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.

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.

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.

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.

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.

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.

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.

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.

The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles, and inter-ovulatory intervals.

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17beta (E(2)), and progesterone (P(4)) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.

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.

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.

Dopamine receptors in equine ovarian tissues.

Dopamine (DA) agonist and antagonist treatments can affect ovarian reproductive events in the mare. To support our theory that DA produces these effects by acting directly on the ovary, we analyzed equine ovarian tissues for the presence of dopamine receptor-1 (D1r) and dopamine receptor-2 (D2r) mRNA by reverse transcription polymerase chain reaction (RT-PCR) and D1r and D2r proteins by Western blot and immunohistochemistry (IHC). RT-PCR was performed on RNA isolated from ovarian cortex, medulla, granulosa/theca or corpus luteum (CL) tissues and from pituitary (D2r control) and renal artery (D1r control). D1r and D2r specific primers were designed from partial DNA sequences known for the horse (D2r) or conserved sequences from other species (D1r). Western blot analyses were conducted on CL, cortex and granulosa/theca samples and IHC was performed on CL tissues using D1r or D2r specific antibodies. The incidence of positive D2r mRNA was high in CL and ovarian cortex, low in granulosa/theca, and not detectable in ovarian medulla. Dopamine D1r mRNA incidence was high (50%) only in CL tissues. D1r and D2r antibody staining was positive for each tissue type analyzed by Western blot procedures. All CL tissues prepared by IHC showed positive staining for D1r and D2r proteins. Both DA receptor proteins appeared uniformly distributed throughout the CL tissue. These results indicate that equine ovarian tissues do possess D1r and D2r, and suggests that DA can act directly on ovarian tissues through its interaction with DA receptors.

Circulating antagonist of luteinizing hormone in association with infertility in stallions.

Using a LH radioligand receptor assay (RRA) previously validated for use in serum and an equine monoclonal RIA, we have distinguished a subset of subfertile stallions with an elevated RRA/RIA ratio. After purification of the active moiety by anion exchange chromatography and immunoprecipitation with the equine LH (eLH) monoclonal antibody, RRA activity remained in the supernatant. This activity was also recognized by a polyclonal LH antibody (GDN 15) with wide cross-species recognition. This active fraction was further purified by gel filtration chromatography and shown to displace labeled eLH in a dose-dependent fashion in the RRA with an inhibition slope of 2.8 compared with a slope of 1.1 for native eLH. This fraction also inhibited the LH-stimulated steroidogenesis of Leydig cells in vitro in a dose-dependent fashion, but had no effect on basal (minus LH) steroid production. Polyacrylamide gel electrophoresis and electroelution of this material demonstrated RRA activity in a fraction with a mol wt between 45-66 kDa. We conclude that this substance 1) competitively inhibited binding of eLH and hCG to the LH receptor, 2) antagonized LH-stimulated steroidogenesis in vitro, and 3) may represent a LH isoform found in association with infertility in these animals.

A supersensitive immunofluorometric assay for rat luteinizing hormone.

We have developed an immunofluorometric assay (IFMA) for rat (r) LH, which is based on two monoclonal antibodies, one to bovine and the other to human LH. Signal detection occurs by time-resolved fluorescence evoked by a europium label (Delfia, Wallac). The method is fast in comparison to the standard RIA with the NIDDK reagents (4 h vs. 3 days). The sensitivity of the IFMA assay (0.75 pg/tube; NIDDK rLH RP-2) is over 30-fold higher than that of the NIDDK RIA (usual detection limit, 20-30 pg/tube). Using 25-microliters serum samples, the sensitivity of IFMA is 0.03 micrograms/liter; with 100-microliters samples, it is 0.0075 micrograms/liter. The cross-reactivity of the IFMA assay is 0.3% with rFSH, 3% with rTSH, and less than 0.05% with rGH, rPRL, and the rat alpha-subunit. A linear correlation between IFMA and RIA values is seen at serum levels above 0.4 micrograms/liter. Below this level, only IFMA is able to detect concentration differences between samples. In practice, this means that only IFMA is able to provide meaningful measurements of suppressed levels of serum LH. The correlation coefficient between IFMA and the mouse interstitial cell in vitro bioassay for LH in randomly selected rat pituitary homogenates was 0.93 (n = 47). The serum concentration of LH determined by IFMA is 0.57 +/- 0.10 micrograms/liter in intact adult male rats (mean +/- SEM; n = 12) and 0.41 +/- 0.10 micrograms/liter (n = 10) in randomly cycling females. The level in hypophysectomized rat serum is 0.035 +/- 0.0033 micrograms/liter (n = 8), if the limit of sensitivity (0.03 microgram/liter) is assigned to unmeasurable levels. One-week treatment of male rats with 2-cm Silastic implants containing testosterone suppressed serum LH, measured by IFMA, from 0.56 +/- 0.057 to 0.086 +/- 0.057 micrograms/liter (P < 0.01). The suppression of LH measured in the same samples by RIA was lower, from 0.73 +/- 0.057 to 0.44 +/- 0.048 micrograms/liter (P < 0.01). A 5-day starvation of intact male rats suppressed serum LH from 0.57 +/- 0.10 to 0.30 +/- 0.05 microgram/liter by IFMA (P < 0.01), whereas the decrease determined by RIA was not significant (0.80 +/- 0.07 vs. 0.66 +/- 0.13 micrograms/liter).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

Effects of lipoprotein, equine luteinizing hormone, equine follicle-stimulating hormone, and equine prolactin on equine testicular steroidogenesis in vitro.

