Nerve Growth Factor Receptors in the Peripubertal Rat Ovary

Gregory A. Dissen, Diane F. Hill, Maria E. Costa, Ying Jun Ma, and Sergio R. Ojeda Division of Neuroscience (G.A.D., D.F.H., M.E.C., S.R.O. Oregon Regional Primate Research Center Beaverton, Oregon 97006 Department of Physiology (Y.J.M.) Oregon Health Sciences University Portland, Oregon 97201

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization. Immunohistochemical examination of the ovary using monoclonal antibody 192 immunoglobulin G, which recognizes the low affinity form of NGFrec, localized the NGFrec protein to ovarian nerve fibers and, unexpectedly, to thecal cells of developing follicles; some immunoreactive material was also detected in follicular granulosa cells but not in luteal cells of newly formed corpora lutea. Detection of NGFrec mRNA by RNase protection assay confirmed the 0888-8809/91/1642-1650$03.00/0 Molecular Endocrinology Copyright© 1991 by The Endocrine Society

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ability of granulosa cells to synthesize NGFrec. Hybridization histochemistry using the same probe used for RNA blots confirmed the thecal cells as a site of NGFrec synthesis and verified the relative absence of NGFrec synthesis in the corpus luteum. The results demonstrate that the gene encoding the predominant low affinity class of NGFrec is expressed in a subset of endocrine steroid-producing cells of the developing ovary and raise the possibility that NGF and other members of the NGF family may act in endocrine glands as neuroendocrinotropic factors that functionally link the nervous and the endocrine systems. (Molecular Endocrinology 5: 1642-1650, 1991)

INTRODUCTION As in other sympathetically innervated tissues (1, 2), development of ovarian innervation requires the presence of target-derived nerve growth factor (NGF) (3, 4). Neutralization of NGF actions by administration of antibodies to the peptide during the early postnatal period prevented the sympathetic innervation of the gland and reduced its sensory innervation (3). Immunosympathectomized animals exhibited conspicuous reproductive abnormalities, the most overt being delayed sexual development and irregular estrous cyclicity. A detailed examination of the hypothalamic-pituitary-ovarian axis in these animals indicated that reproductive dysfunction induced by neonatal NGF deficiency is primarily due to ovarian failure (3). The ovaries of animals treated with NGF antibodies exhibited stunted follicular development and reduced estradiol secretion in the face of elevated or normal serum gonadotropin levels (3). It appears that, as shown in other tissues innervated by sympathetic fibers (5, 6), NGF produced in the ovary is transported in a retrograde fashion by the innervating fibers (4) to reach the neuronal perikarya where its tropic effects are initiated. The transport process has been postulated to involve a two-step mechanism (7, 8) by which NGF first binds to low affinity (fast) recep-

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tors (9), which then transfer the peptide to high affinity (slow) receptors (9,10) synthesized in the soma of the innervating neurons. While it has only recently been shown (11) that the formation of high affinity NGF binding sites requires the interaction of NGF with both the low affinity receptor and the tyrosine kinase product of the trk protooncogene (12, 13), the low affinity receptor has been known for some time to be a 77kilodalton (kD) transmembrane protein encoded by a 3.8-kilobase (kb) mRNA, the product of a single gene (14, 15). This receptor is the most abundantly expressed of the two classes; it is found in both neuronal and nonneuronal tissues and until now was thought not to mediate NGF responses (16). Recently, however, evidence has been presented showing that the low affinity NGF receptor is indeed involved in signal transduction when coupled with the tyrosine kinase product of the trk protooncogene (11,17). Although both NGF and its relative, neurotropin-3 (NT-3) have been found in the ovary (4, 18) nothing is known about the presence in the gland of low affinity NGFrec, which, in addition to NGF, can also bind NT-3 (19). The present experiments demonstrate that these receptors are not only present in ovarian nerves but are also synthesized in endocrine cells of developing follicles. In the latter, receptor expression is preferential in thecal cells and declines strikingly upon thecal differentiation into luteal cells after ovulation. A partial report of these findings has appeared (20).

