Download - Effect of anti-NGF on ovarian expression of alpha1- and beta2-adrenoceptors, TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Effect of anti-NGF on ovarian expression of �1- and �2-adrenoceptors, TrkA,p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Luigi Manni,1,2 Agneta Holmang,1 Stefan Cajander,3

Thomas Lundeberg,4 Luigi Aloe,2 and Elisabet Stener-Victorin1,5,6

1Cardiovascular Institute, Wallenberg Laboratory, Sahlgrenska Academy, Goteborg University, Goteborg, Sweden;2Institute of Neurobiology and Molecular Medicine, Consiglio Nazionale delle Ricerche, Rome, Italy;3Department of Pathology and Cytology, Akademiska Sjukhuset, Uppsala; 4Rehabilitation Medicine,Danderyds Hospital, Stockholm; and 5Department of Obstetrics and Gynecology and 6Institute ofOccupational Therapy and Physical Therapy, Sahlgrenska Academy, Goteborg University, Goteborg, Sweden

Submitted 4 February 2005; accepted in final form 26 September 2005

Manni, Luigi, Agneta Holmang, Stefan Cajander, ThomasLundeberg, Luigi Aloe, and Elisabet Stener-Victorin. Effect ofanti-NGF on ovarian expression of �1- and �2-adrenoceptors,TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries. Am J Physiol Regul Integr CompPhysiol 290: R826–R835, 2006. First published September 29, 2005;doi:10.1152/ajpregu.00078.2005.—Estradiol valerate (EV)-inducedpolycystic ovaries (PCO) in rats are associated with higher ovarianrelease and content of norepinephrine, decreased �2-adrenoceptors(ARs), and dysregulated expression of �1-AR subtypes, all precededby an increase in the production of ovarian NGF. The aim of this studywas to further elucidate the role of NGF in the ovaries by blocking theaction of NGF during development of EV-induced PCO in rats.Control and EV-injected rats were treated with intraperitoneal injec-tions of IgG (control and PCO groups) or with anti-NGF antibodies(anti-NGF and PCO anti-NGF groups) every third day for 5 wkstarting from the day of PCO induction. Rat weight, estrous cyclicity,ovarian morphology, ovarian mRNA, and protein expression of�1-AR subtypes, �2-AR, the NGF receptor tyrosine kinase A (TrkA),p75 neurotrophin receptor (p75NTR), and tyrosine hydroxylase (TH)were analyzed. Ovaries in both PCO and PCO anti-NGF groupsdecreased in size as well as in number and size of corpora lutea.mRNA expression of �1a-AR and TrkA in the ovaries was lower,whereas expression of �1b- and �1d-AR and TH was higher, in thePCO group than in controls. Protein quantities of �1-ARs, TrkA,p75NTR, and TH were higher in the PCO group compared withcontrols, whereas the protein content of �2-AR was lower. Anti-NGFtreatment in the PCO group restored all changes in mRNA and proteincontent, except that of �1b-AR and TrkA mRNAs, to control levels.The results indicate that the NGF/NGF receptor system plays a role inthe pathogenesis of EV-induced PCO in rats.

nerve growth factors; sympathetic nervous system; ovarian innerva-tion; tyrosine kinase A; p75 neutrophin receptor

POLYCYSTIC OVARY SYNDROME (PCOS) is a heterogeneous con-dition of uncertain etiology that affects between 5% and 10%of women of reproductive age (41). PCOS is characterized bya myriad of symptoms and signs, which include ovulatorydysfunction, the presence of polycystic ovaries (PCO), abdom-inal obesity, hyperandrogenism, and in many cases hyperten-sion and insulin resistance (41). Several theories have beenproposed to explain the pathogenesis of PCOS, such as anenhanced production of ovarian androgens, an alteration in the

metabolism of cortisol resulting in enhanced production ofadrenal androgens, a defect in the action and secretion ofinsulin, and a neuroendocrine defect that causes an exaggera-tion in the pulse and frequency amplitude of LH (41).

The involvement of the sympathetic nervous system in theetiology and maintenance of PCOS is suggested by bothclinical and experimental findings (5, 20, 22, 24, 25). Ovarianfunction is known to be regulated by both hormonal andintraovarian signaling. It is also known that the sympatheticinnervation of the ovary is involved in follicular development(23) and in ovarian steroidogenesis (1). That the sympatheticnervous system is involved in the pathophysiology of humanPCOS is supported by the fact that the innervation of thecatecholaminergic nerve fibers in the PCO of women withPCOS is more dense than in normal ovaries (18, 35).

Chronic anovulation and PCO can be induced by a singleintramuscular injection of estradiol valerate (EV) in female rats(6). We demonstrated previously in a dose-response study (38)that rats injected with a dose of 4 mg of EV dissolved in 0.2 mlof oil exhibit a progressive decrease in the number of primaryand secondary follicles after 30 days and that 60 days after theEV injection true cystic follicles were found and the well-defined PCO was fully developed in accordance with previousreports by Brawer et al. (6). The EV-induced rat PCO modelreflects some endocrinologic and morphological characteristicsof human PCOS, and it is assumed that activity in the ovariansympathetic nerves of the rats in this model is higher than inthe nerves of normal rats (5, 22). This is evidenced by a higherrelease and content of norepinephrine (NE) in the ovaries,together with a lower expression of �2-adrenoceptors (ARs) inthe ovarian compartment (5, 22). Transection of the sympa-thetic nerves arriving at the ovary via the superior ovariannerve reduces steroid response, increases �2-AR expression tomore normal levels, and restores estrous cyclicity and ovula-tion in rats with EV-induced PCO to normal levels (5).

It appears that the higher activity of the ovarian sympatheticnerves of rats with EV-induced PCO is related to an increasedsynthesis of ovarian NGF—a target-derived neurotrophin—and its low-affinity receptor, p75 neurotrophin receptor(p75NTR) (20, 37, 38). Intraovarian blockade of NGF/p75NTR

action with an antiserum to NGF and an antisense oligode-oxynucleotide to p75NTR restores estrous cyclicity and ovula-

Address for reprint requests and other correspondence: L. Aloe, Institute ofNeurobiology and Molecular Medicine (CNR), Via del Fosso di Fiorano 64,00143 Rome, Italy (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 290: R826–R835, 2006.First published September 29, 2005; doi:10.1152/ajpregu.00078.2005.

0363-6119/06 $8.00 Copyright © 2006 the American Physiological Society http://www.ajpregu.orgR826

tory capacity in PCO rats (20). In the ovary, NGF and both ofits receptors, p75NTR and the high-affinity receptor tyrosinekinase A (TrkA), are synthesized in thecal cells (13, 15). It isalso known that p75NTR facilitates transfer of NGF from itssites of production to NGF-sensitive fibers (8) as well ascollaborating with the high-affinity TrkA receptor to potentiatecellular response to neurotrophin (17). However, the expres-sion of TrkA has not, to our knowledge, been investigated inthe ovary of rats with steroid-induced PCO.

