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J. K. Hodges

Summary. The positive-feedback action of oestradiol-17β on LH release was studied in gonadectomized male and female and intact male marmoset monkeys. Positive feedback was observed in normal intact males in response to a single subcutaneous injection of 35 μg oestradiol benzoate. The sustained elevation in oestradiol-17β levels achieved by the injections resulted in a marked suppression of circulating testosterone concentrations. Subcutaneous injections (0·5 mg/injection) of testosterone or dihydrotestosterone to gonadectomized males and females immediately following and 8 h after oestradiol benzoate failed to inhibit positive feedback. Similar treatment with progesterone (0·0 mg/injection) tended to standardize both positive and negative components of the feedback response. In contrast, progesterone implants (achieving progesterone concentrations similar to those obtained with the injections), maintained during and for 8 days before the oestradiol benzoate treatment, effectively inhibited positive feedback. LH responses to the various treatments were similar in gonadectomized males and females. These data suggest a relative unimportance of testicular secretions in suppressing oestrogen-induced LH release in the adult marmoset and indicate similarities between the control of positive feedback in male and female primates.

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J. E. Hindle and J. K. Hodges

Summary. 14C-Labelled oestradiol-17β and progesterone (50 μCi each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92·4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41·2% and 51·4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20α-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17β and oestradiol-17α in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17α and oestradiol-17β. The results have established the major excreted metabolites of oestradiol-17β and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.

Keywords: metabolism; oestradiol-17β; progesterone; urine; faeces; rhinoceros

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J. K. Hodges and J. P. Hearn

Summary. Subcutaneous injections of oestradiol benzoate in oil, resulting in a sustained elevation of circulating oestradiol levels, induced an initial suppression of LH secretion, followed by a positive discharge of LH in castrated male and female and in intact male marmosets. Oestrogen-induced LH release (producing maximum LH concentrations 24 h after the injection) was observed in 75% of castrated males and females. A positive discharge of LH occurred in 50% of intact males 28–36 h after oestrogen administration.

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J. Rosenbusch, A. Bellmann, and J. K. Hodges

In this study, the molecular masses and isoelectric characteristics of pituitary LH and FSH in two species of callitrichid primate, the common marmoset (Callithrix jacchus) and the cotton-top tamarin (Saguinus oedipus), were determined. Comparative data for urine samples from Callithrix jacchus are also presented. The separation of gonadotrophins from pituitary extracts and urine was performed under nonreducing conditions using SDS–PAGE and isoelectric focusing procedures. Hormone activity in gel eluates was determined by in vitro bioassays for LH and FSH and by a microtitre plate enzymeimmunoassay for LH. The molecular masses of pituitary and urinary proteins were between 36 and 37 kDa for LH and FSH, and were similar in both species. A dimer form of pituitary LH with a molecular mass of 33 kDa was also found in the cotton-top tamarin, but not in the marmoset. Guanidine–HCl dissociation of gonadotrophins from marmoset and tamarin pituitaries before electrophoresis gave proteins of 16 and 28 kDa, and 16 and 25 kDa range, respectively. Isoelectric focusing revealed numerous peaks of bioactivity for both LH and FSH, indicating the presence of multiple molecular variants (isoforms) of each hormone. In both species pituitary FSH eluted over a narrower and more acidic pH range than LH. Isoelectric focusing profiles for pituitary and urinary LH in the marmoset were similar (pH range 5.0–8.5), whereas urinary FSH demonstrated a more acidic profile than the pituitary protein. These results give comparative information on the properties of New World primate gonadotrophins, which should be useful in studies of their physiological action and in aiding the development of improved reagents and assays for their detection.

