Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.     

Neuroendocrine mechanisms underlying the control of gonadotropin secretion by steroids

There is considerable evidence that although estradiol may trigger the preovulatory surge of gonadotropins, progesterone is required for its full magnitude and duration and that glucocorticoids bring about selective follicle-stimulating hormone release. The luteinizing hormone-releasing hormone (LHRH) neuron does not have steroid receptors and is regulated by excitatory amino acid neurotransmission. Steroids do not appear to modulate excitatory amino acid receptors directly but increase release of glutamate in the preoptic area. This may be due to the suppression by steroids of the enzyme glutamatic acid decarboxylase67 that converts glutamate into GABA. NMDA receptors colocalize with nitric oxide synthase-containing neurons that surround the LHRH neurons in the preoptic area and intersect the LHRH fibers in the median eminence. Other potential novel pathways of LHRH release that are currently being explored include carbon monoxide generated by the action of heme oxygenase-2 on heme molecules and bradykinin acting via bradykinin B2 receptors.     

Evidence that the mediobasal hypothalamus is the primary site of action of estradiol in inducing the preovulatory gonadotropin releasing hormone surge in the ewe

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.     

Regulation of prolactin secretion by adrenal steroids in oestrogen-treated ovariectomized rats: participation of endogenous opioid peptides

The purpose of the present study was to determine whether glucocorticoid inhibition of prolactin (PRL) release in oestrogen-treated ovariectomized (OVX) rats is mediated by endogenous opioid peptides (EOPs). All the animals were OVX and given oestradiol benzoate (OB, 20 microg/rat, s.c.) 2 weeks later (day 0). On day 3 they received vehicle, mifepristone (MIF, 10 mg/kg, s.c.) or hydrocortisone (HYD, 2 mg/rat, s.c.), in combination with the opioid antagonist naloxone (NAL, 2 mg/kg, i.p.) or vehicle. Serum PRL concentration was then measured by RIA at 13.00 and 18.00 hr, to include assessment of diurnal variation of PRL secretion. At 13.00 hr either MIF or NAL alone increased PRL secretion with no additional effect when NAL was combined with MIF. HYD had no significant inhibitory effect, but NAL with HYD increased PRL secretion. At 18.00 hr serum PRL concentration was higher than at 13.00 hr, and not affected significantly by MIF or NAL alone, although PRL secretion was increased by treatment with both. HYD inhibited PRL secretion and this inhibition was prevented by NAL. In a second experiment to distinguish antiglucocorticoid and antiprogesterone effects of MIF, we administered progesterone (2 mg/rat, s.c.) or a specific progesterone antiserum. In contrast with MIF, the progesterone antibody had no effect on PRL secretion at 13.00 hr, nor on the stimulation by NAL, while progesterone (unlike HYD) increased PRL secretion and NAL attenuated this response; this was opposite to the effect of NAL with HYD. Similarly, at 18.00 hr the interaction of MIF and NAL was not explained by antagonism of progesterone. Together, these results indicate inhibition of PRL by glucocorticoids but not progesterone, mediated in part by EOPs. At 18.00 hr endogenous glucocorticoids do not regulate oestrogen-stimulated PRL release, although HYD is inhibitory through EOPs.     

Regulation of chorionic gonadotropin gene expression

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 microliters of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate ("e-carnitine") as the internal standard, standard curves (10-300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.     

Importance of the gonadotropin-releasing hormone (GnRH) surge for induction of the preovulatory luteinizing hormone surge of the ewe: dose-response relationship and excess of GnRH

The preovulatory LH surge in the ewe is stimulated by a large sustained surge of GnRH. We have previously demonstrated that the duration of this GnRH signal exceeds that necessary to initiate and sustain the LH surge. The objective of the present study was to determine whether a similar excess exists for amplitude of the GnRH surge. Experiments were performed using an animal model in which GnRH secretion was blocked by progesterone, which in itself does not block the pituitary response to GnRH. To assess the amplitude of the GnRH surge needed to induce the LH surge, we introduced artificial GnRH surges of normal contour and duration but varying amplitudes. Twelve ewes were run through 3 successive artificial follicular phases (total of 36). Six of these artificial follicular phases were positive controls, in which progesterone was removed, the estradiol stimulus was provided, and vehicle was infused. In these control cycles, animals generated endogenous LH surges. In the remaining artificial follicular phases, progesterone was not withdrawn, the estradiol stimulus was provided, and either vehicle (negative control) or GnRH solutions of varying concentrations (experimental) were infused. The circulating GnRH concentrations achieved by infusion were monitored. No LH surges were observed in negative controls, whereas LH surges were induced in experimental cycles provided a sufficient dose of GnRH was infused. A highly significant dose-response relationship was observed between the amplitude of the GnRH surge and both the amplitude of the LH surge and the area under the curve describing the LH response, but no such relationship existed between the amplitude of the GnRH surge and the duration of the LH response. In numerous cases, LH surges similar to those in the positive control animals resulted from infusion of amounts of GnRH estimated to be considerably less than those delivered to the pituitary during the endogenously generated GnRH/LH surge. These findings indicate that, in the ewe, increased GnRH secretion drives the preovulatory LH surge in a dose-dependent fashion, and they provide evidence that the amplitude of the GnRH surge may exceed that needed to generate the LH surge.     

