Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK 3 R and KISS1R in the human female genital tract

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1 Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK 3 R and KISS1R in the human female genital tract Antonio Cejudo Roman, J.D., a Francisco M. Pinto, Ph.D., a Idaira Dorta, J.D., b Teresa A. Almeida, Ph.D., b,c Mariano Hernandez, Ph.D., b,c Matilde Illanes, M.D., Ph.D., d Manuel Tena-Sempere, Ph.D., e and Luz Candenas, Ph.D. a a Instituto de Investigaciones Químicas (IIQ), CSIC, Universidad de Sevilla, Seville, Spain; b Area de Biología Celular, Facultad de Biología, and c Instituto de Enfermedades Tropicales y Salud Publica, Universidad de La Laguna, San Cristobal de La Laguna, Tenerife, Canary Islands; d Departamento de Anatomía Patologica, Facultad de Medicina, Universidad de Sevilla, Seville, Spain; and e Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Cordoba, IMIBIC and CIBER Fisiopatología de la Obesidad y Nutricion, Cordoba, Spain Objective: To investigate the presence of neurokinin B (NKB)/NK 3 receptor (NK 3 R) and kisspeptin/kiss1 receptor (KISS1R) messenger RNA (mrna) and proteins throughout the human female genital tract. Design: In vitro study. Setting: Academic research laboratories and academic hospitals. Patient(s): Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. Intervention(s): Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. Main Outcome Measure(s): Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK 3 R and kisspeptin/kiss1r in target tissues. Result(s): Expression of the genes encoding NKB (TAC3) and NK 3 R(TACR3), and kisspeptin (KISS1) and its receptor (KISS1R) was found in the uterus, ovary, and oviduct. Both NKB and NK 3 R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK 3 R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. Conclusion(s): NKB/NK 3 R and kisspeptin/kiss1r are present in female peripheral reproductive tissues with colocalization of both systems in some nonneuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Human female reproductive tract, KISS1 receptor, kisspeptin, neurokinin B, NK 3 receptor Neurokinin B (NKB) is a 10 amino acid peptide that belongs to the family of tachykinins. Three tachykinin receptors are actually known, named NK 1, NK 2, and NK 3, and the effects of NKB are predominantly mediated by activation of the NK 3 receptor (NK 3 R) (1 4). A recent report showed that mutations in TAC3 and TACR3,the genes encoding NKB and NK 3 R, were associated with human normosmic hypogonadotrophic hypogonadism, a disease characterized by the failure of sexual maturation, impaired gametogenesis, and infertility (5, 6). These important findings, which have Received December 20, 2011; revised January 24, 2012; accepted February 13, 2012; published online March 14, A.C.R. has nothing to disclose. F.M.P. has nothing to disclose. I.D. has nothing to disclose. T.A.A. has nothing to disclose. M.H. has nothing to disclose. M.I. has nothing to disclose. M.T.-S. has nothing to disclose. L.C. has nothing to disclose. Supported by grants from Junta de Andalucía (P08-CVI-04185) and Ministerio de Ciencia e Innovacion (CTQ /BQU), Spain, with joint financing by FEDER and FSE funds from the European Union. Reprint requests: Luz Candenas, Ph.D., Instituto de Investigaciones Químicas, Avenida Americo Vespucio 49, Isla de La Cartuja, Sevilla, Spain ( luzcandenas@iiq.csic.es). Fertility and Sterility Vol. 97, No. 5, May /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert been confirmed by independent studies (7, 8), provide indubitable evidence for a key role of the NKB/NK 3 R ligandreceptor pair in the regulation of human reproduction. Strikingly similar essential reproductive roles were earlier suggested for kisspeptin (encoded by the KISS1 gene) and its canonical receptor, KISS1R, on the basis of human and experimental animal data (9 13). Investigations prior to the publication by Topaloglu et al. (5) had shown that NKB is synthesized in discrete neuronal populations within the hypothalamus, where it participates in the regulation of gonadotropin-releasing hormone (GnRH) secretion (14 16). The expression of NKB in these neurons was highly increased in postmenopausal women or ovariectomized primates and VOL. 97 NO. 5 / MAY

