Buserelin Treatment Research Articles

Buserelin Promotes the Differentiation and Function of Macrophage-Colony-Stimulating Factor-Producing T Helper Cells

ABSTRACT Background Buserelin has been used to treat central precocious puberty (CPP). However, it could potentially result in immune dysregulation to undermine patients’ health. Therefore, it is necessary to elucidate the effects of buserelin on immune cells. Here we explored buserelin-induced impacts on the differentiation and function of macrophage-colony-stimulating factor-producing T helper (ThGM) cells to uncover the immunoregulatory role of buserelin. Methods Rat CPP was induced by danazol injection followed by buserelin treatment. The frequencies of ThGM cells in the spleen and lymph nodes were evaluated by flow cytometry. ThGM cell generation and function were analyzed in cell culture assays. Cell signaling was measured by Immunoblotting. Results Buserelin increased the frequencies of splenic and lymph node ThGM cells. Buserelin promoted the in vitro differentiation and proliferation of ThGM cells. Buserelin-treated ThGM cells showed stronger supportive effects on other effector T helper cells. Buserelin induced the activation of the nuclear factor of activated T cells and extracellular signal-regulated kinase 1/2 in ThGM cells. Conclusion Buserelin enhances the differentiation and function of pro-inflammatory ThGM cells, thus increasing the risk of autoimmune or inflammatory disorders. Therefore, it is necessary to monitor ThGM cells in buserelin-treated children to prevent latent immune dysregulation.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4μg/day for 4weeks followed by 8μg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Endocrine changes induced by GnRH immunization and re-stimulation of testicular function with a GnRH agonist in stallions

Resumption of testicular function after GnRH immunization varies among animals and some stallions regain fertility only after a prolonged time (Janett et al., 2009, Anim Reprod Sci 115:88e102). This study evaluated endocrine effects of GnRH immunization and subsequent re-stimulation with a GnRH agonist. We hypothesized that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. Shetland stallions were assigned to a GnRH vaccination group (Improvac; 400 µg/animal in weeks 0 and 4; n=6) or a saline control group (n=6). Blood was collected weekly for hormone analyses. Each vaccinated stallion was hemi-castrated together with an age-matched control when testosterone concentration was <0.3 ng/ml for two consecutive weeks (C1). Treatment of vaccinated stallions with the GnRH agonist buserelin (4 µg/day for 4 weeks and 8 µg/day for 6 weeks) started three weeks thereafter. Irrespective of treatment, the remaining testicle was removed when testosterone concentration increased to >0.5 ng/ml for two consecutive weeks in vaccinated stallions (C2; 9-10 weeks after start of buserelin treatment). Testicular tissues were stored at – 80°C and fixed in formalin for RT-qPCR and histology. Statistical comparisons were made by repeated measures ANOVA with group as between and time as within subject factor. Data on semen characteristics in these stallions have been published recently (Gautier et al., 2022, Theriogenology 194:27-34). Plasma testosteroneconcentration decreased after GnRH-vaccination and returned to control stallion concentrations during buserelin treatment (time p<0.001, time x group p<0.001, group p=0.001). Serum LH was undetectable after the second vaccination, increased during buserelin treatment but remained lower than in control stallions (time p=0.003, time x group p<0.001). Serum FSH concentration decreased after vaccination and increased with buserelin treatment in vaccinated but even more in control stallions after both hemi-castrations (time p<0.001, time x group p<0.001, group p=0.008). Relative mRNA abundance for LH receptor (LH-R) and aromatase (CYP19A1) were lower in testicular tissue of vaccinated than control stallions at C1 (p<0.001). At C2, LH-R mRNA abundance no longer differed between groups, whereas CYP19A1 mRNA remained lower in vaccinated than in control stallions (p<0.001). Relative FSH receptor (FSH-R) mRNA abundance was higher in vaccinated than in control stallions at C1 (p<0.001) and similar in both groups at C2. Immunolabelling for LH-R was lower in testes of GnRH-vaccinated compared to control stallions at C1 and C2 (p=0.007), whereas FSH-R protein expression at no time differed between groups. Aromatase immunostaining was reduced at C1 in vaccinated stallions and increased until C2 (time p=0.035, time x group p<0.0031, group p<0.001). In conclusion, endocrine testicular function can be advanced in GnRH-vaccinated stallions by daily low dose buserelin treatment.

Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 μg/day for 4 weeks followed by 8 μg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function.

Effects of buserelin administration on testicular blood flow and plasma concentrations of testosterone and estradiol-17β in rams

This research aimed to examine for the first time the impact of single dose administration of gonadotropin-releasing hormone (GnRH) analog buserelin acetate on the testicular blood flow measurements (peak systolic velocity [PSV], end-diastolic systolic velocity [EDV], resistive index [RI], and pulsatility index [PI]) and the plasma steroids (testosterone and estradiol-17β) concentrations in rams. For this purpose, twelve adult Ossimi rams were randomly assigned into the buserelin group (n = 8) and were injected intravenously (iv) with buserelin acetate (0.008 mg/ram), whereas the remaining rams (n = 4) were injected with normal saline iv and served as a control group. Blood sampling and testicular pulsed-wave Doppler scanning were conducted immediately before (0) and 1, 3, 6, 24, 48, 72, 120, and 168 h after treatment. The control group did not reveal any substantial changes (P > 0.05) in the examined parameters, except for the EDV (P < 0.05). In the buserelin-treated group, a marked reduction in RI and PI values (P < 0.05) occurred 1 to 3 h after administration of buserelin. Besides, there was a significant increase in testosterone plasma concentrations following buserelin treatment. In conclusion, the administration of buserelin triggered a series of substantial changes in the testicular blood perfusion and steroidogenesis that could have a positive effect on testicular function in rams.

Buserelin Inhibits the Immunosuppressive Activity of Regulatory T Cells through the Protein Kinase A Signaling in a Central Precocious Puberty Model

ABSTRACT Background Gonadotropin-releasing hormone analogs (GnRHas) are used for treating central precocious puberty (CPP). However, their roles in the regulation of immune cells especially regulatory T cells (Tregs) remains elusive. Therefore, we characterized buserelin-induced phenotypical and functional changes of Tregs. Methods A rat CPP model was established followed by administration of buserelin acetate. Flow cytometry was used to evaluate the expression of functional molecules in splenic Tregs. The suppressive activity of Tregs was determined by the suppression assay. GnRHR expression in Tregs was assessed by flow cytometry analysis and Immunoblotting. Normal Tregs were then stimulated and treated with buserelin acetate in vitro. After that, Foxp3 expression, Treg proliferation, and cytokine production were analyzed by flow cytometry. Intracellular signaling was evaluated by Immunoblotting, and Treg function was determined by the suppression assay. Results After in vivo buserelin treatment, the frequency of splenic Tregs was decreased, with the reduction in the expression of Foxp3, IL-10, and TGF-β. The suppressive activity of Tregs was weakened. Buserelin down-regulated Foxp3 expression while promoting the expression of RORγt and IL-17 in Tregs through activating the protein kinase A (PKA) pathway in vitro. The PKA inhibitor H-89 abolished the effect of buserelin and enhanced Treg function. Conclusion Buserelin impaired the immunosuppressive activity of Tregs through the PKA signal pathway. Buserelin-induced activation of PKA signaling down-regulated Foxp3 expression while promoting RORγt expression in Tregs, and subsequently weakened Treg function. Our study indicates the necessity of monitoring Treg activity in CPP patients to avoid potential autoimmunity or inflammation.

Role of GnRH in the ontogeny and regulation of the fetal hypothalamo-pituitary-gonadal axis in sheep