Nicotine given intracerebroventricularly does not inhibit the preovulatory surge of LH and PRL secretion in female rats

Twenty-three cases of Little Leaguer's shoulder were reviewed including the history and physical examination findings, as well as bilateral internal and external rotation anteroposterior comparison radiographs of the proximal humerus. The average follow-up was 9.6 months (range, 1.5 to 54), and all patients were observed until they had either returned to baseball or their symptoms had resolved. The average age of the patients in this series was 14 years. The chief complaint in all patients was pain localizing to the proximal humerus during the act of throwing. The average duration of symptoms was 7.7 months. Nineteen patients (83%) were pitchers. Physical examination revealed tenderness to palpation over the proximal humerus in 20 patients (87%), with 16 (70%) demonstrating specific tenderness over the lateral aspect of the proximal humerus. Swelling, weakness, atrophy, and loss of motion were uncommon findings. All 23 patients demonstrated radiographic widening of the proximal humeral physis of the throwing arm on internal and external rotation comparison anteroposterior radiographs of the shoulder. All patients were treated with rest from baseball throwing for an average of 3 months. Twenty-one of the 23 patients (91%) returned to playing baseball and were asymptomatic. The classic radiographic finding of widening of the proximal humeral physis can easily be seen on bilateral anteroposterior internal and external rotation radiographs of the proximal humerus. Rest from throwing for at least 3 months is recommended, followed by a gradual return to throwing in an asymptomatic shoulder.     

Suppression of apoptosis by gonadotropin, 17beta-estradiol, and epidermal growth factor in rainbow trout preovulatory ovarian follicles

In this study we present the first evidence for the occurrence of apoptotic cell death in ovarian follicles from teleost fish. Preovulatory ovarian follicles from mature hatchery-raised rainbow trout (Oncorhynchus mykiss) were collected and either immediately frozen in liquid nitrogen or incubated in serum-free medium at 18 degrees for 24 hr. The extent of ovarian apoptotic DNA fragmentation was determined using 3'-end labeling of DNA with [32P]dideoxy-ATP, size fractionation by agarose gel electrophoresis, and quantification of low-molecular-weight (<15 kb) DNA using autoradiography and liquid scintillation counting. The extent of apoptotic DNA fragmentation was eightfold greater in immediately frozen preovulatory follicles than in previtellogenic ovarian follicles collected from immature rainbow trout (P < 0.05), suggesting differences in the degree of apoptosis at different stages of follicular development. In preovulatory trout follicles, the extent of apoptotic DNA fragmentation was fivefold greater in follicles incubated for 24 hr. Treatment of incubated preovulatory follicles with either partially purified salmon gonadotropin SG-G100 (1 microg/ml) or epidermal growth factor (EGF; 100 ng/ml) suppressed apoptotic DNA fragmentation by 31 and 41%, respectively, in comparison to untreated incubated follicles (P < 0.01). Treatment of incubated follicles with 17beta-estradiol (1-100 ng/ml) caused a concentration-dependent suppression of apoptotic DNA fragmentation (P < 0.05). These results suggest that apoptosis is involved in teleost ovarian development and that several of the hormonal factors acting as follicle survival factors in mammalian and avian ovaries may play a similar role in teleost ovarian follicles.     

