Effects of an LHRH agonist on endocrine function, LHRH receptor and LH/hCG receptor in the pituitary-gonadal axis of male rats

The effects of an LHRH agonist (LHRHa), [D-Ser (tBu)]6 des-Gly-NH210) ethylamide, on endocrine function and the LHRH and LH/hCG receptors in the pituitary-gonadal axis were examined. The LHRHa was injected at 100 ng/100 g body weight into male rats once a day for 4 weeks and its effects were observed until 2 weeks after the end of treatment. Due to LHRHa treatment, the plasma LH concentration began to increase on day 3, reached a peak on day 7, and then decreased, although it remained above the control level during the treatment. The pituitary LH content decreased on day 1, reached a minimum (about 40% of the control) between days 3 and 7, and then was maintained at 60% of the control level until week 4. In contrast, the pituitary LHRH receptor concentration increased only on day 3, and the association constant (Ka) remained unchanged during the observation period. The testis weight and plasma testosterone concentration began to decrease on day 3, reached the minimum on day 7 and remained at this level until week 4, and their levels were not completely restored to normal 2 weeks after cessation of treatment. The testicular LH/hCG receptor concentration was decreased on day 1, and markedly decreased to 10-15% of the control value between day 7 and week 4, but the Ka value was slightly increased during the treatment. However, these values had completely recovered 2 weeks after the cessation of treatment. The testicular LHRH receptor concentration increased between days 1 and 7, returned to the control level in weeks 2 and 4, and then decreased 2 weeks after cessation of treatment. Its Ka value was reduced in weeks 2 and 4. These data suggest that the inhibitory effect of LHRHa on the gonad in male rats is not due to reduced pituitary LH release, but to changes in the number and Ka values of gonadal receptors for LH/hCG and LHRH.     

Effect of neonatal castration on the content of hypothalamic LHRH, pituitary LH and plasma LH in developing male rats.

The content of hypothalamic LHRH and concentration of LH in pituitary and plasma were measured on day 5, 7, 10, 14, 17, 22, 25, 30, 45, 52 and 60 in male rats which were bilaterally castrated on day 2. The levels of plasma LH were significantly higher in all the groups of castrated rats than in normal male rats of corresponding ages. The concentration of plasma LH did not rise progressively but showed day to day fluctuation apparently due to alteration of sexual differentiation of the hypothalamus. The concentration of pituitary LH was significantly lower in neonatally castrated rats compared to normal male rats except on days 17, 25 and 30. The content of hypothalamic LHRH declined initially following castration, but from day 17 onwards significantly higher levels of hypothalamic LHRH were maintained in neonatally castrated rats than in intact control. Initial decline in the content of hypothalamic LHRH may be because of stimulation of release of LHRH which exceeds maximal rate of synthesis and subsequent increase in the content of hypothalamic LHRH may be due to enhanced LHRH synthesis as a result of castration.     

Correlation of hypothalamic LHRH, pituitary LH and plasma LH in developing rats.

Hypothalamic LHRH, pituitary LH and plasma LH levels were measured in rats of both sexes from day 5-60 after birth. The content of hypothalamic LHRH was very high in one-week-old male and female rats. It declined gradually till day 17 in the female rat and sharply on day 10 in the male rat. Subsequently the content of hypothalamic LHRH increased and showed peak values on day 25 in the female rat and on day 45 in the male rat. It decreased markedly at respective times of puberty in both sexes (day 37 in the female rat and day 52-60 in the male rat). Results of the study suggest that maturation of hypothalamo-hypophyseal-axis proceeds in three distinct stages. Observations on days 17, 25 and 37 in the female rat and on days 5, 7, 10 and 22 in the male rat clearly show an inverse relationship between hypothalamic LHRH and plasma LH and a parallel relationship between pituitary and plasma LH. Marked decline in the content of hypothalamic LHRH at respective times of puberty in both sexes indicates that the release of threshold levels of LHRH from the hypothalamus may apparently be the event initiating the pubertal changes in rat.     

