Effects of hypothalamic neurohormones on prolactin release from pituitary allografts in the hamster

Recent reports indicate that luteinizing hormone-releasing hormone (LHRH) releases prolactin (PRL) under some circumstances. We examined the chronic effects of LHRH, growth hormone-releasing hormone (GHRH), and corticotrophin-releasing hormone (CRH) on the release of PRL, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary allografts in hypophysectomized, orchidectomized hamsters. Entire pituitary glands removed from 7-week-old-male Golden Syrian hamsters were placed under the renal capsule of hypophysectomized, orchidectomized 12-week-old hamsters. Beginning 6 days postgrafting, hamsters were injected subcutaneously twice daily with 1 microgram LHRH, 4 micrograms GHRH, or 4 micrograms CRH in 100 microliter of vehicle for 16 days. Six hosts from each of the four groups were decapitated on Day 17, 16 hr after the last injection. Prolactin, LH, and FSH were measured in serum collected from the trunk blood. Treatment with LHRH significantly elevated serum PRL levels above those measured in the other three groups, which were all similar to one another. Serum LH levels in hosts treated with vehicle were elevated above those measured in the other three groups. Serum FSH levels in hosts treated with LHRH were greater than FSH levels in any of the other three groups. These results indicate that chronic treatment with LHRH can stimulate PRL and FSH release by ectopic pituitary cells in the hamster.     

Effect of LHRH immunoneutralization on follicular development, the LH surge and luteal function in the stumptailed macaque monkey (Macaca arctoides)

A dose of 100 microliter of a potent ovine LHRH gamma globulin inhibited ovulation in the cyclic rat when administered at 12:00 h on the day of pro-oestrus. A dose of 10 ml of the preparation was administered i.v. to female stumptailed macaques to achieve circulating antibody titres 3-4-fold higher than in the rat. In an ovariectomized macaque, this caused a marked fall in serum concentrations of LH to less than 10% of pretreatment values and also a significant, though less pronounced, fall in FSH. Six monkeys were treated with the LHRH gamma globulin during the mid-late follicular phase of the cycle. In 2 monkeys in which serum oestradiol concentrations were less than 100 pg/ml at the time of antibody administration, the rising oestradiol levels were abruptly suppressed and the normal mid-cycle LH surge failed to occur. Serum concentrations of LH and FSH declined to low levels for 8-10 days after which time normal follicular development occurred. In the remaining 4 monkeys in which follicular development was more advanced as indicated by serum oestradiol concentrations of greater than 100 pg/ml, the antibodies induced either a transient decline or had no effect on the rising serum concentration of oestradiol. An LH/FSH surge followed by a rise in serum progesterone occurred in these macaques. When the antibodies were administered to a further 6 macaques, which had also been treated with oestradiol benzoate during the early follicular phase to induce an LH surge, the neutralization of LHRH again failed to block the surge even when the dose of antibody was increased to 20 ml. The results show that LHRH antibodies were unable to block the LH surge in the macaque. They contrast with results obtained with LHRH immunoneutralization in the sheep, rat, hamster, mouse and bird and suggest that the ability of oestrogen to induce an LH surge by acting directly on the LHRH-primed anterior pituitary gland is more dominant in the primate.     

Monoclonal antibodies to luteinizing hormone-releasing hormone: production, characterization, and immunocytochemical application

Luteinizing hormone-releasing hormone (LHRH) was conjugated to bovine thyroglobulin and used to immunize a BALB/c mouse. Spleen lymphocytes were subsequently fused to SP2/0 myeloma cells and two of the resulting hybridoma clones were found to produce high titer antibodies to LHRH (HU4H and HU11B); both belonged to the IgG1 subclass. Characterization of the monoclonal antibodies revealed that HU4H and HU11B have conformational and sequential specificity to LHRH, respectively, and that neither one shows significant immunoactivity with pro-LHRH. The value of these antibodies in immunocytochemical applications is demonstrated by their ability to cause intense specific staining of LHRH neuronal cell bodies and fibers in brain sections from several mammalian species.     

