)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/ije/2015/982726.pdflines have...

Research ArticleExpression Analysis of Gnrh1 and Gnrhr1 inSpermatogenic Cells of Rat

Vincenza Ciaramella1 Rosanna Chianese1 Paolo Pariante1 Silvia Fasano1

Riccardo Pierantoni1 and Rosaria Meccariello2

1Dipartimento di Medicina Sperimentale Sezione ldquoF Bottazzirdquo Seconda Universita di Napoli Via Costantinopoli 1680138 Napoli Italy2Dipartimento di Scienze Motorie e del Benessere Universita di Napoli Parthenope Via Medina 40 80133 Napoli Italy

Correspondence should be addressed to Rosaria Meccariello rosariameccariellouniparthenopeit

Received 29 November 2014 Accepted 2 March 2015

Academic Editor Dario Acuna-Castroviejo

Copyright copy 2015 Vincenza Ciaramella et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hypothalamic Gonadotropin Releasing Hormone (GnRH) via GnRH receptor (GnRHR) is the main actor in the control ofreproduction in that it induces the biosynthesis and the release of pituitary gonadotropins which in turn promote steroidogenesisand gametogenesis in both sexes Extrabrain functions of GnRH have been extensively described in the past decades and in maleslocal GnRH activity promotes the progression of spermatogenesis and sperm functions at several levels The canonical localizationof Gnrh1 and Gnrhr1mRNA is Sertoli and Leydig cells respectively but ligand and receptor are also expressed in germ cells Herewe analysed the expression rate of Gnrh1 and Gnrhr1 in rat testis (180 days old) by quantitative real-time PCR (qPCR) and by insitu hybridization we localized Gnrh1 and Gnrhr1 mRNA in different spermatogenic cells of adult animals Our data confirm thetesticular expression ofGnrh1 andofGnrhr1 in somatic cells andprovide evidence that their expression in the germinal compartmentis restricted to haploid cells In addition not only Sertoli cells connected to spermatids in the last steps ofmaturation but also Leydigand peritubular myoid cells express Gnrh1

1 Introduction

One of the most intriguing matters in reproductive endo-crinology is that in both mammalian and nonmammalianvertebrates molecules originally identified in the brain asneuropeptidesneurohormones exert their activity in extra-brain tissues in particular in the gonads which expressthe corresponding receptors [1 2] Notable examples areNeuropeptide Y Corticotropin Releasing Factor Gonadotro-pin Inhibiting Factor and Kisspeptin [1 3 4] The activityof Gonadotropin Releasing Hormone (GnRH) via GnRHreceptor (GnRHR) was the first to be reported in ovary andtestis from fish to mammals [2 5] as from the end of 1970s

GnRH is the gatekeeper of reproductive functions in bothsexes This decapeptide is synthesized in the hypothalamusand is released in the median eminence to trigger thedischarge of pituitary gonadotropins (Follicle Stimulating

Hormone and Luteinizing Hormone (FSH and LH resp))Through the main circulation FSH and LH reach the gonadsand sustain gametogenesis and steroid biosynthesis [5]GnRH was originally isolated in the lsquo70s from the hypothala-mus of pig and sheep [6 7] Nowadays GnRH saga comprisesat least 25 GnRHmolecular forms and three GnRHRs seven-transmembrane G-coupled receptors that exhibit a widerange of subtypes [5 8ndash11] In mammals including humantwo GnRH molecular forms (GnRH1 and GnRH2) and twoGnRHRs (GnRHR1 and GnRHR2) have been characterized[5 11] Whether GNRHR2 in humans is a functional or aremnant gene is still an unresolved matter of debate [9]However the main role of GnRH1 is the communicationbetween basal hypothalamus and pituitary gland By contrastGnRH2 besides a supposed hypophysiotropic activity ismainly produced in the hindbrain and exerts a neurotrans-mitter andor neuromodulatory role in the control of food

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2015 Article ID 982726 8 pageshttpdxdoiorg1011552015982726

2 International Journal of Endocrinology

intake energy balance sexual behavior and stress in responseto many environmental cues A third GnRH molecular formexhibiting neuromodulator activity has been only detectedin the telencephalon of teleost fish Three different GnRHRshave been detected in fish and in amphibians [5 8 11]

Spermatogenesis is a complex process in which mitosismeiosis and differentiation coexist in order to produce highquality sperm under the control of hormonal milieu Data inmammalian and nonmammalian animal models and in celllines have provided evidence that steroid biosynthesis andgonocyte as well as spermatogonia proliferation spermato-genesis progression germ cell apoptosis sperm release andfertilization all require the local activity of GnRH (for reviewsee [2 5 12ndash14]) Thus in the testis GnRH and GnRHR aredeeply involved in the autocrine and paracrine routes thatmodulate the communications between somatic and germcells [2] acting in current with several local biomodulators[5 15ndash21]

