Netherlands Central Institute for Brain Research, Ijdijk 28, Amsterdam – 0, The Netherlands

(Received 6 November 1974)

INTRODUCTION

Although immunohistochemistry is considered to provide a specific and sensitive tool for the localization of hypothalamic hormones (Zimmerman, Hsu, Ferin & Koslowski, 1974), attempts to localize vasopressin and oxytocin by immunofluorescence have raised questions about the specificity of this technique. Homozygous Brattleboro rats were used in our experiments as a control for vasopressin immunofluorescence since their hypothalamo-neurohypophysial system (HNS) does not contain any measurable amount of this hormone (Valtin, Sawyer & Sokol, 1965). Despite this, bright fluorescence was observed in the HNS of these animals, not only using antibodies against oxytocin, but also with all tested antibodies raised against lysine- or arginine-vasopressin. In addition, immunofluorescence was observed beyond the HNS of Wistar and heterozygous Brattleboro rats, i.e. in the suprachiasmatic nucleus.

Because of these findings and the fact that the commonly used

Netherlands Central Institute for Brain Research, Ijdijk 28, Amsterdam-0, The Netherlands

(Received 3 July 1975)

The 'classical' view on the distribution of oxytocin and vasopressin producing cells suggests that the paraventricular nucleus (PVN) is predominantly or entirely responsible for oxytocin production and the supraoptic nucleus (SON) synthesizes mainly vasopressin. However, recent hormone assays and electrophysiological studies indicate the presence of each hormone in both nuclei (for references see Burford, Dyball, Moss & Pickering, 1974). We report an immunofluorescence study in the SON and the magnocellular part of the PVN (Bodian & Maren, 1951) using antibodies to oxytocin (produced in our laboratory) and to vasopressin (produced by Drs Hollemans, Schellekens and Touber), purified by absorption with arginine-vasopressin and oxytocin respectively (Swaab & Pool, 1975).

Frontal cryostat serial sections of glyoxal-prefixed hypothalami from five male Wistar rats weighing 200 g were studied. Out of each group of six sections, the first was

SUMMARY

We have studied whether endogenous α-MSH has a function in stimulating intra-uterine growth in the rat. The approach used was to determine whether or not this hormone is present during the intra-uterine growth spurt, and if binding of endogenous foetal α-MSH by antibodies would inhibit this growth.

Antibodies against α-MSH or ACTH 1-24, either purified or non-purified, induced immunofluorescence in the intermediate lobe of adult male control rats. Using purified anti-α-MSH, fluorescence appeared in the foetal intermediate lobe on day 18 of pregnancy, the day that biologically active MSH was first seen. A negative correlation was observed between the pituitary MSH content and foetal body weight only on day 19 of pregnancy. Injection of purified anti-α-MSH induced a drop in foetal body weight, but no effect on placental weight was observed. Purified anti-ACTH 1-24 had no effect upon body weight but caused an increase in placental weight.

These results support our previous findings and indicate that endogenous MSH has a function in the stimulation of foetal growth.

SUMMARY

Labour was studied in 69 primiparous and multiparous rats by continuous observation and by the recording of intra-uterine activity. The effect of electrical stimulation of the neurohypophysis with stimulation parameters selected to create a pulsatile release of oxytocin was investigated. Stimulation was applied to the neurohypophysis through chronically implanted electrodes every 5 min, in 45 min sessions, from noon on Day 21 of gestation and at 3 h intervals thereafter.

Electrical stimulation successfully promoted (or induced) the onset and facilitated the course of labour. Stimulation at 12.00 h on Day 21, or at a subsequent stimulation session 3, 6, 9 or more hours later, promoted an immediate increase in the frequency and amplitude of uterine contractions. Overt signs of abdominal straining followed within 5–30 min and the first pup was delivered shortly thereafter. These 'induced' deliveries were almost identical to those displayed by control rats; labour continued to completion despite the termination of the stimulation session after 45 min. By contrast, one third of the stimulated animals displayed an interrupted pattern of labour in which events virtually ceased for 30–60 min when stimulation was terminated. Stimulation, however, only advanced labour by 1–2 h in relation to control animals; this was not statistically significant.

Stimulation accelerated the delivery of the first 5 pups in each litter. In both stimulated and control animals, the birth intervals declined over these first few deliveries to reach the lowest values of 5–6 min throughout the remainder of labour. The most common litter size was 12 pups.

The distribution of labour on Day 21 and 22 was bimodal. Seventy per cent of the animals gave birth between 12.00 and 18.00 h on Day 21, a few gave birth during the following night and the remainder formed a second peak on Day 22. All litters of less than 6 pups were born during this later period.

The implications of these results in the context of spontaneous labour are discussed. We conclude that endogenous oxytocin (with perhaps other neurohypophysial hormones) released in pulses of 1–3 mu. every 5 min can promote a pattern of labour on Day 21 of gestation that is almost indistinguishable from that which occurs naturally.

ABSTRACT

The rat kidney and brain are major target organs for vasopressin (VP). A procedure was developed for immunocytochemical staining of VP and its binding sites in the kidney. This procedure involved preincubation of kidney sections with the ligand, followed by immunocytochemical detection of VP.

