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. 2010 Jun 30;24(9):1863–1871. doi: 10.1210/me.2009-0530

Research Resource: Gonadotropin-Releasing Hormone Receptor-Mediated Signaling Network in LβT2 Cells: A Pathway-Based Web-Accessible Knowledgebase

Marc Y Fink 1,a, Hanna Pincas 1,a, Soon Gang Choi 1,a, German Nudelman 1, Stuart C Sealfon 1
PMCID: PMC2940478  PMID: 20592162

Abstract

The GnRH receptor (GnRHR), expressed at the cell surface of the anterior pituitary gonadotrope, is critical for normal secretion of gonadotropins LH and FSH, pubertal development, and reproduction. The signaling network downstream of the GnRHR and the molecular bases of the regulation of gonadotropin expression have been the subject of intense research. The murine LβT2 cell line represents a mature gonadotrope and therefore is an important model for the study of GnRHR-signaling pathways and modulation of the gonadotrope cell by physiological regulators. In order to facilitate access to the information contained in this complex and evolving literature, we have developed a pathway-based knowledgebase that is web hosted. At present, using 106 relevant primary publications, we curated a comprehensive knowledgebase of the GnRHR signaling in the LβT2 cell in the form of a process diagram. Positive and negative controls of gonadotropin gene expression, which included GnRH itself, hypothalamic factors, gonadal steroids and peptides, as well as other hormones, were illustrated. The knowledgebase contains 187 entities and 206 reactions. It was assembled using CellDesigner software, which provides an annotated graphic representation of interactions, stored in Systems Biology Mark-up Language. We then utilized Biological Pathway Publisher, a software suite previously developed in our laboratory, to host the knowledgebase in a web-accessible format as a public resource. In addition, the network entities were linked to a public wiki, providing a forum for discussion, updating, and error correction. The GnRHR-signaling network is openly accessible at http://tsb.mssm.edu/pathwayPublisher/GnRHR_Pathway/GnRHR_Pathway_ index.html.

A pathway-based knowledgebase of the LβT2 gonadotrope literature is openly web accessible. Entities link to a public wiki for discussion.

The GnRH receptor (GnRHR) plays a pivotal role in the neurohormonal control of reproductive function. Expressed at the surface of pituitary gonadotrope cells, the receptor binds hypothalamic neuropeptide GnRH and thus stimulates the synthesis and pulsatile release of gonadotropins LH and FSH; in turn, LH and FSH regulate gonadal functions, including gametogenesis, steroidogenesis, and ovulation. GnRH is secreted by hypothalamic neurons in a pulsatile manner, and the individual gonadotropin genes respond differentially to GnRH pulse frequency, as previously demonstrated in vivo (1,2). Thus, the intracellular signaling network activated upon binding of GnRH to its receptor must decipher the information received to ensure an appropriate physiological response of the gonadotrope cell. The pivotal role of GnRHR in the physiology of reproduction is further evidenced by the existence ofGnRHR mutations that cause hypogonadotropic hypogonadism in humans (3). Furthermore, GnRH analogs are widely used in the treatment of male and female infertility as well as hormone-dependent cancers, such as breast and prostate cancer.

The GnRHR belongs to the family of G protein-coupled receptors (GPCRs), the largest group of membrane receptors. GPCRs transduce extracellular stimuli via heterotrimeric GTP-binding proteins (G proteins). The GnRHR possesses the unique structural feature of lacking an intracellular carboxy-terminal domain. Therefore, in contrast to other GPCRs, it is characterized by poor internalization and lack of rapid desensitization (4,5).

The GnRHR intracellular signaling pathway has been the subject of intense experimental investigation for the past 25 yr, by means of both in vivo and in vitro approaches. In vitro models have mainly consisted of pituitary primary cultures, heterologous cell systems, and the gonadotrope cell lines αT3-1 and LβT2. Because pituitary gonadotropes account for only 5–10% of the anterior pituitary cell population, using primary cultures as a model for the study of gonadotrope-specific intracellular signaling cascades is challenging. The immortalized gonadotrope cell lines αT3-1 and LβT2 were obtained by directed oncogenesis in transgenic mice (6,7): the αT3-1 cell line represents an immature gonadotrope that expresses the glycoprotein hormone α-subunit gene, responds to GnRH, but does not express the LHβ and FSHβ subunits; in contrast, the LβT2 cell line displays the characteristics of a fully differentiated pituitary gonadotrope: in addition to the glycoprotein hormone α-subunit, it expresses LHβ, FSHβ, GnRHR, activin, inhibin, follistatin, and steroid receptors (8,9,10,11,12,13). Consequently, LβT2 cells represent a useful model for the study of GnRHR-mediated signaling, as well as intracellular transduction pathways triggered by physiological modulators of the gonadotropin response.