Foetal expression of GnRH1 and GnRHR1 has beenreported during the ontogenesis of rat gonads [22] and inmouse testis high GnRH1 activity has been observed frompubertal stages to the adult with a decline during senescence[3] Leydig cells are the main target of GnRH activity [1223 24] GnRH1 has been detected in the interstitial fluid ofrat testis [25] and competitive binding studies immunohisto-chemistry and in situ hybridization have suggested that Ser-toli cells may represent the main source of GnRH (for reviewsee [5 12 26]) Besides the canonical localization in somaticcells in rat and human Gnrh1GNRH1 and Gnrhr1GNRHR1transcripts have also been localized in germ cells [27] Inhuman a GNRHR2 transcript is expressed in haploid post-meiotic cells and in mature sperm as well [28] Consistentlyin mouse and rat Northern blot analysis has revealed thepresence of several forms of Gnrhr1 mRNA in isolated germcells [29] In particular in rat spermatogenic cells of someseminiferous tubules also express Gnrh1 and Gnrhr1 [27]However at present little is known about the localizationof GnRH1 and its receptor in different spermatogenic cellsHere we fill this gap by reporting the localization of Gnrh1and Gnrhr1 mRNA during spermatogenesis in adult ratsfocusing on the localization of Gnrh1 and Gnrhr1 transcriptsduring the steps of the spermiogenesis

2 Materials and Methods

21 Animals and Tissue Collection Wistar rats (Rattusnorvegicus) were housed under definite conditions (12D 12L)and given free access to standard food and water ad libi-tum Animals at 180 days postpartum (dpp) were sacrificedby decapitation under ketamine anesthesia (100mgkg ip)in accordance with local and national guidelines coveringexperimental animals For each animal testes were dissectedone testis was fixed in Bouinrsquos fluid and processed for insitu hybridization one was quickly frozen by immersion inliquid nitrogen and stored at minus80∘C until RNA extractionAdditionally adult rat brain was also dissected frozen andstored at minus80∘C to be used as a positive control in qPCRanalysis This project was approved by the Italian Ministry

of Education University and Research (MIUR) Proceduresinvolving animal care were conducted in accordance withthe Guide for Care and Use of Laboratory Animals (NationalInstitutes of Health guide)

22 Total RNA Extraction and cDNA Preparation Total RNAwas extracted from R norvegicus tissues (testis and brain)usingTrizol Reagent (1mL50ndash100mg tissue) (InvitrogenLifeTechnologies Paisley UK) according to the manufacturerrsquosinstructions Total RNA was treated for 30min at 37∘C withDnase I (10Usample) (Amersham Pharmacia Biotech) toeliminate any contamination of genomic DNA RNA purityand integrity were determined by spectrophotometer anal-yses at 260280 nm and by electrophoresis ComplementaryDNA (cDNA) was obtained by reverse transcription using5 120583g of total RNA 05120583g of oligo dT

(18) 05mM dNTP mix

5mM DTT 1x first-strand buffer (Invitrogen Life Technolo-gies) 40U RNase Out (Invitrogen Life Technologies) and200U SuperScript-III RnaseHminus Reverse Transcriptase (Invit-rogen Life Technologies) in a final volume of 20120583L followingthe manufacturerrsquos instructions As negative control totalRNA not treated with reverse transcriptase was used

23 Cloning of Gnrh1 and Gnrhr1 To clone Gnrh1 andGnrhr1 1 120583L of diluted (1 5) cDNA was used for standardPCR analysis in combination with 10 pmol of oligonucleotideprimers designed on R norvegicus nucleotide sequence(Gnrh1 51015840-agcactggtcctatgggttg-31015840 and 51015840-gggccagtgcatct-acatct-31015840 Gnrhr1 51015840-cagctttcatgatggtggtg-31015840 and 51015840-ttcagctgt-agtttgcgtgg-31015840) The predicted amplificate sizes were 221 and370 bp for Gnrh1 and Gnrhr1 respectively PCR conditionswere 94∘C 5min 1 cycle 94∘C 30 s 58∘C 30 s 72∘C45 s 40 cycles 72∘C 7min PCR products were subclonedin pGEM-T Easy Vector (Promega Corp Madison WI)DH5120572 high-efficiency competent cells were transformed andrecombinant colonies were identified by bluewhite colourscreening Recombinant plasmid DNA was extracted byusing the QIAprep SpinMiniprep kit (Qiagen Valencia CA)and the insert sizes were controlled by restriction analysiswith EcoRI (Fermentas St Leon-Rot Germany) and thenthey were sequenced on both strands by Primm SequenceService (Primm Srl Naples Italy)

24 Riboprobes Synthesis and In Situ Hybridization Spe-cific riboprobes were synthesized by in vitro transcriptionusing cDNA fragments of rat Gnrh1 and Gnrhr1 221 and370 bp respectively cloned previously in pGEM-T EasyVector (Promega Corp) The sense (control) and antisensecomplementary RNA (cRNA) probes were transcribed withT7 and SP6 RNA polymerases on plasmids and linearizedwith the appropriate restriction enzymes (SalI or NcoI) usingDIG-uridine triphosphate (UTP) RNA labeling mix (RocheDiagnostics Mannheim Germany) as recommended by themanufacturer

For histological observation rat testes were fixed inBouinrsquos fluid dehydrated in ethanol cleared in xylene andembedded in paraffin Tissue sections (5120583m) were stainedwith hematoxylin-eosin to assess sample quality In situ

International Journal of Endocrinology 3

0

05

1

15

2

B T

lowastlowast

Gnrh

1 no

rmal

ized

fold

expr

essio

n

(a)

Gnrh

r1 n

orm

aliz

ed fo

ld ex

pres

sion

0

2

4

6

8

B T

lowastlowast

(b)

