Gamma-ray sources imaging and test-beam results with MACACO III Compton camera.

Hadron therapy is a radiotherapy modality which offers a precise energy deposition to the tumors and a dose reduction to healthy tissue as compared to conventional methods. However, methods for real-time monitoring are required to ensure that the radiation dose is deposited on the target. The IRIS group of IFIC-Valencia developed a Compton camera prototype for this purpose, intending to image the Prompt Gammas emitted by the tissue during irradiation. The system detectors are composed of Lanthanum (III) bromide scintillator crystals coupled to silicon photomultipliers. After an initial characterization in the laboratory, in order to assess the system capabilities for future experiments in proton therapy centers, different tests were carried out in two facilities: PARTREC (Groningen, The Netherlands) and the CNA cyclotron (Sevilla, Spain). Characterization studies performed at PARTREC indicated that the detectors linearity was improved with respect to the previous version and an energy resolution of 5.2 % FWHM at 511 keV was achieved. Moreover, the imaging capabilities of the system were evaluated with a line source of 68Ge and a point-like source of 241Am-9Be. Images at 4.439 MeV were obtained from irradiation of a graphite target with an 18 MeV proton beam at CNA, to perform a study of the system potential to detect shifts at different intensities. In this sense, the system was able to distinguish 1 mm variations in the target position at different beam current intensities for measurement times of 1800 and 600 s.

Influence of the background in Compton camera images for proton therapy treatment monitoring.

Objective. Background events are one of the most relevant contributions to image degradation in Compton camera imaging for hadron therapy treatment monitoring. A study of the background and its contribution to image degradation is important to define future strategies to reduce the background in the system.Approach. In this simulation study, the percentage of different kinds of events and their contribution to the reconstructed image in a two-layer Compton camera have been evaluated. To this end, GATE v8.2 simulations of a proton beam impinging on a PMMA phantom have been carried out, for different proton beam energies and at different beam intensities.Main results. For a simulated Compton camera made of Lanthanum (III) Bromide monolithic crystals, coincidences caused by neutrons arriving from the phantom are the most common type of background produced by secondary radiations in the Compton camera, causing between 13% and 33% of the detected coincidences, depending on the beam energy. Results also show that random coincidences are a significant cause of image degradation at high beam intensities, and their influence in the reconstructed images is studied for values of the time coincidence windows from 500 ps to 100 ns.Significance. Results indicate the timing capabilities required to retrieve the fall-off position with good precision. Still, the noise observed in the image when no randoms are considered make us consider further background rejection methods.

Joint image reconstruction algorithm in Compton cameras.

Objective.To demonstrate the benefits of using an joint image reconstruction algorithm based on the List Mode Maximum Likelihood Expectation Maximization that combines events measured in different channels of information of a Compton camera.Approach.Both simulations and experimental data are employed to show the algorithm performance.Main results.The obtained joint images present improved image quality and yield better estimates of displacements of high-energy gamma-ray emitting sources. The algorithm also provides images that are more stable than any individual channel against the noisy convergence that characterizes Maximum Likelihood based algorithms.Significance.The joint reconstruction algorithm can improve the quality and robustness of Compton camera images. It also has high versatility, as it can be easily adapted to any Compton camera geometry. It is thus expected to represent an important step in the optimization of Compton camera imaging.

MACACO II test-beam with high energy photons.

The IRIS group at IFIC Valencia is developing a three-layer Compton camera for treatment monitoring in proton therapy. The system is composed of three detector planes, each made of a [Formula: see text] monolithic crystal coupled to a SiPM array. Having obtained successful results with the first prototype (MACACO) that demonstrated the feasibility of the proposed technology, a second prototype (MACACO II) with improved performance has been developed, and is the subject of this work. The new system has an enhanced detector energy resolution which translates into a higher spatial resolution of the telescope. The image reconstruction method has also been improved with an accurate model of the sensitivity matrix. The device has been tested with high energy photons at the National Accelerator Centre (CNA, Seville). The tests involved a proton beam of 18 MeV impinging on a graphite target, to produce 4.4 MeV photons. Data were taken at different system positions of the telescope with the first detector at 65 and 160 mm from the target, and at different beam intensities. The measurements allowed successful reconstruction of the photon emission distribution at two target positions separated by 5 mm in different telescope configurations. This result was obtained both with data recorded in the first and second telescope planes (two interaction events) and, for the first time in beam experiments, with data recorded in the three planes (three interaction events).