Recently, electrical stimulation of the splanchnic nerve hasbeen shown to decrease ovarian blood flow (OBF) via theactivation of �-AR in the ovarian blood vessels (42); this wasconfirmed by an injection of phentolamine, an �1-AR agonist,and indicates an involvement of the �1-AR in the regulation ofovarian function. In a recent study (27) we showed for the firsttime that all �1-AR subtypes are expressed in the ovaries ofhealthy rats and that the mRNA and protein contents of these�1-AR subtypes are dysregulated after induction of PCO inrats.

In the present study, the aim was to determine the role ofNGF in the mRNA and protein expression of ovarian �1a-,�1b-, and �1d-AR, �2-AR, p75NTR, TrkA, and tyrosine hydrox-ylase (TH) and in ovarian morphology by blocking ovarianNGF action with repeated intraperitoneal injections of antibod-ies against NGF in the development of EV-induced PCO.

MATERIALS AND METHODS

Animals. Forty virgin adult cycling female Wistar rats (CharlesRiver Wiga, Uppsala, Sweden) weighing 205–230 g were housed fourto each cage at a controlled temperature of 22°C with a 12:12-hlight-dark cycle for at least 1 wk before and throughout the experi-mental periods. The rats had free access to pelleted food and tapwater. The rats were divided into four experimental groups: 1) controlgroup (n � 10), 2) anti-NGF group (n � 10), 3) PCO group (n � 10),and 4) PCO anti-NGF group (n � 10). Twenty rats, those in the PCOgroups, were given a single intramuscular injection of 4 mg of EV(Riedeldehaen) in 0.2 ml of oil to induce well-defined PCO (6).Twenty rats, those in the control group and the anti-NGF group,received a single intramuscular injection of 0.2 ml of oil (arachidisoleum, Apoteket, Umea, Sweden) only. Thirty to thirty-three daysafter intramuscular injection of EV, the rats were killed by decapita-tion. Before decapitation, the rats were anesthetized with 125 mg/kgbody wt of thiobutarbarbital sodium (Inactin, RBI, Natick, MA). Theexperiments were carried out according to the principles and proce-dures outlined in the National Institutes of Health (NIH) Guide for theCare and Use of Laboratory Animals and were approved by the localanimal ethics committee at Goteborg University.

Vaginal smear. Estrous cyclicity was monitored by vaginal smearobtained between 8:00 AM and 12:00 PM for 10 consecutive daysbefore the experiment to measure ovarian functionality. The differentstages of the estrous cycle were determined according to the predom-inant cell type present in the vaginal smears. The control and anti-NGF groups were killed in estrus because the estradiol levels are lowat that stage, and the PCO and PCO anti-NGF groups were killed inestrus or in a pseudoestrus stage as described previously (5).

Anti-NGF and IgG treatment. NGF plays a crucial role in themaintenance of sympathetic neuron phenotype and metabolic func-tions and in the regulation of sympathetic innervation and plasticity(33). We previously described (26, 40) the use of anti-NGF antibodiesto block NGF action and/or induce biological effects opposite to thoseelicited by NGF. In the present study, the control groups (control andPCO groups) were given 0.5-ml intraperitoneal injections of preim-mune goat IgG (500 �g/rat) produced and purified in our laboratory.The experimental groups (anti-NGF and PCO anti-NGF groups) were

given on 0.5-ml intraperitoneal injection of goat anti-NGF IgG (500�g/rat) produced and purified as previously described (3). Injectionwas given every third day, beginning on the day of PCO induction, intotal 8–10 treatments/rat, and the experiment was finalized within 3days after the last immunization.

Tissue collection. At the completion of the experiment, the ratswere decapitated in estrus or pseudoestrus, as described under Vaginalsmear, and the ovaries were quickly dissected on dry ice. One ovarywas divided in two pieces, weighed, snap frozen in liquid nitrogen,and stored at �80°C until extraction. The second ovary was weighedand fixed in buffered 4% formaldehyde for at least 24 h for morpho-logical analyses.

Ovarian morphology. The ovaries were dehydrated after 24 h offormaldehyde fixation and imbedded in paraffin. They were partiallyserially sectioned (4 �m, every 10th section mounted on the glassslide) and stained with hematoxylin and eosin. The sections, blindedto grouping, were analyzed under a conventional birefringence lightmicroscope. There was no intention to quantify the number of corporalutea or growing or atretic follicles but rather to establish whetherovulation with corpora lutea formation had occurred within the giventime frame. According to morphometric (stereological) and statisticalprinciples, there is no need to perform a statistical analysis in thissituation.

Real-time PCR for AR, TH, and TrkA. Total RNA from the ovarywas extracted with RNeasy Mini kits (Qiagen, Hilden, Germany).PCR analysis was performed with the ABI Prism 7700 SequenceDetection System (PE Applied Biosystems, Stockholm, Sweden) andFAM-labeled probes specific for the �1a-AR (Rn00567876m1), the�1b-AR (ADRA A1B-EX 152027A02), the �1d-AR (Rn00577931ml),the �2-AR (Rn00560650s1), TH (NM_012740), and TrkA(NM_021589) (PE Applied Biosystems). Designed primers and aVIC-labeled probe for GAPDH (NM_031144) were included in thereactions as an internal standard. The cDNA was amplified under thefollowing conditions: 1 cycle at 50°C for 2 min and 95°C for 10 min,followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Theamount of mRNA of each gene was calculated with the standard curvemethod (following the instructions in PE Applied Biosystems UserBulletin no. 2) and adjusted for the expression of GAPDH.

