The goal of this program for the next 5 years is to find the progesterone (P) target neurons in the hypothalamus which mediate an increase in prolactin (PRL) secretion in primates. Progesterone increases prolactin secretion only in estrogen (E)-primed monkeys. We have proven that progesterone receptors (PR) are absent in lactotropes. Together, the data suggest that E must first induce PR in a neuronal population and then P acts via PR in these target neurons to increase PRL secretion. Therefore, it is now inevitable to ask """"""""which neurons?' We know- (1) the neurons must respond to estradiol with an induction of PR, (2) the PR-containing neuron must either directly influence PRL secretion or synapse on a neuronal population that increases PRL secretion, and (3) there are several candidate neuronal populations which have been strongly implicated in the regulation of PRL in rodents and primates. We will compare PR immunocytochemistry (ICC) in the brains of spayed and E-treated monkeys to learn where PR is induced. Candidate neurohormones which regulate PRL secretion are dopamine (DA), serotonin (5HT), oxytocin (OT), vasoactive intestinal peptide (VIP), and substance P. We will determine if any of these candidate populations are progesterone target neurons. Using fluorescent modifications of the ICC techniques developed in pituitary, these populations of neurons will be double-labeled for their cytoplasmic transmitter and for nuclear PR in macaque hypothalami and brainstem. Simultaneously, OT, VIP, substance P, DA, and 5HT will be measured in discrete brain punches from control spayed, E- and E+P-treated monkeys. Identification of a candidate neuronal population as PR positive or as receiving synaptic input from PR positive neurons, plus changes in the hypothalamic content of the candidate neurohormone upon E+P treatment would provide rationale to test whether the P-induced increase in PRL can be blocked with an antagonist to the identified neurohormone. In another approach, we will use subtractive hybridization to identify unique mRNA species induced by progesterone. Messenger RNA will be isolated from PR positive hypothalamic areas in E-treated monkeys and E+P-treated monkeys. cDNA libraries will be made from the mRNA and probed with single-stranded cDNA remaining after a -subtraction hybridization.- Subtraction hybridization will be conducted with radiolabelled cDNA made from reverse-transcribed MRNA extracted from the hypothalamus of an E+P-treated monkey hybridized with excess mRNA extracted from the hypothalamus of an E-treated monkey. The non-hybridizing single-stranded cDNAs remaining represent a greatly enriched mix of cDNAs unique to P-induction. Probing a cDNA library from an E+P-treated monkey hypothalamus with this mix will identify CLones which represent unique cDNAs from P-induction of unique mRNAs. The cDNAs will later be cloned, sequenced, and translated for the identification of protein products which are induced by progesterone. The results of this research will ultimately determine how progesterone increases prolactin secretion in primates.
