LH Release In Response Research Articles

PSIV-16 Factors Affecting the Lh Release and Ovulatory Response to a Gnrh Treatment in Lactating Dairy Cows on Day 7 of the Estrous Cycle

Abstract This study evaluated factors affecting LH release and ovulation in response to a treatment with a conventional dose of gonadorelin (GnRH), in lactating dairy cows on day 7 of the estrous cycle. Factors evaluated were: GnRH preparation (ready-to-use vs. lyophilized), body condition score (BCS), parity, days in milk (DIM), milk yield and dominant follicle (DF) diameter, at the time of GnRH treatment. Seven days after an artificial insemination, preceded by a synchronization protocol, lactating Holstein cows were evaluated by ultrasound and those with a corpus luteum (CL) and a DF ≥10 mm were randomly assigned to receive 100 µg GnRH ready-to-use solution (Fertagyl; MSD, Brazil) or lyophilized (Profertil; J.A. Animal Health, Brazil) diluted just before its administration. Overall, 106 cows (BCS = 3.1 ± 0.1; DIM = 96.7 ± 4.2; 39.7 ± 0.7 kg of milk/d) were assigned to GnRH treatments. Ovulation was determined by ultrasound examinations 2 d after GnRH treatment, by disappearance of the DF, and confirmed 5 d later by the presence of an accessory CL. In a subset of cows (n = 33), blood samples were collected just before the GnRH treatment, and 2 and 4 h later, to evaluate circulating P4 and LH concentrations. Data were analyzed by GLIMMIX (SAS 9.4). Treatment effect and its interaction were included in the analyses of all other factors. Differences were declared when P ≤ 0.05 and tendency when 0.053.0 (1.4 ± 0.2 vs 0.7 ± 0.1 ng/mL; Table 1). Moreover, cows with DF ≤12 mm had greater LH peak than cows with DF >12 mm (1.3 ± 0.2 vs. 0.9 ± 0.1 ng/mL; Table 2). However, these factors did not affect ovulation. Neither milk yield nor DIM affected LH peak or ovulation. In conclusion, although no differences were detected on LH peak, primiparous cows on day 7 of the estrous cycle had greater ovulation when treated with Profertil compared with Fertagyl. Moreover, regardless of treatment, greater BCS and larger DF (>12 mm) at the time of GnRH treatment were associated with decreased LH peak, however, without impairing ovulation.

Influence of GnRH analog and dose on LH release and ovulatory response in Bos indicus heifers and cows on day seven of the estrous cycle

This study evaluated the influence of GnRH analogs (gonadorelin vs. buserelin) and doses (single vs. double) on LH release and ovulatory response in Bos indicus (Nelore) females on Day 7 of the estrous cycle. Cycling heifers and non-lactating cows were pre-synchronized: Day −10: progesterone (P4) implant insertion plus 2 mg of estradiol benzoate; Day −2: implant removal and 0.53 mg of cloprostenol sodium (PGF); Day 0: 25 μg of lecirelin (GnRH). Over four replicates, heifers (n = 57) and cows (n = 53) that ovulated to the GnRH treatment on Day 0, having a visible corpus luteum (CL) and a dominant follicle (DF) ≥ 8.5 mm, were allocated to receive the following GnRH treatments on Day 7: G-Single (100 μg of gonadorelin); G-Double (200 μg of gonadorelin); B-Single (10 μg of buserelin); and B-Double (20 μg of buserelin). At GnRH treatment, a P4 implant was inserted in heifers (0.5 g) and cows (1 g). Ultrasound examinations were done on Days −10, −2, 0, 2, 7, 9, 12, and 14 to evaluate DF diameter, ovulation and presence of CL. Blood samples were collected on Day 7 at 0, 2, and 4 h from GnRH treatment, to evaluate circulating P4 and LH concentrations. On Day 12, the P4 implant was removed, females received two PGF treatments (24 h apart), and 2 d later, 25 μg of GnRH was given to start the next replicate. In both heifers and cows, P4 concentrations were elevated on Day 7, and similar among groups (3.9 and 4.2 ng/mL, respectively). In heifers, buserelin induced greater LH peak (9.5 vs. 2.6 ng/mL; P < 0.01) and greater ovulation (88.9 [24/27] vs. 16.7% [5/30]; P < 0.01) than gonadorelin treatments, regardless of the dose. Similarly, in cows, buserelin induced greater LH peak than gonadorelin (9.9 vs. 4.9 ng/mL; P < 0.01). However, ovulation was only increased in cows from the B-Double group (90.9% [10/11]), whereas in the other groups the ovulatory response was similar (35.7% [15/42]). Regardless of treatment, heifers had similar P4 concentrations (P = 0.22), but smaller DF (P < 0.01) than cows on Day 7. Only in G-Double group the LH peak was lower (P = 0.05) in heifers than in cows, with no difference within other groups. In heifers, but not in cows, the single dose of buserelin resulted in high ovulatory response, equivalent to that produced by the double dose. In conclusion, in Bos indicus heifers and cows on Day 7 of the cycle, with elevated P4 concentrations, buserelin induced greater LH release and ovulatory response than gonadorelin treatments. Double doses increased the LH release, however, only resulted in greater ovulation in females treated with buserelin. Finally, although circulating P4 concentrations did not differ between parities, heifers were more likely to ovulate in response to a GnRH-induced LH peak than cows.