We have constructed a comprehensive, annotated map of the molecular interactions in LβT2 cells described in the literature as a resource for research on GnRHR signaling. We have made this knowledgebase readily accessible to the scientific community, with the goal of shared contribution by experimentalists through a wiki.

Results

General characteristics of the GnRHR-signaling map

We manually curated a comprehensive pathway map for GnRHR-mediated signaling. The pathway construction workflow, which is summarized in Fig. 1, began with a literature-mining phase, using the general PubMed search terms gonado* ANDLbetaT2, more specific terms such as LbetaT2 AND PACAP, or searches by authors’ names. Following a pathway construction step, the curators discussed the contents of their diagrams, carefully reexamining the literature if necessary. This process led to a unified diagram, the layout of which was then optimized. Upon web publication of the pathway, we expect to receive feedback from experts in the research community through the gonadotrope pathway wiki pages. We will keep updating the pathway based on the latest publications in the field in addition to experts’ suggestions.

Figure 1.

Figure 1

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Pathway construction workflow. A workflow diagram summarizes the main stages of pathway assembly from literature mining to pathway update. Boxes represent the pathway construction steps, which are connected by orange arrows. The red arrow indicates feedback to a previous construction step. In the initial phase of literature mining, more specific terms include: LbetaT2 AND PACAP, or searches by authors’ names.

The network diagram was initially created using CellDesigner (http://celldesigner.org/), a free process diagram editor for gene-regulatory and biochemical networks (14). This allows users to draw networks that are stored in Systems Biology Mark-up Language (SBML), a computer-readable format for representing models of biological processes (http://sbml.org/) (15). Furthermore, to enable real-time sharing of the GnRHR knowledgebase, we have used a tool previously developed in our laboratory, Biological Pathway Publisher (BioPP), which converts CellDesigner-SBML formatted pathways into a web-accessible format (16). Accordingly, the knowledgebase is deposited into a public repository endowed with a pathway navigator, which facilitates browsing of the uploaded pathway. Each species in the network is annotated with a complete list of interactions in which it participates, and PubMed references [PubMed Identifications (PMIDs)] supporting those interactions. Entities are also linked to National Center for Biotechnology Information (NCBI) Entrez Gene pages and to a public GnRH wiki, which is provided as a discussion forum for the community. The GnRH wiki pages are generated in an automated fashion by BioPP. Furthermore, users may download the GnRHR pathway diagram from the BioPP web site in an .xml format, which allows them to edit and/or expand it in CellDesigner, based upon their own experimental data or knowledge. The GnRHR-signaling pathway map is accessible at http://tsb.mssm.edu/pathwayPublisher/GnRHR_Pathway/GnRHR_Pathway_index.html.

As illustrated in Fig. 2, the map includes G protein-mediated signal transduction pathways initiated in response to GnRH binding to its receptor, ultimately leading to the synthesis and secretion of gonadotropins LH and FSH. These intracellular signaling events include protein kinase C (PKC) via the activation of phospholipase Cβ, calcium mobilization, and the protein kinase A (PKA)-dependent pathway. GnRHR stimulation also results in activation of phospholipase D and phospholipase A2, the latter leading to the formation of leukotrienes and prostaglandins. Conventional PKCs activate MAPK cascades, whereas some novel PKC isoforms can activate the PKA-dependent pathway. MAPK cascades include ERK1/2, c-Jun N-terminal kinases 1/2, and p38 MAPKs. From a physiological standpoint, rapid gonadotropin secretion is primarily attributed to calcium mobilization by inositol triphosphate (17), whereas activation of MAPKs is thought to be involved in differential gonadotropin expression (18,19).

