Abstract
Individuals and populations time annual events such as migration and reproduction to match favorable times in their environment. Physiological preparations for reproduction rely on predictive cues such as day length to accurately time reproduction. In birds, preparation typically begins with light reception by the hypothalamus, which initiates multiple central and peripheral responses. We studied two closely related populations of a songbird, the dark-eyed junco, that live in a common winter environment but diverge in their timing of reproduction as spring approaches. One population is resident and initiates reproduction earlier than the other, which migrates northward prior to reproducing. We caught resident and migrant juncos from the field during early spring and collected hypothalamic and pituitary tissues. We used isobaric tandem mass tag (TMT) labeling to identify differentially expressed proteins (DEPs) as possible regulators of the seasonal divergence in reproductive timing. We found 3038 unique proteins expressed in the hypothalamus and pituitary proteome, among which we identified 75 DEPs. These were associated with hormones, neurotransmitter secretion, transport, neuropeptide synthesis, prohormone synthesis, neurogenesis, GnRH synthesis, release and stability, food intake, locomotion, and social behavior. Some of these proteins were associated with early breeding in resident juncos, and others were associated with increased food intake, fat metabolism, locomotor activity and phenology in migratory juncos. Our results provide new insight into the neuroendocrine regulation of the timing of reproduction and migration. This study provides the first evidence of a relationship between functional protein variation in the neuroendocrine tissues and seasonal divergence in reproductive timing.
Introduction
Each year as spring approaches and the climate warms in the north temperate zone, birds begin to breed or migrate northwards to their breeding grounds. This annual breeding or migration of birds reflects the seasonal recurrence of biological activities and is associated with the physiology of migration and reproduction. Previous research has shown that endogenous clocks are entrained by annual changes in day length (photoperiod), resulting in photo-induced phenotypes that correspond to seasonal life history states (LHSs; Helm et al., 2013). Each LHS is accompanied by characteristic changes in behavior, neural activity, physiology and hormones (Gwinner & Helm, 2003; Trivedi, Kumar, Rani, & Kumar, 2014). In species with a broad geographic range, closely related populations may differ in when they breed and whether they migrate. In some cases, populations co-occur geographically during winter, but because they differ in migration strategy, they also differ geographic distribution when breeding. In late winter early spring, the populations that do not migrate initiate reproductions while living side by side with the migratory populations that delay reproduction This system of differential response to the same environment thus provides a powerful model to investigate mechanistic differences at the neuroendocrine level associated with differences in the timing of reproduction.
The annual activation of seasonal reproduction is primarily regulated through the interaction of day length with the hypothalamic-pituitary-gonadal (HPG) axis (Cho, Hahn, MacDougall-Shackleton, & Ball, 1998). Annual change in day length varies with latitude such that birds breeding at higher latitudes often require longer days to initiate gonadal recrudescence (Dawson, 2013; Fudickar et al., 2016a, Fudickar, Greives, Atwell, Strocker, & Ketterson, 2016b; Singh et al., 2019). Photoperiodic responses depend on light perceived via encephalic photoreceptors falling during the stimulatory phase of a daily rhythm of sensitivity (Ball & Balthazart, 2003; Follett, Kumar, & Juss, 1992; Brandstätter, 2003; Yasuo, Watanabe, Okabayashi, Ebihara, & Yoshimura, 2003; Nakane et al., 2010). The neuropeptides and hormones synthesized and released from the hypothalamic-pituitary (HP) complex are critical to the initiation of gonadal recrudescence. A vital step in the initiation of reproduction is the release of gonadotropin releasing hormone 1 (GnRH1), a key molecule released from the hypothalamus that binds to gonadotrophic cells located in the pituitary to stimulate release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Cho et al., 1998). Gonadotropins stimulate gonadal growth and the production and release of sex steroids. Synthesis of GnRH1 is regulated at the level of transcription, translation, and posttranslational modification to maintain the stability of active hormone. Hence investigating molecules involved in all of these different processes may provide explanations for differences in the reproductive timing of songbirds breeding at different latitudes.
