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).