2 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY was down-regulated by estrogen (14, 15). A recent major observation shows that NKB is coexpressed with kisspeptin and dynorphin in a population of neurons in the arcuate nucleus, named the KNDy (Kisspeptin/NKB/Dynorphin) neurons (9, 15, 16). Because these three peptides seem to jointly operate as key modulators of GnRH secretion, KNDy neurons are thought to serve as a central pacemaker that drives pulsatile GnRH secretion and thus gonadotropin release (9, 13, 16). In addition to its role at the central level, many experimental data obtained in the past few years by our group and others argued for an important role of NKB and NK 3 R in the regulation of peripheral reproductive functions (4, 17 21). Both NKB and NK 3 R are present in the placenta, the ovary, the testes, and the prostate (18, 21 24). They are also expressed in different types of reproductive cells including oocytes and mature spermatozoa (21, 25). The genes encoding NKB (TAC3/Tac2), NK 3 R (TACR3/Tacr3), and the tachykinin-metabolizing enzyme neprilysin are expressed in human, mouse, and rat uteruses, and their expression and function vary with age, during the ovarian cycle, and during the course of pregnancy (20, 22, 26 30). In the mammalian uterus, Tacr3 expression is strongly downregulated under conditions of estrogen dominance (17, 24, 27 31). This is, to our knowledge, the only mechanism described so far of regulation of the tachykinin NK 3 receptor. In spite of these previous data, little is known about the types of cells that express NKB and NK 3 R in peripheral reproductive organs. Similarly, information regarding the patterns of distribution of kisspeptin and KISS1R in the human reproductive tract remains scarce, and only a few studies have investigated their presence in the mammalian ovary (rat, primate, human), the rat oviduct, and human spermatozoa (32 35). Our study analyzed the presence of NKB and NK 3 R mrna and proteins in the human ovary, fallopian tube, and uterus. We also investigated the expression pattern of kisspeptin and KISS1R and their possible coexpression with NKB and NK 3 R, throughout the human female genital tract. MATERIALS AND METHODS Study Participants This study was approved by the institutional ethics committees (CSIC, Universidad de la Laguna and Hospital), and all patients gave informed written consent. RT-PCR Experiments Samples of fresh ovary, oviduct, and uterus were obtained from nonpregnant women of different ages (range: 36 to 77 years old). Ovarian and oviductal tissue samples were obtained from five postmenopausal women who underwent oophorectomy for treatment of nonovarian disease. Samples of whole uterus were obtained from six mid-reproductiveage women and six postmenopausal women who underwent hysterectomy for benign uterine disease. Menstrual cycle phases were determined by histologic evaluation. Tissue samples from macroscopically normal uterine regions were immersed in RNAlater (Ambion) and stored at 80 C until use. Reverse-transcription polymerase chain reaction (RT- PCR) assays were performed as previously described elsewhere (21, 29). Total RNA from approximately 30 mg of human tissue was isolated using TriReagent (Sigma), and complementary DNA (cdna) was synthesized using the Quantitect Reverse Transcription kit (Qiagen). Aliquots of the resulting cdna samples were amplified by PCR with specific oligonucleotide primer pairs designed to span intron/exon borders. The sequence of the primer pairs used were [1] human TAC3, forward 5 0 -CCAGTGTGTGAGG GGAGCA-3 0 and reverse 5 0 -TCCAGAGATGAGTGGCTTTT GA-3 0, to amplify a PCR product of 266 base pairs (bp); [2] human TACR3, forward 5 0 -TTGCGGTGGACAGGTATATGG- 3 0 and reverse 5 0 -GGCCATTGCACAAAGCAGAG-3 0, giving a PCR product of 178 bp; [3] human KISS1, forward 5 0 -CCA CTTTGGGGAGCCATTAG-3 0 and reverse 5 0 -CCAGTTGTAGTT CGGCAGGTC-3 0, giving a PCR product of 295 bp; and [4] human KISS1R, forward 5 0 -GGACGTGACCTTCCTCCTGT-3 0 and reverse 5 0 -GTACCAGCGGTCCACACTCA-3 0, giving a PCR product of 166 bp. Amplification of b-actin (ACTB, forward, 5 0 -TCCCTGGAGAAGAGCTACGA-3 0 and reverse, 5 0 -ATCTGCTGGAAGGTGGACAG-3 0 ) served as control for the RT-PCR reactions. The expected size of the PCR product was 362 bp. A pool of cdna from 20 different human tissues (human total RNA master