General and Comparative Endocrinology 250 (2017) 58–69

Contents lists available at ScienceDirect

General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

Identification of an oxytocinase/vasopressinase-like leucyl-cystinyl (LNPEP) in teleost fish and evidence for hypothalamic mRNA expression linked to behavioral social status ⇑ Emma A. Elkins, Kayla A. Walti, Kathryn E. Newberry, Sean C. Lema

Biological Sciences Department, Center for Coastal Marine Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA article info abstract

Article history: The vasotocin/vasopressin and isotocin/mesotocin/ family of nonapeptides regulate social Received 17 March 2017 behaviors and physiological functions associated with reproductive physiology and osmotic balance. Revised 3 June 2017 While experimental and correlative studies provide evidence for these nonapeptides as modulators of Accepted 4 June 2017 behavior across all classes of vertebrates, mechanisms for nonapeptide inactivation in regulating these Available online 6 June 2017 functions have been largely overlooked. Leucyl-cystinyl aminopeptidase (LNPEP) – also known as vaso- pressinase, oxytocinase, placental leucine aminopeptidase (P-LAP), and -regulated aminopeptidase Keywords: (IRAP) – is a membrane-bound zinc-dependent that inactivates vaso- Aminopeptidase pressin, oxytocin, and select other cyclic polypeptides. In humans, LNPEP plays a key role in the clearance Vasotocin Vasopressin of oxytocin during pregnancy. However, the evolutionary diversity, expression distribution, and func- Isotocin tional roles of LNPEP remain unresolved for other vertebrates. Here, we isolated and sequenced a full- Oxytocin length cDNA encoding a LNPEP-like polypeptide of 1033 amino acids from the ovarian tissue of Fish Amargosa pupfish, Cyprinodon nevadensis. This deduced polypeptide exhibited high amino acid identity Behavior to human LNPEP both in the protein’s active domain that includes the peptide binding site and zinc cofac- Aggression tor binding motif (53.1% identity), and in an intracellular region that distinguishes LNPEP from other (70.3% identity). Transcripts encoding this LNPEP enzyme (lnpep) were detected at high- est relative abundance in the gonads, hypothalamus, forebrain, optic tectum, gill and skeletal muscle of adult pupfish. Further evaluation of lnpep transcript abundance in the brain of sexually-mature pupfish revealed that lnpep mRNAs were elevated in the hypothalamus of socially subordinate females and males, and at lower abundance in the telencephalon of socially dominant males compared to dominant females. These findings provide evidence of an association between behavioral social status and hypothalamic lnpep transcript abundance and suggest that variation in the rate of VT/IT peptide inactivation by LNPEP may be a contributing component in the mechanism whereby nonapeptides regulate social behavior. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction have emerged as key regulators of social behaviors including aggression, social affiliation, and courtship (Godwin and Neurohypophysial nonapeptides of the arginine vasopressin Thompson, 2012; Goodson and Bass, 2001; Goodson and (VP)/arginine vasotocin (VT) and the oxytocin (OT)/mesotocin Thompson, 2010; Heinrichs and Domes, 2008; Insel and Young, (MT)/isotocin (IT) families regulate several physiological and 2000; Heinrichs et al., 2009; Young and Flanagan-Cato, 2012). Evi- behavioral functions in vertebrates (Banerjee et al., 2017). In mam- dence from such studies comes largely from experimental manip- mals, VP and OT peptides have well established peripheral effects ulations of nonapeptide concentrations (e.g., Goodson, 1998; Lema on water homeostasis (e.g., Nielsen et al., 1995), vasoconstriction and Nevitt, 2004a; Meylan et al., 2017; Semsar et al., 2001) or from and blood pressure (e.g., Pittman et al., 1982), and the stimulation findings that link variation in hypothalamic expression of non- of pituitary adrenocorticotropin (ACTH) secretion in response to apeptides or their receptors to behavioral variation at the individ- stressors (e.g., Gibbs, 1986). More recently, however, nonapeptides ual, sex, and species levels (e.g., Ferris et al., 1997; Greenwood et al., 2008; Larson et al., 2006; Lema et al., 2015). ⇑ Corresponding author. While the preponderance of experimental studies exploring the E-mail address: [email protected] (S.C. Lema). physiological and behavioral effects of VP/VT and OT/MT/IT have http://dx.doi.org/10.1016/j.ygcen.2017.06.002 0016-6480/Ó 2017 Elsevier Inc. All rights reserved. E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 59 focused on nonapeptide production and secretion, the enzymatic length lnpep cDNA from the ovarian tissue of Amargosa pupfish, degradation of VP/VT and OT/MT/IT peptides may also play an and then characterized the protein structure of this enzyme, its important role in regulating nonapeptide concentrations. More expression distribution across tissues in adult pupfish, and its phy- than two decades ago, a leucine aminopeptidase enzyme belonging logenetic diversity across Actinopterygiian fishes and other verte- to the M1 subfamily of aminopeptidases was identified in the brates. We then examined how lnpep gene transcript abundance maternal placenta (Mizutani and Tomoda, 1992; Tsujimoto et al., in hypothalamus and telencephalon regions of the brain of pupfish 1992; Tsujimoto and Hattori, 2005; Rogi et al., 1996). This leucyl- associates with aggressive behaviors, and observed sex- and cystinyl aminopeptidase (LNPEP) – also alternatively referred to individual-level variation in expression linked to social dominance as placental leucine aminopeptidase (P-LAP), oxytocinase, vaso- status. pressinase, or cysteine aminopeptidase – enzymatically hydrolyzes small peptides with a N-terminal ring structure, including both OT and VP (Matsumoto et al., 2000; Tsujimoto et al., 1992). This pla- 2. Materials and methods centa LNPEP enzyme was subsequently determined to be identical to insulin-regulated aminopeptidase (IRAP) (Hattori et al., 1999; 2.1. Amplification and sequencing of full-length lnpep cDNA Kandror et al., 1994; Keller et al., 1995; Ross et al., 1996), which has an unresolved function in insulin signaling but is translocated Ovarian tissue was dissected from an adult female C. n. amar- to cell membranes with insulin-responsive glucose transporter 4 gosa pupfish (36.90 mm standard length; 1.49 g body mass) col- ° 0 00 (GLUT4) during insulin-stimulated glucose trafficking (e.g., lected on 9 May 2014 from Tecopa Bore (35 53 08.42 N, ° 0 00 Mastick et al., 1994). Beyond OT and VP, LNPEP has been shown 116 14 03.01 W) – a small, thermal spring habitat adjacent to to cleave N-terminal amino acids with neutral or basic properties the Amargosa River near Tecopa, California, USA. The ovarian tissue ° from other peptides including angiotensin III, somatostatin, Met- was flash frozen in liquid N2 and stored at 80 C until RNA extrac- enkephalin, and neurokinin A (Herbst et al., 1997; Matsumoto tion. Total RNA was extracted using Tri-Reagent (Molecular et al., 2000, 2001). Notably, however, the enzymatic activity of Research Center, Inc.) with bromochloropropane as the phase sep- LNPEP does not extend to all peptides with a N-terminal ring struc- aration reagent. The resulting RNA was DNase I treated (TURBO ture, but rather appears to be restricted to select peptides, perhaps DNA-free Kit, Ambion) and then quantified using a P300 NanoPho- based on molecular size (e.g., Nomura et al., 2005). tometer (Implen, Inc.) (260:280 ratio = 2.01). Total RNA was reverse transcribed in a 20 ll reaction containing 5 ll of RNA Initially, the LNPEP enzyme was thought to be present only in l l the placenta and maternal serum as a mechanism for degrading (250 ng/ l), 1 l dNTPs (10 mM, Promega Corp., Madison, WI, USA), 1 ll oligo(dT) primers (500 lg/ml; Promega Corp.), 0.25 ll OT and maintaining this hormone at appropriate concentrations l across fetal/placental/maternal tissues during pregnancy recombinant RNasin ribonuclease inhibitor (40 u/ l; Promega l l l (Mizutani et al., 1976; Mizutani and Tomoda, 1992; Titus et al., Corp.), 4.75 l nuclease-free H2O, and 4 l5 buffer, 3 l MgCl2, l TM 1960). While LNPEP expression indeed plays a critical role in main- and 1 l GoScript reverse transcriptase (Promega Corp.). The taining appropriate OT signaling dynamics during pregnancy reverse transcription reaction was run according to the thermal ° ° ° (Mizutani et al., 2007; Nomura et al., 2005; Tian et al., 2016), profile of 25 C for 5 min and 42 C for 1 h, followed by 70 C for LNPEP protein and mRNAs are also expressed in a variety of other 15 min to inactivate the reverse transcriptase. tissues including brain, kidney and heart (Lausten et al., 1997; Gene specific primers were designed to concensus regions of a Nagasaka et al., 1997; Rogi et al., 1996). That broad distribution 5005 bp nucleotide predicted mRNA sequence for a lnpep mRNA of LNPEP expression implies functions for LNPEP beyond preg- from the sheepshead minnow, Cyprinodon variegatus (GenBank nancy, and indeed recent studies provide evidence for roles of accession No. XM_015373062), which we aligned to genomic LNPEP in lactation, cognition, kidney diuretic function, hyperten- DNA scaffold sequences for the lnpepe gene (JSUU01003752 and sion, and stress responses (Banegas et al., 2010; Hernández et al., JSUU01025998) that we identified from Cyprinodon nevadensis pec- 2015; Kim et al., 2016; Ramírez et al., 2011; Segarra et al., 2016; toralis (South Scruggs Spring population) – a closely related sub- Tobin et al., 2014; Villarejo et al., 2012). Hernández et al. (2015), species to C. n. amargosae that also inhabits the Death Valley for instance, observed a decrease in LNPEP activity in the amygdala region of California, USA (Soltz and Naiman, 1978). Nucleotide of adult male rats following a 1 h restraint stress, suggesting that sequences for these primers were as follows: (forward) 50-GTGTG changes in VP/OT degradation rates in this brain region may occur CTTGTGGGGTGAGGC-30 and (reverse) 50-GAGAACTGCGGAG as part of the acute physiological response to perceived stressors. GAGGTTTGTTCG-30. These gene specific primers were designed to Even though research exploring the possible functions of LNPEP the putative 50 and 30 UTRs to generate a predicted 3515 bp nucleo- beyond pregnancy is only in the early stages, the initial results of tide product that would span the complete coding region of the these studies – coupled with the broad expression distribution of pupfish lnpep gene. LNPEP across several tissues (Lausten et al., 1997; Nagasaka Complementary DNA was amplified in a 50 lL reaction com- et al., 1997; Rogi et al., 1996) – suggests that the metabolic degra- prised of 25 lL of GoTaqÒ Long PCR Master Mix (Promega Corp.), dation of nonapeptides may be an important, but overlooked, com- 1 lL each of forward and reverse primer (10 lM), 20 lLof ponent in regulating the availability of these hormones in target RNase-free H2O, and 3 lL of cDNA under a thermal profile of tissues. 