Figure 2.

Figure 2

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Snapshot of the GnRHR-signaling pathway map. This map was created using CellDesigner version 4.0.1 and version 4.0.α (http://celldesigner.org/). A total of 187 species and 206 reactions were included. The main symbols are those implemented by CellDesigner version 4.0.1 and version 4.0.α. Interactions are color coded: black solid arrows, stimulatory reactions; red bar-headed lines, inhibitory reactions; black solid and white solid arrows with a bar, transport and trigger reactions, respectively; round-headed lines, catalysis reactions. The presence of a question mark or a dashed line denotes that, whether the reactions are direct or indirect, is unknown. Transcription reactions are represented by dashed and double-dotted lines, whereas translation reactions are symbolized by dashed and single-dotted lines. The following cellular compartments are illustrated on the diagram, as indicated: cytoplasm, endoplasmic reticulum, and nucleus. The size and color of each module were configured by us. Briefly, round angle green squares signify cell-signaling proteins, whereas green and yellow circles represent small molecules and ions, respectively; round angle blue squares symbolize transcription factors, and round angle purple squares strictly correspond to nuclear receptors; yellow/green rectangles designate genes, on which some regulatory regions or response elements are depicted as small white squares. The area of the signaling pathway map that is framed in black is shown in more detail in Fig. 3. The map can be best viewed on the web at http://tsb.mssm.edu/pathwayPublisher/GnRHR_Pathway/GnRHR_Pathway_index.html.

The current GnRHR-signaling network is based on the molecular interactions documented in more than 100 publications accessible from PubMed. It comprises 187 species or entities and 206 reactions or edges. A species is an entity that can participate in reactions. A reaction describes some transformation that can change the amount or state of one or more species. The database was limited to entities and transitions about which specific information existed in the literature. The following species participate in the GnRHR-signaling network: 119 proteins, three ions, 13 simple molecules, 16 RNAs, 16 genes, 10 complexes, one degraded entity, and seven phenotypes. Among the protein species, 16 are receptors, two are ion channels, and 34 are transcription factors, which are represented in blue or in light purple (nuclear receptors). The reactions can be classified into 101 state transitions (which include four catalyzes), 42 unknown transitions, five transport reactions, 21 inhibitions, nine unknown inhibitions, 16 transcriptions, and 12 translations.

Control of the GnRHR-signaling network

Activating processes are represented by black arrows, whereas inhibiting interactions are represented by red inhibition symbols. The biosynthesis and release of LH and FSH are regulated by hypothalamic factors, mainly GnRH, by gonadal steroids and peptides, as well as other hormones. The cell-signaling and molecular mechanisms that underlie the differential regulation of pituitary gonadotropins in various physiological situations, such as estrous cycle, childhood, and puberty, have been the subject of intense scrutiny and yet are still not fully understood. A number of modulators of the gonadotrope function are depicted on this pathway map. Steroid hormone feedback has been implicated in the regulation of gonadotropin gene expression. In fact, steroid hormone receptors are expressed in the gonadotropes (20,21,22). As illustrated here, estrogen, acting through the estrogen receptor, enhances GnRH-stimulated transcription of LHβ and α-subunit genes, by repressing the expression of the suppressive transcription factor Zeb1 (23). Androgen via its androgen receptor suppresses GnRH-stimulated LHβ transcription, whereas it activates FSHβ transcription (24,25,26,27). As shown in Fig. 3, whereas progesterone [PR (progesterone receptor)] and glucocorticoids [GR (glucocorticoid receptor)] directly activate FSHβ expression, they indirectly suppress LHβ gene expression (26,28).The circulating gonadal proteins, activin, inhibin, and follistatin are also key regulators of the gonadotrope function. These hormones are produced by the gonadotropes, suggesting some autocrine/paracrine effects on gonadotropin expression. It is shown here that activin stimulates the transcription of FSHβ, LHβ, and the GnRHR gene, and that activin action is blocked by follistatin and inhibin (29,30).

Figure 3.