panel; Clontech) was used as a positive control of amplification. The PCR products were separated by agarose gel electrophoresis, and the amplicon sizes were verified by comparison with a DNA mass ladder. Product identity was established by DNA sequence analysis. Immunohistochemistry Samples of fresh myometrium were obtained from six reproductive-aged women (three women in the secretory phase, two women in the proliferative phase, and one woman in menstrual phase) and one postmenopausal woman who underwent hysterectomy for symptomatic uterine leiomyomas at Hospital Universitario de Canarias and Hospital La Colina (Tenerife, Spain). Ovarian tissue samples were obtained from three reproductive-age women (in different stages of the menstrual cycle) and four postmenopausal women who underwent oophorectomy for treatment of nonovarian disease. Tissue samples were fixed in 10% buffered formaldehyde, embedded in paraffin, cut into 3 mm sections and mounted on microscope slides. Sections were deparaffinized and rehydrated in Tris buffer saline (TBS, Trizma Base 0.05 M NaCl 0.9%, ph 7.4), which was used for all further incubations and washes. The sections were incubated overnight at room temperature with a primary polyclonal antibody designed to recognize NKB (sc-14109; Santa Cruz Biotechnology), NK3R (Sigma), kisspeptin (sc-15400; Santa Cruz), or KISS1R (sc-48220; Santa Cruz Biotechnology) diluted 1:30 in TBS buffer containing 0.2% Triton X-100. After rinsing, sections were incubated with a biotinylated secondary antibody (1:1,000) followed by a streptavidin-peroxidase 1214 VOL. 97 NO. 5 / MAY 2012

3 Fertility and Sterility conjugate (1:1,000) (Jackson ImmunoResearch), both for 60 minutes at room temperature. Peroxidase activity was detected using 4-chloro-1-naphthol (Sigma) and 0.01% hydrogen peroxide. The specificity of these antibodies in human cells was previously assessed by our groups (25, 33, 35). In addition, the specificity of the immunostaining was assessed by replacing the specific antisera by normal serum, omitting one step of the reaction, or following preabsorption of the antisera with the corresponding antigen. For mast cell identification, sections were stained with toluidine blue. Briefly, paraffin sections were deparaffinized, hydrated, and rinsed in deionized water. Sections were placed in 1% acid alcohol for 3 to 5 minutes followed by a transfer to 0.5% toluidine blue solution for 15 minutes. The sections were then rinsed in deionized water, dehydrated in acetone, cleared in acetone-xylene, and finally mounted in Permount and coverslipped. Immunohistochemical analysis of the expression of NKB, NK 3 R, kisspeptin, and KISS1R in the human oviduct and uterine endometrium were performed with archival material obtained from the files of the Department of Anatomic Pathology, University of Seville, Spain. Fallopian tube sections were from six women (age range: 37 to 77 years old) who underwent surgery for benign or malign uterine diseases without any affectation of the oviduct. Specimens of uterine endometrium were obtained from six women (age range: 36 to 77 years old) who had required a hysterectomy for benign uterine disease. Only optimally preserved material was included in the analysis. Consecutive slides of the selected blocks were treated with a primary antibody for NKB, NK 3 R, kisspeptin, or its receptor (30 minutes at room temperature, working dilution 1:100) by use of the Ultra- View Universal DAB detection kit (Ventana Medical Systems) in an automatized immunostainer (BenchMark XT). Negative control slides were not exposed to the primary antibody and were processed in the same conditions as the test slides. RESULTS Gene Expression Analyses of TAC3, TACR3, KISS1, and KISS1R The RT-PCR analysis revealed that TAC3, TACR3, KISS1, and KISS1R genes were expressed in the human ovary, the oviduct, and the uterus (Fig. 1). When considered individually, all ovarian and oviductal samples (obtained from five women) were positive for TAC3, TACR3, KISS1, and KISS1R. With respect to uterine tissues, TAC3, KISS1, and KISS1R were expressed in all samples (obtained from 12 women). The TACR3 transcript was absent in three reproductive-aged women (two with proliferative endometrium and one with secretory endometrium) and in three postmenopausal women. The mrna of