94 °C for 2 min followed by 37 cycles of 94 °C for 30 s and 65 °C To date, however, studies of LNPEP have been limited to only a for 4 min 30 s, and then 72 °C for 7 min. The resulting PCR product few mammals, and no study has yet examined LNPEP expression or showed a single band on a 0.8% ethidium bromide gel, and was function in a non-mammalian vertebrate. Here, we isolated and subsequently cleaned (QIAquick PCR Purification Kit, Qiagen) and sequenced a full-length cDNA encoding a lnpep-like enzyme from Sanger sequenced (Molecular Cloning Lab, Inc., South San Fran- the desert Amargosa pupfish (Cyprinodon nevadensis amargosae) cisco, CA, USA). Sequencing of the full cDNA product was con- as an initial step toward exploring the evolutionary and functional ducted with the forward and reverse primers provided above diversity of LNPEP in non-mammalian taxa. The Amargosa pupfish coupled with primer walking using several additional sequencing has been studied previously for the role of VT and IT in regulating primers (Online Supplemental Materials, Table S1). The resulting social behaviors and osmoregulatory function (e.g., Lema et al., sequences were assembled into a single contig assembly using 2010, 2015; Lema, 2010; Lema and Nevitt, 2004a). Using reverse Sequencher v5 software (Gene Codes Corp., Ann Arbor, MI, USA), transcription-PCR (RT-PCR), we isolated and sequenced a full- and the assembly was then compared for identity against previ- 60 E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 ously published lnpep sequences in the National Center for (10 ml), and fluorescently-labeled probe (45 ml). The primers and Biotechnology Information BLAST program (http://blast.ncbi.nlm. probe for lnpep from C. n. amargosae amplified a product 117 bp nih.gov/). Additional characterization and comparative analyses in length and were designed to span an intron boundary, as iden- of the deduced polypeptide generated by the resulting cDNA was tified from a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast. conducted using ProtScale (Gasteiger et al., 2005) and ClustalX cgi) of the genome of the closely-related subspecies C. n. pectoralis software (Larkin et al., 2007). (GenBank accession No. GCA_000776015). Nucleotide sequences for the primers and probe for lnpep were as follows: (forward) 50- 2.2. Phylogenetic analysis CATCAGTGGTTTGGAAACTTGG-30, (reverse) 50- GTCGAGGTCGGGC ATTATTT-30, and (probe) 50-TGGAATGACCTGTGGCTCAACGAG- 30. The deduced amino acid sequence for LNPEP from C. n. amar- The relative mRNA level of elongation factor-1a (ef1a, GenBank gosae was aligned using ClustalX (Larkin et al., 2007) to available accession No. EU906930) was also quantified as a normalizing ref- LNPEP sequences predicted from the genome sequences of other erence gene using primers and probes described previously (Lema, fishes, as well as predicted protein sequences deduced from mRNA 2010). All quantitative PCR reactions were run on a CFX96TM Real- or DNA of select taxa from superclass Tetrapoda. Phylogenetic Time PCR Detection System (BioRad Laboratories, Inc.) under a analysis was constructed using MEGA v.7 software (Kumar et al., thermal profile of 95 °C for 2 min and 45 cycles of 95 °C for 10 s 2016), using the Neighbor-Joining method and a p-distance model and 60 °C for 30 s. Standard curves for qRT-PCR were made for each for tree construction (Saitou and Nei, 1987). Positions containing tissue from a pool of RNA from samples representing all tissues alignment gaps were deleted only in pairwise comparisons (pair- from both sexes. Average PCR efficiencies were 97.90% for lnpep wise deletion of gaps) and uniform rates were assumed among and 100.44% for ef1a. Correlation coefficients (r2) were > 0.99 for sites. Confidence values for nodes were obtained by bootstrapping the standard curve for each gene. Elongation factor-1a (ef-1a) (1000 replicates). was quantified as a tissue reference gene. Reported levels of ef- 1a varied significantly across tissues, so lnpep levels for the tissue 2.3. Tissue distribution pattern of lnpep gene transcript abundance distribution comparisons were not normalized to ef-1a, and instead were normalized to total RNA concentration from each tissue. Select tissues were dissected from sexually-mature female and Select PCR products for each transcript assay were subsequently male C. n. amargosae pupfish (n = 3 for each sex) that had been col- purified and Sanger sequenced (Molecular Cloning Lab, Inc., South lected using minnow traps on 27 June 2015 from the Amargosa San Francisco, CA, USA) to further confirm product identity. River (N 35°51.2750, W 116°13.8330) near Tecopa, California, USA. Fish were maintained in captivity in 38 L tanks at approximately 30 °C and 3.5 ppt salinity at California Polytechnic State University, 2.4. Relationships between brain lnpep mRNA levels and pupfish San Luis Obispo, California, USA, for nearly 5 months prior to tissue behavior collection. Holding conditions represented physical parameters in the Amargosa River on the day of collection. Fish were fed ad lib As an initial step toward examining whether LNPEP has a role in with a 1:1 mixture of commercial spirulina (Aquatic Eco- regulating behavior, we examined whether relative lnpep mRNA Systems, Inc., Apopka, FL) and brine shrimp (San Francisco Bay levels varied in the hypothalamus or telencephalon of adult C. n. Brand, Inc., Newark, CA, USA) flake feeds. Mean (±SD) body sizes amargosae in patterns related to sex (male or female) or behavioral for these fish were as follows: females: 43.40 ± 1.68 mm (standard social status (socially dominant or subordinate). The fish used for length [SL]), 3.15 ± 0.84 g (body mass); males: 41.13 ± 4.48 mm this purpose were also described in Lema et al. (2015), a study that (SL), 2.15 ± 0.82 g (mass). Sex identity was determined by sexually examined patterns of variation in brain VT, IT and nonapeptide dimorphic body coloration and visual confirmation of gonadal sex receptor relative to social status and aggression. A detailed descrip- upon tissue dissection. tion of the experimental design is provided in Lema et al. (2015),so Fish were euthanized using tricaine methanesulfonate (MS222, only a summarized description of the behavioral methods will be Argent Chemical Laboratories, Redmond, WA, USA), and the follow- provided here. ing tissues were dissected, flash frozen in liquid N2, and stored at In brief, adult male and female C. n. amargosae pupfish were col- 80 °C: brain, pituitary gland, gills, heart, liver, gonads, kidney, lected by minnow trap on 9 September 2012 from the Amargosa gastrointestinal (GI) tract, skeletal muscle (collected from the left River, San Bernardino County, California, USA (N 35°50.9760,W side of the caudal peduncle). The brain was further subdivided into 116°13.8500). Fish were transported to California Polytechnic State the telencephalon, hypothalamus, optic tectum, cerebellum, and University, San Luis Obispo, CA, and maintained in 208 L closed hindbrain (medulla oblongata) regions, as described in detail in system tanks at 24-26 °C and 0.4 ppt salinity. Three male and three Lema et al. (2015). Total RNA from these tissues was extracted female pupfish were then assigned to mixed-sex groups in 114 L using TriReagent (Molecular Research Center, Inc.) with bro- tanks (n = 14 replicate tanks; 90 cm long 45 cm wide 30 cm mochloropropane. The RNA was then DNase I treated (TURBO high) under conditions of 25.64 ± 0.34 °C (mean ± SD) and 0.4 ppt DNA-free Kit, Ambion) and quantified using a P300 NanoPhotome- salinity. Prior to transfer to the experimental tank, each fish was ter (Implen, Inc.), and the total RNA from each fish and tissue was anesthetized in MS222, weighed (±0.01 g), measured (standard then diluted to the same concentration of 28.5 ng/lL. The total length; ±0.05 mm), and marked with a visible elastomer tag RNA was then reverse transcribed in 12 ll reactions containing (Northwest Marine Technologies, Seattle, WA, USA). Fish were 6 ll of RNA (28.5 ng/ll), 0.6 ll dNTPs (Promega), 0.6 ll random selected so that each tank contained a similar distribution of vary- primers (500 lg/ml; Promega), 0.1 ll recombinant RNasinÒ ing body sizes. Each tank’s social group therefore contained one ribonuclease inhibitor (40 u/ll; Promega), and 2.4 ll 5x buffer, fish in each of the following sex-size categories: large male: 40– TM 1.8 ll MgCl2, and 0.5 ll GoScript reverse transcriptase (Promega). 44 mm SL; 2.00–3.40 g body mass; medium male: 35–41 mm, We then used quantitative real-time PCR (qPCR) with TaqManÒ 2.00–3.10 g; small male: 31–40 mm, 0.90–3.40 g; large female: probes to examine variation in the relative lnpep mRNA levels 35–44 mm, 1.00–2.70 g; medium female: 33–41 mm, 1.00– among tissues and between sexes. All qPCR reactions were run as 2.00 g; and small female: 31–39 mm, 0.80–1.90 g. The purpose of 16 ml volumes containing 8 ml of iTaqTM Universal Probes Supermix this variation in body size was to increase the probability of fish