Image reconstruction for a multi-layer Compton telescope: an analytical model for three interaction events.

Compton Cameras are electronically collimated photon imagers suitable for sub-MeV to few MeV gamma-ray detection. Such features are desirable to enable in vivo range verification in hadron therapy, through the detection of secondary Prompt Gammas. A major concern with this technique is the poor image quality obtained when the incoming gamma-ray energy is unknown. Compton Cameras with more than two detector planes (multi-layer Compton Cameras) have been proposed as a solution, given that these devices incorporate more signal sequences of interactions to the conventional two interaction events. In particular, three interaction events convey more spectral information as they allow inferring directly the incident gamma-ray energy. A three-layer Compton Telescope based on continuous Lanthanum (III) Bromide crystals coupled to Silicon Photomultipliers is being developed at the IRIS group of IFIC-Valencia. In a previous work we proposed a spectral reconstruction algorithm for two interaction events based on an analytical model for the formation of the signal. To fully exploit the capabilities of our prototype, we present here an extension of the model for three interaction events. Analytical expressions of the sensitivity and the System Matrix are derived and validated against Monte Carlo simulations. Implemented in a List Mode Maximum Likelihood Expectation Maximization algorithm, the proposed model allows us to obtain four-dimensional (energy and position) images by using exclusively three interaction events. We are able to recover the correct spectrum and spatial distribution of gamma-ray sources when ideal data are employed. However, the uncertainties associated to experimental measurements result in a degradation when real data from complex structures are employed. Incorrect estimation of the incident gamma-ray interaction positions, and missing deposited energy associated with escaping secondaries, have been identified as the causes of such degradation by means of a detailed Monte Carlo study. As expected, our current experimental resolution and efficiency to three interaction events prevents us from correctly recovering complex structures of radioactive sources. However, given the better spectral information conveyed by three interaction events, we expect an improvement of the image quality of conventional Compton imaging when including such events. In this regard, future development includes the incorporation of the model assessed in this work to the two interaction events model in order to allow using simultaneously two and three interaction events in the image reconstruction.

Comparison of different regimens of estradiol benzoate treatments followed by long-acting progesterone to prepare noncycling mares as embryo recipients.

The present study evaluated the influence of different regimens of estradiol benzoate (EB) treatments followed by a single dose of long-acting progesterone (LA P4) on plasma estrogen and P4 concentrations in noncyclic mares prepared as embryo recipients. Twenty-one anestrous mares were distributed into three groups (n = 7 mares per group), according to the EB dose received (single dose of 2.5 mg, total of 5 mg in decreasing doses, and total of 10 mg in decreasing doses), which was followed by a single administration of 1500 mg of LA P4 in all groups. Mares were reevaluated during the ovulatory phase and seven of them became part of the cyclic nontreated control group. Ultrasonography was performed to monitor endometrial edema, and blood samples were collected to measure estradiol (E2), estrogen conjugate (EC), and P4 by RIA. Maximum uterine edema was achieved 24 hours after administration of EB in all treated groups. Maximum E2 concentrations were observed 24 hours after the first EB injection in treated groups and there were no differences (P > 0.05) among treatments. Maximum EC concentration was observed 24 hours after the single EB injection in the 2.5-mg group, whereas in the 5- and 10-mg groups EC peaks were observed 48 hours after the first EB administration. Maximum P4 concentrations were detected 24 hours after LA P4 injection, although higher P4 concentrations were observed in the group treated with 2.5 mg of EB than in that treated with 10 mg of EB (P < 0.05). Because P4 concentrations were reduced after administration of high doses of EB, we also measured 17α-hydroxyprogesterone (17-OH-P) to test the hypothesis that high concentrations of EB would accelerate the conversion of P4 to 17-OH-P. However, 17-OH-P concentrations paralleled P4 profile in all groups, irrespective of EB doses. In summary, the three EB treatment regimens induced similar E2 peaks, although the observation of EC peaks 24 hours after E2 peaks in the 5- and 10-mg groups indicate that an excess of E2 was given, which was converted into EC to be inactivated. Administration of 10 mg of EB reduced P4 concentrations 24 hours after LA P4 was given. We demonstrated that the mechanism by which this reduction occurred was not by an increase in P4 metabolism to 17α-OH-P. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 appears to be a more appropriate regimen to treat noncyclic mares, although additional studies are needed to verify embryo survival with this treatment dose.

Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares.

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches.

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Acute and chronic effects of a contraceptive compound RTI-4587-073(l) on testicular histology and endocrine function in miniature horse stallions.

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI-4587-073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI-4587-073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p < 0.05), while the number of immature germ cells increased in ejaculates from treated animals (p < 0.05). Serum concentrations of inhibin and follicle-stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p < 0.05) after administration of RTI-4587-073(l), and increased several days later (p < 0.05). Testicular content of testosterone and estradiol 17-ß was lower in treated stallions than in control stallions on Day 1 after treatment (p < 0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1 day, 2 and 4 weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI-4587-073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI-4587-073(l) has significant but transient effects on Leydig cell function in stallions.

Stimulation of Sertoli cell proliferation: defining the response interval to an inhibitor of estrogen synthesis in the boar.

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11-16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares.

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.

A synergistic effect of insulin-like growth factor (IGF-I) on equine luteinizing hormone (eLH)-induced testosterone production from cultured Leydig cells of horses.

Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1±0.1 year; 1-1.5 year) and eight post-pubertal (2.88±0.35 years; 2-4 years) stallions during routine castrations at the UC Davis Veterinary Hospital. Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated without (control) or with increasing concentrations of purified pituitary-derived eLH and/or recombinant human IGF-I (rhIGF-I) and incubated under 95% air: 5% CO(2) at 32°C for 24h. After 24h, culture media was collected and frozen at -20°C until analyzed for testosterone by a validated radioimmunoassay (RIA). In pubertal stallions, treatment with both increasing concentrations of rhIGF-I and 5ng/ml of eLH failed to demonstrate a significant difference in testosterone production compared with 5ng/ml of eLH only. However, in post-pubertal stallions, a significant increase in the concentration of testosterone in culture media was observed from Leydig cells treated with various concentrations of rhIGF-I and 1 or 5ng/ml of eLH compared with 1 or 5ng/ml of eLH only. In conclusion, IGF-I has a synergistic effect on eLH-induced testosterone production in cultured equine Leydig cells from post-pubertal but not pubertal stallions.

Use of a gonadotrophin-releasing hormone vaccine in headshaking horses.

The aim of this study was to investigate the use of a gonadotrophin-releasing hormone (GnRH) vaccine in the treatment of headshaking in horses. Fifteen geldings received two doses of the GnRH vaccine four weeks apart. Serum was collected before and after vaccination to measure concentrations of luteinising hormone (LH) (10 horses) and follicle-stimulating hormone (FSH) (six horses). Owners recorded the frequency of seven common headshaking behaviours using a visual analogue scale (VAS) before vaccination and at two, four, eight, 12, 16 and 20 weeks after vaccination. Serum LH (P=0.008) and FSH (P=0.03) concentrations decreased significantly following vaccination. Although approximately one-third of the owners reported a subjective improvement in headshaking, serial scoring did not indicate a reduction in headshaking behaviours following vaccination with a commercial GnRH vaccine. Vaccination reactions were observed in four of 15 horses (27 per cent), including one case of severe, presumed immune-mediated, myositis.

Localization of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) in equine testes.

The insulin-like growth factor-I (IGF-I) is a key regulator of reproductive functions. IGF-I actions are primarily mediated by IGF-IR. The main objective of this research was to evaluate the presence of IGF-I and IGF-I Receptor (IGF-IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF-I and IGF-IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF-I and IGF-IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti-human polyclonal antibodies against IGF-I and IGF-IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre-pubertal and pubertal stages, IGF-I immunolabelling was present in spermatogonia and Leydig cells. At post-pubertal, adult and aged stages, immunolabelling of IGF-I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF-IR was observed in spermatogonia and Leydig cells at the pre-pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post-pubertal stage. Strong immunolabelling of IGF-IR was observed in spermatogonia and Leydig cells at post-puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF-I and IGF-IR was not observed in Sertoli cells. In conclusion, IGF-I is localized in equine spermatogenic and Leydig cells, and IGF-IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF-I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.

Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis.

Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may have potential as a treatment regime to prevent testicular degeneration. The present study was designed to evaluate the anti-apoptotic effect of IGF-I on cultured equine Leydig cells. Testes were collected from 5 post-pubertal stallions (2-4 years old) during routine castrations. A highly purified preparation of equine Leydig cells was obtained from a discontinuous Percoll gradient. Purity of equine Leydig cells was assessed using histochemical 3ß-HSD staining. Equine Leydig cells and selected doses of recombinant human IGF-1 (rhIGF-I; Parlow A.F., National Hormone and Peptide Program, Harbor-UCLA Medical Center) were added to wells of 24 or 96 well culture plates in triplicate and cultured for 24 or 48 h under 95% air:5% CO(2) at 34°C. After 24 or 48 h incubation, apoptotic rate was assessed using a Cell Death Detection ELISA kit. Significantly lower apoptotic rates were observed in equine Leydig cells cultured with 5, 10, or 50ng/ml of rhIGF-I compared with control cells cultured without rhIGF-I for 24h. Exposure to 1, 5, 10 or 50 ng/ml of rhIGF-I significantly decreased apoptotic rate in equine Leydig cells cultured for 48 h. After 48 h incubation, cells were labeled with Annexin V and propodium iodine to determine the populations of healthy, apoptotic, and necrotic cells by counting stained cells using a Nikon Eclipse inverted fluorescence microscope. As a percentage of the total cells counted, significantly lower numbers of apoptotic cells were observed in cells treated with 10 (9%) or 50 ng/ml (10%) of rhIGF-I compared with cells cultured without rhIGF-I (control, 22%). In this study, the results from the two assays indicated that rhIGF-I protected equine Leydig cells from undergoing apoptosis during cell culture for 24h or 48 h. In conclusion, IGF-I may be an important paracrine/autocrine factor in protecting equine Leydig cells from undergoing apoptosis.

Differential luteolytic function between the physiological breeding season, autumn transition and persistent winter cyclicity in the mare.

There is a well-documented increase in luteolytic failure, resulting in spontaneously prolonged corpus luteum (SPCL) function, during estrous cycles of horses in autumn. The cause of this phenomenon may be due to seasonal alterations in PGF(2alpha) and/or in prolactin (PRL) secretion around luteolysis. To investigate this, progesterone (P4), 13, 14-dihydro, 15-keto PGF(2alpha) (PGFM) and PRL concentrations were compared between summer and autumn estrous cycles during natural luteolysis and luteolysis induced by benign uterine stimulation. A single estrous cycle from mares in June-July (n=12) was compared to multiple estrous cycles from these 12 mares plus 8 additional mares in September through December. Reproductive behavior was monitored by bringing a stallion in close proximity to the mare and ovarian events by ultrasonography. Blood was collected via jugular cannula every 6h from d 13 to 17 post-ovulation in untreated control mares (n=8 summer, n=9 autumn). In treated mares, blood collection occurred at 0, 15, 30, 45, 60, 90, 120, 180 and 240min followed by 6h intervals for a total of 5d following intrauterine saline infusion on d 7 (n=4 summer, n=11 autumn). Mares failing to return to estrus for 30d received intrauterine saline and the described intensive blood sampling protocol on d 30. Progesterone and PRL were determined on daily samples and PGFM on frequent plasma collections by RIA. Duration of ovarian luteal and follicular phases, P4 and PRL concentrations and PGFM secretion around luteolysis were compared between treatments and seasons by ANOVA. Mean P4 declined from June to December in all groups. Pulses of PGFM were detected on d 13-17 in controls and d 7-11 in saline-infused mares. Pulse patterns were not different between groups. The incidence of SPCL increased during autumn in the control group. PGFM pulses were absent on d 13-17 in mares with SPCL, but PGFM pulses could be induced in these mares by saline infusion at d 30. Autumn PGFM profiles were unchanged during spontaneous or saline-induced luteolysis compared with summer. Circulating PRL increased around natural or induced luteolysis. These results provide evidence that changes in luteal function during the autumn transition are not the result of alterations in the ability of the uterus to produce PGF(2alpha) nor due to changed CL sensitivity to PGF(2alpha). We conclude that seasonal changes in luteolytic function are caused by an alteration in the signal for PGF(2alpha) release.

Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model.

