Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons

Abstract

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

Keywords: GnRH receptors; Kallmann syndrome; Testosterone.

Fig. 1.

GnRH antibodies recognize cells in the anterior preoptic region of the adult brain. Brain sections (A,B, cryostat sections; C-G, paraffin sections) of different animals immuno-stained with different antibodies against GnRH: (A,B) anti-GnRH (LRH13); (C,D) anti-GnRH (Hu11B); (E,F) anti-mGnRH (Sigma-Aldrich); (G,H) anti-sGnRH (BB8). All antibodies show immunoreactivity in neurons (arrows) localized in the hypothalamic POA (diagram, red). Scale bars: 50 μm.

Fig. 2.

Neurospheres can be generated from the hypothalamus of adult zebrafish. (A) Neural progenitors were disaggregated (black cells), plated, and maintained in proliferation media for 7 day (A1 to A3 neurospheres, −7 to 0 days). Cells were then seeded (A4 to A6, upper panel) on coated chamber wells and changed to differentiation media for 7 days (0 to +7 days). Alternatively, neurospheres (A3) were disaggregated and seeded (A4 to A6, bottom panel) on coated chamber wells and changed to differentiation media for 7 days with or without hormonal treatment (0 to +7 days). (B-D) 7 days in vitro bright field images of neurospheres (A3). Scale bar 30 μm.

Fig. 3.

Hypothalamic neurospheres express neural progenitor markers. Undifferentiated 7 days in vitro neurospheres. Cells expressing nestin and GFAP are located principally at the center of the neurospheres, (A-C, arrows). Sox2 and vimentin, markers of undifferentiated neural progenitors, are expressed in the neurospheres (D-F, arrows). Neurospheres are highly proliferative, expressing PCNA (green) in many of the GFAP positive (red) cells (G-I, arrows). Undifferentiated neurospheres reactive to GFAP show a reduced number of neurons (positive for HUC) (J-L, arrows). Scale bar 25 μm; n=3 plates derived from different cultures.

Fig. 4.

Hypothalamic progenitors generate glial and neuronal cells. (A-F) Neurospheres differentiated after 7 days in vitro. (A-C) Differentiated cells surround the core of cells positive for nestin (arrow head) and extend cellular projections that are reactive to GFAP (arrows). (D-F) The reduced number of Sox2 positive cells (arrows) and the high number of cell processes reactive to Vimentin (arrow heads) suggests that progenitors have differentiated into glial cells. (G-L) Neurospheres reactive to GFAP show (G-I) a reduced number of cells positive for PCNA (arrows), (J-L) high number of neurons (arrows) and differentiated glial cells (arrow heads). Scale bar 25 μm, n=3 plates derived from different cell cultures.

Fig. 5.

Hypothalamic progenitors can differentiate GnRH neurons. (A-C) Neurospheres differentiated for 7 days in vitro without hormonal stimulus. Neurospheres generate GnRH neurons (arrows). (D-F) Differentiated 7 days in vitro neurospheres, stimulated for 5 days with 10 μM testosterone. Testosterone increases the number of GnRH neurons in NSC cultures (arrows). (G-I) Differentiated 7 days in vitro neurospheres, stimulated for 5 days with 10 nM GnRH increases the number of GnRH neurons in NSC culture (arrows). Scale bar 25 μm.

Fig. 6.

Hormonal treatment increases the number of GnRH neurons. (A) Neurospheres differentiated 7 days in vitro, and stimulated for 5 days with 10 μM testosterone showed an increase in the number of GnRH neurons. (B) Differentiated 7 days in vitro neurospheres stimulated for 5 days with 10 nM GnRH increase the number of neurons, GnRH cells and GnRH neurons. (C-E) Dose response plot of differentiated 7 days in vitro neurospheres, stimulated for 5 days with GnRH. All parameters analyzed show a bell-shaped dose response curve, with the maximum response at 10 nM. *P<0.05, ***P<0.0001. A,B: n=12 plates derived from 3 different cell cultures; C-E: n=10 plates derived from 4 different cell cultures. Error bars represent s.e.m.

Fig. 7.

GnRH receptors are expressed in undifferentiated neurospheres. RT-PCR analyses of the four zfGnRHRs and zfAR. All zfGnRHRs are expressed in the brain of adult zebrafish. zfGnRHR1, zfGnRHR2 and zfGnRHR4 are expressed in undifferentiated neurospheres (7 days in vitro). UNS, neurospheres; RT+, positive reverse-transcription; RT−, negative reverse transcription. β-actin and Gapdh: house-keeping genes.

Fig. 8.

The hypothalamus of the adult zebrafish contains newly differentiated GnRH cells. Horizontal sections showing the anterior region of the parvocellular preoptic nucleus (PPa), with DAPI labeling (blue). (A-C) HuC labeling in a HuC:GFP background (green, arrows) and vimentin labeling (red, arrowhead) do not co-localize. The vimentin positive cells line the edges of the ventricles. (D-F) HuC (green) and Sox2 labeling (red, arrows) co-localizes in some cells (arrows) located closer to the border of the ventricle (arrows). (G-I) Section showing GnRH cell body labeling (green, box, asterisk) and labeling of processes (green, arrows), Sox2 labeling (red, box, asterisk) in cells located several cell diameters from the border of the ventricle (arrows) co-localizes with the GnRH labeling (merge). All Images: anterior is to the left. Scale bar=30 μm.