Estradiol Priming Potentiates the Kisspeptin-Induced Release of LH in Ovariectomized Cows.

Simple SummaryThe magnitude and duration of the pre-ovulatory release of LH is important in controlling the timing of ovulation in estrus synchronization and fixed-time artificial insemination (FTAI) protocols in cows. The neuropeptide kisspeptin (KISS1) stimulates GnRH neurons in the brain which in turn induce LH release from the pituitary gland. The present study used ovariectomized Nelore cows as a model to examine whether priming with the steroid estradiol benzoate (EB) for 12 h increased both the peak and duration of LH release after treatment with KISS1. It was found that cows pre-treated with EB for 12 h showed a greater LH peak and longer duration of LH release in response to KISS1 compared with cows that received EB at the same time as KISS1, or KISS1 alone. The incorporation of EB priming and KISS1 in estrus synchronization-FTAI protocols could improve the control of LH release and ovulation, and potentially increase fertilization and conception rates in cows. This would have important practical application in assisted breeding in beef and dairy cows.The present study examined whether priming with estradiol benzoate (EB) for 12 h increased both the peak and duration of LH release in response to kisspeptin (KISS1, KP) in cows. In a Latin square design, ovariectomized Nelore cows (n = 8) received: Control, i.m. 4 mL of 0.9% saline; KP, i.m. 4 mg murine KISS1-10; EBKP, i.m. 4 mg KISS1-10 + i.m. 2 mg EB simultaneously; EB12KP, i.m. 4 mg KISS1-10 + i.m. 2 mg EB 12 h before KISS1-10. Concentrations of LH were determined in blood samples obtained at time 0 (treatment), 20, 40, 60, 90, 120, 150, 180, 210 and 270 min. Concentrations of LH were analyzed by Proc GLIMMIX for repeated measures. In case of significance, the adjusted Tukey test was used to test for differences among treatments. GraphPad 8.0 PRISM® was used to determine the area under the LH-response curve (AUC) after injection of KISS1-10. Plasma LH remained relatively constant throughout sampling after treatment with saline. The peak in LH after injection of KISS1-10 occurred at 20 min in Groups KP and EBKP and at 40 min in Group EB12KP. The peak LH response (∆LH, ng/mL) was greater (p < 0.01) in Group EB12KP (5.6 ± 0.9) than in Groups KP (2.4 ± 0.9) and EBKP (3.5 ± 0.9), which did not differ. AUC (LH ng/mL*min) was greater (p = 0.02) in Group EB12KP (439 ± 73) than in Groups KP (176 ± 73) and EBKP (241 ± 73), with the latter two groups not differing. The findings indicated that 12 h priming with EB increased both the peak and duration of the LH response to treatment with KISS1. The incorporation of EB priming and KISS1 could improve the efficiency of estrus synchronization with fixed-time AI in cows. This would have an important practical application in assisted breeding in beef and dairy cattle.

Lame cows have a subdued hypothalamus-pituitary axis as revealed by LH release in response to exogenous endocrine challenges

