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.     

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

The present study aims at ascertaining the influence of α₁‐adrenoreceptors on arginine vasopressin (AVP) release in vitro and determine whether E₂ modulates the α₁‐adrenoreceptor and AVP interaction. Ten minutes after ewe killing, sagittal midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus with the median eminence, 2 mm thick, 2 per sheep) 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 E₂ (24 pg/ml). After 4 h equilibration, 10 min fractions were collected for 4 h interposed with 10 min exposure at 60 min to a specific α₁‐adrenoreceptor agonist or antagonist at various doses (0.1–10 mm ). At the end of all perifusions, slices responded to KCl (100 mm ) with AVP efflux (p < 0.05). Release of AVP was enhanced (p < 0.05) by the α₁‐adrenoreceptor agonist (methoxamine 10 mm ; no E₂, n = 7 perifusion chambers: from 14.3 ± 2.7 to 20.9 ± 3.9, with E₂, n = 10: from 10.7 ± 1.2 to 18.4 ± 3.4 pg/ml) or the antagonist (thymoxamine 10 mm ; no E₂, n = 5: from 9.5 ± 3.1 to 30.4 ± 6.0, with E₂, n = 10: from 10.8 ± 0.9 to 39.1 ± 6.3 pg/ml). With the agonist, the response occurred only at 80 min (p < 0.05) both in the presence and absence of E₂. Whereas, after the antagonist, values were higher (p < 0.05) throughout the post‐treatment period (80–170 min) without E₂, but declined by 150 min in the presence of E₂. Furthermore, the response to the α₁‐adrenoreceptor antagonist was greater (p < 0.05; 90–140 min) than the agonist only in the presence of E₂. In conclusion, these results reveal direct α₁‐adrenoreceptor‐mediated control of the hypothalamic AVP neuronal system which is modulated by E₂.     

Oestradiol stimulates the release of AVP and GnRH from the ewe hypothalamus in vitro.

Oestradiol (E(2)) sensitizes the stress and reproductive axes in vivo. Our current aim is to investigate whether E(2) directly influences hypothalamic AVP and GnRH release in vitro. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to mediobasal hypothalamus, 2 mm thick, two per sheep) were dissected, placed in oxygenated MEM-alpha at 4 degrees C and within next 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 150 microl/min) alone (vehicle; n = 15), with low (6 pg/ml; n = 14) or high E(2) (24 pg/ml; n = 13). After 5 h equilibration, 10 min fractions were collected for 3 h with exposure to 100 mm KCl for 10 min within the last hour. Concentrations of AVP and GnRH were measured by RIA. Baselines for AVP and GnRH were 7.0 +/- 1.1 and 17.4 +/- 0.8 pg/ml respectively. Basal values with low E(2) were similar to vehicle for AVP (7.5 +/- 1.2 pg/ml) and GnRH (17.5 +/- 1.1 pg/ml). However, high E(2) increased basal AVP (11.7 +/- 1.4 pg/ml; p < 0.05) and GnRH (23.7 +/- 1.4 pg/ml; p < 0.05). After KCl, AVP and GnRH respectively, increased (p < 0.05) to 25.6 +/- 7.5 and 38.2 +/- 5.6 (vehicle), 26.3 +/- 7.5 and 23.6 +/- 2.1 (low E(2)) and 24.1 +/- 5.4 and 41.3 +/- 6.6 pg/ml (high E(2)). After KCl, maximum values of AVP occurred at 20 and GnRH at 30 min. In conclusion, high E(2) concentration augments AVP and GnRH release by direct action on the ewe hypothalamus.     

γ-Amino Butyric Acid Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims to ascertain the influence of gamma-amino butyric acid (GABA)(A or B) receptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates GABA-AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)-alpha at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (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 a specific GABA(A or B) receptor agonist or antagonist at various doses (0.1-10 mm). GABA(A) (muscimol; no E(2), n = 7 perifusion chambers, with E(2), n = 11) or GABA(B) (baclofen; no E(2), n = 8, with E(2), n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20-30 min after exposure to 10 mm GABA(A or B) antagonists (bicuculline, no E(2), n = 7: from 4.6 +/- 0.7 to 33.0 +/- 0.4, with E(2), n = 17: from 11.9 +/- 1.4 to 32.8 +/- 6.0; CGP52432, with E(2), n = 14: from 14.0 +/- 2.6 to 28.8 +/- 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABA(B) but not GABA(A) antagonist-stimulated AVP release was enhanced in the presence of E(2). In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E(2) through GABA(B) receptors in vitro.     

