Skin-nervous system interactions.
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Animals↗
Biomedical subjects
A H Kaynard
Publications and source records attributed to A H Kaynard.
Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones.
Prodynorphin is expressed by neurons of the hypothalamus and gonadotrophs of the anterior pituitary gland (AP) and plays a role in the negative feedback regulation of the reproductive neuroendocrine axis. The present study examined whether gonadal steroid hormones are capable of modulating pituitary prodynorphin expression in immature, female rats. Steroids were administered via subcutaneous Silastic implants and rats were killed at 29 days of age. Northern blot analysis was used to measure AP prodynorphin, luteinizing hormone-beta (LH beta), follicle-stimulating hormone-beta (FSH beta), and common alpha-subunit mRNA levels (normalized to 18S ribosomal RNA). Treatment groups (n = 5-6) consisted of control (CNT; empty implants), estradiol (E2; 4 days), E2 + progesterone (E2 + P4; 8 days and 4 days, respectively), and dihydrotestosterone (DHT; 4 days). Pituitary prodynorphin mRNA was significantly suppressed in only the DHT-treated animals (26 +/- 10% of CNT, p < 0.01). LH beta mRNA was suppressed by all steroid treatments (p < 0.01), FSH beta was lower in only the E2 group, and alpha-subunit was reduced in both the E2 + P4 and DHT groups (p < 0.01). Serum LH was suppressed by all steroid treatments but FSH was reduced in only the E2 and E2 + P4 groups (p < 0.01). Treatment of prepubescent rats with continuous high levels of gonadal steroids is known to severely reduce endogenous hypothalamic gonadotropin releasing hormone (GnRH) release and this is supported by our observation of reduced gonadotropin-subunit gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Ovarian 3 beta-hydroxysteroid dehydrogenase and sulfated glycoprotein-2 gene expression are differentially regulated by the induction of ovulation, pseudopregnancy, and luteolysis in the immature rat.
The present studies were conducted to elucidate the effects of gonadotropin-induced alterations in ovarian status on expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), an enzyme which plays a crucial role in steroidogenesis, and sulfated glycoprotein-2 (SGP-2), a heterodimeric protein which is highly expressed by cells undergoing programmed death (i.e. apoptosis). Prepubescent female rats were used to reduce the influence of endogenous gonadotropins and to avoid the presence of preexisting, degenerating corpora lutea in the ovaries. 3 beta-HSD, cholesterol side-chain cleavage cytochrome P450, and SGP-2 messenger RNA (mRNA) levels were measured by Northern analysis of total ovarian RNA. Rats which received PMSG (20 IU) followed 48 h later by human CG (hCG) (10 IU) to induce ovulation and pseudopregnancy exhibited a significant increase in ovarian 3 beta-HSD mRNA 1 day later (164%, P less than 0.01 vs. saline control). The most dramatic change in 3 beta-HSD expression was the rise seen 2 days after hCG (262%, P less than 0.01), after which levels remain constantly elevated throughout pseudopregnancy. In contrast, ovarian cholesterol side-chain cleavage cytochrome P450 mRNA was greatly elevated (i.e. 15-fold) 48 h after PMSG treatment alone (P less than 0.01). Thus, gonadotropic stimulation which induces ovulation and luteogenesis is required to observe a potent stimulatory effect on ovarian 3 beta-HSD expression. The slow time course of induction is indicative of a differentiation-dependent expression. These observations are consistent with luteal cell expression of the 3 beta-HSD gene and suggest that this expression is correlated with the high progestin secretion and 3 beta-HSD activity characteristic of luteal cells. Interestingly, the pattern of regulation of ovarian SGP-2 expression was markedly different than that observed for 3 beta-HSD. PMSG treatment alone (48 h), and in combination with hCG, dramatically reduced SGP-2 mRNA to 12-27% of controls (P less than 0.01). SGP-2 levels were not elevated until 7 days after hCG; levels then remained constant through day 14 of pseudopregnancy. Since luteal progesterone secretion begins to diminish 5-7 days after hCG, the increased expression of SGP-2 on day 7 may be related to the initiation of the regression/degeneration of luteal cells which occurs during luteolysis. Thus, this study demonstrates that alterations in SGP-2 expression by the ovary may precede or occur simultaneously with cellular events initiating luteolysis and suggests a role for this glycoprotein as an early marker for luteolysis and implicates it in yet another instance of programmed cell death.