RT-PCR-ELISA for p75NTR. The expression of p75NTR mRNA wasevaluated with a RT-PCR ELISA protocol exactly as described byTirassa and coworkers (39). Total RNA was extracted from theovaries by the method of Chomczynski and Sacchi (9) as modified inthe TRIzol kit (Invitrogen, Lidingo, Sweden). Complementary DNAwas synthesized from 1 �g of total RNA, using 200 U of Moloneymurine leukemia virus reverse transcriptase (Promega Italia, Milan,Italy) in 20 �l of total volume reaction. p75NTR and GAPDH geneswere coamplified in a single-tube PCR reaction (35 cycles: 1 min at95°C, 1 min at 55°C, 2 min at 72°C), using 5�-biotinylated specificprimers to generate biotinylated PCR products detectable by digoxy-genin-labeled probes in an immunoenzymatic assay. Primer/probesequences are as follows: p75NTR biotinylated forward: 5�-CGTGT-TCTCCTGCCAGGACA-3�; p75NTR reverse: 5�-GAGATGCCACT-GTCGCTGTG-3�; p75NTR digoxygenin-labeled probe: 5�-ACAG-CAGCCAAGATGGAGCAATAGACAGG-3� (GenBank M14764;relative positions of forward and reverse primers and of the probe:518–537, 1020–1039, 873–901, respectively; predicted size of thefragment: 521 bp); GAPDH biotinylated forward: 5�-CACCACCAT-GGAGAAGGCC-3�; GAPDH reverse: 5�-GATGGATGCCTTGGC-CAGG-3�; GAPDH digoxygenin-labeled probe: 5�-ACAATCTT-GAGTGAGTTGTCATATTTCTCG-3� (GenBank M32599; relativepositions of forward and reverse primers and of the probe: 346–365,517–536, 451–480, respectively; predicted size of the fragment: 290bp). The amount of amplified products was measured at an opticaldensity (OD) of 450/690 nm (OD 450/690), using a Dynatech ELISAReader 5000. A GAPDH level of OD 450/690 was used to normalizethe relative differences in sample size, differences in integrity of the

R827EFFECT OF ANTI-NGF TREATMENT IN STEROID-INDUCED PCO

AJP-Regul Integr Comp Physiol • VOL 290 • MARCH 2006 • www.ajpregu.org

individual RNA, and variations in reverse transcription efficiency. Forexact methodological details see Tirassa et al. (39).

Western blotting analysis for ARs, TH, TrkA, and p75NTR proteins.Commercially available antibodies were used for detection by West-ern blotting of the �1a-AR (�1a-AR [C-19]: sc-1477, Santa Cruz), the�1b-AR (�1b-AR [C-18]: sc-1476, Santa Cruz), the �1d-AR (�1d-AR[H-142]: sc-10721, Santa Cruz), the �2-AR (�2-AR [M-20]: sc-1570,Santa Cruz), TH (AB5986, Chemicon International), TrkA (sc-118,Santa Cruz), and �-actin (sc-8432, Santa Cruz). The Western blottingdetection of p75NTR was carried out with monoclonal anti-p75 anti-body (clone 192; Ref. 7) purified in our laboratory.

Tissue samples were homogenized in lysis buffer (0.01 M Tris �HClbuffer pH 7.6, containing 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA,2 mM PMSF, 50 �M leupeptin, 100 �g/ml pepstatin, and 100 �g/mlaprotinin) at 4°C. After centrifugation at 8,000 g for 20 min, thesupernatants underwent Western blotting. Samples (30 �g total pro-tein) were dissolved with loading buffer (0.1 M Tris �HCl buffer pH6.8, containing 0.2 M DTT, 4% SDS, 20% glycerol, and 0.1%bromophenol blue), separated by 12.5% SDS-PAGE, and electro-phoretically transferred to polyvinylidene difluoride membranes for3 h. The membranes were incubated for 40 min at room temperaturewith blocking buffer (10% nonfat dry milk, 10 mM Tris pH 7.5, 100mM NaCl, 0.1% Tween 20). Membranes were washed three times for10 min each at room temperature in 10 mM Tris pH 7.5, 100 mMNaCl, 0.1% Tween 20 (TTBS), followed by an incubation for 1 h atroom temperature with primary antibodies. Membranes were washedthree times for 10 min each at room temperature in TTBS andincubated for 1 h with horseradish peroxidase-conjugated anti-rabbitIgG, horseradish peroxidase-conjugated anti-goat IgG, or horseradishperoxidase-conjugated anti-mouse IgG as the secondary antibody. Theblots were developed with enhanced chemiluminescence (AmershamBioscience) as the chromophore. Similar results were obtained in fiveindependent Western blot runs. Band densitometry evaluation, ex-pressed as arbitrary units of gray level, of five different gel runs/blot,was made on a Macintosh computer with the public domain NIHImage program (developed at NIH, Bethesda, MD; available at http://rsb.info.nih.gov/nih-image/), which determines the OD of the bandswith a gray scale thresholding operation. The OD of �-actin bandswas used as a normalizing factor.

Statistical analyses. All data were analyzed with the softwarepackage StatView for Macintosh (Abacus Concepts, Berkeley,CA). The effect of EV injection and/or anti-NGF treatment on theweight of the rats was analyzed with ANOVA considering therepeated measures (5 tests) and the treatments (4 levels: control,anti-NGF, PCO, and PCO anti-NGF). mRNA expression, OD datafor Western blot analysis, and ELISA of �1- and �2-ARs, TH,TrkA, and p75NTR in the ovaries were evaluated by two-wayANOVA and Fisher’s paired least significant difference post hoctest. All results are reported as means � SE. A P value �0.05 wasconsidered significant.

RESULTS

Decreased body weight in EV-induced PCO rats is counter-acted by anti-NGF. Body weights during the experimentalperiod are presented in Fig. 1. Before the EV and vehicleinjection, there were no differences in body weight between thegroups. Between weeks 2 and 5, the PCO group weighedsignificantly less than the control groups. The PCO-anti-NGFgroup weighed less than the control groups, although signifi-cantly only at week 2 after injection.

Estrous cyclicity. In Fig. 2A, the estrous cyclicity of fourrepresentative rats for each experimental group is illustrated.Vaginal smears were taken daily, starting 20 days after PCOinduction. The control group rats displayed a normal 4-dayestrous cycle. Estrous cyclicity was unaffected in the anti-NGFgroup. In the PCO group, estrous cyclicity was disrupted withpersistent proestrus/estrus phases. The disrupted estrous cyclewas partially restored in the PCO anti-NGF group, where therats underwent diestrus phases of the cycle.

Ovarian weight and morphology. Mean � SE ovarianweights (mg) were 55 � 4 in the control group, 49 � 5 in theanti-NGF group, 36 � 4 in the PCO group, and 35 � 2 in thePCO anti-NGF group. Ovarian weight in the PCO group wassignificantly lower than in the control groups (P � 0.001), andanti-NGF treatment did not affect the weight of the ovary.

