A description of the upper thoracic autonomic nervous system in the rhesus monkey (Macaca mulatta)

The purpose of this study was to describe the autonomic innervation of the carotid sinus and heart in the rhesus monkey. Nine male rhesus monkeys (Macaca mulatta) and one male crab-eating macaque (M. fascicularis) were carefully dissected from the origin of the vagus nerves and superior cervical ganglia to the level of the fourth thoracic ganglion. The specimens were either freshly killed or obtained no later than 24 hours post mortem. The macaque monkeys were found to possess an innervation pattern that displayed features common to dog (connections between the vagus nerves and middle cervical ganglia), baboon (distinct cervical sympathetic and cervical vagal nerve trunks), and man (nerves projecting from the middle cervical and stellate ganglia to the heart). Distinct inferior cervical and first thoracic ganglia were never seen, but rather, large and well defined stellate ganglia were found. The macaque innervation pattern, when considered as a whole, most closely resembled the baboon.     

Placental transfer of synthetic and endogenous estrogen conjugates in the rhesus monkey (Macaca mulatta)

The placental transfer of conjugated and nonconjugated estrogens was compared in the pregnant rhesus monkey. Placement of catheters in the maternal and fetal circulation allowed for the sampling of blood after the administration of radiolabeled naturally occurring and synthetic estrogens to mother or fetus. In all cases, nonconjugated-estrogen placental transfer was greater than conjugated-estrogen transfer. Comparison of the conjugated estrogens suggested that diethylstilbestrol-monoglucuronide (DESG) was transferred less efficiently than the estrone-sulfate (E, S). High-performance liquid chromatog-raphy (HPLC) of selected plasma samples revealed that 50–90% of the E, S observed in the maternal circulation was cleaved after fetal administration. In contrast, HPLC of maternal or fetal plasma samples after DESG administration revealed only intact DESG. These results emphasize differences in the placental transfer of the synthetic and naturally occurring estrogen hormones.     

The available time window for embryo transfer in the rhesus monkey (Macaca mulatta)

Much effort has been focused on improving assisted reproductive technology procedures in humans and nonhuman primates (NHPs). However, the pregnancy rate after embryo transfer (ET) has not been satisfactory, indicating that some barriers still need to be overcome in this important procedure. One of the key factors is embryo–uterine synchronicity, which is little known in NHPs. The objective of this study was to investigate the available ET time window in rhesus monkey (Macaca mulatta). Eighty-two adult female rhesus monkeys were superovulated with recombinant human FSH. Ovarian phases were identified according to estrogen (E2) and progesterone (P4) levels as well as ovarian examination by ultrasonography and laparoscopy. A total of 259 embryos were transferred by the laparoscopic approach into the oviducts of 63 adult female monkeys. Ovarian phases were divided into late follicular and early luteal phases. Similar pregnancy rates (30–36.4%) were obtained from recipients receiving ET either in their late follicular or early luteal phases, regardless of embryo developmental stages. This study indicates that the available time window for ET in rhesus monkeys is from the late follicular to early luteal phases.     

Expression of prostasin and protease nexin-1 in rhesus monkey (Macaca mulatta) endometrium and placenta during early pregnancy.

Serine proteases have been documented to play key roles in uterine matrix turnover and trophoblastic invasion during implantation. Roles of prostasin serine protease in these processes, however, are currently unclear. The present study was first conducted to investigate the colocalization of prostasin and its cognate serpin, protease nexin-1 (PN-1), in rhesus monkey endometrium and placenta on days 12, 18, and 26 of pregnancy by using in situ hybridization (ISH) and immunohistochemistry. With ISH, expression of prostasin mRNA was intensely localized in the glandular epithelium on days 12 and 18 and in the placental villi, trophoblastic column, trophoblastic shell, and fetal-maternal border on days 18 and 26. With the progress of pregnancy, expression level in the glandular epithelium was significantly decreased, and the accumulation in the placental compartments was further increased. In addition, the stroma and arterioles exhibited modest levels of prostasin signals. However, expression level of PN-1 in these compartments on adjacent sections in the three stages of early pregnancy was weak or below the level of detection. Prostasin protein expression in the endometrium was found to be consistent with the distribution patterns revealed in the ISH experiments. It may be suggested from these results that prostasin is involved in endometrial epithelial morphology establishment, tissue remodeling, and trophoblastic invasion during early pregnancy. The cognate serpin PN-1 was not coordinately expressed along with prostasin, creating a tissue environment favorable for proteolytic activities of prostasin during early pregnancy events.     