Regulation of growth hormone-releasing hormone and somatostatin from perifused,bovine hypothalamic slices. III. Reciprocal feedback between growth hormone-releasing hormone and somatostatin

An in vitro perifusion system for bovine hypothalamic tissue was used to determine if growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) modulate each other's release, and whether SRIF mediates D₁-agonist-induced suppression of GHRH in cattle. Up to three sagittal slices (600 μm) of bovine hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4° C, placed in 5 cc syringe barrels, and perifused at 37° C with oxygenated minimum essential medium-α at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Perifusion of SRIF at 10⁻⁶ M and 10⁻⁴ M decreased AUC for GHRH from 86.3 (control) to 65.4 and 59.5 ± 6.3 ng · ml⁻¹ min, but 10⁻⁸ M SRIF was ineffective. Relative to controls, 10⁻⁸, 10⁻⁶, and 10⁻⁴ M GHRH increased release of SRIF 190, 675, and 1,135%, respectively. Activation of D₁ receptors with 10⁻⁶ M SKF 38393 increased AUC for SRIF from 12.5 ng · ml⁻¹ min (control) to 484.9 ng · ml⁻¹ min and decreased AUC for GHRH from 36.4 ng · ml⁻¹ min (control) to 18.2 ng · ml⁻¹ min. Blockade of SRIF action with a SRIF antagonist, cyclo-[7-aminoheptanoyl-phe-d-trp-lys-thr(bzl)], increased release of GHRH 1.9-fold. In addition, the SRIF antagonist blocked SKF 38393-induced suppression of GHRH. We concluded that GHRH and SRIF interact within the bovine hypothalamus/pituitary stalk to modulate the release of the other. Moreover, SRIF mediates the inhibitory effects of activation of D₁ receptors on release of GHRH in cattle.     

13,14‐Dihydro‐15‐Keto Prostaglandin F2α Release in Response to Oxytocin Challenge Early Post‐Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post‐partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre‐ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF₂αand prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 IU of eCG, immediately after PGF₂α treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 ± 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 ± 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14‐dihydro‐15‐keto prostaglandin F₂α (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre‐ovulatory period was not effective in inhibiting PGFM release, which was lower in P4‐primed than in non‐primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4‐primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.     

Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS

Background: Immune stress induced by lipopolysaccharide(LPS) influences the gonadotropin-releasing hormone(GnRH)/luteinizing hormone(LH) secretion. Presence of LPS interacting Toll-like receptor(TLR) 4 in the hypothalamus may enable the direct action of LPS on the GnRH/LH secretion. So, the aim of the study was to investigate the influence of intracerebroventricular(icv) injection of TLR4 antagonist on GnRH/LH secretion in anestrous ewes during LPS-induced central inflammation. Animals were divided into three groups icv-treated with: Ringer-Locke solution, LPS and TLR4 antagonist followed by LPS.Results: It was demonstrated that TLR4 antagonist reduced LPS-dependent suppression of GnRH gene expression in the preoptic area and in the medial basal hypothalamus, and suppression of receptor for GnRH gene expression in the anterior pituitary gland. It was also shown that TLR4 antagonist reduced suppression of LH release caused by icv injection of LPS. Central administration of LPS stimulated TLR4 gene expression in the medial basal hypothalamus.Conclusions: It was indicated that blockade of TLR4 prevents the inhibitory effect of centrally acting LPS on the GnRH/LH secretion. This suggests that some negative effects of bacterial infection on the hypothalamic-pituitary-gonadal axis activity at the hypothalamic level may be caused by central action of LPS acting through TLR4.     