Adult reproductive ability is to a large extent determined by the appropriate development of the reproductive axis during fetal life. Studies have investigated the role of the fetal hypothalamus in the ontogeny and regulation of pituitary gonadal function during fetal development in sheep. Using immunocytochemistry, we examined the ontogeny of gonadotroph development in the pituitary of female sheep fetuses. At day 70 of gestation (term = 145 days), only immunopositive LH beta cells were present. The number and intensity of staining of these LH beta cells had increased by day 100 but had declined again by day 130. Immunopositive alpha-subunit and FSH beta cells appeared at day 100 of gestation and had further increased in number and staining intensity by day 130 of gestation. Treatment of fetuses with the GnRH agonist buserelin resulted in desensitization of the fetal pituitary gonadotrophs, inhibition of pituitary LH beta and FSH beta mRNA expression and a reduction in the number of immunopositive gonadotrophin-containing cells. Pulsatile GnRH treatment resulted in pituitary-gonadal activation and an increase in LH, FSH and testosterone secretion in males. Thus, the synthesis and secretion of the gonadotrophins during fetal development is critically dependent on the secretion of GnRH from the fetal hypothalamus. Inhibition of fetal gonadotrophins by buserelin treatment from day 70 of gestation resulted in a 40% reduction in the size of the fetal testis at birth, and there were no effects on the fetal ovaries. This reduction in testis size was due to a 45% reduction in the number of Sertoli cells. However, when buserelin was given between day 70 and day 110 of gestation, there were no effects on testis size or morphological development of the testis, suggesting that gonadotrophins regulate testicular development during a 'critical window' late in gestation. Taken together, these studies provide convincing evidence that GnRH plays a central role in the ontogeny and regulation of pituitary-gonadal function during fetal life.

Identification of beta-nerve growth factor in dromedary camel seminal plasma and its role in induction of ovulation in females

The main objective of this study was to demonstrate the effect of seminal plasma ovulation inducing factor (OIF) on ovulation in female camels. Seminal plasma was fractionated using gel filtration chromatography, and two protein peaks were obtained and analysed by western blotting. The effect of the bioactive protein fraction (OIF/β-NGF) was tested by intramuscular injection (1 ml) in synchronized females with the following treatments: PBS (negative control group, n = 3); 20 µg Buserelin (GnRH analogue, positive control group; n = 3); and purified OIF with doses of 1 and 2 mg (n = 5, each treatment). Blood samples were collected every two days from day 0 until 14 days post-treatment, and the progesterone concentration was assessed. The obtained results showed that the OIF is highly present in the seminal plasma of dromedary camels as a protein with a molecular mass of approximately 14 kDa. It was detected as a beta-Nerve Growth Factor, named Cam-β-NGF. The effect of this molecule on ovulation was clearly demonstrated by the significant increase of the plasma progesterone concentration in the treated female groups (1 and 2 mg of Cam-β-NGF) as observed in Buserelin group. In summary, intramuscular injection of β-NGF isolated from dromedary camel seminal plasma induces ovulation in females with similar rate to Buserelin treatment.

Long‑term follow‑up of buserelin‑induced enteric neuropathy in rats.

A few patients have been shown to develop severe abdominal pain and gastrointestinal dysmotility during treatment with gonadotropin-releasing hormone (GnRH) analogs. A rat model of enteric neuropathy has been developed by administration of the GnRH analog buserelin to rats. Loss of enteric neurons and ganglioneuritis throughout the gastrointestinal tract has been described, without other histopathological changes. The aim of the present study was to investigate the long-term effects of this rat model on body weight, and on morphology and inflammatory changes in the gastrointestinal tract. Rats were administered subcutaneous injections of buserelin or saline once daily for 5 days and allowed to recover for 3 weeks. This regimen was repeated four times. The rats were weighed weekly and were sacrificed 16 weeks after the fourth treatment. The bowel wall was measured by morphometry, and the presence of enteric neurons, mast cells, eosinophils and T-lymphocytes was evaluated. Buserelin-treated rats were shown to have a lower body weight at sacrifice, as compared with the controls (P<0.05). Compared with controls, buserelin treatment caused loss of myenteric neurons in the ileum and colon (P<0.01), a thinner circular muscle layer in ileum (P<0.05) and longitudinal muscle layer in colon (P<0.05), increased number of eosinophils in the submucosa of the ileum (P<0.05), and an increased number of T-lymphocytes in the submucosa and circular muscle layer of the fundus (P<0.01 and P<0.05, respectively) and circular muscle layer of the colon (P<0.05). Mast cells were equally distributed in the two groups. Thus, long-term follow-up of buserelin-induced enteric neuropathy reveals reduced body weight, loss of myenteric neurons, thinning of muscle layers, and increased numbers of eosinophils and T-lymphocytes in the gastrointestinal tract.

