Radioimmunoassay of polypeptide hormones using immunochemically coated plastic tubes

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Comparative Analyses of Age- and Sex-Specific Patterns of Vocal Behaviour in Four Species of Old World Monkeys

Sound recordings and behavioural data were collected from four primate species of two genera (Macaca, Presbytis). Comparative analyses of structural and behavioural aspects of vocal communication revealed a high degree of intrageneric similarity but striking intergeneric differences. In the two macaque species (Macaca silenus, Macaca radiata), males and females shared the major part of the repertoire. In contrast, in the two langurs (Presbytis johnii, Presbytis entellus), many calls were exclusive to adult males. Striking differences between both species groups occurred with respect to age-specific patterns of vocal behaviour. The diversity of vocal behaviour was assessed from the number of different calls used and the proportion of each call in relation to total vocal output for a given age/sex class. In Macaca, diversity decreases with the age of the vocalizer, whereas in Presbytis the age of the vocalizer and the diversity of vocal behaviour are positively correlated. A comparison of the data of the two genera does not suggest any causal relationship between group composition (e.g. multi-male vs. one-male group) and communication system. Within each genus, interspecific differences in vocal behaviour can be explained by differences in social behaviour (e.g. group cohesion, intergroup relation, mating behaviour) and functional disparities. Possible factors responsible for the pronounced intergeneric differences in vocal behaviour between Macaca and Presbytis are discussed.

Hemiorchidectomy leads to dramatic and immediate alterations in pituitary follicle-stimulating hormone secretion and the functional activity of the remaining testis in the adult male bonnet monkey (Macaca radiata)

The aim of the present study was to examine the effect of hemiorchidectomy (HO) on serum FSH, LH, testosterone (T), and inhibin (INH) concentrations as well as on the testicular volume (TV) and on changes in the kinetics of germ cell turnovers in the remaining testis of adult male bonnet monkeys. Blood samples collected at 2200 h at various times before and after HO and testicular biopsies obtained at different periods were subjected to hormone analysis and DNA flow cytometry. Though serum T levels were lowered (p < 0.05) at 12 h after HO, T levels rapidly returned to intact control concentrations by Day 5. While serum LH remained unaltered, serum FSH increased markedly within 2 days of HO and remained significantly (p < 0.05) elevated over the next 90 days. Though serum INH showed a significant decrease (p < 0.05) by 15 min of HO, it returned to approximately 80% of intact levels within one week. The TV of the remaining testis showed maximal increment by Day 30 (p < 0.05) of HO. DNA flow cytometric analysis 24 days after HO showed increases (p < 0.05) in spermatogonia (2C) and primary spermatocytes (4C). These cell types by Day 45 had transformed to round (1C) and elongate (HC) (by 38%, p < 0.001) spermatids. Overall spermatogenesis (conversion of 2C to 1C and HC) showed significant enhancement at Days 110 and 175, suggesting that the spurt in spermatogenic activity is not confined to a single spermatogenic cycle.

Correlation of seasonal changes in sperm output with endocrinological changes in the adult male bonnet monkey, Macaca radiata

We have examined the monthly variations in sperm output and attempted to correlate the profiles of endocrine hormones secreted with the sperm counts throughout the ,year in the adult male bonnet monkey. As previously reported, there was a distinct spurt in sperm output beginning September through December months. A concomitant increase in serum testosterone and prolactin concentrations were also noted during September through November (mid and post-monsoon season). Although there was a marked increase in gonadotropin releasing hormone stimulated testosterone secretion, the peak testosterone concentrations post gonadotropin releasing hormone injection did not vary significantly (P>0.05) throughout the year. Basal serum follicle stimulating hormone concentrations did not vary significantly (P>0.05) during April to June months compared to September-November months. Serum inhibin concentration remained unaltered throughout the year, except in the month of March. The results of this study provide evidence for annual rhythms in prolactin and testosterone secretion and a distinct seasonality in the sperm output of the adult male bonnet monkey, but the pituitary responsiveness to exogenous gonadotropin releasing hormone remains unaltered throughout the year. Because of the existence of seasonality as noted in the present study, future studies which utilize the adult male bonnet monkey as an experimental model need to take into consideration the seasonal effects on reproductive function in this species.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signal during early pregnancy in primates. CG has close structural and functional similarities to pituitary luteinizing hormone (LH) which is regulated by gonadotrophin releasing hormone (GnRH). To study the regulatory mechanism of CG secretion in primate embryos, we examined the production and timing of secretion of GnRH in peri-implantation embryos of the rhesus monkey. In-vivo fertilized/developed morulae and early blastocysts, recovered from non-superovulated, naturally-bred rhesus monkeys by non-surgical uterine flushing, were cultured in vitro to hatched, attached and post-attached blastocyst stages using a well-established culture system. We measured GnRH and CG in media samples from cultured embryos with a sensitive radioimmunoassay and bioassay, respectively. The secretion of GnRH (pg/ml; mean +/- SEM) by embryos (n = 20) commenced from low levels (0.32 +/- 0.05) during the pre-hatching blastocyst stage to 0.70 +/- 0.08 at 6-12 days and 1.30 +/- 0.23 at greater than or equal to 13 days of hatched blastocyst attachment and proliferation of trophoblast cells. GnRH concentrations in culture media obtained from embryos (n = 5) that failed to hatch and attach were mostly undetectable (less than or equal to 0.1). Samples that did not contain detectable GnRH failed to show detectable CG. Immunocytochemical studies, using a specific monoclonal anti-GnRH antibody (HU4H) as well as polyclonal antisera (LR-1), revealed that immunopositive GnRH cells were localized in pre-hatching blastocysts (n = 4), in blastocysts (n = 2) after 5-10 days of attachment and in monolayer cultures (n = 4) of well-established embryonic trophoblast cells. GnRH positive staining was seen only in cytotrophoblasts but not in syncytiotrophoblasts. Similarly, cytotrophoblast, but not syncytiotrophoblast, cells of the rhesus placenta were immunopositive. In controls, either in the absence of antibody or in the presence of antibody pre-absorbed with GnRH, these cells failed to show stain. These observations indicate, for the first time, that an immunoreactive GnRH is produced and secreted by blastocysts during the peri-attachment period and by embryo-derived cytotrophoblast cells in the rhesus monkey.

