General and Comparative Endocrinology 168 (2010) 326–332

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General and Comparative Endocrinology

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Age-dependent variation in the RFRP-3 neurons is inversely correlatedwith gonadal activity of mice

Sumit Sethi a, Kazuyoshi Tsutsui b, Chandra Mohini Chaturvedi a,*

a Department of Zoology, Banaras Hindu University, Varanasi 221 005, UP, Indiab Department of Biology, Waseda University, Tokyo 162-8480, Japan

a r t i c l e i n f o

Article history:Received 19 January 2010Revised 13 March 2010Accepted 16 April 2010Available online 28 April 2010

Keywords:RFRP-3GnIHDMHTestosteroneTestisReproductionMice

0016-6480/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ygcen.2010.04.011

* Corresponding author. Fax: +91 542 2368323.E-mail address: cmcbhu@yahoo.com (C.M. Chaturv

a b s t r a c t

The present study analyzed changes in the expression of RFamide-related peptide-3 (RFRP-3; a mamma-lian ortholog of avian gonadotropin-inhibitory hormone), in the brain and correlated it with testicularactivity of mice of different age groups (day-old, 1-, 3-, 5-, 7-, 9-, 11-, 13-week and 1.5-year-old). Testic-ular activity after a progressive increase up to 13-week of age declined in the old mice. On the other hand,while immunoreactive (ir) RFRP-3 neurons were not seen in the day-old mice, few appeared in 1-week-old mice, their number and size increased drastically at 3-week of age. This condition remained unaltereduntil 7-week of age followed by a progressive decline up to the age of 13-week and thereafter increasedagain in the old age. The present findings indicate that hyperactivity of the ir-RFRP-3 neurons of dorso-medial nucleus of hypothalamus (DMH) observed in prepubertal mice declines in reproductively activemice and increases again in the old mice having declined reproductive performance. It is concluded thataging mice exhibits inverse correlation of RFRP-3 neurons and gonadal activity suggesting that functionof RFRP-3 is not initiated until 1-week of age and thereafter it could participate in the regulation of gona-dal development.

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1. Introduction

Progressive development and growth of gonad occur in parallelwith somatic growth until the attainment of puberty in some non-seasonal breeders. However, remarkable changes in the gonadalactivity (spermatogenesis, testosterone level, etc.) occur in a shortspan of time just before and around the puberty in these species.Thereafter, sperm production and testosterone concentration re-main at the maximum level during the reproductively active peri-od of the species’ life span. Regressive/degenerative changes startappearing in the seminiferous tubules with aging. Such changeshave been reported in men, rats, oxen, mice and cats but theircauses remain unclear (Lützen and Ueberberg, 1973; Humphreyand Ladds, 1975; Gosden et al., 1982; Elcock and Schoning, 1984;Hatakeyama et al., 2008). Similar degenerative changes are re-ported to be induced by irradiation, artificial cryptorchidism andexperimental autoimmunization in laboratory animals (Nebeland Murphy, 1959; Sato et al., 1981; Jegou et al., 1983). Regressivetesticular changes, such as those in aged men and animals, can alsooccur spontaneously in young men resulting in infertility, althoughthe cause remains unknown (Wong et al., 1973).

Reproduction is under the control of the hypothalamic decapep-tide gonadotropin-releasing hormone (GnRH), which is secreted

ll rights reserved.

edi).

