Immunoreactive and bioactive LH release from pituitaries of intact or castrated male rats: effect of in vitro GnRH and KCl administration

The in vitro immunoreactive (i-LH) and bioactive (b-LH) LH release from hemipituitaries of intact adult male rats (INT) or rats castrated 7 days earlier (CAS) was studied. Hemipituitaries were incubated for 30 min (time 1) plus an additional 30 min (time 2) with GnRH (10 nM) and/or KCl (50 mM), according to one of the following protocols: media alone (C), KCl+KCl (K/K), GnRH+GnRH (G/G), KCl+GnRH (K/G), GnRH+KCl (G/K). All of the hemipituitaries were further incubated in media alone for 120 min (time 3). I-LH, b-LH and i-FSH were assayed on the media. In both models, the highest bioactive:immunoactive (b/i) ratio was noted during time 1; however, CAS always secreted more b-LH than INT at any given time of the study. In INT, GnRH--but not KCl--administration during time 2 resulted in blunted i-LH. During the same time, the b/i ratios decreased in all groups but G/K. LH secretion recovered during time 3 in all groups. In CAS, i-LH levels comparable to those of time 1 were sustained by either stimulus during time 2, while the b/i ratios were markedly decreased. LH secretion recovered in the K/K group during time 3. These results suggest that: 1) promptly releasable pools of b-LH are available in both models; 2) CAS always secrete more b-LH; 3) in INT, desensitization occurs involving parallel changes in both i-LH and b-LH, while changes in b-LH rather than i-LH are noted in CAS; 4) prolonged KCl administration might play a role in new gonadotropin synthesis and/or release.     

Apoptosis occurs independently of the release of interleukin-1 beta in the anterior pituitary of end-lactating rats

The mechanism of Interleukin-1 beta (IL-1 beta) release remains unknown. Because of the absence of typical peptide signal on the precursor, IL-1 beta is not secreted through the classical pathway. The aim of this study was to determine whether IL-1 beta is released during apoptosis, as has been reported for activated macrophages. We chose anterior pituitary cells of end-lactating rats because of their capacity to produce IL-1 beta spontaneously and because this organ undergoes cellular degeneration. The combination of two techniques, reverse haemolytic plaque assay (RHPA) and terminal transferase dUTP nick end labelling (TUNEL), allowed us to observe simultaneously the release of IL-1 beta and apoptosis. Our results show that in these conditions apoptosis is not the mechanism of IL-1 beta release.     

A possible role of the pineal gland in acute immobilization-related suppression of naloxone-induced LH release in ovariectomized estrogen-primed rats

Prostaglandin (PG) D2, PGJ2 and delta12-PGJ2 are antiproliferative eicosanoids. We investigated the production of PGD2 by murine bone marrow-derived mast cells (BMMC) taking into consideration metabolism of PGD2 to PGD2 and delta12-PGJ2. PG-metabolites were quantified by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). Stimulated with calcium ionophore A23187 BMMC released eight-fold more PGJ2 and delta12-PGJ2 than PGD2. Conversion of endogenously produced PGD2 to PGJ2 and delta12-PGJ2 proceeded rapidly in contrast to metabolism of exogenously added PGD2. The antiproliferative potency of these prostaglandins is demonstrated in vitro. We conclude that determination of PGD2 production by mast cells must take into consideration rapid conversion to active derivatives, which may play a significant role in growth regulation.     

LH-RH-dependent synthesis of protein necessary for LH release from rat pituitary glands in vitro

Anterior pituitary glands of intact diestrous and gonadectomized female rats were incubated with a supramaximally active dose of LH-RH. Puromycin (P) and cycloheximide (C) did not consistently affect the LH release from glands of ovariectomized rats. In intact rats, LH release induced by LH-RH occurred in two phases, an initial one (relatively slow rate of release) and a secondary one (high rate of release). P and C had no effect on the initial phase, but prevented the secondary one. Pre-incubation with LH-RH prevented the effect of P and C added after 4 hours. Since P and C did not affect the total amount of LH present in media and hypophyses, the LH-released during the P- and C-sensitive phase is not newly formed LH. It is concluded that LH-RH induces the synthesis of protein which is necessary for LH-RH-induced LH release. The initial phase of LH release is made possible by protein already present in the glands at the beginning of the experiment.     

