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G. P. Adams - One of the best experts on this subject based on the ideXlab platform.
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lh release and ovulatory response after intramuscular intravenous and intrauterine administration of β nerve growth factor of seminal plasma origin in female Llamas
Theriogenology, 2015Co-Authors: Mauricio Silva, G. P. Adams, A Fernandez, Cesar Ulloaleal, M A Berland, M H RattoAbstract:Abstract 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.
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luteotrophic effect of ovulation inducing factor nerve growth factor present in the seminal plasma of Llamas
Theriogenology, 2014Co-Authors: G. P. Adams, O.a. Bogle, Cesar Ulloaleal, M H RattoAbstract:The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female Llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 Llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all Llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of Llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of Llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.
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107 detection of biotinylated ovulation inducing factor oif in cerebrospinal fluid and its ability to induce ovulation
Reproduction Fertility and Development, 2013Co-Authors: M A Berland, G. P. Adams, O.a. Bogle, Montserrat Guerra, K Vio, M H RattoAbstract:Ovulation-inducing factor (OIF) is a protein in the seminal plasma of Llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, Llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all Llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both Llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits. Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.
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The nerve of ovulation-inducing factor in semen
Proceedings of the National Academy of Sciences, 2012Co-Authors: Marcelo H Ratto, Y. A. Leduc, X. P. Valderrama, K. E. Van Straaten, R A Pierson, Louis T. J. Delbaere, G. P. AdamsAbstract:A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from Llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in Llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC(12)) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in Llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.
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dose response of female Llamas to ovulation inducing factor from seminal plasma
Biology of Reproduction, 2011Co-Authors: Valeria M Tanco, Marcelo H Ratto, Maura Lazzarotto, G. P. AdamsAbstract:The present study was designed to determine if the dose of purified ovulation-inducing factor (OIF) from llama seminal plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal ejaculate and to test the hypothesis that corpus luteum (CL) form and function are affected by OIF in a dose-dependent manner. Female Llamas were assigned randomly to five groups (n ¼ 10 per group) and given a single i.m. dose of 500, 250, 125, or 60 l go f purified OIF (representative of the amount present in 1/25th to 1/200th of a normal ejaculate) or 1 ml of PBS (control). Ovulation and CL development were monitored by transrectal ultrasonography. Blood samples were taken to measure plasma progesterone concentrations and to determine changes in plasma concentrations of luteinizing hormone (LH). The high dose of OIF (500 lg) was associated with the highest incidence of ovulation (P , 0.05), the greatest maximum CL diameter (P , 0.05), and the largest day-to-day profiles of CL diameter (P , 0.05) and plasma progesterone concentrations (P , 0.01). A rise in plasma LH concentration was apparent in all Llamas that ovulated and was most rapid and highest in the high-dose group (P , 0.01). The low dose of OIF (60 lg) was minimally effective for induction of ovulation and the least luteotrophic, as evidenced by the smallest maximum CL diameter and the smallest day-to-day profiles for CL diameter and plasma concentrations of progesterone and LH. Responses were intermediate for the middle-dose groups (125 and 250 lg). We conclude that OIF from llama seminal plasma has a dosedependent effect on ovulation rate and CL form and function in Llamas and that the biological effect of OIF is evident at physiologically relevant doses (i.e., as little as 1/100th of that present in an ejaculate). corpus luteum, gonadotropins, ovary, ovulation, seminal plasma
Jack H Ladenson - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of a thermally stable recombinant anti caffeine heavy chain antibody fragment
Analytical Chemistry, 2006Co-Authors: Ruth C Ladenson, Dan L Crimmins, Yvonne Landt, Jack H LadensonAbstract:We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.
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isolation and characterization of a thermally stable recombinant anti caffeine heavy chain antibody fragment
Analytical Chemistry, 2006Co-Authors: Ruth C Ladenson, Dan L Crimmins, Yvonne Landt, Jack H LadensonAbstract:We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (VHH) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific VHH fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these VHH fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 °C. In addition, VSA2 was able to bind caffeine at 70 °C. A competition caffeine ELISA was dev...