Figure 3

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Web-accessible GnRHR pathway navigator. This selected part of the GnRHR-signaling pathway map notably displays the promoters of the gonadotropin β-subunit genes (FSHβ and LHβ subunits), their response elements, and transcription factors that bind to them and regulate gene transcription. Indirect regulators of gene transcription are also illustrated. Entities are clickable, such that corresponding annotations, namely the interactions in which those entities are involved, and the related PubMed references are displayed in the bottom left-hand frame. Additionally, other frames include an interaction list, a protein list, a gene list, and an RNA list. Hyperlinks to the relevant NCBI Entrez Gene page(s) and GnRH wiki pages are also provided. A zoom rectangle located in the upper left corner of the image facilitates navigation through the pathway (16).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important modulator of gonadotropin synthesis and secretion, acting both alone and in concert with GnRH (31). PACAP is not only secreted by hypothalamic neurons, but it is also expressed in the pituitary gonadotropes, suggesting its autocrine/paracrine role in the gonadotrope cells (32,33,34,35). Herein, we illustrate how PACAP, acting through its cell membrane receptor, activates the PKA pathway, thereby stimulating gonadotropin expression in cooperation with GnRH, as well as GnRHR gene expression. Expression of the PACAP gene itself is regulated by both PKC and PKA pathways.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that were found to be expressed in pituitary tumors and thus may represent a molecular target for treating patients with the disease (36). In LβT2 cells, PPARα and PPARγ down-regulate the transcription of the gonadotropin β-subunits as well as that of GnRHR (30).

Bone morphogenetic proteins, which belong to the TGF-β superfamily, play critical roles in the differentiation of pituitary gonadotropes (37). In contrast to PPARs, bone morphogenetic proteins activate FSHβ transcription (38,39).

Dopamine D2 receptor, which is highly expressed in the pituitary lactotropes, has been shown to be present in gonadotrope cells as well. Similarly, prolactin receptors are expressed in gonadotropes (40). Both dopamine and prolactin block GnRH-induced LHβ transcription (41). Moreover, dopamine acts as a negative regulator of the α-subunit gene transcription induced by the cAMP-dependent pathway (42).

The involvement of the epidermal growth factor receptor (EGFR) in the GnRHR-signaling network exemplifies the cross talk between GPCRs and receptor tyrosine kinases in gonadotrope cells. Studies revealed that members of the matrix metalloproteinase family are responsible for GnRH-mediated EGFR transactivation via activation of PKC, which subsequently results in rapid engagement of the ERK cascade (43,44).

Another example of cross talk is depicted on the network between the GnRHR- and Wnt-signaling pathways, wherein GnRH mediates the inactivation of glycogen synthase kinase 3, resulting in β-catenin stabilization and accumulation, T-cell factor activation, and subsequent up-regulation of Wnt target genes such as c-Jun (45). Furthermore, another study revealed that β-catenin is required for maximal activation of LHβ transcription in response to GnRH (46).

GnRH stimulates the induction of cyclooxygenase-2, a key enzyme involved in the synthesis of prostaglandins, which are autocrine regulators in many tissues (47). In the LβT2 cell line, prostaglandins have been shown to down-regulate GnRH-induced expression of GnRHR and LHβ (48).

GnRH increases the production of nitric oxide (NO) by NO synthase type I in LβT2 cells (49). In fact, chronic treatment with GnRH inhibits activin-induced expression of FSHβ via activation of NO synthase type I (50), which is consistent with the fact that chronic administration of GnRH inhibits FSHβ in vivo and in vitro (51,52).

Adiponectin, a factor secreted by the adipose tissue, is thought to be linked to reproduction, because a previous study demonstrated that female mice overexpressing adiponectin are infertile (53). In LβT2 cells, adiponectin binds to adiponectin receptors and attenuates LH secretion via the induction of AMP-activated protein kinase (54). This finding further supports the notion that adiponectin may be an important modulator of the gonadotrope function.

Graphic notations of the GnRHR-signaling map

The main symbols used to represent reactions and entities in the map are provided in the legend of Fig. 2, as well as on the web-accessible GnRHR-signaling map. Those symbols were obtained through CellDesigner version 4.0.1 and version 4.0.α and originally derive from the earlier proposals of Kitano et al. (15). They are also listed in Appendix 1 of the CellDesigner version 4.0 Startup Guide at http://celldesigner.org/documents/StartupGuide40.pdf.