Regulation of the endogenous NO pathway by prolonged inhaled NO in rats

Nitric oxide (NO) modulates the endogenous NO-cGMP pathway. We determined whether prolonged inhaled NO downregulates the NO-cGMP pathway, which may explain clinically observed rebound pulmonary hypertension. Rats were placed in a normoxic (N; 21% O2) or hypoxic (H; 10% O2) environment with and without inhaled NO (20 parts/million) for 1 or 3 wk. Subsequently, nitric oxide synthase (NOS) and soluble guanylate cyclase (GC) activity and endothelial NOS (eNOS) protein levels were measured. Perfusate cGMP levels and endothelium-dependent and -independent vasodilation were determined in isolated lungs. eNOS protein levels and NOS activity were not altered by inhaled NO in N or H rats. GC activity was decreased by 60 +/- 10 and 55 +/- 11% in N and H rats, respectively, after 1 wk of inhaled NO but was not affected after 3 wk. Inhaled NO had no effect on perfusate cGMP in N lungs. Inhaled NO attenuated the increase in cGMP levels caused by 3 wk of H by 57 +/- 11%, but there was no rebound in cGMP after 24 h of recovery. Endothelium-dependent vasodilation was not altered, and endothelium-independent vasodilation was not altered (N) or slightly increased (H, 10 +/- 3%) by prolonged inhaled NO. In conclusion, inhaled NO did not alter the endogenous NO-cGMP pathway as determined by eNOS protein levels, NOS activity, or endothelium-dependent vasodilation under N and H conditions. GC activity was decreased after 1 wk; however, GC activity was not altered by 3 wk of inhaled NO and endothelium-independent vasodilation was not decreased.     

Steroid productions by co-cultures of granulosa cells with inner and outer theca cells in preovulatory follicles of gonadotropin stimulated calves

Granulosa, interna and externa theca cells were isolated from large follicles of equine-chorionic-gonadotropin (eCG)-primed calves and co-cultured during 3 days in the absence or in the presence of dehydroepiandrosterone (DHEA). Co-cultures were performed by adding defined numbers of theca and/or granulosa cells which represented 0, 10, 20, 50 or 100% of total cells per well. Secretion of oestradiol-17beta (E2), androstenedione (A4) and progesterone (P4) depended on the type of theca cells (P < 0.001), on the percentage of seeded granulosa cells (P < 0.001) and on the day of culture (P < 0.001). DHEA increased (P < 0.001) E2 and A4, but not P4 (P > 0.05) productions. Interactions existed between these factors (P < 0.01). On day 1, A4 production was nil in granulosa cells alone. E2 production was negligible in theca cells alone but it increased when granulosa cells were added. E2 and A4 varied in an opposite manner according to the percentage of granulosa cells and with the type of theca cells. On day 3, without DHEA, E2 and A4 were low. On day 3 with DHEA, E2 production was maintained in granulosa cells alone but not with any combination of theca cells. In these conditions, A4 production was maintained in the presence of theca cells but not in granulosa cells alone. Granulosa cells alone secreted more P4 than theca cells. P4 increased as a function of the percentage of granulosa in co-cultures with externa but not interna theca cells with which it remained low. In conclusion, theca cells in culture have two effects in relation to the granulosa cells, which differ according to the steroid concerned and to the cell combination. Both types of theca cells have an inhibitory effect on E2 secretion whereas only interna theca cells are able to alter P4 production.     

Changes in densities of hypothalamic mu opioid receptor during cupric acete-induced preovulatory lh surge in rabbit

Decrease in activity of hypothalamic beta-endorphin (beta-EP) is an important factor for inducing the preovulatory LH surge. To study whether hypothalamic mu opioid receptor is involved in this process, changes in densities of hypothalamic mu opioid receptors were observed in this study by autoradiography and image process during cupric acetate (CuAC)-induced preovulatory LH surge in rabbits. New Zealand female rabbits were injected 1% CuAC 0.9 ml or saline 0.9 ml and sacrificed at different times after the injection. The densities of mu opioid receptor in the medial basal hypothalamus (MBH) and the medial preoptic area (MPO) were measured. A transient increase in densities of MPO mu opioid receptor were observed 1 h after CuAC injection (P < 0.05). The densities of MPO mu opioid receptor decreased significantly before the onset of the LH surge (P < 0.05) and remained at a low level during the surge. The change in densities of mu opioid receptor in the MBH was similar to those in the MPO. No change was observed in the saline control group. There was a negative correlation between the changes in densities of MBH mu opioid receptor and serum LH levels in the process of LH surge. The results suggest that the decrease of hypothalamic mu opioid receptor may be involved in the preovulatory LH surge.     