/ AS kD

A. f 2 ! f f

«• -200

m -92.5

[i26l]NGF-

Coomassie blue

crossKnked receptor Fig. 1. Identification of NGFrec Molecules in Prepubertal Rat Ovaries (28 Days of Age) by Immunoprecipitation of CrossLinked [125I]NGF-Receptor Complexes Followed by SDSPAGE Autoradiography Addition of an NGF excess (2 HM) to the binding reaction (Ov + NGF) eliminated labeling of the approximately 90-kD NGFrec species. The absence of NGFrec in liver (Lv) is also depicted. Panel A shows the Coomassie blue-stained gel that provided the autoradiogram depicted in B. Ov, Ovary.

kb -4.7

RESULTS Identification of Ovarian NGFrec Protein

-2.9

125

Cross-linking of [ I]NGF to ovarian membranes followed by immunoprecipitation (CLIP) with monoclonal antibody 192 immunoglobulin G (IgG) (21) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of the radiolabeled receptor molecules demonstrated the presence in the ovary of an approximately 90-kD molecular species (Fig. 1B) that corresponds in size to the low affinity NGFrec (77 kD) cross-linked to a [125I]NGF monomer (22). Addition of an excess of unlabeled NGF before the binding reaction eliminated cross-linking of [125I]NGF so that no radioligand-labeled complexes were immunoprecipitated (Fig. 1B). Moreover, antibody 192 IgG failed to immunoprecipitate proteins radiolabeled by cross-linking of [125I]NGF to liver membranes, which do not contain NGFrec. Figure 1A depicts the Coomassie bluestained gel that provided the autoradiogram shown in B, to demonstrate that all lanes were equally loaded.

-1.8 -1.5 Ov

St

Cb

Lv Lu

Kd

Fig. 2. Detection of NGFrec mRNA in Juvenile (28-Day-Old) Rat Ovaries by RNA Blot Hybridization Using a Rat 32P-Labeled cRNA Probe Each lane equals 5 ng A+ RNA. Ov, Ovary; St, striatum; Cb, neonatal cerebellum; Lv, liver; Lu, lung; Kd, kidney. Ribosomal RNA mol wt standards (kb) are indicated on the right axis.

and neonatal cerebellum, two brain regions known to express high levels of NGFrec mRNA (Fig. 2) (23, 24). A much fainter signal was found in lung and kidney, but no NGFrec mRNA was detected in liver. Changes in Ovarian NGFrec mRNA Expression During Prepubertal Development

Identification of Ovarian NGFrec mRNA RNA blot hybridization of polyadenylated RNA to a 32Plabeled rat NGFrec antisense RNA probe demonstrated the presence in the ovary of a 3.8-kb mRNA species of identical size as NGFrec mRNA detected in the striatum

The ovarian content of NGFrec mRNA increased moderately during juvenile-peripubertal development (i.e. between postnatal day 23 and early proestrus; Fig. 3A). Early proestrus is the phase of puberty during which the first signs of increased ovarian secretory activation

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1.0 —

23

A

EP BO AO PROTEIN

Fig. 3. A and B, Changes in Ovarian NGFrec mRNA Content During Juvenile-Peripubertal Development of the Rat The lower blots show levels of cyclophilin mRNA, which is constitutively expressed in the ovary, used to normalize NGFrec mRNA levels for statistic analysis. Panel C depicts the drop in NGFrec mRNA levels that occurs after first ovulation, and the tendency of NGFrec protein levels to decline concomitantly. 23, Juvenile 23-day-old rats; A, anestrus, juvenile 28-day-old rats; EP, early proestrous phase of puberty (see text); LP, late proestrus, the day when the first preovulatory surge of gonadotropins occurs; E, first estrus, the day of first ovulation; D^ first diestrus; BO, before ovulation; AO, after ovulation. In Panel C numbers in parentheses represent numbers of individual observations per group.

