Journal of Reproduction and Development, Vol. 50, No. 6, 2004

—Original—

Expression of Nerve Growth Factor (NGF), and Its Receptors trkA and p75 in Ovaries of the Cyclic Golden Hamster (Mesocricetus auratus) and the Regulation of Their Production by Luteinizing Hormone

Zhanquan SHI1,2), Wanzhu JIN1,2), Gen WATANABE1,2), Akira, K. SUZUKI3), Shinji TAKAHASHI4) and Kazuyoshi TAYA1,2)

1)Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501–1193, 2)Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183–8509, and 3)PM2.5/DEP Research Project, and 4)Environmental Dioxin Project Group, National Institute of Environmental Studies, Ibaraki 305–9506, Japan

Abstract. In the present study, changes in localization of nerve growth factor (NGF) and its receptors,trkA and p75 in the ovary were investigated during the estrous cycle of the golden hamster. The effectof LH surge on changes in localization of NGF, trkA and p75 in the ovary was also investigated. NGFand its receptors trkA and p75 were localized in oocytes, granulosa cells and theca cells of variousstages of follicles throughout the estrous cycle. NGF and its two receptors were also present innumerous interstitial cells and luteal cells. The number of interstitial cells staining positively for NGFand its two receptors was greater in ovaries of day 1 (day 1=day of ovulation) than the other daysduring the estrous cycle. Treatment with the antiserum against luteinizing hormone releasinghormone (LHRH-AS) at 1100 h on day 4 completely blocked ovulation. There were few positivereactions for NGF and its two receptors in interstitial cells 24 hr after LHRH-AS injection. The effect ofLHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. Thedistinct widespread distribution of NGF and its two receptors in the ovary of golden hamsters suggestthat NGF may be an important growth factor for regulation of ovarian function. Furthermore, the LHsurge may be an important factor for inducing production of NGF and its two receptors in interstitialcells of the cyclic golden hamster.Key words: Estrous cycle, Golden hamster, Ovary, Nerve growth factor, TrkA, P75

(J. Reprod. Dev. 50: 605–611, 2004)

he neurotrophins (NTs) are target-derivedgrowth factor required for differentiation,

survival and development of discrete neuronalpopulations in the central and peripheral nervoussystems [1, 2]. It is also believed that NTs not onlyhave an effect on the nervous system, but also playan important role in regulating the development of

non-neuronal cells, including cellular subsets of theimmune, cardiovascular and endocrine systems [3–5]. The mammalian ovary is innervated by bothsympathetic and sensory neurons of the peripheralnervous system [6]. It has been demonstrated thatthe survival and differentiation of the neuronsi n n e r v a t i n g t h e o v a r y a r e s u p p o r t e d b yneurotrophic substances produced in the gland [7,8 ] . To da te , bes ide s NGF, bra in -der ived

Accepted for publication: July 27, 2004Correspondence: K. Taya (e-mail: taya@cc.tuat.ac.jp)

SHI et al.606

neurotrophic factor (BDNF) [9, 10], neurotrophin-3and neurotrophin-4/5 [12, 13], and also theirrespective receptors trkA (for NGF), p75 (for allNTs), trkB (for BDNF and NT-4/5) and trkC (forNT-3) [11], for each of the four neurotrophins havebeen identified in the ovary. In recent years,evidence has begun to accumulate that, NGF andits receptors trkA and p75 have important roles insexual development [8], folliclular developmentand ovulation [14–16]. In mammals, throughoutthe functional lifespan of the ovary, the process offollicle recruitment, follicular development andovulation, secretion of steroid hormones is cyclica n d d e p e n d s o n t h e c y c l i c s e c re t i o n o fgonadotropins from the pituitary [17]. Perez-Poloet al. [18] reported that, NGF activity and contentwere increased by estradiol stimulation in a gliomacell line culture. The level of NGF’s receptor trkAmRNA in the medium septum (MS) in thehippocampus showed significant fluctuationsduring the estrous cycle. In ovariectomized rats,estrogen administration significantly increased thetrkA mRNA content in the MS [19], thoughexpression of NGF, trkA and p75 in the ovary didnot show any clear change during the estrous cycle.The purpose of the present study was to determine,1) immunolocalization of NGF and its receptorstrkA and p75 in each ovarian cell type during thenormal estrous cycle in golden hamsters; and 2) thephysiological role of the LH surge on theexpression of NGF and its receptors trkA and p75in the ovary of cyclic golden hamsters.

Materials and Methods

AnimalsAdult female golden hamsters (Mesocricetus

auratus) were housed under controlled temperature(23–25 C) and lighting (14 h of light, 10 h ofdarkness, lights on from 0500–1900 h). Food andwater were available ad libitum. The 4-day estrouscycle was determined by the presence of acharacteristic vaginal discharge on the morning ofthe day of ovulation (day 1). Golden hamsters withat least two consecutive 4-day estrous cycles wereused in the present study. The experimentalprotocol was in accordance with the Guide for the

Care and Use of Laboratory Animals prepared byTokyo University of Agricultural and Technology.

