Current Biology Magazine two. Ethologist Nikolaas Tinbergen A related question is whether Correspondence emphasized the functional role of animals and humans perceive musical song in mate selection and territorial sounds in a similar manner. Although Vectored antibody defense, whereas ornithologist W.H. research in this fi eld is still in its early Thorpe distinguished between songs stages, studies on pitch, timbre, and gene delivery and calls on the basis of duration and rhythm perception in animals suggest complexity, considering the longer that vocal learning species may have mediates long-term and more complex vocalizations to better auditory discrimination abilities contraception be songs. More recently, cognitive than non-vocal learning species, scientists such as Tecumseh Fitch and that, in certain contexts, non- have focused on whether a vocal human species do appear to have Juan Li1, Alejandra I. Olvera1, behavior is learned or innate, treating preferences for some musical sounds Omar S. Akbari1, Annie Moradian2, all learned vocalizations as songs over others. Encouragingly, interest Michael J. Sweredoski2, Sonja Hess2, and all innate vocalizations as calls, for this topic, and more generally and Bruce A. Hay1,* regardless of their aesthetic qualities. for a comparative approach to This ‘vocal learning’ model has zoomusicology, is steadily growing. Development of non-surgical become infl uential, in part because While it may not be possible to methods of long-term or permanent vocal learning species tend to be determine conclusively whether animal contraception remains a challenge. those that most frequently display song is music, getting musicologists, Towards this objective, we show proto-musical behaviors. Moreover, scientists, and philosophers to join that intramuscular injection of a rhythmic entrainment has until now forces in zoomusicological inquiry will replication-incompetent, recombinant only been reported in vocal learning surely lead to a better understanding adeno-associated virus (rAAV) species. of animal song, and to a better designed to express an antibody Only a few animal clades are thus understanding of human music as well. that binds gonadotropin-releasing far known to include vocal learning hormone (GnRH), a master regulator species — among birds, parrots, Where can I fi nd out more? of reproduction in vertebrates, hummingbirds, and oscine songbirds, Doolittle, E.L., and Brumm, H. (2012). O Canto do results in long-term infertility in Uirapuru: Consonant intervals and patterns and among mammals, humans, in the song of the musician wren. J. Interdisc. male and female mice. Female mice cetaceans, pinnipeds, elephants, Music Stud. 6, 55–85. are also rendered infertile through Doolittle, E.L., Gingras, B., Endres, D.M. and and bats. Animals of these species Fitch, W.T. (2014). Overtone-based pitch rAAV-dependent expression of an must be exposed to representative selection in hermit thrush song: Unexpected antibody that binds to the zona adult songs in order to develop a convergence with scale construction in pellucida (ZP), a glycoprotein matrix human music. Proc. Natl. Acad. Sci. USA 111, typical adult song. One consequence 16616–16621. that surrounds the egg and functions of songs being a learned rather Fitch, W.T. (2006). The biology and evolution of as a sperm-binding site. Many music: A comparative perspective. Cognition than an innate behavior is that a 100, 173–215. proteins known or suspected to be number of songbird species display Gingras, B. (2014). Cross-species comparisons. In important for reproduction can be geographically-based dialects. This is Music in the Social and Behavioral Sciences: targeted, potentially reversibly, using An encyclopedia, vol. 1, W.F. Thompson, ed. also the case for humpback whales, (Thousand Oaks, CA: SAGE Publications). pp. this approach, which we refer to as whose songs tend to be similar within 287–290. vectored contraception (VC). Guinee, L.N., and Payne, K.B. (1988). Rhyme-like a group but vary across geographic repetitions in songs of humpback whales. Mature GnRH is a 10-amino-acid areas. Ethology 79, 295–306. peptide of identical sequence in Hauser, M.D., and McDermott, J. (2003). The most mammals, produced in the evolution of the music faculty: A comparative What lies ahead for zoomusicology? perspective. Nat. Neurosci. 