General and Comparative Endocrinology xxx (2014) xxx–xxx
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Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species Christopher Tubbs a,⇑, Caitlin E. McDonough a, Rachel Felton a, Matthew R. Milnes b,⇑ a b
San Diego Zoo Institute for Conservation Research, 15600 San Pasqual Valley Road, Escondido, CA 92027, United States Mars Hill University, PO Box 6671, 100 Athletic Street, Mars Hill, NC 28754, United States
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Endangered species Hormone receptors In vitro approaches Endocrine disruption
a b s t r a c t Among the numerous societal benefits of comparative endocrinology is the application of our collective knowledge of hormone signaling towards the conservation of threatened and endangered species – conservation endocrinology. For several decades endocrinologists have used longitudinal hormone profiles to monitor reproductive status in a multitude of species. Knowledge of reproductive status among individuals has been used to assist in the management of captive and free-ranging populations. More recently, researchers have begun utilizing molecular and cell-based techniques to gain a more complete understanding of hormone signaling in wildlife species, and to identify potential causes of disrupted hormone signaling. In this review we examine various in vitro approaches we have used to compare estrogen receptor binding and activation by endogenous hormones and phytoestrogens in two species of rhinoceros; southern white and greater one-horned. We have found many of these techniques valuable and practical in species where access to research subjects and/or tissues is limited due to their conservation status. From cell-free, competitive binding assays to full-length receptor activation assays; each technique has strengths and weaknesses related to cost, sensitivity, complexity of the protocols, and relevance to in vivo signaling. We then present a novel approach, in which receptor activation assays are performed in primary cell lines derived from the species of interest, to minimize the artifacts of traditional heterologous expression systems. Finally, we speculate on the promise of next generation sequencing and transcriptome profiling as tools for characterizing hormone signaling in threatened and endangered species. Ó 2014 Published by Elsevier Inc.
1. Introduction Comparative endocrinology, defined here as the study of hormone signaling across a variety of taxa, provides numerous benefits to society. Most apparent among these benefits may be the insight into evolutionary history that the similarities and differences in hormone signaling mechanisms across major taxonomic groups provide. For instance, comparative studies of the protein sequences of nuclear steroid receptors has revealed numerous functional and phylogenetic relationships among extant vertebrates species (Thornton, 2001). In addition, these sequences have been used to reconstruct the ancestral steroid receptors giving rise to this broadly significant gene family and to examine the evolutionary forces driving receptor–ligand specificity (Eick et al., 2012; Thornton et al., 2003). Comparative endocrinology research also ⇑ Corresponding authors. Fax: +1 760 291 5428 (C. Tubbs). E-mail addresses: [email protected] (C. Tubbs), [email protected] (M.R. Milnes).
finds itself at the forefront of novel scientific discovery. One example is mPRa, the first membrane bound, non-classical steroid hormone receptor identified for any vertebrate species. First cloned from the ovary of the spotted seatrout (Cynoscion nebulosus), a common estuarine fish, mPRa is an intermediary in progestin induction of oocyte maturation (Zhu et al., 2003b). In other species, including humans, mPRa and related receptors appear to be involved in a wide array of physiological functions, many of which are likely of biomedical importance (Dressing et al., 2012; Thomas, 2012; Thomas and Pang, 2013; Zhu et al., 2003a). Perhaps not as widely recognized is the contribution that comparative endocrinology has made, and continues to make, towards the conservation of threatened or endangered species. For example, hormone concentrations are used to assess the current reproductive status of an individual based on previously characterized endocrine profiles for the reproductive cycle of that species. Determination of the reproductive status of a representative sample of a population can be especially useful for resource managers attempting to model changes in population size over time. Threats to
http://dx.doi.org/10.1016/j.ygcen.2014.02.013 0016-6480/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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long-term population viability can be more readily identified when the data are compared to endocrine data collected previously or from similar populations. Furthermore, a reliable method of assessing fecundity leads to more effective utilization of limited resources – particularly in intensively managed populations, such as those found in zoos, aquariums, and some parks and reserves. The utility of using hormone concentrations to assess and/or manage populations is well demonstrated in the African elephant (Loxodonta africana). The female African elephant reproductive cycle has been well characterized in terms of changing hormone concentrations over the course of the ovarian cycle and gestation (Hildebrandt et al., 2011). This knowledge is routinely applied in the form of serum, urine, or fecal progestagen monitoring at zoos housing mixed-sex herds to detect reproductive activity. Recently, Benavides Valades et al. (2012) utilized our collective understanding of elephant reproductive endocrinology to evaluate immunocontraception of free-ranging cows on a South African game reserve. The efficacy of a gonadotropin-releasing hormone (GnRH) vaccine was evaluated based on fecal progestagen concentrations as an indicator of luteal activity. Interestingly, the vaccine, which has been used successfully in a wide range of mammals, failed to induce anestrus in African elephants, further underscoring the need for more comparative studies. Historically, comparative endocrinologists have focused largely on associations between various hormone concentrations and physiological phenomena. This is especially true for those studying endangered species, for which biological samples are often limited to those that can be collected non-invasively, such as urine, feces or saliva. However, more recent work has begun to investigate other aspects of hormone signaling responsible for maintaining homeostasis and regulating essential functions of life – in particular, hormone receptors. Characterizing hormone receptors, elucidating the mechanisms regulating their expression, and understanding the cellular responses to hormone receptor–ligand interactions have shed new light on hormone-mediated processes. For instance, androgen-dependent territorial behavior in spotted antbirds (Hylophylax naevioides) persists year-round despite lower testosterone concentrations during the nonbreeding season. Elevated mRNA