into the hypophyseal portal system to stimulate synthesis andsecretion of the gonadotropins. This releasing hormone originallyisolated from mammals (Matsuo et al., 1971) and subsequentlyfrom birds (King and Millar, 1982; Miyamoto et al., 1982, 1984)and other vertebrates, is the primary factor responsible for hypo-thalamic control of gonadotropin secretions from the pituitary. Go-nadal sex steroid and inhibin can also modulate gonadotropinsecretion via feedbacks from the gonads. But, a neuropeptideinhibitor for gonadotropin secretion was unknown in vertebratesuntil the discovery of hypothalamic dodecapeptide (SIKP-SAYLPLRF-NH2) termed gonadotropin-inhibitory hormone (GnIH),which directly inhibits gonadotropin release from the culturedquail anterior pituitary (Tsutsui et al., 2000). Ubuka et al. (2003)also analyzed developmental changes in the expressions of GnIHprecursor mRNA and the mature peptide GnIH during embryonicand posthatch ages in the quail diencephalon including the para-ventricular nucleus and median eminence. The gene of the mam-malian RFamide-related peptides (RFRPs) is orthologous to theavian GnIH gene. This RFRP gene gives rise to two biologically ac-tive peptides; RFRP-1 and RFRP-3 (Hinuma et al., 2000; Ukenaet al., 2002; Kriegsfeld et al., 2006; Clarke et al., 2008). Untilnow, these mammalian GnIH orthologues have been identified inthe bovine, rat, mouse and human brains (Fukusumi et al., 2001;Ukena et al., 2003; Yoshida et al., 2003; Ubuka et al., 2009). RFRPsare mainly expressed in the neurons of dorsomedial hypothalamicnucleus (DMH) and/or paraventricular nucleus (PVN) in mammals

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and are directly or indirectly known to have inhibiting influence onthe gonadal function (Hinuma et al., 2000; Fukusumi et al., 2001;Ukena and Tsutsui, 2001; Yano et al., 2003; Kriegsfeld et al.,2006; Johnson et al., 2007; Ubuka et al., 2009; Smith and Clarke,2010).

In spite of the identification, characterization and localization ofthis inhibitory peptide in mammalian system, relatively few re-ports are focused on the physiological aspects of this mammalianGnIH ortholog. Recently, Quennell et al. (2010) observed an in-crease in the hypothalamic RFRP gene expression of male rats be-tween 2 and 4 weeks of age. Sethi et al. (2010) also reported thehigh levels of the expression of RFRP-3 neurons observed in sexu-ally immature mice decreased in the sexually mature condition.Hence, in the present study, age-dependent variations were moni-tored in the expression of ir-RFRP-3 in mice brain. Testicular activ-ity and immunohistochemistry of RFRP-3 neurons of mice ofdifferent age groups (ranging from day-old to prepubertal, postpu-bertal and 1.5-year-old mice) were studied to assess the possiblecorrelation between the gonadal function (development, matura-tion and regression) and the expression of RFamide peptide inaging mice. The present findings indicate an inverse relation be-tween two systems and suggest a functional role of RFRP-3 as akey neuropeptide involved in mammalian reproduction.

2. Materials and methods

2.1. Animals

Male laboratory mice (Mus musculus) of the Parkes (P) strainwere obtained from a colony maintained in our laboratory. Themice were housed under hygienic conditions in a well-ventilatedphotoperiodically controlled room (light:dark 12:12), and wereprovided with commercial food (Pashu Aahar Kendra, Varanasi, In-dia) and tap water ad libitum. All the experiments were conductedin accordance with institutional practices, and within the frame-work of the revised Animals (Scientific Procedures) Act of 2002of the Government of India.

2.2. Source of tissues

Tissues for the aging experiment obtained from day-old, 1-, 3-, 5-,7-, 9-, 11-, 13-week and 1.5-year-old mice (n = 5 per group) wereweighed. Day of delivery/birth was designated as day-old. Small ani-mals (day-old, 1-week and 3-week) were killed by cervical disloca-tion whereas large animals (5-, 7-, 9-, 11-, 13-week and 1.5-year-old) were killed under ether anesthesia prior to dissection. Bloodwas collected from the heart into a heparinized tube and centrifugedat 4000 rpm for 20 min at 4 �C to separate plasma and stored at -20 �C for the radioimmunoassay of testosterone. Mice were perfusedtranscardially with phosphate-buffered saline (PBS) followed byZamboni’s fixative [4% paraformaldehyde in 0.1 M sodium phos-phate buffer (PB); pH 7.4]. The length and width of the left testiswas measured in situ with dial calipers, and the testicular volumewas calculated using Bissonett’s formula 4/3pab2 (a = 1/2 of the longaxis; b = 1/2 of the short axis) (Jaiwal and Chaturvedi, 1991).