SamplingOvaries were removed at 1100 h on each day of

the estrous cycle under light ether anesthesia, andfixed in 4% paraformaldehyde in 0.05 M PBS andkept for later use.

Immunohistochemical detection of NGF, trkA and p75 in ovaries

The paraffin-embedded ovarian tissues wereserially sectioned at 6 µm thickness and mountedon poly-L-lysine coated glass slides for more than24 h at 32 C. The sections were deparaffinized withxylene and rehydrated in graded ethanol, thenexposed to autoclave at 121 C for 15 min in sodiumcitrate buffer (0.01 M, pH 6.0) for antigen retrieval,followed by incubation in 3% H2O2 methanol atroom temperature for 1 h and then in 0.5% casein-Tris saline (0.05 M Tris-HCL with 0.15 M NaCL, pH7.6; CTS) at 37 C for 1 h. Then the tissue sectionswere incubated overnight at 4 C with polyclonalantibodies against NGF (0.4 µg/ml, M-20), trkA (4µg/ml, 763) and p75 (4 µg/ml, H-92) (Santa CruzBiotechnology, Santa Cruz, CA, USA) to identifyNGF, trkA and p75, respectively. The binding sitesof antibodies were visualized by the VECTASTAINABC Kit (Vector Laboratories, Inc., Burlingame,CA94010, USA), and 0.05% diaminobenzidine in 10mM Tris-buffered saline containing 0.01% H2O2.The specificity of the antibodies was examinedusing normal rabbit serum (NRS) instead ofprimary antibodies.

Effect of immunoneutralization of circulating luteinizing hormone releasing hormone (LHRH)

The antiserum against LHRH (LHRH-AS) usedin the present study was the antiserum previouslydescribed [20]. Golden hamsters were given asingle i.v. injection of 200 µl LHRH-AS at 1100 h onday 4, and followed by an another injection of 10 IUhuman chorionic gonadotropin (hCG, Sankyo ZokiLtd., Tokyo, Japan) or saline at 1700 h on the sameday. Ovaries were collected 24 hr after treatmentw i t h L H R H -A S , a n d w er e p r ep a r e d fo rimmunohistochemistry.

607NGF, TRKA AND P75 IN THE HAMSTER OVARY

Results

Immunohistochemical localization of NGF, trkA and p75 in ovaries of cyclic golden hamsters (Table 1 and Fig. 1)

Immunopositive reaction of NGF and its highaffinity receptor trkA and low affinity receptor p75showed same the distribution in ovaries of cyclicgolden hamsters during the estrous cycle.Immunopositive reactions of NGF and its tworeceptors were found in oocytes, theca cells andgranulosa cells of different sized follicles. Positivereactions for NGF, trkA and p75 were also presentin interstitial cells and lutein cells during theestrous cycle. Stronger positive staining for NGFand its two receptors in interstitial cells was foundin the ovary of day 1 as compared to other days ofthe estrous cycle. The sections incubated with NRSinstead of primary antibodies against NGF, trkAand p75 did not show any specific immunostainingreaction in any cell (data not shown).

Effect of LHRH-AS on immunolocalization of NGF, trkA and p75 in the ovaries (Fig. 2)

The treatment of LHRH-AS and saline at 1100 hon day 4 completely blocked ovulation on day 1and the characteristic vaginal discharge did notoccur in the animals 24 hr after treatment. On theother hand, LHRH-AS treatment followed by hCGinjection restored ovulation and the vaginaldischarge. In both groups of golden hamsters, apositive reaction for NGF and its two receptorswere observed in granulosa cells of various sizedfollicles at 24 hr after treatment with LHRH-AS as

well as in the ovaries obtained from intact cyclicanimals at 1100 h on day 1. In the ovaries ofanimals treated with LHRH-AS and saline,however, a positive reaction for NGF and its tworeceptors was very weak in interstitial cells, unlikethose in the intact animals. On the other hand, aclear immunopositive reaction for NGF and its tworeceptors was found in interstitial cells of theanimals to which 10 IU hCG was given afterLHRH-AS treatment. Immunopositive reactionsfor NGF and its two receptors in the LHRH-AS plushCG treated animals were similar to those in intactanimals at 1100 h on day 1.