3-Hydroxysteroid Dehydrogenases↗
Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.
Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.
Animals↗
Modulatory role of substance P on gonadotropin and prolactin secretion in the rabbit.
Substance P (SP) is present in large quantities in the brainstem and hypophysiotropic areas of the brain, but its roles in gonadotropin and prolactin secretion are controversial. The aim of this study was to measure luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) release from the pituitary after either intracerebroventricular (ICV) injection or infusion of SP or its C- and N-terminal fragments in intact (INT) and ovariectomized (OVX) conscious rabbits. A single injection of SP into the 3rd cerebral ventricle (3CVT) in INT and OVX rabbits augmented plasma LH concentrations, especially when SP was applied during the initial phase of an LH peak. Injection of SP during the declining phase of LH release was not effective. Injection of SP into the 3CVT was followed by increased plasma PRL concentrations in OVX but not in INT rabbits. Both SP 1-11 and SP 1-7 failed to alter LH, FSH, and PRL secretion when the peptides were slowly infused into the 3CVT, although ICV infusion of SP 6-11 did cause a delayed increase in LH release. The results support a stimulatory role of SP on LH and prolactin release. The results further indicate that although the stimulatory effect of SP on LH is ovarian steroid-independent, in the absence of ovarian steroids, SP is stimulatory only during the rising phase of an LH pulse. A dual role of SP-ergic transmission in modulating LH secretion is discussed.
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A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates.
Studies of the transcriptional activity of gene promoters have been greatly assisted by the widespread use of the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. Previous techniques for assaying CAT enzymatic activity have utilized radioactive substrates or cofactors with the resulting complications of handling radioactive materials. We report here the development of fluorescent substrates for the CAT enzyme which form the basis of a CAT enzyme assay of enzyme kinetic parameters (Km and Vmax) and sensitivity similar to those based on radioactive substrates. Fluorescent substrates were designed as analogs of chloramphenicol and were based on the structure-function requirements of the enzyme. Several fluorophores were used to derivatize chloramphenicol base; one of the most effective was the borondipyrromethene difluoride (BODIPY) fluorophore. One BODIPY-chloramphenicol analog was found to have a Km for the purified CAT enzyme of 2 microM (compared to 12 microM for 14C-labeled chloramphenicol) and a Vmax of 120 pmole/min (compared to 180 pmol/min for the radioactive substrate). To verify its usefulness, a BODIPY--chloramphenicol-based CAT assay was used to measure transient transfection of primary cultures of ovarian granulosa cells in serum-free medium. This experimental system requires a highly sensitive assay for detecting transfected CAT gene activity. Robust expression of CAT activity was easily detected in crude cellular extracts using FluoReporter FAST CAT, a kit containing the BODIPY-chloramphenicol analog. The expression was precisely quantified by methanol extraction of the substrate and products from TLC plates and subsequent measurement of fluorescence using excitation-emission spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
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Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter.
Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
Animals↗
Effects of neuropeptide Y on the in vitro release of gonadotropin-releasing hormone, luteinizing hormone, and beta-endorphin and pituitary responsiveness to gonadotropin-releasing hormone in female macaques.
The objectives of these studies were to examine the release of gonadotropin-releasing hormone (GnRH) and beta-endorphin-like activity (beta-EP) from macaque hypothalami, and the release of luteinizing hormone (LH) and GnRH-induced LH from macaque anterior pituitaries in response to neuropeptide Y (NPY) treatment. Anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) blocks of tissues and the adenohypophysis were bisected along the midline into two equal-sized fragments. Fragments were superfused with medium for 3 h, followed by 3 h of either NPY (80 nM) or medium alone. In a separate experiment, adenohypophyseal (AP) fragments were superfused in accordance with the same protocol (3 h medium - 3 h NPY or medium) except that exogenous GnRH (352 nM) was added for 30 min at the beginning of hour 3 and again at the beginning of hour 6. Immunoactive GnRH, beta-EP, and LH levels were measured in superfusate samples (400 microliters) collected at 10-min intervals. GnRH levels rose within 20-30 min of initiation of NPY treatment, and elevated GnRH release was sustained for the duration of NPY exposure of both AH and MBH fragments from ovarian intact (INT) rhesus (Macaca mulatta: n = 8; p less than 0.05) or Japanese (Macaca fascicularis; n = 4; p less than 0.01) macaques. NPY treatment had no effect on either AH or MBH fragments isolated from ovariectomized (OVX) rhesus macaques (n = 4 for AH, and n = 5 for MBH). In AP fragments isolated from INT rhesus macaques (n = 8), NPY stimulated LH release within 1 h of treatment (p less than 0.05), whereas NPY had no effect on pituitaries from OVX animals (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)
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Immunoneutralization of neuropeptide Y suppresses luteinizing hormone secretion in rabbits.