Evidence that luteinizing hormone-releasing hormone statin from ovine rete testis fluid is immunologically related to alphaC inhibin

LHRH Statin is a putative gonadal protein that increases the interval between two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of the alphaC subunit of inhibin. Thus, rete testis fluid (RTF) proteins were purified by immunoaffinity chromatography using antibodies against residues 1-7 plus 7-30 (experiment 1, A-fractions) and 14-28 of the alphaC inhibin subunit (experiment 2, B-fractions), and the LHRH Statin activity of the fractions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fractions that bound to the immunoaffinity column with low affinity were eluted with 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequentially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 microg, n = 4) and highly bound fractions (A-F3, 30 ng, n = 8, 150 ng, n = 3; A-F4, 120 ng, n = 5) decreased LH mean plasma levels between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respectively (P<0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 microg, n = 4) had no activity. In experiment 2, RTF (100 microg, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels by 48% and 38%, respectively (P<0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 microg, n = 4) had no effect. A fraction obtained from B-F3 by gel filtration had significant LHRH Statin activity (63%, n = 6, P<0.001). PAGE with colloidal gold staining revealed 3 high molecular weight bands and 5 low molecular weight bands in B-F3. The 3 high molecular weight bands were shown to belong to the clusterin family and did not appear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-alphaC inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14-28 alphaC inhibin sequence.     

Endocrine Effects of Heat Stress in Boars

The purpose of this study was to describe the temporal changes in peripheral plasma levels of testosterone and Cortisol in boars during and after heat stress. A total of 8 boars were utilized, 4 of them were exposed to 35°C, for 100 h in a climatic room, and 4 served as controls and were kept at 20 °C for 100 h in the climatic room. Blood samples were obtained via permanent vein catheters 3 times daily from 5 days before heat stress until 20 days after termination of heat stress. Testestorone levels were determined by radioimmunoassay and Cortisol by a competitive protein binding technique. For both hormones the pre-exposure levels were similar in both groups of boars. The control boars had significantly higher testosterone levels, while being in the climatic room, than during any other period. The experimental boars had slightly increased testosterone levels during the first day of heat stress and thereafter continuously decreased levels. In the control boars the testosterone levels returned to pre-exposure levels immediately after removal from the climatic room, whereas in the experimental boars the testosterone levels were dramatically increased during the first 5 days after exposure. The differences in Cortisol levels, between the 2 groups of boars were restricted to the period spent in the climatic room. During this period the experimental boars had significantly higher Cortisol levels.     

Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs

The objective of this study was to improve normal fertilization, male pronuclear formation and embryonic development following intracytoplasmic injection of dithiothreitol (DTT)-treated boar spermatozoa. To determine the effect of DTT treatment, frozen-thawed boar spermatozoa were treated with DTT for 0, 10, 30, and 60 min, and injected into porcine oocytes. The effects of DTT and male difference on normal fertilization and embryonic development were investigated. The mean normal fertilization rate in the groups treated with DTT for 30 min (73.8%) and 60 min (74.9%) was higher (P < 0.05) than that in the control group (49.3%). The mean blastocyst formation rate in the group treated with DTT for 30 min (23.2%) was higher (P < 0.05) than that in the other groups (8.7-10.9%). Among boars there was no difference in normal fertilization, but there was a significant difference between the non-treated and the DTT-treated groups. The mean rate of blastocyst formation was different (P < 0.05) among boars, and between the non-treated and DTT-treated groups. The mean number of cells in blastocysts was similar among the boars and between the non-treated and the DTT-treated groups. In conclusion, DTT treatment for 30 min increased the rate of normal fertilization and embryonic development to the blastocyst stage. Furthermore, the rate of blastocyst formation of oocytes injected with spermatozoa differed among boars.     

Effect of 3-week treatment with [D-Trp6, des-Gly-NH10(2)]LHRH ethylamide, aminoglutethimide, ketoconazole or flutamide alone or in combination on testicular, serum, adrenal and prostatic steroid levels in the dog

Adult male mongrel dogs were treated with the LHRH agonist [D-Trp6, des-Gly-NH10(2)]LHRH ethylamide, aminoglutethimide, ketoconazole or flutamide alone or in combination for 21 days before measurement of steroid levels in the testes, prostate, adrenals and serum. Ketoconazole alone caused a marked stimulation of the intra-testicular concentration of pregnenolone, 17OH-pregnenolone, progesterone and 17OH-progesterone with no or little change of androstenedione, testosterone and dihydrotestosterone. Aminoglutethimide caused a 30-95% inhibition in the concentration of all steroids in the tests while treatment with the LHRH agonist caused a near complete inhibition of all testicular steroids. When administered concomitantly with the LHRH agonist, ketoconazole partly prevented the inhibitory effect of the LHRH agonist on testicular steroid levels. Serum levels of dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione and androstane-3 alpha, 17 beta-diol were 75 to 95% inhibited by the LHRH agonist while serum testosterone and dihydrotestosterone concentrations were reduced below detection limits by the same treatment. Moreover, treatment with the LHRH agonist caused a 70-95% reduction in the intraprostatic concentration of testosterone and dihydrotestosterone in all the groups although maximal effect was observed when the LHRH agonist was combined with any of the three other agents. The present data show that while treatment with ketoconazole, aminoglutethimide or Flutamide alone has only partial inhibitory effects on androgen levels, combination with an LHRH agonist provides maximal inhibition. In addition to its direct blockade of the androgen receptor, some of the effect of Flutamide could be related to its blockade of testicular 3 beta-hydroxy-steroid dehydrogenase activity.     