Discussion

The present study is the first report the presenceof immunoreactive NGF and its two receptors trkAand p75 in the ovary of cyclic golden hamsters. Theimmunolocalization of the three components, NGF,trkA and p75 are distributed in oocytes, theca cells,granulosa cells and interstitial cells, as well as inlutein cells; and the cellular localization of NGF,trkA and p75 in the ovary of cyclic golden hamstersseems to be a species-specific characteristic. In them o u s e o v a r y , i t w a s r e p o r t e d t h a timmunoreactivity of trkA is located in thecal,interstitial and granulosa cells and oocytes,whereas p75 was only found in thecal andinterstitial compartments [15]. In the rat ovary,immunoreactive NGF and trkA protein wereexpressed in thecal and interstitial cells [14].Immunohistochemical detection of NGF and trkA

Table 1. Immunohistochemical staining of NGF, trkA and p75 in ovaries of cyclic golden hamsters

Components and stages NGF trkA p75

of the estrous cycle day1 day2 day3 day4 day1 day2 day3 day4 day1 day2 day3 day4

Granulosa cellsPreantral follicles + + + + + + + + + + + +Healthy antral follicles + + + + + + + + + + + +Early atretic follicles + + + + + + + + + + ++Advanced atretic follicles + + + + + + + + + + ++

OocytesPreantral follicles + + + + + + + + + + + +Healthy antral follicles + + + + + + + + + + + +

Theca cells + + + + + + + + + + + +Interstitial cells +++ ++ ++ + +++ ++ ++ + +++ ++ ++ +Corpus luteum + ++ + No CL + ++ + No CL + ++ + No CL

The immunohistochemical staining was determined as positive (+), strongly positive (++), negative (–).No CL: There are no corpus luteum in the ovary of the cyclic golden hamster on day 4 of the estrous cycle.

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Fig. 1. Localization of NGF, trkA and p75 in the ovary during the estrous cycle of the golden hamster.The staining of each section is as follows: a–d stained with NGF antibody; e-h stained with trkAantibody; and i–l stained with p75 antibody. The stage of the estrous cycle is as follows: day 1 (a,e, i); day 2 (b, f, j); day 3 (c, g, k); day 4 (d, h, l). Ovarian structures are shown as follows: HAF,healthy antral follicle; EAF, early atretic follicle; AAF, advanced atretic follicle; PF, preantralfollicle; CL, corpus luteum. The solid bar in the panel (a) represents 100 µm. All sections werephotographed using the same magnification.

609NGF, TRKA AND P75 IN THE HAMSTER OVARY

Fig. 2. Changes in localization of NGF, trkA and p75 in the ovary 24 h after treatment with antiserumagainst LHRH (LHRH-AS). The animals were given 10 IU hCG (a, b, c) or saline (d, e, f) 6 hr after theLHRH-AS treatment. The staining of each section is as follows: a, d stained with NGF antibody; b, estained with trkA antibody; c, f stained with p75 antibody. IC: interstitial cells. The solid bar in thepanel (a) represents 100 µm. All sections were photographed using the same magnification.

SHI et al.610

in the bovine ovary showed their presence in thecaland granulosa cells [21]. It was also reported thatp75 immunoreactivity is present in interstitial cellsin the human fetal ovary [22], and NGF bioactivityin sheep antral follicular fluid [23].

The presence of NGF and its receptors trkA andp75 in all cell types in the golden hamster ovarysuggests their physiological roles in folliculardevelopment, ovulation and oocyte maturation.Previous studies in other animals showed criticalroles for NGF and its receptors in ovarian function.In peripubertal rats, immunoneutralization of NGFaction or pharmacological blockade of trkA activityresulted in inhibition of ovulation [14]; NGF nullmutant mice exhibited a markedly reduced numberof primary and secondary follicles, and an increasein number of naked oocytes [15]. Expression ofNGF in oocytes and induction of oocyte meioticmaturation by NGF were reported in Xenopus [24,25]. The stimulatory effect of NGF on oocytematuration was also found in mouse and ovineoocytes [26–28].

The stronger positive staining of NGF, trkA andp75 in interstitial cells of the golden hamster ovaryon day 1, and their very weak immunostainingafter LHRH-AS treatment, together with therecovered stronger positive staining following hCGinjection after LHRH-AS treatment, indicates aninteraction among the three proteins and the cyclicLH surge. Dissen et al. [14] reported that in juvenilerats treated with equine chorionic gonadotropin,

significantly elevated trkA mRNA levels in theovary were found after the first preovulatory peakof the LH surge, but p75 mRNA levels were notaffected. NGF mRNA contents in the ovary alsoreached peak levels coinciding with the trkAmRNA peak, and the increased trkA mRNAexpression was LH dependent, as verified byculture of thecal and interstitial cells treated withhCG. Our present results clearly showed that NGFand its two receptors, trkA and p75, in ovarianinterstitial cells were all regulated by LH during theestrous cycle of golden hamsters.

I n s u m m a r y , w e f o u n d t h eimmunohistochemical localization of NGF and itsreceptors trkA and p75 in the golden hamsterovary. The results demonstrate that LH is animportant regulator of production of NGF, and itsreceptors trkA and p75, in ovarian interstitial cell ofcyclic golden hamsters. These results suggest thatNGF has important autocrine or paracrineregulatory roles in ovarian function of cyclichamsters.

Acknowledgement

This work was supported by a Grant-in-Aid forCOE Scientific Research (The 21st Century Centerof Excellence Program, E-1) from the Ministry ofEducation, Culture, Sport, Science and Technologyof Japan.

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