Human Reproduction, Vol.29, No.12 pp. 2736–2746, 2014 Advanced Access publication on October 14, 2014 doi:10.1093/humrep/deu247

ORIGINAL ARTICLE Reproductive biology

Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells J. Garcı´a-Ortega 1,†, F.M. Pinto 2,†, M. Ferna´ndez-Sa´nchez 1, N. Prados1, A. Cejudo-Roma´n2, T.A. Almeida 3, M. Herna´ndez 3, M. Romero4,5,6, M. Tena-Sempere4,5,6,*, and L. Candenas2,* 1

Instituto Valenciano de Infertilidad, Seville, Spain 2Instituto de Investigaciones Quı´micas, CSIC, Seville, Spain 3Instituto de Enfermedades Tropicales y Salud Publica, Universidad de La Laguna, Tenerife, Canary Islands 4Departamento de Biologı´a Celular, Fisiologı´a e Inmunologı´a, Universidad de Cordoba, Cordoba, Spain 5CIBER Fisiopatologı´a de la Obesidad y la Nutricio´n, Cordoba, Spain 6ISCiii and Instituto Maimo´nides de Investigacio´n Biome´dica de Co´rdoba/Hospital Universitario Reina Sofia, Cordoba, Spain *Correspondence address: Instituto de Investigaciones Quı´micas, Avenida Americo Vespucio 49, Isla de La Cartuja, 41092 Sevilla, Spain. Tel: +34-95-4489565; Fax: +34-95-4460565; E-mail: [email protected] (L.C.)/Physiology Section, Faculty of Medicine, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain. Tel: +34-957-218281; Fax: +34957-218288; E-mail: fi[email protected] (M.T.-S.)

Submitted on May 16, 2014; resubmitted on August 10, 2014; accepted on August 28, 2014

study question: Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? summary answer: The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. what is known already: The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their wellrecognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown.

study design, size, duration: This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval.

participants/materials, setting, methods: The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus –oocyte complexes were collected after transvaginal ultrasoundguided oocyte retrieval. RT– PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca2+ levels, [Ca2+]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. main outcome and the role of chance: NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca2+]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca2+]i. However, the [Ca2+]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB.

limitations, reasons for caution: The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells.

wider implications of the findings: Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells.

study funding/ competing interest(s): This work was supported by grants from Ministerio de Economı´a y Competitividad

†

These authors contributed equally to this work.

& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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(CTQ2011-25564 and BFI2011-25021) and Junta de Andalucı´a (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare. Key words: neurokinin B / kisspeptin / tachykinin NK3 receptor / kisspeptin receptor / human granulosa cells

Introduction Fertility problems are increasingly common in industrialized countries. It is estimated that, in these countries, 15 –20% of couples of reproductive age have problems conceiving children naturally and the use of assisted reproductive treatment accounts for 1–3% of annual births (de los Santos et al., 2012). Among the causes of infertility, the female factor accounts for 40% of cases. The study of the physiology of all cell types involved in reproductive function is vital to identify the causes of growing infertility. Granulosa cells are specialized ovary cells that play an essential role in the regulation of follicular dynamics and therefore female fertility (de los Santos et al., 2012; Ferrero et al., 2012; Fragouli et al., 2014). In the ovary, these cells surround the oocyte forming functional units called follicles (Fragouli, et al., 2014). At the end of follicular development, the oocyte remains surrounded by a layer of granulosa cells, named cumulus oophorus cells (CCs), which are functionally different from other granulosa cells, namely mural granulosa cells (MGCs). MGCs and CCs are involved in the nutrition of the oocyte and produce estrogen, progesterone, and a large number of cytokines implicated in the development of the follicle and, particularly, of the oocyte. Adequate hormone production is essential for ovulation and formation of the corpora lutea, as well as for preparation of the female reproductive tract for conception and implantation (de los Santos et al., 2012; Fragouli et al., 2014). Therefore, the study of granulosa cells and the elucidation of their functional properties can provide essential data for the advancement of reproductive medicine (de los Santos et al., 2012; Ferrero et al., 2012; Fragouli et al., 2014). Neurokinin B (NKB) is a bioactive peptide that belongs to the family of tachykinins (TKs) (Almeida et al., 2004; Page, 2005; Satake et al., 2013). Its biological effects are mediated by specific membrane tachykinin receptors that belong to the superfamily of G-protein coupled receptors (Maggi, 2000; Candenas et al., 2005; Page, 2005; Satake et al., 2013). Tachykinin receptors include NK1 (NK1R), NK2 (NK2R) and NK3 (NK3R), with NK3R being the preferred receptor of NKB (Maggi, 2000; Candenas et al., 2005; Page, 2005; Satake et al., 2013). The evidence accumulated in recent years suggests that NKB may regulate reproductive functions by acting at both central (hypothalamic–pituitary) and peripheral (reproductive tract) levels (Pinto et al., 1999; Candenas et al., 2005; Rance, 2009; Topaloglu et al., 2009; Gianetti et al., 2010; Lehman et al., 2010; Young et al., 2010; Pinilla et al., 2012; Yang et al., 2012). In this context, recent studies have demonstrated the association between human normosmic hypogonadotrophic hypogonadism (nHH) and mutations in the genes encoding NKB (TAC3) and NK3R (TACR3), thus confirming the important role of this system in the regulation of reproduction (Topaloglu et al., 2009; Gianetti et al., 2010; Young et al., 2010). Several years before the disclosure of the link between mutations in the NKB/NK3R pathway and nHH, the essential roles of kisspeptins in the control of reproductive function were identified. Kisspeptins are a family of structurally related peptides encoded by the KISS1 gene. The