Lameness is a serious concern in cows due to its multilevel effects and increasing incidences. The ability of anterior pituitary and hypothalamus to release LH, a prime reproductive hormone, is unknown in lame cows. Our study tested this ability by exogenous endocrine challenge using Buserelin acetate (GnRH analogue) and estradiol valerate (EV). The study included moderately lame (L; n=7) either cyclic (LC; n=3) or anestrus (LA; n=4); and normal cyclic, non–lame (NL; n=5) cows. Progesterone blocks LH release, so it was lowered by injecting PG in cyclic cows. After 14 h, all cows were given a walk for 5 min and immediately thereafter injected with 0.008 mg GnRH and 4 h later with 1 mg of EV. Blood plasma progesterone, cortisol and LH were quantified, periodically. There was a significant delay in the onset (105.0 vs 75.0±7.0 vs 54.0±9.0 min) and duration (22.5±5.3 vs 80.0±10.8 vs 117.0±2.7 min) of LH surge in LA vs LC vs NL cows; the surge was absent in two LA cows. The altered LH profile was attributed to a 3 fold (in L) vs 1.6 fold (in NL) rise in cortisol. LH released (ng/ml) in response to EV was also low in LA (31.5±2.4) and LC (69.9±7.2) than NL (79.2±7.0) cows. In conclusion, lame cows subjected to walk exhibited reduced pituitary response to release LH following exogenous GnRH as also a reduced ability of hypothalamus to stimulate LH release in response to exogenous estradiol.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of β-nerve growth factor of seminal plasma origin in female llamas

The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of β-nerve growth factor (β-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF–treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that β-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110β, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110β, p110γ, and p110δ). Importantly, p110β and p110γ, but not p110α or p110δ, can be directly activated by the Gβγ heterodimer of Gαβγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110β, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110β only regulate basal LH secretion. However, p110β and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110β and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gβγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

The Release of LH in Response to Kiss-10 Is Dependent to the Age and Influenced by Serum Concentrations of Estradiol (E2) in Heifers in Tropical Regions.

The objective of the study was to evaluate the influence of the age of heifers on the release of LH in response to Kiss-10. Heifers (4, 7, and 11 months old) received an i.v. injection of kiss-10 (5 micrograms/kg). Serum concentrations of LH, FSH and estradiol (E2) were quantified by radioimmunoassay and data were subjected to analysis of variance. Serum concentrations of LH and FSH were analyzed in a repeated measurements design in blood samples collected every 15 minutes from 4 hours before to 4 hours after administration of Kiss-10. Serum concentrations of E2 were analyzed in a completely randomized design from a blood sample collected just before treatment with Kiss-10. Serum concentrations of FSH were similar (P > 0.3) in heifers of 4, 7, and 11 months old (21 ± 2.2, 18 ± 2.2, and 15 ± 2.4 ng/ml, respectively) and administration of Kiss-10 increased (P < 0.01) secretion of FSH compared to the concentrations during the pre-treatment period (33 ± 1.9 vs. 18 ± 1.3 ng/ml), and the magnitude of the stimulatory effect of Kiss-10 was similar among age groups. Similarly, serum concentrations of LH did not differ (P > 0.1) in heifers of 4, 7, and 11 months old (1.4 ± 0.26, 2.3 ± 0.41, and 2.3 ± 0.4 ng/ml, respectively). In all three age groups LH increased (P < 0.01) in response to Kiss-10 compared to the pre-treatment period (3.7 ± 0.60 vs. 1.1 ± 0.21 ng/ml). However, the stimulatory effect of Kiss-10 on LH secretion was higher (P < 0.01) in the 11 versus the 4 month old heifers when evaluated as either mean LH (4.8 ± 0.88 vs. 2.6 ± 0.41 ng/ml) or area under the curve of LH (36.5 ± 7 vs. 18.9 ± 3 arbitrary units). The greater LH secretion induced by Kiss-10 detected in 11 month old heifers was associated with higher (P < 0.06) serum concentrations of E2 compared to 4 month old heifers (1.68 ± 0.51 vs. 0.43 ± 0.11 pg/ml). We speculate that increased ovarian activity that occurs as the heifers approach puberty increases sensitivity of the hypothalamic-pituitary axis to Kiss-10. (poster)

Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.