Effect of GnRH antagonists on phorbol ester-induced LH release from rat pituitary gonadotrophs

We previously reported that a blockade of GnRH receptor activation inhibited the already-initiated C-kinase pathway(s). We tried to investigate whether this finding is a general phenomenon or not. In this study, we employed three GnRH antagonists, [D-Phe2,Pro3,D-Phe6]-GnRH, [Ac-D-Nal-Ala1,D-pCl-Phe2,D-Ser(Rha)6]-GnRH, and [Ac-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]-GnRH (referred to as #1-, #2-, #3-GnRH antag., respectively). Each antagonist was examined for its potency against GnRH by analyzing its inhibitory effect on LH release from pituitary gonadotrophs as well as on the increase in the cytosolic Ca2+ concentration. As a result, the #1-GnRH antag. was found to be weaker than the other two compounds. Consistent with a previous study, the #3-GnRH antag. inhibited the action of TPA on LH release. However, independently of their potency as GnRH-antagonists, the two other antagonists had no inhibitory effect on TPA-induced LH release. While it is generally accepted that the C kinase pathway plays a major role in the GnRH-induced LH release, not all GnRH antagonists can inhibit LH release by blocking the already-activated C kinase system.     

Decline of sexual behavior in castrated male rats: effects of female precopulatory behavior

Ventricle cells from neonatal rats were cultured in a medium preventing cell proliferation on a modified Roller apparatus with a defined pericellular oxygen partial pressure (pO2) of 38 or 0.6 mm Hg for about one week. The cells were harvested after the second and eighth day of culture (corresponding to one or seven days of culture under a defined pO2) and prepared for electron microscopy. Electron micrographs of this material were examined by stereologic techniques. The obtained results showed some deviations of mitochondrial fine structure of heart muscle cells depending on oxygen supply. During cultivation with a pO2 of 0.6 mm Hg (hypoxic conditions) we observed a proportional increase in mitochondria without cristae and an increase in size accompanied by a more irregular shape. On the contrary, the mitochondria of cells cultured with a pericellular pO2 of 28 mm Hg, which we consider to be a normoxic condition, did not show any deviation of inner membrane arrangement, but an increase in number and a decrease of size during cultivation was apparent. The mitochondria-myofibrils ratio decreased during cultivation in both conditions of oxygen supply. The ratio between sarcoplasmatic reticulum and myofibrils decreased markedly at a pO2 of 0.6 mm Hg.     

Reversal of ethanol-induced testosterone suppression in peripubertal male rats by opiate blockade

Teenage drinking is a major problem in the United States, as well as abroad. Besides psychosocial implications, ethanol (EtOH) has detrimental effects on the reproductive system. Clinical problems associated with reduced reproductive hormones include osteoporosis, decreased muscle function, anemia, altered immune function, prostate involution, and decreased reproductive abilities. Education coupled with strategies aimed at preventing these deleterious consequences even in the face of continued EtOH intake is extremely important. We have tested the possibility that naltrexone, a drug currently used in patients to decrease alcohol craving, might also prevent the fall in the male hormone, testosterone, caused by EtOH exposure. Rats aged 35 days old (prepubertal), 45 days old (midpubertal), and 55 days old (late pubertal) were injected (intraperitoneally) with either saline, EtOH, naltrexone, or EtOH plus naltrexone. In the two older age groups, EtOH significantly suppressed testosterone, which was prevented by administration of naltrexone. In the youngest animals, there was no treatment effect presumably due to low basal levels of testosterone. EtOH similarly reduced luteinizing hormone (LH), but this suppression was not prevented by naltrexone. There was no consistent effect of any treatment on hypothalamic concentration of pro-LH releasing hormone (RH) (LHRH), LHRH, or on steady-state levels of LHRH mRNA. We conclude that, as animals progress through puberty, EtOH suppresses LH and testosterone. The testosterone decline can be prevented by opiate blockade with naltrexone, an effect primarily seen at gonadal level. Thus, naltrexone, a drug already used clinically to reduce EtOH intake, also has protective physiological effects on the endocrine system.     