M H Ratto - One of the best experts on this subject based on the ideXlab platform.
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effect of mating on mrna and protein expression of beta nerve growth factor and its receptor trka in the oviduct of llama lama glama
Molecular Reproduction and Development, 2020Co-Authors: Luciana M Sari, M H Ratto, Renato Zampini, Martin E Arganaraz, Silvana Andrea ApichelaAbstract:Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of β-nerve growth factor (β-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated Llamas. β-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both β-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative β-NGF abundance was higher in the UTJ of copulated females (p < .05). β-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the β-NGF/TrKA system and that an increase in β-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in Llamas.
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a comparative study of the effects of intramuscular administration of gonadorelin mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified β ngf on luteal development in Llamas
Reproduction in Domestic Animals, 2017Co-Authors: Mauricio Silva, Felipe Urra, Cesar Ulloaleal, M H RattoAbstract:Contents The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta-nerve growth factor from seminal origin (spβ-NGF) on ovulation rate and corpus luteum (CL) development and function in Llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ-NGF contained in 4 ml of PBS (phosphate-buffered saline) (spβ-NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spβ-NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spβ-NGF influences its luteotrophic effect in Llamas.
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lh release and ovulatory response after intramuscular intravenous and intrauterine administration of β nerve growth factor of seminal plasma origin in female Llamas
Theriogenology, 2015Co-Authors: Mauricio Silva, G. P. Adams, A Fernandez, Cesar Ulloaleal, M A Berland, M H RattoAbstract:Abstract 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.
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luteotrophic effect of ovulation inducing factor nerve growth factor present in the seminal plasma of Llamas
Theriogenology, 2014Co-Authors: G. P. Adams, O.a. Bogle, Cesar Ulloaleal, M H RattoAbstract:The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female Llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 Llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all Llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of Llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of Llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.
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107 detection of biotinylated ovulation inducing factor oif in cerebrospinal fluid and its ability to induce ovulation
Reproduction Fertility and Development, 2013Co-Authors: M A Berland, G. P. Adams, O.a. Bogle, Montserrat Guerra, K Vio, M H RattoAbstract:Ovulation-inducing factor (OIF) is a protein in the seminal plasma of Llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, Llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all Llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both Llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits. Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.
Ruth C Ladenson - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of a thermally stable recombinant anti caffeine heavy chain antibody fragment
Analytical Chemistry, 2006Co-Authors: Ruth C Ladenson, Dan L Crimmins, Yvonne Landt, Jack H LadensonAbstract:We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.
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isolation and characterization of a thermally stable recombinant anti caffeine heavy chain antibody fragment
Analytical Chemistry, 2006Co-Authors: Ruth C Ladenson, Dan L Crimmins, Yvonne Landt, Jack H LadensonAbstract:We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (VHH) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific VHH fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these VHH fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 °C. In addition, VSA2 was able to bind caffeine at 70 °C. A competition caffeine ELISA was dev...
Marcelo H Ratto - One of the best experts on this subject based on the ideXlab platform.
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Nerve growth factor from seminal plasma origin (spβ-NGF) increases CL vascularization and level of mRNA expression of steroidogenic enzymes during the early stage of Corpus Luteum development in Llamas
Theriogenology, 2017Co-Authors: Mauricio Silva, X. P. Valderrama, Cesar Ulloa-leal, Gregg P. Adams, O.a. Bogle, Marcelo H RattoAbstract:Abstract The objectives of the study were to determine the effect of seminal plasma β-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 μg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spβ-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 Llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all Llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 Llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1 /P450scc and STAR expression by RT-PCR. Ovulation was similar between Llamas treated with GnRH or spβ-NGF and CL diameter did not differ between GnRH or spβ-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P CYP11A1 /P450scc was upregulated 3 folds at day 4 and 10 by spβ-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spβ-NGF. In conclusion, the luteotrophic effect of spβ-NGF could be related to an increase of vascularization and up regulation of CYP11A1 /P450scc and STAR transcripts enhancing progesterone secretion.
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A comparative study of the effects of intramuscular administration of gonadorelin, mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified β-NGF on luteal development in Llamas.