expression of androgen receptor (AR) and estrogen receptor a (ESR1) in the brain during the nonbreeding season suggests increased sensitivity to sex steroids plays a role in maintaining aggressive behavior in this species (Canoine et al., 2007). Although pioneering work on hormone binding sites in wildlife species was conducted in the 1970s (e.g., Licht and Midgley, 1976), only recently have the tools required to study hormone receptor–ligand interactions at the molecular level become widely accessible to the comparative and conservation-oriented endocrinologist. These tools include molecular cloning, recombinant protein expression, cell culture, and various measures of mRNA, protein, and/or metabolite expression. In this review we provide a survey of the application of molecular endocrinology aimed at increasing our understanding of hormone receptor signaling in wildlife species. In addition, we will present a few of the successes and potential shortcomings of various approaches that we have employed in our efforts to examine the role of dietary phytoestrogens in the poor reproductive success of captive southern white rhinoceros (SWR, Ceratotherium simum simum). 2. Molecular cloning and recombinant receptors Molecular cloning technology has revolutionized our ability to biochemically and functionally characterize hormone receptors in threatened and endangered species. Traditional methods of characterizing hormone receptors began by isolating and purifying receptors from relatively large quantities of tissue. The availability of the tissue required for such methods is generally limited in
species that are listed as threatened or endangered. On the other hand, small amounts of tissue expressing the mRNA of the receptor of interest can be used to isolate complete coding sequences and generate unlimited quantities of recombinant receptors. Small quantities of high quality tissues can be obtained through blood samples and tissue biopsies with minimal risk to the subject. The recombinant receptors generated from these samples can then be used to characterize ligand binding and receptor activation. This approach has been shown to be particularly useful in the arena of endocrine disruption, where inappropriate hormone signaling can be caused by the interaction of hormone receptors and exogenous ligands (Katsu et al., 2013; Naidoo et al., 2008; Tubbs et al., 2012). 2.1. Recombinant receptor binding assays Identifying potential ligands of a receptor and determining the affinity of that receptor for those ligands provides valuable insight into the receptor’s function. Competitive binding assays, in which labeled and unlabeled ligand compete for a limited quantity of recombinant receptor, can be used to partially assess the sensitivity of the receptor to various ligands relative to some standard, which is typically the putative endogenous ligand. This is particularly useful for screening exogenous compounds for species- and receptor-specific endocrine-disrupting capabilities (Rider et al., 2009). Competitive binding assays using recombinant receptors in cellfree systems were used to screen potential endocrine-disrupting chemicals for interactions with alligator and human ESR1 (Rider et al., 2010). Several xenoestrogens exhibited species-specific affinities for ESR1, with p,p0 -dicofol displacing 50% of the estradiol tracer from alligator ESR1 at 4 lM, an order of magnitude lower than the concentration required to do the same in human ESR1. The ability of an exogenous compound to inhibit the binding of a bona fide ligand to its receptor does not necessarily confer agonistic or antagonistic activity to that compound. However, this approach is useful for rapidly screening a large number of compounds or complex mixtures for potential endocrine-disrupting activity. If a compound is capable of inhibiting the binding of a known ligand, further investigation is required to determine if the compound stimulates or inhibits receptor-dependent activity at physiologically relevant concentrations. We have employed this approach with white rhinoceros and greater one-horned rhinoceros (Rhinoceros unicornis; GOHR) ESRs to quantify receptor binding by phytoestrogens and estrogenic substances in extracts of components of captive diets (i.e., commercial pellets and hays) and extracts of grasses predominantly consumed by these species in the wild (Tubbs et al., 2012) (Tubbs et al., unpublished obs.). 2.2. Recombinant receptor activation assays Recombinant receptor activation studies are an emerging tool for studying the comparative endocrinology of endangered species in vitro. In contrast to receptor binding assays, activation assays allow one to discern agonist or antagonist capability of potential ligands. Whereas binding assays are relatively inexpensive and cell-free ligand–receptor binding reaches equilibrium in hours, activation assays are dependent upon transcription and translation occurring in cultured cell lines, adding to both their time and cost. Nevertheless, receptor activation assays are able to detect speciesspecific differences in receptor function that are not detectable by binding assays alone. For example, when investigating phytoestrogen interactions with rhinoceros ESRs, we found no difference in binding of coumestrol or daidzein between SWR and GOHR receptors. However, activation of SWR ESR1 by daidzein and SWR ESR1 and estrogen receptor b (ESR2) for coumestrol was significantly higher than the respective GOHR ESRs (Tubbs et al., 2012). In
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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captivity many SWR and GOHR receive similar phytoestrogen rich diets. While SWR suffer from poor fertility and exhibit pathologies associated with exposure to estrogenic substances such as phytoestrogens, GOHR reproduce relatively well. Our receptor activation findings have therefore established a potential link between reproductive success and phytoestrogen sensitivity at the receptor-level, prompting institutions to work towards reducing or eliminating phytoestrogen sources from captive rhino diets. One potential caveat to performing receptor activation assays is that they measure transcription of a reporter enzyme (typically luciferase) under the control of a ‘canonical’ vertebrate hormone response element (HRE) specific for the receptor of interest. Whether these HREs are in fact present in the species of interest, or if species differences in receptor interactions with those HREs exist is often unclear (Kohno et al., 2008). Below are some considerations to make when addressing these potential issues regarding receptor activation assays. 