Scotostimulation of reproductive neural pathways and gonadal maturation are not correlated with hypothalamic expression of deiodinases in subtropical spotted munia

Abstract

Circannual rhythm regulates the annual timing of reproduction in spotted munia, with sex differences in its relationship with the external photoperiod environment. Interestingly, munia show an atypical photosensitivity and exhibit gonadal maturation when acutely exposed to an unnatural short photoperiod (eg 3 hours of light per day; ie a long scotoperiod). The proximate mechanisms regulating scotoperiod-induced hypothalamic-pituitary-gonadal (HPG) activation are unclear. Because thyroid hormone signalling plays a central role in photoperiodic induction, we hypothesised the involvement of similar mechanism, comprising alterations in hypothalamic deiodinases, under long scotoperiod-induced HPG activation. To test this, several endpoints of cellular and molecular correlates were assayed in male and female munias after 1 and 4 weeks of exposure to an 3:21 hour light/dark cycle (3L:21D), with controls on a 21:3 hour light/dark cycle (21L:3D). We measured the hypothalamic expression of mRNA and protein of light-sensitive (neuropsin, OPN5) and reproductive (vasoactive intestinal peptide [VIP], neuropeptide Y [NPY], gonadotrophin-releasing hormone [GnRH], gonadotrophin-inhibiting hormone [GnIH]) neuropeptides by quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. In addition, we also measured mRNA expression of types 2 (DIO2) and 3 (DIO3) deiodinases that regulate triiodothyronine-mediated GnRH release and gonadal maturation in photoperiodic species. The quantitative PCR and immunohistochemistry results were consistent. Higher OPN5 levels under 21L:3D than under 3L:21D suggested its role in sensing the length of the light period. Similarly, low VIP and high NPY expression under 3L:21D than under 21L:3D were consistent with their roles as cellular correlates of photic and nonphotic environment, respectively. High GnRH-I/low GnIH levels and gonadal recrudescence under 3L:21D, and an inverse pattern under 21L:3D, confirmed the scotostimulation of HPG axis in spotted munia. However, DIO2 and DIO3 mRNA levels did not differ between 2 scotoperiods, in contrast to their reciprocal expression pattern found during long-day photostimulation. We demonstrate for the first time sex-dependent scotostimulation of reproductive neural pathways and suggest the involvement of molecules other than hypothalamic deiodinases in the regulation of gonad development cycle in 'nonphotoperiodic' seasonally breeding vertebrates.

Changes in brain peptides associated with reproduction and energy homeostasis: Putative roles of gonadotrophin‐releasing hormone‐II and tyrosine hydroxylase in determining reproductive performance in response to daily food availability times in diurnal zebra finches

“…For this, birds were quickly decapitated to ensure the optimal determination of transcript expression . The hypothalamus was dissected out as described previously and stored at −80°C. We extracted total RNA using Tri reagent (AM9738; Ambion, Austin, TX, USA) in accordance with the manufacturer's instructions.…”

“…We performed immunohistochemistry (IHC) for ZENK (as a marker of neuronal activity) and GnRH‐I, GnRH‐II, GnIH, NPY, CART and TH (neuropeptides associated with reproduction and metabolism), using brain sections from five birds of each sex, as per standardised procedures in our laboratory . Briefly, birds were deeply anaesthetised with ketamine‐xylazine solution (0.003 mL g ‐1 body weight) and transcardially perfused successively with 50 mL saline (pH 7.4) and 40 mL of fixative (4% paraformaldehyde in 0.1 mol L ‐1 phosphate buffer, pH 7.4).…”

“…A bin of sections containing the hypothalamic region was processed for double‐labelled IHC of CART/NPY, GnRH/GnIH, or ZENK/TH. We used a standard avidin‐biotin peroxidase protocol that has been standardised in our laboratory . Briefly, PBS sections washed three times (pH 7.4) were successively treated with hydrogen peroxide (0.3% dissolved in methanol, 30 minutes) and bovine serum albumin (1% dissolved in PBS containing 0.3% Triton‐X‐100 [PBSBT], 60 minutes) to block endogenous peroxidase activity and non‐specific binding, respectively.…”

“…The primary antibodies that we used are specific and have been used for IHC in passerine birds, including zebra finches . Because GnRH antiserum recognises both GnRH‐I and GnRH‐II isoforms, we identified them based on immunoreactions with reference to their location in the brain (POA: GnRH‐I; midbrain: GnRH‐II) …”

“…In response to thyroid‐stimulating hormone release from the pars tuberalis of the pituitary gland, the ependymal (tanycytes) DIO2/DIO3 ‘genetic switch’ alters the local triiodothyronine concentration, which controls the synthesis and/or release of gonadotrophin‐releasing hormone (GnRH) . This may not be true, however, for ‘non‐photoperiodic’ birds such as zebra finches in which gonadal recrudescence occurs in response to social stimulation and in the subtropical spotted munia ( Lonchura punctulata ) in which a very long scotoperiod (hence short light period) is gonadostimulatory . Regardless of the differential cue sensitivity between photoperiodic and non‐photoperiodic species, hypothalamic GnRH directly activates the HPG axis and regulates gonadal maturation and reproductive behaviour in breeding birds .…”