Fig. 2. A: partial restoration of estrous cyclicity in PCO rats injected with anti-NGF IgG. Left: estrous cycle of 4 representative control rats receiving goat IgG.Most of the rats displayed a normal 4-day cycle. The injection of anti-NGF in control animals had no affect on estrous cyclicity. The estrous cycle of PCO rats,however, was disrupted, with persistent proestrous/estrous phases. The normal estrous cycle was partially restored in the PCO anti-NGF group, with most of therats showing the occurrence of diestrous phases of the cycle. The different stages of the estrous cycle (depicted on vertical axis) were determined according tothe predominant cell type present in vaginal smears (P, proestrus; E, estrus; D1, diestrus day 1; D2, diestrus day 2). B–D: ovarian morphology: a typical rat ovaryfrom the control group with sequentially higher magnifications in B through D. Boxes indicate where the next higher power view was taken. In this ovary thereare growing nonatretic follicles and fresh corpora lutea (CL) with vascularization and luteal cell maturation still going on as demonstrated by the incompletecentral development. In B a group of oocytes are observed in the dilated tube (only seen under higher magnification) at top left (arrow). E–G: a typical ovaryfrom the PCO group. When compared with the corresponding photos from the control group (magnifications similar in the vertical columns), it is obvious thatthis ovary is smaller in size. Antral follicles are mainly atretic and often cystic. CL and luteal cells are smaller than in control ovaries, and signs of regressionwith foam cells can be observed. No young CL are observed. H–J: a typical ovary from the PCO anti-NGF group. CL are larger than in the PCO group, andcystic fluid-filled atretic follicles are missing. CL appear younger than in the PCO group, with larger lipid-containing lutein cells; sometimes these CL were aslarge as in the oil control group, but fresh CL with eggs in the tube or ampullae were not found.

Fig. 1. Body weight of rats after injection with estradiol valerate (EV) or oiland throughout the experiment. EV injection decreased the body weight of therats in the polycystic ovary (PCO) group at week 1 and reduced weight gainfrom week 2 to week 5 compared with the control group. Repeated anti-NGFtreatment did not alter body weight in the anti-NGF group but did counteractthe effect of EV injection in the PCO anti-NGF group. Data are expressed asmeans � SE. *P � 0.05 vs. control group.

R828 EFFECT OF ANTI-NGF TREATMENT IN STEROID-INDUCED PCO


As shown in Fig. 2, B–D, the ovaries in the oil-treatedgroups were normal in appearance, with large corpora luteaand follicles in different stages of growth and regression. Theovaries in both the PCO (Fig. 2, E–G) and the PCO anti-NGF(Fig. 2, H–J) groups displayed decreased ovarian size as wellas a decrease in number and size of the corpora lutea comparedwith the ovaries in the oil-treated groups. The corpora lutea inthe PCO anti-NGF group were slightly larger than in the PCOgroup, and there were more “healthy” nonatretic follicles. Asillustrated in Fig. 2, there were more cystically dilated atreticfollicles in the PCO group than in the PCO anti-NGF group.Compared with the control ovaries, we never observed freshcorpora lutea or signs of recent ovulations with eggs in the tubein either of the PCO groups.

Low �1a-AR mRNA expression and high protein levels inovaries of rats with EV-induced PCO are restored to controllevels by anti-NGF treatments. As seen in Fig. 3A, the mRNAexpression of �1a-AR was lower in the PCO group than in thecontrol group. mRNA expression of �1a-AR in the ovaries ofPCO rats that had undergone repeated anti-NGF treatment (thePCO anti-NGF group), however, was higher than in the PCOgroup and differed nonsignificantly from expression in thecontrol group.

The amount of ovarian �1a-AR protein (Fig. 3B) was higherin the PCO group than in the control group. The amount of�1a-AR protein in the PCO rats that had undergone repeatedanti-NGF treatment was similar to the values in the controlgroup.

High �1b-AR mRNA expression and protein levels are mod-ulated by anti-NGF treatments in rats with EV-induced PCO.mRNA expression of �1b-AR is shown in Fig. 4. The expres-sion of �1b-AR was significantly higher in the PCO groupcompared with the control group. Anti-NGF treatment influ-enced the expression of �1b-AR mRNA in neither the anti-NGF nor the PCO anti-NGF group compared with the controlgroup and the PCO group, respectively.

As shown in Fig. 4B, the amount of ovarian �1b-AR proteinwas higher in the PCO group than in the control group. Theamount of �1b-AR protein in the ovaries of PCO rats that hadundergone repeated anti-NGF treatment, however, was similarto levels in the control groups.

High �1d-AR mRNA expression and protein levels are mod-ulated by anti-NGF treatments in rats with EV-induced PCO.The expression of �1d-AR mRNA (see Fig. 5A) was signifi-cantly higher in the PCO group compared with the control

Fig. 4. Ovarian �1b-AR mRNA (A) and protein (B) contents in control andPCO rats treated with anti-NGF IgG. A: expression of �1b-AR mRNA washigher in the PCO groups than in the control group. Anti-NGF treatmentinfluenced the expression of �1b-AR mRNA in neither control nor PCO rats.Values are given as means � SE (n � 10 in each experimental group) andnormalized to GAPDH. B: protein contents of �1b-AR were higher in the PCOgroup than the control group, whereas no significant differences were foundbetween the control and PCO anti-NGF groups. A representative Western blotis shown at bottom. Values are means � SE (n � 5) and are normalized for�-actin. *P � 0.05 vs. control group.

Fig. 3. Ovarian �1a-adrenoceptor (AR) mRNA (A) and protein (B) contents incontrol and PCO rats treated with anti-NGF IgG. A: �1a-AR mRNA decreasedin the PCO group. An increase in mRNA expression was induced by anti-NGFin PCO rats. Values are given as means � SE (n � 10 in each experimentalgroup) normalized to GAPDH. B: protein contents of �1a-AR were higher inthe PCO group than in the control group, and anti-NGF treatment restored theprotein contents in the PCO anti-NGF group to control levels. A representativeWestern blot is shown at bottom. Values are means � SE (n � 5) and arenormalized for �-actin. *P � 0.05 vs. control group; #P � 0.05 vs. PCO group.



group. mRNA expression of �1d-AR in the ovaries of PCO ratsthat had undergone repeated anti-NGF treatment, however, wassignificantly lower than in the PCO group.

The ovarian protein amount of �1d-AR in the PCO groupwas significantly higher compared with that in the controlgroup (Fig. 5B). A reduction in �1d-AR content to controlvalues was found in the PCO anti-NGF group.

Low levels of �2-AR protein are modulated by anti-NGFtreatments in rats with EV-induced PCO. mRNA expression of�2-AR is shown in Fig. 6A. Expression of �2-AR was un-changed in the PCO group compared with the control group.Anti-NGF treatment did not alter the mRNA expression of�2-AR in PCO rats.

The ovarian amount of �2-AR protein (see Fig. 6, B and C)was lower in the PCO group compared with the control group.Anti-NGF treatment restored levels of �2-AR protein in thePCO anti-NGF group to those in the control group.

High levels of ovarian p75NTR protein are lower afteranti-NGF treatment in rats with EV-induced PCO. mRNAexpression of p75NTR (Fig. 7A) was unaltered in the PCOgroup compared with the control group. mRNA expression of

p75NTR in the ovaries of PCO rats who had undergone repeatedanti-NGF treatment (the PCO anti-NGF group) differed non-significantly from that in the control and PCO groups.

