Distribution of prolactin receptor immunoreactivity in ovine skin and changes during the wool follicle growth cycle.

Prolactin is believed to mediate seasonal cues entraining seasonal reproductive and hair follicle growth cycles. Prolactin receptor binding activity and prolactin receptor gene expression in mammalian skin have recently been described. In this report, prolactin receptor immunoreactivity is identified in sheep skin using a monoclonal antibody against the rat liver prolactin receptor. Western blotting analysis of microsomal membrane proteins from skin showed major bands corresponding to molecular weights of 87 and 71 kDa and minor bands at 101 and 21 kDa. RNase protection analysis revealed the presence of mRNA species coding for long and short forms of the prolactin receptor. Formalin-fixed sections, exposed to the monoclonal antibody and stained by an immunogold method, revealed prolactin receptor-immunoreactivity in the dermal papilla, germinal matrix, outer root sheath, lower regions of the inner root sheath and connective tissue sheath of wool follicles. Staining was absent from keratinised cell populations. In all samples, the interfollicular epidermis, sebaceous and sweat glands were positively stained. The distribution of prolactin receptor is described in both growing and inactive wool follicles and related to postulated cycle-specific actions of circulating prolactin in the control of seasonal fibre growth.

Inhibitory effect of increased photoperiod on wool follicle growth.

The relationships between circulating prolactin (PRL), wool follicle growth and daylength were investigated in 24 New Zealand Wiltshire ewes housed indoors from September 1989 to May 1991. Twelve control (C) ewes were maintained under natural photoperiod. Two other groups were held in short days (SD; 8 h light: 16 h darkness) commencing from the winter solstice (22 June 1990) for either three (group SD3, n = 7) or six (group SD6, n = 5) months before reversion to natural daylength. Skin was sampled at one- to four-week intervals for histological determination of percentages of growing primary and secondary follicles. Hourly blood samples over 24 h were collected via jugular cannulae from C sheep in March and July and then monthly from all animals until December 1990 for estimation of mean monthly PRL concentrations for each treatment group. Between autumn (March 1990) and winter (July) primary follicle activity (PFA) and secondary follicle activity (SFA) declined in C ewes (PFA: 97 to 43%, SFA: 100 to 57%). Follicle regrowth during July and August in eight C ewes preceded the initial rise in plasma PRL from the winter minimum (1.6 ng/ml). Across the three groups, four instances of decreased follicle activity were observed, closely following or concurrent with increases in plasma PRL concentrations. The resumption of spring growth in four C sheep was temporarily checked by falls in follicle activities during September and October as PRL concentrations began to increase (3.4 to 8.9 ng/ml). Follicle activity also declined in November and December in eight C sheep, coincident with the rapid rise in PRL to a seasonal maximum in late November (165.4 ng/ml). The increase in SD3 follicle activity over spring was not delayed by short days but during October, after release from treatment, PRL concentrations rose (1.8 to 12.0 ng/ml) and follicle activity declined (PFA: 65 to 38%, SFA: 68 to 43%). In SD6 ewes, PRL concentrations were suppressed (2.1 ng/ml) and relatively constant levels of follicle activity (PFA: 73%, SFA: 95%) were maintained throughout short-day treatment. Release of SD6 ewes into summer photoperiod in January 1991 temporarily interrupted follicle growth (PFA: 68 to 17%, SFA: 96 to 19%) and caused out-of-season shedding in March and April. Contemporary C follicle activities were high (PFA: 95%, SFA: 98%). These data suggest that natural and experimental increases in daylength have a short-term inhibitory effect on growing wool follicles which could be mediated through rising concentrations of plasma prolactin.

Localisation of receptors for prolactin in ovine skin.