are detected (25). NGF mRNA levels remained elevated on the day of the first preovulatory surge of gonadotropins (LP) and decreased markedly on the day of first estrus (Fig. 3B), i.e. within 12 h after ovulation. Densitometric analysis of several blots demonstrated that the periovulatory decline in NGFrec mRNA was highly significant (Fig. 3C). In contrast, a significant decrease in NGFrec protein did not occur (Fig. 3C). Though initially surprising, this result was found to be entirely consistent with the distribution of the NGFrec protein observed in subsequent experiments (see below). Sites of NGFrec Expression Immunohistochemical examination of the ovary at different phases of puberty revealed the presence of NGFrec immunoreactive material in nerve fibers surrounding blood vessels and coursing across the interstitial tissue, as well as in fibers associated with follicles in different stages of development (Fig. 4). Surprisingly, NGFrec immunoreactivity was also observed in thecal cells of antral and preantral follicles, regardless of the phase of puberty at which the ovaries were collected (Fig. 5, A-C). Not all follicles were immunopositive, and not all positive follicles had the same intensity of thecal staining (Fig. 5, A-C). Granulosa cells also contained NGFrec immunoreactive material, but considerably less than in thecal cells (Fig. 5D). Although newly formed corpora lutea exhibited NGFrec-like material (Fig. 5C), closer examination showed that most of the immunoreactivity was associated with nerve fibers accompanying the neovasculature (Fig. 5E). Hybridization histochemistry using the same antisense RNA probe employed in the RNA blots confirmed thecal cells as a site of NGFrec synthesis (Fig. 6). Expression of NGFrec mRNA was not limited to the

Fig. 4. NGFrec-lmmunoreactive Nerve Fibers around an Ovarian Blood Vessel (Arrow), Coursing into the Interstitial Tissue (Double Arrows), or Associated with an Antral Follicle (Arrowheads). Bar, 20 /*m.

theca of large antral follicles (Fig. 6, A and B) but was also evident in small preantral follicles (Fig. 6C). The finding that NGFrec protein was selectively expressed in the theca of some follicles but not in others was confirmed by the in situ hybridization experiments, which clearly demonstrated that even adjacent follicles have widely different levels of NGFrec mRNA expression (Fig. 6, D and E). Expression of NGFrec in Granulosa Cells Because the presence of NGFrec in granulosa cells detected by immunohistochemistry or in situ hybridization was suggestive but not convincing, two additional experiments were conducted in which granulosa cells were separated from the rest of the ovary (henceforth,

NGF Receptors in Rat Ovary

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B

Fig. 5. NGFrec Protein Detected by Immunocytochemistry in Thecal Cells of Different Size Follicles throughout Puberty A, Anestrus; B, late proestrus; C, estrus. Notice the intense immunoreactivity in the theca of large (B, double arrows), medium, and small follicles (A and C, single arrows), and the lower level of immunoreactive material in corpora lutea (C, arrowheads). Bars, 500 Mm. NGFrec-like material is also detected in granulosa cells (D, single arrowheads) in addition to the theca (double arrowhead) and nerve fibers (arrow), whereas most of the immunoreactivity in the corpus luteum appears localized to blood vessel-associated nerve fibers (E, single arrowheads). Bars, 100 nm.

called residual ovary) and the NGFrec protein and mRNA were measured. The NGFrec protein was detected by CLIP and NGFrec mRNA was detected by RNase protection assay using the same cRNA previously employed in RNA blots and hybridization histochemistry experiments. Figure 7 shows that both the NGFrec protein and the mRNA that encodes it are

expressed in granulosa cells, albeit at levels lower than those seen in the residual ovary.

DISCUSSION It has long been known that the mammalian ovary is a target field for peripheral sympathetic neurons. As such,

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Fig. 6. Identification of NGFrec mRNA in Thecal Cells (Arrowheads) of Large Antral (A) and Small Follicles (C) A brightfield view (B) of the localization of NGFrec mRNA in thecal cells of a developing follicle. NGFrec mRNA expression varies widely in different follicles (D and E). D shows a darkfield view of different size follicles exhibiting a strong intermediate or no NGFrec mRNA signal (single, double, and triple arrowheads, respectively). Panel E depicts the same area counter-stained with thionin. A, C, D and E bars, 100 ^m; B bar, 20 nm.

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kD

A.