The amount of p75NTR protein in the ovary (Fig. 7B) washigher in the PCO group compared with the control group. Theovarian amount of p75NTR protein in PCO rats who hadundergone anti-NGF treatment (the PCO anti-NGF group)differed nonsignificantly from that in rats in the control group.

High levels of ovarian TrkA protein are lower after anti-NGF treatment in rats with EV-induced PCO. The mRNAexpression of TrkA (Fig. 8A) was significantly lower in thePCO group compared with the control group. The mRNAexpression of TrkA in the ovaries of PCO rats that hadundergone repeated anti-NGF treatment (the PCO anti-NGFgroup) was still significantly lower than in the control groupand differed nonsignificantly from that in the PCO group.

The ovarian amount of TrkA protein (Fig. 8B) was signifi-cantly higher in the PCO group compared with the controlgroup. PCO rats who had undergone anti-NGF treatment had a

Fig. 6. Ovarian �2-AR mRNA (A) and protein (B) contents in control and PCOrats treated with anti-NGF IgG. A: mRNA expression of �2-AR was notsignificantly different in the PCO group compared with the control group, andanti-NGF treatment did not alter the mRNA expression of �2-AR in eithercontrol or PCO rats. Values are given as means � SE (n � 10 in eachexperimental group) and normalized to GAPDH. B: protein contents of �2-ARwere lower in the PCO group than in the control group, and anti-NGFtreatment restored protein contents in the PCO anti-NGF group to basal proteinlevels. A representative Western blot is shown at bottom. Values are means �SE (n � 5) and are normalized for �-actin. *P � 0.05 vs. control group; #P �0.05 vs. PCO group.

Fig. 5. Ovarian �1d-AR mRNA (A) and protein (B) contents in control andPCO rats treated with anti-NGF IgG. A: expression of �1d-AR mRNA washigher in the PCO group than the control groups. Anti-NGF treatment restoredthe expression of �1d-AR mRNA to control levels. Values are given asmeans � SE (n � 10 in each experimental group) and normalized to GAPDH.B: �1b-AR protein contents were higher in the PCO group than the controlgroup, and anti-NGF treatment restored protein contents in the PCO anti-NGFgroup to control levels. A representative Western blot is shown at bottom.Values are means � SE (n � 5) and are normalized for �-actin. *P � 0.05 vs.control group; #P � 0.05 vs. PCO group.



lower amount of TrkA protein than PCO rats who had not (thePCO group) but the same protein level as rats in the controlgroup.

High mRNA expression and protein levels of TH are down-regulated by anti-NGF treatment in rats with and withoutEV-induced PCO. Expression of TH mRNA (see Fig. 9A) wassignificantly higher in the PCO group than in the control group.Although the effect of repeated anti-NGF treatment on themRNA expression of TH in PCO rats (the PCO anti-NGFgroup) was not significant compared with in PCO rats who hadnot undergone anti-NGF treatment (the PCO group), the dif-ference in TH mRNA expression between the PCO anti-NGFgroup and the control group was no longer significant.

We found that the ovarian protein amount of TH (Fig. 9B)was significantly higher in the PCO group compared with thecontrol group. Although the TH protein level in PCO rats thathad undergone anti-NGF treatment was lower than in PCOrats, the TH protein level in the PCO anti-NGF group was stillsignificantly higher than in the control group.

DISCUSSION

The present study aimed to investigate the role of endoge-nous NGF in the ovarian expression of the sympathetic mark-ers �1-AR, �2-AR, TH, p75NTR, and TrkA. This was achievedby repeated intraperitoneal treatments with neutralizing anti-NGF antibodies in control and EV-treated rats. The novelfindings obtained in the present study were that repeatedsystemic anti-NGF treatments in EV-injected rats modulatemost of the measured sympathetic markers. Furthermore, theestrous cycle, which had been disrupted by EV injection, wasalso partially restored as well as the ovarian morphology ofPCO rats that underwent anti-NGF treatment.

It has been reported that rats with EV-induced PCO developovarian follicular cysts that are preceded by an increasedsynthesis of NGF in the ovary (20). In the present study, wefound a difference in ovarian morphology between the controland PCO groups, where the latter displayed a decrease both inovarian size and in the number and size of corpora lutea.Interestingly, ovarian morphology was partially restored afterrepeated anti-NGF treatments in the PCO anti-NGF group

Fig. 8. Increased ovarian tyrosine kinase A (TrkA) protein levels are de-creased after treatment with anti-NGF IgG in rats with EV-induced PCO. A:expression of TrkA mRNA was significantly lower in the PCO group com-pared with the control group. Repeated treatment with anti-NGF IgG furtherdecreased the mRNA expression of TrkA in the PCO anti-NGF group com-pared with the PCO group. Values are given as means � SE (n � 10 in eachexperimental group) and normalized to GAPDH. B: protein content of TrkA inthe ovaries was significantly higher in the PCO group compared with thecontrol group. A decrease in TrkA content to control levels was found in thePCO anti-NGF group. A representative Western blot is shown at bottom.Values are means � SE (n � 5) and are normalized for �-actin. *P � 0.05 vs.control group.

Fig. 7. Ovarian p75 neurotrophin receptor (p75NTR) mRNA (A) and protein(B) contents in control and PCO rats treated with anti-NGF IgG. A: nosignificant differences in p75NTR mRNA were found between the PCO andcontrol groups. After repeated anti-NGF treatments, the mRNA expression ofp75NTR was not significantly altered in the PCO anti-NGF group comparedwith the PCO group. Values are given as means � SE (n � 10 in eachexperimental group) and normalized to GAPDH. B: p75NTR protein contentswere higher in the PCO group, and repeated anti-NGF treatments restoredprotein contents in the PCO anti-NGF group to control levels. A representativeWestern blot is shown at bottom. Values are means � SE (n � 5) and arenormalized for �-actin. *P � 0.05 vs. control group.



compared with the PCO group. No difference was foundbetween the groups regarding ovarian weight. However, therewere more atretic follicles in the PCO group, and many of themwere dilated or even cystic compared with the PCO anti-NGFgroup. This, together with the observation of larger corporalutea in the PCO anti-NGF group than in the PCO group,means that there is more cellular and active (according to ourother data) tissue in the anti-NGF-treated PCO ovaries becausethe ovarian weights were equal in the two groups. The fluid-filled, dilated atretic follicles in the PCO group might be the

reason for the lack of ovarian weight reduction found inprevious studies (19).