2.2.1. Chimeric receptors – Gal4-fusion proteins One means of reducing species-specific differences in receptor– HRE interactions is to produce fusion proteins consisting of some or all of the steroid receptor of interest and a common transcription activator, such as the yeast GAL4 (Saccharomyces cerevisiae). Once activated by ligand, chimeras promote transcription of reporter enzymes through a GAL4 binding site upstream of the reporter gene. There are numerous advantages to this approach. First, since transcription is driven through the GAL4 promoter, fusion proteins can be created with any hormone receptor that functions as a transcription factor, such as members of the nuclear receptor super family, eliminating the need to identify receptor-specific response elements. Second, fusion proteins can be synthesized with partial receptor sequences and therefore do not require the cloning of full-length receptors. This approach was used to establish a common framework from which to screen orthologs of the pregnane X receptor (PXR, NR1I2) across more than a dozen species representing four vertebrate classes (Milnes et al., 2008). In that study, the fusion protein included all functional domains downstream from the DNA-binding domain of the PXR orthologs. Species-specific activation of PXR by xenobiotics was observed, and overall similarity in patterns of PXR activation reflected sequence similarity and evolutionary relationships, indicating that the results of toxicological screenings are most applicable across closely related species. However, some caution must be observed even among closely related species, as the magnitude of activation by a particular ligand frequently varied among species sharing 94% or greater amino acid identity in the ligand binding domain (LBD), such as human and non-human primates. Studies with turtle ESR1 have shown that inclusion of the LBD and the E/F domains into GAL4 fusion proteins is sufficient for ligand dependent gene transcription (Katsu et al., 2008a). However, as more of the full-length receptor is included in the construct, up to the N-terminal region of the protein, activation in response to ligand increases up to twofold, allowing for greater assay sensitivity. 2.2.2. Full-length receptors A concern that is always present when using an in vitro approach is whether information gained is representative of the physiology within the context of the whole organism. Although the use of GAL4-receptor fusion proteins circumvents some of the concerns regarding receptor–HRE interactions, they have the potential to introduce artifacts specific to the construct and assay protocol. When using fusion proteins with partial receptor sequences one must consider the potential consequences of not including all of the functional domains that may affect the overall signaling of that receptor. For example, GAL4-LBD constructs typically lack the A/B domain of that particular receptor, which plays
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an important role in transcription by interacting with co-regulatory proteins through the AF-1 activation function (Webb et al., 1998). An alternative approach to the use of hormone receptor fusion proteins is to employ activation assays using native full-length hormone receptors, which has been successful for numerous vertebrate species (Katsu et al., 2008a,b, 2010a,b, 2013; Tubbs et al., 2012). Even though uncertainty about the receptor–HRE interactions mentioned previously will be present, the fact that full-length receptor assays are conducted with native receptor proteins means they quite possibly provide the most accurate prediction of in vivo hormone signaling for a particular species. Nevertheless, fulllength receptor assay are not without shortcomings. First they require the cloning of entire open reading frames for the receptor of interest using techniques such as 50 and 30 rapid amplification of cDNA ends (RACE). Techniques like RACE are routinely performed when high quality RNA is obtainable. However, RNA obtained from endangered species can be sub-optimal for a multitude of reasons, the most common of which is the amount of time that passes between death and tissue collection (Milnes and Tubbs, unpublished obs.). Another potential drawback of full-length receptor activation assays is that fold increases in receptor activation is often less robust compared to assays using GAL4 or other fusion protein systems. Thus, total assay sensitivity can be reduced and difference in potency between different ligands can be more difficult to ascertain. In summary, these in vitro approaches are often the best, if not only, option when studying endocrinology of endangered or threatened species at the receptor level. No system is perfect and therefore care should be taken to consider the advantages and disadvantages of each as they relate to particular project in mind. Regardless, as these powerful techniques become more commonplace in the field of conservation research, they can be applied to the study of comparative endocrinology of endangered species to address questions with new and exciting depth. 2.3. Primary cell cultures from endangered species Proper steroid hormone signaling relies on the coordination of receptors, numerous co-activators, co-repressors and components of the transcriptional machinery such as RNA polymerase to associate with promoter regions and regulate the transcription of target genes. The integrity of these transcriptional complexes is maintained by protein–protein interactions, which have profound impacts on the efficacy of transcription and subsequent physiological outcomes (Malovannaya et al., 2011; Tsai and O’Malley, 1994). In heterologous expression systems, like those described above, receptors from one species are activated in the presence of the co-regulators and transcriptional machinery endogenously expressed in the cells from another species. It is therefore possible that transcriptional responses to ligands observed in vitro are not reflective of those that occur in vivo, particularly when transfected receptors and host cell lines are from more distantly related species. Recently, fibroblasts from endangered species have received considerable attention for their potential application as tools in the field of conservation genomics (Ben-Nun et al., 2011). Grown from small biopsies of tissue collected during veterinary procedures, field immobilizations or necropsy, fibroblasts represent a unique source of biomaterial from endangered species that can be collected in a minimally invasive manner and cryopreserved (Wong et al., 2012). We have also explored fibroblasts, isolated from skin biopsies of rhinos as described previously (Houck et al., 1994), as in vitro models of hormone signaling and environmental toxicology. RT-PCR performed using RNA extracted from skin-derived fibroblast lines of 4 individual rhinoceros (2 SWR and 2 GOHR) reveals detectable expression of steroid hormone receptor,
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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A
B
Fig. 1. Expression of endocrine-related genes in southern white rhinoceros (SWR) and greater one-horned rhinoceros (GOHR) skin-derived fibroblasts. RT-PCR analysis of select steroid hormone receptors (ESR1 = estrogen receptor alpha; PR = progesterone receptor; AR = androgen receptor), co-regulators (SRC: steroid receptor co-activators 1 and 3; NCOR1: nuclear receptor co-repressor 1; p300 = acetyltransferase p300) and estrogen responsive genes (c-fos = proto-oncogene c-fos; IGF1: insulin-like growth factor 1) in 4 cell lines from 4 individuals.