6, 663–668. hypothalamus. GnRH is released into Although zoomusicology has already Hoeschele, M., Merchant, H., Kikuchi, Y., Hattori, the median eminence, from which it Y., and ten Cate, C. (2015). Searching for given us many intriguing fi ndings, the the origins of musicality across species. diffuses into the hypophyseal portal fi eld is still in its infancy. There are Philos. Trans. R. Soc. Lond. B. Biol. Sci. 370, capillary system, which carries it to the more than 4000 species of birds, and 20140094. anterior pituitary, where it stimulates Martinelli, D. (2009). Of Birds, Whales and Other a growing list of mammals, recognized Musicians: An Introduction to Zoomusicology the release of luteinizing hormone (LH) as vocal learners. Each has its own (London: University of Scranton Press). and follicle-stimulating hormone (FSH) Patel, A.D., Iversen, J.R., Bregman, M.R., and pattern of song acquisition and Schulz, I. (2009). Experimental evidence into the general circulation. FSH and structure, and must be studied for synchronization to a musical beat in a LH promote the formation of gametes individually to be understood. Basic nonhuman animal. Curr. Biol. 19, 827–830. and the production of sex steroids. Rothenberg. D, Roeske, T.C., Voss, H.U., Naguib, pitch and rhythmic analysis constitute M., and Tchernichovski, O. (2014). Investigation Thus, loss of GnRH results in male an obvious fi rst approach to analyzing of musicality in birdsong. Hear. Res. 308, and female infertility, and loss of other animal songs, and more advanced 71–83. steroid-hormone-dependent traits [1]. methods of structural, spectral Inhibition of GnRH is considered to 1 and timbral analysis, including Music Department, Cornish College of the be an attractive strategy in animals Arts, 1000 Lenora St., Seattle, WA, USA. computational approaches originating when the goal is to inhibit fertility 2Department of Cognitive Biology, University from the burgeoning fi eld of music of Vienna, Althanstrasse 14, Vienna, Austria. and sex-steroid-dependent traits, information retrieval, potentially hold E-mail: [email protected], such as aggression and territoriality. even greater promise. [email protected] Vaccination with GnRH can inhibit
R820 Current Biology 25, R811–R826, October 5, 2015 ©2015 Elsevier Ltd All rights reserved Current Biology Magazine
st nd Control Anti-GnRH (SMI41) fertility, but these effects are often 19 st 9(1 ) 9*(2 ) 8 13* (1 ) nd A 42 females 42 with no progeny 3(2 ) 2+1 nd temporary [2]. no mate 1st mate 23 6(2 ) 5+1 C D P progeny CL Af We used mass spectrometry to 104 CL P determine the protein sequence of the 103 2 10 * #17
#12 Ovary anti-GnRH monoclonal antibody SMI41 #21 #6 101 #15 A ZPr [3] (see Supplemental Experimental 100 A * A 200µm 200µm Procedures and Figure S1A in the 10-1
-2 Supplemental Information). Gene Antibody titer (µg/ml) 10 048121620 24 28 32 36 40 44 48 E F synthesized heavy and light chains Weeks post-AAV injection were shown to produce a high- S
st nd affi nity anti-GnRH antibody (Figure 15 9(1st) 6(1 ) 6(2 ) 6 B 21 males 21 with no progeny 3(2nd) 3 L nd no mate 1st mate 6 6(2 ) 6 Testis S1B) and were introduced into an progeny AAV2/8 vector [4], generating AAV- 104 3 µ µ SMI41 (Supplemental Experimental 10 100 m 100 m 102 G H Procedures). Viral particles were 101 administered to mice at several doses 100 through injection of the gastrocnemius 10-1 -2