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M. Heistermann, S. Tari, and J. K. Hodges

Measurement of immunoreactive progesterone, pregnanediol and oestradiol in faeces collected throughout ovarian cycles in three species of callitrichid primates is reported. Faecal hormone concentrations were compared with plasma progesterone profiles during PGFcontrolled (n = 7) and natural (n = 8) cycles in Callithrix jacchus and Saguinus fuscicollis, respectively, and with urinary oestrone conjugates during five cycles in Saguinus oedipus. Unconjugated steroids, which predominated over enzyme hydrolysable conjugates in samples from all species, were used to generate cycle profiles. According to results from HPLC, oestrone and oestradiol accounted for virtually all oestrogen immunoreactivity, and oestradiol most often predominated, whereas large amounts of nonspecific immunoreactivity were detected by both progesterone and pregnanediol assays. Faecal progestins were excreted in a cyclic manner in all species; luteal phase values were on average five- to tenfold higher than corresponding follicular phase values. Significant increases in mean amounts of faecal progestins were seen within 48 h of the post-ovulatory rise in plasma progesterone. Although a similar trend was also seen for faecal oestradiol, a clear and consistent luteal phase increase was seen only in Callithrix jacchus and this generally occurred later than that of progestins. The results indicate that faecal progestin analysis provides a useful method for noninvasive reproductive assessment in callitrichid primates. In particular, measurement of immunoreactive pregnanediol enables a multispecies application of a single assay methodology for comparative studies on callitrichid reproductive function.

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J. E. Hindle, E. Möst, and J. K. Hodges

Summary. The development of a sensitive enzyme-immunoassay for 20α-dihydroprogesterone (20α-DHP) and its use in determining reproductive status in black and white rhinoceroses is reported. 20α-DHP in hydrolysed urine diluted in parallel to standards, and high-performance liquid chromatography (HPLC) confirmed the presence of 20α-DHP and the absence of pregnanediol-3α-glucuronide (PdG) in urine collected from rhinoceroses after oestrus. Conjugated oestrone was identified by HPLC as the major urinary oestrogen in the black rhineroceros and conjugated oestradiol-17β was the most abundant in the white rhinoceros. In African species, the black (Diceros bicornis), and northern (Ceratotherium simum cottoni) and southern (Ceratotherium simum simum) white rhinoceroses, excretion of 20α-DHP and oestrogen followed a cyclic pattern. Excretion of 20α-DHP was low before mating, at the time of peak oestrogen excretion, but high after oestrus. In the black rhinoceros, the follicular phase was 3–4 days and the luteal phase was 18 days, suggesting a cycle of 21–22 days. The interoestrus interval in the northern subspecies of white rhinoceros was 25 days, which correlated well with the interval between peaks of oestradiol-17β excretion. The interval between urinary oestrogen peaks in the southern subspecies of white rhinoceros suggested a cycle length of 32 days. This paper provides the first description of the pattern of excretion of urinary oestrogens and progesterone metabolites in African rhinoceroses.

Keywords: ovarian cycles; enzyme-immunoassay; urine; steroid metabolites; rhinoceros

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C. A. Niemuller, H. J. Shaw, and J. K. Hodges

The development of an enzymeimmunoassay for 5β-pregnanetriol and its use for non-invasive monitoring of reproductive cycles in Asian elephants is described. Gas chromatography–mass spectrometry (GCMS) and high performance liquid chromatography (HPLC) confirmed the presence of 5β-pregnane-3α,17α,20α/β-triols as the two most abundant urinary progesterone metabolites. The assay developed used the antiserum anti-5β-pregnane-17α,20α-diol-3α-γl glucuronide but was designed to measure the free steroid in urine samples after hydrolysis and extraction. HPLC confirmed the presence of immunoreactive pregnanetriol in urine, but indicated that the measurement was nonspecific. Immunoreactive pregnanetriol concentrations were significantly correlated with the concentrations of both progesterone (r = 0.98, n = 269, P < 0.01) and 17α-hydroxyprogesterone (r = 0.95, n = 205, P < 0.01), the metabolic precursor of pregnanetriol. The mean ± sem deviation of cycles as determined by measurements of plasma progesterone, 17α-hydroxyprogesterone and urinary pregnanetriol, respectively, were 15.54 ± 1.5 (n = 23, where n = number of cycles), 15.21 ± 1.7 (n = 15) and 15.45 ± 0.94 weeks (n = 20). These results demonstrate that it is possible to monitor ovarian function in Asian elephants by the measurement of urinary immunoreactive pregnanetriol concentrations.