Event Abstract Back to Event Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus. Joshua G. Pemberton1, James L. Stafford1, Yi Yu1 and John P. Chang1* 1 University of Alberta, Biological Sciences, Canada Two endogenous gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) stimulates LH and GH secretion via Ca2+-dependent intracellular signalling pathways in goldfish, Carassius auratus. Whether phosphoinositide 3-kinase (PI3K) mediates GnRH-stimulated LH and GH release and associated intracellular Ca2+ increases ([Ca2+]i) were investigated using hormone release studies with primary cultures of dispersed goldfish pituitary and Fura2 fluorescence Ca2+ imaging in morphologically identified goldfish pituitary somatotropes and gonadotropes. Western blot showed the presence of immunoreactive PI3K p85α, the regulatory subunit for conventional Class IA PI3Ks, in goldfish pituitary and brain tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. Selective PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM) significantly reduced sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of mixed pituitary cells and [Ca2+]i increases in identified gonadotropes. Surprisingly, both PI3K inhibitors reduced the GH release response to the two GnRHs but only attenuated the [Ca2+]i response to cGnRH-II. The effects of PI3K inhibition on hormone release responses were specific to GnRHs and not the result of general decreases in the releasable hormone pool or sensitivity to [Ca2+]i changes as both wortmannin and LY294002 had no effect on Ca2+ ionophore-evoked LH and GH secretion. These results indicate that PI3K is involved upstream of [Ca2+]i increases in mediating LH and GH responses in a cell- and GnRH isoform-specific manner and is the first to implicate PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) individual Discovery Grants to J.P. Chang and J.L. Stafford. Financial support for J.G. Pemberton was provided by the Province of Alberta Queen Elizabeth II Graduate Scholarship, NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS M), and the University of Alberta as a recipient of the Dr. Richard E. Peter Memorial Scholarship, Walter H. Johns Graduate Fellowship and Department of Biological Sciences Teaching Assistantship. Y. Yu was a recipient of a F.S. Chia PhD Recruitment Scholarship from the University of Alberta. We thank Alan Mar and Herman Cortes for their assistance with this study. Keywords: fura2 fluorescence calcium imaging, gonadotropes and somatotropes, gonodotropin-releasing hormone, luteinizing and growth hormone secretion, Phosphoinositide 3-kinase, wortmannin and LY294002 Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Intracellular signaling Citation: Pemberton JG, Stafford JL, Yu Y and Chang JP (2011). Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00079 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Prof. John P Chang, University of Alberta, Biological Sciences, Edmonton, Alberta, T6G2E9, Canada, john.chang@ualberta.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Scholar Joshua G Pemberton James L Stafford Yi Yu John P Chang PubMed Joshua G Pemberton James L Stafford Yi Yu John P Chang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Dissociation of prolactin and LH release responses after stimulation within the preoptic-suprachiasmatic region in male rats.

The relative efficiency of unilateral electrical stimuli applied within the preoptic-suprachiasmatic region (POA-Sch) to induce the release of prolactin and/or LH was studied in male rats under pentobarbital anesthesia. It was observed that unilateral stimulation with 10 Hz, 1.0 msec width pulses applied in the ventral half of the POA-Sch resulted primarily in the release of prolactin rather than LH. Fifty Hz stimuli applied at this same locus resulted also in LH release. It is concluded that the probability to dissociate the induced release of prolactin and LH from the anterior pituitary in male rats after stimulation from a given locus within the ventral POA-Sch is significantly affected by the characteristics of the applied stimulus.

The Modulating Effect of Estrogens on Luteinizing Hormone Release in complete Androgen Insensitivity Syndrome before and after Gonadectomy and Cyclic Steroid Application

The response of LH release to exogenous estrogens was studied in three patients with androgen insensitivity syndrome (AIS) before and after gonadectomy and a prolonged treatment with a sequential pill. Estrogen provocation tests were performed in which 0.05 mg/kg body weight estradiol benzoate was administered intramuscularly and serum FSH and LH levels were assessed every 12 hours for 96 hours after gonadectomy, three months after a prolonged treatment with a sequential pill (16 cycles: 9 days mestranol 100 micrograms, 12 days mestranol 80 micrograms and chlormadinone acetate 2 mg) and in one patient before gonadectomy. The control group consisted of 10 normal females during the follicular phase. A positive feedback effect was induced in normal females and in AIS patients after prolonged treatment with a sequential pill.

Influence of Neurotransmitters on Sexual Differentiation of Brain Structure and Function

Newborn rats received daily subcutaneous treatment with compounds which influence serotoninergic, cholinergic, alpha-adrenergic and beta-adrenergic activity. In adulthood luteinizing hormone (LH) secretion pattern, female sexual behavior, and the volume of the sexually dimorphic nucleus of the preoptic are (SDN-POA) were determined. Postnatal administration of l-tryptophan increased the volume of the SDN-POA significantly when given alone or when given simultaneously with testosterone propionate (TP). Para-chlorophenyl-alanine (pCPA) also increased SDN-POA volume, but did not potentiate the stimulating influence of TP. Clonidine had no effect per se on SDN-POA development, but it significantly potentiated the effect of TP in females. Salbutamol increased SDN-POA volume in females and in males. Postnatal treatment of female rats with the alpha-adrenergic receptor antagonists prazosine and yohimbine or with the nicotin receptor antagonist mecamylamine had permanent potentiating effects on the pattern of LH secretion, whereas postnatal treatment with beta-adrenergic compounds reduced the LH-release response to gonadal steroids in adulthood. Postnatal treatment with clonidin or l-tryptophane inhibited differentiation of the capacity for lordosis behavior. Beta-receptor agonists postnatally had a potentiating effect on the capacity for lordosis behavior in female and male rats. Cholinergic stimulation postnatally inhibited differentiation of the capacity for lordosis behavior in female rats, but prevented the inhibitory effect of postnatal androgenization. There was no correlation between SDN-POA volume and any of the two functional parameters.