Although prolactin (PRL) receptors are found in many mammalian tissues, specific PRL binding to mammalian skin has not been demonstrated. In view of the temporal relationships observed between photoperiod, circulating PRL and pelage replacement in seasonally responsive mammals, we sought to provide evidence of PRL receptors in ovine skin. Cryosections of skin from New Zealand Wiltshire ewes were incubated with radiolabelled human GH (125I-hGH) and ovine PRL (125I-oPRL) in the presence and absence of excess unlabelled hormones (hGH, oPRL or ovine GH (oGH)). Binding was inhibited by unlabelled oPRL and hGH but not by oGH. In microautoradiographs, both radioligands were localised most strongly in the dermal papillae of wool follicles in the anagen (growth) phase of the hair cycle and in apocrine sweat glands. Higher levels of specific binding to dermal papilla cells, compared with the follicle epithelial matrix and the surrounding dermis, were confirmed by measurement of microautoradiograph silver grain density (respectively, 34.1 +/- 3.0, 11.4 +/- 1.0 and 5.5 +/- 0.5 grains per 100 microns2 (mean +/- S.E.M., n = 10)). Total binding for 125I-hGH and 125I-oPRL radioligands to follicle dermal papilla was not significantly different (34.1 +/- 3.0 vs 43.6 +/- 2.5 grains per 100 microns2, n = 10) but the level of non-specific binding of 123I-oPRL was higher than for 125I-hGH (18.9 +/- 1.4 vs 6.1 +/- 0.6 grains per 100 microns2, n = 10; P < 0.001). Binding assays of receptors in crude microsomal membranes extracted from ovine skin were used to ascertain binding capacity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Fiber growth initiation in hair follicles of goats treated with melatonin.

The sequence of structural changes in goat hair follicles was investigated using melatonin implants to advance and synchronize spring hair growth. Ten pasture fed cashmere wethers each received a controlled release formulation of 70 mg of melatonin on September 1 1989, and showed plasma melatonin elevated above physiological levels over 14 days post-treatment (914 +/- 154 pg/ml [mean +/- SEM] on day 14). In ten untreated animals, daytime plasma melatonin was 19.9 +/- 4.7 pg/ml. Histological examination of skin biopsies taken over the 14 days from the start of the experiment showed that primary hair follicles of goats with manipulated hormone levels had initiated fiber growth (entered proanagen), whereas primary follicles of untreated goats largely remained in the quiescent phase (telogen). A standardized terminology was used to describe the sequence of events during induced proanagen. Structural reorganization of follicles began in treated animals between days 6 and 12 post-treatment, and emergent fibers grew by day 24. Advancement of spring fiber growth was associated with a suppression of the normal rise in plasma prolactin concentration. Prolactin levels in untreated goats increased from 7.4 +/- 1.8 ng/ml on day 1 to 12.8 +/- 1.6 ng/ml on day 14, but declined in treated goats from 6.3 +/- 2.3 ng/ml to 2.2 +/- 0.8 ng/ml over the same period.

Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species.

Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivites as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny.

Arginine vasopressin and oxytocin in the porcine corpus luteum.

The relationship between ovarian oxytocin (OT), vasopressin and progesterone, was determined in porcine corpora lutea collected at various stages of the estrous cycle. Fractionation by reverse phase high performance liquid chromatography (HPLC) of extracts from mid-cycle corpora lutea showed the presence of arginine vasopressin-like (AVP) material, but not lysine vasopressin-like (LVP) activity. The tissue content of progesterone reached maximal amounts at mid-luteal phase, and decreased rapidly at the end of the cycle. However, tissue concentration of progesterone was the highest in early luteal phase. OT content was unchanged throughout the luteal phase of the cycle but declined at the end of the cycle. Tissue concentration of OT decreased from early in the cycle but was still measurable in the corpus albicans. Changes of AVP content and concentration were substantially the same as that of OT, except for a significant increase in AVP concentrations at the end of the cycle. These data confirm the presence of OT, indicate the dominant presence of AVP and suggest that both hormones may be involved in porcine CL function.

Levels of oxytocin-like activity and progesterone in follicular fluid from in vitro fertilization cycles.

Oxytocin-like immunoreactivity, estradiol, and progesterone were measured in follicular fluid collected during oocyte collection in an in vitro fertilization program in which clomiphene citrate was used to stimulate follicular development. Follicles which yielded morphologically normal embryos after fertilization of the oocyte had oxytocin concentrations ranging from less than 10 to 600 ng/liter. Oxytocin concentrations did not differ between follicles from 12 pregnancy cycles (median, 169; N = 21) and follicles from 12 nonpregnancy cycles (median, 110; N = 18). Oxytocin concentrations were correlated negatively with progesterone concentrations (Spearman's rank correlation coefficient r = -0.50; P = 0.001). In cycles with some follicles having progesterone concentrations less than 10 and some greater than 10 mumol/liter, oxytocin concentrations were higher in the less progestogenic follicles in 15 of 16 cases.