B.

nt 400 (322) 280 (270)

200-

92.5-

-

GC RO

PC

155

GCRO

Fig. 7. A, Identification of NGFrec Protein in Granulosa Cells (GC) and Residual Ovary (RO) of the Prepubertal Rat Ovary by Immunoprecipitation and SDS-PAGE Autoradiography of Cross-Linked [125I]NGF-Receptor Complexes Molecular markers (kD) are shown on the left vertical axis. B, Detection of NGFrec mRNA in granulosa cells and residual ovary by RNase protection assay. P, 32P-Labeled cRNA probe; C, unprotected probe digested by ribonucleases. The cRNA probe is 322 nucleotides (nt) in length, of which 270 nt are complementary to NGFrec mRNA and 52 nt correspond to vector sequences. RNA molecular markers are shown on the right vertical axis. Numbers in parentheses indicate the size of the cRNA probe (322 nt) and the protected mRNA sequences (270 nt).

it produces NGF, which accumulates in the gland after nerve transection (4), suggesting that ovarian NGF is transported in a retrograde fashion by the innervating libers toward the neuronal perikarya. This process was conclusively demonstrated earlier in other tissues innervated by sympathetic and sensory neurons (5, 6). Transference of NGF from its site of synthesis to the innervating fibers appears to require the involvement of both low and high affinity receptors (7, 8). According to this concept (8, 9), low affinity receptors synthesized in nonneuronal cells bind NGF and present it to high affinity receptors located on nerve fibers (10). These receptors then sequester, internalize, and transport the peptide to the neuronal perikarya (6, 9). Indeed, the low affinity form of NGFrec is widely expressed in tissues innervated by NGF-sensitive neurons and, interestingly, also in some other tissues that do not receive sympathetic innervation (23, 26). The present study provides molecular, biochemical, and immunohistochemical evidence for the presence of low affinity NGFrec in the ovary. The receptors were found in nerve fibers surrounding blood vessels, traveling in the interstitial tissue, and associated with follicles in different developmental stages. Unexpectedly, a major fraction of the total receptor population was found in endocrine cells of developing follicles—namely, thecal cells—and to a lesser extent, granulosa cells. In situ hybridization and RNase protection assays confirmed these cells as the sites of NGFrec synthesis.

Transection of the ovarian nerves led to a decrease in total ovarian NGFrec protein detected by CLIP but affected neither the presence of NGFrec mRNA in follicular thecal cells (assessed by in situ hybridization) nor total ovarian NGFrec mRNA levels (determined by RNA blot hybridization) (data not shown). Thus, NGFrec gene expression in the ovary does not require the presence of an intact innervation, a conclusion in keeping with the finding that the appearance of NGFrec in target fields of the peripheral nervous system occurs independently of the arrival of innervating axons (8). It is plausible, however, that even though expression of NGFrec mRNA in ovarian follicles does not require follicular innervation, the relative level of expression may be regulated by the extent of the innervation, as shown in cutaneous targets of NGF-sensitive neurons (8). Of particular interest was the observation that luteinization of thecal cells after the first ovulation was accompanied by a striking decline in NGFrec synthesis. This indicates that expression of NGFrec in thecal cells is tightly linked to gonadotropin-dependent cytodifferentiation and provides a rather compelling argument that NGF functions in the ovary are intimately associated with follicular development and its attendant cellular activities. If one accepts the view that the low affinity NGFrec acts as a relay station to transfer NGF from the target field to innervating fibers (8, 9), thereby increasing the effective concentration of NGF in the target, the assumption may be made that the degree of innervation received by a given follicle at any given phase of development would depend on the level of low affinity receptor expression. This would be in keeping with the concept that a function of the low affinity NGFrec in target fields is to select the areas of innervation by restricting the availability of NGF to the projecting neurons (8). Because the level of receptor expression varies considerably between follicles, the above concept would also imply that follicular innervation is a much more dynamic process than originally anticipated and that it is closely regulated by the NGFrec-dependent availability of NGF. It is conceivable that, in addition to its role in initiating the neurotrophic action of NGF, ovarian NGFrec are involved in other functions, perhaps of an endocrine or paracrine nature. The presence of NGFrec in Sertoli cells of the testis and the regulation of its expression by androgens (27) support this possibility. Moreover, we have found recently that the level of NGFrec expression in thecal cells is inversely correlated to the degree of follicular atresia (Dissen, G. A., A. N. Hirshfield, and S. R. Ojeda, unpublished observations), and that immunoreactive NGF receptors are present in primordial ovarian cells before initiation of folliculogenesis (Dissen, G. A., S. Malamed, J. Gibney, and S. R. Ojeda, unpublished observations). The low affinity NGFrec recently has been shown to be required for NGF-initiated signal transduction, and the receptor domains responsible for this activity have been defined (17). Evidence also has been presented that binding of NGF to both the low