Ovulation and spawning induction in Caspian kutum Rutilus frisii kutum by administration of GnRHa and catecholaminergic pharmaceutical compounds with Ovaprim

The acceleration of final oocyte maturation and ovulation is recognized as a principal process in Caspian kutum Rutilus frisii kutum, a commercial and valuable species in Caspian Sea. The aim of this study was to evaluate the effects of catecholaminergic pharmaceutics which include Salbutamol (β2-adrenergic receptor agonist), Metoprolol (β1-adrenergic receptor antagonist), Clozapine and Olanzapine (the third generation of D4 and D2 dopamine receptor antagonists) which can be more efficient than the first and second generations, in combination with Buserelin ([D-Ser(tBu)6,Pro9-NEt]-GnRHa) and Ovaprim (D-Ala6,Pro9-Net)-sGnRH+Domperidone on ovulation and spawning induction in Caspian kutum. One hundred eight adult females were injected once with Buserelin Acetate (BUS) 5 μg kg−1 BW, Ovaprim 0.5 mL kg−1 BW, Salbutamol Sulphate (SLB) 4 mg kg−1 BW, Olanzapine (OLZ) 5 mg kg−1 BW, Clozapine (CZ) 12 mg kg−1 BW and Metoprolol Tartrate (MTP) 5 mg kg−1 BW, being divided into 12 groups: group 1, intact (negative control); group 2, Ovaprim (positive control); group 3, OLZ+SLB; group 4, OLZ+MTP; group 5, OLZ+BUS; group 6, CZ+SLB; group 7, CZ+MTP; group 8, CZ+BUS; group 9, OLZ+BUS+SLB; group 10, OLZ+BUS+MTP; group 11, CZ+BUS+SLB and group 12, CZ+BUS+MTP (N = 9). The results showed that the highest mean value in ovulation success, ovulation index, fertilization success, relative fecundity and the number of eggs belonged to Ovaprim treatment. On the other hand, spawning was successful in OLZ+BUS+SLB and CZ+BUS treatments (P < 0.05), whereas it was lower in CZ+MTP, CZ+BUS+MTP and OLZ+BUS than the other treatments. Therefore, it could be concluded that Clozapine and Olanzapine potentiated the effect of Buserelin treatment in ovulation and spawning induction, while Metoprolol blocked the stimulatory effects of GnRHa, Clozapine and Olanzapine. Salbutamol also can amplify stimulatory effect of all mentioned pharmaceutics.

Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum.

BackgroundThe gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior.ResultsSixty rats were given buserelin (20 μg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior.ConclusionsAlthough buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1800-x) contains supplementary material, which is available to authorized users.

Effect of gonadorelin, lecirelin, and buserelin on LH surge, ovulation, and progesterone in cattle

Analogs of gonadoliberin (GnRH) are widely used in cattle to synchronize estrus and to induce ovulation, as well as for the treatment of ovarian cysts. The aim of this study was to compare the plasma profiles of LH and progesterone and the follicular dynamics in response to the administration of gonadorelin, lecirelin, or buserelin at the dose recommended to induce ovulation. In addition, the biological response to a half dose of lecirelin was assessed. Twelve healthy Holstein female cows were divided into four sequence groups, according to a Latin square design and received the four treatments during the four periods of the study. Before each period, the estrous cycle was synchronized, and on Day 6 or 7 of the ensuing cycle, the time at which it was most likely to have a dominant follicle, 100 μg of gonadorelin, 25 μg of lecirelin, 50 μg of lecirelin, or 10 μg of buserelin was administered to the cows. Blood samples were regularly collected for up to 4 days after the GnRH administrations. The plasma LH response was evaluated for up to 6 hours after administration, and the plasma progesterone response and ovarian follicular dynamics were evaluated for up to 4 days. There was a significantly lower LH release after gonadorelin treatment compared to lecirelin at the doses of 25 or 50 μg and the buserelin treatment. The mean maximal LH concentration after gonadorelin treatment was 2.5 lower than after lecirelin or buserelin treatment and was reached 1 hour earlier. Four days after the GnRH administration (i.e., at Days 10–11 of the estrous cycle), the overall mean increase in plasma progesterone concentration was 70% and did not differ between the treatment groups. The percentage of disappearance of the dominant follicle (84.8% of ovulation and 4.3% of luteinization) after GnRH treatment was high (73%, 82%, 100%, and 100%, for gonadorelin, lecirelin at the doses of 25 and 50 μg, and buserelin, respectively) and did not differ between the GnRH treatments. The follicle disappearance was followed by the emergence of a synchronous follicle wave within 2 days in almost all the heifers. Altogether, our data show that the three GnRH analogs, at the doses indicated for the induction of ovulation or at a half dose for lecirelin, are almost equally effective to induce the disappearance of the dominant follicle at Day 6 to 7 of the estrous cycle.