Figure 1 Gnrh1 (a) and Gnrhr1 (b) expression by qPCR in R norvegicus tissues B brain T testis Data are reported as mean fold changeplusmn SD over the value one arbitrarily assigned to brain sample Asterisks indicate statistically significant differences (119875 lt 001) and data arerepresentative of three separate experiments at least (119899 = 6)

hybridization was performed as follows In brief sectionswere subjected to the treatment with 10 120583gmL proteinase K(Sigma Aldrich) in 20mM Tris-HCl then hybridization wasperformed overnight at 60∘C in a humidified chamber using100 120583L hybridization buffer (40 deionized formamide 5xSSC 1x Denhardtrsquos solution 100 120583gmL sonicated salmonsperm DNA 100120583gmL tRNA and 100 ng digoxigenin-labeled cRNA probe) Finally sections were incubated for30min at 37∘C in RNase buffer (05MNaCl 10mMTris-HClpH 7 and 5mMEDTA) containing 20120583gmLRNase A Slideswere observed under a lightmicroscope (Leica LEITSDMRBLeica Microsystem Milan Italy) and images were capturedusing a high-resolution digital camera (Leica MC 170 HDSoftware Leica Application Suite 43)

25 Quantitative Real-Time PCR (qPCR) QuantitativemRNA analysis was conducted on testis and brain toevaluate Gnrh1 and Gnrhr1 expression All qPCRs wereprepared in a final volume of 20120583L containing 1 120583L of 1 5diluted cDNA 05 120583M of each primer and 10 120583L of SSoFast EvaGreen supermix (Bio-Rad) Assays were run twicein duplicate using the Mastercycler CFX-96 (Bio-Rad) anegative control in which cDNA was replaced by waterwas also included All assays included a melting curveanalysis for which all samples displayed single peaks foreach primer pair Genes of interest were normalized tothe reference gene 120573-actin [51015840-agatgacccagatcatgtttgaga-31015840and 51015840-accagaggcatacagggacaa-31015840 119879 (∘C) annealing 56∘Camplificate size 86 bp] the relative quantification of themRNA levels was performed using the comparative Cqmethod with the formula 2minusΔΔCq Data were then reportedas mean fold change plusmn SD over the minimal value arbitrarilyassigned to a reference sample (brain sample) ANOVAfollowed by Duncanrsquos test for multigroup comparison wascarried out to assess the significance of differences

3 Results

31 Expression of Gnrh1 and Gnrhr1 Transcripts in R norvegi-cus Standard RT-PCRwas preliminarily carried out on adultrats in order to clone Gnrh1 and Gnrhr1 cDNA fragmentsBands of the predicted size (221 and 370 bp for Gnrh1 andGnrhr1 resp) were observed in the testis Therefore theabundance of Gnrh1 and Gnrhr1 was evaluated by qPCRanalysis on total RNA from adult male gonads and brain of Rnorvegicus (Figures 1(a) and 1(b)) The analysis revealed thepresence of the transcripts Gnrh1 and Gnrhr1 in the testisas well as in the brain (positive control) In particular theexpression of Gnrh1 in the testis was significantly lower thanin the brain (119875 lt 001) (Figure 1(a)) and that of Gnrhr1increased 546-fold in the testis compared to the brain (119875 lt001) (Figure 1(b))

32 Localization of Gnrh1 and Gnrhr1 in R norvegicusTestis Gnrh1 and Gnrhr1 transcripts were localized by insitu hybridization in rat testis at 180 dpp (Figure 2) duringthe spermatogenesis The stages were classified according toLeblond and Clermont 1952 [30]

Gnrh1 mRNA was strongly localized in the interstitialLeydig cells (LC) (Figure 2(b)) and in spermatids (SPT)in acrosome phase Stages IXndashXII (Figure 3(b)) the signalwas also detected in Sertoli cells (SC) connected with SPTduring the maturation phase Stages VndashVII (steps 17ndash19)(Figures 3(b) and 3(c)) Besides Gnrh1 mRNA outlinedeach seminiferous tubule marking peritubular myoid cells(Figures 3(b) and 3(c))

Gnrhr1 mRNA was mainly localized in SPT from StagesIII to V (steps 16-17) (Figure 3(d)) when SPT are in mat-uration phase until Stages XII-XIII when SPT are in theacrosome phase (Figures 3(e) and 3(f)) a weaker signalwas also observed in the interstitial compartment (LC)(Figure 2(c))


(a)

LC

(b)

LC

LC

(c)

Figure 2 Section of adult R norvegicus testis analyzed by hematoxylin-eosin staining (a) as well as by in situ hybridization for Gnrh1 (b) andGnrhr1 (c) treated with antisense ((b) (c)) and sense ((b) (c) insets) probes Localization ofGnrh1 and Gnrhr1 was observed in the interstitialLeydig cells (LC) and inside the seminiferous tubule Scale bars 20 120583mThe results are representative of one of three assays

SC

Gnrh

1

SC

SC

Gnrh

r1

(a) (b) (c)

(d) (e) (f)

Figure 3 Localization of Gnrh1 and Gnrhr1 by in situ hybridization in rat testis during the stage of the spermatogenetic cycle ((a)ndash(f)) Theblue staining indicates the positive cells few of them representative of the different cell types are pointed out by white arrowhead SPT ((a)ndash(f)) black arrow peritubular myoid cells ((b) (c) (e) and (f)) SC Sertoli cellsThe insets are shown in 100xmagnification Scale bars 20 120583mThe results are representative of one of three assays


