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

Cortés-Campos C, Letelier J, Ceriani R, Whitlock KE - Biol Open (2015)

Bottom Line: Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Centro Interdisciplinario de Neurociencia de Valparaíso (CINV), Facultad de Ciencias, Universidad de Valparaíso, Pasaje Harrington 269, Valparaíso 2340000, Chile Whitehead Institute for Biomedical Research (WIBR), 9 Cambridge Center, Cambridge, MA 02142, USA.

No MeSH data available.


Related in: MedlinePlus

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.
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BIO010447F6: 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.

Mentions: In order to examine in vitro the potential effects of hormones used in the treatment of Kallmann syndrome patients (Raivio et al., 2007), we applied testosterone and GnRH to the neurosphere cultures to determine whether there was an increase in GnRH-positive neurons. For the hormone exposure experiment, GnRH3 was used, as it is currently the proposed potential endocrine form of GnRH in zebrafish. First, using values from the literature (Cheung and Wong, 2008) we exposed cells to control (vehicle) or 10 nM GnRH during the differentiation phase (Fig. 2A3-6, bottom panel) by supplementation every 2 days (days 3, 5, 7). In separate experiments cells were differentiated in the presence of 10 µM testosterone (T) (Mouriec et al., 2009) using the same protocol. The resulting differentiated cells showed an increase in the GnRH-positive neurons following exposure to either testosterone (Fig. 5D-E, arrows) or GnRH (Fig. 5G-I, arrows) relative to the control (Fig. 5A-C, arrows). The increase caused by testosterone was small but significant (Fig. 6A; P<0.05, n=12); in contrast, cells treated with GnRH showed a significant increase in the number of neurons and GnRH positive neurons (Fig. 6B; P<0.0001 for all neurons, n=12). Next, to determine whether there was a dose response in the number of GnRH cells generated, neurospheres were exposed to varying concentrations of GnRH during the differentiation phase and analyzed (Fig. 6C-E; n=10). The maximal effect was obtained using a final concentration of 10-20 nM (Fig. 6E), in agreement with values reported in the literature (Cheung and Wong, 2008).Fig. 6.


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

Cortés-Campos C, Letelier J, Ceriani R, Whitlock KE - Biol Open (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582115&req=5

BIO010447F6: 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.
Mentions: In order to examine in vitro the potential effects of hormones used in the treatment of Kallmann syndrome patients (Raivio et al., 2007), we applied testosterone and GnRH to the neurosphere cultures to determine whether there was an increase in GnRH-positive neurons. For the hormone exposure experiment, GnRH3 was used, as it is currently the proposed potential endocrine form of GnRH in zebrafish. First, using values from the literature (Cheung and Wong, 2008) we exposed cells to control (vehicle) or 10 nM GnRH during the differentiation phase (Fig. 2A3-6, bottom panel) by supplementation every 2 days (days 3, 5, 7). In separate experiments cells were differentiated in the presence of 10 µM testosterone (T) (Mouriec et al., 2009) using the same protocol. The resulting differentiated cells showed an increase in the GnRH-positive neurons following exposure to either testosterone (Fig. 5D-E, arrows) or GnRH (Fig. 5G-I, arrows) relative to the control (Fig. 5A-C, arrows). The increase caused by testosterone was small but significant (Fig. 6A; P<0.05, n=12); in contrast, cells treated with GnRH showed a significant increase in the number of neurons and GnRH positive neurons (Fig. 6B; P<0.0001 for all neurons, n=12). Next, to determine whether there was a dose response in the number of GnRH cells generated, neurospheres were exposed to varying concentrations of GnRH during the differentiation phase and analyzed (Fig. 6C-E; n=10). The maximal effect was obtained using a final concentration of 10-20 nM (Fig. 6E), in agreement with values reported in the literature (Cheung and Wong, 2008).Fig. 6.

Bottom Line: Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Centro Interdisciplinario de Neurociencia de Valparaíso (CINV), Facultad de Ciencias, Universidad de Valparaíso, Pasaje Harrington 269, Valparaíso 2340000, Chile Whitehead Institute for Biomedical Research (WIBR), 9 Cambridge Center, Cambridge, MA 02142, USA.

No MeSH data available.


Related in: MedlinePlus