Pituitary responsiveness to GnRH, hypothalamic content of GnRH and pituitary LH and FSH concentrations immediately preceding puberty in gilts

To determine whether pituitary concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) or hypothalamic content of gonadotropin releasing hormone (GnRH) change before puberty, 40 prepubertal gilts averaging 7 mo of age were slaughtered before or on the second, third or fourth day after relocation and boar exposure. Some gilts responded to relocation and boar exposure as indicated by swollen vulvae, turgid uteri and enlarged ovarian follicles at the time of slaughter. Pituitary concentrations of LH and FSH and hypothalamic content of GnRH were similar between gilts that responded to relocation and boar exposure and gilts that did not respond. In addition, boar exposure and relocation had no effect on pituitary concentrations of LH and FSH or on hypothalamic content of GnRH. To determine whether pituitary responsiveness to GnRH changes before puberty, a third experiment was conducted in which 72 gilts were injected with 400 micrograms of GnRH either before or on the second, third or fourth day after relocation and boar exposure. In gilts that subsequently responded (i.e., ovulated) as a result of relocation and boar exposure, pituitary responsiveness to GnRH was reduced as compared with gilts that failed to ovulate after relocation and boar exposure. Peak concentrations of serum LH after GnRH injection were 4.6 +/- 1.3 vs 9.8 +/- .8 ng/ml for responders vs nonresponders. Peak serum FSH after GnRH injection was also lower for responders than for nonresponders (29.5 +/- 4.2 vs 41.2 +/- 2.4 ng/ml). When compared with controls, relocation and boar exposure did not significantly affect GnRH-induced release of LH and FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of an endocrine challenge test to investigate subfertility in ewes

Simultaneous or sequential injection of 250 ng gonadotrophin releasing hormone (GnRH) and 25 micrograms oestradiol benzoate, with luteinizing hormone (LH) measurements at 0, +20 min (after GnRH) and +16 h (after oestradiol), enabled investigation of the positive feedback effects on the hypothalamus and pituitary. Control ewes had pretreatment LH values of 3.1 +/- 1.2 ng/ml with an increment of 3.2 +/- 2.3 ng/ml 20 min after GnRH. Subfertile ewes, in spite of elevated pretreatment LH concentrations (15.8 +/- 9.5 ng/ml) in eight out of 10 ewes, had increments of 1.4-84 ng/ml after GnRH. Control ewes had LH increments of 3-75 ng/ml 16 h after oestradiol. Subfertile ewes with pretreatment LH concentrations less than 15 ng/ml also responded to oestradiol whereas those with initial LH concentrations 16-40 ng/ml had no further LH increment. Subsequent administration of 1000 iu pregnant mares' serum gonadotrophin (PMSG), with measurement of LH and oestradiol at 0, +24, +30, +48, +54, and +72 h, allowed assessment of ovarian response and hypothalamus-pituitary function. Five control ewes were sampled up to 30 h post-PMSG and only 1 had oestradiol concentrations greater than 10 pg/ml. Sampling up to 72 h in another five control ewes resulted in oestradiol concentrations greater than 10 pg/ml. Increments in LH concentration greater than 3 ng/ml were recorded in control and subfertile ewes with oestradiol concentrations greater than 10 pg/ml. The use of these endocrine challenge tests enabled positive diagnosis of abnormality on 8 out of 10 occasions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Central Effect of β‐Endorphin and Naloxone on The Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The Hypothalamic‐Pituitary Unit of Follicular‐Phase Ewes

The effects of prolonged, intermittent infusion of β‐endorphin or naloxone into the third cerebral ventricle of follicular‐phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme‐linked immunoabsorbent assay. Activation or blockade of μ‐opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with β‐endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that β‐endorphinergic system in the hypothalamus of follicular‐phase ewes affects directly or via β‐endorphin‐sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic‐pituitary axis.     

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.     