Spermatogenic stages

Figure 4 Schematic representation of Gnrh1 and Gnrhr1 expression in the germinal epithelium during a complete spermatogenetic cycle(modified from [45]) Columns show the sequence of germ cell generations observed in rat seminiferous tubule at that stage (IndashXIV Stages)Spermatogonia A (type A) In (intermediate) B (type B) R (resting final DNA replication) and m (mitosis of spermatogonia) Primaryspermatocytes L (leptotene) Z (zygotene) P (pachytene) Di (diakinesis) and II (secondary spermatocytes) Phases 1ndash19 indicate spermatidsdifferentiation (spermiogenesis) Horizontal lines indicate localizationwindowofGnrh1 andGnrhr1 blue line and red dotted line respectivelyVertical green lines indicate the expression window of Gnrh1 in Sertoli cells

Quiescent primary SPG (ISPG) and pachytene or lep-totene spermatocytes (SPC) did not reveal any positivity Nolabeling for Gnrh1 or Gnrhr1 was detected in slides incubatedwith sense cRNA probes (insets in Figures 2(b) and 2(c))

A schematic representation of Gnrh1 and Gnrhr1 expres-sion in the germinal epithelium during the spermatogeneticcycle is represented in Figure 4

4 Discussion

Local activity of GnRH has been reported in male andfemale gonads in several animal models from molluscs tovertebrates human included [2 5 13 14 26] In this respectthe use of GnRH agonists and antagonists in cell lines invivo and in vitro has provided evidences of GnRH involve-ment in the control of Leydig cells ontogenesis and activityspermatogenesis progression sperm release and function([2 5 13 14 26] and references therein)

Present data confirm earlier findings that bothGnrh1 andGnrhr1 are expressed in rodent testis as well as in brain used aspositive control In our study total RNA was extracted fromwhole brain and testis and the same amount of cDNA wasused for cDNA preparation Since it is well known that inthe brain Gnrh1 and Gnrhr1 are expressed in discrete brainareas and not all over the tissue [5] a dilution effect on

the expression of Gnrh1 and Gnrhr1 might be postulated inthe brain Despite that present data confirm that the brainis the major source of GnRH1 By contrast the expressionlevels of Gnrhr1 here observed are higher in the testis thanin the whole brain Consistently previous results obtained byNorthern blot a less sensitive technique than qPCR revealedthat in rodents Gnrhr1 is highly expressed in the pituitarygland the tissue mainly responsive to GnRH1 but also inwhole testis and germ cells [29 31]

By in situ hybridization we have localized Gnrh1 andGnrhr1 inside the testis throughout the spermatogenic cyclewhich in rat comprises XIV different cellular associationswith newly formed and older spermatids overlapping duringthe first eight stages of development [30]

In general the communications between Leydig andSertoli cells and between Sertoli and germ cells ensure anoptimal environment for the progression of germ cells andare fundamental tools to gain a successful spermatogenesis[12] Hypogonadal (hpg) mice a natural model with arrestedreproductive development due to a congenital deficiencyin GnRH synthesis leading to markedly reduced produc-tion of gonadotropins are infertile [32] The administrationof androgens is sufficient to induce spermatogenesis inhpg mice but the administration of recombinant humanFSH whose main targets in testis are Sertoli cells induces


the proliferation of spermatogonia and spermatogenesis pro-gression until spermatocytes [33 34] Similarly the lack ofLH signalling mainly occurring via Leydig cells also causesspermatogenesis arrest [35 36] The detection of GnRH-likematerial in the fluid surrounding Leydig cells that expressGnrhr1 and the finding of Gnrh1 mRNA in Sertoli cells [27]suggest that GnRH1 may be produced by Sertoli cells andmay act as a paracrine factor to modulate the activity ofLeydig cells Here by in situ hybridization we demonstratethat in adult testisGnrh1 is expressed in Sertoli cells in Leydigcells and in peritubularmyoid cells confirming previous dataconcerning the activity of GnRH in the control of steroidoge-nesis and germ cells development as well as its release intothe lumen of seminiferous tubules [2 5] Consistently Gnrh1mRNAhas also been detected in the interstitial compartmentof frog testis [15] and GnRH1 positive immunostaining hasrecently been reported in the interstitial compartment ofmouse testis during different stages of aging with high levelsobserved during reproductively active stages [3] Interest-ingly the expected expression of Gnrh1 in Sertoli cells is notwidespread all over the tubules but is restricted to Stages VndashVII of the spermatogenic cycle with Sertoli cells connectedto the spermatids in the last step of the maturation phase

Present data confirm the expression of both Gnrh1 andGnrhr1 in germ cells [27 29] since the correspondingtranscripts have been specifically localized in haploid cells butnot in mitotic cells or in meiotic cells Consistently in mouseGnRH1 is an autocrine factor for gonocyte that expresses bothligand and receptors [3] but in the adult testis mitotic cellsare devoid of any GnRH1GnRHR1 immunoreactivity [3] Bycontrast in amphibians which exhibit an annual reproduc-tive cycle characterized by periods of spermatogenic arrestand periods of spermatogenesis onset and resumption gnrh1expression has been observed in quiescent and proliferatingspermatogonia as well [15]