The testes from both sides were excised and weighed, and thegonado-somatic index (GSI) was calculated as weight of the pairedtestes/100 g body weight. The brain was also dissected out andpost-fixed to be processed for the immunohistochemistry ofRFRP-3.

2.3. Histological preparations

The testes removed after perfusion from each age group micewere weighed and re-fixed overnight in the same fixative men-

tioned above for histological studies. Testes were then dehydratedin an ascending series of alcohol, treated with xylene and embed-ded in paraffin wax. 6-lm-thick sections were cut by a Weswox ro-tary microtome (Western Electric & Scientific Works, AmbalaCantt, India), and stained with haematoxylin–eosin. Histologicalsections of the testis were viewed under a microscope (Axioskop2 Plus; Carl Zeiss AG, Oberkochen, Germany) and images were cap-tured with a digital camera. Seminiferous tubule diameter wasdetermined in 10 sections per mouse testis by using the image ana-lyzer software Motic Images 2000 version 1.3 (Sethi and Chaturv-edi, 2009).

2.4. Hormone assay

A radioimmunoassay (RIA) of plasma testosterone was per-formed using a commercial RIA kit (Immunotech, Marseille,France) according to the manufacturer’s instructions. The antise-rum used in the assay was specific for testosterone; cross-reactiv-ity was less than 0.03% with estradiol, 0.03% with progesterone,0.01% with dehydroepiandrosterone, and 0.6% with androstenedi-one. Sensitivity of the assay was 0.025 ng/ml. The intra- and in-ter-assay coefficients of variation were 14.8% and 15%,respectively.

2.5. Immunohistochemistry of RFRP-3

Brains, dissected out after whole body perfusion, and post-fixa-tion were passed through a graded series of alcohol and embeddedin paraffin wax. Six micrometers thick coronal sections of the brainpassing through the region of the dorsomedial nucleus of hypo-thalamus (DMH) were deparaffinized in xylene, rehydrated andrinsed in PBS (0.02 M, pH 7.4). Immunohistochemistry of RFRP-3was performed by the method of Sternberger and Sternberger(1986) with some modifications. In brief, endogenous peroxidaseactivity was eliminated from the sections by incubation with0.3% H2O2 in absolute methanol for 20 min. After blocking nonspe-cific binding components with 5% normal goat serum in PBS for 1 hat room temperature, the sections were immersed in 1:1000 dilu-tion of primary antiserum raised against quail GnIH (Tsutsui et al.,2000) for 16–20 h at 4 �C. Slides were rinsed in PBS and incubatedwith 1:1000 dilution of horseradish-peroxidase-conjugated sec-ondary antibody, followed by another set of washes. Sections werethen incubated for 1 h in avidin–biotin complex (Vectastain EliteKit, Vector Laboratories, Burlingame, CA, USA). The resulting com-plex was visualized using 0.03% 3,30-diaminobenzidine in 0.05 MTris–HCl (pH 7.4) with 0.03% H2O2 for 10–20 min. After washingwith Tris–HCl, the staining was stopped by washing in distilledwater. Sections were dehydrated through an ethanol series,cleaned in xylene and mounted using DPX (a mixture of distyrene,a plasticizer and xylene). Slides were viewed under a microscope(Axioskop 2 Plus; Carl Zeiss AG, Oberkochen, Germany) and imageswere captured using a digital camera.

2.6. Image analysis

The number of immunoreactive RFRP-3 cell bodies was countedfrom the centre region of DMH using a X20 objective lens and Mac-Biophotonics ImageJ software. Around 25–35 sections were ob-tained from the dorsomedial nucleus and the RFRP-3 neuronalnumbers were counted in the middle 5 sections only. Once imagesof these sections containing RFRP-3 neurons were captured, back-ground threshold levels were adjusted to allow for automaticcounting of ir-neurons (not fibres) in these sections by the softwarepackage. In addition, the ImageJ software placed a dot on each neu-ron counted, allowing the observer to verify accuracy during thecounting process.