HPLC separation of vasopressin-like hormones in chicken neurohypophysial extracts.

HPLC techniques were used to identify peptides that possess vasopressin-like immunoreactivity in the chicken neurohypophysis. The presence of arginine vasotocin (AVT) was confirmed together with arginine vasopressin (AVP). That the presence of AVP may be confined to the chicken, and not other avian species, was concluded from the absence of the hormone in the neurohypophysis of the duck and turkey. The chicken thus resembles some members of the suiformes and metatheria in possessing two vasopressin-like peptides.

Isolation and properties of sheep neurohypophysial nerve terminals.

Nerve terminals were isolated from sheep posterior pituitary lobes using a collagenase digestion technique. Freshly dispersed terminals, and terminals cultured for up to 3 days were examined by immunofluorescence microscopy. Immunohistochemical results suggested the presence of immunoreactive-neurophysin, -oxytocin and -vasopressin in membrane-bound structures. Membrane depolarization induced by high concentrations of potassium ions stimulated oxytocin release. This release was attenuated in the absence of calcium ions. The calcium ionophore, A23187, was also an effective stimulator of oxytocin secretion. These data suggest that neurohypophysial nerve terminals prepared by a collagenase dispersion procedure may be suitable for the investigation of posterior pituitary secretion mechanisms.

Isolation and sequence analysis of oxytocin from the sheep corpus luteum.

Studies have revealed that the corpus luteum (CL) of the sheep releases oxytocin (OT) -like immunoreactivity under normal and physiological conditions. We have now purified and completely characterized the OT-like species from ovine CL and established by Edman degradation and comparative reverse-phase HPLC its identity with hypothalamic oxytocin. On the basis of radioimmunoassay, the characterized oxytocin was the only peptide possessing OT-like immunoreactivity. This study represents the first identification by sequence analysis of oxytocin outside the central nervous system.

Evidence for the pulsatile release of PGF-2 alpha inducing the release of ovarian oxytocin during luteolysis in the ewe.

Frequent blood samples were removed from a utero-ovarian vein, a jugular vein and a femoral artery of 5 ewes during luteolysis. Analysis of these samples for oxytocin-associated neurophysin revealed a significant venous-arterial difference across the ovary and uterus but not across the head. This occurred during the pulsatile surges as well as when levels were basal and confirms the corpus luteum as a major source of the pulsatile surges of oxytocin-associated neurophysin and oxytocin that occur during CL regression and also of the elevated luteal phase concentrations of both hormones. The pulsatile surges of oxytocin-associated neurophysin measured in the utero-ovarian vein were accompanied by the release of an approximately equimolar amount of oxytocin. The concentration of PGF-2 alpha in the utero-ovarian vein samples began to increase before the levels of oxytocin and oxytocin-associated neurophysin started to increase. This suggests that uterine PGF-2 alpha initiates the release of ovarian oxytocin and oxytocin-associated neurophysin during luteolysis in the ewe.

Bulbospinal serotonin neurons and hypotensive effects of methyldopa in the spontaneously hypertensive rat.

Systemic methyldopa administration in genetically hypertensive rats evoked a hypotension which was attenuated after prior treatment with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) given either intracerebroventricularly, to produce a generalised ablation of serotonin nerves in brain and spinal cord, or by injection into the cervical spinal cord, to cause a selective destruction of descending serotonin pathways. Direct microinjection of methyldopa into the ventrolateral medulla in the area of the B1 and B3 serotonin cells was also effective in lowering arterial pressure. The hypotensive response to this medullary administration of methyldopa was again attenuated by 5,7-DHT given either intracerebroventricularly or by intraspinal injection. On the other hand, prior treatment intracerebroventricularly with 6-hydroxydopamine did not affect the hypotensive effect of methyldopa injected into the region of the ventrolateral B1 and B3 cells, supporting the suggestion that this effect of methyldopa is mediated by bulbospinal serotonin neurons and not by descending catecholamine nerves. The fact that methyldopa injection into the ventrolateral medulla in a region coinciding with the B1 and B3 cells lowers blood pressure is consistent with previous studies demonstrating that these neurons serve to maintain or elevate arterial pressure.