(BioRad Laboratories, Inc.), 3.5 ml of nuclease-free H2O, 1.5 mlof of both sexes exhibiting variation in social status and associated cDNA, and 1 ml each of forward primer (10 ml), reverse primer aggressive behaviors. Each tank also contained rocks of similar size E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 61 and dimensions placed in the same locations to provide structural these factors, or the interaction between these factors. We then ran complexity for territory establishment and defense. Fish were fed Tukey’s HSD tests to assess pairwise differences among sex-body ad libitum a diet of 1:1 mixed spirulina (Aquatic Ecosystems) and size categories for lnpep mRNA levels for each brain region. brine shrimp (San Francisco Bay Brand) flake feeds to satiation twice daily. 3. Results Focal behavioral observations were made on four of the six fish in each tank (the largest male and female and the smallest male 3.1. Identification and sequence analysis of LNPEP-like cDNA from and female) over 10 min time periods on day 5, day 10, day 18, pupfish and day 30 after the introduction of fish to the 114 L experimental tanks. Quantified behaviors included aggressive ‘charges,’ ‘nips,’ A single 3376 bp cDNA (Genbank accession No. KY290248) and ‘displays,’ as described previously in this species of pupfish encoding a LNPEP-like enzyme was amplified and sequenced from (Lema and Nevitt, 2004a; Lema et al., 2010). The individual that the ovary of an adult female C. n. amargosae pupfish. This cDNA behaved aggressively toward another fish, as well as the fish that consists of 11 bp of 50 UTR, 263 bp of 30 UTR, and 3102 bp of open received the aggression, was recorded for each aggressive interac- reading frame encoding a deduced protein of 1033 amino acid resi- tion. Aggressive behaviors were analyzed using one-factor ANOVA dues. BLAST analysis of the deduced polypeptide sequence con- models followed by orthogonal contrasts to make specific compar- firmed high sequence similarity to human leucyl-cystinyl isons among specific treatment groups to test for body size effects aminopeptidase (LNPEP), with 49.95% residue identity to H. sapiens within each sex, as well as sex differences in behavior. Due to cor- relations between several of the measured behaviors, we used LNPEP splice variant isoform 1 (NP_005566) and 49.85% identity to Principal Components Analysis (PCA) to generate composite variant LNPEP isoform 2 (NP_787116). This pupfish LNPEP-like behavioral variables that more efficiently characterized the behav- enzyme exhibited lower deduced amino acid identity values of ioral phenotypes of individual pupfish. Details on the criteria used 42.62% to adipocyte-derived leucine aminopeptidase (AAF07395) to determine the number of principal components (PCs) retained and 42.51% to endoplasmic reticulum aminopeptidase-1 are provided in Lema et al. (2015). (XP_005272072)ofH. sapiens, suggesting that the pupfish After 32 d of social interaction and behavioral observation, the aminopeptidase enzyme encoded by this newly sequenced cDNA largest and smallest males and the largest and smallest females is evolutionary homologous to the LNPEP – or vasopressinase/oxy- were collected from each tank. Within 2 min of netting, pupfish tocinase – enzyme previously identified in the placenta of humans were euthanized with MS222, and weighed and measured. The (Mizutani and Tomoda, 1992; Tsujimoto et al., 1992; Rogi et al., brain of each fish was dissected, subdivided into the telencephalon 1996). and hypothalamus, flash frozen with liquid N2, and stored at More detailed examination of the structure of this deduced 80 °C. polypeptide encoded by this cDNA from C. n. amargosae further Total RNA was extracted from the hypothalamus and telen- confirmed the identity of the deduced protein as homologous to cephalon using TriReagent (Molecular Research Center, Inc.) with human placental LNPEP. The pupfish LNPEP-like enzyme exhibited bromochloropropane for phase separation. Total RNA was then similarities to human placental LNPEP in several key functional DNase treated (TURBO DNA-free Kit, Ambion), quantified by spec- regions. Evaluation of hydrophobic regions of the deduced trophotometry (P300 NanoPhotometer, Implen; 260:280 ratios LNPEP-like enzyme using a Kyte and Doolittle plot revealed a 21 >1.98), and diluted to 15 ng/ml before being reverse transcribed residue region of high hydrophobicity located comprised of amino TM in 10 ml reactions containing 2 mlof5 iScript Reaction Mix acid positions 119–139 (Fig. 1a) (Gasteiger et al., 2005). This (BioRad Laboratories, Inc., Hercules, CA, USA), 2 ml of nuclease- hydrophobic region shows 52.4% residue identity to the 21 amino m free H2O, and 6 l of RNA template (90 ng) under a thermal profile acid transmembrane region of human LNPEP. The pupfish LNPEP- of 5 min at 25 °C and 20 min at 46 °C, followed by 1 min at 95 °C like protein is also comprised of a 118 amino acid region toward for reverse transcriptase inactivation. Real-time qPCR to measure the N-terminus from this putative transmembrane region (Fig. 1a). TM relative lnpep levels was run in 16 ml volumes on a CFX96 Real- The length and amino acid identity of this N-terminus region cor- Time PCR Detection System (BioRad Laboratories, Inc.) with the responds most closely to the intracellular, cytoplasmic region of same reaction mix composition and thermal cycling conditions as placental LNPEP of humans (70.3% amino acid identity), and exhib- described above. Primers and probes designed to ef1a and 60S ited lower identities of 37.5% to both adipocyte aminopeptidase ribosomal protein L8 (rpl8)(KJ719257) from Amargosa pupfish and endoplasmic reticulum aminopeptidase (ERAP) from were used as endogenous reference genes (see Lema et al., 2015). H. sapiens, both of which have a truncated N-terminus (Fig. 1b). Relative transcript abundance values for lnpep were normalized Human placental LNPEP has an extracellular domain toward the to the geometric mean of ef1a and rpl8 mRNA levels, which did C-terminus of its hydrophobic transmembrane region. This extra- not vary among sexes or body size categories (Lema et al., 2015). cellular domain contains the active, catalytic region of the LNPEP The expression of lnpep was subsequently expressed as a relative enzyme with the peptide substrate binding site and a zinc cofactor level by dividing mean values for each sex and social status cate- binding motif characterized by the following sequence: HEXXH gory by the mean value of the socially subordinate, small-bodied (X)18E (e.g., Hermans et al., 2015; Nomura et al., 2005; Tsujimoto female group. The mean intra-assay% CV for the qPCR reactions and Hattori, 2005). Amino acid composition of the substrate bind- was 1.17%. Correlation coefficients (r2) were >0.98 for the standard ing site (residue sequence: GAMEN) is conserved across all three curve of each gene. Select products from the PCR reactions were human aminopeptidases (placental LNPEP, adipocyte aminopepti- again purified (QIAquick PCR Purification Kit, Qiagen) and Sanger dase, and ERAP-1), as well as the pupfish LNPEP-like polypeptide sequenced (Molecular Cloning Labs, South San Francisco, CA, (Fig. 1a,b). The zinc binding motif was also identified in this pup- USA) to confirm product identities. fish LNPEP-like enzyme and conserved across all three human To examine whether either telencephalic or hypothalamic lnpep aminopeptidases (Fig. 1b). Unlike human adipocyte aminopepti- mRNA levels varied in patterns associated with sex or social status, dase and ERAP, the pupfish LNPEP-like enzyme also shares a con- we first ran two-factor ANOVA models with ‘sex’ and ‘body size served di-leucine (LL83,84) amino acid sequence in the N-terminus category’ to examine whether relative lnpep gene transcript abun- intracellular region with placental LNPEP; this region has been dance in the telencephalon or hypothalamus varied with either of demonstrated to function in the rapid internalization of human 62 E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69