Condylar translation and the function of the superficial masseter muscle in the rhesus monkey (M. mulatta).

The relationship between translation of the mandibular condyle during symmetrical mandibular rotation, i.e., symmetrical jaw depression and elevation, and the function of the superficial masseter muscle was examined in light of relative torque and the length-tension relationship for muscle. Lateral cephalograms of live adult rhesus monkeys (Macaca mulatta) were analyzed using two models: (1) Model A, normal symmetrical jaw rotation accompanied by condylar translation; and (2) Model B, mandibular rotation about an axis fixed at the position of the condyles during centric occlusion. The decrease in relative torque and the excursion of the superficial masseter at mouth-open positions are significantly greater in Model B than in Model A. Symmetrical rotation of the jaw about a fixed axis would result in a 35% greater loss of maximum producible tension at maximum gape than rotation associated with condylar translation. These results suggest that condylar translation during mandibular depression and elevation functions to minimize reduction in relative torque and excursion of superficial masseter muscle, thereby maintaining optimal potential for exerting maximum tension during jaw closure.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

The effects of extended time in the laboratory on selected aspects of reproduction in the female rhesus monkey (Macaca mulatta)

Reproduction data from 60 wild-caught and 16 captive-born, hand-reared female rhesus monkeys (Macaca mulatta) were examined. Both groups had been maintained in a controlled laboratory environment, the wild-caught for a minimum of 10 years and the captive-born for a minimum of 5 years. All were bred to wild-caught males. Animals of both sexes were individually caged unless being bred. Data from 662 pregnancies indicated that, although seasonal breeding became attenuated in the laboratory, it did not disappear. Neither pregnancy outcome nor number of matings necessary for conception was affected by increasing parity or prior occurrence of fetal wastage or hysterotomy. Nor did hysterotomy affect the potential for a subsequent vaginal delivery. The number of matings necessary for conception were shown to be a useful predictor of animals that should be culled from the breeding colony. Birthweights of infants of wild-caught females, but only male infants of house-born females, increased with parity of the mother. Parity had only minimal effect on gestation length. Conception was shown to occur infrequently at less than 100 days postpartum even when animals were not lactating and were rebred begining as early as 56 days postpartum. Summary data were presented for pregnancy outcome, gestation length, infant birth weight, and sex for both groups of animals.     

Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies

Three different fragments of microneme 1 protein termed, r-MIC1ex2, r-MIC1ex34 and r-MIC1 of Toxoplasma gondii, were expressed in Escherichia coli as fusion proteins containing six histidyl residues at N- and C-terminal. After purification by metal affinity chromatography, these recombinant proteins were tested for their usefulness as antigens in an enzyme-linked immunosorbent assay for the detection of immunoglobulin G. Ninety-eight sera from patients with different stages of invasion and 24 sera from seronegative patients were examined. There was no significant difference observed in the antigenicity for human serum samples from patients with acute toxoplasmosis between three recombinant types of MIC1 antigen (96.1% for r-MIC1ex2 antigen and 100% for both r-MIC1ex34 and r-MIC1 proteins). Sera from chronic infections (with low titers of IgG antibody) showed significant lower sensitivity, especially for r-MIC1ex34 and r-MIC1 antigens (75%, 52.7% and 36.1% for r-MIC1ex2, r-MIC1ex34 and r-MIC1, respectively). These results indicate that the strongest antigenic region of the MIC1 is encoding by the second exon of mic1 gene. When r-MIC1ex2 (N-terminal fragment of protein) was combined with MAG1 (matrix antigen) and MIC3 (microneme 3 protein), the sensitivity increased to 88.9%. This result was comparable to an ELISA using a Toxoplasma lysate antigen (TLA) and two combinations of recombinant antigens: M1 (GRA1+GRA7+SAG1) and M2 (P35+SAG2+GRA6) with the sensitivity for serum samples tested 94.4%, 88.9% and 94.4%, respectively.     

Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti-serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.     

An enzyme-linked immunosorbent assay for the Amblyospora sp. of Culex salinarius (Microspora: Amblyosporidae)

The first enzyme-linked immunosorbent assay for a microsporidian is described. The assay detects as little as 2 ng of spore homogenate protein and as few as 2000 intact spores. Several time-saving and reagent-conserving modifications of traditional ELISA protocols are employed.     

