Decreased hypothalamic thyrotropin-releasing hormone gene expression in patients with nonthyroidal illness.

Changes in hypothalamus-pituitary-thyroid function occur in patients with a variety of illnesses and are referred to as the euthyroid sick syndrome or nonthyroidal illness (NTI). In NTI, serum concentrations of T3 decrease to low, or even undetectable, levels without giving rise to elevated concentrations of TSH. We hypothesized that decreased activity of TRH-producing cells in the paraventricular nucleus (PVN) contributes to the persistence of low TSH levels. To test this hypothesis, we collected a series of formalin-fixed, paraffin-embedded hypothalami of patients whose plasma concentrations of T3, T4, and TSH had been measured in a blood sample taken less than 24 h before death. Quantitative TRH messenger RNA in situ hybridization (intraassay coefficient of variation: 13%) was performed in the PVN. Total TRH messenger RNA in the PVN showed a positive correlation with serum T3 (r = 0.66; P < 0.05) and with logTSH (r = 0.64; P < 0.05), but not with T4 (r = -0.02; P = 0.95). This is the first study to correlate premortem serum concentrations of thyroid hormones with postmortem gene expression of identified neurons in the human hypothalamus. The results suggest an important role for TRH cells in the pathogenesis of NTI.

Unchanged amounts of vasopressin mRNA in the supraoptic and paraventricular nucleus during aging and in Alzheimer's disease.

The paraventricular (PVN) and supraoptic nucleus (SON) demonstrate a striking stability with respect to cell numbers during aging and Alzheimer's disease (AD). Vasopressin (AVP) neurons even become activated during aging as judged from several parameters for neuronal activity, such as increased AVP plasma levels, enlarged nucleolar as well as cell size and an increased size of the Golgi apparatus in AVP-neurons. The activation possibly occurs as compensation for an age-related loss of AVP-receptors in the kidney. As a specific marker for AVP synthesis, we used quantitative in situ hybridization and estimated total amounts of AVP-mRNA in the entire SON and PVN of 14 control subjects and 14 AD patients that were matched for age, fixation time, postmortem delay and storage time of the tissue in paraffin. Following quantification, no differences were observed in total amounts of AVP-mRNA in the SON or PVN between young and old controls or between young and old AD patients, nor between the entire group of controls and AD patients. A significant negative correlation was found between the volume of the AVP-mRNA signal in the AD SON and age while the total amount of mRNA remained the same. This suggests a redistribution of cells or cell compartments in aging. A significant positive relation in both SON and PVN of AD patients was found between storage time of the paraffin-embedded tissue and the total amount of AVP-mRNA. A significant positive relation was present in the PVN, but not SON between pH of the cerebrospinal fluid, which is a marker for agonal state and the total amount of AVP mRNA. The present unchanged AVP-mRNA levels in SON and PVN confirm earlier observations on the stability of cell numbers in these nuclei in aging and AD. Although on the basis of other parameters, AVP-mRNA upregulation was expected, gradual, chronic stimulation over prolonged periods of time may, possibly, induce alternative mechanisms of regulation such as changes in translatability or in mRNA stability.

Fos and Jun expression in rat supraoptic nucleus neurons after acute vs. repeated osmotic stimulation.

Using a double-label immunofluorescence method, we analyzed the time course of the appearance of Fos and Jun in the nuclei of supraoptic nucleus (SON) neurons following intraperitoneal injection of hypertonic saline. Fos and Jun immunostaining appeared within 30 min, peaked at 90-120 min, and disappeared 4-5 h later. At all time points (30, 60, 120, 180, 240 min postinjection), colocalized Fos and Jun immunostaining was observed (> 90% colocalized staining in labeled neurons). At 4 h post-saline injection, some rats received a second injection of normal or hypertonic saline. A second injection of normal saline resulted in no Fos/Jun immunostaining 90 min later, while hypertonic saline induced combined Fos/Jun staining in only 17% of SON neurons. Of the remaining SON cells, 23% had staining to Fos alone and 4% of the cells stained for Jun only. In spite of the delivery of an effective second osmotic stimulus, determined by assessment of plasma osmolality and sodium content, SON neurons exhibited less colocalized Fos/Jun immunostaining, dramatically less Jun expression, and substantial, but attenuated, immunostaining for Fos. These results are discussed in the context of known negative feedback mechanisms that control the re-expression of these transcription factors.

Thyrotropin-releasing hormone gene expression in the human hypothalamus.