Regulation of cyclooxygenase activity in airway epithelium by endogenous nitric oxide

The role of nitric oxide (NO) in the activity of cyclooxygenase (COX) in cultured canine tracheal epithelium was studied. Tracheal epithelium spontaneously released prostaglandin E2 (PGE2), which is a product of COX. The release of PGE2 was increased by bradykinin and was decreased by two NO synthase inhibitors: NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. That decrease was reversed in the presence of L-arginine. Chrolpromadin, but not aminoguanidine, inhibited PGE2 production, which suggests that constitutive NO synthase is involved. Two stable NO donors, sodium nitroprusside and S-nitroso-N-acetyl DL-penicillamine, also increased the production of PGE2. These effects were abolished by coincubation with hemoglobin, which binds and inactivates NO, but not by methylene blue, an inhibitor of soluble guanylate cyclase. NADPH diaphorase histochemistry of cultured tracheal cells revealed activity in the periphery of the cytoplasm. These results suggest that, in cultured canine tracheal epithelium, NO directly interacts with COX to regulate PGE2 production.     

Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras

Clinical teaching behaviour is a critical determinant for quality clinical learning experiences of student nurses. It is believed that a better understanding of the perceptions of clinical teaching behaviours between student nurses and nurse educators will enhance clinical teaching. This study examined the perceptions of effective clinical teaching behaviours of nurse educators by student nurses (n = 81) and nurse educators (n = 10) in a hospital-based 3-year general nurse training programme in Hong Kong. Knox & Mogan's Nursing Clinical Teacher Effectiveness Inventory (NCTEI) (1985) was adopted. The respondents were asked to rate the importance of each discrete behaviour on a seven-point scale. It was found that there was greater agreement in the 10 most important behaviours than the 10 least important behaviours among the four groups: students, junior students, senior students and nurse educators. No statistically significant difference could be identified in the perceptions between the nurse educators and students as well as between the junior and senior students regarding the five behavioural categories. The nature and the student status of the nursing programme was accountable for most of the discrepancies between the findings of this study and those of past studies.     

The midcycle gonadotropin surge in normal women occurs in the face of an unchanging gonadotropin-releasing hormone pulse frequency

The midcycle gonadotropin surge is a critical event in normal reproductive cycles and requires functional integration of the hypothalamus, pituitary, and ovary. To determine whether a change in GnRH frequency occurs coincident with the onset or termination of the surge in normal women, 20 studies were performed at a sampling interval of every 5 min for up to 36 h. The frequency of pulsatile GnRH secretion was assessed by the use of two surrogate markers of its secretion, LH and free alpha-subunit (FAS). The timing of the studies was prospectively determined by serial ultrasound and previous cycle history, whereas measurements of LH, FSH, estradiol, and progesterone in daily blood samples were used retrospectively to locate the frequent sampling study in relation to the day of ovulation in each individual. The frequent sampling studies were divided into late follicular phase (LFP; days -4 to -2) and early, mid-, and late portions of the midcycle surge (days -1 to 1) in relation to the 95% confidence limits of the LH peak derived from daily samples in 69 normal ovulatory women. The patterns of LH and FAS secretion were pulsatile at all times during the midcycle surge. The amplitude of LH pulsations increased from the LFP and early surge to the midportion of the midcycle surge (5.9 +/- 6 and 15.1 +/- 5 vs. 39.0 +/- 3 IU/L; P < 0.0001) and decreased from the mid- to the late portion of the surge (13.4 +/- 5 IU/L; P < 0.0001). Likewise, the amplitude of FAS pulse increased from the LFP and early surge to the midportion of the surge (82.4 +/- 59 and 153.1 +/- 50 vs. 421.4 +/- 35 ng/L; P < 0.0001) and decreased from the mid- to the late portion of the surge (190.8 +/- 49 ng/L; P < 0.0002). Although there was excellent concordance of pulsatile secretion of LH and FAS, significantly more pulses of FAS were detected than of LH (P < 0.0001). There was no change in frequency (expressed as interpulse interval) between the LFP and the early and midportions of the surge for LH (70.0 +/- 8, 67.5 +/- 7, and 65 +/- 5 min, respectively) or FAS (55.1 +/- 7, 54.6 +/- 6, and 60.0 +/- 4 min). However, there was an increase in LH interpulse interval (decrease in pulse frequency) in the late portion of the surge (87.0 +/- 6 min) compared to the early and midportions of the surge (P < 0.02 and P < 0.0005, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)     

Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.