effects of kisspeptins are mediated by activation of the KISS1 receptor (KISS1R), also known as GPR54, which is encoded by the KISS1R gene (Navarro and Tena-Sempere, 2012; Pinilla et al., 2012). Like mutations in the genes encoding NKB or NK3R, inactivating mutations of KISS1R are associated with nHH in humans (de Roux et al., 2003; Seminara et al., 2003), and this phenotype is also observed in mice carrying inactivating mutations of Kiss1 or Kiss1r genes (Seminara et al., 2003; d’Anglemont de Tassigny et al., 2007; Lapatto et al., 2007). Altogether, these findings stress the indispensable role of kisspeptins as gatekeepers of reproductive maturation and function. Kisspeptins are primarily synthesized in discrete neuronal populations within the hypothalamus where they modulate GnRH secretion and, thereby, gonadotrophin release (Oakley et al., 2009; Navarro and TenaSempere, 2012; Pinilla et al., 2012). Interestingly, in a subset of these neurons, located in the arcuate nucleus (ARC) of the hypothalamus (or its equivalent infundibular region in primates), kisspeptins are co-localized with NKB and the opioid peptide, dynorphin. For this reason, these cells have been called KNDy neurons (Lehman et al., 2010; Navarro and Tena-Sempere, 2012; Pinilla et al., 2012). Of note, co-localization of the three peptides has been documented in a very high proportion of ARC KNDy neurons in various mammalian species, including the monkey, sheep, mouse and rat (Lehman et al., 2010; Navarro and Tena-Sempere, 2012). Yet, KNDy neurons, co-expressing kisspeptins, NKB and dynorphin, seem to be less abundant in the infundibular nucleus of young men (Hrabovszky et al., 2012), in which a substantial proportion of NKB neurons do not co-express kisspeptins or dynorphin. Notwithstanding, up to 75% of kisspeptin-positive neurons in the infundibular region of young men do express NKB also (Hrabovszky et al., 2012). Besides their expression at hypothalamic levels, different reports have shown that NKB, NK3R, KISS1 and KISS1R mRNAs or proteins are present in mammalian peripheral reproductive tissues, including the testes (Pinto et al., 2012), the uterus (Pinto et al., 1999; Patak et al., 2003; Pinto et al., 2009, 2012; Cejudo Roman et al., 2012; Canete et al., 2013), the oviduct (Gaytan et al., 2007; Cejudo Roman et al., 2012) and the ovary (Loffler et al., 2004; Castellano et al., 2006; Gaytan et al., 2009; Lasaga and Debeljuk, 2011; Cejudo Roman et al., 2012; Peng et al., 2013). We have recently shown that both NKB/ NK3R and KISS1/KISS1R systems are co-expressed in the human ovary (Cejudo Roman et al., 2012). Furthermore, the immunoreactivity observed for the components of these systems in human ovarian tissue sections was stronger in the corpus luteum than in the early stages of follicular development (Gaytan et al., 2009; Cejudo Roman et al., 2012), which demonstrates the existence of changes in their expression levels depending on the maturation degree of the follicle. However, the potential effects of these peptide/receptor systems on the female gonads and, particularly, their possible roles in the regulation of human granulosa cell function remain largely unknown. In the present study, we have characterized the presence of the NKB/ NK3R and KISS/KISS1R systems in human ovarian MGCs and CCs, both

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at the mRNA and protein levels. Because of its expression in KNDy neurons in the brain of certain mammalian species, we also examined the expression of dynorphin (prodynorphin, PDYN) in these cells. In parallel, we analyzed the effects of kisspeptin and NKB on intracellular free Ca2+ levels, [Ca2+]i, in MGCs, in an attempt to investigate the functional role of these peptides in the regulation of human granulosa cell function.

streptomycin (Sigma). Cells were seeded into 24-well plates in the absence or in the presence of FF for subsequent RNA collection (MGCs) or onto sterile poly-L-lysine-treated coverslips for immunofluorescence assays (MGCs and CCs) and maintained for 18 – 48 h at 378C under 5% CO2.