In vivo release of interleukin-1 beta into hypothalamic extracellular fluid in rats: effects of repeated sampling

Interleukin-1 beta (Il-1 beta) concentrations in extracellular fluid (ECF) withdrawn at 10-min intervals through a push-pull cannula (PPC) located in the hypothalamus were studied in freely behaving male rats for 1 h at 24 and 72 h and again at 7 days after PPC implantation. Il-1 beta concentrations in ECF were similar in the latter. However, when ECF was sampled at 3 h and again 7 days after PPC implantation, Il-1 beta concentrations were greatly elevated at 7 days when compared to all other intervals. These results demonstrate how the relationships between Il-1 beta measured in ECF and the conditions of measurement appear to be integral parts of a whole intracerebral system: cytokine concentrations appear to be inextricably bound to intrahypothalamic conditions created by the sampling device presence and frequency of use.     

Ventromedial hypothalamus and short-term feeding suppression by caerulein in male rats.

A series of 6 experiments with 88 male Sprague-Dawley rats examined the neural loci responsible for caerulein's suppression of eating. Caerulein is a decapeptide chemically and physiologically similar to cholecystokinin, a naturally occurring gut hormone in rats. Ss with lesions in the ventromedial hypothalamus (VMH) showed reduced sensitivity to caerulein (1 mug/kg); Ss with lateral hypothalamic (LH) destruction showed heightened sensitivity. Microinjections of caerulein into the VMH, but not into the LH, limited feeding. Finally, tritiated carulein was selectively bound to tissue in the VMH. Results are discussed in terms of the hypothesis that the VMH manages postprandial inhibition in the rat. (26 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Androgen action during male sex differentiation includes suppression of cranial suspensory ligament development

The cranial suspensory ligament is located on the border of the cranial (mesonephric) mesentery in adult female mammals, which runs between the cranial pole of the internal genitalia and the dorsal abdominal wall. Absence of the cranial suspensory ligament in male mammals depends upon exposure of its primordium to fetal testicular androgens and is a prerequisite for testis descent. Female rats were exposed to 5alpha-dihydrotestosterone propionate at different stages of genital development, and cranial suspensory ligament development was studied in neonatal and in adult animals. Androgens suppressed cranial suspensory ligament development when exposure started during the early stages of genital development, until day 19 postconception (pc). Androgen receptor expression was immunohistochemically detected in the cranial mesentery of both sexes from day 16 pc onwards. A decrease of androgen receptor expression in female fetuses from day 18 pc onwards coincided with the appearance of a differentiated cranial suspensory ligament, as evidenced by the expression of two cell differentiation markers: alpha-smooth muscle (alpha-SM) actin and desmin. alpha-SM actin was located on the outer border of the cranial mesentery of both sexes at day 17 pc, and expression increased only in female fetuses. On day 19 pc, desmin expression was also detectable in the a-SM actin-positive cells. Proliferation and apoptosis indices of cells in the cranial mesentery, as analysed by 5'-bromodeoxyuridine incorporation and by detection of DNA strand breaks (TUNEL method) respectively, did not show any difference between the sexes, neither on day 17 nor on day 18 pc. Since primordial cells of the cranial suspensory ligament highly express the androgen receptor during the period of gestation when androgens can suppress cranial suspensory development, altered morphogenesis of these cells may be a direct consequence of androgen action.     