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata)

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type-the Sertoli cells-to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction (>40%; P < 0.05) in [H-3] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition (>50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block (<90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8-11%), S-phase (6-9%), and 4C (6-9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31-46%, HCI:7-20% and and HC2:11-25%) they represented the bulk of germ cells (70-80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to IC (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, for e.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation

Evidence for follicle-stimulating hormone mediation in the hemiorchidectomy-induced compensatory increase in the function of the remaining testis of the adult male bonnet monkey (Macaca radiata)

Hemiorchidectomy (HO) in the adult male bonnet monkey results in a selective increase in circulating concentrations of FSH and testosterone, and this is accompanied by compensatory increase in sperm production by the remaining testis. We investigated the possible role of increased FSH concentration that occurs after HO in the compensatory increase in the activity of the remaining testis. Of eight adult male bonnet monkeys that underwent HO, four received i.v. injections every other day for 30 days of a well-characterized ovine FSH antiserum (a/s) that cross-reacts with monkey FSH. The remaining four males received normal monkey serum (NMS) as control treatment in a protocol similar to that employed for ais-treated males. Blood samples were collected between 2100 and 2200 h before and 1/2, 1, 3, 5, 7, 14, 22, and 29 days after HO. Testicular weight, number of 3 beta-hydroxy steroid dehydrogenase-positive (3 beta-HSD+) cells, and DNA flow cytometric analysis of germ cell populations were obtained for testes collected before and at the termination of NMS or ais treatment. In NMS-treated males, circulating serum FSH concentrations progressively increased to reach a maximal level by Day 7 after HO (1.95 +/- 0.3 vs. 5.6 +/- 0.7 ng/ml on Days -1 and 7, respectively). Within 30 min of ais injection, FSH antibodies were detected in circulation, and the antibody level was maintained at a constant level between Day 7 and end of treatment (exhibiting 50-60% binding to I-125-hFSH). Although circulating mean nocturnal serum testosterone concentration showed an initial decrease, it rose gradually to pre-HO concentrations by Day 7 in NMS-treated males. In contrast, nocturnal mat serum testosterone concentrations in a/s-treated males remained lower than in NMS-treated controls (p < 0.05) up to Day 22 and thereafter only marginally increased. Testicular weights increased (p < 0.05) over the pre-HO weight in NMS- but not in ais-treated males. After HO, the number of 3 beta-HSD+ cells (Leydig cells) was markedly increased but was significantly (p < 0.05) higher in NMS-treated males compared to a/s-treated males. A significant (p < 0.05) reduction in the primary spermatocyte population of germ cells was observed in ais-treated compared to NMS-treated males. These results suggest that the increased FSH occurring after HO could be intimately involved in increasing the compensatory functional activity of the remaining testis in the male bonnet monkey.

The effect of malathion, an organophosphate, on the plasma FSH, 17 beta-estradiol and progesterone concentrations and acetylcholinesterase activity and conception in dairy cattle

The effect of malathion on jugular plasma concentrations of follicle-stimulating hormone (FSH), estradiol (E2), progesterone (P4) and acetylcholinesterase (AchE) on conception in dairy cattle during a cloprostenol (prostaglandin F2-alpha analogue, PG)-induced estrus was studied. Malathion (1 mg/kg, intraruminally) given at the onset of estrus (48 h after PG) did not alter the plasma FSH or E2 concentrations but significantly (P < 0.05) inhibited plasma P4 concentration. The mean P4 concentration in the malathion-treated group on days 8 and 12 were 0.8 +/- 0.4 and 1.0 +/- 0.5 ng/ml, as compared to 2.6 +/- 0.0 and 2.4 +/- 0.3 ng/ml in the control group. There was a nonsignificant (P > 0.05) inhibition of plasma AchE activity in malathion-treated cattle. Conception was 16.6% in malathion-treated cows and 50% in controls. Inhibition of progesterone secretion and poor conception occurred after the single intraruminal dose of malathion at the onset of estrus.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata)

Administration of Tamoxifen via the Alzet pump at a rate of 50 mu g hr(-1) for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Susceptibility of sperm chromatin to acid denaturation in situ: A study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata)

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

Mutual inhibition and capacity sharing during parallel preparation of serial eye movements

Many common activities, like reading, scanning scenes, or searching for an inconspicuous item in a cluttered environment, entail serial movements of the eyes that shift the gaze from one object to another. Previous studies have shown that the primate brain is capable of programming sequential saccadic eye movements in parallel. Given that the onset of saccades directed to a target are unpredictable in individual trials, what prevents a saccade during parallel programming from being executed in the direction of the second target before execution of another saccade in the direction of the first target remains unclear. Using a computational model, here we demonstrate that sequential saccades inhibit each other and share the brain's limited processing resources (capacity) so that the planning of a saccade in the direction of the first target always finishes first. In this framework, the latency of a saccade increases linearly with the fraction of capacity allocated to the other saccade in the sequence, and exponentially with the duration of capacity sharing. Our study establishes a link between the dual-task paradigm and the ramp-to-threshold model of response time to identify a physiologically viable mechanism that preserves the serial order of saccades without compromising the speed of performance.