Materials and Methods

These assays were performed essentially as previously described (Patak et al., 2003; Pinto et al., 2009; Canete et al., 2013). Total RNA was extracted from fresh MGCs and CCs, and from 18 and 48-h cultured MGCs using TriReagent (Sigma) and residual genomic DNA was removed by incubating the RNA samples with RNase-free DNase I and RNasin (Promega, Madison, WI, USA). Complementary DNAs (cDNAs) were synthesized using the Quantitect Reverse Transcription kit (Qiagen, Venlo, The Netherlands). The sequence of the primer pairs used for PCR were: (i) TAC3, forward 5′ -CCAGTGTGTGAGGGGAGCA-3′ and reverse 5′ -TCCAGAGATGAG TGGCTTTTGA-3′ , giving a PCR product of 266 base pairs (bp); (ii) TACR3, forward 5′ -TTGCGGTGGACAGGTATA TGG-3′ and reverse 5′ -GGCCATTGCACAAAGCAGAG-3′ , giving a PCR product of 178 bp; (iii) KISS1, forward 5′ -CCACTTTGGGGAGCCATTAG-3′ and reverse 5′ -CCAGTTGTAGTTCGGCAGGTC-3′ , giving a PCR product of 295 bp; (iv) KISS1R, forward 5′ -GGACGTGACCTTCCTCCTGT-3′ and reverse 5′ -GTACCAGCGGTCCACACTCA-3′ , giving a PCR product of 166 bp and (v) PDYN, forward 5′ -ATGTTCCCCTCCACCACAG-3′ and reverse 5′ -CTGGCATCTCTCCCATTCC-3′ , giving a PCR product of 150 bp. The genes encoding b-actin (ACTB); hypoxanthine phosphoribosyl-transferase 1 (HPRT1), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and polymerase (RNA) II (DNA directed) polypeptide A (POLR2A) were chosen as housekeeping genes for normalizing the PCR data, on the basis of our previous studies on human tissues (Patak et al., 2003; Pinto et al., 2009; Canete et al., 2013). The sequences of the forward and reverse primers used were: ACTB, 5′ -TCCCTGGAGAAGAGCTACGA-3′ and 5′ -ATCTGCTGG AAGGTGGACAG-3′ ; HPRT1, 5′ -GCCAGACTTTGTTGGATTTGA-3′ and 5′ -GGCTTTGTATTTTGCTTTTCC-3′ ; GAPDH, 5′ -CAATGCCTCCTG CACCAC-3′ and 5′ -CCTGCTTCACCACCTTCTTG-3′ and POLR2A, 5′ -ACATCACTCGCCTCTTCTACTCC-3′ and 5′ -GTCTTGTCTCGGGC ATCGT-3′ . Real-time quantitative polymerase chain reaction (qPCR) was used to quantify the expression of the test genes in CCs and MGCs from the same patients, and was carried out by using the 22DDCT method, as described previously (Patak et al., 2003; Canete et al., 2013). qPCR was performed on a Bio-Rad iCycler iQ real-time detection apparatus (Bio-Rad Laboratories, Hercules, CA, USA) using a FastStart SYBR Green Master (Roche Diagnostics GmbH, Manheim, Germany). The parameters of PCR amplification were: 10 s at 948C, 20 s at 608C and 30 s at 728C, for 50 cycles. The identity of each product was established by DNA sequence analysis and the specificity of PCR reaction was confirmed by melting curve analysis of the products and by size verification of the amplicon in a conventional agarose gel. The fold change of the target gene expression was expressed relative to the geometric mean mRNA expression of the four reference genes in each sample, as described in detail elsewhere (Vandesompele et al., 2002). Each assay was performed in triplicate and three negative controls were run for each assay: no template, no reverse transcriptase and no RNA in the reverse transcriptase reaction.

Study population Approval for this work was obtained from the institutional Ethics Committee of Hospital Virgen Macarena (Sevilla, Spain) and informed consent was obtained from each patient at the time of oocyte retrieval for IVF. Human MGCs and CCs from preovulatory follicles were collected at the time of oocyte retrieval from 56 oocyte donors having IVF treatment at IVI Centre for Reproductive Care. The selected donors had a normal BMI (18– 24 kg/m2) and all were under 35 years old.

Stimulation protocol Patients were treated with a long or short luteal protocol of GnRH agonist or antagonist (Abbott Laboratories, Montreal, QC, Canada) and daily doses of recombinant FSH (75– 225 IU; Merck Serono, Geneva, Switzerland) followed by ovulation induction with hCG (Merck Serono). Doses were adjusted according to ovarian response as judged by ultrasound and by serum estradiol concentrations.

Human MGC collection MGCs were collected from follicular fluid (FF) obtained via transvaginal ultrasound-guided oocyte retrieval, which was performed 36 h after hCG administration. After removal of oocyte – cumulus complexes, the remaining follicular aspirates from each patient were pooled and transported to the research laboratory and MGCs collected as described elsewhere (Ferrero et al., 2012) with minor modifications. Briefly, MGCs were separated from erythrocytes by density gradient centrifugation on 5 ml HISTOPAQUE 1077 (v/v, Sigma, St. Louis, MO, USA) for 20 min at 400g. The middle layer was collected, washed by centrifugation for 5 min at 600g and re-suspended in Red Blood Cell lysing buffer (Hybri-max, Sigma) for 15 min at 378C, for lysis of residual erythrocytes. After centrifugation, the recovered MGCs were suspended in 1 ml HTF and incubated with 20 ml of magnetic beads coated with monoclonal anti-human CD45 antibody (Dynabeads pan mouse IgG, Invitrogen, Eugene, OR, USA) for 20 min under continuous agitation. The plastic tube containing the mixture was then placed next to a fixed magnet for 2 min. The unlabeled cells of interest were then collected while the immune cells remained associated with the beads and retained to the plastic walls closest to the magnet.

Human cumulus cell collection CCs were obtained from the same donors from whom MGCs were collected. After follicular aspiration, the CCs surrounding the oocyte were removed by using cutting needles, by subsequent treatment of cumulus – oocyte complexes (COCs) for 1 min with Sydney IVF Hyaluronidase (80 IU/ml, K-SIHY Cook Medical, Brisbane, Australia) and finally, by carefully removing the CCs of the corona radiata with very thin glass pipettes (Swemed denudation pipette, 0.134 – 0.145 mm, Vitrolife, Goteborg, Sweden).