Lists of entities, annotations, and links to wiki pages

The pathway navigator component of the BioPP suite allows users to browse the pathway and search for specific information. Lists of entities include hyperlinked lists of all proteins, genes, RNAs, and simple molecules/ions/phenotypes. As described above, annotations for entities include hyperlinked lists of interactions, which themselves provide relevant literature references in conjunction with their hyperlinked PMIDs. Entities may be associated with one or more GeneIDs, depending on the extent of experimentally based literature findings (Fig. 3). Finally, the rationale for associating the GnRHR-signaling pathway diagram with a mediawiki is to provide a community-based curation effort, allowing members of the scientific community to add comments or suggest new additions to the network. Hence, as new experimental data are being published, this editorial interface contributes to a perpetual improvement of the network.

Discussion

In this report, we present a comprehensive network map of the GnRHR signaling in the LβT2 gonadotrope cell line. Based on all the relevant articles published in the field, we manually curated a pathway map of GnRHR signaling in LβT2 cells. In addition to GnRH itself, regulators of the gonadotrope include gonadal and adrenal steroids, gonadal peptides, and hypothalamic neuropeptide PACAP. Steroid hormones are known to play a fundamental role in the control of GnRH expression in the hypothalamus, on the one hand, and in the regulation of expression of pituitary gonadotropins, on the other hand. Hence, under the influence of sex steroid feedback, transcription levels of the gonadotropin subunits vary during the estrous cycle in females (55,56,57). This network illustrates the intracellular cascades that are elicited by the interaction of GnRH and its receptor, as well as by other known regulators of gonadotrope function. The map is intended to be comprehensive and help researchers to unravel the signal transduction pathway and gene response mechanisms occurring in the pituitary gonadotrope. However, it is not necessarily exhaustive. We anticipate updating the map regularly using data drawn from newly published studies, as well as through exchanges with researchers whose area of expertise is the hypothalamic-pituitary-gonadal axis in general, and/or the pituitary gonadotrope cell in particular. Those exchanges will be made possible by the availability of the GnRH wiki, which will allow experts to suggest corrections or additions through their feedback and comments.

Building a thorough signaling network is challenging and thus may inevitably result in a visually dense and complex pathway diagram. For instance, the number of nodes and edges comprised in the signaling networks of the toll-like receptor [444 and 652, respectively; (58)] and of macrophage activation [295 and 272, respectively; (59)] are such that all interactions cannot be observed on the same page. This is in contrast with other existing GnRH-signaling networks, such as those of the KEGG PATHWAY database (http://www.genome.jp/kegg/pathway.html), and of Ingenuity Systems (a commercial knowledgebase; http://www.ingenuity.com/index.html). Our signaling network is considerably more detailed and referenced than its KEGG counterpart, because it notably depicts the effects of various hormonal regulators on the gonadotrope cell and numerous elements of the transcriptome. Although Ingenuity Systems offers a great diversity of annotations, including literature references from various biological models and many other database resources, it presents a fairly basic GnRH-signaling network comprised of only 50 entities; moreover, the Ingenuity network depicts a hypothetical GnRH-responsive cell rather than a true reflection of the gonadotrope cell: for instance, it displays the activation of transcription factor nuclear factor-κB by protein Gαi, a finding that was reported in human melanoma cells, but never in gonadotrope cells (60). Herein, we provide a web-published GnRHR-signaling diagram, which represents both an online resource and an opportunity for community-wide collaboration. Each node is clickable and links to a list of interactions in which it is involved in the LβT2 cell line; interactions themselves are supported by hyperlinked PMIDs; in the case of transcription factors, which typically bind to gene promoters, hyperlinked PMIDs are directly associated with those entities. Any entity that belongs to either a protein, RNA, or gene category is linked to a wiki page, which allows for contributions from experts in the field. Moreover, an .xml version of the pathway is available for download on the BioPP web site, allowing individual biologists to modify and expand it in CellDesigner in accordance with their own experimental observations. This .xml file (also available in the Supplemental data published on The Endocrine Society’s Journals Online web site http://mend.endojournals.org) includes the PMIDs supporting the interactions and the GeneIDs describing the entities. Finally, researchers who wish to generate a web-accessible version of their network diagrams are encouraged to do so by following the guidelines posted on the BioPP web site.