co-activator, co-repressor and ESR-regulated genes (Fig. 1). Expression levels of the different genes varied across cell-lines, suggesting that each cell line possess a unique transcriptional profile. We have also assessed the functionality of fibroblasts in receptor activation studies by comparing phytoestrogen activation of ESR2 in fibroblasts to ESR2 activation HEK293 cells. To do this, SWR fibroblasts were transfected in a manner identical to previous experiments with HEK cells with a plasmid containing SWR ESR2 (SWR ESR-pcDNA3.1(+)), a luciferase reporter plasmid (pGL23XERE; Hall and McDonnell, 1999) and b-galactosidase plasmid (pCMV-b-Gal) (Tubbs et al., 2012). Overall, the two cell lines perform comparably in activation assays with similar levels of mRNA expression of the transfected ESR2 plasmid and fold activation by 1 nM E2 ranging from 20 to 40-fold in HEK cells and 18 to 50-fold in fibroblasts (Tubbs, unpublished obs.). In HEK cells, SWR ESR2s show higher maximal activation than GOHR ESR2s in response to the phytoestrogens coumestrol and daidzein (Tubbs et al., 2012). There were no differences in receptor activation in response to genistein. In SWR, similar species differences in response to coumestrol and daidzein are observed (Fig. 2A and B). In contrast to HEK cells, however, genistein is a more potent activator of SWR ESR2 than GOHR ESR2 in the SWR fibroblast cell line expressing rhino-specific steroid hormone co-regulators (Fig. 2C). These data support our previous studies showing an overall higher sensitivity to phytoestrogens for SWR ESRs compared to GOHR ESRs. Moreover, they suggest that species-specific differences in sensitivity to phytoestrogens in vivo may be different than those detected in our in vitro heterologous expression system used previously. 3. Future directions Within recent years, the decreased costs and increased precision of next generation sequencing (NGS) technologies has led to
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Fig. 2. Activation of southern white rhinoceros (SWR) and greater one-horned rhinoceros (GOHR) estrogen receptors beta (ESR2) transfected into SWR fibroblasts. In previous studies performed in HEK293 cells only significant differences in maximal activation by coumestrol and daidzein were observed (Tubbs et al., 2012). However, in SWR fibroblasts genistein was also a more potent activator of SWR ESR2 compared to GOHR ESR2 (⁄p < 0.05 as determined using a Student’s t-test).
a dramatic increase in the number of species whose genomes have been sequenced. This trend seems only likely to continue with large-scale genome sequencing efforts like Genome 10K, which seeks to obtain whole genome sequences for 10,000 individual species (Genome, 2009). Such efforts have already led to exciting discoveries, and have provided novel insight into the evolutionary history of vertebrates. For example, whole genome analysis of the African coelacanth (Latimeria chalumnae) has recently challenged our long-standing belief that this species is the closest living tetrapod relative (Amemiya et al., 2013). NGS has already contributed greatly to the advancement of the field of conservation genomics (see Steiner et al., 2013 for recent review). The primary use of NGS within this field typically involves the identification of genome-wide microsatellite loci or single nucleotide polymorphisms (SNPs) within individuals of a population. This
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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information can then be practically applied to the management of both wild and captive populations of endangered species to address potential issues dealing with gene flow between groups, adaptive genetic variation leading to increased fitness, inbreeding depression and hybridization events (Steiner et al., 2013). However, despite the relative ease in generating whole-genome data for a particular species, post-sequencing analysis still presents numerous challenges, particularly for non-model species. The largest obstacle is the lack of well-suited, annotated reference genomes to which newly generated sequence can be compared, thus often requiring that sequences be assembled de novo. Nevertheless, as de novo assembly pipelines become more refined and more species genome sequences become available, these obstacles should be easier to overcome. In addition to aiding the field of conservation genomics, NGS also has the potential to be a valuable tool in studying the comparative endocrinology of endangered species. For example, performing 30 and 50 RACE is a common strategy for identifying and cloning novel full-length receptor coding sequences. Although a standard practice, 50 RACE in particular can be troublesome, requiring high quality RNA and limiting researchers to the identification of one receptor at a time. An alternative approach is to perform transcriptome profiling using RNA-seq on a tissue, or cell-line, of interest that would permit the simultaneous identification of coding sequences of multiple receptors, or any expressed gene of interest. Moreover, this approach would permit the relative quantification of different receptor transcripts as well as identify receptor splice variants that may be overlooked or undetectable using traditional RACE methods. The finding that fibroblasts express many endocrine related genes (Fig. 1) presents another intriguing possible application of NGS that may reduce the limitations that arise when conducting in vitro studies in endangered species. Specifically, cell lines that express endogenous steroid hormone receptors, such as the SWR1 line expressing ESR1, shown in Fig. 1, could potentially serve as a system that is more reflective of in vivo conditions. Treatment of such lines with ligands followed by RNA-seq would allow for the assessment of relative ligand potency in cells that are not overexpressing the steroid hormone receptor of interest, that express many of the species-specific co-activators and that are altering the rate of transcription of target genes under the control of native promoters instead of reporter enzyme downstream of a synthetic HRE. Moreover, techniques such as ChiP-seq, which have proven valuable in studying the endocrine regulation of reproductive tissues in model species (Hewitt et al., 2012; Rubel et al., 2012), could be applied to fibroblasts of endangered species to provide genome wide profiles of hormone receptor binding sites. Whether the endocrine responses of fibroblasts reflect in vivo responses, particularly of reproductive tissues is unclear, but we are currently investigating this question in our lab using SWR fibroblasts.
4. Concluding remarks Here we present a case for the use of in vitro methods in studying the comparative endocrinology of threatened or endangered species. We believe these approaches are valuable for two primary reasons. First, they avoid many of the challenges associated with studying species for which in vivo or invasive studies are difficult or impossible to conduct, particularly when studying endocrine disruption. Second, they allow the conservation-oriented endocrinologist to work at the interface of basic and applied science by exploring hormone signaling in unique species at new mechanistic depths, while addressing specific issues of conservation concern and generating knowledge that can inform the management of both wild and captive populations of endangered species. As more sophisticated tools and techniques become available, the creative
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application of primarily in vitro studies to conservation-specific problems seems likely to increase, bringing with it novel and exciting discoveries while advancing the field of comparative endocrinology as a whole.
Acknowledgments We are especially grateful to Julie Fronczek, Marlys Houck and the Genetics Division of the San Diego Zoo Institute for Conservation Research for assistance with fibroblast cultures and Dr. Barbara Durrant for reviewing the manuscript. This work was supported in part by a fellowship awarded to C.M. by the J.W. Sefton Foundation.