Fig. 1. (a) Deduced amino acid sequence of LNPEP from Amargosa pupfish (Genbank accession No. KY290248). Putative domains indicated here include the intracellular domain (overline; residues 1–118), hydrophobic membrane region (dark gray highlight; residues 119–139), and enzyme active region (bold underline; residues 183–625).

Within the active region, the peptide substrate binding region sequence ‘GAMEN’ (black highlight) and zinc cofactor binding motif HEXXH(X)18E (gray highlight) are indicated. Asterisks denote key conserved residues in the zinc binding motif. (b) Schematic representation of the polypeptide structures of pupfish LNPEP, human placental LNPEP, human adipocyte aminopeptidase, and human endoplasmic reticulum aminopeptidase-1 (ERAP-1). Pupfish LNPEP had an extended N-terminus intracellular domain containing the conserved di-leucine (LL) amino acids similar to those in placental LNPEP and shown to be important for enzyme internalization from the plasma membrane (Johnson et al., 2001).

LNPEP from the plasma membrane and recycling of the protein exhibited high conservation between the human and pupfish back to the cell surface (Johnson et al., 2001). A second, acidic resi- LNPEP sequences, although this region exhibited a few residue due region in human LNPEP identified by Johnson et al. (2001) also changes from the sequence DEDEEDYES64–72 in human to E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 63

DDDEEDCES71–79 in C. n. amargosae pupfish. Taken as a whole, identified homologous, predicted LNPEP-like proteins across most structural similarity in the intracellular domain and high amino classes of vertebrates including fishes of the classes Sarcopterygii acid sequence identity across the polypeptide suggests that the C. (e.g., West Indian Ocean coelacanth, Latimeria chalumnae, n. amargosae LNPEP-like enzyme encoded by this newly sequenced XM_014498363) and Chondrichthyes (e.g., elephant shark, Cal- cDNA is evolutionarily homologous to human placental LNPEP. lorhinchus milii, XM_007893103). Alignment and phylogenetic analysis of the pupfish LNPEP-like Interestingly, all of the LNPEP-like polypeptide sequences in the deduced amino acid sequence with human placental LNPEP, adipo- phylogeny in Fig. 2 had conserved amino acid composition in the cyte aminopeptidase, and ERAP isoform 1 (ERAP-1) – as well as zinc binding motif HEXXH(X)18E, and all but one species also had putative LNPEP-like enzymes from other vertebrates identified by the conserved sequence of ‘GAMEN’ as the peptide substrate bind- BLAST search of the NCBI database (https://www.ncbi.nlm.nih.- ing region. That sole exception was the putative LNPEP-like gov/) – again indicated structural similarity of the pupfish enzyme from the snake Python bivittatus (XM_007430066). The LNPEP-like enzyme to human placental LNPEP (Fig. 2). BLAST deduced LNPEP-like polypeptide sequence from python has an searches using the deduced LNPEP-like polypeptide from pupfish

Fig. 2. Neighbor-joining (p-distance, pairwise deletion of gaps) phylogenetic tree of putative LNPEP-like enzymes in select vertebrates. Phylogenetic analysis was conducted using deduced amino acid sequences aligned in ClustalX and the assembled into a tree in MEGA. The C. n. amargosae pupfish sequence identified here is indicated by the red box. Bootstrap values (1000 replicates) are indicated at each node for relationships with <50% confidence. GenBank accession Nos. for each sequence are provided with each species. The tree was rooted using sequences for adipocyte-derived LNPEP and endoplasmic reticulum aminopeptidase-1 (ERAP-1) from humans. Note that all sequences shown above for LNPEP are predicted based on genome sequences, except pupfish (KY290248), human (NM_005575), mouse (NM_172827), and rat (NM_001113403). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 64 E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69

‘AGMEN’ amino acid sequence in the putative substrate-binding region rather than ‘GAMEN.’ Given that this Python sequence was predicted from a genome sequence, we conducted further BLAST searches to identify LNPEP-like enzymes from other snakes, and found identical ‘AGMEN’ residue sequences in putative LNPEP enzymes from two other species, the Taiwan habu (Protobothrops mucrosquamatus, XM_015831937) and common garter snake (Thamnophis sirtalis, XM_014056281), indicating that the LNPEP enzyme in snakes (suborder Serpentes, order Squamata) may have different binding specificities than in other vertebrates. What is more, a putative python ERAP-like enzyme (XM_007437799) exhibited the conserved ‘GAMEN’ active binding site, suggesting that evolutionary, and possibly functional, changes in aminopepti- dase structure in snakes may have occurred specifically in the LNPEP enzyme. Future studies should confirm this LNPEP amino acid sequence divergence in reptiles of suborder Serpentes, and explore whether that variation in primary structure has any conse- quences for LNPEP substrate specificity and function.

3.2. Comparison of tissue expression patterns for LNPEP enzyme mRNAs

Relative levels of lnpep mRNAs, as measured by qPCR, varied across tissues and organs in sex-dependent patterns (Fig. 3). Gene transcripts for lnpep were most abundant in the ovary, with lnpep mRNA levels more than 450% higher in the ovarian tissues of sexually-mature females than in the testis of sexually-mature males. Transcripts encoding the LNPEP-like enzyme were also observed at high relative abundance in the forebrain, hypothala- mus, and optic tectum brain subdivisions, as well as in the gill and skeletal muscle tissues in both sexes. Transcripts encoding lnpep were at very low abundance in the pituitary gland of both females and males.

3.3. Variation in brain lnpep mRNA levels associated with social status

As described previously in Lema et al. (2015), pupfish in the mixed-sex social groups varied in aggressive behaviors (ANOVA models: aggression toward females, F3,54 = 27.675, p < 0.0001; aggression toward males, F3,54 = 18.240, p < 0.0001). Male pupfish behaved more aggressively than females (p < 0.0001), and fish Fig. 3. Tissue distribution of relative lnpep mRNAs in sexually-mature (a) female from the larger body size category behaved more aggressively than and (b) male pupfish (C. n. amargosae). Relative mRNA levels were measured using their counterparts of smaller body size and similar sex (p < 0.0001) real-time quantitative reverse transcription PCR using Taqman probes (n = 3 fish (Fig. 4a). For our discussion from this point onward, we refer to the per sex). Data are plotted as mean ± SEM values. large-bodied fish of each sex as being socially dominant, and small- bodied fish as socially subordinate. Notably, however, the extent of behavioral variation associated with body size varied between the were recipients of more frequent female aggression and more often sexes. Large, dominant male pupfish were observed to be signifi- behaved aggressively toward other females. We therefore inter- cantly more aggressive toward both females and other males com- preted pupfish with higher PC2 values as more frequently engaging pared to smaller, subordinate male pupfish, but pairwise in agonistic interactions with females compared to fish with lower comparisons did not indicate significant differences in aggression PC2 values. Average PC2 values were observed to be highest in sub- between female pupfish from the two body size categories. ordinate female pupfish (Fig. 4c). Additional descriptions of the Principal Components Analysis (PCA) identified two governing identification and analysis of these PCs are provided in Lema PCs: 1) PC1 that explained 47.15% of the behavioral variation et al. (2015). observed, and 2) PC2 that explained 13.96% of the behavioral vari- Hypothalamic lnpep mRNA abundance was at higher relative ation (Table 1). PC1 represented the ‘aggressiveness’ of the focal levels in the subordinate pupfish, who were smaller in body size individual, with offensive aggressive behaviors (e.g., charging by compare to dominant fish of the same sex (size category effect: focal fish, nipping by focal fish) showing robust positive loadings F1,43 = 9.117, p = 0.0042). Pairwise statistical comparisons within for PC1, and aggression performed at the focal fish (e.g., charges each sex revealed this size category effect to be more pronounced by males at the focal fish, nips by males at the focal fish) loading in female pupfish (Fig. 5a). Subsequent analyses failed to show any negatively to PC1. Accordingly, PC1 values varied across sexes in relationships between individual variation in hypothalamic lnpep patterns dependent on social status, with higher PC1 values in transcript abundance and either body mass (g) or body length the more aggressive fish interpreted as being socially dominant (SL, mm) within each sex-size category, suggesting that the (Fig. 4b). PC2 represented the frequency of female-female aggres- observed effect of ‘size category’ on hypothalamic lnpep mRNA sive interactions, and pupfish exhibiting higher PC2 values both levels is not simply attributable to fish body size, but may instead E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 65

Fig. 4. (a) Behavioral differences in aggression (sum of charges, nips and displays) among male and female pupfish in mixed-sexed social tests. Distribution of behavioral principal components (b) PC1 and (c) PC2 for both sexes of socially subordinate and dominant pupfish from the social groups. Behaviors that loaded onto each PC variable are provided in Table 1. Values are shown as mean ± SEM, with individual fish values also provided in (b) and (c). Asterisks indicated orthogonal comparisons with *p < 0.05 and **p < 0.0001. Data adapted with permission from Lema et al. (2015).