Reproduction in the vervet monkey (Cercopithecus aethiops): I. Testicular volume,testosterone, and seasonality

To test whether the male could contribute to the birth peak seen in both wild and captive vervets, testicular volume and peripheral testosterone concentration were measured monthly in nine adults throughout a 14-month period. Volume was an average of 15% greater during the months of June to September, the period of natural breeding activity in the wild, than at other times of the year. Testosterone concentration rose throughout the time period but did not correlate with testicular volume. Quarterly biopsies of the right testis in animals either used for or withheld from breeding revealed the presence of spermatozoa and the existence of spermatogenesis throughout the year. There was no correlation of testicular volume with breeding status or biopsy. If these results from individually caged animals are representative of group-caged and wild animals, then such small seasonal changes are unlikely to affect year-round breeding. Therefore, a physiological contribution by the male to natural birth peaks seen in the wild and captivity is not readily apparent.     

In situ hybridization assay for the diagnosis of chagas myocarditis and orchitis in a rhesus macaque (Macaca mulatta): A case report

We present the first documented case of Trypanosoma cruzi‐induced orchitis in a rhesus macaque. Additionally, we describe an in situ hybridization–based assay to confirm T. cruzi infection in formalin‐fixed tissues.     

Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)

Antibodies to a low molecular weight folate binding protein isolated from human milk were raised in rabbits and used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for immunological characterization of human folate binding proteins (FBPs). The high and low molecular weight FBPs from human milk were immunologically indistinguishable. Furthermore, the FBPs in human urine and cerebrospinal fluid showed a cross-reactivity of 70% and 30%, respectively. No cross-reactivity of the FBP from cow's milk was observed.     

Postnatal changes in the blood of Japanese monkeys (Macaca fuscata): I. Immunoglobulins Ig G,Ig M and Ig A

The levels of immunoglobulin Ig G, Ig M and Ig A in the plasma of Japanese monkeys (Macaca fuscata) from birth to 180 days of age were examined. The level of Ig G reached a minimum at 50 to 80 days of age, when the neonates weighed 600–800 g. Anti streptolysin O antibody decreased from birth with age and was not detected from day 80 to 150. The level of Ig M showed a temporary peak at 20–30 days of age and increased again from 60–80 days. The concentration of Ig A increased gradually to reach 20% of the adult level at day 180.     

On the reliability of a simple method for scoring phenotypes to estimate heritability: A case study with pupal color in Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae)

In this paper, two methods for assessing the degree of melanization of pupal exuviae from the butterfly Heliconius erato phyllis, Fabricius 1775 (Lepidoptera, Nymphalidae, Heliconiini) are compared. In the first method, which was qualitative, the exuviae were classified by scoring the degree of melanization, whereas in the second method, which was quantitative, the exuviae were classified by optical density followed by analysis with appropriate software. The heritability (h(2)) of the degree of melanization was estimated by regression and analysis of variance. The estimates of h (2) were similar with both methods, indicating that the qualitative method could be particularly suitable for field work. The low estimates obtained for heritability may have resulted from the small sample size (n = 7-18 broods, including the parents) or from the allocation-priority hypothesis in which pupal color would be a lower priority trait compared to morphological traits and adequate larval development.     

A new species: Gerres microphthalmus (Perciformes: Gerreidae) from Japan with notes on limited distribution, included in the “G. filamentosus complex”

 Gerres microphthalmus sp. nov., described on the basis of the holotype and 38 paratypes, has a limited distribution in Japan, including Tanega Island, southeastern Kyushu, southern Shikoku, and southern Kii Peninsula, central Honshu. It has not been recorded from the Ryukyu Islands. The species is the fourth member of the “G. filamentosus complex,” characterized by an elongated second dorsal fin spine, the others being G. filamentosus, G. infasciatus, and G. macracanthus. Gerres microphthalmus sp. nov. is most similar to G. filamentosus in overall body appearance, but differs from the latter in having smaller eyes (8%–11% of standard length vs. 10%–16% in the latter), a higher orbit diameter ratio (as % of snout length) (93%–143% of snout vs. 71%–104% of snout), and fewer pored lateral line scales (40–43 vs. 43–46). The limited distribution pattern of the new species is discussed with G. equulus and Lates japonicus (Centropomidae) having a limited distribution similar to that of the new species. Received: June 28, 2001 / Revised: November 13, 2001 / Accepted: December 10, 2001     

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent,human hepatoma cells (Hep G2) using single cell gel electrophoresis

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.