Effects of complete hypothalamic deafferentation on the estrous phase of follicle-stimulating hormone release in the cyclic rat

We investigated whether neural afferents to the medial basal hypothalamus play an acute role in the estrous phase of FSH release in the 4-day cyclic rat. A cannula was inserted into the right atrium of the heart under brief ether anesthesia during the early afternoon of proestrus for subsequent blood collections and injection of LHRH. In some of the rats, the medial basal hypothalamus was surgically isolated from the rest of the brain with a small knife under brief ether anesthesia between 2000 h and 2130 h of proestrus. Control groups consisted of naive rats which were not treated during the night of proestrus and sham-operated animals in which the knife was lowered to the corpus callosum between 2000 h and 2130 h or proestrus. Rats were bled at 2200 h of proestrus and at 0200 h, 0600 h and 1000 h of estrus for radioimmunoassay of plasma FSH and LH. The plasma FSH levels in all 3 groups between 2200 h of proestrus and 1000 h of estrus were elevated above levels observed in other cannulated rats bled to the onset of the proestrous phase of FSH release at 1400 h of proestrus. There were no statistically significant differences in plasma FSH or LH concentrations at any of the time periods between the 3 groups of serially bled rats. The deafferentation procedure did not appear to impair the pituitary gland's ability to secret gonadotrophins as injection of 50 ng of LHRH after the bleeding at 1000 h of estrus caused substantial elevations in plasma FSH and LH concentrations which were not different between the 3 groups. The results suggest that neural afferents to the medial basal hypothalamus play no acute role in the estrous phase of FSH release in the cyclic rat.     

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.     

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.     

Effects of aging on pituitary and testicular luteinizing hormone-releasing hormone receptors in the rat

Aging exerts profound influences on the function of the hypothalamic-pituitary-testicular-axis. This work has been performed in order to verify whether, in male rats, the decreased secretion of LH and testosterone (T) occurring in old animals is reflected by modifications of luteinizing hormone-releasing hormone (LHRH) receptors at the level of the anterior pituitary and of the testes. To this purpose, the affinity constant (Ka) and the maximal binding capacity (Bmax) for the LHRH analog [D-Ser(tBu)6]des-Gly10-LHRH-N-ethylamide were evaluated, by means of a receptor binding assay, in membrane preparations derived from the anterior pituitary and testicular Leydig cells of male rats of 3 and 19 months of age. Serum levels of LH and T were measured by specific RIAs. The results obtained show that, in aged male rats, the concentration of pituitary LHRH receptors is significantly lower than that found in young animals. On the other hand, the concentration of LHRH binding sites is significantly increased on the membranes of Leydig cells of old rats. In no instance the Ka for the LHRH analog is significantly affected. Serum levels of LH and T are significantly lower in old than in young male rats. In conclusion, these results suggest that the reduced secretion of LH in old male rats may be linked, at least partially, to a decrease of the number of pituitary LHRH receptors. The impaired production of testosterone occurring in aged rats is accompanied by a significant increase of the number of testicular LHRH receptors, indicating that also the intratesticular mechanisms controlling testosterone release undergo significant alterations with aging.     