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Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species Christopher Tubbs a,⇑, Caitlin E. McDonough a, Rachel Felton a, Matthew R. Milnes b,⇑ a b
San Diego Zoo Institute for Conservation Research, 15600 San Pasqual Valley Road, Escondido, CA 92027, United States Mars Hill University, PO Box 6671, 100 Athletic Street, Mars Hill, NC 28754, United States
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Endangered species Hormone receptors In vitro approaches Endocrine disruption
a b s t r a c t Among the numerous societal benefits of comparative endocrinology is the application of our collective knowledge of hormone signaling towards the conservation of threatened and endangered species – conservation endocrinology. For several decades endocrinologists have used longitudinal hormone profiles to monitor reproductive status in a multitude of species. Knowledge of reproductive status among individuals has been used to assist in the management of captive and free-ranging populations. More recently, researchers have begun utilizing molecular and cell-based techniques to gain a more complete understanding of hormone signaling in wildlife species, and to identify potential causes of disrupted hormone signaling. In this review we examine various in vitro approaches we have used to compare estrogen receptor binding and activation by endogenous hormones and phytoestrogens in two species of rhinoceros; southern white and greater one-horned. We have found many of these techniques valuable and practical in species where access to research subjects and/or tissues is limited due to their conservation status. From cell-free, competitive binding assays to full-length receptor activation assays; each technique has strengths and weaknesses related to cost, sensitivity, complexity of the protocols, and relevance to in vivo signaling. We then present a novel approach, in which receptor activation assays are performed in primary cell lines derived from the species of interest, to minimize the artifacts of traditional heterologous expression systems. Finally, we speculate on the promise of next generation sequencing and transcriptome profiling as tools for characterizing hormone signaling in threatened and endangered species. Ó 2014 Published by Elsevier Inc.
1. Introduction Comparative endocrinology, defined here as the study of hormone signaling across a variety of taxa, provides numerous benefits to society. Most apparent among these benefits may be the insight into evolutionary history that the similarities and differences in hormone signaling mechanisms across major taxonomic groups provide. For instance, comparative studies of the protein sequences of nuclear steroid receptors has revealed numerous functional and phylogenetic relationships among extant vertebrates species (Thornton, 2001). In addition, these sequences have been used to reconstruct the ancestral steroid receptors giving rise to this broadly significant gene family and to examine the evolutionary forces driving receptor–ligand specificity (Eick et al., 2012; Thornton et al., 2003). Comparative endocrinology research also ⇑ Corresponding authors. Fax: +1 760 291 5428 (C. Tubbs). E-mail addresses: [email protected] (C. Tubbs), [email protected] (M.R. Milnes).
finds itself at the forefront of novel scientific discovery. One example is mPRa, the first membrane bound, non-classical steroid hormone receptor identified for any vertebrate species. First cloned from the ovary of the spotted seatrout (Cynoscion nebulosus), a common estuarine fish, mPRa is an intermediary in progestin induction of oocyte maturation (Zhu et al., 2003b). In other species, including humans, mPRa and related receptors appear to be involved in a wide array of physiological functions, many of which are likely of biomedical importance (Dressing et al., 2012; Thomas, 2012; Thomas and Pang, 2013; Zhu et al., 2003a). Perhaps not as widely recognized is the contribution that comparative endocrinology has made, and continues to make, towards the conservation of threatened or endangered species. For example, hormone concentrations are used to assess the current reproductive status of an individual based on previously characterized endocrine profiles for the reproductive cycle of that species. Determination of the reproductive status of a representative sample of a population can be especially useful for resource managers attempting to model changes in population size over time. Threats to
http://dx.doi.org/10.1016/j.ygcen.2014.02.013 0016-6480/Ó 2014 Published by Elsevier Inc.
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long-term population viability can be more readily identified when the data are compared to endocrine data collected previously or from similar populations. Furthermore, a reliable method of assessing fecundity leads to more effective utilization of limited resources – particularly in intensively managed populations, such as those found in zoos, aquariums, and some parks and reserves. The utility of using hormone concentrations to assess and/or manage populations is well demonstrated in the African elephant (Loxodonta africana). The female African elephant reproductive cycle has been well characterized in terms of changing hormone concentrations over the course of the ovarian cycle and gestation (Hildebrandt et al., 2011). This knowledge is routinely applied in the form of serum, urine, or fecal progestagen monitoring at zoos housing mixed-sex herds to detect reproductive activity. Recently, Benavides Valades et al. (2012) utilized our collective understanding of elephant reproductive endocrinology to evaluate immunocontraception of free-ranging cows on a South African game reserve. The efficacy of a gonadotropin-releasing hormone (GnRH) vaccine was evaluated based on fecal progestagen concentrations as an indicator of luteal activity. Interestingly, the vaccine, which has been used successfully in a wide range of mammals, failed to induce anestrus in African elephants, further underscoring the need for more comparative studies. Historically, comparative endocrinologists have focused largely on associations between various hormone concentrations and physiological phenomena. This is especially true for those studying endangered species, for which biological samples are often limited to those that can be collected non-invasively, such as urine, feces or saliva. However, more recent work has begun to investigate other aspects of hormone signaling responsible for maintaining homeostasis and regulating essential functions of life – in particular, hormone receptors. Characterizing hormone receptors, elucidating the mechanisms regulating their expression, and understanding the cellular responses to hormone receptor–ligand interactions have shed new light on hormone-mediated processes. For instance, androgen-dependent territorial behavior in spotted antbirds (Hylophylax naevioides) persists year-round despite lower testosterone concentrations during the nonbreeding season. Elevated mRNA expression of androgen receptor (AR) and