Contribution of ovarian steroid production to urinary estrone conjugate concentrations in Macaca mulatta.

This study was designed to test the hypothesis that basal estrone conjugate (E1C) profiles do not accurately detect ovarian function when ovarian estrogen production is low or absent. We employed surgical removal of active ovaries from laboratory rhesus macaques to simulate an acute decline in ovarian estrogen production. In the first experiment, urine samples collected prior to and following ovariectomy (Ovx) were subjected to high-performance liquid chromatography (HPLC) separation. Eluates were then assayed for E1C immunoreactive components. The results indicated a modest decrease in total immunoreactive polar conjugates following ovariectomy, with no substantial change in the overall retention profile. In the second experiment, estradiol (E2) cypionate injections were used to replace the E2 component of ovarian estrogen production in the treated (Tx) group, while the control group (C) received only vehicle. Urine samples were hydrolyzed and individual estrogens were separated by celite chromatography prior to immuno-assay. Both the Tx and C groups exhibited similar urinary excretion levels of estrone (E1), E2, and E1C prior to Ovx (Pre-Ovx) and after Ovx (Post-Ovx), but there were significant differences between groups after treatment (Post-Tx). Significant differences were observed in the Tx group's excretion of E1, E2, and E1C in the Pre- vs. Post-Ovx samples and in the Post-Ovx and Post-Tx samples. The C group also showed the expected significant differences in the Pre- vs. Post-Ovx samples, as well as in the Pre-Ovx and Post-Tx samples. The results indicate that the use of E1C measurements is clearly a suitable method for monitoring ovarian function in intact, cycling animals, but urinary E2 measurements are required to verify loss of follicular activity.

Noninstrumented enzyme-linked immunosorbant assay for detection of early pregnancy in macaques.

A practical, noninstrumented enzyme-linked immunosorbant assay (NELISA) for the measurement of urinary monkey chorionic gonadotropin (mCG) has been developed for the detection of early pregnancy in macaque monkeys for use in both the laboratory and the field. Five rhesus monkeys (Macaca mulatta) and six crab-eating monkeys (Macaca fascicularis) were tested for the presence of mCG in urine on gestational days (GDs) 12 to 35. The mCG NELISA detected pregnancy as early as GD 14, with an average earliest detection at GD 16.5 +/- 1.4 (n = 11). Out of 90 tests, 27 false-negative and zero false-positive tests were obtained, for an accuracy of 70.0%. Without the aid of a spectrophotometer, the presence of mCG in pregnant monkey samples was indicated by a dark green color change. Nonpregnant monkey urine samples, on the other hand, exhibited no color change. These findings suggest that the simple, economical, and reliable urinary mCG NELISA may be useful for diagnosing early pregnancy in these and related species. Because capture and restraint are unnecessary for collecting urine samples, the mCG NELISA has widespread potential for confined and free-ranging animals.

Application of an enzyme immunoassay for urinary follicle-stimulating hormone to describe the effects of an acute stressor at different stages of the menstrual cycle in female laboratory macaques.

An enzyme-linked immunosorbent assay (ELISA) for human urinary beta follicle-stimulating hormone (FSH) subunit was validated for use in the laboratory macaque (Macaca mulatta and Macaca fasicularis). This ELISA is based on the dissociation of the FSH heterodimer in urine and the subsequent measurement of the beta subunit as a representation of total urinary FSH. This assay was then used to describe the gonadotropin escape following ovarian senescence in post-menopausal macaques. In addition, the assay was used to observe the impact of an acute stressor on the pituitary-gonadal axis and how the impact of this stressor varies when experienced at different stages of the menstrual cycle. The study design involved the measurement of ovarian steroids and FSH in urine collected daily during a period of time when animals experienced a well-defined event on two occasions consisting of capture, restraint, and anesthesia. This unique study design was made possible by the ability to monitor both ovarian and pituitary function in the absence of confounding daily captures and restraint for blood collection. There was a high correlation between urinary FSH measured in macaques with the beta FSH subunit ELISA and serum FSH measured in paired blood samples by radioimmunoassay (n=39, r2=0.878, P<0.001) and the composite urinary FSH profile obtained from normal, premenopausal macaques exhibited the expected dynamics with a transient rise of FSH during the luteal-follicular transition as well as an acute rise of FSH at mid-cycle. This pattern was lost in castrate and post-menopausal monkeys in which FSH levels were significantly increased (P<0.0001) above those of intact males and young females, respectively. In the stress study, we found that stressors occurring during the luteal-follicular transition not only resulted in acute perturbations of FSH but also led to abnormalities in the subsequent menstrual cycle in 50% of the cases.

ELISA for the measurement of serum and urinary chorionic gonadotropin concentrations in the laboratory macaque.