Obesity and the Emergence of Sleep-Wake Gonadotropin Secretion in Girls during Early Pubertal Development

It has been clearly established that obesity is associated with reduced gonadotropin secretion in men and women. In normal women, early studies involving relatively few subjects had shown that serum LH levels were significantly reduced in obese individuals compared with levels of normal-weight controls (1, 2). Subsequent larger studies that examined the relationship of LH secretory dynamics to body weight included women with polycystic ovary syndrome (PCOS) as well as normal women (3, 4). In particular, LH secretion in both groups of obese women was characterized by reduced basal levels and decreased pulse amplitude, whereas LH frequency appeared unaffected. Recently, it was demonstrated that morbidly obese women with normal menstrual function exhibited a similar pattern of LH suppression compared with that of normal women (5). In adolescent girls, there are essentially no data concerning the influence of obesity on the rate and amplitude of LH secretion. However, during puberty the progressive maturation of gonadotropin secretion in normal girls through various stages of development has been well documented (6). Given the observed impact of obesity on LH secretion in adults, it seems logical to investigate this relationship at a time when activation of hypothalamic-pituitary interaction is emerging. This notion is underscored by the association of obesity with an earlier onset of puberty and precocity, increased risk of chronic anovulation with menstrual irregularities, and greater likelihood of PCOS. The last of these conditions commonly manifests clinical symptomatology during or soon after puberty. In this issue of JCEM, McCartney et al. (7) have extended their efforts to define the role of obesity on gonadotropin secretion during pubertal development. In previous studies using a cross-sectional design, they showed that during early puberty (Tanner 1–3), obese girls had significantly increased serum total and free testosterone levels compared with nonobese controls accompanied by corresponding decreases of circulating LH concentrations (8, 9). In addition, obese girls exhibited hyperinsulinemia and increased homeostasis model of assessment values compared with that of normal-weight controls, which may have accounted, in part, for hyperandrogenemia in the presence of lowered serum LH. In early pubertal girls, diurnal variation was observed for both testosterone and progesterone with levels lower at night and rising in the early morning hours. The incremental change in both hormones was significant in nonobese girls but failed to achieve statistical significance in obese girls likely because of too few numbers. Nevertheless, the heightened level of progesterone in obese girls may have contributed to reduced LH levels in early puberty. This notion relates to the insensitivity of the GnRH pulse generator to progesterone negative feedback in women with PCOS. In normal women, progesterone administration inhibits LH secretion, e.g. pulse frequency. Prior studies by this group have shown that androgen may interfere with this feedback mechanism as suggested by reduction of rapid LH release in response to progesterone administration in women with PCOS treated with an antiandrogen, which implies that hyperandrogenemia may contribute to increased LH secretion in this disorder. This proposed mechanism is suggested in young adolescents because some girls with PCOS treated with progesterone failed to exhibit a reduced rate of LH release (10). Because their earlier studies had been restricted to basal serum LH measurements, McCartney et al. (7) proceeded to conduct an extensive frequent sampling study of obese [body mass index (BMI) 95th percentile] and nonobese (BMI 95th percentile) girls at Tanner stages 1–5 and found differences in LH secretion within and between groups during early puberty (11). The experimental design employed a sampling interval of 1900–2300 h to denote daytime secretion and 2300–0300 h and 0300– 0700 h to reflect nighttime release. As expected, LH pulse frequency, amplitude, and mean concentrations in nonobese Tanner 1–2 girls were, in general, greater with sleep than values observed in awake subjects. In Tanner 1–2 obese girls, there was little change in LH secretion over the same intervals of study.