Cell culture Following isolation, MGCs and CCs were suspended in Sydney IVF Fertilization Medium (K-SIFM 50, Cook Medical, Brisbane, Australia) supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin and 50 mg/ml

RNA extraction and real-time quantitative polymerase chain reaction

Immunofluorescence CCs and MGCs were seeded onto sterile poly-L-lysine-coated coverslips and cultured for 18 h. Cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 20 min and the membranes permeabilized with 2% Triton X-100 in PBS for 20 min. After blocking for 120 min with 2% casein in PBS, slides were incubated with one or two

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(double immunostaining experiments) of the following primary antibodies: goat anti-human NKB (sc-14109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human NK3R (sc-28952, Santa Cruz), rabbit antihuman kisspeptin (sc-15400, Santa Cruz), goat anti-human KISS1R (sc-48220, Santa Cruz) or rabbit anti-human prodynorphin (sc-67043, Santa Cruz). All these primary antibodies were incubated overnight at 48C and used at a dilution of 1:200 in PBS containing 2% casein. The specificity of the primary antibodies for NK3R and KISS1R was confirmed by western blot analysis, which showed the presence of the band of the expected size for each protein (see below for further details of western blot methods). The specificity of anti-human NKB and anti-human kisspeptin had been previously assessed by our group in sections of human ovary, uterus and oviduct (Cejudo Roman et al., 2012) by omitting the primary antibody or by preabsorption with the corresponding immunogenic peptides; procedures that abolished specific immunostaining. Negative control slides were not exposed to the primary antibody and were incubated with PBS in the same conditions as the test slides. Samples were extensively washed and incubated for 60 min with appropriate FITC- or Tx-red conjugated secondary antibodies (Santa Cruz). In addition, double immunofluorescence labeling experiments were performed to analyze the immunolocalization of NKB and NK3R, kisspeptin and KISS1R, or NKB and kisspeptin, in the same cell. Slides were further washed in PBS, mounted using Prolong Gold antifade reagent with or without DAPI (Invitrogen, Molecular Probes) and examined with an Olympus BX-51 fluorescence microscopy (Tokyo, Japan) using a 60× oil immersion objective.

Western blot experiments Western blotting was used to analyze the presence of NK3R and KISS1R in MGCs and CCs and was performed essentially as described previously (Pinto et al., 2012). Briefly, total proteins were extracted from fresh cells by boiling for 15 min in Laemmli buffer and 20 mg of protein were loaded on 10% sodium dodecyl sulfate (SDS) –PAGE gels. Proteins were separated by electrophoresis, transferred to polyvinyldifluoride membranes and non-specific staining blocked by incubation with 1% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TTBS). The membranes were incubated overnight either with rabbit anti-human NK3R antibody (sc-28952), goat anti-human KISS1R (sc-48220) or rabbit anti-human actin antibody (sc-1616, Santa Cruz). Immunoreactivity was detected by treatment with appropriate HRP-conjugated secondary antibody and developed with the Amersham advance enhanced chemiluminescence kit (Buckinghamshire, UK). Primary antibody dilution was 1:10 000 and for the secondary antibody it was 1:50 000 (goat anti-rabbit IgG) or 1:20 000 (rabbit anti-goat IgG). Membranes were stripped and incubated with anti-human actin antibody for protein quantification, using ImageLab Software (Bio-Rad), and NK3R and KISS1R protein expression was normalized to actin expression.

Measurements of intracellular Ca21 For measurement of intracellular Ca2+, [Ca2+]i, fresh MGCs were incubated with the acetoxymethyl ester form of Fura-2 (Fura-2/AM, 8 mM, Molecular Probes, Invitrogen) for 60 min at room temperature in the presence of the non-cytotoxic detergent pluronic acid (0.1%, Molecular Probes). After loading, the cells were washed, re-suspended in HTF solution at a concentration of 150 000 cell/ml and used within the next 1 – 5 h, following previously described procedures (Pinto et al., 2012). Cell aliquots (1 ml) were placed in the quartz cuvette of a spectrofluorometer (SLM Aminco-Bowman, Series 2, Microbeam, Barcelona, Spain) and magnetically stirred at 378C. Cell suspensions were alternatively illuminated with two excitations wavelengths (340 nm and 380 nm) and the emitted fluorescence was measured at 510 nm. Changes in [Ca2+]i were monitored using the Fura-2 (F340/F380) fluorescence ratio as previously described (Pinto et al., 2012). Calibration

of [Ca2+]i was achieved according to the equation of Grynkiewicz, adding 20 ml of Triton X-100 (5%), to obtain the maximal response, followed by addition of 100 ml of EGTA (40 mM) to obtain the minimal response. Kisspeptin (1 or 10 mM), NKB (10 mM), or the corresponding vehicle, was added to the cuvette 10 min after initiation of [Ca2+]i measurement, with only one treatment being assayed in each MGC aliquot. In addition, kisspeptin effects were studied in aliquots pretreated for 5 min with NKB (10 mM), or with the selective NK3R agonist, senktide (10 mM), or the corresponding vehicle. In specific experiments, cells were preincubated with the tachykinin receptor antagonists SR 140333 (NK1 receptorselective, 0.1 mM), SR 48968 (NK2 receptor-selective, 0.1 mM) and SB 222200 (NK3 receptor-selective, 1 mM), or with SB 222200 alone (1 mM), or with the corresponding vehicle for 45 min at 378C before addition of NKB and kisspeptin. SB 222200 was from Sigma and SR 140333 and SR 48968 were a generous gift of Sanofi Recherche (Montpellier, France).

Statistical analysis Values (means + SEM) were obtained by pooling individual data. Unless otherwise indicated, n represents the number of experiments in MGC or CC samples from n different donors. Statistical analyses used Mann – Whitney’s U (for comparison of mean ranks between two groups) or Kruskal – Wallis (to compare more than two groups) non-parametric tests. These procedures were undertaken using GRAPHPAD PRISM (version 5.0) program. P , 0.05 values were considered significant.