Importance of central serotonin neurons in the hypotensive action of methyldopa in the rat.

Normotensive (WKY) and stroke prone spontaneously hypertensive (SHR-SP) rats were given methyldopa (200 mg/kg i.p.) daily for five days and their brains were then sectioned and processed with the Faglu method for catecholamine fluorescence. This treatment with methyldopa induced a green fluorescence not seen in control animals, in cells coinciding with the B1-B9 groups of serotonin neurons in the brainstem. Pretreatment with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, i.c.v.), which is relatively specific for serotonin neurons, prevented the appearance of this green fluorescence in the serotonin cell groups of rats given methyldopa. Pretreatment with 5,7-DHT, i.c.v. approximately halved the magnitude of the hypotensive response to a single dose of methyldopa (80 mg/kg i.p.). We suggest that central serotonin nerves contribute to the hypotensive action of methyldopa. It is our hypothesis that methyldopa is taken up by these serotonin cells and that the green fluorescence reflects the production of alpha-methyldopamine, as a result of decarboxylation by the ubiquitous enzyme, L-aromatic amino acid decarboxylase.

Neuroendocrine pacemaker for growth, development and ageing.

The hypothesis is formulated that ageing is mediated through alteration in a pacemaker system located in the hypothalamus and dependent on serotonin-thyroid hormone interactions. Data in support of this hypothesis include: imbalance in central nervous system (CNS) neurotransmitters with ageing, with serotonin dominance; impairment of hypothalamic neurosecretion; increased heterogeneity of thyrotropin forms secreted from the aged pituitary; and drug and dietary manipulation of CNS serotonin with consequent effects on endocrine function and homeostasis.

Maturation of the hypothalamo-neurohypophysial system. II. Neurophysin, vasopressin and oxytocin in the median eminence of the developing rat brain.

Transverse sections of the median eminence from fetal and neonatal rats were examined by the immunoperoxidase technique to detect the presence of oxytocin, vasopressin and neurophysin. Neurophysin was observed in the 18-day fetus. Vasopressin and oxytocin were not detected until after birth, on the 4th and 8th days respectively. There was an accumulation of material crossreactive with neurophysin and vasopressin antibodies in the palisade layer of the median eminence between the 4th and 9th days after birth. This distribution of immunoreactive material in the palisade layer was suggestive of neurosecretory substances localized in two fibre tracts on either side of the median eminence. The data are consistent with the accumulation of corticotropin releasing factor and an associated neurophysin in this area. It is suggested that the accumulation of material occurs because of the relative immaturity of the capillary loops that constitute the primary plexus of the hypophysial portal system.

Maturation of the hypothalamo-neurohypophysial system. I. Localization of neurophysin, oxytocin and vasopressin in the hypothalamus and neural lobe of the developing rat brain.

Sections of the hypothalamus, median eminence and pituitary from fetal and neonatal rats were examined with the immunoperoxidase staining technique and light microscopy. Purified antisera raised against vasopressin and oxytocin, and antisera cross-reactive with rat neurophysin were used to localize these antigens in the hypothalamo-neurohypophysial system (HNS). Neurophysin was detected throughout the HNS of the 18-day fetal rat. Vasopressin was present in the hypothalamus and pituitary of the 19-day fetus, and in the median eminence of the 4-day neonate. Oxytocin was not detected in the pituitary until 1--2 days after birth, in the hypothalamus after 4 days, and in the median eminence after 8 days. During the first days after birth the supraoptic nucleus was more mature than the paraventricular nucleus. The HNS did not approach maturity until at least 7 days after birth. The relative maturity of the supraoptic nucleus compared with the paraventricular nucleus, and the detection of vasopressin before oxytocin are evidence for the one-neuron-one-hormone theory. The data do not exclude the possibility that the fetal hypothalamo-neurohypophysial system, and perhaps the fetal hormone, vasotocin, affect the initiation and course of parturition.