Effects of gonadotropin‐releasing hormone on in vitro gonadotropin release in testosterone‐treated immature rainbow trout

The control mechanism of gonadotropin‐releasing hormone (GnRH) on gonadotropin (GTH) release was studied using cultured pituitary cell or cultured whole pituitary obtained from Testosterone (T) treated and control immature rainbow trout. The release of FSH was not changed by salmon type GnRH (sGnRH), chiken‐II type (cGnRH‐II), GnRH analogue ([des‐Gly10D‐Ala6] GnRH ethylamide) and GnRH antagonist ([Ac‐3, 4‐dehydro‐Pro1, D‐p‐F‐Phe2, D‐Trp3,6] GnRH) in cultured pituitary cells of T‐treated and control fish. Indeed, FSH release was not also altered by sGnRH in cultured whole pituitary. All tested drugs had no effect on the release of LH in both culture systems of control fish. The levels of LH, in contrast, such as the pituitary content, basal release and responsiveness to GnRH were increased by T administration in both culture systems. In addition, the release of LH in response to sGnRH or cGnRH‐ll induced in a dose‐dependent manner from cultured pituitary cells of T‐treated fish, but which is not significantly different between in both GnRH at the concentration examined. Indeed, LH release was also increased by sGnRH in cultured whole pituitary of T‐treated fish. GnRH antagonist suppressed the release of LH by sGnRH (10–8 M) and GnRH analogue (10–8 M) stimulation in a dose‐dependent manner from cultured pituitary cells of T‐treated fish, and which were totally inhibited by 10–7 M GnRH antagonist. These results indicate that the sensitivity of pituitary cells to GnRH is elevated probably through the T treatment, and that GnRH is involved in the regulation of LH release. GnRH‐stimulated LH release is inhibited by GnRH antagonist in a dose‐dependent manner. The effects of gonadal steroids on FSH levels are less clear.

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

Effects of kisspeptin-10 on the electrophysiological manifestation of gonadotropin-releasing hormone pulse generator activity in the female rat.

Kisspeptins are extraordinarily potent in stimulating gonadotropic hormone secretion via an action on the hypothalamic GnRH neural system. Because the physiological frequency of the GnRH pulse generator is a critical component of the control system that governs reproductive processes, the aim of this study was to examine the effect of kisspeptin-10 on pulsatile LH secretion and on the electrophysiological manifestation of GnRH pulse generator activity to determine frequency modulatory effects. Adult Sprague Dawley rats were ovariectomized and chronically implanted with electrodes in the arcuate nucleus to record the characteristic increases in hypothalamic multiunit electrical activity volleys coincident with the initiation of each LH pulse measured in peripheral blood and/or indwelling cardiac catheters for the collection of blood samples (25 microl) every 5 min for 6-7 h for the measurement of LH. Intravenous infusion of kisspeptin-10 (7.5, 35, and 100 nmol) induced a dose-dependent increase in LH secretion. The stimulatory effect of kisspeptin-10 (100 nmol) on LH secretion was blocked by the GnRH antagonist cetrorelix, precluding a singular action on gonadotropes. Unexpectedly, however, the marked increase in LH release in response to kisspeptin-10 (100 nmol) administration was not accompanied by any change in multiunit electrical activity volley frequency. It seem unlikely, therefore, that kisspeptin-10 has an appreciable frequency modulatory effect on GnRH pulse generator activity in the female rat.

Effect of Continuous Intravenous Administration of Human Metastin 45–54 on the Neuroendocrine Activity of the Hypothalamic-Pituitary-Testicular Axis in the Adult Male Rhesus Monkey (Macaca mulatta)

In agonadal juvenile male monkeys, continuous administration of human metastin 45-54 (hu metastin 45-54) leads to desensitization of its receptor, G protein-coupled receptor 54 (GPR54), and decreased LH. The present study extended this observation to the adult male monkey, a more preclinically relevant model in which robust activity in the hypothalamic-pituitary-testicular axis is present. Continuous iv infusion of hu metastin 45-54 at either 200 or 400 microg/h elicited a marked rise in circulating LH that peaked 2-3 h after initiation of treatment. Thereafter, levels declined, and by 24 h, LH in metastin 45-54-infused animals was similar to control. LH release in response to an iv bolus of hu metastin 45-54 (10-30 microg) during the final 3 h of continuous infusion was truncated or abolished (low and high peptide dose, respectively). GPR54 desensitization by the high-dose metastin 45-54 infusion was associated with compromised pituitary response to a bolus GnRH injection (0.3 microg). LH pulse amplitude and pulse frequency were markedly suppressed during high-dose metastin 45-54 treatment. Surprisingly, the fidelity of the relationship between circulating testosterone (T) and LH was distorted during the high-dose peptide infusion. Thus, for a given concentration of LH, T levels were invariably higher during the high-dose metastin 45-54 infusion than during vehicle, suggesting that the peptide may exert direct actions on the testis to amplify T production. These findings support the notion that GPR54 is desensitized by continuous exposure to ligand, and they raise the possibility of an intratesticular role of GPR54.