Mediation by gonadal steroids of plasma beta-endorphin and LH in castrated female and male rats

Immunoreactive beta-endorphin (IR-BE) was significantly decreased and luteinizing hormone (LH) significantly increased in female rats castrated for four weeks. Forty eight hours after a single injection of estradiol benzoate (EB), IR-BE levels increased, and LH levels were reduced. On the afternoon following the administration of a second injection of EB given six hours earlier, IR-BE levels were reduced below control values, whereas LH levels were significantly elevated. There was no change in IR-BE levels during the remainder of that afternoon whereas LH levels decreased over time. Similar to female rats, IR-BE was diminished and LH increased in castrated male rats. IR-BE was increased significantly above those values observed in intact animals 24 hr after a single injection of TP and returned to control levels by 48 hr after administration of TP. Injection of TP reduced LH to levels observed prior to castration. These findings suggest that gonadal steroids exert a feedback on the release of IR-BE from the pituitary of female and male rats opposite to their feedback effect on the release of pituitary gonadotropins.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Effects of a long-acting LHRH agonist preparation on plasma gonadotropin and prolactin levels in castrated male rats and on the release of prolactin from ectopic pituitaries

We have examined the effects of a single subcutaneous injection of an LHRH agonist, D-Trp-6-LHRH, in biodegradable microcapsules of poly(DL-lactide-co-glycolide) on plasma gonadotropin and prolactin (PRL) levels in castrated and in castrated-hypophysectomized-pituitary grafted (CAST-APX-GRAFT) male rats. The results were compared to the effects of daily injections of the same LHRH agonist dissolved in saline. In castrated rats, there were no significant alterations in plasma LH or PRL levels during the 10 days following the injection of LHRH agonist microcapsules, while FSH levels were generally reduced. In castrated males given daily injections of 6 micrograms of LHRH agonist in saline, plasma LH levels were significantly reduced while plasma PRL levels were not changed. In CAST-APX-GRAFT rats, both D-Trp-6-LHRH microcapsules and daily LHRH agonist injections appeared to increase plasma PRL levels. The pattern of changes in PRL release in both groups was similar, with levels on day 6 being significantly higher than those measured on days 1, 3 and 10 after onset of treatment. As expected, LH and FSH levels in these animals were extremely low. Immunoreactive D-Trp-6-LHRH was consistently detectable in the plasma of CAST-APX-GRAFT animals after microcapsule administration, whereas in animals given daily injections of this agonist in saline, its plasma concentrations were often below the detectability limit of the employed assay. These findings suggest that the LHRH agonist, D-Trp-6-LHRH, is capable of causing a short term stimulation of PRL release from ectopic pituitaries. Elevation of plasma LH levels is apparently not required for this effect.     

Comparative effects of LHRH agonist and ovine LH administration on testicular steroidogenesis in intact adult rat

In order to better understand the effects of LHRH administration on testicular function in adult rat, we compared the inhibitory effects of LH and the LHRH analogue [D-Ser-(TBU)⁶, des-Gly-NH₂ ¹⁰]LHRH ethylamide upon testicular steroidogenesis and LH, FSH and prolactin receptor contents. Administration of LH as well as LHRH analogue resulted in a marked decrease of LH receptor levels, accompanied by a blockage at the level of 17-hydroxylase activity. We have been able to demonstrate that multiple LH administration can achieve a testicular desensitization comparable to that observed after LHRH agonist treatment.     

LH response (in vivo and in vitro) to an LHRH agonist administered to domestic male cats

We investigated plasma luteinizing hormone (LH) concentration in domestic male cats challenged with Luteinizing Hormone Releasing Hormone Analog (LHRH-A) [des Gly 10, (DTrp6)-LHRH ethylamide] that mediates the function of the hypothalamic-pituitary-gonadal axis (HPG). Plasma LH concentrations in cats treated daily with LHRH (10 microg/100 microl/kg/day, subcutaneously-s.c.) for 19 days (LHRH group) and in controls treated with saline (NaCl-0.9%, same volume-SAL group) were chronically studied. LHRH administration (s.c.) for 15 days induced a significant fall (P < 0.05) in plasma LH concentrations during the chronic study. After the 15th day of treatment the groups were divided once more into animals treated with LHRH (10 microg/100 microl/kg) or saline (i.v.), and a time course study (300 min) was performed (acute study). Next, four groups of cats were compared in an acute study involving the s.c./i.v. administration of SAL/SAL, SAL/LHRH, LHRH/SAL, and LHRH/LHRH. The responses of the SAL animals challenged by acute i.v. administration of LHRH (group SAL/LHRH) were significantly higher (P < 0.01) than those of animals treated with LHRH (sc) (group LHRH/LHRH). LH release was also significantly increased in the latter group (P < 0.05), although the effect was short lasting, being recorded only at the first observation (45 min). An in vitro study with the pituitaries was also performed on day 20. Mean (+/-SEM) LH concentrations in the culture medium containing pituitaries with LHRH (10(-7) M) or saline were determined. In vitro analysis of these pituitaries demonstrated a significantly reduced response (P < 0.05) by animals treated sc with LHRH for 19 days. This study represents a source of data for the domestic cat going beyond its own physiology. Serving as a model, this animal provide important information for the study of reproductive physiology in other members of its family (Felidae), almost all of them threatened with extinction.     

Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells

The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells.     

Demonstration and topographical distribution of LHRH receptors in the central nervous system in the normal and castrated male rat

The topographical distribution of [125I]-LHRH binding sites was studied on brain sections of adult male rat by quantitative autoradiography. High density of sites was observed in the hippocampus, amygdala and entorhinal cortex (4-7 fmol of LHRH bound/mg protein). Lower density of sites was observed in the septum and frontal cortex. The receptor density was not significantly modified at day 5 following castration. Under the same conditions the pituitary receptors were significantly increased. The presence of specific LHRH binding sites in the limbic system may explain the behavioural effect observed following intracerebroventricular injection of LHRH. However, their functions under physiological conditions remain to be elucidated.     

Reversible suppression of pituitary-testicular function by a sustained-release formulation of a GnRH agonist (leuprolide acetate) in dogs

Pituitary-testicular function was studied in 15 dogs following treatment with a sustained-release formulation of a GnRH agonist, leuprolide acetate (LA). Adult male dogs were treated with a single subcutaneous injection of microencapsulated LA (0.1 or 1 mg/kg). Treatment with LA at a dose of 1 mg/kg resulted in decreased (P<0.001) ejaculatory volume and disappearance of morphologically normal spermatozoa within 8 wk and the effect persisted for 6 wk, while the 0.1 mg/kg dose was not adequate to effect suppression of spermatogenesis. The larger dose treatment (1 mg/kg) caused a transient rise in plasma levels of LH and testosterone followed by a marked decline to below the normal level by 2 wk, the low levels being maintained for at least 5 wk, indicating a prolonged effect of LA treatment on pituitary-gonadal axis. Twenty weeks after treatment with LA, a complete return to normal spermatogenesis was observed. The full reversibility of spermatogenesis in the dog after LA treatment suggests that this peptide could be used as a reversible method of male contraception.     

Alterations in plasma concentrations of testosterone, LH, and prolactin associated with mating in the male rat.

The hormonal response of the male rat to sexual activity was investigated in two studies. In the first, no evidence of a chronic elevation in plasma levels of testosterone (T), LH, or prolactin (PRL) was observed in sexually experienced rats compared to naive controls. Both groups showed an acute increase in plasma levels of all three hormones following mating, but the increases shown by the experienced group were more pronounced. In the second study, plasma levels of T, LH and PRL rose in sexually experienced male rats following exposure to a mating arena whether it contained an estrous female, an anestrous female, or no other animal. However, the increases were considerably larger in the group exposed to estrous females. It is suggested that plasma hormones rise in anticipation of mating, although not to the same extent as following mating, and that the anticipatory rise may function to initiate or facilitate mating behavior.     

Blockade of pulsatile LH, FSH and testosterone secretion in rams by constant infusion of an LHRH agonist

Adult Soay rams were infused for 21 days with 50 micrograms buserelin/day, using s.c. implanted osmotic mini-pumps. The continuous treatment with this LHRH agonist induced a supraphysiological increase in the blood concentrations of LH (15-fold) and testosterone (5-fold) followed by a decrease below pre-treatment values after 10 days. The blood concentrations of FSH showed only a minimal initial increase but the subsequent decrease was dramatic, occurring within 1 day. By Day 10 of treatment, the blood concentrations of all 3 hormones were low or declining, LH pulses were absent in the serial profiles based on 20-min blood samples and the administration of LHRH antiserum failed to affect the secretion of LH or testosterone. By Day 21, the secretion of FSH, LH and testosterone was maximally suppressed. The i.v. injection of 400 ng LHRH was totally ineffective at stimulating an increase in the blood concentrations of LH while the i.v. injection of 50 micrograms ovine LH induced a normal increase in the concentrations of testosterone; this confirmed that the chronic treatment with the LHRH agonist had desensitized the pituitary gonadotrophs without markedly affecting the responsiveness of the testicular Leydig cells. The ratio of bioactive: radioimmunoactive LH did not change during the treatment. The long-term effect of the infusion was fully reversible as shown by the increase in the blood concentrations of FSH, LH and testosterone and the return of normal pulsatile fluctuations in LH and testosterone within 7 days of the end of treatment.     

