbital (PhB), on pulsatile LH and FSH release in OVX rats. Three to 4 weeks after ovariectomy, blood samples were obtained at 10-min intervals for 3 h, after which LHRH was injected and sampling continued for an additional hour. In control OVX rats, periodic increases in plasma LH and FSH levels occurred approximately every 30 to 60 min, respectively. Treatment of OVX rats with PhB several hours earlier resulted in a suppression of mean plasma levels and pulse frequencies of both LH and FSH. Interestingly, PhB suppressed the pulse amplitude of LH, but not of FSH. Phenobarbital increased pituitary LH responses to LHRH, but did not alter the FSH responses. When ALHRH was given to OVX rats 24 h before blood sampling, mean plasma LH levels as well as LH pulse frequency and amplitude were severely diminished. In striking contrast, ALHRH did not affect the frequency or amplitude of FSH pulses. However, mean plasma FSH levels were suppressed to 31% of levels measured in control OVX rats. These results demonstrate that in contrast to LH secretion, FSH secretion in OVX rats appears to be regulated by two distinct neuroendocrine mechanisms: an LHRH-dependent mechanism controlling the nonepisodic component of FSH secretion (baseline secretion) and a LHRH-independent mechanism controlling pulsatile FSH release.     

Evidence for decreased luteinizing hormone-releasing hormone pulse frequency in men with selective elevations of follicle-stimulating hormone

To examine the hypothesis that the frequency of endogenous pulsatile LHRH stimulation controls the relative secretion of FSH and LH from the pituitary, we studied men with elevated FSH levels and normal LH levels to determine whether they have an altered frequency of pulsatile LHRH secretion compared to normal men. Because peripheral blood measurements of LHRH do not reflect the pulsatile characteristics of hypothalamic LHRH secretion, and it is generally accepted that the pulse frequency of LH secretion is an index of the frequency of endogenous LHRH pulsation, we used LH pulse frequency as the indicator of LHRH pulse frequency. Frequent blood sampling was performed to characterize LH pulse patterns in five men with selective elevations of FSH and seven age-matched normal men. Beginning at 0800-0930 h, blood samples were obtained every 10 min for 24 h through an indwelling iv catheter. Serum LH and FSH levels were measured by RIA in each sample, and the pattern of LH secretion was determined. Testosterone (T), estradiol, sex hormone-binding globulin, and free T were measured in a pooled serum sample from each man. Men with selective elevations of FSH had fewer LH pulses per 24 h (mean +/- SEM, 10.6 +/- 0.5) than the control group (12.9 +/- 0.6; P less than 0.01). There was no statistically significant difference in LH pulse amplitude (23 +/- 4 vs. 17 +/- 3 ng/ml). There were no statistically significant differences in T (4.9 +/- 0.5 vs. 6.1 +/- 0.5 ng/ml), estradiol (23 +/- 7 vs. 31 +/- 5 pg/ml), sex hormone-binding globulin (7.7 +/- 1.4 vs. 7.7 +/- 1.2 ng bound dihydrotestosterone/ml), or free T (0.16 +/- 0.02 vs. 0.23 +/- 0.04 ng/ml) in these men vs. normal subjects. We conclude that 1) compared to normal men, men with selectively elevated FSH levels have decreased LH pulse frequency, which suggests decreased LHRH pulse frequency; and 2) the relative secretion rates of LH and FSH by the pituitary may be regulated by the frequency of pulsatile LHRH secretion from the hypothalamus.     