Table 1 be related to variation in behavioral phenotype. To test this idea Factor loadings for principal components analysis (PCA) performed on measured further, we examined associations between individual variation agonistic behaviors. Bold loadings indicate axis of strongest loading for the measured in behavior and hypothalamic lnpep mRNA levels. PC2 behavioral variable. Reproduced with permission from Lema et al. (2015). values, which are positively indicative of the frequency of Behavior PC1 PC2 female-female aggressive interactions (Table 1). Behavior PC2 Focal charge at male 0.7480 0.1607 was found to be correlated negatively with individual variation Focal nip at male 0.7910 0.1445 in hypothalamic lnpep expression in small, subordinate female Focal charge at female 0.8362 0.2126 pupfish (Fig. 6). The strength and direction of that correlation sug- Focal nip at female 0.6543 0.4434 gests that individual small-sized females that engaged more fre- Male charge at focal 0.8458 0.0434 Male nip at focal 0.5621 0.0917 quently in female-female aggressive interactions (higher PC2 Female charge at focal 0.6729 0.5127 values) had lower relative levels of lnpep mRNAs in the hypothala- Female nip at focal 0.3313 0.8248 mus compared to small females performing or experiencing less Display by focal 0.5809 0.1193 aggression with other females. No other significant associations 66 E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69

Fig. 6. Aggressive behavior principal component PC2, which explained 13.96% of the observed variation in behavior, plotted against the relative abundance of lnpep mRNAs in the hypothalamus of subordinate female pupfish.

dase enzyme that metabolizes and inactivates both OT and VP, as well as several other peptides including angiotensin III and Met- enkephalin (Matsumoto et al., 2000; Tsujimoto et al., 1992; Tsujimoto and Hattori, 2005). LNPEP may therefore be an impor- tant mediator for the degradation of neurohypophysial nonapep- tides in non-mammalian vertebrates. However, to our knowledge, all studies on LNPEP expression and function to date have been conducted in mammals, even though nonapeptides of the VP/VT and OT/MT/IT families have established physiological and behavioral functions across vertebrate taxa (e.g., Goodson and Bass, 2001; Goodson and Thompson, 2010). Here, we isolated and sequenced a full-length cDNA encoding a LNPEP-like enzyme from a teleost fish, the Amargosa pupfish. The deduced protein encoded by this cDNA is homologous to mam- malian LNPEP and shares 50% residue identity with LNPEP isoform 1 of humans. Structural domain analysis of the pupfish LNPEP-like protein to human LNPEP and the evolutionarily-related adipocyte aminopeptidase and ERAP-1 of humans confirmed that the pupfish Fig. 5. Variation in relative lnpep gene transcript abundance in the (a) hypotha- lamus and (b) telencephalon brain regions of socially subordinate and dominant LNPEP protein has highest structural similarity to H. sapiens pla- pupfish of both sexes. Values are shown as mean ± SEM, and letters indicate cental LNPEP. While critical functional regions including the significant difference among groups (Tukey HSD tests). Sample sizes are n =10to13 ‘GAMEN’ residue substrate binding site and the zinc cofactor bind- fish per group. ing motify ‘HEXXH(X)18E’ are conserved in all four aminopeptidase enzymes, the pupfish LNPEP-like enzyme showed greater struc- tural similarity to placental LNPEP in the enzymes’ N-terminus between individual behavior (PC1 or PC2) and hypothalamic lnpep intracellular domain. That similarity includes conservation of mul- mRNA levels were detected. tiple acid residue regions shown previously to be critical to the In the telencephalon, relative lnpep transcript abundance also internalization and recycling of placental LNPEP (Johnson et al., varied with pupfish body size, but that relationship differed ⁄ 2001). Given those structural similarities, we interpret this newly between females and males (sex size category interaction: identified lnpep cDNA from pupfish as encoding an aminopeptidase F1,43 = 9.130, p = 0.0044). The greatest difference in telencephalic homologous to human LNPEP. lnpep mRNA levels was observed between dominant males and Subsequent BLAST analyses using this pupfish LNPEP protein females, with dominant females exhibiting higher telecephalic identified homologous LNPEP-like proteins in taxa spanning major lnpep mRNA abundance that dominant males (Fig. 5b). We did evolutionary groups of fishes (e.g., Actinopterygii, Sarcopterygii, not observe any associations between individual behavior (PC1 Chondrichthyes), as well as tetrapods. The deduced LNPEP-like or PC2 values) and lnpep mRNA abundance in the telecephalon. proteins from those taxa all shared the zinc cofactors binding motif

‘HEXXH(X)18E,’ as well as the substrate binding site ‘GAMEN,’ with 4. Discussion the sole exception of snakes, which had a modified putative sub- strate binding site motify of ‘AGMEN.’ That structural conservation The regulation of social and sexual behaviors by the VP/VT and across classes of vertebrates implies strong evolutionary conserva- the OT/MT/IT families of nonapeptides is well established, but tion of the peptide substrate and zinc cofactor binding sites, but whether those behavioral effects are in part mediated by regula- also raises interesting questions concerning the potential evolu- tory changes in nonapeptide degradation and clearance is not tionary diversification of LNPEP function in snakes that should be known. In humans, LNPEP has been identified as an aminopepti- explored in future studies. E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 67