Antibody titer (µg/ml) 10 muscle (Table S1). Figure 1A,B, Figure 048121620 24 28 32 36 40 44 48 S1C and Table S1 summarize the Weeks post-AAV injection Epididymis 100µm 100µm results of mating experiments. Females 7 Control Anti-ZP2 (IE3) I 9(2nd) 9 12 females 12 with no progeny with titers <200 g/ml showed dose- no mate 1st mate 5 CL P J K A Af progeny A CL dependent subfertility (reduced progeny 100 CL CL numbers; Figure S1C), while females 10 Af CL with titers >200 g/ml were, with IC Ovary CL 1 several exceptions, infertile, as was one P A Af animal with a fi nal titer of 129.7 g/ml 0.1 P Antibody titer (µg/ml) µ 048121620 24 28 32 36 200µm 200 m (#17) (Figure 1A). The four exceptional Weeks post-AAV injection females (#6, #12, #15, #21) had titers >200 g/ml at the beginning of the Figure 1. Vectored expression of the anti-GnRH antibody SMI41 or the anti-ZP2 antibody IE3 four-week period in which pregnancy results in dose-dependent long-term infertility. was scored, but each experienced (A) SMI41 titers in females are plotted over time. Red line segments indicate four-week in- a signifi cant drop in titer by the end tervals during which a female became pregnant; black line segments indicate intervals dur- of week four (#6, 266.264.6 g/ ing which no pregnancies occurred. Individuals are labeled as in Table S1. Vertical dashed lines indicate points at which AAV-SMI41 females were introduced to new breeding partners. ml; #12, 201.7186.0 g/ml; #15, The number of females maintained with either the fi rst male mating partner (1st mate, green 219.230.3 g/ml; #21, 379.745.6 g/ regions), or a second mating partner (2nd, purple rectangles), is indicated. Asterisks identify ml), which may explain the restoration females who died. Note that some infertile females were euthanized for histology at weeks of fertility (Figure 1A, Table S1). 32 and 40 (Supplemental Experimental Procedures). (B) SMI41 titers in males, characterized Males with titers >100 g/ml were as in (A), but with female mating partners. (C) PAS-stained section from the ovary of control infertile (Figure 1B). Control animals 12-month-old female. Antral follicles (A), atretic follicles (Af) and multiple corpora lutea (CL) tested for fertility at 4–6 months, and are labeled. (D) PAS-stained section of ovary from sterile anti-GnRH-expressing 12-month- old female (#8). Primary follicles (P) and numerous remnants of the zona pellucida, associ- 12–14 months of age were, with one ated with follicle atresia (ZPr, arrows) are indicated. (E) H&E-stained section from testis of a exception, fertile (Figure S1D). 14-month-old control male. Sperm in the tubule lumen (S) and large Leydig cell islands (L) Ovaries of untreated females between tubules are marked. (F) H&E-stained section from 14-month-old AAV-SMI41 male contained multiple stages of (#48). (G) Section through the cauda epididymis of control male from (E). (H) Section through oogenesis, had few atretic follicles, the cauda epididymis of AAV-SMI41 male #48. (I) IE3 antibody titers of female mice injected and contained multiple corpora lutea, with AAV-IE3 are plotted over time as above. The blue line indicates a female (#74) who gave birth twice. (J) PAS-stained section from the ovary of a 7-month-old control female. (K) PAS- indicative of regular oocyte maturation stained section of ovary of 7-month-old AAV-IE3 female #75. and ovulation (Figure 1C). In contrast, ovaries from infertile anti-GnRH- controls ranged from 0.11 ng/ml epididymis (Figure 1G). In contrast, expressing females contained many to 8.01 ng/ml (n = 11; mean ± SEM, testes of high-titer infertile males atretic follicles, and preovulatory 1.65 ± 0.77). In contrast, testosterone lacked late-stage spermatids (Figure follicles and corpora lutea were was undetectable in infertile SMI41 1F), and sperm were absent from the not observed (Figure 1D). These males (n = 8). Testes of infertile males epididymis (Figure 1H), phenotypes phenotypes, indicative of a failure were also signifi cantly smaller than similar to those of males lacking GnRH to complete follicle development, those of age-matched controls (n = [1] or the subunit of LH [5]. resemble those of mice lacking 15; 12.57 ± 1.25 mm, vs n = 18; 30.2 ± The ZP consists of a matrix of GnRH [1]. 