Structural and functional consequences of buserelin-induced enteric neuropathy in rat.

BackgroundWomen treated with gonadotropin-releasing hormone (GnRH) analogs may develop enteric neuropathy and dysmotility. Administration of a GnRH analog to rats leads to similar degenerative neuropathy and ganglioneuritis. The aim of this study on rat was to evaluate the early GnRH-induced enteric neuropathy in terms of distribution of neuronal subpopulations and gastrointestinal (GI) function.MethodsForty rats were given the GnRH analog buserelin (20 μg, 1 mg/ml) or saline subcutaneously, once daily for 5 days, followed by 3 weeks of recovery, representing one treatment session. Two weeks after the fourth treatment session, the animals were tested for GI transit time and galactose absorption, and fecal weight and fat content was analyzed. After sacrifice, enteric neuronal subpopulations were analyzed. Blood samples were analyzed for zonulin and antibodies against GnRH and luteinizing hormone, and their receptors.ResultsBuserelin treatment transiently increased the body weight after 5 and 9 weeks (p < 0.001). Increased estradiol in plasma and thickened uterine muscle layers indicate high estrogen activity. The numbers of both submucous and myenteric neurons were reduced by 27%–61% in ileum and colon. The relative numbers of neurons containing calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-related transcript (CART), galanin, gastrin-releasing peptide (GRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP) or vesicular acetylcholine transporter (VAchT), and their nerve fiber density, were unchanged after buserelin treatment, but the relative number of submucous neurons containing somatostatin tended to be increased (p = 0.062). The feces weight decreased in buserelin-treated rats (p < 0.01), whereas feces fat content increased (p < 0.05), compared to control rats. Total GI transit time, galactose absorption, zonulin levels in plasma, and antibody titers in serum were unaffected by buserelin treatment.ConclusionsA marked enteric neuronal loss with modest effects on GI function is found after buserelin treatment. Increased feces fat content is suggested an early sign of dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-014-0209-7) contains supplementary material, which is available to authorized users.

Comparison between the effect of hCG and GnRH analogue (buserelin) treatment post laparoscopic insemination on reproductive performance of ewes

Comparison between the effect of hCG and GnRH analogue (buserelin) treatment post laparoscopic insemination on reproductive performance of ewes

The effect of short-term treatments of a gonadotropin- releasing hormone analog (buserelin) on sciatic nerve regeneration

Summary Gonadotropin-releasing hormone (GnRH) has neuromodulatory roles in central and peripheral nervous systems. The purpose of this study is to evaluate the effect of GnRH analog (buserelin) on peripheral nerve regeneration. Forty adult male rats were divided into buserelin-treated, normal saline, sham surgery, and castrated + buserelin groups. The left sciatic nerve was crushed by a fine forceps and all animals were evaluated by sciatic functional index (SFI), electrophysiology, histology and immunohistochemistry testing. On post operation days 21 and 28, the difference between buserelin and normal saline groups was statistically significant (P 0.05). At the 28th day after operation, the diameters (µm) of the regenerated myelinated fibers of the buserelin group were significantly greater than those of the normal saline group (P<0.05). Although nerve conduction velocity (NCV) of the buserelin group was faster than the normal saline group, the difference was not statistically significant. The present study suggests that buserelin treatment might accelerate peripheral nerve regeneration.