The effect of estrogen on episodic secretory patterns of luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) in hypergonadotropic hypogonadism

The secretory dynamics of plasma luteinizing hormone-releasing hormone (LHRH) and serum luteinizing hormone (LH) were studied in three hypogonadal women before and after chronic administration of mestranol. Blood samples were obtained through an indwelling iv line every 15 min over 3 hours, and plasma levels of LHRH and LH were measured by radioimmunoassay. LHRH and LH pulses were defined as rising from nadir to peak that exceed 2 times the intraassay coefficient of variation. All patients showed pulsatile LHRH and LH release before mestranol administration. The mean LH levels (89 +/- 20 mIU/ml) and pulse amplitude (33 +/- 14 mIU/ml) were significantly reduced after mestranol administration. On the other hand, the mean LHRH levels (1.87 +/- 0.49 pg/ml) and pulse amplitude (0.92 +/- 0.41 pg/ml) did not change significantly after mestranol administration. Pulse frequency (2 approximately 3 times/3 hrs) of LHRH and LH did not change after mestranol administration. These data show that the chronic administration of estrogen to such patients cause a decrease in mean LH levels and amplitude of LH pulse without a decrease of pulsatile LHRH secretions. These results suggest that the chronic negative feedback action of estrogen on episodic LH release in women may be at the level of the pituitary gland and estrogen may change the pituitary sensitivity to LHRH.     

Pulsatile luteinizing hormone release, and the inhibitory effect of estradiol-17 beta in gonadectomized male and female rats: effects of neonatal androgen or exposure to constant light

The characteristics of pulsatile LH release and the acute inhibition of LH release by estradiol-17 beta (E2) were studied in long term (21 days) gonadectomized female and male Wistar rats. Three groups of female rats were examined; animals exposed either to summer lighting (14-h on; 10-h off; LD) or continuous illumination (LL) and animals treated neonatally with testosterone propionate (TP) and exposed to LD. The mean plasma LH concentrations and interpulse intervals were similar in both male and LD female rats. However, treatment of female rats with TP or exposure to LL reduced the mean plasma LH concentration in female rats and increased the interpulse interval when compared with LD female or male rats. The amplitude of the LH pulses was significantly greater in the LD female rats compared with those in the male rats; since pituitary responsiveness to a single iv injection of 50 ng LHRH/100 g BW was similar in the two groups, this suggests that the amount of LHRH released per pulse of LH is greater in the female than in the male. The greater amplitude but similar frequency of LH pulses in the LD female compared with the LD male suggest that the MCR of LH may be greater in the female. The pulse amplitude in the TP-treated rats was similar to that in the rats exposed to LL and since pituitary responsiveness to LHRH was significantly greater in the TP rats, there was probably more LHRH released per pulse of LH in the LL-treated rats. Pituitary responsiveness to LHRH was significantly lower in the LL- and TP-treated rats compared with males and LD females. The timing of the inhibition of LH secretion by E2 was similar in all four groups of animals, and in LD females was not affected by a 92% depletion of serotonin, 100% depletion of 5-hydroxyindoleacetic acid, or a 33% depletion of dopamine in the hypothalamus produced by the administration of parachlorophenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that a decrease in opioid tone on proestrus changes the episodic pattern of luteinizing hormone (LH) secretion: implications in the preovulatory LH hypersecretion