It has been shown that an ovary-specific increase in NGFavailability leads to disruption of the estrous cycle and tochanges in follicular dynamics similar to some of the alter-ations produced by EV treatment (14). Furthermore, estrouscyclicity in EV-treated rats was restored by the ovarian appli-cation of anti-NGF antibodies and a p75NTR antisense mRNAoligodeoxynucleotide. The present data on the partial restora-tion of estrous cyclicity in PCO rats by intraperitoneal anti-NGF treatment confirm the importance of NGF in the regula-tion of follicle development and indicate the pathogenetic roleof the neurotrophin in rat PCO. It is worth mentioning that weobtained results similar to those achieved by Lara’s group (20),although they administered anti-NGF treatment locally insteadof systemically. This implies that both a local and a systemiccomponent of the NGF/NGF receptor system may be active inthe regulation of ovarian pathophysiology. It is known thatNGF can directly affect ovarian function through the activationof both the p75NTR and TrkA receptors expressed by severaltypes of ovarian cells (13, 20). Nevertheless, it cannot beexcluded that intraperitoneal injections of NGF antibodiestrigger a systemic response, modulating both central sympa-thetic outflow and the sympathetic drive to the ovaries, whichin turn affects estrous cyclicity (16, 22).

PCO induction in adult rats by EV injection causes adecrease in body weight gain. A variety of studies havedemonstrated that activation of the sympathetic nervous systemcan increase metabolic rate and fat consumption, leading to areduction in body weight (30, 32). The NGF/NGF receptorsystem could participate in this response to EV injection. Theevidence that NGF is overexpressed in the brain of PCO rats(4) and that intracerebroventricular administration of NGF hasan anorectic effect (44) provides reasonable support for thehypothesis that the NGF/NGF receptor system expressed in thecentral nervous system, in particular by hypothalamic neurons,participates in this behavioral/metabolic response. The findingthat the systemic blockade of endogenous NGF counteracts theanorectic effect of EV injection in the present study supportsthis hypothesis.

A novel result of the present study is that intraperitonealinjection of highly purified anti-NGF IgG in rats with EV-induced PCO causes significant changes toward normal levelsin the �1-AR protein subtypes and gene expression in theovaries. In a recent study (27) we found that the ovarianexpression of the three �1-AR subtypes was affected by EVinjection in the PCO model used in this study. The presentstudy confirms and extends our previous data. Here we de-scribe—for the first time to the best of our knowledge—evidence of control exerted by endogenous NGF on the ovarianexpression of �1-ARs. Thus a reasonable hypothesis is that thesympathetic nervous system regulates the expression and con-tent of the �1-AR subtypes because systemic blockade of theNGF action normalizes the EV-induced changes. The in-creased expression of ovarian �1a-AR in PCO rats is in linewith the finding that spontaneously hypertensive rats exhibitincreased central �1a-AR expression and decreased �2a-ARexpression that are correlated with elevated blood pressure andincreased sympathetic activity (31).

The functional significance of the �1-ARs in the ovaries ofPCO rats has not been clearly identified. The function of these

Fig. 9. Increased tyrosine hydroxylase (TH) mRNA expression and proteinlevels are downregulated by anti-NGF in both control and PCO groups. A:expression of TH was significantly higher in the PCO group compared with thecontrol group. Repeated anti-NGF treatments did not significantly decrease themRNA expression of TH in the PCO anti-NGF group compared with the PCOgroup. B: protein content of TH in the ovaries was significantly higher in thePCO group compared with the control group. A significant decrease in TH wasfound in the PCO anti-NGF group compared with the PCO group. A repre-sentative Western blot is shown at bottom. Values are means � SE (n � 5) andare normalized for �-actin. *P � 0.05 vs. control group; #P � 0.05 vs. PCOgroup.



types of ARs has been characterized in ovarian physiology asbeing primarily related to the regulation of OBF (45). Electri-cal stimulation of the splanchnic nerve has been shown todecrease OBF via activation of �-adrenergic receptors in theovarian blood vessels (42). We have also demonstrated (36)that modulation of the sympathetic drive to the ovaries differ-ently affects OBF in normal and PCO rats. Our present datasuggest that PCO-induced alterations in the expression of the�1-ARs could, at least in part, be responsible for these effectsand possibly for the dysregulation of OBF described in humanPCOS (2). It was also demonstrated recently that �1-ARs couldparticipate in the control of progesterone secretion by granu-losa cells in vitro (43). Thus �1-ARs could most probably beinvolved in the upregulation of progesterone release that hasbeen described in the EV-induced PCO ovary and related to�2-AR activation (5). Moreover, our present data demonstratethat both �1- and �2-AR expression are under NGF control,indicating that the involvement of the neurotrophin in thesteroidogenic activity of the ovaries could be exerted boththrough a direct activation of neurotrophin receptors (23, 29)and through the modulation of ovarian responsiveness to sym-pathetic inputs. Further studies are necessary to investigate thishypothesis.

Unaltered �2-AR mRNA expression and reduced �2-ARprotein levels after EV injection were also found in PCO rats,whereas inhibition of the biological activity of endogenousNGF counteracts the EV-induced reduction of ovarian �2-ARprotein. The downregulation of ovarian �2-AR mRNA in PCOrats has been reported previously (21, 22), as has �2-ARhyperresponsiveness to challenges by �2 agonists (5). It is alsoworth mentioning that the activity of the sympathetic nervoussystem and the ovarian NE content regulate the expression of�2-AR after the induction of PCO (22). Thus the presentobservations point to the hypothesis of an involvement of NGFin the regulation of �2-AR in this PCO model. Studies byothers report that challenging �2-AR with the selective agonistclenbuterol induces NGF synthesis in vitro (34) and that �2-ARagonists exert neuroprotective effects in rat models of focalcerebral ischemia through the induction of NGF synthesis (10).These findings, together with the data presented here, areconsistent with the hypothesis of a regulatory feedback loopbetween �2-AR and NGF in physiological and physiopatho-logical conditions.

The finding that the TrkA mRNA expression decreased afterEV treatment appears to be in line with previous studiesdemonstrating that ovarian TrkA is under LH control (15) andthat LH levels are lower than normal in EV-treated rats (6).Nevertheless, it has been demonstrated that NGF is able toregulate TrkA expression at several levels (46) and that theearly increase of ovarian NGF is a cardinal feature of thesteroid-induced PCO model (20, 37, 38). In the present studywe demonstrate that TrkA protein increased in EV-treated ratsand both TrkA mRNA and protein were downregulated byanti-NGF treatment in EV-treated rats, indicating that NGFitself participates in the regulation of its receptor expression inthe ovary. The activation of ovarian p75NTR synthesis after EVinjection and the knowledge that p75NTR can collaborate withTrkA receptors in the formation of high-affinity binding sitesand in enhancing cellular responses to NGF (17) are in linewith the present findings of elevated TrkA protein levels thatare counteracted by repeated anti-NGF treatment.