MOL ENDO-1991 1648

affinity receptor and to the tyrosine kinase product of the trk protooncogene is necessary for the formation of high affinity NGF binding sites (11). We have recently found by Northern blot analysis a trk mRNA-like species in the rat ovary (Dissen, G. A., and S. R. Ojeda, unpublished observations). This suggests that the ovary possesses the receptor molecules required for NGF action. At present, the site of NGF synthesis in the ovary has not been established. In preliminary experiments we have detected NGF immunoreactive material in granulosa cells. This and the recent finding of NT-3 in granulosa cells (18) implicate this follicular compartment as a site of NGF synthesis. NT-3 is a member of the NGF family recognized by the low affinity NGFrec (19). While NGF is recognized by the trk tyrosine kinase receptor, NT-3 binds to the trkB gene product (28, 29). Since both trk and trkB tyrosine kinase receptors appear to be expressed in the ovary (our observation; see above and Ref. 30), the possibility exists that NGF and NT-3 act in concert to regulate ovarian innervation and perhaps endocrine function. The presence of NGF, NT3, and their receptors in ovarian endocrine cells suggests that, in addition to their neurotropic activity, the polypeptides may be functioning in the ovary as neuroendocrinotropic factors. This would provide a mechanism for reciprocal communication between the nervous and the endocrine reproductive systems.

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mM phenylmethylsulfonylfluoride (a protease inhibitor) or RNasin (a ribonuclease inhibitor, 20 U/ml), depending on whether the cells were to be used for protein or mRNA analysis. Nucleic Acid Probes NGFrec mRNA was detected with an antisense RNA probe transcribed from a BamHI -EcoRI cDNA fragment derived from the 5'-region of a rat NGFrec cDNA (clone 5A; Ref. 24). Preparation of the template for transcription and the transcription procedure itself were performed as reported (4, 32). The cRNA transcript was radiolabeled with [32P]cytidine triphosphate for RNA blot hybridizations and RNase protection assays, and with [35S]uridine triphosphate for hybridization histochemistry. Cyclophilin mRNA, which is constitutively expressed in rat tissues (33), was used as an internal standard to normalize the results of RNA blot hybridization. Cyclophilin mRNA was detected with a 32P-labeled 700-basepair Sa/r?H1-Psfl cDNA fragment derived from p1 B15 (34), a cDNA complementary to the coding region of cyclophilin mRNA. The cDNA was labeled by the random primers method (35), as reported (4, 32). Preparation of RNA Total RNA was prepared by the acid-phenol method (36), and polyadenylated RNA (A+ RNA) was isolated by a microbatch procedure (37) as described earlier (4, 32). RNA Blot Hybridization

MATERIALS AND METHODS

The procedures used for RNA blotting and hybridization have been described in detail in previous publications (4, 32). The RNA blots were first hybridized to the NGFrec probe, and after autoradiography they were probed with p1B15 cDNA.

Animals

RNase Protection Assay

Female rats of the Sprague-Dawley strain were purchased from Bantin and Kingman (Fremont, CA). They were housed in groups of five per cage in a room with controlled temperature (23-25 C) and photoperiod (10 h darkness/14 h light, with lights on from 0500-1900 h). The animals had ad libitum access to tap water and pelleted rat chow (Purina Laboratory Chow, Ralston-Purina, St. Louis, MO).