At the end of meiosis haploid round spermatids undergodynamic morphologic changes which include acrosomeformation nuclear shaping elongation and tail formationin order to produce sperm cells Hence the functionalmeaning of mRNA detection in haploid cells might openseveral interpretations Some mRNAs repressed in earlyspermatids might be recruited toward the polysomes fortranslation in late spermatids whereas some other mRNAsmight be transcribed in a specific time window to be imme-diately translated By contrast untranslated mRNA might bedegraded at specific steps during spermiogenesis or storedin sperm in order to contribute to fertilization and embryodevelopment with the chromatoid body thought to be themain actor in the capture of mRNAs for degradation orstorage [37] Here we demonstrate that in rat Gnrh1 is onlyexpressed in spermatids during the acrosome phase (StagesIXndashXII) whereas Gnrhr1 is expressed in spermatids from theacrosome phase (Stages XII-XIII) throughout the maturationphaseThis is consistent with the recent finding that GnRHR1is localized in elongating spermatids in reproductively activemouse [3] However the expression of ligand and receptor inthe same cell type during a specific time window supportsthe hypothesis that GnRH1 may be an autocrine modulatorof acrosome biogenesis By contrast the detection of Gnrh1

mRNA in peritubular myoid cells and in Sertoli cells duringthe last step of thematuration phase (Stages VndashVIII) in paral-lel toGnrhr1 detection in late spermatids suggests thatGnRH1may be a paracrinemodulator in sperm release and transportConsistently in a nonmammalian vertebrate the anuranamphibian Pelophylax esculentus a species that expresses twoGnRH molecular forms (GnRH1 and GnRH2) and threeGnRHRs (GnRHR1 R2 and R3) [38] with a functionalportioning in testis [15] gnrhr2 mRNA has been localizedin Sertoli cells connected to elongating spermatids whichexpress gnrh2 [15] In this experimental model buserelin aGnRH agonist induces spermiation [39]

GNRHR1 signaling is important for sperm binding tothe human zona pellucida [40] and for the inhibition of invivo and in vitro fertilization in rodents exerted by GnRHantagonists [41] Furthermore GnRH-like substances havebeen detected in human seminal fluid and related to theacquisition of sperm functions [42] the prostate being themajor candidate for its production [43 44]

5 Conclusions

In rat Gnrh1 and Gnrhr1 are expressed in the testis insomatic cells and in haploid germ cells The expressionof Gnrh1Gnrhr1 at specific steps of spermiogenesis and inSertoli cells connected to spermatids in the late phase ofmaturation suggests a deep involvement in functions relatedto the production of high quality sperm

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Financial support was provided by the Ministero ItalianodellrsquoIstruzione dellrsquoUniversita e della Ricerca (Prin-MIUR201011) to Rosaria Meccariello

References

[1] N L McGuire and G E Bentley ldquoNeuropeptides in the gonadsfrom evolution to pharmacologyrdquo Frontiers in Pharmacologyvol 1 article 114 Article ID Article 114 2010

[2] R Meccariello R Chianese T Chioccarelli et al ldquoIntra-testicular signals regulate germ cell progression and productionof qualitatively mature spermatozoa in vertebratesrdquo Frontiers inEndocrinology vol 5 article 69 2014

[3] S Anjum A Krishna R Sridaran and K Tsutsui ldquoLocalizationof gonadotropin-releasing hormone (GnRH) gonadotropin-inhibitory hormone (GnIH) kisspeptin and GnRH receptorand their possible roles in testicular activities from birth tosenescence in micerdquo Journal of Experimental Zoology Part AEcological Genetics and Physiology vol 317 no 10 pp 630ndash6442012

[4] R Chianese V Ciaramella S Fasano R Pierantoni and RMeccariello ldquoKisspeptin receptor GPR54 as a candidate for


the regulation of testicular activity in the frog Rana esculentardquoBiology of Reproduction vol 88 no 3 article 73 pp 1ndash11 2013

[5] R Pierantoni G Cobellis R Meccariello and S Fasano ldquoEvo-lutionary aspects of cellular communication in the vertebratehypothalamo-hypophysio-gonadal axisrdquo International Reviewof Cytology vol 218 pp 69ndash141 2002

[6] M Amoss R Burgus R Blackwell W Vale R Fellows andR Guillemin ldquoPurification amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasingfactor (LRF) of ovine originrdquo Biochemical and BiophysicalResearch Communications vol 44 no 1 pp 205ndash210 1971

[7] H Matsuo Y Baba R M G Nair A Arimura and AV Schally ldquoStructure of the porcine LH- and FSH-releasinghormone I The proposed amino acid sequencerdquo Biochemicaland Biophysical Research Communications vol 43 no 6 pp1334ndash1339 1971

[8] J A King and R P Millar ldquoEvolutionary aspects of gonadotro-pin-releasing hormone and its receptorrdquo Cellular andMolecularNeurobiology vol 15 no 1 pp 5ndash23 1995

[9] R P Millar Z-L Lu A J Pawson C A Flanagan K Morganand S R Maudsley ldquoGonadotropin-releasing hormone recep-torsrdquo Endocrine Reviews vol 25 no 2 pp 235ndash275 2004

[10] K Morgan and R P Millar ldquoEvolution of GnRH ligand pre-cursors and GnRH receptors in protochordate and vertebratespeciesrdquo General and Comparative Endocrinology vol 139 no3 pp 191ndash197 2004

[11] O Kah C Lethimonier G Somoza L G Guilgur C Vaillantand J J Lareyre ldquoGnRH and GnRH receptors in metazoa ahistorical comparative and evolutive perspectiverdquoGeneral andComparative Endocrinology vol 153 no 1ndash3 pp 346ndash364 2007