328 S. Sethi et al. / General and Comparative Endocrinology 168 (2010) 326–332

For measurement of the RFRP-3 neuronal area (visible immuno-reactive area), up to 50 randomly selected cells were measured. Foranalysis, the cell body area was averaged within a group. Using aX100 objective lens in 1 plane of focus only, 5 cells nearest tothe centre of the screen that were in focus had their perimeterstraced by using a mouse with image analyzer software (MoticImages 2000 version 1.3). The fibres sprouting from the cell bodywere excluded by continuing tracing in an arc defined by theperimeter of the cell body on either side of the region that the fi-bres emanated from (i.e. continuing as if the fibre was not presentand the cell body was uniform in shape at the fibre’s origin). Thisprovided a measure of the area of each cell, which was storedand used in the later analysis. If the whole perimeter of a cell bodywas not clearly visible, then that cell was not measured.

2.7. Statistical analyses

For each parameters measured, the data was calculated asmeans ± standard error. One-way analysis of variance – ANOVA(to compare all the groups) was used followed by multiple pair-wise comparisons (Tukey Alpha). For this (pair-wise comparison)all the groups were compared (i) with their previous age groupand (ii) with 3-week-old mice. The nonparametric Spearman’srho was used for correlation coefficient of RFRP-3 neuronal areaand plasma testosterone. Significance was assumed at the levelof p < 0.05. Statistical analyses were performed with SPSS 12.0 soft-ware. Error bars in the graphs represent means ± standard error ofthe mean (SEM).

3. Results

There was a significant increase in the body weight of mice withadvancing age when compared to the previous age group. Bodyweight of day-old to 13-week-old mice ranged from 2 to 30 gand was 37 to 42 g in 1.5 year-old/aged mice (Fig. 1A). The gona-

Fig. 1. Age-dependent variation in the (A) body weight; (B) gonado-somatic index (GSI)means ± SE (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; significance of difference from the p3-week-old group.

do-somatic index (mg weight of testes/100 g body weight) also in-creased significantly from day-old to aged mice when compared tothe previous age group (Fig. 1B). However, for the testicular vol-ume, which also increased with increasing age, the significant dif-ference was observed only among 3-week vs 1-week and 11-weekvs 9-week and old vs 13-week mice (Fig. 1C). Plasma testosteronealso showed increasing trend from young to maturing and maturedmice but decreased in the old mice (Fig. 1D).

The testes of day-old mice had smaller loosely arranged ill de-fined seminiferous tubules containing only one layer of restingspermatogonial cells (Type A). Abundant interstitial tissue was alsovisible in the intertubular spaces (Fig. 2A). However, the testes of1-week-old mice contained well defined seminiferous tubules con-taining 2–3 layers of primitive type A spermatogonial cell and Ser-toli cells around the central lumen (Fig. 2B). The continual growthof the seminiferous tubules and the progression of spermatogene-sis were noted from 3-week onward. Progressively, more numberof spermatocytes were observed in the testis of both 3- and 5-week-old mice. Further, while haploid round spermatids were seenin the testes of 3-week-old mice (Fig. 2C), elongation of spermatidswas evident at 5-week of age (Fig. 2D). Thereafter, the completespermatogenic complement was observed in the seminiferous epi-thelium of 7-, 9-, 11- and 13-week-old mice testes with spermato-zoa from 9-week onward (Fig. 2E–H). Seminiferous tubules of oldmice showed a condition of degeneration. The tubules containedfew layers of spermatogonial cells and some primary spermato-cytes. Most of the tubules exhibited marked depletion and exfolia-tion of germ cells, intraepithelial vacuolation due to degenerationof Sertoli cells and loosening of the germ cells along with the for-mation of giant cells in some tubules (Fig. 2I). The seminiferous tu-bule diameter also increased significantly with the increasing agefrom day-old to 13-week-old mice, but at old age (1.5 years), diam-eter of the seminiferous tubule decreased (Fig. 5A).

