The rat ovary is innervated by sympathetic nerve fibers. Since the development and survival of peripheral sympathetic neurons innervating nonreproductive organs have been shown to depend on the production of nerve growth factor (NGF) by the innervated tissues, the present experiments were undertaken to determine if the immature rat ovary has the capability of synthesizing NGF. Blot hybridization of ovarian polyadenylated RNA (A+-RNA) to a NGF cRNA probe revealed the presence of a 1.3- to 1.4-kilobase (kb) mRNA species similar to mature NGF mRNA detected in mouse submaxillary gland, a source rich in NGF. Quantitation of NGF protein by a sensitive and specific two-site enzyme immunoassay demonstrated the presence of NGF in juvenile ovaries at levels comparable to those found in other sympathetically innervated tissues. Neither denervation of the ovary nor treatment with gonadotropins (hCG and FSH) or somatomammotropins (PRL and GH) affected the levels of NGF mRNA. However, denervation significantly increased NGF levels, suggesting that, as in other target tissues, denervation prevents the retrograde transport of NGF by the sympathetic terminals and leads to accumulation of the protein at its site of production. It is concluded that 1) the developing ovary is able to both transcribe the NGF gene and translate its mRNA into NGF protein; and 2) the NGF content in the ovary is regulated by its innervation. The results provide the biochemical basis for the concept, elaborated in the companion paper, that NGF through its trophic actions on ovarian sympathetic neurons contributes to the regulation of ovarian development and, hence, to the acquisition of female reproductive capacity.

Evidence exists that a norepinephrine/ prostaglandin E2 (PGE2)/cAMP pathway is involved in the regulation of luteinizing hormone-releasing hormone (LHRH) secretion. The aim of the present experiments was to determine if release of LHRH from the immature rat hypothalamus could also be stimulated by activation of protein kinase C. Median eminences from 28-day-old female rats were incubated in vitro with either dioctanoylglycerol (a synthetic diacylglycerol that selectively activates protein kinase C in intact cells) or 4fi-phorbol 12(-myristate 13a-acetate (another protein kinase C activator). Both agents increased LHRH release, the response to dioctanoylglycerol being more pronounced than that to the phorbol ester. This direct activation ofprotein kinase C was not accompanied by changes in PGE2 formation. Activation of the PGE2/cAMP pathway by either norepinephrine, PGE2, or forskolin (a stimulator of adenylate cyclase) increased LHRH release. Dioctanoylglycerol or phorbol ester in conjunction with either norepinephrine, PGE2, or forskolin resulted in an additive effect on LHRH release suggesting coexistence of both pathways. Phospholipase C, which activates protein kinase C via formation of diacylglycerol, increased the release of both LHRH and PGE2. This suggests that an increase in endogenous phospholipase C activity caused by neurotransmitter inputs may lead to both activation of protein kinase C and PGE2 formation. Blockade of cyclooxygenase activity by indomethacin obliterated phospholipase C-induced PGE2 release. The same treatment reduced the LHRH response by only 50% indicating that protein kinase C activation can cause LHRH release in the absence of PGE2 synthesis. It is suggested that the median eminence of the rat possesses a protein kinase Cdependent pathway that is coupled positively to LHRH release and complements PGE2/cAMP-dependent mechanisms. Norepinephrine, however, does not appear to be the neurotransmitter responsible for activating the protein kinase C pathway. Simultaneous activation of both pathways may provide a mechanism by which a large increase in LHRH secretion occurs, such as in the afternoon of first proestrus.The intracellular mechanism by which norepinephrine (NE) elicits luteinizing hormone-releasing hormone (LHRH) release involves the activation of prostaglandin E2 (PGE2) and cAMP formation (1-3). Many neurotransmitters, however, are known to provoke phosphatidylinositol degradation upon binding to their specific membrane receptors and evoke a cascade of events (4) that, independently of cAMP, leads to regulation of cellular function. A key product of this cascade is diacylglycerol, which appears to control phosphorylation processes through activation of protein kinase C, a Ca2' activated, phospholipid-dependent kinase, which in the presence of diacylglycerol becomes attached to membranes and acquires enhanced enzymatic activity (4, 5). Brain tissue is particularly rich in protein kinase C, which is, to a large extent, localized in the synaptosomal fraction (6, 7).These ...

Experiments were performed to examine whether estradiol (E₂) can influence some of the intraneuronal mechanisms involved in luteinizing hormone-releasing hormone (LHRH) release during the onset of puberty in the female rat. The capacity of median eminence (ME) nerve terminals to secrete LHRH, as determined by both their basal release of LHRH and by their response to prostaglandin E₂ (PGE₂) in vitro, increased significantly during the juvenile-early peripubertal periods of development (postnatal days 22–34). Ovariectomy (OVX) on day 22 led to a striking reduction in LHRH response to PGE2 on day 34. E₂ administered via s.c. Silastic capsules, at a dose that reproduces juvenile serum E₂ levels, restored the response. Simulation of first proestrous serum E2 levels in late juvenile (28-day-old) female rats enhanced both the sensitivity and the responsivenes of LHRH-containing terminals to PGE₂. Furthermore, E₂ enhanced the sensitivity and the responsiveness of LHRH terminals to norepinephrine (NE). This effect appeared to be related to both the increased LHRH response to PGE₂ and an enhanced sensitivity of the PGE₂-synthesizing pathway to NE. This is because MEs from E2-treated rats showed a marked increase in PGE₂ release in response to a NE concentration which was barely effective in untreated controls. It is suggested that one of the mechanisms by which E₂ activates the first preovulatory discharge of LHRH release in the female rat is by facilitating the occurrence of two different but sequentially related biochemical events: the stimulation of PGE₂ formation by NE and the enhancement of LHRH release by PGE₂. In addition, it appears that maintenance of LHRH responsiveness to PGE₂, which has been implicated as an obligatory component of NE-induced LHRH release, is E=-dependent.

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