Regressive changes occurring in the pituitary gland of the rat after removal of litters were studied. Pituitary glands of lactating rats were characterized by the presence of numerous hypertrophied lactotrophs. Interruption of lactation caused a blockade of prolactin synthesis and secretion, followed by degeneration of lactotrophs. Morphometric analysis of pituitary glands revealed that lactotrophs accounted for about 50% of the total hypophysial cell count in lactating rats. This percentage decreased progressively and reached pre-pregnant levels 7 days after removal of litters; the decrease was inversely correlated with an increase in the number of degenerating lactotrophs which comprised 30% of all lactotrophs 72 h after removal of litters. The morphological changes found in lactotrophs were closely related to changes in the prolactin content of serum and the pituitary gland. Regression of lactotrophs appeared to be the most important cause inducing the reversal of hypophysial lactotrophic activity to pre-pregnant conditions.

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.

Two pools of prolactin (Prl) were isolated from the pituitary gland. One readily soluble after homogenization and the other sedimented with secretory granules. In ovariectomized-oestrogen treated rats the latter represents the major Prl fraction of pituitary and can be recovered as immunoreactive Prl after extraction in 2.5 m urea. By correlation of electron microscopy, chromatography and RIA, evidence is gathered that the soluble fraction is constituted by monomeric (little) Prl and that secretory granules contain Prl in a polymerized form (big).

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