In general, RFRP-3 neurons located in the dorsomedial nucleusof hypothalamus (DMH) region are observed on either side of thethird ventricle (Fig. 3). RFRP-3 neurons were not detectable in

; (C) testicular volume and (D) and plasma testosterone concentrations. Values arerevious age group. a, p < 0.05; b, p < 0.01; c, p < 0.001; significance of difference from

Fig. 2. Transverse section of the testes of mice of different age groups. (A) Testis of day-old mice showing abundant interstitial cells along with smaller, scattered and illdefined seminiferous tubules with only one layer of quiescent spermatogonial cells. (B) Testis of 1-week-old mice showing well defined but smaller seminiferous tubulescontaining more than one layer of the spermatogonial cells around the lumen. (C) Testis of 3-week-old mice showing increased size of seminiferous tubules containingprimary spermatocytes and haploid spermatids. (D) Testis of 5-week-old mice showing the presence of elongating spermatids. (E–H) Testis of 7-, 9-, 11- and 13-week-oldmice showing more or less complete spermatogenic complement in the seminiferous tubule with bunches of spermatozoa in the lumen and Leydig cells in the triangularinterstitial spaces. (I) Testis of old (1.5 year) mice showing degenerative/regressive changes in the relatively smaller seminiferous tubules (note, the loosening of the germinalepithelium and the depletion and exfoliation of germ cells) (scale bar = 50 lm).

Fig. 3. Low and high magnification coronal sections of the brain of mice passingthrough the DMH region showing ir-RFRP-3 in the 3-week and 13-week old mice.V3, third ventricle; DMH, dorsomedial hypothalamus; VMH, ventomedialhypothalamus.

S. Sethi et al. / General and Comparative Endocrinology 168 (2010) 326–332 329

the coronal section of day-old mouse brain passing through DMH(Fig. 4A). The RFRP-3 stained neurons in 1-week-old mouse werefewer in number and weakly immunostained (Fig. 4B). Dense pop-ulation of large sized RFRP-3 neurons observed at the age of 3-week continued till 7th week followed by gradual decline in theseparameters until 13-week of age. The immunoreactivity was also

low in DMH neurons of these mice which declined gradually fromthe age of 9- to 13-week. These immunoreactive cells tended to beof various shapes like round, oval, unipolar/bipolar or even irregu-lar but their fibres were short (Fig. 4). At old age intense immuno-reactive RFRP-3 neurons were observed with larger cell bodieswhose number increased significantly compared to previous agegroup (i.e. 13-week-old) (Fig. 4I). Coronal section of brain of day-old mice showed no immunoreactivity of RFRP-3 although someimmunoreactivity was observed at the age of 1-week in few neu-rons. The RFRP-3 neuronal area increased only up to 3-week andthe neuronal number up to 5-week, which declined thereafter.Again RFRP-3 neuronal area and neuronal number are increasedin old age mouse brain (Fig. 5B and C).

4. Discussion

A number of avian studies reported that hypothalamic RFamidepeptide containing neurons project to the median eminence (ME)as well as GnRH neurons. This peptide named GnIH inhibits gona-dotropin release both in vitro and in vivo in birds (Tsutsui et al.,2000; Ciccone et al., 2004; Osugi et al., 2004; Bentley et al.,2006; Ubuka et al., 2006). RFRP-3, a mammalian GnIH orthologhas been also identified in the rat, mouse, bovine and human (Yos-hida et al., 2003; Bentley et al., 2006; Tsutsui and Ukena, 2006;Ubuka et al., 2009; Sethi et al., 2010; Smith and Clarke, 2010). BothGnIH and RFRP-3 share a common C-terminal LPXRFamide motifand are functionally the same (Tsutsui and Ukena, 2006). Both ofthese peptides have been shown to suppress gonadotropin releasein vivo in rodents (Kriegsfeld et al., 2006; Johnson et al., 2007).Therefore, it is considered that RFRP-3, which is a mammalianGnIH ortholog, might be an important factor in inhibiting gonado-tropin release in mammals, similar to the action of GnIH in birds.