We have studied the LH secretion pattern evoked by diminution in the opioid tone produced by iv naloxone (NAL) infusion between 1100-1400 h on proestrus, the LH secretion pattern occurring spontaneously between 1430-1730 h on proestrus and the LH secretion pattern produced by exogenous LHRH administered either as a 10 ng/pulse at 20-, 30-, or 60-min intervals or infused continuously at a rate of 30 ng/h between 1200-1700 h in rats given pentobarbital at 1100 h on proestrus. Infusion of 0.5 ng NAL/h raised plasma NAL levels to 200-300 ng/ml and augmented LH secretion, as evident by increments in pulse amplitude and frequency discharge to one every 37.5 min from an average of one every 75 min in saline-infused control rats. A 4-fold increase in circulating NAL levels, produced by 2 mg/h NAL infusion, further augmented the frequency of LH episodes to 30-33 min and induced a surge-like LH secretion pattern which resembled that seen on the afternoon of proestrus. Further analysis of the secretory pattern of the preovulatory LH surge (n = 7) showed LH pulses of increased amplitude during the basal phase (n = 4), ascending phase (n = 2), and plateau and descending phases (n = 3); in two rats the LH rise was steep, and no LH pulses were identified. A LHRH pulse (10 ng/pulse) delivered at 20- or 30-min intervals or continuous infusion of LHRH at a rate of 30 ng/h produced LH surges, with peak levels reaching the range seen on the afternoon of proestrus. Further, despite the fact that 10 ng LHRH/pulse at 20-min intervals reproduced a proestrous-type LH surge, only 40% of the LHRH pulses were followed by identifiable LH pulses. Surprisingly, despite the observations that NAL evoked robust LH episodes, the basal pattern of FSH secretion in these rats was not altered. These findings show that a decrease in opioid tone on proestrus accelerates episodic LH discharge to the range that occurs after gonadectomy. A quantitative relationship between the degree of restraint on the opioid tone imposed by NAL and the magnitude of the LH response can be demonstrated. The evidence suggests that the preovulatory LH surge may occur in an episodic fashion and that it can be reproduced by LHRH delivered at a frequency rate of LH pulses seen in ovariectomized rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endogenous inhibin suppresses only basal follicle-stimulating hormone secretion but suppresses all parameters of pulsatile luteinizing hormone secretion in the diestrous female rat

The purpose of these studies was to ascertain which parameters of pulsatile gonadotropin secretion are regulated by endogenous inhibin in the intact diestrous female rat. This was determined by examining the changes in the secretion parameters of FSH and LH that resulted from immunoneutralizing endogenous inhibin in diestrous I female rats. Passive immunoneutralization of endogenous inhibin was achieved using specific, high titer ovine antiserum generated against the alpha-subunit of the recently described inhibin molecule. The optimal times after inhibin immunoneutralization to observe the changes in FSH secretion were determined in initial experiments. Pulsatile secretion of both FSH and LH was observable in the diestrous female. Two hours after inhibin immunoneutralization, the mean trough level, mean peak level, and overall mean level of FSH began to increase. The maximal increase and plateau of these parameters were observed 5 h after antiserum injection. During the period of increase, mean FSH pulse amplitude was also increased, but returned to the level observed in control (normal sheep serum-injected) animals when the parameters of trough, peak, and overall mean FSH reached their plateau levels. FSH pulse frequency was not changed at any time. These results indicate that endogenous inhibin affects only the basal parameters of FSH secretion without affecting pulsatile FSH secretion. The transient increase in FSH pulse amplitude resulted from FSH pulses being superimposed on the increasing basal FSH secretion. In contrast, immunoneutralization of endogenous inhibin rapidly increased all parameters (i.e. pulse amplitude and frequency, mean trough and peak levels, and mean plasma levels) of LH secretion. In addition, pituitary sensitivity to an exogenous LHRH challenge was increased in inhibin-immunoneutralized females in terms of stimulated LH secretion. As a result of the already increased rate of basal secretion, the actual quantity of FSH released in response to the LHRH challenge was greatly increased in the inhibin-immunoneutralized rats compared with the normal sheep serum-injected controls; however, the increase in the rate of FSH secretion stimulated by the LHRH challenge was the same in both groups. The observations from these studies collectively demonstrate that inhibin acts endogenously to suppress those parameters of gonadotropin secretion that are regulated by LHRH.     

Ontogeny of pulsatile gonadotrophin secretion and pituitary responsiveness in male puberty in man: a mixed longitudinal and cross-sectional study