The observation that systemic injections of NGF antibodiescounteract the EV-induced increase not only of ovarian p75NTR

but also of ovarian TH indicates that NGF regulates p75NTR

and TH synthesis during the development of PCO. This inter-pretation is supported by previous experiments that showedthat the expression of both p75NTR and TH in mature sympa-thetic neurons is under NGF control (12, 28). Indeed, theenhanced activity of TH in the ovaries of PCO rats (22) and thehigher content of TH mRNA in catecholaminergic cells of theceliac ganglion that selectively project to the ovaries (20) areconsidered reliable biochemical and molecular markers ofactivation of the sympathetic nervous system after EV injec-tion. As for the mechanism responsible for the effects of NGF,our findings indicate that the action of NGF exerted on ovariantissues expressing TH is most probably due to sympatheticnerve fibers and intrinsic catecholaminergic cells (11), both ofwhich express p75NTR (20). As stated above, the hyperactiva-tion of ovarian sympathetic nerves in EV-induced PCO aremost likely related to an overproduction of NGF, which in turnregulates the expression of ovarian sympathetic markers suchas �1- and �2-ARs and TH.

In conclusion, the data obtained with this animal modelsuggest a major role for NGF in the pathogenesis of PCO inrats. Whether NGF plays a similar role in analogous processesin humans remains to be investigated.

ACKNOWLEDGMENTS

The laboratory assistance provided by Britt-Marie Larsson is gratefullyacknowledged.

GRANTS

This study was supported by grants from Wilhelm and Martina Lundgrens’sScience Fund, the Hjalmar Svensson Foundation, The Royal Society of Art andSciences in Goteborg and Magnus Bergwall’s stiftelse, the Novo NordiskFoundation, The Goteborg Medical Society, the Medical Research Council(Project Nos. 12206, 2004-6399, and 2004-6827), and the Swedish Heart LungFoundation. The contribution of L. Aloe and L. Manni is supported by ProgettiStrategici Fondo Integrativo Speciale per la Ricerca/Neurobiotecnologie andby Fondazione Cass di Risparmio di Bologna, Bologna, Italy.

REFERENCES

1. Aguado LI, Petrovic SL, and Ojeda SR. Ovarian beta-adrenergic recep-tors during the onset of puberty: characterization, distribution, and cou-pling to steroidogenic responses. Endocrinology 110: 1124–1132, 1982.

2. Aleem FA and Predanic M. Transvaginal color Doppler determination ofthe ovarian and uterine blood flow characteristics in polycystic ovarydisease. Fertil Steril 65: 510–516, 1996.

3. Aloe L and Levi-Montalcini R. Comparative studies on the effectselicited by pre- and postnatal injections of anti-NGF, guanethidine, and6-hydroxydopamine in chromaffin and ganglion cells of the adrenalmedulla and carotid body in infant rats. Adv Biochem Psychopharmacol25: 221–226, 1980.

4. Bai YH, Lim SC, Song CH, Bae CS, Jin CS, Choi BC, Jang CH, LeeSH, and Pak SC. Electro-acupuncture reverses nerve growth factorabundance in experimental polycystic ovaries in the rat. Gynecol ObstetInvest 57: 80–85, 2004.

5. Barria A, Leyton V, Ojeda SR, and Lara HE. Ovarian steroidalresponse to gonadotropins and beta-adrenergic stimulation is enhanced inpolycystic ovary syndrome: role of sympathetic innervation. Endocrinol-ogy 133: 2696–2703, 1993.

6. Brawer JR, Munoz M, and Farookhi R. Development of the polycysticovarian condition (PCO) in the estradiol valerate-treated rat. Biol Reprod35: 647–655, 1986.

7. Chandler CE, Parsons LM, Hosang M, and Shooter EM. A monoclo-nal antibody modulates the interaction of nerve growth factor with PC12cells. J Biol Chem 259: 6882–6889, 1984.



8. Chao MV and Hempstead BL. p75 and Trk: a two-receptor system.Trends Neurosci 18: 321–326, 1995.

9. Chomczynski P and Sacchi N. Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. Anal Bio-chem 162: 156–159, 1987.

10. Culmsee C, Stumm RK, Schafer MK, Weihe E, and Krieglstein J.Clenbuterol induces growth factor mRNA, activates astrocytes, and pro-tects rat brain tissue against ischemic damage. Eur J Pharmacol 379:33–45, 1999.

11. D’Albora H, Lombide P, and Ojeda SR. Intrinsic neurons in the ratovary: an immunohistochemical study. Cell Tissue Res 300: 47–56, 2000.

12. DeCouto SA, Jones EE, Kudwa AE, Shoemaker SE, Shafer AJ,Brieschke MA, James PF, Vaughn JC, and Isaacson LG. The effects ofdeafferentation and exogenous NGF on neurotrophins and neurotrophinreceptor mRNA expression in the adult superior cervical ganglion. BrainRes Mol Brain Res 119: 73–82, 2003.

13. Dissen GA, Hill DF, Costa ME, Ma YJ, and Ojeda SR. Nerve growthfactor receptors in the peripubertal rat ovary. Mol Endocrinol 5: 1642–1650, 1991.

14. Dissen GA, Lara HE, Leyton V, Paredes A, Hill DF, Costa ME,Martinez-Serrano A, and Ojeda SR. Intraovarian excess of nervegrowth factor increases androgen secretion and disrupts estrous cyclicityin the rat. Endocrinology 141: 1073–1082, 2000.

15. Dissen GA, Mayerhofer A, and Ojeda SR. Participation of nerve growthfactor in the regulation of ovarian function. Zygote 4: 309–312, 1996.

16. Erskine MS and Weaver CE Jr. The role of ovarian sympatheticinnervation in the control of estrous responsiveness in the rat. Horm Behav22: 1–11, 1988.

17. Hantzopoulos PA, Suri C, Glass DJ, Goldfarb MP, and YancopoulosGD. The low affinity NGF receptor, p75, can collaborate with each of theTrks to potentiate functional responses to the neurotrophins. Neuron 13:187–201, 1994.

18. Heider U, Pedal I, and Spanel-Borowski K. Increase in nerve fibers andloss of mast cells in polycystic and postmenopausal ovaries. Fertil Steril75: 1141–1147, 2001.

19. Krauchi K, Wirz-Justice A, Morimasa T, Willener R, and Feer H.Hypothalamic alpha2- and beta-adrenoceptor rhythms are correlated withcircadian feeding: evidence from chronic methamphetamine treatment andwithdrawal. Brain Res 321: 83–90, 1984.

20. Lara HE, Dissen GA, Leyton V, Paredes A, Fuenzalida H, Fiedler JL,and Ojeda SR. An increased intraovarian synthesis of nerve growth factorand its low affinity receptor is a principal component of steroid-inducedpolycystic ovary in the rat. Endocrinology 141: 1059–1072, 2000.

