Pituitary LH response to LHRH in male rats following pentobarbital anesthesia

Plasma LH concentrations were determined in unanesthetized or pentobarbital (PB) anesthetized male rats following several doses of LHRH administered into the lateral ventricle of jugular vein. Regardless of the route of injection of LHRH, plasma LH concentrations were similar whether animals received PB anesthesia or not. No evidence was found that PB enhanced or diminished the response of the pituitary to LHRH in male rats.     

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.     

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus)

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.     

Effects of androgen administered to neonatal male rats on hypophyseal gonadotropin content, secretion and response to LHRH at adulthood

Supraphysiologic doses (1.75-3.50 mg) of testosterone propionate (TP) administered to male rats on the day of birth and 24 h later resulted in markedly reduced serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in adult males castrated for 16 days. These effects diminished as androgen was injected on succeeding postnatal days. Since exogenous dihydrotestosterone and testosterone were similarly effective, aromatization to estrogen is not required to elicit these effects. No build-up of either gonadotropin occurred in the pituitaries of TP-treated animals; pituitary LH content was appreciably reduced, while FSH remained unchanged. These data imply that hypophyseal synthesis and secretion of gonadotropins are curtailed in adult castrated males who have been androgenized neonatally. Pituitaries of such neonatally treated animals, however, were capable of increased secretion of LH in response to a challenge of luteinizing hormone-releasing hormone. These findings are compatible with a model in which an androgen suppressible event occurs at a suprahypophyseal level, e.g., hypothalamus or higher brain centers, in the male rat during a restricted neonatal period, which is responsible for programming the development of mechanisms involved in accumulation and secretion of gonadotropins.     

Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

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.     

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.     

Annual cycle in LH and testosterone release in response to GnRH challenge in male woodchucks (Marmota monax)

Testosterone and LH concentrations were determined in serum samples obtained before and 15 min after injections of GnRH (1 microgram kg-1) administered at 4-7 week intervals over 20 months to groups of male woodchucks (n = 6-7) born and maintained in Northern Hemisphere (boreal) versus Southern Hemisphere (austral) simulated natural photoperiods, beginning at 18-24 months of age. Nadir and peak unstimulated testosterone (0.1 +/- 0.01 and 7.0 +/- 0.1 ng ml-1, respectively) and LH (0.8 +/- 0.2 and 8.1 +/- 1.1 ng ml-1, respectively) concentrations did not differ in boreal versus austral males. In the five boreal and five austral males that were confirmed to be photoentrained, basal (pre-GnRH) concentrations of LH and testosterone were lowest in summer, increased simultaneously in late autumn or early winter, and declined in the spring. GnRH stimulated some LH release throughout the year except for a 1-4 month period in the summer. The initial annual increase in the LH response to GnRH occurred in early autumn, and in 17 of 20 cycles it occurred 1-2 months before the initial increase in basal LH was detected. In the three free-running males not entrained to the photoperiod, the endocrine patterns were similar but were advanced by several months. The results demonstrate that in woodchucks there is a late autumn increase in LH secretion associated with the onset of testicular recrudescence, and an early autumn increase in pituitary response to GnRH before a detectable increase in serum testosterone.     

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.     

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.     

Hypothalamic luteinizing hormone-releasing hormone (LHRH) and LH secretion in aging female rats

Age-related changes in hypothalamic luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) secretion were studied in young (6 months), middle-aged (12 months) and old (18 months) female rats. The LHRH levels in the mid-hypothalamic area were higher in intact middle-aged and old females than in young ones. Additionally, there was no age difference in the hypothalamic LHRH levels in male rats. In order to clarify the significance of this age-related increase in female rats, we examined the effects of progesterone treatment in estrogen-primed ovariectomized young and old rats on the LHRH levels in the median eminence (ME) and on plasma LH levels. We found phasic changes in ME-LHRH and plasma LH levels in estrogen-primed rats following progesterone treatment in rats of both ages, but the progesterone-induced change in ME-LHRH levels tended to be delayed in old rats compared with young females. This delay may correspond to the delayed onset, slow and low magnitude of plasma LH increase in old females. The ME-LHRH levels were generally higher in old rats than in young rats. Nevertheless, we found that the increase in plasma LH in response to progesterone treatment in estrogen-primed ovariectomized females was smaller in old rats than young rats. These results suggest that the LHRH secretory mechanism changes with age in female rats. Such alterations may result in the accumulation of LHRH in the mid-hypothalamic area and an increase in ME-LHRH.     

Artificial insemination in domestic cats (Felis catus)

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 degrees C or after cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50x10(6) spermatozoa, while, in another report, the conception rate was 78% after AI with 80x10(6) spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2x10(6) and 8x10(6) spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4x10(6) spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50x10(6) frozen/thawed spermatozoa resulted in a conception rate of 57%. Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.     

Downward regulation of plasma LH by LHRH agonist, leuprolide acetate, resulting in inhibited renal growth and function in the castrated male rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.     

Changes in serum luteinizing hormone (LH) concentrations in response to luteinizing hormone releasing hormone (LHRH) in bull calves that attained puberty early or late

The objectives of this study were to determine if the response to luteinizing hormone releasing hormone (LHRH) could be used to select bull calves capable of early sexual maturation and to establish the optimum route and dose of LHRH. In Trial 1, at 4, 10 and 20 week of age, 20 calves were treated iv with 2 microg/kg body weight of LHRH 1 and 5h after commencing a 9-h period of blood sampling. Bulls were separated into early and late maturing (n=10), based on age at puberty (scrotal circumference (SC) of >or=28 cm). At 4 and 20 week of age, peak serum LH concentrations and area under the LH response curve in response to LHRH were lower (P<0.05) in early- versus late-maturing bulls. In Trial 2, calves at 20 week of age were given LHRH as follows: 2 microg/kg body weight iv (n=6), im (n=6) or sc (n=6); 5 microg/kg im (n=6), or ischio-rectally (ir, n=6) or sc (n=6); and 10 microg/kg im (n=6) or sc (n=6). Serum LH concentrations were at a plateau from 30 to 165 min after treatment with 5 microg/kg of LHRH (im or ir; P>0.05). We concluded that the LH responses to LHRH in calves at 4 and 20 week of age could facilitate the development of a simple test (one blood sample prior to treatment with LHRH and a second during the period of sustained response to LHRH) to select early-maturing bulls.     

Immunohistochemical study of the responsiveness of LH cells of fetal rats to synthetic LHRH in vitro

Summary Fetal rat pituitaries on days 17–19 of gestation were maintained in serum-free Medium 199 for 24 h in the presence of 1, 10 and 100 ng/ml of synthetic LHRH. Immunohistochemical examination of such stimulated tissue reveals a complete depletion of immunoreactive material in most of the LH cells, irrespective of the LHRH concentrations tested, though some cells remain weakly immunopositive in the pituitaries of later developmental stages. Once discharge has occurred, there is little reaccumulation of secretory material in LH cells during prolonged incubation for 48 h in LHRH-free medium containing 10% calf serum. The LHRH treatment causes no immunohistochemical change in TSH cells.It is concluded that in fetal rats recently differentiated LH cells can release the secretory product if they are stimulated by hypothalamic 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.