• Bethea, Cynthia (PI)

    Project: Research project

    Project Details


    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.
    Effective start/end date1/1/8312/31/00


    • National Institutes of Health


    • Medicine(all)


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