A rapid, sensitive, enzyme-linked immunosorbant assay (ELISA) for the measurement of chorionic gonadotropin (CG) in serum and urine samples of laboratory macaques is reported. The ligand (CG) is captured by a readily available, widely used, and well-characterized monoclonal antibody (Mab, 518B7) generated against the beta subunit of bovine luteinizing hormone (LH). This Mab, while specific for LH, shows very little species specificity, and has been shown to detect LH and CG by radioimmunoassay (RIA) in both human and non-human primates. A polyclonal antiserum raised in rabbits against human chorionic gonadotropin (hCG) is conjugated to horseradish peroxidase, and is used as the second antibody signal. This anti-hCG antiserum cross reacts with CG secreted by both the human (hCG) and macaque (mCG). The ELISA utilizes hCG as the standard, and results are based on the relative concentrations of mCG in serum and urine. Total assay time is less than 5 hours. Range of the standard curve is 0.002 to 0.5 ng hCG/well, and the least detectable concentration of hCG is 0.0023 +/- 0.0007 ng/well. Pregnancy was detected in early pregnant macaques (M. fascicularis) on 9 (N = 1/16), 10 (N = 1/16), 11 (N = 1/16), 12 (N = 6/16), 13 (N = 1/16), 14 (N = 4/16), and 15 (N = 2/16) days following the pre-ovulatory urinary estrone conjugate peak. The detection of pregnancy by urinary mCG occurred approximately 24 to 72 hours after its detection in serum.

Pregnancy detection by ultrasound and chorionic gonadotropin during the peri-implantation period in the macaque (Macaca fascicularis).

The goal of these studies was to correlate sonographic evidence of pregnancy during the peri-implantation period with the timing of the rise in monkey chorionic gonadotropin (mCG) as measured with an enzyme-linked immunosorbent assay. Animals were time-mated at mid-cycle, and ultrasound examinations were performed on postovulation days 12-15 (n = 77). Pregnancy was sonographically identified in 48 of 77 animals (62.3%), of which 28 had correlative ultrasound/endocrine data collected. For these animals, blood samples were obtained on postovulation days 12-15 for mCG assay. Pregnancy was identified by ultrasound on postovulation days 12 (6/28; 21.4%), 13 (6/28; 21.4%), 14 (8/28; 28.6%) or 15 (8/28; 28.6%). Seven of the 28 (25.0%) were found to have mCG levels consistent with pregnancy (> or = 1 ng/ml) on the same day as ultrasound confirmation, 12 of 28 (42.9%) were sonographically detected as pregnant 1 (n = 6), 2 (n = 3) or 3 (n = 3) days earlier than by mCG, and nine of 28 (32.1%) were found to have elevated mCG levels 1 (n = 7), 2 (n = 1) or 3 (n = 1) days earlier than ultrasound confirmation of pregnancy. The results of these studies have demonstrated (1) the utility of anatomical and endocrine techniques for detecting pregnancy approximately 3 days after the onset of implantation, and (2) the variation in the timing of implantation and the rise in circulating mCG in individual animals.

An enzyme immunoassay for serum and urinary levonorgestrel in human and non-human primates.

A microtiter plate enzyme immunoassay (EIA) is reported for the measurement of levonorgestrel (LNG) in serum and urine samples of human and non-human primates, and the results are compared to data obtained by radioimmunoassay (RIA). Rabbit polyclonal antibodies were raised against the bovine serum albumin conjugate of the 3-O-carboxymethyl oxime (CMO) derivative of LNG. The enzyme label was produced by the conjugation of horseradish peroxidase to LNG at the 3-position by the same CMO bridge used for the immunogen. The assay requires 2.5 hours to perform using 2.2-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt as the chromogenic substrate. Serum (100 microliters) is extracted with petroleum ether prior to assay, whereas urine samples (25 microliters) are diluted and measured directly. The sensitivity of the assay is 0.25 pg/well with a 50% displacement of label at 7.5-9.5 pg and a linear response through 250 pg/well. Minimum levels of 8.7 and 10.0 pg/ml can be detected in serum and urine samples, respectively. Changes in serum LNG concentrations were measured in women and non-human primates following LNG implantation or injection. In the non-human primate study, serum LNG concentrations began to rise rapidly following i.m. injection of LNG, with peak levels occurring on days 3 to 5, then decreasing to approximately 25-35% of peak levels for the duration of the study. Circulating concentrations of 1.86 +/- 0.18 ng/ml LNG were reached in women the first week post-insertion of Norplant implants and decreased by 50% at 7-10 days, 75% after 14-21 days, followed by a steady decrease during the next 60-70 days to constant low levels that exhibited a high individual variation. Correlation coefficients of EIA and RIA results were 0.988 for human serum, 0.926 for human urine, and 0.972 for non-human primate serum.

Studies on the metabolism of testosterone trans-4-n-butylcyclohexanoic acid in the cynomolgus monkey, Macaca fascicularis.

Metabolism of intravenously administered testosterone trans-4-n-butylcyclohexanoate (T bucyclate), a potent, long-acting androgen, was studied in cynomolgus monkeys (Macaca fascicularis). About 5% of the radioactivity of a dose of doubly labeled ester (14C, 3H) was excreted via the gastrointestinal tract. Most of the administered radioactivity was excreted in the urine within 120 h. No intact T bucyclate was recovered from either compartment. Tritium attributed to bucyclic acid and its metabolites was excreted rapidly (peak excretion was at 6h after injection), while 14C excretion, attributed to testosterone and its metabolites, extended over 4 days. Testosterone metabolites were excreted predominantly as sulfate esters. Analysis of urinary products derived from the bucyclic acid moiety of T bucyclate showed no products susceptible to glucuronidase treatment, and showed a mixture of unidentified solvolyzable and unconjugated products. No unmetabolized trans-4-n-butylcyclohexanoic acid was detected in urine or feces. It is concluded that metabolism of testosterone bucyclate is initiated in vivo in cynomolgus monkeys by hydrolysis of ester to testosterone and bucyclic acid. The bucyclate side chain is rapidly cleared, and the testosterone is retained in the circulation.