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin

Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.     

Specific activity of LHRH and TRH degrading enzymes in various tissues of normal and castrated male rats.

The chlorophyll composition and Hill activity of the leaf, developing seed parts and pod have been studied in three species of legumes, $\text{Lathyrus latifolius}$, $\text{Pisum sativum}$ and $\text{Vicia faba}$. The studies indicate that in all the species, the level of chlorophyll, on mg/g fresh weight basis, is maximum in the leaf. However, Hill activity studies show that the cotyledonary chloroplasts in all the cases have a higher Hill activity than the leaf chloroplasts. Thus, the Hill activities of the cotyledonary chloroplasts are 340% of the leaf chloroplasts in $\text{Lathyrus}$$\text{latifolius}$, 144% of the leaf chloroplasts in $\text{Pisum}$$\text{sativum}$ and 200% of the leaf chloroplasts in $\text{Vicia}$$\text{faba}$.     

Long-term reversible suppression of oestrus in bitches with nafarelin acetate, a potent LHRH agonist

Adult cyclic beagle bitches were treated for up to 18 months with nafarelin acetate via subcutaneously implanted osmotic pumps, starting during the first week of a pro-oestrous vaginal discharge. The imminent ovulation appeared to be unaffected by treatment, but doses of 8 or 32 micrograms analogue/day reduced the integrated luteal progesterone values. No new oestrus was detected in 3 bitches during 18 months of treatment with 32 micrograms/day, which resulted in mean plasma levels of 0.4 ng analogue/ml. A return to oestrus was observed in all 3 bitches between 3 and 18 weeks after cessation of treatment: 2 of the bitches mated at those times and produced normal litters. Another 2 bitches were similarly treated with 32 micrograms analogue/day; they were mated at the oestrus at start of treatment and dosing was continued for about 63 days. One of the bitches conceived and produced a normal litter. Nafarelin acetate treatment begun during anoestrus resulted in an induced heat 1-2 weeks after the start of treatment. The induced heat consisted of pro-oestrous vaginal discharge, oestrous vaginal cytology, and ovulation (judged by increased circulating levels of progesterone). Three bitches mated at the induced heat and treated for the normal duration of gestation did not litter. Nafarelin treatment of 3 bitches before puberty did not induce signs of oestrus and prevented the occurrence of oestrus through 18 months of treatment. The first oestrus in these bitches occurred 3.5-4 months after cessation of treatment, but mating at that time did not result in pregnancy. These studies have established the feasibility of and dosage requirement for the use of the LHRH agonist as a contraceptive in the bitch.     

Photoperiodicity and circannual levels of LH, FSH, and testosterone in normal and castrated male, white-tailed deer

To establish the relation between photoperiodicity and the levels of LH, FSH, and testosterone (T) in plasma, three intact and three castrated adult male white-tailed deer were sampled once a month for 2-3 years. The rang of average LH levels in controls varied between 0.8 and 2.0 ng/mL; the levels in castrates were considerably higher, 3.4 to 8.9 ng/mL. Average levels of FSH varied in controls between 25 and 112 ng/mL and in castrates between 141 and 240 ng/mL. A significant correlation between the seasonal time course of LH and FSH was found in castrated, but not in intact bucks. In castrates both gonadotropins exhibit two major elevations coinciding with spring and fall equinoxes in March and September. The seasonal time course of FSH in castrates correlates highly with seasonal levels of FSH in controls. However, the time course of the LH curve in controls is substantially different from the curve in castrates, presumably owing to feedback mechanisms. A possible role of testicular estradiol in this feedback is discussed. In controls, peak T levels are reached in December, i.e., 3 months after maximum levels of FSH and 5 months after peak levels of LH were detected. It appears that male deer undergo two periods of reproductive stimulation (one in the spring, the other in the fall). However, the organism responds with the full range of gonadal and behavioral mechanisms leading to the initiation of the rut only during the fall.