The onset of puberty is characterized by a sleep-associated increase in pulsatile LH secretion which is not observed in adults. The ontogeny of gonadotrophin secretion during pubertal maturation may reflect changes in endogenous LHRH secretion, pituitary sensitivity to LHRH and/or alterations in gonadal steroid feedback. To understand the interplay between these mechanisms, we have examined the pulsatile pattern of plasma LH, FSH, testosterone, oestradiol and prolactin between 20.00 and 09.00 h and the pituitary response to repeated exogenous LHRH stimulation in 16 boys with delayed puberty (age 16.3 +/- 2.7 (S.E.M.) years) on one to four occasions in a mixed longitudinal/cross-sectional analysis. Physical maturity was determined by Tanner G staging (1-5) and clinical progress followed for a mean duration of 22.4 +/- 8.5 months during which 33 hormone profiles were obtained. Nocturnal (23.00-09.00 h) LH pulse frequency increased to a peak of 0.54 +/- 0.03/h at stage 2 which was followed by a gradual decline to 0.42 +/- 0.04/h at stage 5. The appearance of LH pulses in the evening (20.00-23.00 h), probably representative of the rest of the day, was delayed until mid-puberty from which point frequency increased to a peak of 0.53 +/- 0.08/h at stage 5. LH pulse amplitude showed a linear increase from stages 1 to 5, with nocturnal pulse amplitudes being higher than evening pulses throughout. FSH did not show a clear pulsatile pattern. The LH:FSH ratio reversed from less than 1 to greater than 1 at stage 2. The LH response to exogenous LHRH increased in parallel with LH pulse amplitude. There was no difference in the pattern of LH response to repeated LHRH stimulation as puberty advanced; the first stimulus always elicited a greater response than subsequent doses. In contrast, the FSH response to LHRH was maximal at stage 1 and became attenuated thereafter. The estimated mean nocturnal LHRH concentration or amplitude did not show any increase during pubertal maturation from 20.42 +/- 11.57 at stage 1 to 35.96 +/- 20.83 ng/l at stage 5. In conclusion, the sequential changes in this study suggest that the sleep-entrained increase in LHRH pulse frequency plays a key role at the onset of puberty. By enhancing pituitary responsiveness and setting in motion a cascade of events, this peripubertal augmentation of LHRH pulse frequency can account for most of the subsequent changes in LH, FSH and testosterone secretion during pubertal development in the male without any apparent alteration in LHRH pulse amplitude.     

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-stimulated LH surges in pentobarbital-blocked proestrous rats

Recent evidence suggests that hypothalamic neurosecretion of neuropeptide Y (NPY) may be required for the preovulatory LH surge in female rats. Results of immunoneutralization and portal blood collection studies have suggested that NPY may serve to enhance the response of gonadotropes to the stimulatory action of LHRH. To directly test this hypothesis, the effects of NPY on LHRH-stimulated LH secretion were assessed in proestrous rats that were anesthetized with pentobarbital (PB) to block endogenous LHRH neurosecretion. Female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 0900-2100 h. At 1330 h, rats received PB (40 mg/kg BW) or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15, 150, or 1500 ng/pulse) or saline along with concurrent pulses of NPY (1 or 10 micrograms/pulse). Plasma samples were analyzed by LH RIA. In PB-treated rats receiving vehicle pulses only, LH surges were completely blocked. Pulsatile LHRH treatments at 15, 150, and 1500 ng/pulse produced subphysiological, physiological, and supraphysiological LH surges, respectively. Simultaneous administration of NPY pulses with 15 ng/pulse LHRH produced significant dose-related potentiations of LHRH-stimulated LH surges (P less than 0.0001). Administration of NPY pulses with 150 ng LHRH/pulse also significantly enhanced LHRH-induced LH surges (P less than 0.05). NPY RIA of plasma confirmed NPY increments after treatments. These results demonstrate that NPY administration can potentiate pituitary responsiveness to LHRH stimulation, and are consistent with the hypothesis that one function of NPY is to operate as a neurohormonal modulator at the level of the gonadotrope during generation of the preovulatory LH surge.     