TAC3, TACR3, KISS1, and KISS1R genes were also detected in the cdna pool used as a positive control (see Fig. 1). Conversely, no PCR product was detectable in negative controls when samples were amplified without the RT step (not shown) or when RT-PCR was performed with no added RNA (see Fig. 1). Immunohistochemical Analyses of NKB, NK 3 R, Kisspeptin, and KISS1R In the human myometrium, NKB and NK 3 R immunoreactivity was observed in vascular smooth muscle cells, connective cells, and myometrial smooth muscle cells (Fig. 2A and D). Mast cells specifically stained using toluidine (see inset in Fig. 2B) were identified as connective cells positive for NK 3 R immunoreactivity. Tissue obtained from patients in the proliferative phase of the menstrual cycle showed weak NK 3 R staining in smooth muscle cells (see Fig. 2B) compared with samples from the secretory phase (see Fig. 2C) and menstrual phase (not shown). On the contrary, the NKB staining pattern remained unchanged during the different stages of the menstrual cycle (see Fig. 2E and F). Positive immunostaining for kisspeptin and KISS1R was not detected in the human myometrium (not shown). In the endometrium, NKB, NK 3 R, kisspeptin, and KISS1R showed strong immunostaining in both luminal and glandular epithelial cells in all samples assayed (Fig. 3A, a d). No labeling was detected in the stromal cells surrounding the glands. In the oviduct, NKB and NK 3 R immunolabeling was observed in the vast majority of epithelial cells, but no staining could be detected in stromal or muscle cells (see Fig. 3A, e f). Similarly, positive staining for kisspeptin and KISS1R was restricted to epithelial cells (see Fig. 3A, g h). The immunostaining was of low intensity for NKB and of moderate intensity for kisspeptin (see Fig. 3A, e, g). Both NK 3 R and KISS1R were apparently localized in the membrane, which showed moderate staining for KISS1R and stronger staining for NK 3 R (see Fig. 3A, f, h). For all tissues assayed, no staining was detected in any of the negative controls (see Fig. 3A, i l). NKB and NK 3 R Protein Expression in the Human Ovary Both NKB and NK 3 R immunoreactivity was found in different compartments of the ovary (see Fig. 3B). The NK 3 R immunoreactivity was detected in theca and granulosa cells of growing follicles, with low to moderate staining intensity (see Fig. 3B, d). Faint staining was found for NKB in growing follicles (not shown). Both NKB and NK 3 R immunostaining was particularly intense in atretic follicles and corpora lutea (see Fig. 3B a, b, e, f). In all ovaries examined, NK 3 R immunoreactivity was also found in blood vessels, stromal, and connective cells (see Fig. 3B, a d). The NKB immunoreactivity was present in the same cells, but the labeling was of lower intensity. Ovaries from postmenopausal women presented moderate to intense staining for NKB and NK 3 R in vascular cells and low staining in stromal cells (not shown). DISCUSSION Our study shows that NKB/NK 3 R and kisspeptin/kiss1r systems are expressed in the human uterus, ovary, and oviduct at both mrna and protein levels. This provides further evidence for the synthesis and potential local functional roles for these peptides and their receptors in peripheral reproductive tissues. Mutations in TAC3 and TACR3 cause hypogonadotrophic hypogonadism in humans (5 8). This observation adds VOL. 97 NO. 5 / MAY

4 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY FIGURE 1 Agarose gel showing expression of the genes encoding neurokinin B (TAC3), tachykinin NK 3 receptor (TACR3), kisspeptin (KISS1), the kisspeptin receptor (KISS1R), and b-actin (ACTB) in complementary DNA (cdna) obtained from the human uterus, ovary, and fallopian tube (FT). (þ) ¼ expression of all target genes and ACTB in a pool of cdnas from 20 different human tissues, used as a positive control of amplification; M ¼ molecular weight standards; ( ) ¼ negative control with no added RNA. Cejudo Roman. NKB and kisspeptin in female genital tract. Fertil Steril FIGURE 2 Localization of (A C) NK 3 R and (D F) NKB in human myometrium. Vascular smooth muscle cells (asterisks), myometrial smooth muscle cells (arrowheads), and connective cells (arrows) showed immunoreactivity for both markers (A and D). Among connective cells, histologic staining demonstrated that mast cells were also immunopositive for NK3R (A and B). The staining of NK3R was less intense in proliferative phase (B) than in secretory phase myometrium (C). No differences in NKB immunostaining were observed between the proliferative (E) and secretory (F) phases. Scale bar: a, b, and c, 250 mm; d, e, and f, 125 mm. Cejudo Roman. NKB and kisspeptin in female genital tract. Fertil Steril VOL. 97 NO. 5 / MAY 2012