1.159 mm; p < 0.01) (Figure S1E,F). All three or four glycoproteins (ZP1–4) In males GnRH-dependent release stages of spermatogenesis, including synthesized by the growing oocyte, of LH is required for the production elongated spermatids, were present surrounding it and the early embryo. of testosterone by testicular Leydig in testes of control mice (Figure 1E), It serves as an initial binding site for cells. Serum testosterone levels of and sperm were abundant in the sperm, an interaction required for
Current Biology 25, R811–R826, October 5, 2015 ©2015 Elsevier Ltd All rights reserved R821 Current Biology Magazine sperm to penetrate the ZP and fuse ACKNOWLEDGEMENTS Correspondence with the egg plasma membrane [6]. Vaccination with solubilized ZP or We thank Janet Baer and Karen Lencioni for Eurasian isolated ZP proteins can bring about providing advice on a number of aspects of this work relating to mouse husbandry and infertility. Sometimes the effects are reproduction. Shirley Pease provided help reed warblers transient; alternatively, they can be with mouse superovulation and Ken Chan compensate for long-lasting, but may be associated with microscopy of eggs. Alejandro B. Balazs with reproductive tissue damage, due and Rachel Galimidi provided help with virtual magnetic to the induction of antibody- and/or AAV. Jost Vielmetter and the Caltech Protein T-cell-mediated responses [7]. Expression Center provided assistance with displacement We cloned rat IE3, a monoclonal protein production and SPR measurements. antibody that binds the amino terminus Cynthia Besch-Williford (IDEXX Bioresearch) Dmitry Kishkinev1,*, of mouse ZP2, a critical ligand for provided analysis of mouse histology. This Nikita Chernetsov2,3,*, taxon-specifi c sperm binding [8] work did not receive specifi c funding. The Alexander Pakhomov2,4, (Figure S2A). IE3 transiently inhibits Proteome Exploration Laboratory (A.M., 5,6 female mouse fertility when introduced M.J.S., S.H.) was supported by the Gordon Dominik Heyers , 5,6 as ascites fl uid through intraperitoneal and Betty Moore Foundation through Grant and Henrik Mouritsen injection [9]. Recombinant IE3 GBMF775, the Beckman Institute, and the localized to the egg ZP in vitro and NIH, through Grant 1S10RR029594-01A1. A. Displacement studies have shown in vivo (Figure S2B). When mating Olvera was supported by a Gates Millennium that long-distance, night-migrating was initiated 5 weeks after injection Scholars award. songbirds are able to perform true of AAV-IE3, seven out of twelve mice navigation from their fi rst spring expressing IE3 were infertile, while REFERENCES migration onwards [1,2]. True fi ve were fertile, but with reduced navigation requires both a map 1. Cattanach, B.M., Iddon, C.A., Charlton, H.M., litter sizes compared with controls Chiappa, S.A., and Fink, G. (1977). and a compass. Whereas birds (mean ± SEM, 3.8 ± 1.35 vs 14.7 Gonadotrophin-releasing hormone defi ciency are known to have sun, star, and ± 0.94; p < 0.01) (Figure 1I, Figure in a mutant mouse with hypogonadism. Nature magnetic compasses, the nature of 269, 338–340. S2C). However, over subsequent 2. Gupta, S.K., Shrestha, A., and Minhas, V. the map cues used has remained months all AAV-IE3 mice (titer range (2013). Milestones in contraceptive vaccines highly controversial. There is quite development and hurdles in their application. between 2.8 and 19.3 g/ml) failed to Hum. Vaccin. Immunother. 