The procedure employed was based on the method of Gilman (38). After transcription, the NGFrec cRNA probe was isolated by electrophoresis in a 7.1 M urea, 5% polyacrylamide gel followed by elution at 37 C into a solution containing 2 M ammonium acetate, 1 % SDS, and 25 fig/m\ tRNA. The RNA samples (10 Mg total RNA to which 15 ^g tRNA were added to complete a total amount of 25 M9) were dried by vacuum centrifugation, reconstituted in 30 MI hybridization buffer [80% formamide in 40 mM piperazine-A/,A/'-bis(2-ethanesulfonic acid), pH 6.4, 0.4 M NaCI, 1 mM EDTA] containing 500,000 cpm [32P]NGFrec cRNA, and hybridized overnight at 45 C. The next day, nonhybridizing RNA was digested with a mixture of ribonucleases A and T1 (40 and 2 ^g/ml, respectively) for 1 h at 37 C in a 380-MI vol. The digestion reaction was then transferred to a new tube containing 10 MI 20% SDS and 50 Mg proteinase K, incubated for 15 min at 37 C, phenol/ chloroform extracted, and ethanol precipitated. The pellets were resuspended in 90% formamide containing the dyes bromophenol blue and xylene cyanol and electrophoresed in a 7.1 M urea, 5% polyacrylamide gel at 300 V for 2 h. Thereafter, the gel was dried and exposed to Kodak XAR-5 (Eastman Kodak Co., Rochester, NY) film to visualize the protected RNA species.

Tissue Collection The ovaries and, when necessary, other tissues were collected at the beginning (postnatal day 23) and end (postnatal day 28) of the juvenile period (25) or during the peripubertal phase (days 32-40). Upon collection, they were rapidly frozen on dry ice. Peripubertal rats were classified in different phases of puberty according to criteria previously established (25). The anestrous (A) phase corresponds to late juvenile development (postnatal day 28 in this case); early proestrus signals the initiation of increased ovarian estrogen secretion and is characterized by the appearance of uterine intraluminal fluid; late proestrus is the day of the first preovulatory surge of gonadotropins. At this time, accumulation of uterine fluid is maximal (ballooned uterus), and wet uterine wt exceeds 200 mg. The first estrus (E) corresponds to the day of ovulation; at this time the vagina is patent, and most cells found in vaginal lavages are cornified. Finally, E is followed by the first diestrus, which signals the activation of corpus luteum function and is noninvasively characterized by a vaginal cytology showing a preponderance of leukocytes. In some experiments, granulosa cells were isolated from the rest of the ovary (termed residual ovary; 25) using a procedure described earlier (31). The cells were collected in sterile 0.01 M phosphosaline buffer pH 7.4 containing either 1

Hybridization Histochemistry The procedure employed was based on the method reported by Simmons et al. (39). Ovaries were immersion-fixed for 24 h at 4 C in 4% paraformaldehyde-0.1 M sodium phosphate buffer pH 7.4, transferred to 20% sucrose in phosphate buffer (overnight at 4 C), embedded in OCT compound (Miles Inc., Elkhart, IN), frozen on dry ice, and stored at - 8 5 C until sectioned. The cryostat sections (14 Mm) were collected on polylysine-