Results TAC3, TACR3, KISS1 and KISS1R are expressed in mural granulosa and cumulus cells The genes encoding NKB (TAC3), the tachykinin NK3 receptor (TACR3), kisspeptin (KISS1) and the kisppeptin receptor (KISS1R) were all found to be expressed in human MGCs and CCs. In contrast, we failed to detect the specific transcript encoding PDYN, the precursor protein of the endogenous opioid peptide dynorphin. Yet, using the same oligonucleotide primer pair and conditions, the presence of the PDYN product was observed in human testis and sperm, used as positive controls of amplification (data not shown). Real-time RT–PCR analysis of mRNA derived from MGCs and CCs demonstrated that TAC3 expression levels were significantly higher in CCs (4-fold), compared with MGCs from the same donors, a fact that occurred in all CC-MGC pairs assayed (P , 0.05, n ¼ 30, Fig. 1A). Similarly, TACR3 and KISS1R mRNA levels were 3.5-fold higher in CCs, when compared with paired MGCs (Fig. 1B and D). When the expression of the mRNAs in CC–MGC pairs was considered individually, TACR3 mRNA values were higher in CCs in 25 of the 30 patients assayed while KISS1R mRNA values were higher in 29 of 30 patients. In the case of KISS1, there were small differences between the mRNA levels in CCs and their paired MGCs, which did not reach statistical significance (Fig. 1C). The expression of the NKB and kisspeptin systems at the mRNA level was also analyzed in MGCs cultured for 18- or 48-h in the absence or in the presence of FF of the donor from which MGCs were obtained. Compared with expression values observed in the same cells before culture (i.e. at time 0), TAC3 mRNA declined by 10-fold after culture for 18 h, and by 60-fold after culture for 48 h (Fig. 2A). The presence of FF in the medium caused a partial recovery of TAC3 expression, a response

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Figure 1 Expression of TAC3, TACR3, KISS1 and KISS1R genes in human granulosa cells. Quantitative real-time RT– PCR analysis showed that the expression of (A) TAC3, (B) TACR3 and of (D) KISS1R was significantly higher in CCs, compared with MGCs from the same patients. KISS1 mRNA expression (C) did not show a significant variation. Each bar represents the mean with SEM of 30 different experiments. *P , 0.05, ***P , 0.001, significant difference versus mRNA expression in CCs, Mann– Whitney test.

that reached statistical significance in 48-h cultures (Fig. 2A). In turn, the expression of TACR3 mRNA was completely lost after 18 h culture and could not be recovered in cell cultures maintained either in the absence or in the presence of FF (Fig. 2B). In clear contrast, KISS1 mRNA increased by 3-fold in MGCs cultured for 18-h and showed a greater increase (of 20-fold) after 48-h culture (Fig. 2C). KISS1 expression was similar in the absence or presence of FF in the medium (Fig. 2C). KISS1R mRNA levels were also increased, by 5-fold, in 18- and 48-h cultures, and the increase was higher (by 20-fold) in MGCs cultured in the presence of FF (Fig. 2D).

The NKB/NK3R and KISS/KISS1R proteins are expressed in mural granulosa and cumulus cells Immunofluorescence studies demonstrated a positive immunostaining for NKB, NK3R, KISS1 and KISS1R, which was observed in 100% of cells in all samples assayed (n ¼ 6 CC-MGC pair samples from six different oocyte donors, Fig. 3). NKB immunoreactivity (ir) showed a predominant cytoplasmic expression pattern while NK3R-ir was mainly detected in the cytoplasm and the cell membrane (Fig. 3A, a–e). The merged image of NKB and NK3R staining shows the specific localization of each protein, with the nucleus showing a green fluorescence due to NK3R staining of the superimposed cell membrane. Immunoreactivity to KISS1 was preferentially located in the cytoplasm and in the cell nuclei, while KISS1R-ir appeared mainly located in the cytoplasm and the cell membrane (Fig. 3B, a– e). Additional double immunofluorescence labeling experiments confirmed that NKB and kisspeptin were co-localized in the same cell (Fig. 3C). The

cellular localization of each of the proteins assayed was similar in MGCs (Fig. 3) and CCs (data not shown). In keeping with mRNA expression data, immunoreactivity for PDYN was not observed in any MGC or CC cell assayed (n ¼ 4 CC-MGC pairs from four different donors, data not shown). The presence of the NK3R and KISS1R proteins in MGCs and CCs was confirmed by western blot analyses. These studies showed the presence of the specific bands corresponding to the expected molecular weights for the NK3R (53 kDa) and KISS1R (43 kDa), respectively (Fig. 4A). Semi-quantitative analysis of band densitometry showed that the expression of NK3R and KISS1R was stronger in CCs, compared with that in MGCs from the same patients (n ¼ 8 CC-GC pairs from eight different oocyte donors, Fig. 4B).

Effects of NKB and kisspeptin on granulosa cell intracellular Ca21 levels Kisspeptin (1 and 10 mM) caused an increase in intracellular free Ca2+ levels, [Ca2+]i, in human granulosa cells loaded with Fura-2. The increase in [Ca2+]i was observed in six of eight MGC samples assayed, but the responses were of very different magnitude between different MGC suspensions. The responses initiated rapidly after exposure to kisspeptin and consisted mainly of [Ca2+]i oscillations (Fig. 5). In some, but not in all, cell suspensions, [Ca2+]i oscillations were superimposed on an increase in the basal [Ca2+]i levels (Fig. 5), a profile that was only observed with kisspeptin 10 mM. Of note, while the [Ca2+]i responses to kisspeptin were rather homogeneous in different aliquots from the same MGC suspension, there were considerable differences in magnitude when MGC suspensions from different patients were analyzed. For this reason,