Fig. 4. Coronal section of the brain of mice of different age groups passing through the DMH showing ir-RFRP-3 neurons. (A) Day-old mice brain showing the absence of ir-RFRP-3 neurons. (B) 1-week-old mice showing some ir-RFRP-3 neurons having weak immunostaining. (C–E) 3-, 5- and 7-week-old mice brain showing increased populationof RFRP-3 neurons with intense immunostaining. (F–H) 9, 11 and 13-week-old mice brain showing decreased number, size and the staining of ir-RFRP-3 neuronal cell bodies.(I) 1.5-year-old mice brain showing increased number and size of RFRP-3 neurons with intense immunostaining (scale bar = 20 lm). Low-magnification coronal sectionsthrough the DMH showing the localization of ir-RFRP-3 neurons (arrows) in the 1-, 5-, 13-week and 1.5-year-old mice brain (inset) (B, D, H, I). V3, third ventricle; DMH,dorsomedial hypothalamus; VMH, ventomedial hypothalamus (scale bar = 200 lm).

330 S. Sethi et al. / General and Comparative Endocrinology 168 (2010) 326–332

This strongly suggests RFRP-3 would also be active in the directinhibition of gonadotropin release from gonadotropes in mice.The demonstration that RFRP regulates GnRH neuron firing andearly gene expression (Anderson et al., 2009; Ducret et al., 2009;Wu et al., 2009) suggests that the peptide may also be effectiveat this level in mice.

In this study, we observed a correlation between ir-RFRP-3 neu-rons and gonadal development in the mice of different age groups.Our previous findings also indicated that number and size of ir-RFRP-3 neurons as well as immunostaining of the peptide de-creased in the sexually mature mice compared to that of immaturemice (Sethi et al., 2010). Based on our previous and the presentfindings, we hypothesized that the decrease in hypothalamicRFRP-3 content may be involved in the increased activity of gona-dal axis (plasma testosterone concentration, testicular growth andspermatogenesis) observed during sexual development. In accor-dance with this hypothesis, it is likely that dense population ofRFRP-3 in old/aged mice may suppress the plasma testosteroneconcentration resulting in apoptotic cell death in the testis. Thishypothesis is supported by the observations in rodents that testos-terone is a testicular cell-survival factor and a decrease in its levelmay lead to apoptosis (Tapanainen et al., 1993; Woolveridge et al.,1999).

In the present developmental study, in 1-day-old mice, ir-RFRP-3 neurons were not visible. However, few ir-RFRP-3 neurons ap-peared in 1-week-old mice and these were immature judging fromtheir small cell size/neuronal area. In contrast, at 3-week of ageRFRP-3 neurons appear to be mature enough with abundant cellbodies, increased size and intense immunostaining which weremaintained up to 7-week of age followed by a decline at 9-weekof age which continued until 13-week of age attaining minimumwhen testes were at its maximal activity. Interestingly, in old micethe trend got reversed with sharp increase in RFRP-3 neuronal

parameters and significant decrease in testicular activity includingspermatogenesis. On the other hand, from the age of 3-week on-ward there is a sharp increase in the testicular size followed bygradual development in the activity up to 9-week and attainingplateau thereafter.

