Prolonged Elevated Concentrations of Estradiol Do Not Affect Conception Rates in Beef Cattle

Following treatments causing either prolonged elevated concentrations of estradiol associated with development of persistent follicles or inhibited elevated concentrations of estradiol and development of persistent follicles, conception rates were compared. Beef females received either four norgestomet implants for 9 days (day 0 = treatment initiation; n=59) or one norgestomet implant for 7 days and three additional norgestomet implants for 2 days (n=60). All implants were removed on day 9 followed by estrous detection and AI for 7 days. Treatment and day interacted to affect estradiol concentrations from day 0 to day 9 with elevated estradiol in females treated with one norgestomet implant for 7 days. Conception rates to AI were similar across treatments. Prolonged elevated concentrations of estradiol associated with development of persistent ovarian follicles do not affect fertility when persistent ovarian follicles are not allowed to ovulate

Changing Dose of Progesterone Results in Sudden Changes in Frequency of Luteinizing Hormone Pulses and Secretion of 17β-Estradiol in Bovine Females

The aim of the present study was to elucidate the time course according to which changes in circulating concentrations of progesterone influence pulsatile secretion of LH and secretion of 17β-estradiol. Our working hypothesis was that changing the dose of progesterone would result in changes in frequency of LH pulses and secretion of 17β-estradiol within 72 h. Five days after behavioral estrus, thirty-three cows were randomly assigned to one of five groups: 1) control, no treatment (CONT, n = 5); 2) treatment with two progesterone-releasing intravaginal devices (PRIDs) for 11 days (2PRID, 5-6 ng/ml plasma progesterone, n = 7); 3) treatment with a 0.5 PRID for 11 days (0.5PRID, 1-2 ng/ml plasma progesterone, n = 7); 4) treatment with 2 PRIDs for 8 days followed by treatment with a 0.5 PRID for 3 days (2-0.5PRID, n = 7); and 5) treatment with a 0.5 PRID for 8 days followed by treatment with 2 PRIDs for 3 days (0.5-2PRID, n = 7). Cows subject to PRID treatments received injections of prostaglandin F2 on Days 1 and 2 (Day 0 = day of initiation of PRID treatments, fifth day of the estrous cycle in CONT cows) to lyse the existing corpus luteum. Cows were bled for 12 h at 15-min intervals on Day 7.5 of the treatment period (twelfth day of the estrous cycle in CONT cows). The dose of progesterone was changed on Day 8 in cows that were assigned to the 2-0.5PRID and 0.5-2PRID groups, and blood collections continued an additional 72 h to characterize profiles of circulating concentrations of LH and 17β-estradiol. Cows treated with a 0.5 PRID had a greater (p \u3c 0.05) number of LH pulses and higher (p \u3c 0.05) concentrations of 17β-estradiol throughout the entire blood collection period than cows in the 2PRID and CONT groups. An increase in the number of LH pulses was detected within 6 h after the change from the high to the low dose of progesterone (2-0.5PRID), and frequency of LH pulses was similar to that of cows in the 0.5PRID group for the remainder of the period of blood collection. LH pulse frequency declined within 6 h after the shift from the low to the high dose of progesterone (0.5-2PRID) and was similar to that of cows in the 2PRID group by 12 h after the dose was changed. Within 6 h after the dose of progesterone was changed, circulating concentrations of 17p-estradiol increased (p \u3c 0.05) in cows shifted from the high to low dose (2-0.5PRID) and declined (p \u3c 0.05) after the dose of progesterone was changed from low to high (0.5-2PRID). We conclude that changing the circulating concentrations of progesterone concurrently affects frequency of pulsatile LH release and secretion of 17β-estradiol within 6-24 h

Changing Dose of Progesterone Results in Sudden Changes in Frequency of Luteinizing Hormone Pulses and Secretion of 17β-Estradiol in Bovine Females