estrogen receptor a (ESR1) in the brain during the nonbreeding season suggests increased sensitivity to sex steroids plays a role in maintaining aggressive behavior in this species (Canoine et al., 2007). Although pioneering work on hormone binding sites in wildlife species was conducted in the 1970s (e.g., Licht and Midgley, 1976), only recently have the tools required to study hormone receptor–ligand interactions at the molecular level become widely accessible to the comparative and conservation-oriented endocrinologist. These tools include molecular cloning, recombinant protein expression, cell culture, and various measures of mRNA, protein, and/or metabolite expression. In this review we provide a survey of the application of molecular endocrinology aimed at increasing our understanding of hormone receptor signaling in wildlife species. In addition, we will present a few of the successes and potential shortcomings of various approaches that we have employed in our efforts to examine the role of dietary phytoestrogens in the poor reproductive success of captive southern white rhinoceros (SWR, Ceratotherium simum simum). 2. Molecular cloning and recombinant receptors Molecular cloning technology has revolutionized our ability to biochemically and functionally characterize hormone receptors in threatened and endangered species. Traditional methods of characterizing hormone receptors began by isolating and purifying receptors from relatively large quantities of tissue. The availability of the tissue required for such methods is generally limited in
species that are listed as threatened or endangered. On the other hand, small amounts of tissue expressing the mRNA of the receptor of interest can be used to isolate complete coding sequences and generate unlimited quantities of recombinant receptors. Small quantities of high quality tissues can be obtained through blood samples and tissue biopsies with minimal risk to the subject. The recombinant receptors generated from these samples can then be used to characterize ligand binding and receptor activation. This approach has been shown to be particularly useful in the arena of endocrine disruption, where inappropriate hormone signaling can be caused by the interaction of hormone receptors and exogenous ligands (Katsu et al., 2013; Naidoo et al., 2008; Tubbs et al., 2012). 2.1. Recombinant receptor binding assays Identifying potential ligands of a receptor and determining the affinity of that receptor for those ligands provides valuable insight into the receptor’s function. Competitive binding assays, in which labeled and unlabeled ligand compete for a limited quantity of recombinant receptor, can be used to partially assess the sensitivity of the receptor to various ligands relative to some standard, which is typically the putative endogenous ligand. This is particularly useful for screening exogenous compounds for species- and receptor-specific endocrine-disrupting capabilities (Rider et al., 2009). Competitive binding assays using recombinant receptors in cellfree systems were used to screen potential endocrine-disrupting chemicals for interactions with alligator and human ESR1 (Rider et al., 2010). Several xenoestrogens exhibited species-specific affinities for ESR1, with p,p0 -dicofol displacing 50% of the estradiol tracer from alligator ESR1 at 4 lM, an order of magnitude lower than the concentration required to do the same in human ESR1. The ability of an exogenous compound to inhibit the binding of a bona fide ligand to its receptor does not necessarily confer agonistic or antagonistic activity to that compound. However, this approach is useful for rapidly screening a large number of compounds or complex mixtures for potential endocrine-disrupting activity. If a compound is capable of inhibiting the binding of a known ligand, further investigation is required to determine if the compound stimulates or inhibits receptor-dependent activity at physiologically relevant concentrations. We have employed this approach with white rhinoceros and greater one-horned rhinoceros (Rhinoceros unicornis; GOHR) ESRs to quantify receptor binding by phytoestrogens and estrogenic substances in extracts of components of captive diets (i.e., commercial pellets and hays) and extracts of grasses predominantly consumed by these species in the wild (Tubbs et al., 2012) (Tubbs et al., unpublished obs.). 2.2. Recombinant receptor activation assays Recombinant receptor activation studies are an emerging tool for studying the comparative endocrinology of endangered species in vitro. In contrast to receptor binding assays, activation assays allow one to discern agonist or antagonist capability of potential ligands. Whereas binding assays are relatively inexpensive and cell-free ligand–receptor binding reaches equilibrium in hours, activation assays are dependent upon transcription and translation occurring in cultured cell lines, adding to both their time and cost. Nevertheless, receptor activation assays are able to detect speciesspecific differences in receptor function that are not detectable by binding assays alone. For example, when investigating phytoestrogen interactions with rhinoceros ESRs, we found no difference in binding of coumestrol or daidzein between SWR and GOHR receptors. However, activation of SWR ESR1 by daidzein and SWR ESR1 and estrogen receptor b (ESR2) for coumestrol was significantly higher than the respective GOHR ESRs (Tubbs et al., 2012). In
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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captivity many SWR and GOHR receive similar phytoestrogen rich diets. While SWR suffer from poor fertility and exhibit pathologies associated with exposure to estrogenic substances such as phytoestrogens, GOHR reproduce relatively well. Our receptor activation findings have therefore established a potential link between reproductive success and phytoestrogen sensitivity at the receptor-level, prompting institutions to work towards reducing or eliminating phytoestrogen sources from captive rhino diets. One potential caveat to performing receptor activation assays is that they measure transcription of a reporter enzyme (typically luciferase) under the control of a ‘canonical’ vertebrate hormone response element (HRE) specific for the receptor of interest. Whether these HREs are in fact present in the species of interest, or if species differences in receptor interactions with those HREs exist is often unclear (Kohno et al., 2008). Below are some considerations to make when addressing these potential issues regarding receptor activation assays. 