[12] R M Sharpe ldquoParacrine control of the testisrdquo Clinics inEndocrinology and Metabolism vol 15 no 1 pp 185ndash207 1986

[13] R Pierantoni G Cobellis R Meccariello et al ldquoTesticulargonadotropin-releasing hormone activity progression of sper-matogenesis and sperm transport in vertebratesrdquo Annals of theNew York Academy of Sciences vol 1163 pp 279ndash291 2009

[14] M Osada and N Treen ldquoMolluscan GnRH associated withreproductionrdquoGeneral andComparative Endocrinology vol 181no 1 pp 254ndash258 2013

[15] R Chianese V Ciaramella D Scarpa S Fasano R Pierantoniand R Meccariello ldquoAnandamide regulates the expression ofGnRH1 GnRH2 andGnRH-Rs in frog testisrdquo American Journalof Physiology Endocrinology andMetabolism vol 303 no 4 ppE475ndashE487 2012

[16] R Chianese V Ciaramella D Scarpa S Fasano R Pierantoniand R Meccariello ldquoEndocannabinoids and endovanilloidsa possible balance in the regulation of the testicular GnRHsignallingrdquo International Journal of Endocrinology vol 2013Article ID 904748 9 pages 2013

[17] M-C Botte Y Lerrant A Lozach A Berault R Counisand M-L Kottler ldquoLH down-regulates gonadotropin-releasinghormone (GnRH) receptor but not GnRH mRNA levels in therat testisrdquo Journal of Endocrinology vol 162 no 3 pp 409ndash4151999

[18] J P Hapgood H Sadie W van Biljon and K Ronacher ldquoReg-ulation of expression of mammalian gonadotrophin-releasinghormone receptor genesrdquo Journal of Neuroendocrinology vol 17no 10 pp 619ndash638 2005

[19] R Chianese V Ciaramella S Fasano R Pierantoni and RMeccariello ldquoKisspeptin drives germ cell progression in theanuran amphibian Pelophylax esculentus a study carried out in

ex vivo testesrdquoGeneral and Comparative Endocrinology vol 211pp 81ndash91 2015

[20] S Anjum A Krishna and K Tsutsui ldquoInhibitory roles of themammalian GnIH ortholog RFRP3 in testicular activities inadult micerdquoThe Journal of Endocrinology vol 223 no 1 pp 79ndash91 2014

[21] H Y Lee M I Naseer S Y Lee and M O Kim ldquoTime-dependent effect of ethanol on GnRH and GnRH receptormRNA expression in hypothalamus and testis of adult andpubertal ratsrdquo Neuroscience Letters vol 471 no 1 pp 25ndash292010

[22] M-C Botte A-M ChamagneM-C Carre R Counis andM-L Kottler ldquoFetal expression of GnRH andGnRH receptor genesin rat testis and ovaryrdquo Journal of Endocrinology vol 159 no 1pp 179ndash189 1998

[23] A J Hsueh and P B Jones ldquoExtrapituitary actions ofgonadotropin-releasing hormonerdquo Endocrine Reviews vol 2no 4 pp 437ndash461 1981

[24] G Chieffi R Pierantoni and S Fasano ldquoImmunoreactiveGnRH in hypothalamic and extrahypothalamic areasrdquo Interna-tional Review of Cytology vol 127 pp 1ndash55 1991

[25] F Petersson G Emons and M Hammar ldquoTesticular GnRH-receptors and direct effects of a GnRH-agonist on humantesticular steroidogenesisrdquo Scandinavian Journal of Urology andNephrology vol 23 no 3 pp 161ndash164 1989

[26] N Ramakrishnappa R Rajamahendran Y M Lin and P CK Leung ldquoGnRH in non-hypothalamic reproductive tissuesrdquoAnimal Reproduction Science vol 88 no 1-2 pp 95ndash113 2005

[27] J Y Bahk J S Hyun S H Chung et al ldquoStage specific identi-fication of the expression of GnRH mRNA and localization ofthe GnRH receptor in mature rat and adult human testisrdquo TheJournal of Urology vol 154 no 5 pp 1958ndash1961 1995

[28] W van Biljon S Wykes S Scherer S A Krawetz and JHapgood ldquoType II gonadotropin-releasing hormone receptortranscripts in human spermrdquo Biology of Reproduction vol 67no 6 pp 1741ndash1749 2002

[29] P Bull P Morales C Huyser T Socıas and E A CastellonldquoExpression of GnRH receptor inmouse and rat testicular germcellsrdquoMolecular Human Reproduction vol 6 no 7 pp 582ndash5852000

[30] C P Leblond and Y Clermont ldquoDefinition of the stages of thecycle of the seminiferous epithelium in the ratrdquo Annals of theNew York Academy of Sciences vol 55 no 4 pp 548ndash573 1952

[31] S S Kakar K Grantham L C Musgrove D Devor J CSellers and J D Neill ldquoRat gonadotropin-releasing hormone(GnRH) receptor tissue expression and hormonal regulation ofits mRNArdquo Molecular and Cellular Endocrinology vol 101 no1-2 pp 151ndash157 1994

[32] B M Cattanach C A Iddon H M Charlton S A Chiappaand G Fink ldquoGonadotrophin-releasing hormone deficiency inamutantmousewith hypogonadismrdquoNature vol 269 no 5626pp 338ndash340 1977