In the ovarian intact rabbit, neuropeptide Y (NPY) has stimulatory actions on both LH secretion and GnRH release. The present study measured the pattern of tonic, basal LH release in the rabbit after passive immunoneutralization of endogenous NPY. Eight intact rabbits with third cerebroventricular cannulae and venous catheters were subjected to 8 h of blood sampling at 15-min intervals. Intracerebroventricular (icv) infusion of 1 ml of either normal rabbit serum (NRS) or NPY antiserum (NPY-Ab; raised in rabbits against human NPY) was begun after the second hour of basal blood sampling and was continued for the remaining 6 h of the 8-h protocol. A 1-ml matching iv dose of NRS or NPY-Ab was administered at the start of the icv infusion. All rabbits received both NRS and NPY-Ab treatments 2 weeks apart in a Latin square design. Administration of NPY-Ab significantly (P less than 0.05) suppressed plasma LH after 165 min. After 4 h, plasma LH was maximally reduced to 42% of the control value (pretreatment, 0.093 +/- 0.016 ng/ml; 4 h, 0.039 +/- 0.005 ng/ml). A reduction in the rate of pulsatile LH release also occurred during NPY-Ab treatment (P less than 0.05). Treatment with NRS had no effect on LH. The experiment was repeated in four rabbits 2 weeks after ovariectomy. Administration of NPY-Ab suppressed plasma LH after 75 min in ovariectomized (OVX) rabbits (P less than 0.05). The greatest inhibition was seen after 5 h of NPY-Ab treatment, when LH was reduced to 21% of the control level (pretreatment, 0.841 +/- 0.274 ng/ml; 5 h, 0.134 +/- 0.025 ng/ml). Both LH-pulse amplitude and frequency were suppressed in these OVX does. To determine whether central actions of NPY are of predominant importance in maintaining LH secretion, four OVX rabbits were given NPY-Ab icv only. LH was suppressed after 90 min (P less than 0.05) and was maximally inhibited after 3 h of treatment to 22% of the control value (pretreatment, 1.804 +/- 0.711 ng/ml; 3 h, 0.434 +/- 0.221 ng/ml). Although both LH-pulse amplitude and frequency were diminished, neither was decreased significantly (P greater than 0.05). FSH secretion was not affected by NRS or NPY-Ab treatment in either intact or OVX does. These results clearly indicate that in both intact and OVX does, endogenous NPY is in part responsible for maintaining basal, tonic LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
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Gonadotropin-releasing hormone and norepinephrine release from the rabbit mediobasal and anterior hypothalamus during the mating-induced luteinizing hormone surge.