Sexually differentiated role of calcium ions in chicken GnRH-I-stimulated release of LH from anterior pituitary glands from adult domestic chickens

The addition of a muscarinic agonist, carbachol (Carb, 0.1 mM), to a physiological medium markedly increased Ca(2+)-dependent transglutaminase (TG) activity (approximately 10-fold) in isolated rat superior cervical sympathetic ganglia (SCG) following in vitro aerobic incubation for 30 min at 37 degrees C. The Carb-evoked stimulation of ganglionic TG activity was considerably reduced (-51%) in the presence of NG-monomethyl-L-arginine (L-NMMA, 50 microM), a selective inhibitor of nitric oxide (NO) synthase. While the suppressant effect of L-NMMA was completely eliminated by the addition of an excess concentration of L-arginine (0.5 mM), a precursor of NO. These observations imply that Carb-induced TG activation possibly involves NO mediation in SCG tissue. The Carb-induced elevation in ganglionic TG activity was markedly reduced (-84%) at as early as 15 min of incubation in the medium containing hemoglobin (Hb) (20 microM), an agent that scavenges only extracellular NO gas. Thus, it is evident that a large fraction of NO released from inside the neuronal cells to extracellular space could rapidly diffuse back into the same group of cells to induce activation of the tissue TG. Methylene blue (MB), an inhibitor of soluble guanylate cyclase (GC), at 0.5 mM, a concentration which is effective in almost abolishing the Carb-evoked synthesis of cyclic GMP (cGMP), had no effect on ganglionic TG activation induced by Carb. Therefore, an increase in cGMP synthesis mediated by NO might not participate in NO-dependent ganglionic TG activation following the stimulation with Carb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed rats

The present study was undertaken to evaluate the role and possible interaction of the endogenous opioid peptide (EOP) and corticotropin-releasing factor (CRF) in the acute stress-induced suppression of gonadotropin secretion in ovariectomized estrogen-primed rats. An intravenous (i.v.) injection of naloxone (10 or 20 mg/kg), an EOP antagonist, significantly elevated serum luteinizing hormone (LH) levels within 10 min in non-stressed animals. The naloxone-induced LH release was completely eliminated when tested 30 min after the onset of acute immobilization. In a subsequent study, it was found that suppression of the naloxone-induced LH release occurred as early as 5 min after the stress onset, and was still evident 60 min after the end of a 30-min period of immobilization. The effect of naloxone was restored 3 h after liberation of the animal from the 30-min immobilization. An intraventricular (i.c.v.) injection of CRF (1 or 5 micrograms) also significantly suppressed, in a dose-related manner, the effect of a subsequent i.v. injection of naloxone. However, an i.c.v. injection of alpha-helical CRF(9-41) (25 or 50 micrograms), a CRF antagonist, prior to immobilization, could not interfere with the suppressive effect of stress on naloxone-induced LH release. These results suggest that both acute immobilization stress and CRF can inhibit the LH secretory activity without mediation by EOP neurons. However, the stress-related suppression may involve non-CRF mechanism(s).     

Effects of sex steroids on secretory granule formation in gonadotropes of castrated male rats with respect to granin expression

Pituitary gonadotropes show sex-related differences in their ultrastructure. Typical gonadotropes of male rats exhibit both large granules, which contain chromogranin A (CgA), and small granules, which contain secretogranin II (SgII). In contrast, typical female rat gonadotropes show only a very few large granules among the numerous small granules. To clarify the nature of the biogenesis of these secretory granules and the effects of sex steroids, the ultrastructural and immunocytochemical changes in gonadotropes were examined in castrated male rats supplied with a testosterone or estradiol implant. In castrated rats, pituitary expression and plasma levels of LH increased drastically, but the pituitary content of CgA decreased. The majority of gonadotropes then showed features of "castration cells" containing many small secretory granules. A testosterone implant to castrated rats remarkably suppressed the expression and circulating levels of LH and increased the CgA content in the pituitary to near-normal levels. In this situation, immunocytochemical studies demonstrated that gonadotropes again exhibited large and small secretory granules with the respective localization of CgA and SgII. On the contrary, in castrated rats supplied with an estradiol implant, the expression and content of CgA in the pituitary were remarkably suppressed, and large secretory granules disappeared from gonadotropes. These results suggest that the expression of CgA in gonadotropes is regulated differently by male and female sex steroids. These different effects of androgen and estrogen on the expression level of CgA are closely associated with the sex-related differences in the ultrastructure of secretory granules within gonadotropes.     