While enzyme kinetic studies are required to confirm the pep- The function of LNPEP in the mammalian brain remains tide specificity of these LNPEP enzymes in pupfish and other unknown, although there is limited evidence that LNPEP may influ- non-mammalian vertebrates, qRT-PCR analyses did reveal distinct ence cognition and memory (Ebstein et al., 2012). Pharmacological lnpep mRNA expression patterns across tissues, with high levels of LNPEP inhibitors have been observed to enhance spatial and lnpep transcripts in the ovary and brain, and moderate mRNA aversion-related memories in rats (e.g., Albiston et al., 2011; abundance in the gill and skeletal muscle tissues. Transcripts Andersson and Hallberg, 2012). However, given the variety of pep- encoding lnpep were also detected in the liver, gastrointestinal tides metabolized by LNPEP, it remains unresolved whether those tract, and kidney at low abundance, but appear to be at very low cognitive effects of LNPEP inhibition are related to changes in non- relative levels in the pituitary gland. That distributional pattern, apeptide inactivation or to the degradation of other peptides such in part, parallels the known production sites or target tissues for as angiotensin IV or somatostatin (e.g., Gard, 2008; Gard et al., VT in teleost fishes (Balment et al., 2006), including VT’s role in 2007). Interestingly, Pham et al. (2009) did not observe any water retention via the kidney and intestine (Amer and Brown, changes in maternal behaviors including pup feeding, nurturing, 1995; An et al., 2008; Balment et al., 1993; Martos-Sitcha et al., and retrieval in female LNPEP knockout mice. These LNPEP knock- 2015a; Warne et al., 2005), ion transport across the gill epithelium out mice also exhibited a typically gestation period, litter size, lit- (e.g., An et al., 2008; Lema, 2010; Martos-Sitcha et al., 2014, ter number, and time of parturition. Pham et al. (2009) interpreted 2015b), and the modulation of behaviors including aggression, the absence of any detectable changes in behavior or reproductive courtship, and amphibious movements (e.g., Backstrom and processes with LNPEP knockout as potentially indicative of Winberg, 2009; Greenwood et al., 2008; Huffman et al., 2015; taxonomic-level variation in LNPEP function – given that signifi- Kagawa, 2013; Larson et al., 2006, Lema and Nevitt, 2004a; cant LNPEP secretion into maternal serum may only occur in homi- Sakamoto et al., 2015; Santangelo and Bass, 2006; Semsar et al., nids (e.g., Yamahara et al., 2000) – and suggested that rodents may 2001). VT has also been shown to regulate 17b- produc- not be a useful model organism for testing hypotheses about the tion from the teleost fish ovary (Singh and Joy, 2009), as well as functions of LNPEP during human pregnancy. Even so, the absence release of the oocyte maturation hormone 17,20-dihydroxy-4- of detectable changes in behavior in these LNPEP knockout mice pregnen-3-one, presumably via VT’s action through a V1-type suggests that LNPEP enzymatic activity may not be critically receptor in the ovary (Singh and Joy, 2011). Several types of VT important to the performance of maternal care. receptors are present in both the ovarian and testicular tissues of The limited data testing whether LNPEP protein or transcript teleosts (Joy and Chaube, 2015; Lema, 2010; Lema et al., 2012; levels vary with behavioral phenotype stems from the dearth of Rawat et al., 2017). Rawat et al. (2017) recently observed that tran- studies that have examined LNPEP expression in a behavioral con- scripts encoding the teleost V1-type receptors V1a1 and V1a2 are text. To our knowledge, the findings presented here for pupfish predominantly expressed in the follicular layer, while mRNAs for represent the first evidence for variation in hypothalamic lnpep the V2-type receptor V2a were present in oocytes, supporting transcript abundance between socially dominant and subordinate expectations that VT function is likely compartmentalized and individuals of any species. Our results point to subordinate female varying across cell types in the ovary. and male pupfish having higher hypothalamic lnpep mRNA levels In accordance with LNPEP’s role in regulating female pregnancy compared to dominant fish of the same sex. That observed varia- in mammals, LNPEP protein is abundant in the placenta (Nomura tion in relative lnpep mRNA abundance with social status implies et al., 2002, 2005; Small and Watkins, 1975), where a modified, sol- that changes in LNPEP activity may be an overlooked component uble form of LNPEP is released into maternal serum via cleavage of to the nonapeptide regulation of behavior in fishes. In pupfish, membrane-bound LNPEP from syncytiotrophoblast cells (Iwase prior studies have demonstrated that VT regulates aggression in et al., 2001; Yamahara et al., 2000). The rate of LNPEP release into males (Lema and Nevitt, 2004a), and changes in hypothalamic VT the local extracellular environment has been shown to be regu- immunoreactivity and prepro-vasotocin (proVT) mRNA levels – as lated by OT action via OT receptors (Nakamura et al., 2000), indi- well as mRNA levels for select VT receptors – have been linked to cating that – at least in some tissues – nonapeptides themselves variation in aggressive behaviors in pupfish in several contexts, induce LNPEP translocation from intracellular vesicles to the including social dominance rank (Lema et al., 2015), population plasma membrane where cleavage to the soluble form occurs. differences (Lema and Nevitt, 2004b; Lema, 2006; Lema and Indeed, OT stimulation has been observed to increase membrane Kitano, 2013), and diurnal patterns of behavioral variation (Lema LNPEP protein over 3-fold in human vascular endothelial cells et al., 2010). While experimental intraperitoneal administration (Nakamura et al., 2000). of exogenous VT was observed to decrease aggression in male pup- LNPEP protein has also, however, been detected in other fish (Lema and Nevitt, 2004a), socially dominant male pupfish had mammalian reproductive structures including the myometrium higher relative proVT mRNA levels in the hypothalamus compared layers and luminal epithelium of the uterus (e.g., Mustafa to socially subordinate males (Lema et al., 2015). Likewise, individ- et al., 2004), as well as in tissues including the brain, kidney, ual dominant males that behave more aggressively also were heart and intestine (Matsumoto et al., 2001), suggesting broader found to express elevated hypothalamic proVT mRNA levels com- functions for LNPEP beyond placenta regulation of oxytocin pared to dominant males engaging in aggression less frequently levels in maternal serum. In the rat brain, LNPEP protein has (Lema et al., 2015). been localized to the cerebral cortex, cerebellar cortex, and basal Similar associations between hypothalamic proVT mRNAs or VT ganglion and appears to be largely localized to neurons in these protein and social behaviors have been documented in other fishes, region (Matsumoto et al., 2001). In contrast to the placenta, and generally support the idea that VT signaling within the where LNPEP is synthesized as a membrane protein and then hypothalamus is linked to aggression and agonistic interactions. secreted into maternal serum (Iwase et al., 2001; Yamahara For instance, Greenwood et al. (2008) found that territorial male et al., 2000), brain LNPEP protein was strongly associated with Astatotilapia burtoni cichlid that performed more threat displays the intramembrane fractions of neurons (Matsumoto et al., had higher levels of proVT mRNA in gigantocellular neurons of 2001). A subsequent analysis of soluble and membrane fractions the preoptic area of the hypothalamus. In a separate study of the of cultured rat pheochromocytoma PC12 cells from the adrenal anemonefish Amphiprion ocellaris, Yaeger et al. (2014) observed medulla confirmed the presence of LNPEP protein in the mem- that juvenile males injected with the V1-type brane constituent, and not in the soluble, cytoplasmic compo- Manning compound behaved less aggressively and exhibited nent (Matsumoto et al., 2001). reduced numbers of neurons expressing c-Fos protein in the 68 E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 preoptic area, suggesting that inhibition of hypothalamic VT action References may reduce both aggression and hypothalamic neural activity. And, Teles et al. (2016) observed rapid changes in both VT and IT Albers, H.E., 2012. The regulation of social recognition, social communication and aggression: vasopressin and the social behavior neural network. Horm. Behav. peptides in the brain of zebrafish (Danio rerio) following aggressive 61, 283–292. interactions; the direction and extent of change in VT and IT con- Albiston, A.L. et al., 2011. Identification and development of specific inhibitors for centrations, however, varied both across brain regions and depend- insulin-regulated aminopeptidase as a new class of cognitive enhancers. Brit. J. ing on the outcome (win or loss) of a fish’s behavioral interactions. Pharmacol. 164, 37–47. Amer, S., Brown, J.A., 1995. Glomerular actions of arginine vasotocin in the in situ Given the findings of those and other studies, and the associa- perfused trout kidney. Amer. J. Physiol. Reg. Integr. Comp. Physiol. 269, R775– tion between elevated hypothalamic proVT mRNAs and elevated R780. aggression observed in pupfish (Lema et al., 2015), one possible An, K.W. et al., 2008. Cloning and expression of aquaporin 1 and arginine vasotocin receptor mRNA from the black porgy, Acanthopagrus schlegeli: effect of interpretation of our findings here is that subordinate male and freshwater acclimation. Fish Physiol. Biochem. 34, 185–194. female pupfish exhibiting elevated lnpep mRNAs have higher rates Andersson, H., Hallberg, M., 2012. Discovery of inhibitors of insulin-regulated of VT inactivation in the hypothalamus, and thus lower VT avail- aminopeptidase as cognitive enhancers. Intern. J. Hyperten. 2012, 789671. http://dx.doi.org/10.1155/2012/789671. ability for local release within the brain. As prior studies in fishes Backstrom, T., Winberg, S., 2009. Arginine-vasotocin influence on aggressive have shown, however, specific neuronal groupings within the behavior and dominance in rainbow trout. Physiol. Behav. 96, 470–475. hypothalamus can exhibit distinct patterns of VT mRNA or protein Balment, R.J. et al., 1993. Arginine vasotocin and fish osmoregulation. Fish Physiol. Biochem. 11, 189–194. expression, and the relationships between behavior and neural VT Balment, R.J. et al., 2006. Arginine vasotocin a key hormone in fish physiology and peptide availability in each of those groupings is complex and can behaviour: a review with insights from mammalian models. Gen. Comp. be dependent on behavioral or ecological context (e.g., Goodson Endocrinol. 147, 9–16. Banegas, I. et al., 2010. Lateralized response of oxytocinase activity in the medial et al., 2009; Greenwood et al., 2008; Lema and Kitano, 2013; prefrontal cortex of a unilateral rat model of Parkinson’s disease. Behav. Brain Lema, 2006; Lema et al., 2015). Additional studies that develop Res. 213, 328–331. methods for localizing LNPEP protein and quantifying LNPEP activ- Banerjee, P. et al., 2017. Structural and functional diversity of nonapeptide ity in the fish hypothalamus are therefore needed to test this idea hormones from an evolutionary perspective: a review. Gen. Comp. Endocrinol. 241, 4–23. further, and could go a long way toward localizing the specific neu- Burbach, J.P.H. et al., 2006. Oxytocin: synthesis, secretion, and reproductive ral networks wherein LNPEP activity might contribute to variation functions. In: Neill, J.D. (Ed.), Knobil and Neill’s Physiology of Reproduction. in behavior. Even so, however, our observation that hypothalamic Elsevier Inc., St Louis, MO, pp. 3055–3128. Caldwell, H.K. et al., 2008. Vasopressin: behavioral roles of an ‘‘original” lnpep mRNA levels differ between socially dominant and subordi- . Prog. Neurobiol. 84, 1–24. nate pupfish points to the possibility that the degradation rate of Ebstein, R.P. et al., 2012. The contributions of oxytocin and vasopressin pathway nonapeptides may be regulated in some brain regions and con- genes to human behavior. Horm. Behav. 61, 359–379. Ferris, C.F. et al., 1997. Vasopressin/serotonin interactions in the anterior tribute to variation in behavioral phenotype, presumably by alter- hypothalamus control aggressive behavior in golden hamsters. J. Neurosci. 17, ing local VT and IT availability. Given its novelty, that finding 4331–4340. should be confirmed in future studies of fishes and other verte- Gard, P.R., 2008. Cognitive-enhancing effects of angiotensin IV. BMC Neurosci. 9 (suppl. 2), S15. brates that already have established nonapeptide effects on their Gard, P.R. et al., 2007. Interactions of angiotensin IV and oxytocin on behavior in behavior. Such future studies should not only measure gene tran- mice. J. Renin-Angiotensin-Aldosterone Sys. 8, 133–138. http://dx.doi.org/ script abundance but also quantify hypothalamic VT and IT peptide 10.3317/jraas.2007.016. Gasteiger, E. et al., 2005. Protein Identification and Analysis Tools on the ExPASy levels. Associations between nonapeptide production, brain non- Server. In: Walker, J.M. (Ed.), The Proteomics Protocols Handbook. Humana apeptide receptor distribution, and sociosexual behaviors have Press, pp. 571–607. now been demonstrated across vertebrates (e.g., Albers, 2012; Gibbs, D.M., 1986. Vasopressin and oxytocin: hypothalamic modulators of the stress Burbach et al., 2006; Caldwell et al., 2008; Goodson and Bass, response: a review. Psychoneuroendocrinology 11, 131–139. Godwin, J., Thompson, R., 2012. Nonapeptides and social behavior in fishes. Horm. 2001; Goodson and Thompson, 2010; Lim and Young, 2006; Behav. 61, 230–238. Thompson and Walton, 2013). Given that LNPEP homologues also Goodson, J.L., 1998. Territorial aggression and dawn song are modulated by septal appear to be present in all major classes of vertebrates, future stud- vasotocin and vasoactive intestinal polypeptide in male field sparrows (Spizella pusilla). Horm. Behav. 34, 67–77. ies should continue to explore what role, if any, hormone inactiva- Goodson, J.L., Bass, A.H., 2001. Social behavior functions and relates anatomical tion by LNPEP might contribute to the regulation of social and characteristics of vasotocin/vasopressin systems in vertebrates. Brain Res. Rev. affiliative behaviors by nonapeptides. 35, 246–265. Goodson, J.L., Thompson, R.R., 2010. Nonapeptide mechanisms of social cognition, behavior and species-specific social systems. Curr. Opin. Neurbiol. 20, 784–794. Goodson, J.L. et al., 2009. Dynamic neuromodulation of aggression by vasotocin: Acknowledgments influence of social context and social phenotype in territorial songbirds. Biol. Lett. 5, 554–556. This research was supported by funding from California Greenwood, A.K. et al., 2008. Expression of arginine vasotocin in distinct preoptic regions is associated with dominant and subordinate behaviour in an African Polytechnic State University’s College of Science and Mathematics cichlid fish. Proc. R. Soc. B Biol. Sci. 275, 2393–2402. College Based Fee (CBF) undergraduate program awards to E.A.E., Hattori, A. et al., 1999. Molecular cloning of adipocyte-derived leucine K.A.W. and K.E.N., and by a California State University (CSU) Pro- aminopeptidase highly related to placental leucine aminopeptidase/ oxytocinase. J. Biochem. 125, 931–938. gram for Education and Research in Biotechnology (CSUPERB) Heinrichs, M., Domes, G., 2008. and social behaviour: effects of Development Grant to S.C.L. The authors thank two anonymous oxytocin and vasopressin in humans. Prog. Brain Res. 170, 337–350. reviewers for insightful comments that improved the quality of Heinrichs, M. et al., 2009. Oxytocin, vasopressin, and human social behavior. Front. Neuroendocrinol. 30, 548–557. this manuscript. All experimental procedures were approved by Herbst, J.J. et al., 1997. Insulin stimulates cell surface aminopeptidase activity the Animal Care and Use Committee of California Polytechnic State toward vasopressin in adipocytes. Amer. J. Physiol. 272, E600–E606. University (Protocol # 1507). Hermans, S.J. et al., 2015. Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides. Prot. Sci. 24, 190–199. Hernández, J. et al., 2015. Interaction of neuropeptidase activities in cortico-limbic regions after acute restraint stress. Behav. Brain Res. 287, 42–48. Appendix A. Supplementary data Huffman, L.S. et al., 2015. Arginine vasotocin regulates social ascent in the African cichlid fish Astatotilapia burtoni. Gen. Comp. Endocrinol. 212, 106–113. Supplementary data associated with this article can be found, in Insel, T.R., Young, L.J., 2000. Neuropeptides and the evolution of social behavior. Curr. Opin. Neurobiol. 10, 784–789. the online version, at http://dx.doi.org/10.1016/j.ygcen.2017.06. Iwase, A. et al., 2001. Characterization of a secretase activity for placental leucine 002. aminopeptidase. Arch. Biochem. Biophys. 393, 163–169. E.A. Elkins et al. / General and Comparative Endocrinology 250 (2017) 58–69 69