Annual change in day length varies with latitude such that birds breeding at higher latitudes typically require longer days to initiate gonadal recrudescence (Dawson 2013; Fudickar et al., 2016b; Singh et al., 2019). However, birds that breed at higher latitude often migrate from wintering to breeding grounds and delay their reproduction despite increasing day length (Grieves, Fudickar, Atwell, Meddle, & Ketterson, 2016; Fudickar et al., 2016b; Ramenofsky, Campion, Pérez, Krause, & Németh, 2017). Preparations for migration include a range of physiological changes such as increased food intake, fat deposition, increased metabolic activity in flight muscles and liver, and shifting from primarily diurnal behavior to a mix of diurnal and nocturnal activity (Piersma, Perez-Tris, Mouritsen, Bauchinger, & Bairlein, 2005; Bairlein, 2003; Landys et al., 2005; Singh, Trivedi, Rani, & Kumar, 2015; Singh, Swarup, Le, & Kumar, 2018). The regulation of seasonally recurring reproduction and migration potentially involves the interaction of key molecules in the hypothalamus in a daily and annual phase-dependent manner that integrate signals for circadian and circannual time measurement and fine tune physiological and behavioral responses (Singh et al., 2015; Mishra, Bhardwaj, Malik, & Kumar, 2017; Johnston, Paxton, Moore, Wayne, & Smith, 2016).
The dark-eyed junco provides a powerful model system for investigating the neuroendocrine mechanisms that underlie variation in reproductive and migratory timing. In early spring populations of juncos that differ in when they reproduce and whether they migrate are found living together in the same environment. As spring progresses, locally breeding juncos undergo gonadal recrudescence, while migratory individuals that are sympatric during the non-breeding months delay recrudescence to prepare to migrate. In recent studies using this system, microarray and transcriptome sequencing techniques have revealed differential patterns of gene expression between residents and migrants in a limited number of tissues (Fudickar et al. 2016a; Johnston et al., 2016). However, a comprehensive account of the final products of gene expression through profiling of translated proteins is needed for a fuller understanding (Anderson, Matheson, & Steiner, 2000) and can be provided by proteomics, including chains of amino acid sequences, post-translational modifications, characterization and identification of protein with relative or absolute quantification (Domon, & Aebersold, 2010).
Here, we performed a global quantitative proteome analysis of the neuroendocrine tissue of seasonally diverged junco sub-populations that, while in sympatry, express striking differences in physiology and behavior in early spring. To identify differentially expressed hypothalamic and pituitary proteins associated with differences in seasonal timing of reproduction and migration, we first quantified global proteome abundance in neuroendocrine tissues using TMT labeling chemistry, LC-MS/MS, followed by characterization of specific proteins using junco open reading frame reference. Second, we identified differentially expressed proteins using the ShinyGO tool, an application based on large annotation and pathway databases compiled from many different sources (Ge, Jung & Yao, 2020). We searched for differentially expressed KEGG pathways and GO terms associated with reproduction, including GnRH synthesis, release, and stability of reproductive hormones and metabolic pathways associated to migratory processes. The goal was to identify functional correlates of seasonal divergence in reproductive and migratory timing in free-living birds belonging to closely related populations living in sympatry but diverging in behavior and physiology related to annual timing.
Material and Methods
Capture and tissue collection-
We captured adult male migrant and resident dark-eyed juncos (n=5 each) using mist nets on state road 714, Giles County Virginia near Mountain Lake Biological Station (MLBS; 37.37 0N, 80.520W). Migrants (J. h. hyemalis ) and residents (J. h. carolinensis ) were readily identified based on the bill color, plumage color, and difference in wing cord (Ketterson & Nolan, 1976). All birds were caught between 18 and 24 March 2017. A schematic figure shows the sampling site (Fig. 1 a), latitudinal differences in the timing of reproduction in dark-eyed juncos (Fig. 1 b), and a workflow illustrating the steps applied in the proteome study (Fig 1 c, d). We collected the most distal secondary feather of the right wing and claw from each individual at the time of capture for stable hydrogen isotope (δ2H) analysis. We measured indicators of reproduction and migration preparedness, including subcutaneous fat score (FS), cloacal protuberance volume (CPV) and body mass (BM) (Fudickar et al., 2016b; Greives et al., 2016; Singh et al, 2019).
We also collected 50-100 µl of blood by puncturing the wing vein for baseline testosterone hormone measurement. Following blood collection, birds were euthanized using isoflurane for tissue harvesting. Hypothalamus and pituitaries were dissected from the whole brain and flash frozen immediately. The tissues were stored at -800C freezer until processing. Scientific collecting permits were issued by the Virginia Department of Game and Inland Fisheries (permit # 052971) and the USFWS (permit # MB093279). All methods were approved under protocol (# 15-026-17) by the Indiana University Institutional Animal Care and Use Committee.