B. D. Schanbacher

Radioimmunoassay of Ovine and Bovine Serum Progesterone Without Extraction and Chromatography

A procedure for the radioimmunoassay of ovine and bovine serum progesterone is described which does not require extraction and chromatography. Serum samples are assayed directly, and a highly specific antiserum that was prepared in rabbits against 11 alpha-hydroxyprogesterone conjugated to bovine serum albumin is used. Interference from serum binding proteins is alleviated by use of a phosphate buffer containing 5% BSA and separation of bound and free tritiated progesterone by a double antibody procedure. Serum samples are assayed in a mini-vial, the bound fraction (double antibody precipitate) is mixed with scintillation solution and the radioactivity is counted in the same vial. The assay procedure is sensitive (10 pg, 100 pg/ml) and has acceptable accuracy and precision. Because there is no extraction or chromatography, serum progesterone is not lost. Most important, the procedure is specific for progesterone and measures serum progesterone concentrations in the ewe and cow which are comparable with those obtained with conventional assay techniques. The progesterone assay described herein provides a rapid, economical procedure that can facilitate the study of ovarian cyclicity and aid in the early diagnosis of pregnancy.

Pituitary androgen receptors and the resistance of long-term castrated rams to the androgenic control of luteinizing hormone (LH) secretion

Luteinizing hormone (LH) responses of short-term (24 h) and long-term (6 mo) castrated rams to testosterone replacement therapy were investigated. Testosterone filled Silastic capsules which maintained physiological concentrations of testosterone in blood (approximately 3.2 ng/ml) prevented the post-castration rise in serum LH of short-term castrated rams but failed to effectively reduce the established elevated serum LH levels in long-term castrated rams. The LH response to exogenous LHRH was also suppressed in the short-term castrated rams, whereas the response was increased in direct proportion to basal LH levels in long-term castrates. The possibility that a change in pituitary androgen receptors may explain the resistance of long-term castrates to testosterone feedback was examined. For this purpose, a cytosolic androgen receptor binding assay was developed using tritiated methyltrienolone (R 1881) as the labeled ligand and dextran-charcoal to separate free and protein-bound steroid. Stable, high affinity (Kd = 0.3-1.5 nM), saturable binding of R 1881 was demonstrated in pituitary cytosol from both intact and castrate rams. Relative binding specificities in intact rams, however, suggested partial binding to a progestin-binding component, whereas receptor binding in long-term castrates was androgen specific. For this reason, androgen receptor binding was studied after addition of triamcinolone acetonide to occupy apparent progestin binding sites. We observed no differences in either androgen receptor concentration (4.10 +/- 0.70 versus 3.23 +/- 0.45 fmol/mg protein; Mean +/- SD) nor ligand affinity at 0 degrees C (Kd = 0.66 +/- 0.34 versus 0.40 +/- 0.08 nM) in 6 mo compared to 24-h castrate rams. These data indicate that the inability of androgens to suppress gonadotropin secretion in long-term castrate rams is unlikely to be related to specific changes in pituitary cytosol androgen receptors.

Use of hypothalamic releasing hormones to evaluate the reproductive endocrine status of rams

Pituitary and testicular responses to exogenous gonadotrophic hormone releasing hormone (GnRH, 5 ng/kg body weight) and thyrotrophin releasing hormone (TRH, 50 ng/kg body weight) were assessed in four breeds of ram (n = 5/breed) maintained under alternating short (8L:16D) and long (16L:8D) photoperiods each of 12 weeks duration. The amplitudes of LH responses (ΔLH) to a single injection of GnRH were higher (P < 0.01) after 12 weeks of 16L:8D (9.8 ± 1.2 ng/ml, mean ± SEM for all rams) compared with responses after 12 weeks of 8L:16D (5.7 ± 0.8 ng/ml). Plasma testosterone concentrations were higher (P < 0.01) after 12 weeks of 8L:16D (7.2 ± 1.0 ng/ml) than after 16L:8D (0.7 ± 0.1 ng/ml) and the amplitudes of testosterone responses after injection of GnRH were also greater (P < 0.01) under 8L:16D (14.3 ± 1.3 ng/ml) than under 16L:8D (4.6 ± 0.3 ng/ml). Prolactin concentrations were elevated under 16L:8D (166 ± 31 ng/ml) and suppressed under 8L:16D (15 ± 2 ng/ml) and the areas under the prolactin response curves were likewise greater (P < 0.01) under 16L:8D (83 ± 9, arbitrary units) than under 8L:16D (24 ± 5). The results indicate that hypothalamic releasing hormones can be used to obtain information on the endocrine status of rams. The acute testosterone response to GnRH injection is of particular interest since it not only reflects testosterone secretory capacity, but it also provides an index of prevailing LH secretion based on the absolute dependence of steroidogenesis on gonadotrophin support.