Johnson, A.O. et al., 2001. A di-leucine sequence and a cluster of acidic amino acids Nielsen, S. et al., 1995. Vasopressin increases water permeability of kidney are required for dynamic retention in the endosomal recycling compartment of collecting duct by inducing translocation of aquaporin-CD water channels to fibroblasts. Mol. Biol. Cell 12, 367–381. plasma membrane. Proc. Natl. Acad. Sci. U.S.A. 92, 1013–1017. Joy, K.P., Chaube, R., 2015. Vasotocin – a new player in the control of oocyte Nomura, M. et al., 2002. Differential distribution of placental leucine maturation and ovulation in fish. Gen. Comp. Endocrinol. 221, 54–63. aminopeptidase/oxytocinase and aminopeptidase A in human trophoblasts of Kagawa, N., 2013. Social rank-dependent expression of arginine vasotocin in normal placenta and complete hydatidiform mole. Placenta 23, 631–639. distinct preoptic regions in male Oryzias latipes. J. Fish Biol. 82, 354–363. Nomura, S. et al., 2005. Gene regulation and physiological function of placental Kandror, K.V. et al., 1994. The major protein of GLUT4-containing vesicles, gp160, leucine aminopeptidae/oxytocinase during pregnancy. Biochim. Biophys. Acta has aminopeptidase activity. J. Biol. Chem. 269, 30777–30780. 1751, 19–25. Keller, S.R. et al., 1995. Cloning and characterization of a novel insulin-regulated Pham, V. et al., 2009. Reproduction and maternal behavior in insulin-regulated membrane aminopeptidase from Glut4 vesicles. J. Biol. Chem. 270, 23612– aminopeptidase (IRAP) knockout mice. Peptides 30, 1861–8165. 23618. Pittman, Q.L. et al., 1982. Central effects of arginine vasopressin on blood pressure Kim, N. et al., 2016. Vasopressinase activity: a potential early biomarker for in rats. Endocrinology 110, 1058–1060. detecting cardiopulmonary bypass-associated acute kidney injury? Thoracic Ramírez, M. et al., 2011. Bilateral distribution of angiotensinase and oxytocinase

Cardiovas. Surgeon 64, 555–560. http://dx.doi.org/10.1055/s-0035-1564446. activity (IRAP/AT4) in brain of normotensive and hypertensive rats with Kumar, S. et al., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 experimental hemiparkinsonism. Collection Symp. Ser. 13, 111–117. http://dx. for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. doi.org/10.1135/css201113111. Larkin, M.A. et al., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, Rawat, A. et al., 2017. Effects of the fish spawning inducer ovaprim on vasotocin 2947–2948. receptor gene expression in brain and ovary of the catfish Heteropneustes fossilis Larson, E.T. et al., 2006. Aggression and vasotocin are associated with dominant- with a note on differential transcript expression in ovarian follicles. Gen. Comp. subordinate relationships in zebrafish. Behav. Brain Res. 167, 94–102. Endocrinol. 241, 24–32. Lausten, P.G. et al., 1997. The complete amino acid sequence of human placental Rogi, T. et al., 1996. Human placental leucine aminopeptidase: a new member of oxytocinase. Biochim. Biophys. Acta 1352, 1–7. type II membrane-spanning zinc metallopeptidase family. J. Biol. Chem. 271, Lema, S.C., 2006. Population divergence in plasticity of the AVT system and its 56–61. association with aggressive behaviors in a Death Valley pupfish. Horm. Behav. Ross, S.A. et al., 1996. Characterization of the insulin-regulated membrane 50, 183–193. aminopeptidase in 3T3-L1 adipocytes. J. Biol. Chem. 271, 3328–3332. Lema, S.C., 2010. Identification of multiple vasotocin receptor cDNAs in teleost fish: Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for sequences, phylogenetic analysis, sites of expression, and regulation in the reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. hypothalamus and gill in response to hyperosmotic challenge. Mol. Cell. Sakamoto, T. et al., 2015. Neurohypophysial hormones regulate amphibious Endocrinol. 321, 215–230. behavior in the mudskipper goby. PLoS One 10, e0134605. http://dx.doi.org/ Lema, S.C., Kitano, J., 2013. Hormones and phenotypic plasticity: implications for 10.1371/journal.pone.0134605. the evolution of integrated adaptive phenotypes. Curr. Zool. 59, 506–529. Santangelo, N., Bass, A.H., 2006. New insights into neuropeptide modulation of Lema, S.C., Nevitt, G.A., 2004a. Exogenous vasotocin alters aggression during aggression: field studies of arginine vasotocin in a territorial tropical agonistic exchanges in male Amargosa River pupfish (Cyprinodon nevadensis damselfish. Proc. Biol. Soc. B 273, 3085–3092. amargosae). Horm. Behav. 46, 628–637. Segarra, A.B. et al., 2016. Neuropeptidase activities in plasma after acute restraint Lema, S.C., Nevitt, G.A., 2004b. Variation in vasotocin immunoreactivity in the brain stress. Interaction with cortico-limbic areas. Acta Neuropsych., 1–5 of recently isolated populations of a Death Valley pupfish, Cyprinodon Semsar, K. et al., 2001. Manipulations of the AVT system shift social status and nevadensis. Gen. Comp. Endocrinol. 135, 300–309. related courtship and aggressive behavior in the bluehead wrasse. Horm. Behav. Lema, S.C. et al., 2010. Diurnal rhythms of behavior and brain mRNA expression for 40, 21–31. arginine variation, isotocin, and their receptors in wild Amargosa pupfish Singh, V., Joy, K.P., 2009. Relative in vitro seasonal effects of vasotocin and isotocin (Cyprinodon nevadensis amargosae). Mar. Fresh. Behav. Physiol. 43, 257–281. on ovarian steroid hormone levels in the catfish Heteropneustes fossilis. Gen. Lema, S.C. et al., 2012. Variation in gene transcript profiles of two V1a-type arginine Comp. Endocrinol. 162, 257–264. vasotocin receptors among sexual phases of bluehead wrasse (Thalassoma Singh, V., Joy, K.P., 2011. Vasotocin induces final oocyte maturation and ovulation bifasciatum). Gen. Comp. Endocrinol. 179, 451–464. through production of a maturation-inducing steroid in the catfish Lema, S.C. et al., 2015. Arginine vasotocin, isotocin and nonapeptide receptor gene Heteropneustes fossilis. Gen. Comp. Endocrinol. 174, 15–21. expression link to social status and aggression in sex-dependent patterns. J. Small, C.W., Watkins, W.B., 1975. Oxytocinase—immunohistochemical Neuroendocrinol. 27, 142–157. demonstration in the immature and term human placenta. Cell Tissue Res. Lim, M.M., Young, L.J., 2006. Neuropeptidergic regulation of affiliative behavior and 162, 531–539. social bonding in animals. Horm. Behav. 50, 506–517. Soltz, D.L., Naiman, R.A., 1978. The natural history of native fishes in the Death Martos-Sitcha, J.A. et al., 2014. Variations in the expression of vasotocin and isotocin Valley system. Nat. Hist. Mus. Los. Angeles County, Sci. Ser. 30, 1–76. receptor genes in the gilthead sea bream Sparus aurata during different osmotic Teles, M.C. et al., 2016. Agonistic interations elicit rapid changes in brain challenges. Gen. Comp. Endocrinol. 197, 5–17. nonapeptide levels in zebrafish. Horm. Behav. 84, 57–63. Martos-Sitcha, J.A. et al., 2015a. Vasotocin and isotocin regulate aquaporin 1 Thompson, R.R., Walton, J.C., 2013. Social regulatory functions of vasotocin and function in the sea bream. J. Exp. Biol. 218, 684–693. isotocin in fish. In: Choleris, E., Pfaff, D.W., Kavaliers, M. (Eds.), Oxytocin, Martos-Sitcha, J.A. et al., 2015b. AVT and IT regulate ion transport across the Vasopressin, and Related Peptides in the Regulation of Behavior. Cambridge opercular epithelium of killifish (Fundulus heteroclitus) and gilthead sea bream University Press, Cambridge, UK, pp. 75–96. (Sparus aurata). Comp. Biochem. Physiol. A 182, 93–101. Tian, C. et al., 2016. Associations between serum placental leucine aminopeptidase Mastick, C.C. et al., 1994. Characterization of a major protein in GLUT4 vesicles: and pregnancy outcomes. Intern. J. Gynecol. Obstetr. 135, 255–258. concentration in the vesicles and insulin-stimulated translocation to the plasma Titus, M.A. et al., 1960. Plasma aminopeptidase activity (oxytocinase) in pregnancy membrane. J. Biol. Chem. 269, 6089–6092. and labor. Amer. J. Obstetr. Gynecol. 80, 1124–1128. Matsumoto, H. et al., 2000. Characterization of a recombinant soluble form of Tobin, V.A. et al., 2014. Oxytocinase in the female rat hypothalamus: a novel human placental leucine aminopeptidase/oxytocinase expressed in Chinese mechanism controlling oxytocin neurones during lactaction. J. hamster ovary cells. Eur. J. Biochem. 267, 46–52. Neuroendocrinol. 26, 205–216. Matsumoto, H. et al., 2001. Expression of placental leucine aminopeptidase/ Tsujimoto, M., Hattori, A., 2005. The oxytocinase subfamily of M1 aminopeptidases. oxytocinase in neuronal cells and its action on neuronal peptides. Eur. J. Biochim. Biophys. Acta 1751, 9–18. Biochem. 268, 3259–3266. Tsujimoto, M. et al., 1992. Identification of human placental leucine Meylan, S. et al., 2017. Arginine vasotocin inhibits social interactions and enhances aminopeptidase as oxytocinase. Arch. Biochem. Biophys. 292, 388–392. essential activities in male common lizards (Zootoca vivipara). Gen. Comp. Villarejo, A.B. et al., 2012. Angiotensinase and vasopressinase activities in Endocrinol. 243, 10–14. hypothalamus, plasma, and kidney after inhibition of angiotensin-converting Mizutani, S., Tomoda, Y., 1992. Oxytocinase: placental cystine aminopeptidase or enzyme: basis for a new working hypothesis. Horm. Metabol. Res. 44, 152–154. placental leucine aminopeptidase. Sem. Reprod. Endocrinol. 10, 146–153. Warne, J. et al., 2005. Altered plasma and pituitary arginine vasotocin and Mizutani, S. et al., 1976. Placental and non-placental leucine aminopeptidases hypothalamic provasotocin expression in flounder (Platichthys flesus) during normal pregnancy. Clin. Biochem. 9, 16–18. following hypertonic challenge and distribution of vasotocin receptors within Mizutani, S. et al., 2007. Essential role of placental leucine aminopeptidase in the kidney. Gen. Comp. Endocrinol. 144, 240–247. gynecologic malignancy. Expert Opin. Ther. Targets 11, 453–461. Yaeger, C. et al., 2014. Blockade of arginine vasotocin signaling reduces aggressive Mustafa, T. et al., 2004. Oxytocinase/insulin-regulated aminopeptidase is behavior and c-Fos expression in the preoptic area and periventricular nucleus distributed throughout the sheep, female reproductive tract and is regulated of the posterior tuberculum in male Amphiprion ocellaris. Neuroscience 267, by oestrogen in the uterus. Reg. Peptides 122, 85–89. 205–218. Nagasaka, T. et al., 1997. Immunohistochemical localization of placental leucine Yamahara, N. et al., 2000. Placental leucine aminopeptidase/oxytocinase in aminopeptidase/oxytocinase in human placental, fetal and adult tissues. maternal serum and placenta during normal pregnancy. Life Sci. 66, 1401–1410. Reprod. Fertil. Dev. 9, 747–753. Young, L.J., Flanagan-Cato, L.M., 2012. Editorial comment: oxytocin, vasopressin and Nakamura, H. et al., 2000. Oxytocin stimulates the translocation of oxytocinase of social behavior. Horm. Behav. 61, 227–229. human vascular endothelial cells via activation of oxytocin receptors. Endocrinology 141, 4481–4485.