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.