Reproduction in domestic animals = Zuchthygiene, 2017Co-Authors: Mauricio Silva, Felipe Urra, Cesar Ulloa-leal, Marcelo H RattoAbstract:Contents The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta-nerve growth factor from seminal origin (spβ-NGF) on ovulation rate and corpus luteum (CL) development and function in Llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ-NGF contained in 4 ml of PBS (phosphate-buffered saline) (spβ-NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p
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The nerve of ovulation-inducing factor in semen
Proceedings of the National Academy of Sciences, 2012Co-Authors: Marcelo H Ratto, Y. A. Leduc, X. P. Valderrama, K. E. Van Straaten, R A Pierson, Louis T. J. Delbaere, G. P. AdamsAbstract:A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from Llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in Llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC(12)) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in Llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.
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dose response of female Llamas to ovulation inducing factor from seminal plasma
Biology of Reproduction, 2011Co-Authors: Valeria M Tanco, Marcelo H Ratto, Maura Lazzarotto, G. P. AdamsAbstract:The present study was designed to determine if the dose of purified ovulation-inducing factor (OIF) from llama seminal plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal ejaculate and to test the hypothesis that corpus luteum (CL) form and function are affected by OIF in a dose-dependent manner. Female Llamas were assigned randomly to five groups (n ¼ 10 per group) and given a single i.m. dose of 500, 250, 125, or 60 l go f purified OIF (representative of the amount present in 1/25th to 1/200th of a normal ejaculate) or 1 ml of PBS (control). Ovulation and CL development were monitored by transrectal ultrasonography. Blood samples were taken to measure plasma progesterone concentrations and to determine changes in plasma concentrations of luteinizing hormone (LH). The high dose of OIF (500 lg) was associated with the highest incidence of ovulation (P , 0.05), the greatest maximum CL diameter (P , 0.05), and the largest day-to-day profiles of CL diameter (P , 0.05) and plasma progesterone concentrations (P , 0.01). A rise in plasma LH concentration was apparent in all Llamas that ovulated and was most rapid and highest in the high-dose group (P , 0.01). The low dose of OIF (60 lg) was minimally effective for induction of ovulation and the least luteotrophic, as evidenced by the smallest maximum CL diameter and the smallest day-to-day profiles for CL diameter and plasma concentrations of progesterone and LH. Responses were intermediate for the middle-dose groups (125 and 250 lg). We conclude that OIF from llama seminal plasma has a dosedependent effect on ovulation rate and CL form and function in Llamas and that the biological effect of OIF is evident at physiologically relevant doses (i.e., as little as 1/100th of that present in an ejaculate). corpus luteum, gonadotropins, ovary, ovulation, seminal plasma
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comparison of the effect of ovulation inducing factor oif in the seminal plasma of Llamas alpacas and bulls
Theriogenology, 2006Co-Authors: Marcelo H Ratto, W Huanca, J. Singh, Gregg P. AdamsAbstract:Abstract We have recently reported the presence of an ovulation-inducing factor (OIF) in the seminal plasma of Llamas and alpacas—species characterized as induced ovulators. The study was designed to test the hypothesis that the seminal plasma of bulls will induce ovulation in Llamas, and to compare the ovulation-inducing effect of seminal plasma of conspecific versus hetero-specific males. The seminal plasma of alpacas, a closely related induced ovulator ( Lama pacos ), and cattle, a distantly related ruminant species ( Bos taurus ) considered to be spontaneous ovulators, were compared with that of the llama ( Lama glama ). Ovulation and maximum corpus luteum diameter were compared by ultrasonography among female Llamas ( n = 19 per group) treated intramuscularly with 2 mL of phosphate buffered saline (PBS, negative control) and those treated with 2 mL of seminal plasma of bulls, alpacas, or Llamas (conspecific control). The diameter of the preovulatory follicle did not differ among groups at the time of treatment. Bull seminal plasma induced ovulations in 26% (5/19) of Llamas compared to 0% (0/19) in PBS group ( P P B. taurus . The interspecies effects of seminal plasma on ovulation and luteal development provide rationale for the hypothesis that OIF is conserved among both spontaneous and induced ovulating species.
