The membrane androgen receptor ZIP9 (SCL39A9) maintains ovarian homeostasis by mediating post-ovulatory follicle breakdown in zebrafish.

ZIP9 was recently characterized as a membrane androgen receptor in Atlantic croaker granulosa/theca (G/T) cells where it mediates androgen-induced apoptosis in vitro, but the physiological significance of this action has remained unclear. In the current study, we utilized ZIP9 knockout (zip9-/-) zebrafish to investigate the role of ZIP9-mediated androgen-induced G/T cell apoptosis in vivo. We first confirmed ZIP9 mediates apoptosis of zebrafish G/T cells in vitro. Testosterone increased apoptosis, intracellular free zinc, and expression of pro-apoptotic members bax and p53 in wildtype and zip9+/+ zebrafish G/T cells, but not in ZIP9 knockout and knockdown cell models. We hypothesized ZIP9-mediated G/T cell apoptosis may be involved in post-ovulatory follicle (POF) breakdown in vivo. Post ovulation, zip9, bax, and p53 were upregulated in zip9+/+ but not in zip9-/- ovaries. Immunoreactivity of cleaved caspase 3 was also higher in POFs from zip9+/+ ovaries compared to zip9-/-, and POF breakdown was significantly delayed in zip9-/- fish compared to zip9+/+ counterparts. To determine the detrimental consequences of delayed POF breakdown in the zip9-/- model, fish were challenged with repeated ovulation induction. After the challenge, zip9-/- fish exhibited abnormal ovarian lesions that contained debris consistent with atretic or necrotic cellular material. However, no abnormalities were observed in zip9+/+ fish ovaries, indicating that the abnormal phenotype is due to the loss of ZIP9. This study demonstrates an important role for ZIP9 in mediating POF breakdown and maintaining tissue remodeling and homeostasis in the teleost ovary and indicates a role for the ZIP9-mediated androgen-induced apoptotic response in vivo.

Vasopressin mediates nonapeptide and glucocorticoid signaling and social dynamics in juvenile dominance hierarchies of a highly social cichlid fish.

Early-life social experience can strongly affect adult behavior, yet the behavioral mechanisms underlying developmental trajectories are poorly understood. Here, we use the highly social cichlid, Burton's Mouthbrooder (Astatotilapia burtoni) to investigate juvenile social status and behavior, as well as the underlying neuroendocrine mechanisms. We placed juveniles in pairs or triads and found that they readily establish social status hierarchies, with some group structural variation depending on group size, as well as the relative body size of the group members. Next, we used intracerebroventricular injections to test the hypothesis that arginine vasopressin (AVP) regulates juvenile social behavior and status, similar to adult A. burtoni. While we found no direct behavioral effects of experimentally increasing (via vasotocin) or decreasing (via antagonist Manning Compound) AVP signaling, social interactions directed at the treated individual were significantly altered. This group-level effect of central AVP manipulation was also reflected in a significant shift in whole brain expression of genes involved in nonapeptide signaling (AVP, oxytocin, and oxytocin receptor) and the neuroendocrine stress axis (corticotropin-releasing factor (CRF), glucocorticoid receptors (GR) 1a and 1b). Further, social status was associated with the expression of genes involved in glucocorticoid signaling (GR1a, GR1b, GR2, mineralocorticoid receptor), social interactions with the dominant fish, and nonapeptide signaling activity (AVP, AVP receptor V1aR2, OTR). Together, our results considerably expand our understanding of the context-specific emergence of social dominance hierarchies in juveniles and demonstrate a role for nonapeptide and stress axis signaling in the regulation of social status and social group dynamics.

Distributions of hypothalamic neuron populations coexpressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse.

The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFPVglut2 - or EGFPVgat -fluorescence, respectively. EGFPVglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFPVgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFPVgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFPVgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFPVgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFPVgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFPVgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine ß-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.