Structural requirement for the recognition of luteinizing hormone releasing hormone (LHRH) by monoclonal and conventional anti-LHRH antibodies

Immunochemical studies on monoclonal and conventional anti-LHRH antibodies have been reported. The association constants (Ka) were in the order of 10(9)-10(10) L/mol when calculated from Scatchard and Steward-Petty plots. Heterogeneity indices (a) calculated from Sips plot were nearly 1.0, indicating the homogeneous nature of monoclonals. Most of the conventional anti-LHRH produced by monkey, baboon, rabbit, and dog, by immunization using LHRH linked to tetanus toxoid by the carbodiimide condensation method, showed a single slope in Scatchard analysis (except two dog antisera) and a values were nearly 1.0. Monoclonals and conventionals reacted most strongly with native LHRH(NH2). Monoclonals showed poor reactivity with LHRH free acid and LHRH fragments containing free acid. The C-terminus tetrapeptide 7-10 showed 10 times more reactivity than tripeptide 4-6. The heptapeptide 4-10 showed 100 and 1000 times more reactivity than the tetra- and tri-peptide, respectively. Introduction of the tripeptide pGlu-His-Trp-OH to heptapeptide 4-10 caused five times more inhibition in reactivity than the heptapeptide. Conventional anti-LHRH antibodies manifested specificity to the C-terminus of LHRH. These sera did not react with LHRH free acid and LHRH fragments of sequence 4-6, 7-10, and 4-10. The complete loss of reactivity of conventional antibodies and poor reactivity of monoclonal antibodies was partly regained when LHRH free acid was coupled to Lys, Lys-MDP, or (Ala)2-tuftsin, suggesting that for monoclonals and conventionals the antigenic determinant was confined to the conformation involving the C-terminus amide of LHRH.     

The effects of porcine follicular fluid inhibin on gonadotropin secretion in the rabbit

Porcine follicular fluid (pff), treated with charcoal to remove steroids, was used to determine whether inhibin is active in the laboratory rabbit. When pff (5 ml/4 kg body weight) was injected (ip) into does that had been castrated 2 weeks earlier, there was a significant decline in blood follicle-stimulating hormone (FSH) levels; the decline lasted for 8-12 h. Blood levels of luteinizing hormone (LH) were suppressed, but only briefly at 3 h after injection. In other experiments, intact does which had been injected with pff 9 h and 10 min before receiving a single, i.v. injection of luteinizing hormone-releasing hormone (LHRH) (10 micrograms/kg body weight) showed a sharp reduction in the concentration of LH in the blood samples collected 15, 30 and 60 min after LHRH administration. Secretion of FSH responded poorly to LHRH stimulation, and pff had little suppressive action on blood levels. Having established that the pff preparation had inhibin activity, its action on the postovulatory surge of FSH secretion was next examined. This release of FSH, which occurs 6 to 36 h after ovulation, has been hypothesized to be required for the establishment of pregnancy by stimulating the growth of the ovarian follicles supplying the luteotropic estradiol. To test this hypothesis, pff was injected into rabbits every 8 h for the first 5 days of pregnancy and found to block the postovulatory FSH surge. The patterns of secretion of LH and progesterone in the same pff-injected animals were, however, not altered from normal pregnancy patterns by pff.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of feeding level on semen quantity and quality of breeding boars

An experiment was carried out to investigate the influence of feeding level on semen quantity and quality in breeding boars in two successive periods of 12 and 8 weeks. Forty-two Yorkshire boars (13 months old) were fed for 12 weeks ad libitum (H=5.74 kg/day), a medium feeding level (M=3.62 kg/day) or a low feeding level (L=1.92 kg/day). After 8 weeks the number of ejaculated sperm cells for boars on treatment L was lower than for those on treatments H and M (P <0.05). In the last 2 weeks, the M and H boars ejaculated 46 and 69% more sperm cells, respectively, than the L boars. Differences between all three feeding levels were significant (P < 0.05). After this 12-week period the feeding level was altered for the H and the L boars. H boars were fed a low feeding level (HL) and L boars were fed the medium feeding level (LM). The medium-fed boars were kept at the same feeding level (MM). After 8 weeks in the second period, differences in the number of ejaculated sperm cells between the HL and MM boars and between the LM and MM boars were no longer significant (P >0.05). Throughout the experiment, no differences in the quality parameters, percentage of moving sperm cells in the ejaculate, vitality of the moving sperm cells and non-return percentage at 56 days were found between treatment groups.     

Secretion of alpha-subunit and intact gonadotropins after surgical and chemical castration.

In order to investigate the regulatory mechanisms involved in the secretion of glycoprotein hormones, we studied the secretory patterns of LH, FSH and alpha-subunit in hypogonadal men. Three groups of patients with carcinoma of the prostate were studied both before and 15 days after orchiectomy, or the initiation of ketoconazole or LHRH analog therapy. There were significant increases (P less than 0.01) in LH and alpha-subunit levels in the patients treated with orchiectomy and ketoconazole, but FSH levels increased only in the orchiectomized patients. After LHRH analog treatment, LH levels were significantly decreased when assayed with an immunoradiometric assay method which does not cross-react with alpha-subunit. FSH values were significantly lower than pretreatment levels, while alpha-subunit levels remained significantly elevated throughout the study period. These results demonstrate that after both chemical (ketoconazole) and surgical castration, the secretion of alpha subunit follows a pattern which is tightly correlated with that of LH but not of FSH. However, after LHRH analog treatment, alpha-subunit appears to be the sole secretory product of the gonadotroph.     