Interaction of estradiol and LHRH on LH release in rhesus females: evidence for a neural site of action

The effects on LH release of infusing luteinizing hormone-releasing hormone (LHRH 80 mug/20 min) into the third ventricle, the pituitary, and the peripheral circulation were compared in spayed rhesus monkeys. Within 30 min after iv administration, serum LH concentrations increased to twice to preinfusion levels, and by 120 min declined to original values. Intraventricular or intrapituitary infusions of LHRH resulted in similar LH increments, but the peaks occurred somewhat later (70 to 90 min) and the elevations persisted beyond 200 min. Estradiol-17beta (E2) administered by a sc silastic capsule caused a 5-fold increase in serum E2 within 1 h and reduced serum LH levels by 65% within 4 h. The LH release caused by intrapituitary LHRH was significantly suppressed by maintaining for 72 h E2 concentrations near 100 pg/ml, a level inadequate for stimulating an LH surge. A comparable E2 treatment before intraventricular infusion of LHRH, however, did not inhibit LH release. This difference between the effects of intrapituitary and intraventricular LHRH was demonstrable only in E2-treated monkeys. Moreover, the release of LH after intraventricular infusion of LHRH in E2-treated females was blocked (P less than 0.001) by a single iv injection (90 min before LHRH) of haloperidol (1 mg/kg BW) or phentolamine (5 mg/kg), but was not altered by phenoxybenzamine (3 mg/kg) or propranolol (5 mg/kg). Without E2 pretreatment, LH release after intraventricular LHRH was enhanced by each drug. Phentolamine, injected into both E2- and non-E2-treated monkeys 90 min before an intrapituitary infusion of LHRH had no demonstrable effects on the patterns of serum LH. Our interpretation of these data is that E2 at a concentration below the level that triggers an LH surge has a dual action on LHRH-induced LH release in monkeys: an inhibitory effect exerted directly on the pituitary and a stimulatory effect on the brain. Furthermore, the paradoxical effects of the drugs with and without E2 are due to the involvement of two distinct neuronal systems. The postulated neural effects of both E2 and these drugs can be explained either by an increase in the quantity of injected or secreted LHRH which ultimately binds to LH-secreting cells or by the release of additional endogenous LH-stimulating agents together with ventricular LHRH.     

Hypersecretion of LH and FSH by a pituitary adenoma

A 51-year-old man who had a pituitary adenoma that appeared to be hypersecreting LH and FSH is described. Not only were serum LH and FSH concentrations above the normal ranges, but the serum concentrations of testosterone, free testosterone, and dihydrotestosterone were also above normal. Serum LH and FSH concentration increased in response to synthetic thyrotropin-releasing hormone as well as to synthetic gonadotropin-releasing hormone. The elevated hormone concentrations decreased following an initial partial hypophysectomy and decreased further following repeat hypophysectomy.     

Are mu-opioid receptors involved in the control of endothelin-1 release from the pituitary gland in normal and dehydrated rats?

A stereognostic ability test was performed in 60 patients. Forty patients were rehabilitated by means of osseointegrated implants. One group consisted of 20 patients with fixed prostheses on implants in both the upper and lower jaws. The other 20 patients had a maxillary denture while in the mandible an overdenture was retained by means of two implants connected by a bar. They were compared to a group of 20 subjects (controls) with a non-restored natural dentition. For the stereognostic ability test, subjects had to recognise ten different test pieces by manipulating them with two antagonistic incisor teeth, avoiding any contact with other oral structures. Both response time and percentage accuracy of recognition were evaluated. The present findings indicated that subjects with an overdenture on implants did not score significantly different from those with an implant-supported fixed prosthesis. In contrast, subjects with teeth had a significantly better stereognostic ability. The percentage of correct responses was 52% for overdentures, 56% for fixed prostheses on implants and 75% for natural dentitions. From these results, it could be concluded that the stereognostic ability is impaired in subjects rehabilitated with osseointegrated implants by about one-third to one-quarter compared to subjects with natural teeth.