[33] J Singh and D J Handelsman ldquoThe effects of recombinant FSHon testosterone-induced spermatogenesis in gonadotrophin-deficient (hpg) micerdquo Journal of Andrology vol 17 no 4 pp382ndash393 1996

[34] H Baines M O Nwagwu E C Furneaux et al ldquoEstrogenicinduction of spermatogenesis in the hypogonadal (hpg) mouserole of androgensrdquo Reproduction vol 130 no 5 pp 643ndash6542005


[35] I Huhtaniemi P Ahtiainen T Pakarainen S B Rulli F-P Zhang and M Poutanen ldquoGenetically modified mousemodels in studies of luteinising hormone actionrdquoMolecular andCellular Endocrinology vol 252 no 1-2 pp 126ndash135 2006

[36] H Peltoketo F P Zhang and S B Rulli ldquoAnimal models foraberrations of gonadotropin actionrdquo Reviews in Endocrine andMetabolic Disorders vol 12 no 4 pp 245ndash258 2011

[37] K C Kleene ldquoPatterns mechanisms and functions of transla-tion regulation inmammalian spermatogenic cellsrdquoCytogeneticand Genome Research vol 103 no 3-4 pp 217ndash224 2003

[38] R Chianese V Ciaramella S Fasano R Pierantoni and RMeccariello ldquoAnandamide modulates the expression of GnRH-II and GnRHRs in frog Rana esculenta diencephalonrdquo Generaland Comparative Endocrinology vol 173 no 3 pp 389ndash3952011

[39] S Minucci L Di Matteo G C Baccari and R Pierantoni ldquoAgonadotropin releasing hormone analog induces spermiation inintact and hypophysectomized frogs Rana esculentardquo Experi-entia vol 45 no 11-12 pp 1118ndash1121 1989

[40] P Morales E Pizarro M Kong and C Pasten ldquoSperm bindingto the human zona pellucida and calcium influx in response toGnRH and progesteronerdquoAndrologia vol 34 no 5 pp 301ndash3072002

[41] P Morales C Pasten and E Pizarro ldquoInhibition of in vivoand in vitro fertilization in rodents by gonadotropin-releasinghormone antagonistsrdquoBiology of Reproduction vol 67 no 4 pp1360ndash1365 2002

[42] S I Izumi TMakino and R Iizuka ldquoImmunoreactive luteiniz-ing hormone-releasing hormone in the seminal plasma andhuman semen parametersrdquo Fertility and Sterility vol 43 no 4pp 617ndash620 1985

[43] N Azad S Uddin N La Paglia et al ldquoLuteinizing hormone-releasing hormone (LHRH) in rat prostate characterizationof LHRH peptide messenger ribonucleic acid expression andmolecular processing of LHRH in intact and castrated maleratssrdquo Endocrinology vol 133 no 3 pp 1252ndash1257 1993

[44] R B White J A Eisen T L Kasten and R D FernaldldquoSecond gene for gonadotropin-releasing hormone in humansrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 1 pp 305ndash309 1998

[45] B Perey Y Clermont and C P Leblond ldquoThe wave of theseminiferous epithelium in the ratrdquo The American Journal ofAnatomy vol 108 no 1 pp 47ndash77 1961

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Disease Markers


BioMed Research International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


intake energy balance sexual behavior and stress in responseto many environmental cues A third GnRH molecular formexhibiting neuromodulator activity has been only detectedin the telencephalon of teleost fish Three different GnRHRshave been detected in fish and in amphibians [5 8 11]

Spermatogenesis is a complex process in which mitosismeiosis and differentiation coexist in order to produce highquality sperm under the control of hormonal milieu Data inmammalian and nonmammalian animal models and in celllines have provided evidence that steroid biosynthesis andgonocyte as well as spermatogonia proliferation spermato-genesis progression germ cell apoptosis sperm release andfertilization all require the local activity of GnRH (for reviewsee [2 5 12ndash14]) Thus in the testis GnRH and GnRHR aredeeply involved in the autocrine and paracrine routes thatmodulate the communications between somatic and germcells [2] acting in current with several local biomodulators[5 15ndash21]

Foetal expression of GnRH1 and GnRHR1 has beenreported during the ontogenesis of rat gonads [22] and inmouse testis high GnRH1 activity has been observed frompubertal stages to the adult with a decline during senescence[3] Leydig cells are the main target of GnRH activity [1223 24] GnRH1 has been detected in the interstitial fluid ofrat testis [25] and competitive binding studies immunohisto-chemistry and in situ hybridization have suggested that Ser-toli cells may represent the main source of GnRH (for reviewsee [5 12 26]) Besides the canonical localization in somaticcells in rat and human Gnrh1GNRH1 and Gnrhr1GNRHR1transcripts have also been localized in germ cells [27] Inhuman a GNRHR2 transcript is expressed in haploid post-meiotic cells and in mature sperm as well [28] Consistentlyin mouse and rat Northern blot analysis has revealed thepresence of several forms of Gnrhr1 mRNA in isolated germcells [29] In particular in rat spermatogenic cells of someseminiferous tubules also express Gnrh1 and Gnrhr1 [27]However at present little is known about the localizationof GnRH1 and its receptor in different spermatogenic cellsHere we fill this gap by reporting the localization of Gnrh1and Gnrhr1 mRNA during spermatogenesis in adult ratsfocusing on the localization of Gnrh1 and Gnrhr1 transcriptsduring the steps of the spermiogenesis