The release of hypothalamic GnRH in association with the mating-induced LH surge was studied in the rabbit. Push-pull perfusion (PPP) of the mediobasal (MBH) or anterior (AH) regions of the hypothalamus was performed on conscious, unrestrained does for 3 h before and 5 h after exposure to a vasectomized buck. In experiment 1, GnRH concentrations were measured by RIA in 20-min fractions of MBH-PPP. An approximately 100-fold increase in GnRH release was observed within 1 h of coitus (pre, 1.15 +/- 0.29 pg/ml; peak, 106.67 +/- 37.42 pg/ml; n = 6; P less than 0.05). Concomitant surges of LH and PRL in the peripheral circulation were observed. In experiment 2, GnRH and norepinephrine (NE) were measured (the latter by radioenzymatic assay) in 10-min fractions of MBH-PPP. A 218% postcoital rise in NE levels (n = 5; P less than 0.05) in MBH-PPP accompanied an approximately 50-fold peak rise in GnRH in the same samples (pre, 1.57 +/- 0.23 pg/ml; peak, 76.52 +/- 50.14 pg/ml; P less than 0.05). MBH-NE, MBH-GnRH, LH, and PRL release began rising within 10 min of coitus. In experiment 3, GnRH was measured in 20 min fractions of AH-PPP. Coitus induced a marked rise in AH-GnRH release (precoitus, 0.31 +/- 0.03 pg/ml; peak, 2.25 +/- 0.80 pg/ml; n = 4; P less than 0.05) which differed from coitus-induced MBH-GnRH release both quantitatively (i.e. approximately 7-fold increase for AH vs. approximately 50-100-fold increase for MBH; 50-min lag time for AH vs. less than 20 min for MBH) and qualitatively (i.e. AH-GnRH release was discontinuous, while MBH-GnRH release rose sharply, plateaued, and then declined slowly over the 2-5 h following coitus). No changes in MBH-NE, MBH-GnRH, AH-GnRH, LH, FSH, or PRL were observed in sham-mated does (Exp 1, n = 7; Exp 2, n = 3; Exp 3, n = 4). These data support the hypotheses that: 1) hypothalamic GnRH release is a component of reflexive ovulation in the rabbit; 2) increased hypothalamic noradrenergic tone is related to the surge-release of GnRH; and 3) AH-GnRH release is enhanced following coitus.
Animals↗
Third-ventricular infusion of neuropeptide Y suppresses luteinizing hormone secretion in ovariectomized rhesus macaques.
Neuropeptide Y (NPY) has been shown to modulate gonadotropin secretion in an estrogen-dependent manner in the rat and rabbit, and to act centrally in these species to alter GnRH release. The present study examined the ability of centrally administered NPY to affect LH secretion in the primate. Human NPY (hNPY) was administered into the third cerebroventricle of unanesthetized, freely moving, ovariectomized (OVX), or estradiol (E2)-treated OVX rhesus monkeys. LH was measured in blood samples collected remotely at 10-min intervals throughout the experiments. An extensive range of NPY doses was tested in a preliminary study in which OVX monkeys received a 3-h control infusion of Krebs Ringer phosphate buffer (KRP) followed immediately by a 3-h infusion of hNPY (0.1-50 micrograms/h). Only doses greater than or equal to 5 micrograms/h produced a consistent and marked suppression of LH (5 micrograms/h; 35.1 +/- 7.2% reduction, P less than 0.05, n = 4). A longer duration study was performed to better characterize changes in LH pulse frequency and amplitude produced by hNPY treatment. We administered 5 micrograms/h and 15 micrograms/h to OVX (n = 5) and E2-treated OVX (n = 4) monkeys according to the following protocol: 8 h control KRP/8-h hNPY/6-h recovery KRP. In OVX monkeys, LH was suppressed after 2 to 3 h of peptide infusion (P less than 0.01); LH secretion returned to normal after treatment. Both doses of hNPY suppressed mean LH by approximately 55% (P less than 0.05) and LH pulse frequency by approximately 69% (P less than 0.025). LH pulse amplitude was unaffected. In E2-treated OVX monkeys, neither dose of hNPY affected mean LH or LH pulse amplitude. LH pulse frequency was suppressed by approximately 65% (P less than 0.05) during 15-micrograms/h treatment. Because centrally administered hNPY reduced LH pulse frequency and thereby mean LH levels, our results support a central, neural action of NPY to affect the GnRH/LH secretory system. The ability of estrogen feedback to alter the response to NPY treatment supports a physiological role for NPY in controlling reproduction in the primate.
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Endogenous opiates regulate the nocturnal reduction in luteinizing hormone pulse frequency during the luteal phase of the macaque menstrual cycle.