The aim of the present study was to elucidate the time course according to which changes in circulating concentrations of progesterone influence pulsatile secretion of LH and secretion of 17β-estradiol. Our working hypothesis was that changing the dose of progesterone would result in changes in frequency of LH pulses and secretion of 17β-estradiol within 72 h. Five days after behavioral estrus, thirty-three cows were randomly assigned to one of five groups: 1) control, no treatment (CONT, n = 5); 2) treatment with two progesterone-releasing intravaginal devices (PRIDs) for 11 days (2PRID, 5-6 ng/ml plasma progesterone, n = 7); 3) treatment with a 0.5 PRID for 11 days (0.5PRID, 1-2 ng/ml plasma progesterone, n = 7); 4) treatment with 2 PRIDs for 8 days followed by treatment with a 0.5 PRID for 3 days (2-0.5PRID, n = 7); and 5) treatment with a 0.5 PRID for 8 days followed by treatment with 2 PRIDs for 3 days (0.5-2PRID, n = 7). Cows subject to PRID treatments received injections of prostaglandin F2 on Days 1 and 2 (Day 0 = day of initiation of PRID treatments, fifth day of the estrous cycle in CONT cows) to lyse the existing corpus luteum. Cows were bled for 12 h at 15-min intervals on Day 7.5 of the treatment period (twelfth day of the estrous cycle in CONT cows). The dose of progesterone was changed on Day 8 in cows that were assigned to the 2-0.5PRID and 0.5-2PRID groups, and blood collections continued an additional 72 h to characterize profiles of circulating concentrations of LH and 17β-estradiol. Cows treated with a 0.5 PRID had a greater (p \u3c 0.05) number of LH pulses and higher (p \u3c 0.05) concentrations of 17β-estradiol throughout the entire blood collection period than cows in the 2PRID and CONT groups. An increase in the number of LH pulses was detected within 6 h after the change from the high to the low dose of progesterone (2-0.5PRID), and frequency of LH pulses was similar to that of cows in the 0.5PRID group for the remainder of the period of blood collection. LH pulse frequency declined within 6 h after the shift from the low to the high dose of progesterone (0.5-2PRID) and was similar to that of cows in the 2PRID group by 12 h after the dose was changed. Within 6 h after the dose of progesterone was changed, circulating concentrations of 17p-estradiol increased (p \u3c 0.05) in cows shifted from the high to low dose (2-0.5PRID) and declined (p \u3c 0.05) after the dose of progesterone was changed from low to high (0.5-2PRID). We conclude that changing the circulating concentrations of progesterone concurrently affects frequency of pulsatile LH release and secretion of 17β-estradiol within 6-24 h

Differential Regulation of Gonadotropin Synthesis and Release in Ovariectomized Ewes after Treatment with a Luteinizing Hormone-Releasing Hormone Antagonist

Our working hypothesis was that synthesis and release of LH, but not FSH, were solely dependent on LHRH. Twenty ovariectomized (OVX) ewes were randomly assigned to one of five treatments (n = 4 per group). Ewes were administered a low (10 μg/kg) or high (100 μg/kg) dose of LHRH antagonist (LHRH-Ant) at 24-h intervals for 3 or 6 days. Control ewes received vehicle (5% mannitol) at 24-h intervals for 6 days. Blood samples were collected every 15 min for 4 h before LHRH-Ant or vehicle and every 2 h during the period of treatment to determine concentrations of LH and FSH. Twenty-four hours after the last treatment with LHRH-Ant or vehicle, anterior pituitaries were collected and divided in half along the midsagittal plane; the number of receptors for LHRH, pituitary content of LH and FSH, and relative amounts of mRNA for α, LHβ, and FSHβ subunits were determined. Concentrations of LH in serum decreased (p \u3c 0.05) from 25.4 ± 4.3 ng/ml before LHRH-Ant to less than 0.5 ng/ml within 4 h after the first treatment of LHRH-Ant and remained low (\u3c 0.5 ng/ml) throughout the study. Serum concentrations of FSH declined gradually during the 3- or 6-day period of treatment with LHRH-Ant, from 37.3 ± 2.4 and 26.5 ± 4.8 ng/ml to 19.9 ± 1.8 and 13.7 ± 2.1 ng/ml, respectively. The magnitude of decline in serum concentrations of LH and FSH did not differ among ewes treated with low or high doses of LHRH-Ant. Pituitary content of LH was not different (p \u3e 0.10) from that in controls, whereas pituitary content of FSH was greater (p \u3c 0.01) in control ewes compared to ewes treated with LHRH-Ant. Receptors for LHRH were nondetectable (\u3c 0.018 x 10-16 mol receptor/μg protein) in pituitaries after 3 or 6 days of treatment with LHRH-Ant (low or high dose). Relative amounts of mRNA for α, LHβ, and FSHβ subunits were lower (p \u3c 0.01) after 6 days of treatment with LHRH-Ant (low or high dose) than after 3 days of treatment with LHRH-Ant (low or high dose). The LHRH was, therefore, required to maintain steady state amounts of mRNA for FSH and LH and to maintain pituitary stores of FSH but not LH. Our data support the hypothesis that differential regulation of LH and FSH release occurs in ewes. While synthesis and release of LH are dependent on LHRH, synthesis but not release of FSH appears to be dependent on LHRH

Melengestrol Acetate at Greater Doses Than Typically Used for Estrous Synchrony in Bovine Females Does Not Mimic Endogenous Progesterone in Regulation of Secretion of Luteinizing Hormone and 17β-Estradiol