We studied the distribution of mRNA coding for thyrotropin-releasing hormone (TRH) in the human hypothalamus by means of in situ hybridization. In 10% formalin-fixed paraffin-embedded tissue sections of five hypothalami, TRH mRNA-containing cells were found in several nuclei and areas. Numerous TRH mRNA-containing cells were detected in the medial region of the caudal part of the paraventricular nucleus. These neurons were heavily labeled and mainly small to medium-sized. Few, lightly- and medium-labeled, small cells were detected in the suprachiasmatic nucleus. In addition, heavily labeled single cells were found in the perifornical area and the anterior- and lateral hypothalamic regions. In the latter region, occasional heavily labeled cells were found just dorsal to the supraoptic nucleus. Neither in the supraoptic nucleus nor in the sexually dimorphic nucleus of the preoptic area were TRH mRNA-containing cells found. This is the first description of TRH mRNA containing cells in the human hypothalamus.

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Estimation of oxytocin mRNA in the human paraventricular nucleus in AIDS by means of quantitative in situ hybridization.

Recently, a 40% reduction in the total of oxytocin immunoreactive neurons of the paraventricular nucleus of the hypothalamus in AIDS patients was reported. In the present study, we determined whether this decrease is associated with a diminished amount of oxytocin mRNA. We used in situ hybridization combined with densitometric image analysis for the quantitative assessment of oxytocin gene expression in the paraventricular nucleus of a group of AIDS patients (n=10) and a carefully matched control group (n=8). We found no significant difference (P=0.08) in the amount of oxytocin mRNA per total paraventricular nucleus between the two groups. In addition, no significant differences were found in the part of the volume of the paraventricular nucleus that was occupied by hybridized cells (P=0.12) or in the mean signal density (P=0.08). The findings do not support the hypothesis that the extensive decrease in oxytocin immunoreactive neurons of the paraventricular nucleus in AIDS is associated with a decrease in total oxytocin mRNA content in this nucleus. The data are compatible with the suggestion that in AIDS the biosynthesis of oxytocin is changed in an unknown way at the (post)transcriptional level.

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Double immunofluorescence staining of Fos and Jun in the hypothalamus of the rat.

The nuclear factor Fos participates in the transcriptional regulation of genes that contain a functional AP-1 binding site. Hyperosmotic stress induces Fos-like immunoreactivity in neurons of the hypothalamus. Fos appears to depend on the co-expression of the nuclear factor Jun, with which it dimerizes, to complete its regulatory role. The immunocytochemical co-localization of both peptides, however, has not been reported. The present study was designed to analyze the distribution of Fos and Jun by double immunofluorescence staining in Long-Evans rats that were osmotically challenged by a single intraperitoneal injection of 1.5 M NaCl. Non-injected and isotonic saline-injected animals were used as controls. Hypertonic saline injection induced Jun immunoreactivity in cell nuclei in the supraoptic nucleus, paraventricular nucleus, and median preoptic nucleus. The immunofluorescence for Jun was strong in the supraoptic and paraventricular nuclei, but weak in the median preoptic nucleus. The immunofluorescence for Fos in all 3 nuclei followed a similar pattern to that for Jun. Double immunofluorescence staining revealed co-localization of Jun with Fos in 87.4% of the cells of the supraoptic nucleus. Neither Jun nor Fos immunofluorescence was detected in control animals. The data support a role for Jun in transcriptional regulation of genes in hypothalamic neurons during acute hyperosmotic stress. Moreover, the findings are compatible with the suggestion that Fos and Jun act cooperatively in the regulation of gene transcription in neuroendocrine systems involved in the control of water balance during acute hyperosmotic stimulation.

Fos-like immunoreactivity in the brain of homozygous diabetes insipidus Brattleboro and normal Long-Evans rats.

Water deprivation induces the production of the transcription factor Fos in neurons of the neurohypophysial system. These neurons, which are located primarily in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), produce the antidiuretic hormone vasopressin. The present immunocytochemical study has analyzed the distribution of Fos in brain regions involved in osmoregulation and compared the extent of Fos immunoreactivity (Fos-IR) in vasopressin-deficient Brattleboro and normal Long-Evans rats under stimulated and non-stimulated conditions. Rats were osmotically challenged by means of a single intraperitoneal injection of 1.5 M/L NaCl. Since Fos may be induced by the stress of handling of animals, non-injected and isotonic saline-injected rats were used as controls. Faint nuclear Fos immunostaining was found in the organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus (MnPO), subfornical organ (SFO), and SON of non-injected and isotonic saline-injected Brattleboro but not Long-Evans rats. Hypertonic saline injection specifically induced Fos-IR in neurons located in the SFO, OVLT, MnPO, PVN, SON, hypothalamic accessory nuclei (including the nucleus circularis), and arcuate hypothalamic nucleus (Arc) in both Long Evans and Brattleboro rats. No differences in distribution of the induced immunostaining were found between the strains. Stress of handling and (isotonic saline) injection induced Fos-IR in the lateral septal nuclei, central amygdaloid nuclei, medial amygdaloid nucleus, medial preoptic area, the bed nucleus of the stria terminalis, cingulate- and piriform cortex, the lateral hypothalamic area, ventromedial hypothalamic nucleus, and the habenular nucleus. The data are consistent with a role for Fos in the regulation of vasopressin gene expression during acute hyperosmotic stimulation. In addition, this study demonstrated that during chronic osmotic stimulation, as experienced by homozygous Brattleboro rats, Fos-IR is limited but apparently present constantly and that it increased in these animals following acute osmotic challenge. Our observations suggest that c-fos regulatory controls in homozygous Brattleboro rats are different from those in Long-Evans rats.

