GABA Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study examines the involvement of GABA_{A or B} receptors in gonadotrophin‐releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates γ‐aminobutyric acid (GABA)–GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)‐α at 4°C; within 2 h, slices were singly perifused at 37°C with oxygenated MEM‐α (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA_{A or B} receptor ligands (0.1–10 mm ). The GABA_{A or B} agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mm GABA_{A or B} antagonists (bicuculline or CGP52432, respectively). The GABA_A antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus‐pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to the GABA_A antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA_A antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA_A receptor inhibitory control and this is attenuated in the presence of oestradiol.     

Noradrenergic Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study investigates the influence of α₁‐adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates α₁‐adrenoreceptor‐GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media‐α (MEM‐α) at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM‐α (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4‐h equilibration, 10‐min fractions were collected for 4 h interposed with a 10‐min exposure at 60 min to specific α₁‐adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1–10 mm ). The α₁‐adrenoreceptor agonist (10 mm ) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, α₁‐adrenoreceptor agonist (10 mm )‐induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus–pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to α₁‐adrenoreceptor agonist (10 mm ), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 ± 12.3–36.2 ± 21.6 ng/ml) confirming that the α₁‐adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.     

Progesterone and the surge release of gonadotropins in the ewe

On day 12 of an estrous cycle, 4 groups of ewes were treated with either blank (no steroid) or 3 different sizes of progesterone-containing rubber implants to study the effect of maintained progesterone levels on preovulatory events. Following luteolysis progesterone levels were 0.55 ± 0.13 ng/ml in control ewes and 0.62 ± 0.10 ng/ml, 0.99 ± 0.09 ng/ml and 1.85 ± 0.04 ng/ml in the 3 groups of progesterone-treated ewes. Preovulatory surges of LH and FSH occurred in $\text{5}\text{5}\text{,}\text{5}\text{5}\text{;}\text{4}\text{5}\text{,}\text{3}\text{5}\text{;}\text{4}\text{5}\text{,}\text{3}\text{5}$ and $\text{0}\text{5}\text{,}\text{0}\text{5}$ ewes in these 4 groups respectively. Eleven days after implant insertion, all ewes responded to a GnRH challenge. The height of all FSH peaks was depressed by progesterone treatment (P<0.05). Three groups of ewes were ovariectomized at day 6 of a cycle and treated with estradiol-17β and progesterone-containing implants. After 8 days of treatment, progesterone implants were removed in sections to give 3 different rates of decline in serum progesterone levels. The gonadotropin surges occurring following progesterone removal were delayed by the slower rates of progesterone decline. Another group of ewes was treated as in experiment 2, but the progesterone implants were all removed together after 8 days. Subsequent replacement of progesterone implants, after 12 or 18 hours, blocked the gonadotropin surges in all, or 2 of 6 ewes respectively. When replaced after 24 hours, implants producing low progesterone (1.38 ± 0.22 ng/ml) did not block gonadotropin surges, but in 2 of 5 ewes high progesterone did (2.87 ± 0.22 ng/ml). Removal of progesterone implants, 12 or 24 hr after replacement, produced secondary gonadotropin surges of smaller magnitude than the initial peaks (P<0.01).     

Distribution of orexin B and its relationship with GnRH in the pig hypothalamus

Increasing evidence suggests that orexins--hypothalamic neuropeptides--act as neurotransmitters or neuromediators in the brain, regulating autonomic and neuroendocrine functions. Orexins are closely associated with gonadotropin-releasing hormone (GnRH) neurons in the preoptic area and alter luteinizing hormone (LH) release, suggesting that they regulate reproduction. Here, we investigated the distribution of orexin B (immunohistochemical technique) and the relationship between orexin B and GnRH containing fibres and neurons in the pig hypothalamus using double immunofluorescence and laser-scanning confocal microscopy. Orexin B immunoreactive neurons were mainly localized in the perifornical area (PeF), dorsomedial hypothalamic nucleus (DMH), zona incerta (ZI) and the posterior hypothalamic area (PH), with a sparser distribution in the preoptic and anterior hypothalamic area. Immunoreactive fibres were distributed throughout the central nervous system. Approximately 30% GnRH neurons were in close contact with orexin B immunoreactive fibres, among these approximately 6% of GnRH neurons co-localized with orexin B perikarya in the region between the caudal preoptic area and the anterior hypothalamic area. Orexin B may regulate reproduction by altering LH release in the hypothalamus.     