The efficacy of a recently engineered single chain recombinant equine follicle stimulating hormone (reFSH) was investigated in estrous cycling mares whose gonadotropins and follicular activity had been suppressed by concurrent treatment with progesterone and estradiol (P&E). Time of estrus was synchronized in 15 estrous cycling mares during the breeding season with prostaglandins F(2alpha) (PGF(2alpha)). The day after ovulation, mares were treated once daily with P&E for 14 days. Mares received a second injection of PGF(2alpha) on day 6 of the synchronized estrous cycle to induce luteolysis. On day 8 post-ovulation mares were randomly assigned to three groups: small dose reFSH-treatment group (0.5mg reFSH IV, twice daily); large dose reFSH-treatment group (0.85mg reFSH IV twice daily); control group (saline IV, twice daily). reFSH treatment occurred concurrently with the last week of P&E treatment. After a follicle or cohort of follicles reached 35mm in diameter, mares were injected with 0.75mg of recombinant equine luteinizing hormone (reLH) to induce ovulation. Post-treatment ovulation was assessed. Daily blood samples were collected for analysis of FSH, LH, estradiol, progesterone, and inhibin by radioimmunoassay (RIA). On the first day of reFSH/saline treatment, blood samples were collected periodically from 1h prior to treatment to 6h post-injection via an indwelling jugular catheter to determine acute changes in FSH concentrations. Monitoring of follicular activity, estrus, and ovulation was performed daily by utilizing a stallion and transrectal ultrasonography. A difference (por=35mm follicles (days 16-21) than controls. Mares treated with reFSH, at either dose, took less time (average: 2.95+/-0.42 days) to develop 2-3 times more pre-ovulatory follicles than control mares (7.8+/-0.51 days) (p

Hormone profiles of mares affected by the mare reproductive loss syndrome.

While searching for the cause of the Mare Reproductive Loss syndrome (MRLS), we postulated that 1 of 3 tissues in 40-120 D pregnant mares was the likely primary target of the noxious factor that caused early abortions: The corpora lutea (CL), the endometrium or the fetus and/or its membranes. At this stage of gestation, progesterone (P4) is solely produced by luteal tissue, eCG by endometrial cups in the endometrium and oestrogens by the feto-placental unit. We determined whether concentrations of P4, eCG and/or total conjugated oestrogens (CE) would indicate which tissue was targeted during the MRLS. P4, eCG and CE were measured in single serum samples collected from 216 mares, 60-110 D after ovulation during the 2001 MRLS outbreak. All mares had previously been confirmed pregnant by ultrasonography. The following data was obtained from each mare: Interval from ovulation, pregnancy status and normalcy of fetal fluids at the time of sampling, and pregnancy status 3 weeks after sampling and at term. There were no meaningful differences in hormone concentrations between pregnant mares that had normal and excessively echogenic fetal fluids at the time of sampling. CE were lower (p < 0.05) in mares that aborted after sample collection than in mares the carried to term. In 8 mares from which multiple samples were obtained, CE consistently decreased prior to any decreases in P4 or eCG. Arguments are presented that lead to the hypothesis that the fetal trophoblast was the primary target of the MRLS agent.

Analysis of molecular hydrogen formation on low-temperature surfaces in temperature programmed desorption experiments.

The study of the formation of molecular hydrogen on low-temperature surfaces is of interest both because it enables the exploration of elementary steps in the heterogeneous catalysis of a simple molecule and because of its applications in astrochemistry. Here, we report results of experiments of molecular hydrogen formation on amorphous silicate surfaces using temperature-programmed desorption (TPD). In these experiments, beams of H and D atoms are irradiated on the surface of an amorphous silicate sample. The desorption rate of HD molecules is monitored using a mass spectrometer during a subsequent TPD run. The results are analyzed using rate equations, and the energy barriers of the processes leading to molecular hydrogen formation are obtained from the TPD data. We show that a model based on a single isotope provides the correct results for the activation energies for diffusion and desorption of H atoms. These results are used in order to evaluate the formation rate of H2 on dust grains under the actual conditions present in interstellar clouds. It is found that, under typical conditions in diffuse interstellar clouds, amorphous silicate grains are efficient catalysts of H2 formation when the grain temperatures are between 9 and 14 K. This temperature window is within the typical range of grain temperatures in diffuse clouds. It is thus concluded that amorphous silicates are good candidates to be efficient catalysts of H2 formation in diffuse clouds.