A novel, [tyrosyl-3,5-3H]oxytocin binding, uterine cell population in the rat.

The cellular localization of uterine oxytocin binding sites in the rat was studied by means of in vitro receptor autoradiography. Using [tyrosyl-3,5-3H]oxytocin as ligand, binding sites were localized in tissue sections from uteri of estrous, mated, and artificially cervically stimulated rats (n = 4 per group), and specificity of binding was investigated by means of simultaneous incubations with oxytocin, [Gly4,Thr7]oxytocin and [Arg]vasopressin. A previously unidentified type of cell was densely labelled by tritiated oxytocin. The labelled cells were preferentially localized near the endomyometrial border and at the interface of the circular and longitudinal muscle layers. In addition, these cells were found in the muscle layers. The dense labelling of these cells, which did not constitute part of the endometrial epithelium or blood vessels, was abolished when oxytocin or [Arg]vasopressin, but not [Gly4,Thr7]oxytocin, was added to the incubation medium. Binding of the radioligand was also found on muscle cells of the circular and longitudinal layers of the myometrium and cells of the endometrial luminal and glandular epithelium. Whereas incubation with oxytocin and [Gly4,Thr7]oxytocin diminished the labelling in both myometrium and endometrium, incubation with [Arg]vasopressin reduced labelling only in the myometrium. Similar results were obtained in tissues from rats in different reproductive states. This study demonstrates the presence of oxytocin binding sites in three different types of cell in the uterus of the rat. While the sites in the myometrium may be associated with the contractile response of this type of tissue to oxytocin, the functional significance of oxytocin binding sites on the endometrial epithelium and in the densely labelled, scattered cells remains to be elucidated.

Testicular oxytocin: effects of intratesticular oxytocin in the rat.

The long-term effects of oxytocin administration on the testis were studied using intratesticular implants. Adult male rats had an Accurel device containing 20 micrograms oxytocin (releasing approximately 200 ng/day) implanted into the parenchyma of each testis; control animals received empty devices. The animals were killed at weekly intervals for 4 weeks. Some animals were perfused and the testes processed for light and electron microscopy. Blood was collected from the remaining animals for the measurement of testosterone, dihydrotestosterone, LH, FSH and oxytocin; epididymal sperm counts were measured and the testes were extracted and radioimmunoassayed for testosterone, dihydrotestosterone and oxytocin. Long-term administration of oxytocin resulted in a significant reduction in testicular and plasma testosterone levels throughout the 4-week period examined and, after 14 days of treatment, lipid droplets were seen in the Leydig cells of treated but not control animals. Concentrations of dihydrotestosterone in the plasma and testes of the oxytocin-treated animals, however, were significantly elevated after 7 and 14 days and at no time fell below control values. Plasma FSH levels were also lower in the oxytocin-treated animals. Intratesticular oxytocin treatment did not affect LH or oxytocin concentrations in the plasma, epididymal sperm counts or the number of Leydig cells in the testis. Empty Accurel devices had no effect on testicular morphology. This study provides the first evidence that oxytocin in vivo can modify steroidogenesis in the testis.

Characterization and localization of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist.

