Prolactin action on luteal protein expression in the corpus luteum

Although it is well established that PRL is essential for luteal cell steroidogenesis and growth in the rat, its exact mechanism of action remains unknown. Whereas PRL stimulates growth and induces the content of specific proteins in several target tissues, its effect on proteins in the corpus luteum has not been examined. To determine whether PRL affects the synthesis of specific luteal protein(s), corpora lutea were obtained from rats hypophysectomized on day 3 of pregnancy and then treated with or without PRL (125 μ x 2/day) for 4 days. In this rat model, corpora lutea are exquisitely responsive to PRL and produce high levels of progesterone in response to PRL stimulation. Analysis of cytosolic proteins on one- and two-dimensional gel electrophoresis revealed a dramatic inhibitory effect of PRL on a 37,000 mol wt (MW) protein. This PRL-regulated protein (PRP) resolved into five separate isoelectric variants (pIs 5.5, 5.6, 6.15, 6.6, and 6.85). Since differences in phosphorylation state can alter the isoelectric point of proteins, we examined the possibility that the 37,000 MW PRP is a substrate for phosphorylation. Luteal homogenates were incubated with [³²P]ATP in the presence or absence of Ca²⁺, Ca²⁺/calmodulin, Ca²⁺/phospholipid, or cAMP. Phosphorylation of PRP was not affected by PRL treatment or the addition of specific cofactors. The least abundant isoelectric species (pI 6.85) was identified by Western blot analysis as annexin I, a known regulator of phospholipase A₂ and prostaglandin synthesis. To determine whether the other four isoelectric species represent variants of the same protein, we developed a polyclonal antibody to the 6.15 isoelectric species which is remarkably regulated by PRL. In the absence of PRL, the 6.15 isoelectric species (37K) is the major protein of the PRP band. Conversely, PRL treatment resulted in the virtual disappearance of this luteal protein. The antibody recognized the 37K alone, indicating that the other 37,000 MW isoelectric species were distinct proteins. To examine the time course of PRL action on the expression of the 37K, luteal cells from day 3 pregnant rats were cultured with different doses of PRL from 6 h to 5 days. Western blot analysis of luteal cellular proteins indicated that PRL caused a decrease in the expression of the 37K within 6 h of treatment. Although it is well known that estradiol together with PRL is required for optimal growth of the rat corpus luteum, estradiol alone had no inhibitory action on the 37K nor did it affect the inhibitory action of PRL. Developmental studies performed on corpora lutea obtained from rats at different stages of pregnancy have revealed that the 37K is absent from highly active steroidogenic corpora lutea and appears abruptly before parturition during luteolysis. In summary, the results of this investigation demonstrate that PRL substantially down-regulates the expression of the abundant 37,000 MW protein(s) in the corpus luteum. The least abundant of these proteins has been identified as annexin I. The major protein (37K), to which we developed a specific antibody, is highly tissue-specific and is expressed only in the corpus luteum undergoing luteolysis. This 37K protein, which is remarkably regulated by PRL, can serve as a marker for both PRL action in the corpus luteum as well as luteal regression.