Evidence that pulsatile follicle-stimulating hormone secretion is independent of endogenous luteinizing hormone-releasing hormone

Although LHRH can stimulate the release of both LH and FSH from the pituitary, there are a number of instances in which the secretion of LH and FSH are divergent. Previous studies from our laboratory have indicated that pulsatile LH and FSH secretion are independently regulated by gonadal factors. We have, therefore, reexamined the role of LHRH in regulating pulsatile gonadotropin secretion by evaluating the effect of passive LHRH immunoneutralization on LH and FSH secretion in castrate adult male rats. Injection of 500 microliters ovine anti-LHRH serum no. 772 (LHRH-AS) into 2-week-castrate rats caused an 85% suppression of mean plasma LH levels by 2 h, which lasted through 48 h. Mean plasma FSH, however, was reduced by only 19% after 2 h and by only 59% after 48 h. When cannulated 2-week-castrate rats were bled every 10 min, both LH and FSH were secreted in a pulsatile manner. Injection of 500 microliters LHRH-AS caused an immediate abolishment of LH pulses and a rapid reduction in mean plasma LH through 24 h. Pulsatile FSH secretion, as characterized by the parameters of pulse frequency and amplitude, was unaffected by LHRH-AS, although mean plasma FSH levels were significantly reduced. Collectively, the results suggest that pulsatile FSH secretion is regulated by a separate factor(s) distinct from LHRH, but that LHRH is required for the maintenance of elevated FSH levels.     

Pulsatile LH secretion in streptozotocin-induced diabetes in the rat.

This study aimed to determine the effect of streptozotocin (STZ)-induced diabetes on pulsatile LH secretion in the mature male rat. LH pulse frequency was reduced by 56% and pulse amplitude by 54%, with a consequential decrease of 72% in mean LH levels 8 days after i.v. administration of STZ (55 mg/kg) to castrated Wistar rats compared with castrated non-diabetic controls. Twice daily insulin treatment completely reversed all parameters of pulsatile LH secretion to control values. Food-restricted non-diabetic controls, studied to distinguish the metabolic effect of diabetes from that of concurrent weight loss, demonstrated a 34% reduction in LH pulse frequency but no significant changes in LH pulse amplitude or mean LH levels compared with non-diabetic controls given free access to food. To distinguish whether the decreased LH pulse amplitude in diabetes was due to a reduction in either the quantity of hypothalamic gonadotrophin-releasing hormone (GnRH) released per secretory episode or to decreased pituitary responsiveness to GnRH, the responsiveness of the pituitary to exogenous GnRH (1-1000 ng/kg body weight) was tested in diabetic rats after castration, using a full Latin square experimental design. The net LH response (total area under response curve over 40 min following GnRH) was decreased by 33% (P = 0.001) in diabetic compared with control rats. The decreased LH pulse frequency in STZ-induced diabetes therefore suggests that the metabolic effect of diabetes is to decelerate directly the firing rate of the hypothalamic GnRH pulse generator independent of testicular feedback. These effects were fully reversed by insulin treatment and were only partly due to the associated weight loss.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential control of luteinizing hormone and follicle-stimulating hormone secretion by luteinizing hormone-releasing hormone pulse frequency in man

To test the hypothesis that the frequency of pulsatile LHRH stimulation can differentially control LH and FSH secretion in man, we administered low doses of LHRH in pulsatile fashion in several different regimens to men with idiopathic hypogonadotropic hypogonadism (IHH) and presumed endogenous LHRH deficiency. In study 1, four men with IHH received a constant amount of LHRH per day in three different frequencies. After an initial 7-day period of LHRH (5.0 micrograms every 2 h), the men received 2.5 micrograms every 1 h and 7.5 micrograms every 3 h, each for 4 days, in varying order. Frequent blood samples were obtained before LHRH administration and at the end of each regimen. Before LHRH administration, mean serum FSH and LH levels were low [28 +/- 3 (+/- SEM) and 6 +/- 2 ng/mL, respectively], and they increased into the normal adult male range during LHRH treatment. As the frequency of LHRH administration decreased from every 1 to 2 to 3 h, serum FSH levels progressively increased from 99 +/- 33 to 133 +/- 34 to 181 +/- 58 ng/mL (P less than 0.05). Serum LH levels (34 +/- 6, 33 +/- 6, and 34 +/- 5 ng/mL) were significantly higher than those before LHRH administration and did not differ significantly among the three regimens. Total serum testosterone (T), estradiol, and free T levels were increased by LHRH, but were not significantly different during the three regions of LHRH administration. In study 2, three men with IHH received the same amount of LHRH per dose, given in two different pulse frequencies; 2.5 micrograms LHRH were administered in frequencies of every 0.5 h and every 1.5 h, each for 4 days, in varying order. During the 0.5 h frequency, the mean serum FSH level was 42 +/- 13 ng/mL, and it rose to 80 +/- 19 ng/mL during the 1.5 h frequency (P less than 0.05). Corresponding mean serum LH levels were 25 +/- 5 and 27 +/- 4 ng/mL. Serum T and estradiol levels were not significantly different during the two LHRH regimens. We conclude that the frequency of LHRH stimulation can differentially control FSH and LH secretion by the human pituitary gland, and the pattern of hormonal stimulation may be a determinant of target organ response.     