Neurokinin B and kisspeptin in granulosa cells

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Figure 2 Expression of TAC3, TACR3, KISS1 and KISS1R genes in human MGCs cultured for 18- or 48-h in the absence or in the presence of FF of the donor from which MGCs were obtained. (A) Compared with expression values before culture (0 h), TAC3 mRNA declined by 10-fold after culture for 18 h, and by 63-fold after culture for 48 h. The expression was partially recovered in the presence of FF. (B) TACR3 mRNA expression was completely lost after 18 h culture and could not be recovered, either in the absence or in the presence of FF. (C) KISS1 mRNA levels rose by 3-fold in MGCs cultured for 18-h and by 20-fold after 48-h culture, in a manner independent on the presence of FF. (D) KISS1R mRNA levels increased by 5-fold in 18- and 48-h cultures and the increase was higher in MGCs cultured in the presence of FF. Each bar represents the mean with SEM of five different experiments. *P , 0.05, significant difference versus mRNA expression in MGCs before culture, Kruskal– Wallis followed by Dunn’s multiple comparison test. aP , 0.05, significant difference versus mRNA expression in MGC culture in the absence of FF, Mann– Whitney test.

it was not possible to calculate a mean response to kisspeptin and the differences in responses were calculated individually, between different aliquots of a same MGC suspension. The kisspeptin-induced [Ca2+]i changes could be terminated by the addition of 2.5 mM EGTA, which caused a return of [Ca2+]i to basal levels, demonstrating the dependence of these responses on Ca2+ influx. Exposure to NKB (1 and 10 mM) failed to induce any change in [Ca2+]i in human MGCs (Fig. 5A). However, the [Ca2+]i response to kisspeptin was reduced in the presence of NKB (10 mM) (Fig. 5A and B). Intriguingly, the tachykinin NK3R-selective agonist senktide (10 mM) did not fully mimic the effects of NKB in terms of blockade of [Ca2+]i responses to kisspeptin (Fig. 5A and B). Yet, the inhibitory effect of NKB was reverted in the presence of a cocktail of antagonists selective for the tachykinin receptors NK1R (SR 140333, 0.1 mM), NK2R (SR 48968, 0.1 mM) and NK3R (SB 222200 1 mM), but was less affected in the presence of the NK3R-selective antagonist when added alone (Fig. 5B).

Discussion The present findings show that the NKB/NK3R and KISS1/KISS1R systems are present at the mRNA and protein levels in human ovarian granulosa cells. Their expression levels change depending on cell differentiation and both peptide systems can modulate the function of these cells. Altogether, these data suggest that at least part of the effects of kisspeptin and NKB on reproduction might include a direct action at the ovarian level, involving the regulation of granulosa cell function.

In our study, granulosa and cumulus cells were obtained from oocyte donors undergoing controlled ovarian stimulation, following standard protocols used in assisted reproduction treatments, which cause the growth of multiple follicles. In comparison with natural cycles, this ovarian stimulation induces changes in serum and FF hormonal milieu, and these changes may affect gene and protein expression in granulosa cells (de los Santos et al., 2012), which in turn might influence the responses of granulosa and/or cumulus cells per se. While this is the standard procedure for most of research involving human granulosa cells, it must be stressed, though, that in order to minimize the influence of the possible variations in the hormonal environment, this study was performed in healthy women of matched characteristics and the level of expression of each target gene or protein in granulosa cells was compared with its expression in cumulus cells from the same patient. NKB and kisspeptin play a key role in the regulation of gonadal function acting primarily at the hypothalamic level of the gonadotropic axis. These peptides are expressed in discrete neuronal populations within the hypothalamus where they modulate GnRH secretion and gonadotrophin release (Topaloglu et al., 2009; Lehman et al., 2010; Navarro and TenaSempere, 2012; Pinilla et al., 2012). Besides this fundamental role, our present data show that NKB/NK3R and KISS/KISS1R are locally synthesized and are functionally active in mural granulosa and cumulus cells. Previous studies had documented that NKB and NK3R are expressed in human, rat and mouse granulosa cells (Loffler et al., 2004; Candenas et al., 2005; Cejudo Roman et al., 2012). KISS1 and/or KISS1R have also been detected in granulosa cells from humans and rats (Castellano

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Figure 3 Immunolocalization of NKB, tachykinin NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) proteins in human granulosa cells. (A) Double immunofluorescence analysis of NKB (a, red signal) and NK3R (b, green signal) and merged images (d, e) in MGCs stained with goat anti-human NKB and rabbit anti-human NK3R primary antibodies. The merged image shows the specific localization of each protein, with the cytoplasm showing the yellow– orange color that results from overlap of the red NKB staining with the green NK3R staining, while the nucleus shows green fluorescence due to NK3R staining of the superimposed cell membrane. (B) Double immunofluorescence analysis of KISS1R (a, red signal) KISS1 (b, green signal) and merged images (d, e) in MGCs stained with goat anti-human KISS1R and rabbit anti-human KISS1 primary antibodies. The merged image shows the localization of kisspeptin in the nuclei and the yellow –orange staining in the cytoplasm. (C) Double immunofluorescence analysis of NKB (a, red signal) and KISS1 (b, green signal) and merged image (yellow signal, d, e) in MGCs stained with goat anti-human NKB and rabbit anti-human KISS1 primary antibodies. The blue signal shown in c and e corresponds to nuclear staining with DAPI. The differential interference contrast images of the cells analyzed are also shown (f). Experiments were performed at least six times with similar results. Scale bar, 50 mm.