Direct Effects of Luteal Regression on Anterior Pituitary Response To GnRH

The preovulatory period of the ewe is marked by a dramatic decrease in concentrations of progesterone in serum during the late luteal phase, followed by elevated luteinizing hormone (LH) secretion, final follicular maturation and ovulation. This experiment was designed to ascertain the extent to which removal of endogenous progesterone negative feedback at the anterior pituitary gland, independent of effects at the hypothalamus, promotes increased secretion of LH in the hours immediately after induction of luteolysis. Estrus was synchronized in ovary-intact ewes with two injections of prostaglandin F₂α (PGF₂α) analog given 10 d apart (Day 0 = second day after the second PGF₂α injection). Ewes were subjected to hypothalamic-pituitary disconnection (HPD; n = 6) on Day 3 and were pulsed with gonadotropin-releasing hormone (GnRH). Ewes were used during the estrous cycle or received approximately 400 IU pregnant mare serum gonadotropin (PMSG) on Day 2 to stimulate ovulation; there was no difference (P < 0.10) in ovulation rate or progesterone production between these two groups. Luteal regression was induced by injection of PGF₂α analog on approximately Day 10 of the estrous cycle. Blood samples were collected around exogenous GnRH pulses before and at 2- or 4-hr intervals after PGF₂α administration and concentrations of LH and progesterone determined. At 4, 12 and 24 hr after PGF₂α administration, mean serum progesterone levels in all ewes had decreased by 54.7%, 66.2% and 89.4%, respectively (P < 0.05) from pre-injection levels. The decrease in progesterone was associated with an increase (P < 0.01) in LH pulse amplitude with means at 4-hr post-PGF₂α ranging from 190% to 288% of pre-PGF₂α values. Mean serum LH levels were also increased (P < 0.01) within 4 hr of PGF₂α administration and remained elevated at all but the 24-hr time point. The timing of this increase (within 4 hr) indicates that it is independent of changes in serum estradiol concentrations, which do not increase for at least 16 hr after induction of luteolysis. Thus, removal of endogenous progesterone negative feedback at the anterior pituitary gland in the hours immediately after induction of luteolysis seems to play a role in facilitating LH release independently of hypothalamic action.     

Opioid modulation of gonadotropin releasing hormone release from the hypothalamic preoptic area in the pig

Two experiments (Exp) were conducted to examine in vitro the release of gonadotropin releasing hormone (GnRH) from the hypothalamus after treatment with naloxone (NAL) or morphine (MOR). In Exp 1, hypothalamic-preoptic area (HYP-POA) collected from 3 market weight gilts at sacrifice and sagitally halved were perifused for 90 min prior to a 10 min pulse of morphine (MOR; 4.5 × 10⁻⁶ M) followed by NAL (3.1 × 10⁻⁵ M) during the last 5 min of MOR (MOR + NAL; N=3). The other half of the explants (n=3) were exposed to NAL for 5 min. Fragments were exposed to KCl (60 mM) at 175 min to assess residual GnRH releasability. In Exp 2, nine gilts were ovariectomized and received either oil vehicle im (V; n=3); 10 μg estradiol-17β/kg BW im 42 hr before sacrifice (E; n=3); .85 mg progesterone/kg BW im twice daily for 6 d prior to sacrifice (P₄; n=3). Blood was collected to assess pituitary sensitivity to GnRH (.2 μg/kg BW) on the day prior to sacrifice. On the day of sacrifice HYP-POA explants were collected and treated as described in Exp 1 except tissue received only NAL. In Exp 1, NAL increased (P<.05) GnRH release. This response to NAL was attenuated (P<.05) by coadministration of MOR. Cumulative GnRH release after NAL was greater (P<.05) than after MOR + NAL. All tissues responded similarly to KCl with an increase (P<.05) in GnRH release. In Exp 2, pretreatment luteinizing hormone (LH) concentrations were lower (P<.05) in E gilts compared to V and P₄ animals with P₄ being lower (P<.05) than V gilts. LH response to GnRH was lower (P<.05) in E pigs than in V and P₄ animals, while the responses was similar between V and P₄ gilts. NAL increased GnRH release in all explants, whereas, KCl increased GnRH release in 6 of 9 explants. These results indicate that endogenous opioid peptides may modulate in vitro GnRH release from the hypothalamus in the gilt.     

The Possible Involvement of Salsolinol and Hypothalamic Prolactin in the Central Regulatory Processes in Ewes During Lactation