Immunocytochemical study of the hypothalamo-neurohypophysial system. II. Distribution of neurophysin, vasopressin and oxytocin in the normal and osmotically stimulated rat.

Antisera, with cross reactive antibodies removed by affinity chromatography, were used in the immunoperoxidase-bridge technique to study the distribution of oxytocin and vasopressin together with neurophysin in the hypothalamo-neurohypophysial system of the rat. The hormones were demonstrated in different areas of the supraoptic nucleus (SON) and paraventricular nucleus (PVN), in neurosecretory fibres of the hypothalamo-neurohypophysial tract, median eminence, and in nerve terminals of the neurohypophysis. Intact normal and rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain), and rats dehydrated by the administration of oral hypertonic saline were studied. In dehydrated rats the hormone concentration in the neurons, and the number of neurons containing hormone varied according to the time of dehydration stress. The observations support the hypotheses that: 1) oxytocin and oxytocin-neurophysin, and vasopressin and vasopressin-neurophysin are synthesised in different neurons and are transported along different axons; 2) the SON and PVN are functionally indistinguishable in that neurons containing oxytocin or vasopressin are present in both nuclei; and 3) the two types of neurons respond to osmotic stimulation in a way that is qualitatively the same but quantitatively different.

Immunocytochemical study of the hypothalamo-neurohypophysial system. III. Localization of oxytocin- and vasopressin-containing neurons in the pig hypothalamus.

Synthetic oxytocin and [8-arginine]-vasopressin conjugated to bovine thyroglobulin were used to induce specific antibodies in rabbits. The specificity of the anti-oxytocin serum, and the suitability of the anti-[8-arginine]-vasopressin serum for the detection of [8-lysine]-vasopressin, was evaluated by immunofluorescent studies of the respective hormones bound to Sepharose 4B particles. Oxytocin and [8-lysine]-vasopressin were specifically localized in the paraventricular (PVN) and supraoptic (SON) nuclei of the pig hypothalamus using the immunoperoxidase staining technique. After an examination of serial transverse and sagittal sections stained for either of the hormones we observed that: 1. In the rostral SON, oxytocin and vasopressin containing neurons were uniformly distributed; 2. In the caudal SON, most of the neurons contained oxytocin, but there were still a few 'vasopressin' neurons; 3. In the rostral PVN, the two hormones were evenly spread in neurons close to the third ventricle; 4. In the caudal PVN, the oxytocin and vasopressin containing neurons were differentially distributed, with 'oxytocin' neurons adjacent to the third ventricle, and 'vasopressin' neurons lateral to these and concentrated in the dorso-caudal PVN. In the cells of the PVN, there was evidence that the distribution of oxytocin and vasopressin is similar to the distribution of porcine neurophysin-II and porcine neurophysin-I respectively. This similarity is consistent with the one hormone--one neurophysin concept in the pig.

Effect of fixation on the demonstration of neurophysin and "Gomori-positive" substances in neurosecretory granules of the rat hypothalamus.

Proteins reacting with neurophysin antibodies and "Gomori-positive" substances were demonstrated histochemically in hypothalamic neurosecretory material of normal and bilaterally adrenalectomized rats after two different fixations: a) picric acid-formalin (PAF) for 7 days at 37 degrees C; b) Bouin's fluid for 20 h at 4 degrees C. After PAF-fixation anti-neurophysin reactive neurosecretory granules are found in all parts of the supraoptico-hypophysial system and in the suprachiasmatic nucleus of normal and adrenalectomized animals. In the latter they can additionally be demonstrated in the outer layer of the median eminence. Amount and distribution of "Gomori-positive" substances correspond to those described for the immunoreactive material, except for the suprachiasmatic nucleus, in which the substances can not be detected; Following fixation in Bouin's fluid the immunohistochemical reactions are unchanged whereas the staining of "Gomori-positive" substances is remarkably impaired. The amounts of the substances demonstrable in the neural lobe are diminished and in the cells of the supraoptic and paraventricular nuclei as well as in both median eminence layers only traces of the substances are to be seen. The findings indicate that negative results in demonstrating "Gomori-positive" substances may be caused by inappropriate fixation and need to be controlled by immunohistochemical techniques.