Neurokinin B and kisspeptin in granulosa cells

Figure 4 Expression of tachykinin NK3R and kisspeptin receptor (KISS1R) proteins in human granulosa cells. (A) Western blot analysis with specific anti-human NK3R and KISS1R primary antibodies, showing the presence of both proteins in mural granulosa and cumulus cells. The expression of NK3R and KISS1R within each tissue was normalized with respect to actin. (B,C) Semi-quantitative analysis of band densitometry showed that the expression of NK3R (B) and of KISS1R (C) was higher in CCs, compared with MGCs from the same patients. Each bar represents the mean with SEM of eight different experiments. *P , 0.05, significant difference versus protein expression in CCs, Mann – Whitney test.

et al., 2006; Gaytan et al., 2009; Peng et al., 2013). To our knowledge, the present results are the first to show co-expression of NKB/NK3R and KISS1/KISS1R in MGCs and CCs, which strongly suggests that these peptide systems might play a coordinated role in the direct regulation of human granulosa cell function. In a subset of hypothalamic neurons, located in the ARC/infundibular region, kisspeptin and NKB are co-expressed together with the opioid peptide, dynorphin; hence, these neurons have been termed KNDy, for the co-expression of Kisspeptin, NKB and Dynorphin (Lehman et al., 2010; Pinilla et al., 2012). Admittedly, however, the proportion of KNDy neurons seems to vary among species, and these seem to be less abundant in the infundibular region of young men (Hrabovszky et al., 2012). In any event, on the basis of our present results, we investigated whether granulosa cells express dynorphin, therefore constituting a peripheral, nonneuronal population of KNDy cells. However, we were unable to detect the expression of dynorphin, at the mRNA or protein levels, neither in

2743 MGCs nor in CCs. It thus seems that granulosa cells are kisspeptin/NKB (KN)-only cells, as opposite to KNDy cells in the hypothalamus. Notably, neuroanatomical analyses in humans have revealed that, in contrast to other mammalian species, only a few neurons in the infundibular region in young men seem to express also dynorphin (Hrabovszky et al., 2012). The physiological relevance of such a KN-only pattern, and/or of the absence of dynorphin co-expression, remains to be determined. The expression of NKB/NK3R and KISS1/KISS1R showed marked changes depending on the differentiation state of the granulosa cells. Thus, with the exception of KISS1, whose expression was similar in MGCs and CCs, the mRNA levels of TAC3, TACR3 and KISS1R were significantly higher in CCs, when compared with MGCs from the same patients. Western blot analyses confirmed that the expression of NK3R and KISS1R proteins was also higher in CCs than in MGCs. In the same vein, our previous immunohistochemical studies demonstrated that the expression of NKB/NK3R and KISS1/KISS1R in human ovarian tissue sections changed depending on the stage of follicular maturation and these proteins were particularly abundant in corpora lutea (Gaytan et al., 2009; Cejudo Roman et al., 2012). The strong regulation of the expression of NKB and KISS1 systems was further confirmed in 18and 48-h cultures of MGCs. The expression of TAC3 and TACR3 greatly decreased while the expression of KISS1 and KISS1R was significantly enhanced, in MGC cultures. Moreover, the presence of FF differentially affected the expression of these genes. Altogether, these data suggest that subtle differences in the developmental state of the ovarian follicle would result in changes in the expression profiles of TAC3, TACR3, KISS1 and KISS1R. Further studies will help to determine the potential use of these factors as biomarkers of oocyte developmental quality and/or fertilization ability, which may help to improve the effectiveness of the treatments in reproductive medicine (de los Santos et al., 2012; Fragouli et al., 2014). Likewise, the potential correlation between changes in endogenous reproductive hormone levels and the expression of these targets merits future investigation. The presence of NK3R and KISS1R in MGCs and CCs suggest that these cells are not only a local source but are also target cells for NKB and kisspeptin. Previous studies in the protochordate Ciona intestinalis have shown that Ciona tachykinins enhance oocyte growth by acting on specific receptors presents in the cells surrounding the oocyte, named test cells (Aoyama et al., 2008, 2012; Satake et al., 2013). In mammals, it is reasonable to hypothesize that the role of test cells was assumed by granulosa cells, as they play an essential role in the regulation of follicular development, oocyte maturation and ovulation. Regarding kisspeptin, recent studies have shown that this peptide is able to induce progesterone release from rat luteal cells or cultured chicken GCs (Xiao et al., 2011; Peng et al., 2013), therefore suggesting that the KISS1 system could participate in the local control of ovarian steroid secretion. In the same line, very recent findings have shown that haploinsufficiency of the kisspeptin receptor, Kiss1r, causes premature ovarian insufficiency, with a progressive loss of ovarian follicles in mice, despite the fact that these animals were not gonadotrophin deficient (Gaytan et al., 2014). Moreover, Kiss1r null mice, which fail to spontaneously ovulate because of the lack of kisspeptin signaling, display subnormal ovulatory responses following an intense protocol of gonadotrophin priming (Gaytan et al., 2014). As a whole, this recent experimental evidence strongly supports a complementary role of kisspeptin, acting directly in the ovary, in the regulation of ovarian function. This possibility is further supported by very recent data demonstrating the existence of a novel intra-ovarian neurotrophin (NT) pathway, essential for oocyte

