Lisa S. Laughlin

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 anti‐body (Mab, 518B7) generated against the β 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 anti‐body 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. Am. J. Primatol. 41:307–322, 1997.

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, pre‐menopausal 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. Am. J. Primatol. 48:135–151, 1999.

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.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on chorionic gonadotrophin activity in pregnant macaques.

As many as 62% of all human conceptions are lost prior to 12 weeks of pregnancy and it is unknown how many of these losses result from environmental hazards. Previous studies have shown that single doses of 1, 2, and 4 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administrated orally to cynomolgus macaques during the peri-implantation period leads to early fetal loss (EFL) within 10-20 days. TCDD induced EFL is associated with a reduction in the biological activity of monkey chorionic gonadotrophin (mCG) but no change in the immunoreactive mCG profile. These studies are consistent with either a direct effect of TCDD on differentiation of the trophoblast and an indirect effect on mCG synthesis, or a direct effect on mCG synthesis and secretion independent of trophoblast development. The present study was designed to test the hypothesis that the action of TCDD is directly on mCG synthesis rather than on the differentiation of the trophoblast. Female macaques (Macaca fascicularis) were treated with a single dose of TCDD (4 microg/kg b.wt.) on Gestational Day 20, a stage of pregnancy following initial trophoblast differentiation and invasion. Circulating mCG concentrations were monitored for the next 6 days. Compared to the controls, the peak level of serum bioactive mCG was lower in the treated group (P<0.05), with a decrease observed on the day following exposure. The bioactive/immunoreactive mCG ratio was also lower in the treated group compared to the controls (P<0.05). There was no difference in serum immunoreactive mCG levels between the groups. Histological evaluation of the embryo-placental unit showed increased apoptosis and vascular congestion after treatment but was otherwise grossly normal. Because exposure of the conceptus to TCDD following differentiation of the trophoblast decreased the bioactivity of circulating mCG, we conclude that the action of TCDD in the placenta is directly on mCG synthesis.

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.