Different circulating progesterone concentrations during synchronization of ovulation protocol did not affect ovarian follicular and pregnancy responses in seasonal anestrous buffalo cows

Three experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during synchronization of ovulation protocol for timed artificial insemination of seasonal anestrous buffalo cows. In the first trial, ovariectomized cows were randomly allocated into one of three groups: using new P4 devices (G-New; n = 8), using devices previously used for 9 days (G-Used1x; n = 8), and using devices previously used for 18 days (G-Used2x; n = 8). The P4 device was maintained for 9 days, and the circulating P4 concentration was measured daily. The circulating P4 concentrations during the P4 device treatment were the lowest for G-Used2x (1.10 ± 0.04 ng/mL), intermediate for G-Used1x (1.52 ± 0.05 ng/mL), and the highest for G-New (2.47 ± 0.07 ng/mL; P = 0.001). In the second trial, 31 anestrous cows had their ovarian follicular dynamics evaluated after receiving the treatments described previously (G-New [n = 10], G-Used1x [n = 11], and G-Used2x [n = 10]). At insertion of the P4 device, cows were administered 2.0 mg of estradiol benzoate. Nine days later, the P4 device was removed and cows were administered 0.53 mg of cloprostenol sodium plus 400 IU of eCG. Forty-eight hours after P4 device removal, 10 μg of buserelin acetate was administered. There were no differences among the groups (G-New vs. G-Used1x vs. G-Used2x) in diameter of the largest follicle at P4 device removal (9.0 ± 0.8 vs. 10.1 ± 0.9 vs. 8.6 ± 0.8 mm; P = 0.35), in interval from P4 device removal to ovulation (77.1 ± 4.5 vs. 76.5 ± 4.7 vs. 74.0 ± 4.4 hours; P = 0.31), or in ovulation rate (80.0% vs. 81.8% vs. 60.0%; P = 0.51). In experiment 3, 350 anestrous cows were randomly assigned into one of the three treatments described previously (G-New, n = 111; G-Used1x, n = 121; G-Used2x, n = 118) and received a timed artificial insemination for 16 hours after buserelin treatment. The 30-day pregnancy rates did not differ among groups (55.9% vs. 55.4% vs. 48.3%; P = 0.39). Thus, the low circulating P4 concentrations released from a used P4 device efficiently control the ovarian follicular growth and had no detrimental effect on the pregnancy rates of the seasonal anestrous buffalo cows.

Autoantibodies and gastrointestinal symptoms in infertile women in relation to in vitro fertilization

BackgroundPrior reports suggest a link between gonadotropin-releasing hormone (GnRH) and gastrointestinal function. The aim of the study was to prospectively investigate women subjected to in vitro fertilization (IVF) using the GnRH analog buserelin, taking into account gastrointestinal symptoms and antibody development against buserelin, GnRH, luteinizing hormone (LH), and their receptors.MethodsGastrointestinal symptoms were registered by the Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS) before and after IVF treatment, and five years later. Health-related quality of life was evaluated by the 36-item Short-Form questionnaire (SF-36). ELISA was used for antibody analyses before and after treatment. Data were compared with women from the general population.ResultsIn total, 124 patients were investigated before and after IVF, and 62 were re-evaluated after five years. Buserelin treatment led to significant impairment of constipation (p = 0.004), nausea and vomiting (p = 0.035), psychological well-being (p = 0.000), and the intestinal symptoms’ influence on daily life (p = 0.027). At 5-year follow-up, abdominal pain was worsened (p = 0.041), but psychological well-being was improved (p = 0.036), compared to prior treatment, and 15% had an observable deterioration in gastrointestinal symptoms. None developed severe dysmotility. Patients had higher prevalence of IgG antibodies against LH (p = 0.001) and its receptor (p = 0.016), and IgM antibodies against the GnRH receptor (p = 0.001) prior treatment compared with controls, but no antibody development was observed after IVF.ConclusionPatients experience gastrointestinal symptoms during buserelin treatment, and abdominal pain is still increased after five years, but buserelin does not increase antibody formation against GnRH, LH or their receptors.

Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract

Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1-4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.

160 CONCEPTION RATES FOLLOWING TREATMENT OF LACTATING HOLSTEIN RECIPIENTS WITH A GONADOTROPIN RELEASING HORMONE (GnRH) ANALOGUE AT THE TIME OF TRANSFER OF OVUM PICK-UP/IVF-DERIVED EMBRYOS