10, 1–15. strong experimental evidence for the produce progeny, with the exception 3. Knapp, R.J., and Sternberger, L.A. (1984). High involvement of olfactory map cues of one of the initially fertile group of affi nity monoclonal antibodies to luteinizing in pigeon and seabird homing [3]. In hormone-releasing hormone. Preparation and fi ve, who delivered a second litter binding studies. J. Neuroimmunol. 6, 361–371. contrast, the evidence for the use of of one during month three of mating 4. Balazs, A.B., Chen, J., Hong, C.M., Rao, D.S., magnetic map cues has remained Yang, L., and Baltimore, D. (2012). Antibody- (Figure S2D). Importantly, ovaries from based protection against HIV infection by weak and very little is known about infertile females expressing IE3 for 6 vectored immunoprophylaxis. Nature 481, the map cues used by long-distance months (Figure 1, compare panels K 81–84. migratory songbirds. In earlier 5. Ma, X., Dong, Y., Matzuk, M.M., and and J), as with those exposed to IE3 Kumar, T.R. (2004). Targeted disruption of experiments [2,4], we have shown transiently [9], appeared histologically luteinizing hormone beta-subunit leads that Eurasian reed warblers physically normal, and contained follicles at to hypogonadism, defects in gonadal displaced 1,000 km eastward from steroidogenesis, and infertility. Proc. Natl. multiple stages of development, as Acad. Sci. USA 101, 17294–17299. Rybachy to Zvenigorod (Figure 1) well as multiple corpora lutea. This, in 6. Avella, M.A., Xiong, B., and Dean, J. (2013). re-orient towards their breeding The molecular basis of gamete recognition conjunction with the observation that in mice and humans. Mol. Hum. Reprod. 19, destinations by changing their comparable numbers of eggs can be 279–289. orientation in Emlen funnels from the superovulated from control and AAV- 7. Gupta, S.K., Gupta, N., Suman, P., NE to the NW. We have also previously Choudhury, S., Prakash, K., Gupta, T., IE3 females (Figure S2B), suggests Sriraman, R., Nagendrakumar, S.B., and shown that this re-orientation cannot that vectored expression of anti-ZP Srinivasan, V.A. (2011). Zona pellucida-based be explained by a ‘jetlag effect’ [5]. We antibodies can induce long-term contraceptive vaccines for human and animal have now used this model system to utility. J. Reprod. Immunol. 88, 240–246. infertility without disrupting normal 8. Avella, M.A., Baibakov, B., and Dean, J. (2014). show that Eurasian reed warblers use follicular maturation. A single domain of the ZP2 zona pellucida geomagnetic map cues to determine protein mediates gamete recognition in mice and humans. J. Cell Biol. 205, 801–809. their position. SUPPLEMENTAL INFORMATION 9. East, I.J., Mattison, D.R., and Dean, J. (1984). We performed orientation Monoclonal antibodies to the major protein experiments in Emlen funnels with of the murine zona pellucida: effects on Supplemental information, including back- fertilization and early development. Dev. Biol. birds captured during spring migration 104, 49–56. ground, discussion, hurdles that must be in Rybachy (55° 09 N, 20° 52 E; Figure 1). The birds were housed overcome in implementation of vectored 1 Division of Biology and Biological outdoors in wood and cloth-net contraception, possible strategies for bring- 2 Engineering, Beckman Institute Proteome cages, which provided the birds ing about reversibility, experimental proce- Exploration Laboratory, dures, two fi gures and one table, can be California Institute of Technology, Pasadena, with a clear view of the sky and the found with this article online at http://dx.doi. CA 91125, USA. surroundings (see Figure S1 in the org/10.1016/j.cub.2015.08.002. *E-mail: [email protected] Supplemental Information). First,
R822 Current Biology 25, R811–R826, October 5, 2015 ©2015 Elsevier Ltd All rights reserved