Our working hypothesis was that doses of melengestrol acetate (MGA) greater than those typically administered in estrous synchrony regimens would regulate secretion of LH and 17β-estradiol (E2) as endogenous progesterone (P4) does during the midluteal phase of the estrous cycle. We also hypothesized that endogenous P4 from the CL would interact with MGA to further decrease the frequency of LH pulses and E2. Cows on Day 5 of their estrous cycle (Day 0 = estrus) were randomly assigned to an untreated control group (CONT, n = 5) or to one of six MGA treatment groups (n = 5 per group): 1) MGA administered orally each day via a gelatin capsule at a dose of 0.5 mg MGA/cow with the CL present (0.5CL); 2) 0.5 mg MGA/cow daily in the absence of CL (0.5NO); 3) 1.0 mg MGA with CL present (1.OCL); 4) 1.0 mg MGA without CL (1.ONO); 5) 1.5 mg MGA with CL present (1.5CL); 6) 1.5 mg without CL (1.5NO). MGA was administered for 10 days (Day 5 = initiation of treatment). To regress CL, cows assigned to groups without CL received injections of prostaglandin F2α (PGF, 0; 25 mg) on Days 6 and 7 of their estrous cycle. All cows were administered PGF2α. at the end of the 10-day treatment period. During the treatment period, daily blood samples were collected to determine concentrations of E2. Serial blood samples were collected at 15-min intervals for 24 h on Days 8, 11, and 14 to determine pattern of LH secretion. Frequency of LH pulses on Days 8, 11, and 14 was greater (p \u3c 0.05) in cows without CL (0.5NO, 1.ONO, and 1.5NO) than in cows with CL (0.5CL, 1.OCL, 1.5CL, and CONT). Mean concentrations of LH were greater (p \u3c 0.05) in cows from the 0.5NO group on Days 8 and 11 and were greater (p \u3c 0.05) in cows from the 0.5NO, 1.ONO, and 1.5NO groups on Day 14 as compared to cows with CL. Overall mean concentrations of LH across Days 8, 11, and 14 were greatest (p \u3c 0.05) in cows from the 0.5NO group and were also greater (p \u3c 0.05) in cows from the 0.5NO, 1.ONO, and 1.5NO groups as compared to cows in the 0.5CL, 1.OCL, 1.5CL, and CONT groups. Mean concentrations of E2 during the treatment period were greater (p \u3c 0.05) in cows from the 0.5NO group than in cows from either the 1.ONO or the 1.5NO group; these values were also greater (p \u3c 0.05) in cows of the 0.5NO, 1.ONO, and 1.5NO groups as compared to cows of the 1.OCL and CONT groups. Therefore, we reject our working hypothesis because doses of MGA greater than those typically used in estrous synchrony protocols did not suppress LH and E2 to the same extent that endogenous P4 does. In addition, MGA treatment when CL were present did not result in a further suppression of LH pulse frequency or of E2 as compared to the values in control cows with functional CL

Comparison of Circulating Concentrations of Reproductive Hormones in Boars of Lines Selected for Size of Testes or Number of Ovulations and Embryonal Survival to Concentrations in Respective Control Lines

The objectives of this study were to determine whether circulating concentrations of gonadotropins and gonadal hormones of boars were altered as a result of selection of pigs for size of testes or for embryonal survival and(or) number of ovulations. Included in Exp. 1 and 2 were boars with the greatest estimated paired weight of testes (TS) and boars from a control (C) line. Concentrations of FSH were similar ( P \u3e .10) in boars from the TS and C lines. In Exp. 3, 4, and 5, circulating concentrations of FSH and 17β-estradiol (E2) were evaluated in neonates, during pubertal development, and in mature boars of lines selected for an index of number of ovulations and embryonal survival ( I ) , and data were compared to those for boars from a respective C line. Concentrations of E2 were not different in boars from the I line and those from the C line during the early neonatal period but were greater ( P \u3c .05) in boars of the C line than in those from the I line during pubertal development. Concentrations of FSH were greater ( P \u3c .05) in mature boars from the I line than in those from the C line. In summary, selection for size of testes did not influence circulating concentrations of FSH in mature boars. The secretory pattern of E2 in boars before puberty changed as a result of selection for embryonal survival and number of ovulations in females of the I line, and the different patterns of circulating E2 early in life may result in enhanced circulating concentrations of FSH in adult boars of the I line compared with boars of the C line

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ(9)-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).JWH-018, five potential monohydroxylated metabolites (M1-M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [(3)H]CP-55,940, and then for CB1R intrinsic efficacy using an [(35)S]GTPγS binding assay. JWH-018 and M1-M5 bound CB1Rs with high affinity, exhibiting K(i) values that were lower than or equivalent to Δ(9)-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ(9)-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ(9)-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ(9)-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis

Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Intranasal “painless” Human Nerve Growth Factors Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that “painless” hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of “painless” hNGF variants as a new generation of therapeutics for neurodegenerative diseases