Prolactin content in rat pituitary gland. RIA of prolactin after different extraction procedures

Abstract

The prolactin content of rat pituitary varies considerably when determined by RIA, due to incomplete solubilization of prolactin secretory granules with standard procedures for tissue homogenization and centrifugation. Freezing and thawing, Triton X-100 and ultrasonic treatments increased the yield of prolactin significantly but electron microscopy of pellets revealed numerous unmodified secretory granules. Addition of 2.5 m urea produced complete extraction of tissue prolactin confirmed by RIA of supernatants and electron microscopy of pellets. Radioimmunoassay (RIA) has greatly expanded our knowledge of circulating hormones and the secretory activity of endocrine glands. The use of RIA for quantification of prolactin in the pituitary gland, however, has been less successful if judged by the great variation in prolactin content reported by different investigators (Voogt et al. 1970;Dickerman et al. 1972;Relkin 1972;Nicoll et al. 1976). Analysis of these reports suggests that the erractic results in pituitary prolactin content can be ascribed more to the extraction procedure em¬ ployed than to the RIA itself. There is no doubt that hormones stored in pituitary cells are only partially solubilized after routine procedures for cell disruption and homogenization. Furthermore, repeated application of the same procedure, such as freezing and thawing, results in a progressive increase of assayable prolactin. Since a complete extraction of tissue prolactin is critical for studies involving measurements of pituitary hormonal contents, we have tested and compared the me¬ thods used by different investigators as a guideline for future experimental studies.

Choice of Extraction Procedure for Estimation of Anterior Pituitary Hormone Content.

“…Zanini and Giannattasio (1972) compared the efficiency of the extraction media by using electrophoresis and electron microscopy, concluding that 2.5 M urea completely solubilized PRL and GH, while water and phosphate buffer solubilized only a part of these hormones. Haggi and Aoki (1981) effect of urea, and further reported that F-T, sonication, or 1% triton X-100, though enhancing the efficiency of phosphate buffer, did not cause complete solubilization of PRL. In our previous report (Ishikawa et al, 1985), we demonstrated that PRL, which was solubilized with 0.25 M ammonium sulfate, pH 5.5, had isoelectric points quite different from those of PRL which remained in the residue and was solubilized with 60% EtOH pH 9.5.…”

Pituitary Prolactin Concentrations Associated with Daliy Prolactin Surges in Pseudopregnant Rats

“…It has been reported that some PRL present in the pituitary is not extracted into the supernatant when the pituitary is extracted by the common method with a homogenizer [10], and further, that secretory granules containing PRL particles are not disrupted in the buffer with isotonic solution [10,12,221. Therefore, the residue after Polytron extraction was disrupted with a Sonifier.…”

Mechanisms for altered reproductive function in female rats following neonatal administration of raloxifene

“…Pituitaries were dissected, weighed and homogenized in 1 ml physiological saline containing urea (2.5 mol/l) and subjected to ultrasonic treatment (20). Samples were centrifuged for 20 min at 2800 g and the supernatant was frozen at 2 20 8C until analysis for hormone content.…”