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.     

Humoral immunity in the ewe. 3. The influence of adjuvants and immunisation regimes on immune reactivity in the breeding ewe and her progeny

Breeding ewes were immunised with clostridial vaccine using different inoculation schedules. Results, showing differences in the class of antibody produced, were heavily dependent on the vaccination regime used. Immunoglobulin M (IgM) levels were significantly lower in ewes given double doses of vaccine compared to ewes given a single inoculation or no treatment at all (P less than 0.01). Neonatal lambs showed significant de novo IgM production with interference in this antibody production in the lambs of ewes vaccinated with the clostridial vaccine (P less than 0.05). Immunoglobulin G1 (IgG1) levels were significantly increased in all lambs which had mothers vaccinated with the clostridial vaccine prior to or during pregnancy (P less than 0.025). The greatest quantity of IgG1 was transferred to lambs when their mothers were given a double injection with primary inoculation prior to conception and booster prepartum (P less than 0.025). No antigen specific immunoglobulin G2 (IgG2) was detected in the lambs. Ewes were also immunised with BSA and their isotype specific serum antibody response was compared with their respective lambs. There was no detectable anti-BSA IgM in the lambs of all groups of ewes though specific IgG1 antibodies could be readily detected in the lambs of hyperimmunised ewes. The efficiency of transfer was related directly to the ability of the adjuvant to maximise IgG1 production in the ewe. Although immunised ewes produced high levels of IgG2, this was not transferred passively to the lamb.     

Characterisation of IgE-mediated histamine release from equine basophils in vitro

In vitro IgE-mediated histamine release by equine blood basophils was characterised as the basis for a screening test for immediate hypersensitivity responses in horses. The responses are initiated by inducing agents that are capable of crosslinking or bridging the membrane-bound IgE molecules. The release process is complete within 40 mins. In vitro histamine release is dose-dependent, with a submaximal response at less or greater than the optimal dose of inducing agent. Exogenous calcium is required but not magnesium; the optimal release calcium concentration is 1.0 to 1.5 mM. If an IgE-mediated inducing agent is added in the absence of exogenous calcium, the basophils become desensitised. The pH and temperature optima for release are physiological (pH 7.4, 37 degrees C). Histamine release is potentiated by deuterium oxide.     

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.     

Pharmacological characterisation of the electrically evoked release of monoamines from chicken brain in vitro

1. A study was conducted to develop an in vitro model for examining the basal and electrical-stimulation-induced release of [3H]monoamines from chicken hyperstriatal neurones in order to demonstrate the presence of presynaptic autoreceptors for the three main monoamine transmitters: noradrenaline, dopamine and 5-HT. 2. Two sets of experiments were carried out: the first was to evaluate the effect of calcium and tetrodotoxin (TTX, sodium channel conductance inhibitor) in order to demonstrate that evoked release of monoamines was a consequence of exocytotic processes; the second to investigate the effect of selective agonists and antagonists on neurotransmitter release. 3. Ross and Cobb broiler chickens of either sex (approximately 7 to 8 weeks old) were used. Slices of hyperstriatal tissue were preincubated with [3H]noradrenaline, [3H]dopamine or [3H]5-hydroxytryptamine (5-HT), washed, perfused and electrically stimulated at three time points (S1, S2 and S3) which released [3H]noradrenaline, [3H]dopamine and [3H]5-HT, as determined by scintillation spectrometry. 4. When calcium was removed from, or TTX added to, the superfusion medium prior to and including the second period of electrical stimulation (S2) the evoked releases of [3H]noradrenaline, [3H]dopamine and [3H]5-HT at S2 were abolished. 5. In the presence of the selective alpha2-adrenoceptor agonist UK 14304 during the S2 period, the S2/S1 ratio was lower than the control ratio due to a reduction in the stimulated release of [3H]noradrenaline. The selective alpha2-adrenoceptor antagonist RX 821002 blocked the UK 14304-induced reduction of evoked release and the S2/S1 ratio was similar to the control ratio. 6. The D2-like receptor agonist quinpirole reduced the S2/S1 ratio for [3H]dopamine release, an effect blocked by the antagonist AJ 76. The 5-HT1B receptor agonist CP 94253 during S2 reduced the S2/S1 ratio due to a reduction in evoked [3H]5-HT. This effect was blocked by the 5-HT1B receptor antagonist GR 55562. 7. The results demonstrate, for the first time, the functional presence of presynaptic alpha2-adrenoceptors, presynaptic 5-HT1B autoreceptors and presynaptic D2-like autoreceptors in broiler chicken hyperstriatal neurones in vitro.     

