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Previous in vivostudies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitrousing both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro.NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose–response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose–response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitrointerstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.

Previous investigators have reported that specific somatogenic receptors are present in liver membrane preparations from male rats. However, specific binding was less than 3% of total counts per minute added, and the radioreceptor assay using such receptor preparations was impractical to use. Livers from female rats bound 25% of 125I-labeled human growth hormone added, but binding was not specific in that both prolactin and growth hormone competed. Starting with membrane preparations from male rats, we have systematically studied and improved (a) preparation of the fraction, (b) the method of separation of bound and free, (c) time, temperature, and conditions of incubation, (d) the amount of membrane preparation and treatment of rats before sacrifice, and (e) the quality of the labeled growth hormone. As a result, specific binding increased from less than 3 to 60%. This is offered as a model system for optimization of radioreceptor assay systems. The data indicate that 125I-hGH binding to male rat liver membranes was low, as previously reported, because the preparation and assay system had not been optimized, and not because of an inadequate number of receptors in the male liver.

Specific, high titer anti-GnRH antisera were obtained following immunization of rabbits with a conjugate of thyroglobulin and D-[Lys6]-GnRH. D-[Lys6]-GnRH was chosen to take advantage of an ∊-amino group in the center of the peptide chain and three carbons away from the chain as a unique point of conjugation. The RIA developed with this antisera was validated for the measurement of GnRH in plasma and hypothalamic supernatants. Studies of GnRH metabolic clearance indicated that multicompartmental clearance by different organs was a probable explanation for the rapid disappearance of GnRH from the peripheral circulation.

A Mr 26,000 corticotropin (ACTH)-like material is present in glacial acetic acid extracts of all normal rat extrapituitary tissues. In the present study, beta-melanotropin (beta-MSH) immunoactivity was detected in glacial acetic acid extracts of normal rat extrapituitary tissues. beta-MSH immunoactivity was also present in all extracts (mean +/- SEM, fmol/mg of protein): brain, 71.0 +/- 16.3; stomach, 11.5 +/- 1.6; kidney, 8.9 +/- 0.8; colon, 8.2 +/- 1.1; small intestine, 6.5 +/- 1.1; liver, 4.3 +/- 0.5; and heart, 3.2 +/- 0.5. Except in brain extracts, beta-MSH and ACTH immunoactivities of tissue extracts were strongly correlated to each other (r = 0.79; n = 42). When tissue extracts (except brain) were passed through a Sephadex G-75 (superfine) column, ACTH and beta-MSH immunoactivities were eluted in a single peak corresponding to Mr 26,000. In contrast, for brain extracts, the MrS of major peaks of ACTH and beta-MSH immunoactivities were 4,500 and 8,000, respectively; a smaller peak of Mr 26,000 ACTH/beta-MSH-like material was also eluted. Specific anti-ACTH immunocolumns, which did not bind purified synthetic beta-MSH, adsorbed both ACTH and beta-MSH immunoactivities of all tissue extracts except those of brain. One-third of the beta-MSH immunoactivity in brain extracts adsorbed to the anti-ACTH immunocolumn, but two-thirds of beta-MSH immunoactivity passed through the column. We conclude that ACTH and beta-MSH immunoactivities are present in all normal rat extrapituitary tissues and exist in most tissues on the same molecule. This Mr 26,000 molecule is closely related to the pituitary ACTH/beta-lipotropin common precursor.

Serum prolactin concentrations were determined by radioimmunoassay during estrus cycles in 15 dairy heifers bled by repeated jugular punctures or from a long, indwelling jugular catheter. The method of bleeding was found to influence the serum prolactin concentration and was attributed to nonspecific stress. Further confirmation for the stress effect was provided by a controlled study in other heifers, submitted to specific forms of acute and chronic stress stimuli. In cattle, studies of blood prolactin concentrations under various physiological conditions may be performed only when environmental conditions are carefully controlled.

Using antibody-coated test tubes as a detection method, three types of assay for 17β-estradiol (E2) have been investigated. Method A involved the measurement of E2in the dried ether extract of biological fluids. Method B required a chromatographic step after ether extract, prior to measurement of E2. Method C (direct method) measured E2directly in the biological fluid, diluted with assay buffer. Method C was found reliable for the measurement of E2in human follicular fluid, but not in pregnancy serum. Method A was adequate for pregnancy serum, but overestimated E2in serum from normal adult male and female subjects. Method B was reliable to measure E2in all the biological fluids tested.