The effects of GnRH analogue (buserelin) or hCG (Chorulon) on Day 12 of pregnancy on ovarian function, plasma hormone concentrations, conceptus growth and placentation in ewes and ewe lambs

The objectives of this study were to determine the effect of GnRH analogue (buserelin) or human chorionic gonadotrophin (hCG, Chorulon) treatment on Day 12 of pregnancy on ovarian function, plasma hormone concentrations, conceptus growth and placentation in ewes and ewe lambs. After oestrus synchronization with progestagen sponges and eCG, all the animals were mated with fertile rams. Both ewes and ewe lambs (20 per treatment group) were given either normal saline or 4 μg GnRH or 200 IU hCG on Day 12 post-mating. Pre- and post-treatment plasma hormone concentrations were determined in seven pregnant animals per treatment group in samples collected 1 h before and 0, 2, 4, 6, 8, 24, 48 and 72 h after treatment. Overall mean progesterone concentrations were higher ( P < 0.001) in ewes as compared with ewe lambs in saline-treated controls. GnRH or hCG treatment increased ( P < 0.001) mean plasma progesterone concentrations in both age groups, however, post-treatment concentrations were significantly ( P < 0.05) higher in ewes than in ewe lambs. Oestradiol concentrations were similar in the two control groups. In ewes, but not in ewe lambs, both GnRH and hCG treatments significantly ( P < 0.05) increased the mean oestradiol concentrations above pre-treatment levels. Moreover, post-treatment oestradiol concentrations in GnRH- and hCG-treated animals were significantly ( P < 0.05) higher than those in the saline-treated controls. LH release in response to GnRH treatment was greater ( P < 0.05) in ewes than in ewe lambs, whereas FSH release in ewes was less ( P < 0.05) than that of ewe lambs. The effects of GnRH or hCG on conceptus growth and placentation was determined at slaughter on Day 25. In ewes, GnRH treatment increased ( P < 0.05) luteal weight, amniotic sac width and length, and crown-rump length compared with controls, but had no effect on these parameters in ewe lambs. In ewes, hCG treatment also enhanced ( P < 0.05) luteal weight, amniotic sac width and length, crown-rump length, embryo weight and number of placentomes as compared with controls. In ewe lambs, there was no difference ( P < 0.05) between hCG and control groups in luteal weight, embryo weight and amniotic sac width but crown-rump length, amniotic sac length and the number of placentomes forming the placenta were greater ( P < 0.05). In conclusion, GnRH or hCG treatment on Day 12 of pregnancy can increase ovarian function, conceptus growth and placental attachment in ewes. However, these treatments were less effective in ewe lambs.

The Presence and Ancestral Role of Gonadotropin-Releasing Hormone in the Reproduction of Scleractinian Coral, Euphyllia ancora

The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10(-9)-10(-5) M irGnRH) as well as mammalian (m) GnRH agonist (10(-10)-10(-6) M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10(-5) M irGnRH) and mGnRH agonist (10(-6) M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.