The staining in renal tubules from Wistar rats was enhanced by preincubation of tissue sections with increasing concentrations of VP (6–6000 nmol/l). Staining was present in the epithelium of distal convolutions and collecting ducts (medullary and cortical portions) and more pronounced in the apical zone of the tubular epithelium. With high concentrations of VP in the preincubation, staining was also obtained in the thick ascending limb of the loop of Henle. There was no staining under any circumstances in proximal tubules. In the kidney of the Brattleboro rat homozygous for hypothalamic diabetes insipidus (DI) which congenitally lacks VP but responds to the peptide, exactly the same staining pattern was observed after preincubation with VP, but the maximal staining was less intense. The VP binding to the DI rat kidney, after 2 weeks treatment with VP (using Accurel implants), reached levels seen in the Wistar kidney after in-vitro preincubation with high doses of VP.

J. Endocr. (1985) 105, 133–140

ABSTRACT

Postnatal development of the supraoptic nucleus (SON) in the pig hypothalamus was studied morphometrically. The volume of the SON increased from 6·2±0·45 (s.e.m.) mm³ at 7 weeks postnatally to 18·5 ± 1·35 mm³ at 2·5 years of age. A sex difference was found at the development point when the SON volume increased, with earlier SON enlargement in males. This sex difference was 30% at 30 weeks and 50% at 1 year of age. At 2·5 years of age no difference in volume was apparent between the sexes. The number of SON neurones was similar for all age groups concerned (43 500 ± 1475), except for the 2·5year-old females where 40% more were found (55 500 ± 3285). No significant difference was found in neurone number between gonadectomized and sham-operated animals, but the operation caused a 30% reduction in the number of neurones and SON volume. Testosterone supplementation following gonadectomy, during the first 4 weeks postnatally, resulted in sexual dimorphism, the males having more SON neurones than the females. The volume showed only a trend in the same direction. Testosterone supplementation at other ages did not result in any difference when compared with controls.

The results of this study show that the postnatal development of the SON of the pig is sexually dimorphic, and that it continues after puberty in females. In contrast to the vasopressin- and oxytocincontaining nucleus, the development of the SON was not influenced by gonadectomy and only slightly by gonadal steroids.

Journal of Endocrinology (1992) 134, 19–25

The demonstration of vasotocin in the mammalian pineal gland, subcommissural organ and fetal pituitary gland by bioassay has led to hypotheses regarding the function of this hormone in various reproductive processes.

Preliminary examinations of the pineal gland and subcommissural organ with a specific radioimmunoassay failed to show vasotocin immunoreactivity. The presence of vasotocin, vasopressin and oxytocin in the pineal gland, subcommissural organ and fetal neurohypophysis was therefore investigated, using three specific radioimmunoassays. Frog and chicken pituitary glands were used to validate the vasotocin radioimmunoassay.

Direct measurements in diluted homogenates of pituitary glands from frogs, chickens, mid-term fetal sheep and near-term fetal seals revealed the presence of vasotocin only in the frog and chicken pituitary glands, while vasopressin and oxytocin were found in the two fetal pituitary homogenates.

Vasopressin and oxytocin were measured in homogenates of rat and bovine pineal glands and in preparations of the subcommissural organ of rats and rabbits after extraction with Vycor glass powder, but no specific vasotocin immunoreactivity was observed. These results indicate a discrepancy between the reported biological activity of vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland and the immunoreactivity of this material, which can at present only be explained by the presence of a peptide which is structurally closely related to, but not identical with, vasotocin.

Neurohypophysial hormones are thought to be involved in alterations in fluid balance during pregnancy and delivery. In the course of normal pregnancy intravascular volume is increased whereas sodium restriction is thought to reduce plasma volume and cardiac output. In the present study, we measured the effect of long-term severe sodium restriction on vasopressin (AVP) and oxytocin (OT) levels during normal pregnancy and after delivery.

Fifty-nine healthy nulliparous women were randomized either for a low sodium diet (20 mmol sodium daily) or for a normal diet from week 12 of pregnancy onwards. Circulating plasma levels and urinary excretion of AVP and OT, their neurophysins (Np-AVP and Np-OT) and AVP bound to platelets were determined at regular intervals during pregnancy and after delivery. After completion of the study, women on a sodium-restricted diet were compared with control women on a normal diet using repeated measurement ANOVA with adjustment for potentially confounding variables.

After randomization, a reduction in urinary sodium excretion of, on average, 40–82% was found. In general, no effect of sodium restriction could be demonstrated on the various parameters (0·53<P<0·98) with the exception of a significantly lower 24-h urinary AVP excretion by non-smokers with sodium restriction compared with non-smokers having a normal diet (P=0·018) For all parameters, clear changes were found in the course of pregnancy and puerperium (P<0·0001 to P<0·005). Platelet-bound AVP decreased and Np-OT increased during pregnancy. After birth, free plasma AVP, plateletbound AVP, OT, osmolality, sodium and potassium increased, while Np-AVP and Np-OT decreased.

Although elevated Np-AVP and Np-OT levels during pregnancy seem to indicate increased release of neurohypophysial hormones, pregnancy up to 36 weeks of gestation is accompanied by low circulating AVP and OT levels.

Long-term severe sodium restriction diminishes urinary AVP excretion in (non-smoking) pregnant women, without changing circulating levels of AVP and OT, despite the known reduction in circulating volume. The reduced circulating (platelet-bound) AVP levels during pregnancy, whether or not in combination with severe sodium restriction, support the absence of significant non-osmotic stimulation of AVP during pregnancy.

Journal of Endocrinology (1997) 152, 345–354