Twenty-four-hour pulsatile patterns of bioactive luteinizing hormone (LH) and immunoactive estradiol (E2) and progesterone (P4) were measured during the mid-follicular (FP, Days 5, 6, or 7) and mid-luteal (LP, Days 7, 8, 9, or 10) phases of the menstrual cycle in six intact and in four ovariectomized (OVX) rhesus monkeys. Blood samples were collected remotely at 7.5-min (FP) and 15-min (LP, OVX) intervals via catheters in freely moving, vested monkeys. Hormonal patterns were analyzed by the MUNRO computer program. A significant coupling of LH and E2 pulses was observed during the FP and between the LH and P4 pulses during the LP. No diurnal rhythm of LH pulse frequency was observed during the FP or in OVX monkeys. In contrast, day/night differences in frequency were seen during the luteal phases (0.35 +/- 0.03 vs. 0.24 +/- 0.05 pulses/h, day vs. night, p less than 0.05). Naloxone (NAL), an opiate antagonist, was continuously infused (2 mg/h.i.v.) for 12 h at night during the LP. NAL treatment increased nighttime LH pulse frequency to 0.33 +/- 0.07 pulses/h, which was not different from the daytime LH pulse frequency, thereby abolishing the normal diurnal pattern. Interestingly, NAL treatment did not achieve the approximately 1 pulse/h frequency seen in the FP or OVX condition. These results support the hypothesis that P4 modulates the activity of an opioid system that is responsible both for a slowing of LH pulse frequency during the LP and for an additional nightly reduction in hypothalamic LH pulse-generating activity.
Animals↗
Simultaneous induction of neuropeptide Y and gonadotropin-releasing hormone release in the rabbit hypothalamus.
Neuropeptide Y (NPY) can induce the release of endogenous mediobasal hypothalamic gonadotropin-releasing hormone (MBH-GnRH) and pituitary gonadotropins, especially LH. In these studies, we monitored changes in endogenous NPY concentrations at 20-min intervals for 6-8 h during push-pull perfusion (PPP) in both the mediobasal hypothalamus (MBH) and the third cerebroventricle (3VT) of ovarian intact, conscious rabbits. Because previous studies had shown that copper ion can induce hypothalamic GnRH release, cupric acetate (CuAc) was administered either intravenously or intraventricularly during the PPP to manipulate changes in NPY concentrations. Our results show that NPY concentrations in both MBH and 3VT PPP samples were detectable by radioimmunoassay. Administration of CuAc sharply increased hypothalamic NPY release within the same time interval as that for induction of hypothalamic GnRH release. The results are consistent with the hypotheses that NPY may act as a neuromodulator for hypothalamic GnRH secretion, or that common mechanisms drive secretion of these two neuropeptides.
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Suppression of mediobasal hypothalamic gonadotropin-releasing hormone and plasma luteinizing hormone pulsatile patterns by phentolamine in ovariectomized rhesus macaques.
In gonadectomized animals, pulses of LH are secreted concurrently with pulsatile hypothalamic GnRH and it is hypothesized that pulses of GnRH are either driven or modulated by episodes of catecholamine release. The objective of this study was to determine if the alpha-adrenergic antagonist phentolamine (PHEN) can simultaneously block the release of GnRH and LH in ovariectomized (OVX) rhesus macaques. In Exp 1, simultaneous peripheral blood and mediobasal hypothalamic push-pull perfusion (PPP) samples were collected remotely at 10-min intervals for 24 h via a swivel/tether device in eight conscious, freely moving OVX rhesus monkeys. Phentolamine was continuously infused iv for 6 h at the rate of 4 mg/kg BW.h in five animals and 20 mg/kg BW.h in three animals. Infusion started at 6 h after the commencement of PPP. Sampling of PPP and blood continued for 12 h after the cessation of PHEN infusion. Exp 2 was carried out to determine if PHEN affects pituitary responsiveness to exogenous GnRH under conditions similar to those in Exp 1. Exogenous GnRH (5 micrograms, iv) was injected as a single bolus at 10-h intervals before, during, and after either a saline (4 ml/h for 6 h) infusion or, 3 weeks later, a PHEN infusion (4 mg/kgBW.h for 6 h) in three OVX females. The results of Exp 1 show that pulsatile patterns of hypothalamic GnRH and LH were either dampened or abolished by PHEN infusion. During the recovery period after PHEN infusion, pulse amplitudes of LH were enhanced, but pulse amplitudes of endogenous GnRH did not differ, as compared to those of corresponding LH and GnRH before infusion of PHEN. Data from Exp 2 suggested that the alpha-adrenergic blocking agent had no effect on the pituitary LH response to exogenous GnRH administration. These results directly support the hypothesis that adrenergic neuronal activities are critical for the pulsatile release of hypothalamic GnRH which governs the pulsatile release of LH in gonadectomized animals.
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Feedback regulation of pulsatile LH secretion in the ewe: stimulation of frequency by estradiol.