PULSATILE RELEASE OF LH AND OESTRADIOL DURING THE PERIOVULATORY PERIOD IN WOMEN

Blood was collected every 8 h in five women from Day 10 of the menstrual cycle for 5–7 d for the measurement of LH, oestradiol and progesterone. In all women the LH surge started between 2400 and 0800 h before there was any significant decline in the concentration of oestradiol. In order to investigate the pulsatile secretion of LH and estradiol during the mid-cycle surge, blood samples were collected every 5 min for up to 5 h. Immediately before and during the LH surge there were numerous episodic pulses of LH with an interpulse interval (44 ± 7 min) very similar to that observed in two women with hypergonadotrophic hypogonadism (48 min). The concentration of oestradiol fluctuated in a similar manner although it was not always possible to relate each pulse of oestradiol to a corresponding pulse of LH. The mid-cycle surge of LH is characterized by frequent pulses of high amplitude. These results are compatible with the view that the positive feedback effect of oestradiol is due not only to enhanced sensitivity of the anterior pituitary to LHRH but also to the high frequency of LHRH pulses released from the hypothalamus.     

Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids

In sheep, physiological levels of estradiol and progesterone each suppress the pulses of LH characteristics of tonic LH secretion, but do so by completely different mechanisms. Estradiol treatment decreases LH pulse amplitude but not frequency and also inhibits the height of the LH peak resulting from the administration of gonadotropin-releasing hormone (GnRH). In contrast, progesterone decreases the frequency of LH pulses without reducing their amplitude or the response to exogenous GnRH. This suggests that progesterone suppresses tonic LH secretion by acting in the brain to decrease the frequency of GnRH pulses, while estradiol may suppress the response of the pituitary to GnRH and thereby decrease LH pulse amplitude.     

Inhibitory effect of central LHRH on LH secretion in the ovariectomized ewe

The role of central luteinizing hormone releasing hormone (LHRH) in the control of pulsatile LHRH and luteinizing hormone (LH) secretion was investigated in ovariectomized adult ewes. Injection of LHRH (2.1-21 pmol) into the third cerebral ventricle caused a delayed but sustained inhibition of LH secretion. Pulse frequency, pulse amplitude and mean LH levels were reduced significantly when compared with the responses to the control injection of saline (50 microliters). The inhibitory effect of centrally administered LHRH was not accompanied by a reduction in the pituitary responsiveness to intravenous LHRH. In contrast to the effect on LH, plasma levels of follicle-stimulating hormone (FSH) and prolactin were unaffected by central LHRH. The inhibitory action of LHRH was antagonized by prior injection of an LHRH antagonist ([N-Ac-D-Nal(2)1, D-p-Cl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10] LHRH, 69 pmol) into the third ventricle. Central injection of the LHRH antagonist alone (at the same concentration) did not influence any characteristic of pulsatile LH secretion. In conclusion, these data indicate that exogenous administration of LHRH into the brain exerts a dose-related and receptor-mediated inhibition of LHRH pulse generator activity. However, the physiological significance of endogenous LHRH in the regulation of the LHRH pulse generator remains unresolved.     