2.2.1. Chimeric receptors – Gal4-fusion proteins One means of reducing species-specific differences in receptor– HRE interactions is to produce fusion proteins consisting of some or all of the steroid receptor of interest and a common transcription activator, such as the yeast GAL4 (Saccharomyces cerevisiae). Once activated by ligand, chimeras promote transcription of reporter enzymes through a GAL4 binding site upstream of the reporter gene. There are numerous advantages to this approach. First, since transcription is driven through the GAL4 promoter, fusion proteins can be created with any hormone receptor that functions as a transcription factor, such as members of the nuclear receptor super family, eliminating the need to identify receptor-specific response elements. Second, fusion proteins can be synthesized with partial receptor sequences and therefore do not require the cloning of full-length receptors. This approach was used to establish a common framework from which to screen orthologs of the pregnane X receptor (PXR, NR1I2) across more than a dozen species representing four vertebrate classes (Milnes et al., 2008). In that study, the fusion protein included all functional domains downstream from the DNA-binding domain of the PXR orthologs. Species-specific activation of PXR by xenobiotics was observed, and overall similarity in patterns of PXR activation reflected sequence similarity and evolutionary relationships, indicating that the results of toxicological screenings are most applicable across closely related species. However, some caution must be observed even among closely related species, as the magnitude of activation by a particular ligand frequently varied among species sharing 94% or greater amino acid identity in the ligand binding domain (LBD), such as human and non-human primates. Studies with turtle ESR1 have shown that inclusion of the LBD and the E/F domains into GAL4 fusion proteins is sufficient for ligand dependent gene transcription (Katsu et al., 2008a). However, as more of the full-length receptor is included in the construct, up to the N-terminal region of the protein, activation in response to ligand increases up to twofold, allowing for greater assay sensitivity. 2.2.2. Full-length receptors A concern that is always present when using an in vitro approach is whether information gained is representative of the physiology within the context of the whole organism. Although the use of GAL4-receptor fusion proteins circumvents some of the concerns regarding receptor–HRE interactions, they have the potential to introduce artifacts specific to the construct and assay protocol. When using fusion proteins with partial receptor sequences one must consider the potential consequences of not including all of the functional domains that may affect the overall signaling of that receptor. For example, GAL4-LBD constructs typically lack the A/B domain of that particular receptor, which plays
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an important role in transcription by interacting with co-regulatory proteins through the AF-1 activation function (Webb et al., 1998). An alternative approach to the use of hormone receptor fusion proteins is to employ activation assays using native full-length hormone receptors, which has been successful for numerous vertebrate species (Katsu et al., 2008a,b, 2010a,b, 2013; Tubbs et al., 2012). Even though uncertainty about the receptor–HRE interactions mentioned previously will be present, the fact that full-length receptor assays are conducted with native receptor proteins means they quite possibly provide the most accurate prediction of in vivo hormone signaling for a particular species. Nevertheless, fulllength receptor assay are not without shortcomings. First they require the cloning of entire open reading frames for the receptor of interest using techniques such as 50 and 30 rapid amplification of cDNA ends (RACE). Techniques like RACE are routinely performed when high quality RNA is obtainable. However, RNA obtained from endangered species can be sub-optimal for a multitude of reasons, the most common of which is the amount of time that passes between death and tissue collection (Milnes and Tubbs, unpublished obs.). Another potential drawback of full-length receptor activation assays is that fold increases in receptor activation is often less robust compared to assays using GAL4 or other fusion protein systems. Thus, total assay sensitivity can be reduced and difference in potency between different ligands can be more difficult to ascertain. In summary, these in vitro approaches are often the best, if not only, option when studying endocrinology of endangered or threatened species at the receptor level. No system is perfect and therefore care should be taken to consider the advantages and disadvantages of each as they relate to particular project in mind. Regardless, as these powerful techniques become more commonplace in the field of conservation research, they can be applied to the study of comparative endocrinology of endangered species to address questions with new and exciting depth. 2.3. Primary cell cultures from endangered species Proper steroid hormone signaling relies on the coordination of receptors, numerous co-activators, co-repressors and components of the transcriptional machinery such as RNA polymerase to associate with promoter regions and regulate the transcription of target genes. The integrity of these transcriptional complexes is maintained by protein–protein interactions, which have profound impacts on the efficacy of transcription and subsequent physiological outcomes (Malovannaya et al., 2011; Tsai and O’Malley, 1994). In heterologous expression systems, like those described above, receptors from one species are activated in the presence of the co-regulators and transcriptional machinery endogenously expressed in the cells from another species. It is therefore possible that transcriptional responses to ligands observed in vitro are not reflective of those that occur in vivo, particularly when transfected receptors and host cell lines are from more distantly related species. Recently, fibroblasts from endangered species have received considerable attention for their potential application as tools in the field of conservation genomics (Ben-Nun et al., 2011). Grown from small biopsies of tissue collected during veterinary procedures, field immobilizations or necropsy, fibroblasts represent a unique source of biomaterial from endangered species that can be collected in a minimally invasive manner and cryopreserved (Wong et al., 2012). We have also explored fibroblasts, isolated from skin biopsies of rhinos as described previously (Houck et al., 1994), as in vitro models of hormone signaling and environmental toxicology. RT-PCR performed using RNA extracted from skin-derived fibroblast lines of 4 individual rhinoceros (2 SWR and 2 GOHR) reveals detectable expression of steroid hormone receptor,
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A
B
Fig. 1. Expression of endocrine-related genes in southern white rhinoceros (SWR) and greater one-horned rhinoceros (GOHR) skin-derived fibroblasts. RT-PCR analysis of select steroid hormone receptors (ESR1 = estrogen receptor alpha; PR = progesterone receptor; AR = androgen receptor), co-regulators (SRC: steroid receptor co-activators 1 and 3; NCOR1: nuclear receptor co-repressor 1; p300 = acetyltransferase p300) and estrogen responsive genes (c-fos = proto-oncogene c-fos; IGF1: insulin-like growth factor 1) in 4 cell lines from 4 individuals.