Progesterone inhibits prolactin and growth hormone release from fowl pituitary glands in vitro

After preincubation of anterior pituitary glands of broiler fowls for 20 h in either medium alone or medium containing progesterone, their responsiveness to hypothalamic stimulation and to thyrotrophin releasing hormone (TRH) was determined. Following exposure to progesterone the basal rate of release of prolactin was reduced in a concentration-related manner but basal growth hormone release was unaffected. Stimulation of the release of prolactin and growth hormone by both hypothalamic extract and TRH was reduced following incubation with progesterone, and the reduction of the prolactin response to TRH was related to progesterone concentration.     

Influence of reproductive state on pituitary-adrenal activity in the ewe.

Plasma corticosteroid concentrations are altered in pregnancy, during the reproductive cycle and by ovariectomy in many species. This study was designed to examine basal ACTH and cortisol in ewes of four different reproductive statuses: ovariectomized, nonpregnant cycling, nonpregnant noncycling, and pregnant. Blood samples were drawn every 4 hr for 48 hr from ewes during quiet, undisturbed conditions and analyzed for plasma ACTH, cortisol and progesterone concentrations. There were no significant changes in ACTH, cortisol or progesterone over time. Mean progesterone concentrations were significantly greater in the pregnant ewes than in all other ewes, and were greater in cycling ewes than noncycling or ovariectomized ewes. Mean ACTH was significantly greater in pregnant ewes than noncycling ewes, and mean cortisol was significantly greater in cycling ewes than in nonpregnant cycling or noncycling ewes. Ovariectomized ewes also had significantly greater mean cortisol concentrations than cycling ewes. The results demonstrate that there is an increase in basal ACTH and cortisol in ovine pregnancy.     

Akabane virus infection in the pregnant ewe. 2. Pathology of the foetus

A strain of Akabane virus (CSIRO 16) isolated from Culicoides brevitarsis was given three different passage treatments in the laboratory and then inoculated into ewes that were 32 to 36 days pregnant. The foetuses from these ewes were examined between the 69th and 106th days of gestation. The 39 infected ewes produced 55 foetuses of which 44 (80%) had severe developmental defects. Arthrogryposis and agenesis of the brain or hydranencephaly, were present in 43 of the foetuses. Other gross defects affecting variable proportions of foetuses were porencephaly, brachygnathism, scoliosis, hypoplasia of the lungs and agenesis or hypoplasia of the spinal cord. Histopathological findings covered a wide spectrum of defects that have previously been considered to occur over an extended range of foetal ages. These defects included skeletal muscle atrophy and degeneration, and in the brain, particularly in the cerebrum, cystic areas and malacia, general oedema, subependymal gliosis, perivascular cuffing and mineralised plaques. Similar lesions were seen in the pons and cerebellum. Extensive lesions, with and without inflammation were seen in the spinal cord.     