Salsolinol, a dopamine‐related compound and prolactin‐producing cells were found in the ovine hypothalamus. This study was designed to test the hypothesis that salsolinol, acting from the CNS level, is able to stimulate pituitary prolactin release as well as prolactin mRNA expression in the anterior pituitary cells (AP) and in the mediobasal hypothalamus (MBH) in lactating ewes. The intracerebroventricular infusions of salsolinol in two doses, total of 50 ng or 5 μg, were performed in a series of five 10‐min infusions at 20‐min intervals. All infusions were made from 12:30 to 15:00 and the pre‐infusion period was from 10:00 to 12.30 h. The prolactin concentration in plasma samples, collected every 10 min, was determined by radioimmunoassay; prolactin mRNA expression in AP and MBH tissues was determined by real‐time PCR. The obtained results showed that salsolinol infused at the higher dose significantly (p < 0.001) increased plasma prolactin concentration in lactating ewes, when compared with the concentration noted before the infusion and with that in lactating controls. In lactating ewes, the relative levels of prolactin mRNA expression in the AP and MBH were up to twofold and fivefold higher respectively than in non‐lactating ewes (p < 0.05). In our experimental design, salsolinol did not significantly affect the ongoing process of prolactin gene expression in these tissues. We conclude that in ewes, salsolinol may be involved, at least, in the process of stimulation of prolactin release during lactation and that hypothalamic prolactin plays an important role in the central mechanisms of adaptation to lactation.     

Postpartum reproduction in protein restricted beef cows: effect on the hypothalamic-pituitary-ovarian axis

The influence of dietary CP on circulating LH and anterior pituitary and hypothalamic function was examined. In Exp. 1, 28 cows were randomly assigned to four treatment groups: adequate CP (ADQ; .96 kg/d) or deficient CP (DEF; .32 kg/d) beginning at 90, 60 and 30 d before parturition and continued at a 33% increase in feed consumption after parturition. Cows were bled at 15-min intervals for 8 h on d 20, 40 and 60 after parturition. Pituitaries were collected on d 62 to analyze GnRH receptor numbers and gonadotropin content. Frequency of pulsatile LH release increased (P less than .05) from 20 to 60 d in ADQ cows. Basal and mean LH were not affected (P greater than .10) by CP restriction or by days after parturition. Crude protein did not affect pituitary GnRH receptors (P greater than .10), but it did affect pituitary LH content, FSH content and FSH concentration (P less than .05). In Exp. 2, 28 cows were assigned to treatment groups as in Exp. 1. All cows were challenged with GnRH (.22 micrograms/kg BW) at 20, 40 and 60 d after parturition and were bled every 30 min for 6 h. Responsiveness to GnRH increased with increased time after parturition (P less than .07). Deficient CP decreased GnRH-induced LH release (P less than .05). In Exp. 3, 12 cows were randomly assigned to ADQ or DEF CP beginning 120 d before parturition. All cows received 1 mg estradiol-17 beta (E2) on d 19, 39 and 59 after parturition and were bled every 30 min for 14 h beginning 14 h following E2. Response to E2 was unaffected by CP restriction (P greater than .10), whereas time to E2-induced LH peak decreased as time after parturition increased in ADQ cows (P less than .05). Results suggest that delayed return to estrus in CP-deficient postpartum beef cows might be due to reduced gonadotropin release from the anterior pituitary and decreased anterior pituitary responsiveness to GnRH.     

Effect of LPS on Reproductive System at the Level of the Pituitary of Anestrous Ewes

In our research we focused our attention on the effect of the immune stress induced by bacterial endotoxin–lipopolysaccharide (LPS) on the hypothalamic–pituitary–gonadal axis (HPG) at the pituitary level. We examined the effect of intravenous (i.v.) LPS injection on luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) release from the anterior pituitary gland (AP) in anestrous ewes. The effect of endotoxin on prolactin and cortisol circulating levels was also determined. We also researched the effect of immune challenge on the previously mentioned pituitary hormones and their receptors genes expression in the AP. Our results demonstrate that i.v. LPS injection decreased the plasma concentration of LH (23%; p < 0.05) and stimulates cortisol (245%; p < 0.05) and prolactin (60%; p < 0.05) release but has no significant effect on the FSH release assayed during 6 h after LPS treatment in comparison with the control levels. The LPS administration affected the genes expression of gonadotropins’β‐subunits, prolactin and their receptors in the AP. Endotoxin injection significantly decreased the LHβ and LH receptor (LHR) gene expression (60%, 64%; p < 0.01 respectively), increased the amount of mRNA encoding FSHβ, FSH receptor (FSHR) (124%, 0.05; 166%, p < 0.01; respectively), prolactin and prolactin receptor (PRLR) (50%, 47%, p < 0.01; respectively). The presented, results suggest that immune stress is a powerful modulator of the HPG axis at the pituitary level. The changes in LH secretion could be an effect of the processes occurring in the hypothalamus. However, the direct effect of immune mediators, prolactin, cortisol and other components of the hypothalamic pituitary–adrenal (HPA) axis on the activity of gonadotropes has to be considered as well. Those molecules could affect LH synthesis directly through a modulation at all stages of LHβ secretion as well as indirectly influencing the GnRHR expression and leading to reduced pituitary responsiveness to GnRH stimulation.     