5 Fertility and Sterility FIGURE 3 (A) Localization of NKB (a, e), NK3R (b, f), kisspeptin (c, g), and KISS1R (d, h) in human endometrium (a d), and oviduct (e h). Images i and j are negative controls for human endometrium, and k and l are negative controls for human oviduct. (B) Localization of NK3R (a d) and NKB (e g) in human ovary. Images a and e are corpus luteum, and b and f are atretic follicles. Images c and g are blood vessels, with immunoreactivity in vascular smooth muscle cells shown by arrows. Image d shows immunoreactivity for NK 3 R in granulosa (arrows) and theca cells (arrowheads) of growing follicles. Images h and i are negative controls. Scale bar: 250 mm. Cejudo Roman. NKB and kisspeptin in female genital tract. Fertil Steril support to data obtained at both central (14 16) and peripheral (17 22, 26 30) levels indicating an important role for the NKB/NK 3 R ligand-receptor pair in the regulation of reproductive functions. The mrna of NKB and NK 3 R have been found in the placenta, uterus, and ovary of various mammalian species (17 24). Our present data extend and refine those observations by providing the first thorough characterization of the distribution pattern of NKB and NK 3 R throughout the human female genital tract, with clear expression of both ligand and receptor in the epithelial cells VOL. 97 NO. 5 / MAY

6 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY lining the endometrium and oviduct as well as in vascular, smooth muscle, and connective cells in the myometrium and in mast cells. The expression of NK 3 R in myometrium, particularly in smooth muscle cells, showed marked changes depending on the hormonal state. This observation confirms our previous data in human, mouse, and rat uteruses showing that TACR3 gene expression was highly reduced under conditions of estrogen dominance (17, 27 30). Although NKB increased myometrial contractility in various mammalian species, its effects appear to be mainly mediated by activation of NK 2 R (20, 28 30). A role for NK 3 R in mediating the effects of NKB on myometrial contractility could only be demonstrated in aged or early pregnant rats (27, 28). This led us to conclude that NKB and NK 3 R participate in the regulation of uterine functions other than myometrial contractility (29, 30). The presence of NKB and NK 3 R in mast cells as well as in vascular and epithelial cells makes it tempting to speculate that this system could participate in the cross-talk between the uterus and the immune system. In the ovary, NK 3 R and NKB immunoreactivity was of low to moderate intensity in growing follicles and of high intensity in corpora lutea. Weak immunoreactivity in growing follicles might be due to the estrogen dominance that characterizes the initial stages of follicle development and the suppressive effects of E 2 on NKB and NK 3 R expression in different tissues (14, 15, 17, 31). Tachykinin and GnRH receptors are also present in the ovary of Ciona intestinalis, an invertebrate that lacks the hypothalamic-pituitary system (36). One of the tachykinins produced by this ascidian, Ci-TK- I, was shown to enhance the growth of oocytes from vitellogenic to postvitellogenic stages of maturation (37). These data suggest that peripheral reproductive tissues are a primary site of NKB expression and argue for an evolutionary conserved role of tachykinins in the regulation of reproduction acting locally at the level of the gonads. This appears to be the case for many other neuropeptide systems as well, which are synthesized in peripheral reproductive organs (36, 38), where they might be involved in rapid local responses, modulate the peripheral effects of pituitary gonadotropins, or participate in the cross-talk with their brain counterparts. Admittedly however, the physiologic relevance of such local expression and actions remains undefined and needs additional investigation (38). Kisspeptin plays a key role in the central