Glycosaminoglycans in ewe reproductive tracts and their influence on acrosome reactions in bovine spermatozoa in vitro

Glycosaminoglycans (GAG) promote acrosome reactions (AR) in bovine and rabbit spermatozoa in vitro. Female bovine reproductive tract secretions contained GAG and the concentrations and composition of those GAG varied with different regions and stages of the estrous cycle. This study was designed to evaluate the types of GAG found in reproductive tract secretions of ewes at different stages of the estrous cycle and the ability of the secretions to promote AR. Ewes (n = 48) were slaughtered at 0, 12, 24, 36, 72 and 144 h following observations of standing estrus. Reproductive tracts were flushed with a modified Tyrode's medium (TALP: 7 ml) not containing calcium. Concentrations and composition of GAG in the tract were determined by high performance liquid chromatography. Concentrations of GAG decreased anterior to the cervix: 2.28 to .74 mg/100 mg protein (P less than .05). Increases in heparin-like GAG during the estrual phase and chondroitin sulfate GAG during the luteal phase were noted. Bovine sperm were incubated in tract flushings standardized to contain 4 micrograms/ml of GAG and supplemented with calcium. Sperm incubated in estrual flushings for 9 h exhibited a higher incidence of AR than those incubated in luteal flushings or control, 50% vs 36% and 29%, respectively (P less than .0001). It is concluded that during the estrous cycle there were changes in concentrations and composition of GAG in ewe reproductive tracts and the potencies of those female secretions causing AR varied.     

Effect of vasoactive intestinal peptide on the release of growth hormone in perifused pituitary and hypothalamus of the goat

The effect of vasoactive intestinal peptide (VIP) on the release of growth hormone (GH) from the adenohypophysis of the goat was studied in vitro using the perifusion system. Two perifusion systems were employed to differentiate potential direct effects of VIP on the pituitary from indirect effects mediated through the hypothalamus. The first perifusion system used single chambers housing only pituitary fragments. The second system used two chambers in tandem, one containing hypothalamus and the second the pituitary fragments, the flow passing through the hypothalamic chamber first. VIP (10(-6), 10(-7), 10(-8)M) stimulated significant GH release in both perifusion systems (P less than 0.05, P less than 0.01) in a concentration related fashion. The quantity of GH release induced by the 10(-9)M and 10(-10)M VIP groups were not significant. Further, the GH released from the system that perifused the pituitary alone (10(-8)M VIP) was significantly higher than that containing both the hypothalamus and the pituitary fragments (P less than 0.05). The relative lower GH secretory response to the same dose of VIP applied to the hypothalamus-pituitary suggests that the perifused caprine hypothalamus may release an inhibitory factor, such as somatostatin in vitro. These results suggest that VIP stimulates GH release by acting directly on the adenohypophysis of the goat.     

In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells

The growth of Sarcocystis neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 microM was found to release intracellular merozoites with a 40 min treatment at 37 degrees C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.     

Combined administration of ghrelin and GHRH synergistically stimulates GH release in Holstein preweaning calves

Ghrelin is a gut peptide which participates in growth regulation through its somatotropic, lipogenic and orexigenic effects. Synergism of ghrelin and growth hormone-releasing hormone (GHRH) on growth hormone (GH) secretion has been reported in humans and rats, but not in domestic animals in vivo. In this study, effects of a combination of ghrelin and GHRH on plasma GH and other metabolic parameters, and changes in plasma active and total ghrelin levels were studied in Holstein bull calves before and after weaning. Six calves were intravenously injected with vehicle (0.1% BSA-saline), ghrelin (1 microg/kg BW), GHRH (0.25 microg/kg BW) or a combination of ghrelin plus GHRH at the age of 5 weeks and 10 weeks (weaning at 6 weeks of age). Ghrelin stimulated GH release with similar potency as GHRH and their combined administration synergistically stimulated GH release in preweaning calves. After weaning, GH responses to ghrelin and GHRH became greater compared with the values of preweaning calves, but a synergistic effect of ghrelin and GHRH was not observed. The GH areas under the concentration curves for 2h post-injection were greater in weaned than in preweaning calves (P<0.05) if ghrelin or GHRH were injected alone, but were similar if ghrelin and GHRH were injected together. Basal plasma active and total ghrelin levels did not change around weaning, but transiently increased after ghrelin injection. Basal plasma insulin, glucose and non-esterified fatty acid levels were reduced after weaning, but no changes by treatments were observed. In conclusion, ghrelin and GHRH synergistically stimulated GH release in preweaning calves, but this effect was lost after weaning.     

A recombinant feline immunodeficiency virus envelope fusion protein stimulates peripheral blood lymphocytes from naive cats to proliferate in vitro.

A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase. The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system. The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST.