Experiments were initiated to determine whether exogenous estradiol-17beta (E2) and oxytocin (OT) can be used to dilate the cervix and improve transcervical embryo transfer (ET) procedures for sheep. However, there was concern that the E2-OT treatment may alter luteal function and that embryo quality would decrease as the superovulatory response to FSH increased. In Exp. 1, 32 ewes were assigned to a 2 x 2 factorial array of treatments. On d 7, ewes received an i.v. injection of either 100 micrograms of E2 in 5 mL of 1:1 ethanol:saline or 5 mL of 1:1 ethanol:saline; 12 h later, ewes received i.v. injection of either 400 USP units of OT or saline. Jugular blood was collected on d 7, 8, 9, 10, 12, 14, 16, and 18. Progesterone concentrations were unaffected by the treatments. Experiment 2 was conducted to determine the dose of pFSH needed to induce approximately six corpora lutea (CL). Ten-day Norgestomet implants inserted between d 8-12 of the estrous cycle were used to synchronize estrus in Hampshire and Hampshire x Dorset ewes (n = 23). Ewes received a total of either 0, 18, 27, or 36 mg of pFSH, which was injected i.m. at -24, -12, 0, 12, 24, and 36 h relative to implant removal. The dose at each respective time was 19.4, 19.4, 16.7, 16.7, 13.9, and 13.9% of the total. Ewes received 400 IU of PMSG i.m. at -24 h. The CL were counted laparoscopically on d 6 (d 0 = estrus). Number of CL increased linearly (P < .01) with dose of pFSH; there were 1.8, 3.6, 6.3, and 11.2 CL/ewe, respectively. Experiment 3 was conducted to determine the effect of the E2-OT treatment, mode of transfer or the interaction of E2-OT treatment x mode of transfer on embryo survival and development. Experiment 3 was conducted over two breeding seasons and across two trials. In the first trial ewes were assigned to one of three randomized treatments. Procedural limitations that were later overcome prevented a true 2 x 2 factorial design; therefore, transcervical transfer without hormonal treatment was excluded in the first trial. In the second trial, ewes were assigned to a 2 x 2 factorial array of treatments. On d 6 of pregnancy, embryos rating a fair or better were transferred into recipients either transcervically or laparoscopically. Recipients were administered either an E2 (d 6) - OT (d 7) treatment or an ethanol:saline-saline treatment following the same protocol as in Exp. 1. Embryos were recovered on d 12 in Trial 1 and d 14 in Trial 2. Embryos were evaluated morphologically for development and ranked on a scale of one to four; one represented no development and four represented development to the morphological stages associated with the day of collection. The treatments did not affect the percentage of embryos recovered after transfer or the percentage of embryos that showed some developed. However, there was an effect of mode of transfer on mean rank of embryo development; embryos transferred laporscopically developed further than embryos transferred transcervically (P < .01). This may have been an artifact of a technician effect between trials. There was an effect of E2-OT treatment on transcervical transfer (P < .01), indicating that it may be detrimental to transfer embryos transcervically without dilating the cervix. In conclusion, the E2-OT treatment did not affect luteal function, and the E2-OT treatment can be used to dilate the cervix and enhance success of transcervical transfer of embryos. A 400 IU priming dose of PMSG and a total dose of 27 mg of pFSH can be used to induce the target number of six CL. / Master of Science

Experiments were conducted to determine the effects of a treatment used to dilate the cervix for transcervical AI and embryo transfer on pregnancy or fertilization rates in sheep. Experiment 1 was conducted as a 2 × 2 factorial, with the main effects of estradiol-17β (E2) and oxytocin (OT). On d 6 of pregnancy, ewes received (i.v.) 100 μg of estradiol-17β, or saline; 10 h later, ewes received (i.v.) 400 USP units (20 mL) or saline. Blood samples were collected for progesterone analysis. Transrectal ultrasonography was used to determine pregnancy on d 25. Treatment did not affect (χ² , P > .05) pregnancy rates. For nonpregnant ewes, the period × E2 × OT interaction (P < .01) was significant. Experiments were conducted to evaluate the effects of oxytocin and(or) cervical manipulation on fertilization rates. In Exp. 2, nulliparous ewes were assigned to one of two randomized treatment groups: 1) saline or 2) oxytocin. In Exp. 3, multiparous ewes were assigned to one of two randomized treatment groups: 1) saline-cervical manipulation or 2) oxytocin-cervical manipulation. Ewes in Exp. 2 and 3 were laparoscopically inseminated. In Exp. 2 and 3, ewes received (i.v.) either 400 USP units of oxytocin or 20 mL of saline. In Exp. 2 and 3, oocytes and(or) embryos were collected. Treatment did not affect recovery, fertilization, or pregnancy rates in Exp.2 and 3. In conclusion, the treatment used to dilate the cervix in ewes to facilitate the transcervical AI or ET does not seem to affect fertilization or pregnancy rates. / Master of Science

A expressão dos receptores da ocitocina (OTR) em machos, embora estudada em algumas espécies, não foi ainda estabelecida em cães. O objetivo deste estudo foi trazer novas perspectivas para a utilização terapêutica da ocitocina (OT) em patologias reprodutivas em cães já que tem sido utilizada no tratamento da infertilidade em homens. Foi realizada a imunolocalização dos receptores de OTR em amostras de testículo e epidídimo de cães coletadas por cirurgia eletiva de orquiectomia e fixadas em formaldeído tamponado a 10%. Seguiu-se o processamento histológico de rotina, desparafinização, desidratação e recuperação de sítios antigênicos. A incubação foi feita com o anticorpo primário anti-OTR humano policlonal produzido em coelho e o polímero NOVOLINK da Nicherei®. A imunomarcação foi observada nas células mioepiteliais, células da musculatura lisa do estroma e nas células de Leydig no testículo e muito mais intensa nas células da musculatura lisa da região do epidídimo. A expressão dos OTR demonstrou-se maior no epidídimo quando comparada ao testículo. A distribuição dos OTR na imunomarcação e no western blotting reforçam os dados da literatura que sugerem ação da ocitocina no estímulo da contratilidade dos túbulos seminíferos e epidídimo, assim como seu papel na modulação dos níveis de androgênios nestes tecidos estimulando a conversão da testosterona em diidrotestosterona (DHT). Nossos resultados corroboram com a literatura permitindo que o conhecimento da distribuição das OTR no testículo e epidídimo de cães possibilite a utilização terapêutica da ocitocina e novas perspectivas para a área de reprodução e biotecnologia de cães. / The expression of oxytocin receptors (OTR) in the male reproductive tract has been studied in several species with the exception of the dogs. In men, oxytocin (OT) therapy has been successfully used to treat infertility. Therefore, the aim of the present study was to provide new perspectives for the therapeutic use of oxytocin (OT) in reproductive disorders in dogs. We performed the immunolocalization of OTR in samples of testis and epididymides of dogs collected by elective orchiectomy and fixed in 10% buffered formaldehyde. This was followed by routine histological processing, deparaffinization, dehydration and recovery of antigenic sites. Incubation was performed with the human polyclonal anti-OTR primary antibody produced in rabbits and the NOVOLINK polymer (Nichirei, Japan). Oxytocin receptor Immunostaining was observed in myoepithelial, smooth muscle, stromal, and Leydig cells in the testis and, a more intense staining was found in the smooth muscle cells in the region of the epididymis. The distribution of OTR immunostaining and expression on western blotting reinforces the hypothesis proposed by previous studies that oxytocin may stimulate seminiferous tubules and epididymis contractility, and modulate the androgen levels in these tissues, stimulating the conversion of testosterone to dihydrotestosterone (DHT).Results suggest that testicular and epididymal OTR immunolocalization in dogs may be similar to previously studied species. This could indicate that the therapeutic use of oxytocin could be an alternative in infertile dogs, providing new insights to the reproduction and biotechnology in this species.

A expressão dos receptores da ocitocina (OTR) em machos, embora estudada em algumas espécies, não foi ainda estabelecida em cães. O objetivo deste estudo foi trazer novas perspectivas para a utilização terapêutica da ocitocina (OT) em patologias reprodutivas em cães já que tem sido utilizada no tratamento da infertilidade em homens. Foi realizada a imunolocalização dos receptores de OTR em amostras de testículo e epidídimo de cães coletadas por cirurgia eletiva de orquiectomia e fixadas em formaldeído tamponado a 10%. Seguiu-se o processamento histológico de rotina, desparafinização, desidratação e recuperação de sítios antigênicos. A incubação foi feita com o anticorpo primário anti-OTR humano policlonal produzido em coelho e o polímero NOVOLINK da Nicherei®. A imunomarcação foi observada nas células mioepiteliais, células da musculatura lisa do estroma e nas células de Leydig no testículo e muito mais intensa nas células da musculatura lisa da região do epidídimo. A expressão dos OTR demonstrou-se maior no epidídimo quando comparada ao testículo. A distribuição dos OTR na imunomarcação e no western blotting reforçam os dados da literatura que sugerem ação da ocitocina no estímulo da contratilidade dos túbulos seminíferos e epidídimo, assim como seu papel na modulação dos níveis de androgênios nestes tecidos estimulando a conversão da testosterona em diidrotestosterona (DHT). Nossos resultados corroboram com a literatura permitindo que o conhecimento da distribuição das OTR no testículo e epidídimo de cães possibilite a utilização terapêutica da ocitocina e novas perspectivas para a área de reprodução e biotecnologia de cães. / The expression of oxytocin receptors (OTR) in the male reproductive tract has been studied in several species with the exception of the dogs. In men, oxytocin (OT) therapy has been successfully used to treat infertility. Therefore, the aim of the present study was to provide new perspectives for the therapeutic use of oxytocin (OT) in reproductive disorders in dogs. We performed the immunolocalization of OTR in samples of testis and epididymides of dogs collected by elective orchiectomy and fixed in 10% buffered formaldehyde. This was followed by routine histological processing, deparaffinization, dehydration and recovery of antigenic sites. Incubation was performed with the human polyclonal anti-OTR primary antibody produced in rabbits and the NOVOLINK polymer (Nichirei, Japan). Oxytocin receptor Immunostaining was observed in myoepithelial, smooth muscle, stromal, and Leydig cells in the testis and, a more intense staining was found in the smooth muscle cells in the region of the epididymis. The distribution of OTR immunostaining and expression on western blotting reinforces the hypothesis proposed by previous studies that oxytocin may stimulate seminiferous tubules and epididymis contractility, and modulate the androgen levels in these tissues, stimulating the conversion of testosterone to dihydrotestosterone (DHT).Results suggest that testicular and epididymal OTR immunolocalization in dogs may be similar to previously studied species. This could indicate that the therapeutic use of oxytocin could be an alternative in infertile dogs, providing new insights to the reproduction and biotechnology in this species.

Immunocytochemical studies have demonstrated that nerve fibers containing immunoreactive oxytocin and vasopressin project to many areas of the central nervous system, including the hippocampus and the lateral septum (Buijs, 1980; Sofroniew and Weindl, 1978). Biochemical, physiological and behavioral studies of the effects of these peptides on the CNS have indicated that they are involved in functions as diverse as the control of serotonin turnover (Auerbach and Lipton, 1982), the regulation of body temperature (Kasting et. al., 1979) and the retention of conditioned behavior (de Wied et. al., 1974; Koob and Bloom, 1982). The presence of immunoreactive vasopressin (iAVP) in the hippocampus of Wistar rats was confirmed by radioimmunoassay. The vasopressin content of the dorsal hippocampus was 30.3 ± 7.3 pg iAVP/mg soluble protein and that of the ventral hippocampus was 81.4 ± 8.3 pg iAVP/mg soluble protein, while tissue from the cerebral cortex contained no detectable vasopressin. That this immunoreactivity was due to vasopressin was confirmed by the absence of immunoreactivity in hippocampal or cortical tissue from Brattleboro rats, which are genetically unable to synthesize vasopressin. Vasopressin applied by iontophoresis was found to increase the activity of neurones in the lateral septum and in the hippocampus of the anesthetized rat. There was no obvious difference between the response of spontaneously active cells and the response of cells excited by continuous iontophoresis of glutamate or acetylcholine. Repeated application of vasopressin resulted in a decline in the magnitude of the response, but at least part of this decline was due to progressive blockage of the micropipette barrel rather than to tachyphylaxis. Oxytocin, tested only in the septum, was without effect. When applied by superfusion onto rat hippocampal slices, the NHP peptides were found to increase the activity of 88% of spontaneously active cells and to induce activity in many neurones that were not spontaneously active. Arginine vasopressin, lysine vasopressin, arginine vasotocin, and oxytocin were found to be of roughly equivalent potency, producing a dose dependent response in the range 10⁻⁹-10⁻⁶M. Most cells were tested with more than one peptide and were always found to respond either to all or to none of them. There was no decline in responsiveness when cells were subjected to repeated applications of peptide, but continuous application caused the cells to become unresponsive. Following continuous application of oxytocin, a cell failed to respond to both oxytocin and vasopressin, as would be expected if the two peptides were acting on the same receptor. The analogues ddOT, ddAVP, and Gly⁷0T were also active, but the oxytocin fragment PLG had no effect, and the vasopressin fragment DGAVP was extremely weak. The response to the peptides could be blocked by vasopressin antagonists. The peptide sensitive cells appeared to be pyramidal cells rather than interneurones, since the peptide induced activity could be inhibited for about 200-600 msec by electrical stimulation of the stratum radiatum. / Arts, Faculty of / Philosophy, Department of / Graduate

The preparation of 11 new oxytocin analogs is described. The synthesis of the protected peptides were performed using solid phase peptides synthetic methodology. The protected peptides were deprotected and cyclized using sodium in liquid ammonia followed by aqueous potassium ferricyanide treatment. The purification of each peptide was accomplished using partition chromatography followed by gel filtration. Final purity was checked using high-performance liquid chromatography. Several amino-acid derivatives were prepared and incorporated as racemates into the synthetic peptides. The synthetic diastereomeric peptides were separated and purified by high pressure liquid chromatography using aqueous trifluoroacetic acid:acetonitrile mixtures. The oxytocin analogs prepared in this dissertation were divided into 2 classes: oxytocin agonists and oxytocin antagonists. The oxytocin agonist analogs prepared are [2-cycloleucine]oxytocin and [8-cycloleucine]oxytocin. The oxytocin antagonists described in this dissertation are [Pen¹,Cle²]oxytocin, [Pen¹,Cle⁸]oxytocin, [Pen¹,L-TyrMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-PheMe²,Thr⁴,Orn⁸]oxytocin and [Pen¹,D-PheEt²,Thr⁴,Orn⁸]oxytocin. A conformational study of the synthetic peptides was also undertaken in order to determine possible solution conformations for the various peptides. Two biophysical methods were used in the conformational study of these peptides; they include nuclear magnetic resonance spectroscopy (H-1 and C-13) and circular dichroism spectroscopy. Two somewhat different solution conformations were discovered for peptides containing all L-amino acids and for peptides containing a D-amino acid residue in position 2. A possible correlation between biological potency and observed solution conformation is suggested; the proposed models may aid in the design of more potent peptide inhibitor analogs.

This study was conducted to investigate the extent to which affectionate physical contact (i.e., cuddling) affects preschoolers’ and parents’ abilities to engage in theory-of-mind reasoning. We explored the hypothesis that if affectionate contact affected theory-of-mind, then preschoolers and parents who cuddled would outperform those who did not. To test this hypothesis, we recruited 44 preschool aged children (3.8-4.6-year-olds) and their primary caregivers. We found that children who cuddled with their primary caregiver during a storybook reading task performed significantly better on theory-of-mind tasks compared to children who did not receive a cuddle. Importantly, our findings support the contention that affectionate contact affected children’s performance on theory-of-mind related tasks specifically, but not performance on executive functioning or non-mental representation tasks. A secondary goal of this study was to explore whether any effects of affectionate contact would be mediated by functional polymorphisms of the oxytocin receptor gene (OXTR). Although we were unable to obtain a sample size that was sufficient to directly test this hypothesis, we found that parents homozygous for the G allele at rs2254298 were significantly better at decoding the affective mental states of others compared to those who carried at least one A allele. Thus, our results support the hypotheses that affectionate contact promotes children’s theory-of-mind reasoning abilities and that adult’s mental state decoding skills can be predicted by allelic variations on the OXTR gene. This study offers preliminary support for the role of affectionate contact and, separately, the oxytocinergic system on tasks related to theory-of-mind reasoning. These claims are discussed with respect to possible alternative explanations for our findings, as well as future directions to directly test the extent to which such experiential and psychobiological factors can affect theory-of-mind reasoning. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-08-15 14:13:54.174

No description available.

Purpose: The present study investigates the potential anti-inflammatory effects of in vivo oxytocin (OT) infusion on adipose tissue inflammation in the Watanabe Heritable Hyperlipidimic Rabbits (WHHL). Methods: Twenty-eight 3-month-old WHHL were surgically implanted with osmotic minipumps containing OT (n = 14, infusion rate 250 ng/kg/hr) or vehicle (n = 14). Blood samples were taken at baseline, midpoint, and endpoint for lipids and C-reactive protein (CRP). After 16 weeks, animals were sacrificed and samples of adipose tissue (epididiymal, retroperitoneal, mesenteric, pericardial, and subcutanous) were collected and analyzed for pro-inflammatory cytokine (IL-6, TNF-α, and MCP-1) and anti- inflammatory adipokine (adiponectin and IL-10) expression levels by Real Time- Polymerase Chain Reaction. Adipose tissue was also immunohistologically analyzed for macrophage infiltration. Aortas were dissected, formalin-fixed, and stained with oil-red O for en face quantification of lesion area. Student’s t-tests were used to compare group means for all measures. Results: Endpoint OT levels were significantly different (p < .05) between the control ( M = 11.28 pg/ml, SEM = 2.5) and treatment group (M = 132.35 pg/ml, SEM = 8.5). Plasma lipids were not altered by OT infusion. OT-treatment significantly decreased plasma CRP, a marker of systemic inflammation, at midpoint and endpoint compared to controls (p = 0.05). OT-treated animals displayed significantly less atherosclerosis in the thoracic aorta (p < 0.05); a finding similar to our previously published study in a mouse model of atherosclerosis. In some fat depots, there was a trend suggesting adiponectin gene expression increased in the OT-treatment group. There were no significant differences or trends regarding macrophage infiltration in adipose tissue. Conclusions: Oxytocin infusion attenuated thoracic aortic atherosclerosis, plasma CRP, and may affect inflammatory cytokine expression in adipose tissue in the WHHL model.

No description available.