This study tested the hypothesis that estradiol can enhance LH pulse frequency in the ewe by an action which does not depend on other ovarian hormones. Long-term ovariectomized ewes were treated with a small subcutaneous estradiol implant at a time equivalent to the early breeding season (October), and frequent blood samples (6-min intervals) were obtained during sequential 3-hour periods over the next 84 h. All ewes responded with an increase in frequency of LH pulses, a response evident by 60 h and maintained at 84 h after initiation of the estradiol stimulus. Mean (+/- SE) pretreatment frequency was one pulse every 41 +/- 2 min; that at the height of the response was one pulse every 34 +/- 2 min (p less than 0.01). This increased rate was equivalent to the annual mid-winter maximum observed in ovariectomized ewes not treated with estradiol. These findings are consistent with the hypothesis that estradiol can enhance LH pulse frequency by an action which does not depend on other ovarian steroids. It is suggested that this action contributes to the heightened pace of LH pulses during the follicular phase of the estrous cycle.
Animals↗
Hypophyseal actions of pulsatile gonadotropin-releasing hormone in the ewe: development and application of a new experimental model.
Ovariectomy of ewes during seasonal anestrus and immediate replacement with subcutaneous Silastic progesterone implants which maintained a midluteal-phase level of circulating progesterone obliterated pulsatile luteinizing hormone (LH) secretion for up to 2 weeks without preventing a normal response of the pituitary to exogenous pulses of gonadotropin-releasing hormone (GnRH). Consideration was given to the possibility that such 'progesterone-suppressed ewes' would be useful as an animal model for isolating the pituitary from pulsatile GnRH secretion, and for testing the hypophyseotropic actions of exogenous GnRH. Two experiments were conducted using this progesterone-suppressed ewe as an animal model. In the first, the amplitude of LH pulses elicited by episodic delivery of GnRH was found to depend upon the frequency of exogenous GnRH pulses. Hourly frequency produced larger LH pulses than a 30-min frequency of GnRH. In the second experiment, LH surges were induced in progesterone-suppressed ewes by a combined treatment of estradiol and GnRH in patterns designed to approximate those secreted in the follicular phase of the estrous cycle. Our findings suggest that the progesterone-suppressed ewe is a suitable animal model for studying the hypophyseotropic actions of GnRH. Further, they are consistent with two hypotheses concerning the regulation of the tonic and surge modes of LH secretion. (1) The inverse relationship between LH pulse frequency and amplitude observed in a number of situations can be accounted for, at least in part, by a differential response of the pituitary to GnRH. (2) Progesterone can block the LH surge by an action on the brain and an inhibition of pulsatile GnRH release.
Animals↗
Importance of pituitary and neural actions of estradiol in induction of the luteinizing hormone surge in the ewe.
Two experiments were performed to test the importance of both pituitary and neural sites of action of estradiol in inducing the surge of luteinizing hormone (LH) in the ewe. Both experiments were conducted using an animal model in which pulsatile secretion of gonadotropin-releasing hormone (GnRH) and endogenous secretion of ovarian steroids were eliminated by ovariectomy during seasonal anestrus and treatment with Silastic implants which maintained a luteal-phase level of serum progesterone. The hormonal requirements for the surge were then evaluated by systematic application of GnRH and estradiol signals using pulsatile infusion pumps (for GnRH) and Silastic implants (for estradiol). In experiment 1, the circulating level of estradiol and frequency of GnRH pulses were increased either abruptly or progressively (i.e. mimicking the changes in the estrous cycle between luteolysis and just before the LH surge). Abrupt increments led to an LH surge in all ewes; progressive rises to the same absolute levels did not. However, sudden application of a further large increase in GnRH upon the progressive rise elicited an LH surge in every instance. In experiment 2, a GnRH pulse pattern known to be effective in inducing the LH surge was applied under conditions of differing estradiol concentration: no estradiol, basal estradiol, basal rising to peak estradiol. The GnRH signal elicited high-amplitude surges of LH only in the presence of a peak estradiol concentration. Our findings are consistent with the conclusion that two actions are required for a rise in estradiol to elicit a full-amplitude surge of LH in the ewe: an action on the brain to evoke a sudden increase in GnRH release and an action on the pituitary to maximize its response to GnRH.
Animals↗
Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes.
Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