NGF Receptors in Rat Ovary

coated slides and were dehydrated overnight under vacuum. They were postfixed in 4% paraformaldehyde for 15 min at room temperature immediately before hybridization. Prehybridization treatment included exposure to proteinase K (0.2 ng/ ml) for 30 min at 37 C, acetylation with acetic anhydride (0.0025%) in 0.1 M triethanolamine, pH 8.0 (10 min at room temperature), and dehydration in graded alcohols (50%, 60%, 95%, and 100%). The sections were then overlaid with 30 MI hybridization solution containing 1 x 107 cpm/ml [32P] NGFrec cRNA, 50% formamide, 0.4 M NaCI, 10 mM Tris, 10 mM EDTA, 10% dextran sulfate, and 2x Denhardt's solution. After 1720 h hybridization at 53-56 C, the sections were treated with ribonuclease A (20 fig/m\, 30 min at 37 C) and washed in decreasing concentrations of (2x, 1x, 0.5x, and 0.1 x) of sodium citrate-sodium chloride (SSC) buffer, pH 7.0, containing 1 mM dithiothreitol (2x SSC = 0.03 M sodium citrate, 0.3 M sodium chloride). All washes were for 10 min at room temperature, except the last one, which was at 65 C for 30 min. The sections were then dehydrated in graded alcohols and exposed for 24-48 h to Amersham Hyperfilm (Amersham, Arlington Heights, IL) to obtain an initial assessment of the hybridization reaction. Thereafter, the sections were delipidated by successive passage in ethanol and chloroform and dipped in Kodak NTB-2 emulsion for signal detection. The reaction was developed after 2-3 weeks at 4 C, and the sections were counterstained with thionin. Controls included pretreatment of the sections with ribonuclease-A before the hybridization and hybridization with sense [32P]NGFrec RNA. Imimunocytochemistry The ovaries were processed as described for hybridization histochemistry, except that the cryostat sections were dehydrated under vacuum for only 2 h after collection on polylysinecoated slides. The immunohistochemical procedure used was similar to that described by Nilaver and Kozlowski (40). The sections were first rinsed in 0.05 M Tris-0.9% saline, pH 7.6, and then incubated with a 0.3% solution of hydrogen peroxide for1 30 min at room temperature to destroy endogenous peroxidase. After rinsing they were overlaid with the primary antibody (192 IgG, 4 Mg/ml) diluted in 0.05 M Tris-saline buffer containing 0.02% BSA and 0.1% Triton X-100, and incubated overnight at 4 C. The next day, the primary antibody was removed, and the sections were incubated with biotinylated horse-antimouse IgG (rat preadsorbed) (1:250 dilution) for 90 min at room temperature. This was followed by incubation with avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 2 h at room temperature and development of the reaction with diaminobenzidine in the presence of B-D glucose, ammonium chloride, and glucose oxidase to generate the necessary hydrogen peroxide. After 30-45 min the reaction was terminated and the sections dehydrated in graded alcohol and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Controls were those previously recommended by other authors (41), i.e. omission of the primary or secondary antibody and substitution of the primary antibody with an unrelated antibody, in this case, 3H3, a monoclonal antibody directed against an unknown antigen. Immunoprecipitation of NGFrec The NGFrec protein was detected employing the CLIP assay of Yan and Johnson (41) with some modifications (32). Briefly, the ovaries or cells were homogenized in 1 mM phenylmethylsulfonyl fluoride-PBS, pH 6.5, and centrifuged. The pellets were rehomogenized, centrifuged again, and the pooled supernatants were used in the assay. Lactoperoxidase-labeled [125I]NGF (2 nM) was added to the homogenates and allowed to bind to the receptors for 60 min at 35 C. Unlabeled NGF (2 HM) was added to some tubes to competitively displace the labeled NGF and, thus, have a nonspecific binding control. Thereafter, ethyldimethylisopropylaminocarbodiimide (10 mM final concentration) was added to the reaction to cross-link

1649

[125I]NGF to its receptor (20 min, 22 C). After centrifugation and solubilization of the pellets with 2% octyl glucoside in 0.5% BSA, the cross-linked complexes were treated with formalin-fixed Staphylococcus aureus (Pansorbin, Calbiochem, San Diego, CA) to preabsorb radiolabeled protein that may bind nonspecifically to Pansorbin. The samples were then centrifuged, and the supernatants were incubated with 5 M9 192-lgG, a monoclonal antibody to the rat NGF receptor (21). The radioligand-receptor antibody complex was then immunoprecipitated with Pansorbin bound to rabbit antimouse IgG antibodies, and the labeled receptor was identified by SDSPAGE autoradiography. Data Analysis The results of several experiments detecting NGFrec mRNA by RNA blot hybridization or NGFrec protein by CLIP were analyzed by laser densitometric scanning of the autoradiograms followed by statistical assessment of the differences using the Student's f test. Before statistical analysis, values were normalized using the cyclophilin mRNA signal.

Acknowledgments We thank Dr. Eugene M. Johnson (Department of Pharmacology, Washington University School of Medicine, St. Louis, MO) for his generous supply of monoclonal antibody 192 IgG to the NGFrec and Dr. Moses Chao (Department of Cell Biology and Anatomy, Cornell University Medical School, New York, New York) for providing us with a rat NGF receptor cDNA. We are grateful to Dr. Richard B. Simerly (Division of Neuroscience, Oregon Regional Primate Research Center) for his invaluable help in setting up the hybridization histochemistry technique. We also thank Ms. Janie Gliessman for editorial assistance.

Received July 25, 1991. Revision received September 6, 1991. Accepted September 6,1991. Address requests for reprints to: Dr. Gregory A. Dissen, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006. This work was supported by NIH Grants HD-24870, HD18185, and RR-00163. This is publication 1808 of the Oregon Regional Primate Research Center.

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