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Figure 5 Effects of NKB and kisspeptin (KISS1) on intracellular free Ca2+ ([Ca2+]i) levels in human MGCs loaded with Fura-2. (A) Representative traces showing responses to NKB (10 mM) or kisspeptin (10 mM) alone and responses to kisspeptin in the presence of NKB or the tachykinin NK3R-selective agonist senktide (10 mM). The X-axis shows time in seconds and the Y-axis shows [Ca2+]i data expressed by the F340/F380 ratio. (B) Effects of senktide (10 mM) and NKB (10 mM) on kisspeptin (10 mM)-induced changes in [Ca2+]i and effects of NKB on the kisspeptin response in the presence of the NK3R-selective antagonist SB 222200 (1 mM) or of a cocktail of antagonists selective for the tachykinin receptors NK1R (SR 140333, 0.1 mM), NK2R (SR 48968, 0.1 mM) and NK3R (SB 222200). Bars are means with SEM of 4 – 12 different experiments and represent percentage changes in the area of the [Ca2+]i response to kisspeptin relative to the control response to kisspeptin in the presence of the corresponding solvent. *P , 0.05, significant difference versus control [Ca2+]i response to kisspeptin. aP , 0.05, significant difference versus [Ca2+]i response to kisspeptin in the presence of NKB. Kruskal – Wallis test followed by Dunn’s multiple comparison test.

survival after puberty, whose activation is completely dependent on direct kisspeptin actions in the ovary (Dorfman et al., 2014). Because Ca2+ signaling plays a central role in the regulation of many different granulosa cell functions, we analyzed the effects of NKB and

KISS1 on intracellular Ca2+ concentrations in this cell population. Our data show that kisspeptin was able to induce changes in [Ca2+]i dynamics in human MGCs. Of note, NKB per se was incapable of evoking any change in MGC [Ca2+]i levels, but significantly reduced the increase in

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Neurokinin B and kisspeptin in granulosa cells

[Ca2+]i produced by KISS1. This observation, together with our expression data, is strongly suggestive of a possible, local interaction between both peptides that might be relevant for granulosa cell function. In addition to the physiological relevance of these data, for a methodological perspective, our results suggest that MGCs and CCs may provide a useful, simple cellular model for analyzing the integrated effects of NKB and kisspeptin in the control of signal transduction and, eventually, reproductive cell function in humans. The inhibitory effects of NKB on kisspeptin-induced [Ca2+]i responses were not fully mimicked by the NK3R-selective agonist, senktide. Similarly, the effects of NKB were only partly attenuated in the presence of the NK3R-selective antagonist, SB 222200, but were clearly reduced in the presence of a cocktail of antagonists selective for the tachykinin receptors NK1R, NK2R and NK3R. These data strongly suggest that the effects of NKB in granulosa cells involve its molecular interaction with different tachykinin receptor subtypes. It is well known that tachykinin peptides are very promiscuous and can activate any of the three known tachykinin receptors (Maggi, 2000; Almeida et al., 2004; Satake et al., 2013). Thus, the effects of NKB and, more broadly, of the various tachykinins, would depend on the level of expression of the different tachykinin receptors in a given tissue or in a particular cell type. Hence, in the uterus from young women and rats, NKB increases motility by activation of the NK2R (Patak et al., 2003; Candenas et al., 2005), whereas in the uterus of aged rats, NKB-induced contractions are mediated by the NK3R (Candenas et al., 2005). Moreover, in the human fetal placental circulation, NKB causes vasodilation by stimulation of NK1R (Brownbill et al., 2003). In this same context, recent data have shown that the effects of NKB on KNDy neurons in the mouse arcuate nucleus could only be blocked by the simultaneous application of selective antagonists for tachykinin NK1R, NK2R and NK3R (de Croft et al., 2013). All together, these data supports the participation of the whole tachykinin system in the regulation of reproductive function, a feature that might explain the disparate responses observed in different animal species and experimental conditions when comparing the effects of NK3R-selective agonists and NKB itself (Rance et al., 2010; Navarro and Tena-Sempere, 2012; Jayasena et al., 2014). In summary, our present results demonstrate that human granulosa cells are non-neuronal peripheral kisspeptin/NKB producing cells, which also express the major receptors for these peptide systems. Indeed, our current data show that NKB/NK3R and kisspeptin/KISS1R peptide systems are expressed in mural granulosa and cumulus cells in a tightly controlled manner, which depends on the degree of cell differentiation. Furthermore, our results are the first to document functional responses in human granulosa cells following exposure to kisspeptin and NKB. These responses include the ability of NKB, presumably acting via interaction with NK3R and other tachykinin receptors, to inhibit Ca2+ mobilization in response to kisspeptin. All in all, our present findings further demonstrate that, in addition to their indispensable effects at the central nervous system, NKB and kisspeptin are able to act directly on the female gonads, an action that might contribute to the whole spectrum of biological effects whereby these peptide systems carry out their relevant roles in the control of human reproductive function.

Authors’ roles J.G.-O. obtained and processed granulosa and cumulus cell samples, provided patient data, performed PCR and cell culture studies, and

contributed to the draft of the manuscript. F.M.P. processed granulosa and cumulus cells, performed real-time PCR, cell cultures, western blots and immunofluorescence studies, and participated in the study design. M.F.-S. supervised and participated in obtaining granulosa and cumulus cells, and contributed to the design of the study. N.P. participated in obtaining granulosa and cumulus cells, provided patient data and obtained informed consents from patients. A.C.-R. participated in PCR and immunofluorescence analyses and critically revised the manuscript. T.A.A. participated in the design of the study and critically revised the manuscript. M.H. performed the statistical analyses and participated in the draft of the manuscript. M.R. participated in the analysis of samples, especially those involving RNA isolation and PCR assays. M.T.-S. was involved in the experimental design and study planning, participated in the analysis and interpretation of data, revised the first draft of the manuscript, and had an active role in editing and preparation of the final version of the paper (including revision and responses to referees). L.C. conceived the study, analyzed PCR, immunofluorescence and western blot data, and had a leading role in writing the manuscript (in its original version and revision).

Funding This work was supported by grants from Ministerio de Economı´a y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucı´a (P08-CVI-04185), Spain.

Conflict of interest J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.

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