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P. M. Summers, Caroline J. Wennink, and J. K. Hodges

Summary. A single intramuscular injection of 0·5 μg cloprostenol was not luteolytic on Day 6 or 7 of the ovarian cycle (N = 3), but was luteolytic in some animals (3/5) on Day 8 and 9 and luteolytic in all 23 animals treated between Days 10 and 17 of the ovarian cycle, and in 7 animals treated between Days 19 and 43 of pregnancy. Luteal function was monitored by measurement of progesterone in peripheral blood using a simple and rapid non-extraction assay. There was a dramatic fall in peripheral blood progesterone to <10 ng/ml within 24 h of cloprostenol injection; progesterone remained at this low level until the day after post-treatment ovulation. The interval from cloprostenol injection to ovulation in animals treated between Days 8 and 17 was 10·7 ± 0·3 days. A similar interval was found in pregnant animals. Embryos recovered from the uterus after cloprostenol treatment were morphologically normal (23/24).

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J. K. Hodges, Cilla Henderson, and J. P. Hearn

Summary. Concentrations of progesterone, 17α-hydroxyprogesterone, oestrone and oestradiol-17β in peripheral and utero-ovarian vein blood were measured during the first 60 days of pregnancy. The same hormones were also measured in peripheral blood samples from non-fertile cycles. Peripheral levels of 17α-hydroxyprogesterone, oestrone and oestradiol increased gradually during early pregnancy whereas concentrations of progesterone declined. The patterns of secretion of progesterone, 17α-hydroxyprogesterone and oestrone, but not oestradiol, were significantly different in fertile and non-fertile cycles by 15 days after ovulation.

Comparison of hormone values in peripheral and utero-ovarian vein samples from ovaries with and without corpora lutea (Days 7, 9, 13, 21, 40 and 60 of pregnancy) showed that: (a) progesterone was secreted by the corpus luteum until at least Day 40 by which time there was also placental secretion; (2) although 17α-hydroxyprogesterone was secreted by the corpus luteum, the relative contribution of luteal and placental secretion after Day 21 was not clear; (3) oestrone secretion by the corpus luteum was no longer detectable by Day 40, but placental oestrone secretion appeared to be present by this time; (4) the corpus luteum did not secrete significant amounts of oestradiol at any stage of early pregnancy, although there was evidence for placental secretion by Day 40.

These results suggest that progesterone secretion by the corpus luteum of early pregnancy continues beyond the time when oestrogen secretion has declined. The corpus luteum to placental shift in the marmoset appears to occur at a later stage of pregnancy than it does in the macaque monkey and probably also in man.

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J. K. Hodges, Heather Brand, Cilla Henderson, and R. W. Kelly

Summary. The levels of immunoreactive oestrone, oestradiol-17β and oestriol in plasma and urine were measured during early, mid- and late pregnancy in the marmoset monkey. In plasma, unconjugated oestrone remained <2% of total (conjugated plus unconjugated) oestrone throughout gestation, whereas unconjugated oestradiol-17β increased from 3% of the total value in early and mid-pregnancy to 35% in late pregnancy. The reversal in the unconjugated oestrone: oestradiol-17β concentration ratio from early (12:1) to late (0·15:1) pregnancy occurred despite the continuing predominance of oestrone in terms of total hormone. Total oestriol was measurable but in relatively low concentrations. Oestradiol conjugate was the predominant urinary oestrogen metabolite measured at each stage of pregnancy. The pattern of urinary oestrone and oestradiol-17β reflected plasma levels of total hormone, rather than unconjugated hormone, showing no further increase after mid-pregnancy. In contrast, oestriol increased throughout pregnancy and to a proportionately greater extent than oestrone or oestradiol-17β, but at lower absolute levels.

High-pressure liquid chromatography of urine extract indicated the presence of considerable amounts of oestrogen immunoreactivity not accounted for by oestrone, oestradiol-17β and oestriol and with a retention time similar to that of 16α-hydroxyoestrone. Gas chromatography and mass spectroscopy provided further evidence to suggest that 16α-hydroxyoestrone is an abundant urinary oestrogen metabolite during pregnancy in the marmoset monkey.