13 EFFECTS OF PLASMA PROGESTERONE CONCENTRATIONS ON LH RELEASE AND OVULATION IN BEEF CATTLE GIVEN GnRH

Two experiments were conducted to determine the effects of plasma progesterone (P4) concentrations on LH release and ovulation in beef heifers and lactating beef cows given gonadotropin-releasing hormone (GnRH). Previously autoclaved, once-used CIDR inserts (Colazo et al. 2004 Anim. Reprod. Sci. 81, 25-34) were used for experimental purposes to induce differential plasma progesterone concentrations. In Experiment 1, postpubertal heifers received 25 mg of dinoprost i.m. (prostaglandin F (PGF); Lutalyse; Pfizer Animal Health, Montreal, Quebec, Canada). On Day 4 (estrus = Day 0), heifers were randomly assigned (10/group) to receive no treatment (control) or 1 or 2 autoclaved once-used CIDR (Pfizer Animal Health) inserts (1CIDR and 2CIDR, respectively). On Day 5, heifers in the 1CIDR group were given PGF twice 12 h apart. On Day 6, all heifers received 100 �g of GnRH i.m. (Cystorelin; Merial Canada, Inc., Victoriaville, Quebec, Canada). Once daily on Days 4 to 9, a blood sample was collected and ultrasonography was performed. On Day 6, heifers in the control (3.0 � 0.4 ng/mL; mean � SD) and 1CIDR groups (3.0 � 0.3 ng/mL) had lower (P &lt; 0.01) plasma progesterone concentrations than those in the 2CIDR group (5.7 � 0.4 ng/mL). However, the diameter of the dominant follicle was larger (P &lt; 0.001) in heifers in the control and 1CIDR groups than in the 2CIDR group (12.1 � 1.0, 11.5 � 0.7, and 10.1 � 0.7 mm, respectively). More (P &lt; 0.01) heifers ovulated in response to GnRH in the control and 1CIDR groups than in the 2CIDR group (10/10, 9/10, and 3/10, respectively). In Experiment 2, ultrasound-guided follicular ablation (FA) was performed (to synchronize ovarian follicular wave emergence) 4 to 6 days after estrus in 20 postpubertal heifers and 20 mature lactating cows. Cattle were randomly and equally assigned to receive an autoclaved, once-used CIDR, either with no further treatment (High-P4) or with two PGF treatments 12 h apart (Low-P4) given after FA. All cattle received 100 �g of GnRH either 6 days after FA or the day after the dominant follicle reached 9 mm in diameter. Ultrasonography was performed daily (from 4 days after FA to ovulation or to 3 days after GnRH treatment). In three cows and three heifers per group, blood samples were collected every 30 min for 12 h after GnRH. The dominant follicle at GnRH treatment was larger in cows than heifers (11.0 � 1.1 vs. 10.3 � 0.9 mm, respectively; P = 0.05) and tended to be smaller in the High-P4 group vs. the Low-P4 group (10.3 � 1.0 vs. 11.0 � 1.0 mm; P = 0.06). Ovulatory response was not different (P = 0.9) between heifers (77.7%) and cows (78.9%), but combined for heifers and cows, was lower in High-P4 vs. Low-P4 cattle (61.1 vs. 94.7; P &lt; 0.01). The GnRH-induced LH surge did not differ (P = 0.23) between cows and heifers, but it was lower and of shorter duration (P &lt; 0.001) in the High-P4 group than in the Low-P4 group. In summary, higher plasma P4 concentrations resulted in decreased LH release and the proportion of cattle ovulating in response to GnRH treatment. There was no significant difference between heifers and cows in LH release or ovulatory response.

Effects of intracerebroventricular infusion of genistein on the secretory activity of the GnRH/LH axis in ovariectomized ewes

Phytoestrogens, plant derived estrogen like-compounds exert numerous effects on the reproductive functions of animals. The present study was designed to demonstrate if exogenous genistein infused during the breeding season into the third ventricle of the brain of ovariectomized ewes could affect the secretory activity of the GnRH/LH axis. Two-year-old ovariectomized ewes ( n = 8) were infused with vehicle (control, n = 3) or genistein (10 μg/100 μl/h, n = 5) into the third ventricle. The infusions were done from 10.00 to 14.00 h and blood samples collection was performed this day up to 20.00 h and next day from 8.00 to 10.00 h. The animals were slaughtered, thereafter. Immunoreactive (IR) GnRH neurons in the hypothalamus and LH cells in the adenohypophysis were localized by immunohistochemistry. Messenger RNA analyses were performed by nonisotope in situ hybridization using sense and anti-sense riboprobes produced from β subunits of LH cDNA clones. Plasma LH concentrations were measured by radioimmunoassay. Immunohistochemical analysis revealed that genistein infusion affected the morphology of GnRH neurons evoking a visualization of long axons in the GnRH perikarya and visibly diminished IR GnRH stores in the median eminence. The number of IR LH cells and IR material stored in the adenohypophyses increased in genistein-infused animals, which was confirmed by statistical analysis ( P < 0.001). The in situ hybridization analyses showed in these ewes the increase of mRNA LHβ hybridization signal. The changes in LH release in response to genistein infusion had a biphasic character: it decreased within 6 h after infusion and increased 24 h later. Mean concentration of LH and amplitude of pulses measured from the beginning of infusion up to end of the experiment were significantly higher ( P < 0.05) in genistein-infused ewes compared to vehicle-treatment. In conclusion, our data show that genistein, a phytoestrogen, may effectively modulate GnRH and LH secretion in OVX ewes by acting directly on the CNS. The biphasic character of the LH response is similar to that of estradiol during the breeding season in the ewes.