Oxytocin, a male intragonadal hormone.

It is now well recognised that oxytocin is not confined to the hypothalamo-neurohypophysial system but present elsewhere in the body. However, the significance of the peptide in these peripheral sites is still unclear. This paper considers the evidence for oxytocin to be a male gonadal hormone and focusses on three of the criteria which need to be fulfilled for it to earn this title: oxytocin must be produced within the gonads; have a physiological action and be regulated by factors which alter gonadal function.

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.

The vasopressin precursor in the Brattleboro (di/di) rat.

The vasopressin precursor in the rat hypothalamus has been studied, using trypsin to release desglycinamide vasopressin and coupling it to glycinamide (T & G treatment). The resulting amidated nonapeptide was detected and measured with a radioimmunoassay for vasopressin. The "vasopressin" produced in this way had the full immunoreactivity of the authentic peptide but eluted from an hplc column 1 min earlier and appeared to have a larger molecular weight. It was found that T&G treatment generated vasopressin immunoreactivity in extracts of the supraoptic nucleus (SON) of the Brattleboro rat in just the same way as it did in normal animals. Furthermore, this procedure produced vasopressin immunoreactivity in those hplc fractions from Brattleboro SON extracts that corresponded with the elution time of vasopressin precursor. Similar amounts of "vasopressin" could be generated from Brattleboro and normal SONs. These results support the suggestion that the Brattleboro SON synthesizes an aberrant vasopressin precursor which is not processed by the cell.

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.

Two vasopressin-like peptides in the pig testis?

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

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.

LH and testosterone cause the development of seminiferous tubule contractile activity and the appearance of testicular oxytocin in hypogonadal mice.

Immunoreactive oxytocin is present in the testis and it has been shown that this hormone increases the contractility of seminiferous tubules. We have investigated the relationship between testicular oxytocin, tubular movements and the effects of LH and testosterone using, as a model, the hypogonadal (hpg/hpg) mouse, which is deficient in hypothalamic LH-releasing hormone (LHRH). Whilst both testicular oxytocin and seminiferous tubule movements, resembling those seen in the rat, can be found in normal adult mice, neither can be found in hypogonadal mice. After 2 weeks of treatment with LH (200 ng to 100 micrograms daily) low levels of testicular oxytocin and tubular movements were observed. Treatment with large doses of testosterone for 2-12 weeks led to higher concentrations of testicular oxytocin and tubular movements resembling those seen in the normal adult mouse. The results support the evidence that testicular oxytocin modulates seminiferous tubule movements. We suggest that testosterone may play a part in the accumulation of oxytocin in the testis.

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat.

CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats. CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.

Immunocytochemical evidence for the presence of oxytocin in rat testis.

The presence of oxytocin, vasopressin and neurophysin in the testis of adult Wistar and Brattleboro rats has been examined immunocytochemically. After fixation in modified Bouin's solution, or Bouin's sublimate fixative, immunostaining was accomplished with the peroxidase-antiperoxidase method. The presence of immunoreactive oxytocin was demonstrated in 80% of the interstitial cell population of both rat strains while no staining was observed for vasopressin or neurophysin.

Presence of a neurophysin-like precursor in the green turtle (Chelonia mydas).

A glycoprotein of neurohypophysial origin was found to have cofractionated with FSH prepared from pituitary glands of the green turtle, Chelonia mydas. Antiserum raised against this preparation contained high antibody titres and affinity for the neurohypophysial component and allowed development of a specific radioimmunoassay to monitor its purification and distribution in the brain. Immunocytochemistry revealed that the glycoprotein was concentrated in the pars nervosa and associated nerve tracts passing through the median eminence to the supraoptic and paraventricular nuclei; similar distributions were observed in turtles and rats. The antiserum to the turtle material bound radiolabelled rat vasopressin (VP)-neurophysin and precipitated precursors of this neurophysin, but it did not cross-react with rat oxytocin-neurophysin. An amino-terminal alanine was also consistent with the structure of rat VP-neurophysin, but the turtle molecule was larger than the corresponding rat molecule. Limited tryptic digests of the turtle glycoprotein contained two components, one of which bound to lysine VP. Both components contained carbohydrate, but only the one which bound to VP cross-reacted in a radioimmunoassay for rat VP-neurophysin. The apparent surge in plasma immuno-FSH at the time of oviposition previously described in the turtle probably represented release of a neurophysin-like 'carrier' molecule associated with secretion of the neurohypophysial hormone (e.g. arginine vasotocin; AVT) responsible for oviduct contractility. These data suggest that the neurohypophysial glycoprotein represents a partially processed AVT precursor and provide the first biochemical evidence of a mammalian-like biosynthetic pathway for neurohypophysial hormones in a non-mammalian species.

Biosynthesis of oxytocin in the corpus luteum.

In this report we demonstrate that ovine and bovine luteal cells synthesise oxytocin by way of a precursor protein similar to that found in the hypothalamus. Isolated ovine or bovine luteal cells were incubated for up to 12 h with [35S]cysteine. Neurophysin-Sepharose column separation and HPLC of cell extracts demonstrated the presence of [35S]oxytocin. Incorporation of [35S]cysteine was confirmed by performic acid oxidation. Immunoprecipitation of cell extract with anti-rat oxytocin-neurophysin followed by SDS-PAGE yielded 2 radioactive bands of 14 kDa and 11-12 kDa. Immunoprecipitation with anti-oxytocin yielded 1 band at 14 kDa. On SDS-PAGE the 14 kDa band had a similar mobility to rat-hypothalamic oxytocin precursor.

Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy.

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.

Identification of oxytocin and vasopressin in the testis and in adrenal tissue.

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum.

The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.

Non-neuronal cells of rat hypothalamus in dissociated cell culture. Evidence that neurophysin modulates growth and DNA synthesis of non-neuronal cells.

The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture. Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum). Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium. DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium. At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin. This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.