Endogenous opioid peptide regulation of pulsatile luteinizing hormone secretion during pregnancy in the rat

The objective of this study was to determine if endogenous opioid peptides (EOPs) influence the pattern of pulsatile luteinizing hormone (LH) secretion on days 6-8, 14-16 and 22 of gestation in the rat. Unanesthetized animals with two jugular cannulae were initially infused with 0.9% saline during which the control pattern of pulsatile LH release was determined. Possible EOP involvement was then determined by infusion of the EOP receptor antagonist naloxone. Plasma estradiol (E2) and progesterone (P) values increased between days 6-8 and 14-16. While plasma E2 values remained elevated through day 22, plasma P values declined by 90%. As previously reported, mean blood LH levels during the control period on day 22 were higher than on days 6-8 and 14-16 due to an increase in LH pulse frequency. At each stage of gestation naloxone infusion increased mean blood LH levels. This stimulatory action of naloxone was reduced in a dose-dependent fashion by simultaneous infusion with morphine, demonstrating that this effect is mediated via EOP receptors. There was no difference in the in vivo pituitary responsiveness to LH-releasing hormone (LHRH) between rats infused with saline or naloxone at any stage of pregnancy, demonstrating that the stimulatory effect of naloxone was not exerted at the pituitary level. Naloxone increased both the amplitude and frequency of pulsatile LH secretion on days 6-8, and stimulated frequency on days 14-16. The effect on amplitude could not be assessed on days 14-16 because too few rats exhibited pulsatile LH secretion prior to naloxone infusion. The increase in pulse frequency was similar on days 6-8 and 14-16. Although naloxone increased LH pulse amplitude and frequency on day 22, these increases were significantly less than those seen on days 6-8 and 14-16, respectively. Pituitary responsiveness to LHRH was less at all stages of pregnancy in comparison to responsiveness in ovariectomized rats, and progressively declined from days 6-8 through day 22. The lowest responsiveness to LHRH was seen on day 22 and contributed, at least in part, to the diminished increase in LH pulse amplitude in response to naloxone infusion on day 22 compared to days 6-8. The reduced naloxone-induced increment in LH pulse frequency on day 22, occurring coincident with a precipitous decline in plasma P levels, suggests a decreased EOP suppression of pulse frequency at this time.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen inhibits luteinizing hormone (LH), but not follicle-stimulating hormone secretion in hypophysectomized pituitary-grafted rats receiving pulsatile LH-releasing hormone infusions

The differential feedback actions of estrogen (E2) on gonadotropin secretion were studied by means of an in vivo isolated pituitary paradigm. Adult female rats were hypophysectomized (hypox) and the next day received single anterior pituitary transplants (graft) under the kidney capsule. At the same time rats underwent bilateral ovariectomy. On the third day each animal was fitted with a catheter system which allowed for intermittent infusions of LHRH (250 ng/5 min.h) and chronic blood sampling. Rats received LHRH infusions for 7 days. On the sixth day of LHRH infusions blood samples were collected for 4 h 5, 15, 25, 35, 45 min after each hourly LHRH pulse. After 1 h of sampling, animals received sc injections of 2 micrograms estradiol benzoate (EB; n = 5) or oil vehicle (n = 5). Plasma LH, FSH, E2, and PRL levels in samples from all groups were determined by RIA. In hypox/graft rats LH release, but not FSH release, was pulsatile in response to the hourly LHRH infusions. Injection of EB in the hypox/graft rats significantly (P less than 0.05) suppressed LH release within 3 h by 57%, while FSH was unaffected. PRL levels were elevated by approximately 10-fold in the hypox/graft animals compared to those in pituitary-intact rats. These levels, however, were not changed as a function of steroid treatment and, therefore, could not account for the effects of EB on LH secretion. On the basis of these observations we conclude that 1) a major inhibitory effect of an acute injection of EB on LH secretion is exerted by a direct action on pituitary gonadotropes, and 2) E2 can differentially affect the release of LH and FSH by an intrapituitary mechanism. It is hoped that development of this model will allow for further investigation of the cellular mechanisms that mediate feedback actions of E2 on pituitary gonadotropes exposed to intermittent LHRH stimulation.     

Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat

Studies were undertaken to examine the effects of hyperprolactinemia on the frequency and amplitude of pulses of LH, and determine if changes in pituitary sensitivity to LHRH were involved in the prolactin-induced suppression of LH secretion. Rats were bilaterally ovariectomized (day 0). Ovine prolactin (4 mg/kg body weight, subcutaneously) or vehicle was administered every 8 h beginning at 09.00 h on day 4 after ovariectomy and continuing until 09.00 h on day 6. On day 6, between 07.00 and 09.00 h all animals received a right atrial cannula, using ether anesthesia. In experiment I blood samples were taken at 10-min intervals beginning at 12.00 h on day 6, for a total of 180 min. To test the effect of hyperprolactinemia on pituitary responsiveness (experiment II) animals received an intravenous injection of LHRH (25 ng/100 g body weight) after the 180-min and again after the 240-min sample. Blood was drawn every 10 min for a total of 300 min. Serum was assayed for LH. Hyperprolactinemia altered the pattern of pulsatile secretion of LH. Treatment with ovine prolactin produced a decrease in both the frequency and amplitude of the LH pulses compared to values found in control animals. However, no differences in pituitary responsiveness between hyperprolactinemic and control animals were found at the dose of LHRH given. Thus, the prolactin-induced suppression of pulsatile secretion of LH was not apparently a result of alterations in the sensitivity of the pituitary to LHRH. From these studies we suggest that hyperprolactinemia directly affects a hypothalamic site which ultimately alters the LHRH pulse generator, thereby changing the secretion of LHRH.     

Secretion rates and short-term patterns of gonadotrophin-releasing hormone, FSH and LH throughout the periovulatory period in the mare

We have developed a non-surgical technique for long-term collection of pituitary venous blood which consists of slightly diluted hypophysial portal blood into which pituitary hormones have been secreted. In these experiments jugular and pituitary venous blood samples were collected from five unmedicated, ambulatory mares at 5-min intervals for 2-6 h on 11 occasions during the 6 days surrounding the ovulatory LH peak. Jugular blood only was collected from another five periovulatory mares without pituitary cannulae. The duration of oestrus was similar in mares with and without pituitary cannulae and all mares ovulated, showing that the procedure did not affect the reproductive axis. In all pituitary-cannulated mares the secretion of gonadotrophin-releasing hormone (GnRH), FSH and LH occurred almost continuously with broad, concurrent pulses of the three hormones superimposed upon this tonic background. Only 9% of the GnRH pulses appeared to be ineffective in inducing a rise in gonadotrophin levels. When measured in pituitary blood, gonadotrophin pulse frequency varied from 0.45 pulses/h early in the LH surge to 1.87 pulses/h at the time of ovulation. In contrast, mean pulse frequency measured in jugular blood did not exceed 1 pulse/h throughout the periovulatory period in cannulated or non-cannulated mares. The low amplitude of jugular pulses (less than 50% fractional increase) may have caused problems in identifying the pulses. In the two mares in which pituitary venous blood was sampled during more than one period before ovulation, GnRH secretion tended to be lower on the day of ovulation (day 0) than earlier in oestrus (ratio day 0:day -1; mare WV = 0.58, mare LS = 0.66), whereas LH secretion rate was higher on the day of ovulation (ratio day 0:day -1; mare WV = 1.54, mare LS = 6.68). These studies show that the painless and non-invasive collection of pituitary venous blood, which is possible only in horses, can provide a useful tool for studying hypothalamic-pituitary interactions under completely physiological conditions.