Sethi et al. (2010) demonstrated experimentally that theexpression of RFRP-3 in the hypothalamus increases at the onsetof testicular regression in mice treated with 5-HTP and L-DOPAat the interval of 8 h, a condition which induces testicular suppres-sion. Therefore, it is reasonable to suggest that increased expres-sion of RFRP-3 neurons containing GnIH-like activity may be oneof the causes of gonadal regression in old/aged mice. On the otherhand it is also quite possible that this peptide may maintain gona-dal axis in quiescence until puberty is attained. A gradual decreasein the ir-RFRP-3 peptide in developing mice during the attainmentof puberty as well as reproductive potency and an increase in plas-ma testosterone supports the GnIH-like activity of this peptide.Age-related changes in the activity of RFRP 3-producing neuronsalso seems to be associated with testicular regression in old/agedmice since RFRP-3 neurons of sexually immature (3–5 weeks)and old mice were in similar condition although immunostainingwas intense in the later age group mice.

The present study provides profiles of developmental changesin the expression of ir-RFRP-3 peptide in the DMH neurons of miceof different age groups. Since, the expression of RFRP-3 neurons de-creases in the sexually active condition and is at the increased levelin the reproductively quiescent sexually immature as well as oldmice. Obviously, RFRP-3 neuronal expression is negatively corre-lated with gonadal growth and activity in mice (R2 = 0.56;p < 0.01, where R is the coefficient of linear regression) (Fig. 6). Itmay be also suggested although indirectly that similar to birds,in the continuous breeder mice also, RFRP-3 acts as the inhibitoryhypothalamic peptide and is involved in the regulation of testicular

Fig. 6. Relationship between RFRP-3 neuronal area and plasma testosteroneconcentration in mice. The data points represent means ± SE from different agegroups (3-week, 13-week and 1.5-year-old). R2 values for the regressions were 0.56(p < 0.01).

Fig. 5. Age-dependent variation in the (A) seminiferous tubule diameter; (B) RFRP-3 neuronal area and (C) and the number of RFRP-3 neurons in the DMH. Values aremeans ± SE *p < 0.05; **p < 0.01; ***p < 0.001; significance of difference from theprevious group. a, p < 0.05; b, p < 0.01; c, p < 0.001; significance of difference from3-week-old group.

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development and maintenance (Sethi et al., 2010). Thus, RFRP-3neurons also play a role in mammalian reproduction supportingearlier data that RFRP-3 act directly on the pituitary or GnRH neu-rons through RFRP-3 receptor to regulate gonadal function.

Sex steroids and inhibin levels are reported to influence/modu-late HPG axis at the level of the brain (GnRH) and pituitary(gonadotrophs) via negative feedback, and sensitivity to feedbackvaries with reproductive status (Ramirez and McCann, 1965; Leh-man et al., 1997; Richardson et al., 2004). But feedback effect oftestosterone at the level of RFRP-3/GnIH is not yet investigated,although this possibility can not be ruled out. However it is quitepossible that increase in RFRP-3 neuronal activity in aged mice oc-curs via steroid feedback-independent mechanism similar topubertal increase in GnRH (Richardson et al., 2004). Further, thereare many inhibitory neuroendocrine mechanisms involved in the

aging process and RFRP-3 may be one of these factors. Furtherstudies are required to elucidate mechanisms underlying changesin RFRP-3 levels.

In conclusion, this study demonstrates that the gonadal quies-cence in sexually immature mice and a decline in reproductive per-formance in aging mice (concomittent with a low and decreasingtestosterone level respectively) are inversely correlated with in-creased and increasing expression of ir-RFRP-3 in the DMH.Accordingly, it is suggested that RFRP-3 might contribute to theprepubertal sexual quiescence of the gonadal axis as well as duringthe age-related decline in the reproductive performance. Further, itis also quite possible that, decreased level of this peptide may leadto reproductive development and increased gonadal activity,although further experimental study is required to identify suchmechanism of action involving both GnRH- and GnIH-like activityin relation to age-related gonadal development in mammals.

Acknowledgments

This work was supported by funds from the Council of Scientificand Industrial Research (CSIR), New Delhi, India, to C.M.C. (Re-search Project 37/1284/07/EMR-II). A Senior Research Fellowshipto S.S. from the Indian Council of Medical Research (ICMR) is grate-fully acknowledged.

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