regulation of reproduction by acting mainly as a major component of the neuronal networks controlling GnRH secretion (10 13). However, compelling though as yet incomplete evidence has suggested additional roles of kisspeptin on peripheral reproductive tissues, with prominent expression in the placenta (32, 39). Our present data confirm and greatly extend our previous findings on the expression of kisspeptin in rat oviduct (33) and human and rat ovary (32, 34) and show, for the first time, that kisspeptin and KISS1R are also expressed in the human endometrium and oviduct. As is the case for NKB, peripheral expression of kisspeptin may account for, or modulate, some of the reproductive effects of this system. Intriguingly, a recent report has shown that puberty onset can apparently proceed in mice after congenital (but not pubertal) ablation of kisspeptin neurons, thus suggesting some degree of redundancy and compensation for the brain kisspeptin system (40). Whether the peripheral expression of kisspeptin and its receptor reported here and in previous publications may contribute to the rescue of gonadal function in such (experimental) conditions warrants further investigation. Kisspeptin is coexpressed with NKB in KNDy neurons, where they regulate pulsatile GnRH and gonadotropin release (6, 9 16). Apart from our recent data on their expression in human spermatozoa (35), our present findings are the first to show the coexpression of NKB and kisspeptin in non-neuronal reproductive cells at the peripheral level. However, the precise roles and mechanisms of action of these peptides in the female reproductive tract are not well understood. Our data show that NKB/NK 3 R and kisspeptin/kiss1r systems are expressed in epithelial cells lining the lumen of the uterus and the oviduct and are therefore continuously present throughout the female genital tract. These data are strongly suggestive of a role of NKB and kisspeptin in the regulation of epithelial functions. Uterine and tubal epithelial cells are the first to enter in contact with exogenous factors and play an important role in the regulation of reproductive functions such as sperm transport, oocyte fertilization, and blastocyst implantation (41, 42). Epithelial cells in the endometrium and the oviduct produce GnRH (42), and it is possible that NKB and kisspeptin could participate in some way in the peripheral regulation of GnRH secretion and gonadotropin function. In the ovary, immunoreactivity for NKB and NK 3 R (present data) as well as for kisspeptin and KISS1R (34) is particularly intense in corpora lutea, suggesting a potential role for these peptide systems in the local control of ovulation and/or in postovulatory ovarian processes. Similarly, a role of these peptides in the local control of ovarian steroidogenesis cannot be excluded. In fact, a recent report has documented direct stimulatory effects of kisspeptin-10 on progesterone secretion in chicken ovarian granulosa cells (43). Further investigation may clarify whether a similar phenomenon occurs in the mammalian ovary and the eventual involvement, if any, of NKB. Altogether, our present results and those of previous studies (18, 20, 29, 32 34) strongly argue for an involvement of the NKB and kisspeptin systems in the regulation of human fertility acting at the female genital tract. Therefore, it would be of great interest to analyze the expression profiles of NKB/NK 3 R and kisspeptin/kiss1r in reproductive tissues and cells (i.e., the ovary, uterus, and gametes) and assess their potential usefulness as predictors of human fertility and/or as biomarkers in the diagnosis of reproductive diseases. Our study provides a thorough characterization of the wide pattern of distribution of NKB/NK 3 R and kisspeptin/ KISS1R in human female peripheral reproductive tissues. Additionally, we present novel evidence for colocalization of both systems in non-neuronal reproductive cells. Our current findings pave the way for future analyses on the peripheral reproductive roles of NKB and kisspeptin, and strongly suggest that the female reproductive tract is a site of expression and action of these factors, a feature that may contribute to their well-known roles as major regulators of human fertility VOL. 97 NO. 5 / MAY 2012

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