REGULATION OF PROLACTIN

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.