The use of lactating dairy cows as ovum pick-up (OPU)/IVF-derived embryo recipients in large-scale embryo transfer programs may be limited by the effects of low progesterone concentrations on the uterine environment and subsequent embryo development. It was hypothesized that the injection of a GnRH analogue on the day of embryo transfer would induce ovulation and formation of an accessory corpus luteum, increasing progesterone concentrations and, as a result, conception rates. An experiment was designed to evaluate the effects of two different GnRH analogues injected IM on the day of embryo transfer on conception rates of lactating Holstein recipients. Oestrus was not synchronized and was detected continuously by multiple observers during the day and in the evening until midnight. Recipient days in milk varied from 45 to 150, daily milk production ranged from 20 to 45 kg, body condition scores and age ranged from 2.5 to 4.5 and 2.5 to 5.5 years, respectively. Herd pregnancy rates after the first artificial insemination ranged from 15% to 70%, considering the period the experiment was conducted. Embryos were produced by standard in vitro techniques following OPU on Holstein donors. Lactating Holstein recipients (n = 224) were allocated randomly to one of three treatment groups 7 days after detected oestrus: Control (n = 77) = vehicle; Buserelin (Sincroforte®, Ouro Fino, Sao Paulo, Brazil; n = 75) = 10 µg; and Deslorelin (Sincrorrelin®, Ouro Fino; n = 72) = 750 µg. Embryos were transferred nonsurgically 6 to 8 days after observed oestrus. Pregnancy was detected by ultrasonography between 62 and 72 days after embryo transfer. Percent pregnant data were analyzed as a binomial distribution under the GENMOD procedure of SAS (SAS Institute Inc., Cary, NC, USA). Conception rates were similar (P = 0.13) between Control (28%, 22/77), Buserelin (24%, 18/75), and Deslorelin (38%, 28/72) treatment groups. These data demonstrate that conception rates from in vitro produced embryos transferred to lactating dairy cows are not improved by treatment with a GnRH analogue on the day of transfer. Although not critically tested, results indicate that adequate conception rates may be obtained in commercial dairy operations from OPU/IVF-derived embryos, adding an important management tool for reproduction and animal breeding enhancement.

928P - Lhrh Agonist-Induced Suppression of the Androgen Synthesis Pathway in Prostate Cancer

Background: Standard first-line therapy for advanced prostate cancer (PC) includes the use of a luteinizing hormone releasing hormone (LHRH) agonist. While the primary site of action for these agents is the pituitary-hypothalamic-gonadal axis, receptors for LHRH are also present in the membrane of PC cells and mediate a direct anti-tumor effect. The mechanism of this effect has been largely unexplored and may harbor a context of vulnerability. Previously, we have demonstrated that luteinizing hormone (LH) increases de novo steroidogenesis in PC cells and silencing of the LH receptor leads to downregulation of steroidogenesis. Here, we examined the impact of LHRH agonists on this process at the cellular level. Methods: LNCaP PC cells were treated with the LHRH agonist buserelin for 8 hours in the presence or absence of the LHRH antagonist antide. Gene expression of LH and androgen synthesis enzymes was quantified by real-time polymerase chain reaction and protein expression was measured with Western blot. Cell proliferation was assessed in these cells using MTS assays. Results: The LHRH agonist buserelin significantly suppressed the gene expression of LH and the androgen synthesis enzymes AKR1C1, AKR1C2, AKR1C3, CYP11A1 and RDH5 (p<0.05). Protein expression of LH, AKR1C1, AKR1C3 and CYP17A1 was also inhibited by buserelin treatment (p<0.05). Buserelin also suppressed LNCaP cell proliferation in a dose-dependent manner (p<0.05). The suppressive effects of buserelin on gene expression, protein expression and cell proliferation were mitigated by the LHRH antagonist antide. Conclusion: These data implicate the LHRH agonist buserelin in the direct inhibition of de novo tumoral steroidogenesis, which may be mediated by suppression of LH. The LH pathway warrants further investigation as a therapeutic target. The treatment of advanced prostate cancer relies on androgen deprivation, often with an LHRH agonist. These agents induce the downregulation of LHRH receptors in pituitary gonadotrophs that ultimately leads to a decrease in gonadal testosterone synthesis. LHRH receptors are also highly expressed on prostate cancer cells. Unlike their pituitary counterparts, prolonged exposure to LHRH agonists does not lead to their downregulation. The role of these tumoral LHRH receptors remains unclear. Our data suggest that the LH pathway may play a role in tumoral steroidogenesis. Here, we explore the impact of LHRH agonist therapy on LH expression and tumoral steroidogenesis.