co-activator, co-repressor and ESR-regulated genes (Fig. 1). Expression levels of the different genes varied across cell-lines, suggesting that each cell line possess a unique transcriptional profile. We have also assessed the functionality of fibroblasts in receptor activation studies by comparing phytoestrogen activation of ESR2 in fibroblasts to ESR2 activation HEK293 cells. To do this, SWR fibroblasts were transfected in a manner identical to previous experiments with HEK cells with a plasmid containing SWR ESR2 (SWR ESR-pcDNA3.1(+)), a luciferase reporter plasmid (pGL23XERE; Hall and McDonnell, 1999) and b-galactosidase plasmid (pCMV-b-Gal) (Tubbs et al., 2012). Overall, the two cell lines perform comparably in activation assays with similar levels of mRNA expression of the transfected ESR2 plasmid and fold activation by 1 nM E2 ranging from 20 to 40-fold in HEK cells and 18 to 50-fold in fibroblasts (Tubbs, unpublished obs.). In HEK cells, SWR ESR2s show higher maximal activation than GOHR ESR2s in response to the phytoestrogens coumestrol and daidzein (Tubbs et al., 2012). There were no differences in receptor activation in response to genistein. In SWR, similar species differences in response to coumestrol and daidzein are observed (Fig. 2A and B). In contrast to HEK cells, however, genistein is a more potent activator of SWR ESR2 than GOHR ESR2 in the SWR fibroblast cell line expressing rhino-specific steroid hormone co-regulators (Fig. 2C). These data support our previous studies showing an overall higher sensitivity to phytoestrogens for SWR ESRs compared to GOHR ESRs. Moreover, they suggest that species-specific differences in sensitivity to phytoestrogens in vivo may be different than those detected in our in vitro heterologous expression system used previously. 3. Future directions Within recent years, the decreased costs and increased precision of next generation sequencing (NGS) technologies has led to
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Fig. 2. Activation of southern white rhinoceros (SWR) and greater one-horned rhinoceros (GOHR) estrogen receptors beta (ESR2) transfected into SWR fibroblasts. In previous studies performed in HEK293 cells only significant differences in maximal activation by coumestrol and daidzein were observed (Tubbs et al., 2012). However, in SWR fibroblasts genistein was also a more potent activator of SWR ESR2 compared to GOHR ESR2 (⁄p < 0.05 as determined using a Student’s t-test).
a dramatic increase in the number of species whose genomes have been sequenced. This trend seems only likely to continue with large-scale genome sequencing efforts like Genome 10K, which seeks to obtain whole genome sequences for 10,000 individual species (Genome, 2009). Such efforts have already led to exciting discoveries, and have provided novel insight into the evolutionary history of vertebrates. For example, whole genome analysis of the African coelacanth (Latimeria chalumnae) has recently challenged our long-standing belief that this species is the closest living tetrapod relative (Amemiya et al., 2013). NGS has already contributed greatly to the advancement of the field of conservation genomics (see Steiner et al., 2013 for recent review). The primary use of NGS within this field typically involves the identification of genome-wide microsatellite loci or single nucleotide polymorphisms (SNPs) within individuals of a population. This
Please cite this article in press as: Tubbs, C., et al. Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.02.013
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information can then be practically applied to the management of both wild and captive populations of endangered species to address potential issues dealing with gene flow between groups, adaptive genetic variation leading to increased fitness, inbreeding depression and hybridization events (Steiner et al., 2013). However, despite the relative ease in generating whole-genome data for a particular species, post-sequencing analysis still presents numerous challenges, particularly for non-model species. The largest obstacle is the lack of well-suited, annotated reference genomes to which newly generated sequence can be compared, thus often requiring that sequences be assembled de novo. Nevertheless, as de novo assembly pipelines become more refined and more species genome sequences become available, these obstacles should be easier to overcome. In addition to aiding the field of conservation genomics, NGS also has the potential to be a valuable tool in studying the comparative endocrinology of endangered species. For example, performing 30 and 50 RACE is a common strategy for identifying and cloning novel full-length receptor coding sequences. Although a standard practice, 50 RACE in particular can be troublesome, requiring high quality RNA and limiting researchers to the identification of one receptor at a time. An alternative approach is to perform transcriptome profiling using RNA-seq on a tissue, or cell-line, of interest that would permit the simultaneous identification of coding sequences of multiple receptors, or any expressed gene of interest. Moreover, this approach would permit the relative quantification of different receptor transcripts as well as identify receptor splice variants that may be overlooked or undetectable using traditional RACE methods. The finding that fibroblasts express many endocrine related genes (Fig. 1) presents another intriguing possible application of NGS that may reduce the limitations that arise when conducting in vitro studies in endangered species. Specifically, cell lines that express endogenous steroid hormone receptors, such as the SWR1 line expressing ESR1, shown in Fig. 1, could potentially serve as a system that is more reflective of in vivo conditions. Treatment of such lines with ligands followed by RNA-seq would allow for the assessment of relative ligand potency in cells that are not overexpressing the steroid hormone receptor of interest, that express many of the species-specific co-activators and that are altering the rate of transcription of target genes under the control of native promoters instead of reporter enzyme downstream of a synthetic HRE. Moreover, techniques such as ChiP-seq, which have proven valuable in studying the endocrine regulation of reproductive tissues in model species (Hewitt et al., 2012; Rubel et al., 2012), could be applied to fibroblasts of endangered species to provide genome wide profiles of hormone receptor binding sites. Whether the endocrine responses of fibroblasts reflect in vivo responses, particularly of reproductive tissues is unclear, but we are currently investigating this question in our lab using SWR fibroblasts.
4. Concluding remarks Here we present a case for the use of in vitro methods in studying the comparative endocrinology of threatened or endangered species. We believe these approaches are valuable for two primary reasons. First, they avoid many of the challenges associated with studying species for which in vivo or invasive studies are difficult or impossible to conduct, particularly when studying endocrine disruption. Second, they allow the conservation-oriented endocrinologist to work at the interface of basic and applied science by exploring hormone signaling in unique species at new mechanistic depths, while addressing specific issues of conservation concern and generating knowledge that can inform the management of both wild and captive populations of endangered species. As more sophisticated tools and techniques become available, the creative
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application of primarily in vitro studies to conservation-specific problems seems likely to increase, bringing with it novel and exciting discoveries while advancing the field of comparative endocrinology as a whole.
Acknowledgments We are especially grateful to Julie Fronczek, Marlys Houck and the Genetics Division of the San Diego Zoo Institute for Conservation Research for assistance with fibroblast cultures and Dr. Barbara Durrant for reviewing the manuscript. This work was supported in part by a fellowship awarded to C.M. by the J.W. Sefton Foundation.
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