GABA(A) receptor antagonism increases NMDA receptor inhibition by isoflurane at a minimum alveolar concentration

ObjectiveAt the minimum alveolar concentration (MAC), isoflurane potentiates GABA_A receptor currents and inhibits NMDA receptor currents, and these actions may be important for producing anesthesia. However, isoflurane modulates GABA_A receptors more potently than NMDA receptors. The objective of this study was to test whether isoflurane would function as a more potent NMDA receptor antagonist if its efficacy at GABA_A receptors was decreased.Study designProspective experimental study.AnimalsFourteen 10-week-old male Sprague–Dawley rats weighing 269 ± 12 g.MethodsIndwelling lumbar subarachnoid catheters were surgically placed in isoflurane-anesthetized rats. Two days later, the rats were anesthetized with isoflurane, and artificial CSF containing either 0 or 1 mg kg^?1 picrotoxin, a GABA_A receptor antagonist, was infused intrathecally at 1 μL minute^?1. The baseline isoflurane MAC was then determined using a standard tail clamp technique. MK801 (dizocilpine), an NMDA receptor antagonist, was then administered intravenously at 0.5 mg kg^?1. Isoflurane MAC was re-measured.ResultsPicrotoxin increased isoflurane MAC by 16% compared to controls. MK801 significantly decreased isoflurane MAC by 0.72% of an atmosphere in controls versus 0.47% of an atmosphere in rats receiving intrathecal picrotoxin.Conclusions and clinical relevanceA smaller MK801 MAC-sparing effect in the picrotoxin group is consistent with greater NMDA antagonism by isoflurane in these animals, since it suggests that fewer NMDA receptors are available upon which MK801 could act to decrease isoflurane MAC. Decreasing isoflurane GABA_A potentiation increases isoflurane NMDA antagonism at MAC. Hence, the magnitude of an anesthetic effect on a given channel or receptor at MAC may depend upon effects at other receptors.     

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.     

Short-term hormone release from adult female rat hypothalamic and pituitary explants is not altered by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2, 3, 7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has adverse effects on reproduction, in part due to direct actions at the ovary. It is unclear whether effects are further mediated by glands that regulate ovarian function. We investigated whether effects of TCDD are mediated via the hypothalamic-pituitary axis. Hypothalamic and pituitary tissues were cultured in medium with and without TCDD. TCDD did not alter GnRH release from hypothalamic samples. It continued to be pulsatile with no differences in the average peak frequency, average peak amplitude, or baseline GnRH release. TCDD did not alter GnRH-induced release of gonadotropins from pituitary samples. There were no differences in average peak amplitude or baseline release. AhR, ARNT or ER alpha mRNA copy numbers in cultured pituitaries were not affected by TCDD. Our data suggest that TCDD effects on ovarian function are not mediated through the hypothalamic or pituitary release parameters tested in this study.     

Successful use of a gonadotropin-releasing hormone (GnRH) analog for the treatment of tertiary hypogonadism (GnRH deficiency) in a 5-year-old Belgian Blue bull

A bull was referred for a progressive oligoasthenotheratozoospermia that resulted in a unsuitable seminal quality for the cryopreservation. Breeding soundness evaluation results suggested gonadal dysfunction. Because of the lack of normal ranges for these hormones in the bull, in this study, the hypogonadism and the site of the dysfunction (hypothalamus) were diagnosed by the gonadotropin-releasing hormone (GnRH) stimulation test. The evaluation of pituitary and testicular responsiveness by a GnRH stimulating test revealed a responsiveness of the pituitary and testis, thus a secondary hypogonadism (hypothalamic hypogonadism) was postulated and a therapeutic approach based on the subcutaneous administration of GnRH analog was attempted. An increase in semen volume, concentration and sperm characteristics were detected 9 weeks after the start of the treatment, corroborating the hypothalamic origin of the disease and the useful of the GnRH therapy.