Endocrine signaling associated with earlier timing of reproduction
in resident juncos-
The hypothalamus is the top-most locus of the HPG neuroendocrine system
where photoperiodic signals are perceived and transformed into an
endocrine response that leads to HPG axis activation and gonad
recrudescence. In many avian species, an increase in the hypothalamic
neuropeptide GnRH1 levels is known to be a key event that stimulates
release of luteinizing hormone from pituitary and eventually leads to
gonadal stimulation and testosterone synthesis (Cho et al., 1998). Thus,
any regulatory process that influences GnRH transcription, translation,
proteolytic processing to produce mature hormone, stability of peptide,
synthesis and release of hormone could play a critical role in the
process of gonad recrudescence. We consider here the potential role of
proteins known to be related to GnRH that were found in greater
abundance in resident juncos (DUSP1, PURA, CBE, 7B2, GAD2, ITPR3,
AGPAT4, PENK, UCHL1, RP2).
The binding of GnRH molecule to the GnRH receptor triggers a cascade of
mitogen-activated protein kinase C (MAPK) signaling and dual-specificity
phosphatases (DUSP), both immediate early genes (Conn, McArdle, Andrews,
& Huckle, 1987; Stojilkovic, Reinhart, Catt, 1994; Kondoh & Nishida,
2007). DUSP1 is a dephosphorylating enzyme shown to act in the feedback
regulation of phosphorylated MAPK in association with increased GnRH in
the gonadotrophs (Zhang, & Roberson, 2006). DUSP1 has also been
observed in luteinizing hormone beta (LHb) subunit gene expression in
gonadotrophs (Purwana, Kanasaki, Oride, & Miyazaki, 2010). A few
studies in the mouse neuronal GT1-7 cells suggest that enhanced
occupancy of GnRH 1 gene upstream promoter by a transcription factor
purine-rich element-binding protein (PURA) downregulated GnRH 1 mRNA
levels, suggesting PURA as a repressor of GnRH1 gene transcription
(Zhao, Kelm Jr., Fernald, 2010). We note that migratory juncos showed
higher level of PURA protein than residents, which could be related to
delay in their reproduction.
We found another intracellular second messenger protein ITPR3 to be more
abundant in the neuroendocrine tissue of earlier reproducing resident
juncos as compared to migrants. ITPR3 is a member of the inositol
1,4,5-triphosphate (IP3) receptor family known to trigger a signaling
cascade that facilitates the binding of GnRH to its receptor found on
gonadotrophs in the pituitary of huoyan geese (Luan et al., 2017). In
contrast this protein is found in lower levels during the laying period
in a goose, suggesting that later in the reproductive cycle ITPR2a can
also play a role in negative feedback arising from prolonged GnRH or LH
secretion (Luan et al., 2017).
Resident juncos had higher abundance of acylglycerophosphate
acyltransferase family (AGPAT4) protein, which plays an important role
in the synthesis and maintenance of N-methyl-D-asparate (NMDA) and
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.
NMDA and AMPA receptors are essential for reproduction in mammals
(Christian & Moenter, 2010). Glutamic acid decarboxylase (GAD) was also
present in higher abundance in resident juncos. GAD is a rate-limiting
enzyme that converts glutamate to GABA, an inhibitory neurotransmitter,
and the literature emphasizes an inhibitory role of GABA in GnRH
release. Given earlier reproduction by residents in this study, the
finding is surprising, but we know that GnRH release is known to result
from the interaction of many other neurotransmitters present on the cell
membrane (Christian & Moenter, 2010). Interestingly, we also found
higher abundance of a protein, retinitis pigmentosa 2 (RP2) in
residents. RP2 is found in the synaptic region of rod and cone
photoreceptors and acts as an importer in membrane protein trafficking
in mammals (Holopainen et al., 2010). Photoperiod in bird is detected by
deep brain photoreceptors (Nakane et al., 2010), so its presence in high
abundance in resident junco suggests its role in perceiving and
transducing photoperiodic information by hypothalamic deep brain rod
photoreceptors.
Another protein in higher abundance in residents was carboxypeptidase E
(CBE). CBE is an endoproteolytic enzyme located in the secretory
granules of neuroendocrine cells that play a vital role in peptide
hormone and neuropeptide biosynthesis (Fricker, 1988; Cawley et al.,
2012; Ji et al., 2017). Elimination of the CPE gene in mice leads to
infertility associated with a defect in hypothalamic GnRH 1 proteolytic
processing (Wetsel, Liposits, Seidah, & Collins, 1995; Cawley et al.,
2012). Higher expression of CBE protein in residents possibly
accelerates the proteolytic cleavage of pro GnRH to synthesize mature
GnRH hormone. The posttranslational proteolytic process of transforming
inactive neuropeptide hormones to biologically active peptides is
achieved partly through the family of prohormone convertases (PCs)
(Nillni, 2007).
Similar, to CBE, the neuroendocrine protein 7B2 is also expressed at
higher levels in resident juncos. This protein is involved in the
activation of an endoprotease prohormone convertase 2 (PC2). Previous
studies indicate that the large amount of stored 7B2 protein observed in
gonadotroph cells of the mammalian pituitary plays a critical role in
hormone regulation (Westphal et al., 1999; Mbikay, Seidah, & Chrétien,
2001). Finally, ubiquitin C-terminal hydrolase L1 (UCHL1), a
deubiquitinating enzyme expressed in the anterior pituitary, was more
abundant in residents. In mice, UCHL1 genetic knock out mice (akagad mice) exhibit a significant decrease in gonadotropic,
lactotropic cells in the anterior pituitary, suggesting a critical role
for this enzyme in the GnRH stability in gonadotrophs, and activation of
HPG axis (Xu, Hideshima, Ishii, Yoshikawa, & Kyuwa, 2014). In addition
to proteins associated with GnRH synthesis and release, we also found
proenkephalin (PENK) to be more abundant in resident juncos. PENK is an
endogenous opioid peptide that has been suggested to play a role in the
regulation of negative feedback on luteinizing hormone releasing hormone
(LHRH) in domestic fowl (Scanes & Pierzchala-Koziec, 2018).