Some of the binding characteristics of a novel oxytocin receptor ligand 125I-labelled [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin ([125I]OTA) have been determined in the sheep uterus. The compound was subsequently used for the autoradiographic localization of oxytocin receptors in the uterus and oviduct of the ewe. Specific binding of [125I]OTA to crude membrane fractions of ovine endometrium was time-dependent and was unaffected by the addition of cations to incubation media. Endometrial membranes contained a single population of saturable, high-affinity binding sites for the iodinated ligand (dissociation constant (Kd) 0.23 +/- 0.08 nmol/l) and unlabelled oxytocin competed with [125I]OTA for binding sites with high affinity (Kd 1.29 +/- 0.4 nmol/l) in the presence of Mg2+ In contrast, unlabelled OTA was able to compete with high affinity (Kd 1.13 +/- 0.16 nmol/l) in the absence of cation. Competition studies with a number of oxytocin analogues and related peptides and the tissue distribution of [125I]OTA binding sites also indicated that [125I]OTA bound to the ovine oxytocin receptor. This was further validated by autoradiographic studies which showed specific labelling with [125I]OTA to be greater to uterus and oviduct obtained from ewes which had been killed within 2 days of oestrus than to similar tissue from ewes killed during the luteal phase. In both the ampullary and isthmic regions of the oviduct and the myometrium, [125I]OTA binding sites were confined to smooth muscle. Endometrial binding sites for [125I]OTA were consistently located on the luminal epithelium and epithelial cells lining secretory glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig.

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.

Mutant vasopressin precursor in the endoplasmic reticulum of the Brattleboro rat. Ultrastructural evidence from individual "vasopressin" cells localized with the light microscope by use of a new gold/silver method for immunostain enhancement.

This ultrastructural study demonstrates that the vasopressin immunoreactivity found in the occasional, densely stained cells in the hypothalamus of the homozygous Brattleboro rat is localized in the rough endoplasmic reticulum. 50-micron Vibratome sections were stained with anti-vasopressin serum by use of a peroxidase method with 3,3-diaminobenzidine as chromogen. The diaminobenzidine end-product has a specific capability to bind gold particles from a chloroauric acid solution and the bound gold was used to precipitate silver grains from a silver developer. The stained sections were flat embedded in resin and ultrathin sections were cut of areas containing the immuno-identified occasional cells. In these densely stained, vasopressin-immunoreactive cells of homozygous Brattleboro rats the rough endoplasmic reticulum was dilated. The lumen of the reticulum contained both end-products of diaminobenzidine and gold/silver grains, but some parts of the reticulum appeared unstained. No other cell organelles were immunostained and no secretory granules were found. In control rats, gold/silver deposits were found throughout the cytoplasm of vasopressin-immunoreactive cells. In these immunostained cells secretory granules were seen.

Oxytocin in Leydig cells: an immunocytochemical study of Percoll-purified cells from rat testes.

Testicular interstitial cells, from rats aged 35 days, were dispersed with collagenase and separated through Percoll into 5 fractions (I-V); fraction I being the least dense. Measurement of basal testosterone production, histo-enzymological staining for 3 beta-hydroxysteroid dehydrogenase activity and electron microscopy indicated that the majority of Leydig cells were found in fraction IV (corresponding to a density of 1.076-1.097 g/ml). In addition, cells from this fraction responded to hCG treatment in a dose-dependent manner on day 0 and remained responsive after being cultured for 1 day. Immunostaining for oxytocin indicated that this fraction also contained the majority of the oxytocin-immunoreactive cells. On day 1 of culture, 56% of the cell population from fraction IV were positively stained for the steroidogenic enzyme and 75% immunoreactive for oxytocin. This overlap indicates that the Leydig cells were also the oxytocin immunoreactive cells.

Ethan-1,2-dimethanesulphonate reduces testicular oxytocin content and seminiferous tubule movements in the rat.

An oxytocin-like peptide is present in the interstitial cells of the testis, and testicular concentrations of oxytocin have been shown to increase seminiferous tubule movements in vitro. We have used the drug ethan-1,2-dimethanesulphonate (EDS), which depletes the Leydig cell population of the adult rat testis, to examine further the relationships between the Leydig cell, testicular oxytocin and tubular movements. Adult rats were injected i.p. with a single dose of EDS (75 mg/kg) or of vehicle (25% dimethyl sulphoxide). Histological study 3 and 10 days after treatment with EDS showed a reduction in the number of interstitial cells, and levels of oxytocin immunoreactivity were undetectable by radioimmunoassay. Immunostaining revealed very few oxytocin-reactive cells. Spontaneous contractile activity of the seminiferous tubules in vitro was also dramatically reduced, but could be restored by the addition of oxytocin to the medium. Four weeks after EDS treatment, the interstitial cells were similar to those in the control animals both in number and in immunostaining; immunoassayable oxytocin was present and tubular movements were normal. The EDS effect, seen at 3 and 10 days, was not altered by daily treatment with testosterone. However, repopulation of the testes with oxytocin-immunoreactive cells was not seen until 6 weeks in the testosterone-treated animals. We suggest that the Leydig cells are the main source of oxytocin immunoreactivity in the testis and that this oxytocin is involved in modulating seminiferous tubule movements and the resultant sperm transport. The results also imply that testosterone does not play a major role in controlling tubular activity in the mature rat.

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe.

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.