A stallion testicular cell incubation system was developed and used to investigate the regulation of steroidogenesis in stallion testes. Cells isolated from testes of 2- to 4-year-old stallions (n = 6) were cultured for 12 hours in a defined medium with and without varying doses of lipoprotein, equine luteinizing hormone (eLH), human chorionic gonadotropin (hCG), equine follicle-stimulating hormone (eFSH), and/or equine prolactin (ePRL). Estrogen conjugate (EC), testosterone (T), and estradiol-17 beta (E2) production were determined by RIA. Increasing doses of lipoprotein significantly (P < 0.001) increased basal, hCG- and eLH-stimulated EC production, resulting in a maximal fourfold increase in each case. A maximal dose of lipoprotein (3 mg/ml) significantly (P < 0.001) increased basal T production threefold, whereas hCG- and eLH-stimulated T production were increased 76- and 30-fold, respectively. In the presence of 0.5 mg/ml of lipoprotein, increasing doses of eLH significantly (P < 0.001) stimulated EC, T, and E2 production. The increase in T production (5.6-fold) at a physiological dose of eLH (5 ng/ml) was significantly (P < 0.05) greater than the increase in EC or E2 production (2.1- and 2.3-fold, respectively). However, the total mass of EC produced was significantly greater (P < 0.05) than the total amount of T produced at both basal (15 ng vs. 148 pg) or hormone-stimulated (48 ng vs. 2,427 pg at 5 ng/ml eLH) levels. hCG significantly (P < 0.001) stimulated EC and T production and was 82-fold more active in stimulating EC production and 41-fold more active in stimulating T production than was eLH. FSH had no significant effect on steroidogenesis either alone or in the presence of eLH, except at the highest dose tested (50 ng/ml), which was above the physiological level of circulating FSH (4-7 ng/ml) in the stallion. PRL (1-50 ng/ml) had no significant effect on steroidogenesis either alone or in the presence of eLH. These data suggest that in the postpubertal stallion, both estrogen and T production are regulated by LH, and this regulation appears to be dependent on the availability of lipoprotein-derived cholesterol. Furthermore, the observation that testicular cells produced a larger mass of EC than T, but responded to eLH with a larger relative increase in T production, suggests that production of these two steroids may be independently regulated.

Seasonal effects on seminal quality, plasma hormone concentrations, and GnRH-induced LH response in fertile and subfertile stallions.

Seasonal effects on hormonal and seminal parameters in subfertile stallions have not been well documented and could provide information that is needed to understand the underlying endocrine mechanisms associated with testicular dysfunction. Such information may be useful in developing diagnostic tools to identify those stallions who are candidates for treatment. This investigation characterizes and compares the effects of season on endocrine function and seminal quality in fertile and subfertile stallions. Eight fertile and six subfertile stallions between the ages of 5 and 18 years were injected intravenously once every hour for 3 hours with either 1 mL saline on the first experimental day or 5 micrograms gonadotropin-releasing hormone in 1 mL saline on the second experimental day during the nonbreeding and breeding season. Heparinized blood samples were collected periodically through a jugular catheter before and after treatment for analysis of luteinizing hormone, follicle-stimulating hormone, testosterone, and estrogen conjugates by radioimmunoassay. Semen samples were collected twice, 1 hour apart, from all stallions in both seasons for analysis of volume, concentration, motility, pH, and morphology. A series of low intravenous doses (5 micrograms) of gonadotropin-releasing hormone induced a significant luteinizing hormone response (P less than 0.05) compared with saline treatment in both fertile and subfertile stallions. Fertile stallions had a twofold higher (P less than 0.05) net increase in plasma luteinizing hormone levels (peak levels minus baseline levels) in the breeding seasons than in the nonbreeding season. The magnitude of the luteinizing hormone response relative to baseline levels in fertile stallions, however, was one-and-one-half times greater (P less than 0.05) in the nonbreeding season than in the breeding season. In contrast, season did not have an effect on the net increase in plasma luteinizing hormone or the magnitude of the luteinizing hormone response relative to baseline levels in subfertile stallions. The net increase in plasma luteinizing hormone was similar between the two groups of stallions in both seasons. The magnitude of luteinizing hormone response relative to baseline levels, however, was lower (P less than 0.05) in subfertile stallions (141 +/- 14%) than in fertile stallions (235 +/- 46%) in the nonbreeding season; the two groups exhibited similar responses in the breeding season. Compared with fertile stallions, subfertile stallions had twofold to fourfold higher (P less than 0.05) plasma levels of gonadotropins and similar testosterone levels. The number of total progressively motile sperm was lower (P less than 0.05) in subfertile stallions in both seasons.(ABSTRACT TRUNCATED AT 400 WORDS)