The concept of a knowledgebase that receives input from the research community is fairly novel. In fact, a new community-based platform termed “Payao” (http://www.payaologue.org) has just been developed by the Kitano laboratory for sharing pathway models. Payao provides a web-based interface for adding tags and comments to curated pathway models (61). However, whereas Payao assigns privileges to specific community members, the wiki scheme can record any changes made by contributors. We hope that our platform, as well as similar initiatives stemming from other groups, will contribute to the improvement of community-driven pathway enrichment (62).

To conserve space on the pathway map and for ease of readability, we intentionally omitted the following: 1) modification states of proteins, such as phosphorylation, acetylation, and ubiquitination, 2) ligands of nuclear receptors, e.g. steroid receptors. In some instances, a protein-protein interaction may be ambiguous due to conflicting literature results, in which case we typically choose to illustrate the interaction, which we believe is the most strongly supported by experimental data. Nevertheless, we include the contradictory paper(s) among the PubMed references. In fact, this type of scenario occurred with PACAP, which was demonstrated to have a stimulatory effect on glycoprotein hormone α-subunit (CGA) transcription in LβT2 cells (63), whereas another group failed to show any activation of the CGA promoter (64). The discrepancies in the results obtained from those two research teams might be due to a more than 300-bp difference in the length of the CGA gene promoter used in transfection studies, as well as distinct transfection methods and cell culture conditions.

We reckon that the LβT2 cell model offers a valuable representation of the GnRHR-signaling network and a reliable framework for integrating additional experimental data, as has occurred in other systems such as EGFR (65). We also recognize that the choice of a cellular model in a given research study may affect its experimental outcome; hence, Fujii et al. (66) formerly observed opposite results on FSHβ gene expression after treatment of primary pituitary cultures vs. clonal gonadotrope cell line LβT2 with either GnRH or PACAP: namely, FSHβ mRNAs were suppressed in GnRH- or PACAP-treated pituitary cultures and were increased in LβT2 cells; moreover, FSHβ gene transcription was stimulated in transiently transfected LβT2. Therefore, a cautious interpretation of experimental data based upon the selected model and an awareness of the limitations of this model are necessary. We would like to emphasize that the task of assembling a gonadotrope cell-signaling network is a work in progress, and that the support of the community in doing so is crucial. Hence, in the near future, we may envision to integrate data derived from 1) in vitro studies performed in cellular models other than the LβT2, such as other cell lines (αT3-1, etc.) and pituitary primary cultures, 2) in vivo experiments carried out in animals or transgenic rodents.

Materials and Methods

The GnRHR-signaling pathway map was created using CellDesigner version 4.0.1 and version 4.0.α (14), which provide graphic tools, pathway visualization, and navigation. The BioPP suite was employed to allow web publication of the pathway with an easily navigated user interface (16). Links to Entrez PubMed pages, Entrez Gene pages, and the appropriate pages of a public wiki-based discussion forum were supplied. An upgrade of BioPP was implemented in the context of the present work.

Supplementary Material

[Supplemental Data]
me.2009-0530_index.html (938B, html)

Acknowledgments

We thank Dr. Jeremy Seto for establishing the wiki.

Footnotes

This work was supported by National Institutes of Health Grant DK46943.

Disclosure Summary: M.Y.F., H.P., S.G.C., and G.N. have nothing to declare. S.C.S. is an inventor on US Patents 5,985,583 and 5,750,366 and has received royalties for these patents.

First Published Online June 30, 2010

Abbreviations: BioPP, Biological Pathway Publisher; CGA, glycoprotein hormone α-subunit; EGFR, epidermal growth factor; GnRHR, GnRH receptor; GPCR, G-protein-coupled receptor; NO, nitric oxide; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor; SBML, Systems Biology Mark-up Language.

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[Supplemental Data]
me.2009-0530_index.html (938B, html)
me.2009-0530_1.pdf (27.1KB, pdf)
me.2009-0530_ME-09-0530_GnRHR_Pathway.xml (651.4KB, xml)

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