2 Materials and Methods

21 Animals and Tissue Collection Wistar rats (Rattusnorvegicus) were housed under definite conditions (12D 12L)and given free access to standard food and water ad libi-tum Animals at 180 days postpartum (dpp) were sacrificedby decapitation under ketamine anesthesia (100mgkg ip)in accordance with local and national guidelines coveringexperimental animals For each animal testes were dissectedone testis was fixed in Bouinrsquos fluid and processed for insitu hybridization one was quickly frozen by immersion inliquid nitrogen and stored at minus80∘C until RNA extractionAdditionally adult rat brain was also dissected frozen andstored at minus80∘C to be used as a positive control in qPCRanalysis This project was approved by the Italian Ministry

of Education University and Research (MIUR) Proceduresinvolving animal care were conducted in accordance withthe Guide for Care and Use of Laboratory Animals (NationalInstitutes of Health guide)

22 Total RNA Extraction and cDNA Preparation Total RNAwas extracted from R norvegicus tissues (testis and brain)usingTrizol Reagent (1mL50ndash100mg tissue) (InvitrogenLifeTechnologies Paisley UK) according to the manufacturerrsquosinstructions Total RNA was treated for 30min at 37∘C withDnase I (10Usample) (Amersham Pharmacia Biotech) toeliminate any contamination of genomic DNA RNA purityand integrity were determined by spectrophotometer anal-yses at 260280 nm and by electrophoresis ComplementaryDNA (cDNA) was obtained by reverse transcription using5 120583g of total RNA 05120583g of oligo dT

(18) 05mM dNTP mix

5mM DTT 1x first-strand buffer (Invitrogen Life Technolo-gies) 40U RNase Out (Invitrogen Life Technologies) and200U SuperScript-III RnaseHminus Reverse Transcriptase (Invit-rogen Life Technologies) in a final volume of 20120583L followingthe manufacturerrsquos instructions As negative control totalRNA not treated with reverse transcriptase was used

23 Cloning of Gnrh1 and Gnrhr1 To clone Gnrh1 andGnrhr1 1 120583L of diluted (1 5) cDNA was used for standardPCR analysis in combination with 10 pmol of oligonucleotideprimers designed on R norvegicus nucleotide sequence(Gnrh1 51015840-agcactggtcctatgggttg-31015840 and 51015840-gggccagtgcatct-acatct-31015840 Gnrhr1 51015840-cagctttcatgatggtggtg-31015840 and 51015840-ttcagctgt-agtttgcgtgg-31015840) The predicted amplificate sizes were 221 and370 bp for Gnrh1 and Gnrhr1 respectively PCR conditionswere 94∘C 5min 1 cycle 94∘C 30 s 58∘C 30 s 72∘C45 s 40 cycles 72∘C 7min PCR products were subclonedin pGEM-T Easy Vector (Promega Corp Madison WI)DH5120572 high-efficiency competent cells were transformed andrecombinant colonies were identified by bluewhite colourscreening Recombinant plasmid DNA was extracted byusing the QIAprep SpinMiniprep kit (Qiagen Valencia CA)and the insert sizes were controlled by restriction analysiswith EcoRI (Fermentas St Leon-Rot Germany) and thenthey were sequenced on both strands by Primm SequenceService (Primm Srl Naples Italy)

24 Riboprobes Synthesis and In Situ Hybridization Spe-cific riboprobes were synthesized by in vitro transcriptionusing cDNA fragments of rat Gnrh1 and Gnrhr1 221 and370 bp respectively cloned previously in pGEM-T EasyVector (Promega Corp) The sense (control) and antisensecomplementary RNA (cRNA) probes were transcribed withT7 and SP6 RNA polymerases on plasmids and linearizedwith the appropriate restriction enzymes (SalI or NcoI) usingDIG-uridine triphosphate (UTP) RNA labeling mix (RocheDiagnostics Mannheim Germany) as recommended by themanufacturer

For histological observation rat testes were fixed inBouinrsquos fluid dehydrated in ethanol cleared in xylene andembedded in paraffin Tissue sections (5120583m) were stainedwith hematoxylin-eosin to assess sample quality In situ


0

05

1

15

2

B T

lowastlowast

Gnrh

1 no

rmal

ized

fold

expr

essio

n

(a)

Gnrh

r1 n

orm

aliz

ed fo

ld ex

pres

sion

0

2

4

6

8

B T

lowastlowast

(b)




3 Results






(a)

LC

(b)

LC

LC

(c)


SC

Gnrh

1

SC

SC

Gnrh

r1

(a) (b) (c)

(d) (e) (f)







4 Discussion












5 Conclusions




Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

B T

lowastlowast

Gnrh

1 no

rmal

ized

fold

expr

essio

n

(a)

Gnrh

r1 n

orm

aliz

ed fo

ld ex

pres

sion

0

2

4

6

8

B T

lowastlowast

(b)




3 Results






(a)

LC

(b)

LC

LC

(c)


SC

Gnrh

1

SC

SC

Gnrh

r1

(a) (b) (c)

(d) (e) (f)







4 Discussion












5 Conclusions




Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

LC

(b)

LC

LC

(c)


SC

Gnrh

1

SC

SC

Gnrh

r1

(a) (b) (c)

(d) (e) (f)







4 Discussion












5 Conclusions




Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4 Discussion












5 Conclusions




Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions




Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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