Maturation of the hypothalamo-pituitary-gonadal axis of male Syrian hamsters.

Light-microscope immunocytochemistry (ICC) was used to investigate postnatal changes in the morphology of LHRH neurons in the brains of male Syrian hamsters and to relate these changes to more overt maturational developments within the hypothalamo-pituitary-gonadal axis. The animals were maintained under long-day photoperiods (14L:10D), and groups of 6-7 were killed at 10-day intervals from Day 15 to Day 65. Their brains were fixed with 4% paraformaldehyde, sectioned sagittally with a vibratome (75 microns), and processed for ICC using monoclonal LHRH antibody HU4H. Throughout the study period, the hamsters showed a progressive increase in plasma gonadotropin levels, closely followed by an increase in testicular weight and plasma testosterone levels. Histology of the testes revealed that spermatogenesis was already qualitatively completed by Day 35 and quantitative aspects were established by Day 45. Within the brain, LHRH neuronal perikarya were distributed primarily in the medial septal-preoptic area and the diagonal band of Broca; morphologically, these immunopositive neurons were either monopolar or bipolar. The total number of LHRH neurons detected in the areas examined was approximately 440 throughout the developmental period, and the relative proportions of monopolar and bipolar subtypes (86% and 14%, respectively) remained unchanged. In contrast, the area of the perikarya, as determined by autoimage analysis, showed a highly significant age-related increase, both for the monopolar and bipolar neurons. It is suggested that these developmental changes in the LHRH neurons reflect an increase in LHRH synthesis and may, therefore, provide a neuroendocrine trigger for the onset of puberty.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Effect of luteinizing hormone releasing hormone (LHRH) and [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide on alpha-subunit and LH beta messenger ribonucleic acid levels in rat anterior pituitary cells in culture

The effect of incubation with LHRH and its agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide has been measured on the concentrations of mRNAs for the common alpha-subunit of glycoprotein hormones and beta-LH in rat anterior pituitary cells in primary culture. After incubation, total RNA was analyzed by Northern blot or dot blot hybridization with alpha- and LH beta 32P-labeled cRNA probes and mRNA levels were quantified by autoradiography. Short-term treatment (4-6 h) of pituitary cells with 100 nM LHRH led to a marked stimulation of LH release but no effect was observed on alpha-subunit or LH beta mRNA levels. Longer (24-72 h) incubation periods with LHRH led to complete desensitization of the LH response to the neurohormone and induced 2- to 3-fold increases in alpha-mRNA cell content while LH beta mRNA levels remained unchanged. Maximal induction of alpha mRNA accumulation was observed with an LHRH concentration as low as 0.1 nM. Incubation with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide for 24-72 h also increased alpha mRNA but did not modify LH-beta mRNA levels. It is concluded that long-term exposure of anterior pituitary cells to LHRH or to an LHRH agonist positively regulates alpha-subunit gene expression in the absence of change in LH beta mRNA levels. This observation can provide an explanation for the high plasma levels of free alpha-subunits found in patients treated chronically with LHRH agonists.     

Active immunization of boars against gonadotropin releasing hormone. II. Effects on libido and response to testosterone propionate

Of 20 sexually mature Duroc boars showing normal libido, 10 were actively immunized against gonadotropin releasing hormone (GnRH). After immunization against GnRH, boars showed minimal sexual interest in an estrous female, while untreated boars showed normal libido. Eight of the boars actively immunized against GnRH were randomly assigned to treatment (T) or control (C) groups. Boars in the T and C groups were given testosterone propionate or vehicle, respectively, on Days 0, 5, 10, and 15. Boars in both groups were observed for libido in the presence of an estrous female every 4 d for 28 d. Mean libido score for T boars increased gradually until all boars displayed maximum libido on Day 20, but libido returned to low levels on Day 28. In contrast, C boars remained sexually inactive throughout the study. The results of this study indicate that active immunization of sexually mature boars eliminates sexual behavior and that sexual behavior can be restored quickly by administering testosterone propionate.