21. Lara HE, Dorfman M, Venegas M, Luza SM, Luna SL, MayerhoferA, Guimaraes MA, Rosa E Silva AA, and Ramırez VD. Changes insympathetic nerve activity of the mammalian ovary during a normalestrous cycle and in polycystic ovary syndrome: studies in norepinephrinerelease. Microsc Res Tech 59: 495–502, 2002.

22. Lara HE, Ferruz JL, Luza S, Bustamante DA, Borges Y, and OjedaSR. Activation of ovarian sympathetic nerves in polycystic ovary syn-drome. Endocrinology 133: 2690–2695, 1993.

23. Lara HE, McDonald JK, and Ojeda SR. Involvement of nerve growthfactor in female sexual development. Endocrinology 126: 364–375, 1990.

24. Lobo RA. The role of neurotransmitters and opioids in polycystic ovariansyndrome. Endocrinol Metab Clin North Am 17: 667–683, 1988.

25. Lobo RA, Granger LR, Paul WL, Goebelsmann U, and Mishell DR Jr.Psychological stress and increases in urinary norepinephrine metabolites,platelet serotonin, and adrenal androgens in women with polycystic ovarysyndrome. Am J Obstet Gynecol 145: 496–503, 1983.

26. Manni L, Antonelli A, Costa N, and Aloe L. Stress alters vascular-endothelial growth factor expression in rat arteries: role of nerve growthfactor. Basic Res Cardiol 100: 121–130, 2005.

27. Manni L, Lundeberg T, Holmang A, Aloe L, and Stener-Victorin E.Ovarian expression of �1- and �2-adrenoceptors and p75 neurotrophinreceptors in rats with steroid-induced polycystic ovaries. Auton Neurosci118: 79–87, 2005.

28. Miller FD, Speelman A, Mathew TC, Fabian J, Chang E, Pozniak C,and Toma JG. Nerve growth factor derived from terminals selectivelyincreases the ratio of p75 to trkA NGF receptors on mature sympatheticneurons. Dev Biol 161: 206–217, 1994.

29. Ojeda SR, Dissen GA, and Junier MP. Neurotrophic factors and femalesexual development. Front Neuroendocrinol 13: 120–162, 1992.

30. Racotta R, Ramirez-Altamirano L, and Velasco-Delgado E. Metaboliceffects of chronic infusions of epinephrine and norepinephrine in rats.Am J Physiol Endocrinol Metab 250: E518–E522, 1986.

31. Reja V, Goodchild AK, and Pilowsky PM. Catecholamine-related geneexpression correlates with blood pressures in SHR. Hypertension 40:342–347, 2002.

32. Ruffin M and Nicolaidis S. Electrical stimulation of the ventromedialhypothalamus enhances both fat utilization and metabolic rate that precedeand parallel the inhibition of feeding behavior. Brain Res 846: 23–29,1999.

33. Rush RA, Chie E, Liu D, Tafreshi A, Zettler C, and Zhou XF.Neurotrophic factors are required by mature sympathetic neurons forsurvival, transmission and connectivity. Clin Exp Pharmacol Physiol 24:549–555, 1997.

34. Samina Riaz S and Tomlinson DR. Pharmacological modulation ofnerve growth factor synthesis: a mechanistic comparison of vitamin Dreceptor and �2-adrenoceptor agonists. Brain Res Mol Brain Res 85:179–188, 2000.

35. Semenova I. Adrenergic innervation of the ovaries in Stein-Leventhalsyndrome. Vestn Akad Med Nauk SSSR 24: 58–62, 1969 (abstract inEnglish).

36. Stener-Victorin E, Kobayashi R, Watanabe O, Lundeberg T, andKurosawa M. Effect of electro-acupuncture stimulation of differentfrequencies and intensities on ovarian blood flow in anaesthetized rats withsteroid-induced polycystic ovaries. Reprod Biol Endocrinol 2: 16, 2004.

37. Stener-Victorin E, Lundeberg T, Cajander S, Aloe L, Manni L,Waldenstrom U, and Janson PO. Steroid-induced polycystic ovaries inrats: effect of electro-acupuncture on concentrations of endothelin-1 andnerve growth factor (NGF), and expression of NGF mRNA in the ovaries,the adrenal glands, and the central nervous system. Reprod Biol Endocri-nol 1: 33, 2003.

38. Stener-Victorin E, Lundeberg T, Waldenstrom U, Manni L, Aloe L,Gunnarsson S, and Janson PO. Effects of electro-acupuncture on nervegrowth factor and ovarian morphology in rats with experimentally inducedpolycystic ovaries. Biol Reprod 63: 1497–1503, 2000.

39. Tirassa P, Manni L, Stenfors C, Lundeberg T, and Aloe L. RT-PCRELISA method for the analysis of neurotrophin mRNA expression in brainand peripheral tissues. J Biotechnol 84: 259–272, 2000.

40. Torcia M, Bracci-Laudiero L, Lucibello M, Nencioni L, Labardi D,Rubartelli A, Cozzolino F, Aloe L, and Garaci E. Nerve growth factoris an autocrine survival factor for memory B lymphocytes. Cell 85:345–356, 1996.

41. Tsilchorozidou T, Overton C, and Conway GS. The pathophysiology ofpolycystic ovary syndrome. Clin Endocrinol (Oxf) 60: 1–17, 2004.

42. Uchida S, Hotta H, Kagitani F, and Aikawa Y. Ovarian blood flow isreflexively regulated by mechanical afferent stimulation of hindlimb innon-pregnant anesthetized rats. Auton Neurosci 106: 91–97, 2003.

43. Wasilewska-Dziubinska E, Borowiec M, Chmielowska M, Wolinska-Witort E, and Baranowska B. Alfa 1 adrenergic potentiation of proges-terone accumulation stimulated by vasoactive intestinal peptide (VIP) andpituitary adenylate cyclase-activating polypeptide (PACAP) in cultured ratgranulosa cells. Neuro Endocrinol Lett 23: 141–148, 2002.

44. Williams LR. Hypophagia is induced by intracerebroventricular admin-istration of nerve growth factor. Exp Neurol 113: 31–37, 1991.

45. Yousif M, Williams KI, and Oriowo MA. Characterization of alpha-adrenoceptor subtype(s) mediating vasoconstriction in the perfused rabbitovarian vascular bed. J Auton Pharmacol 16: 221–227, 1996.

46. Zhou J, Valletta JS, Grimes ML, and Mobley WC. Multiple levels forregulation of TrkA in PC12 cells by nerve growth factor. J Neurochem 65:1146–1156, 1995.



Download - Effect of anti-NGF on ovarian expression of alpha1- and beta2-adrenoceptors, TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Top Related