Inducible Kiss1 knockdown in the hypothalamic arcuate nucleus suppressed pulsatile secretion of luteinizing hormone in male mice

Abstract

Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

Keywords: Adeno-associated virus; Gonadotropin; Gonadotropin-releasing hormone; Kiss1; Metastin.

Fig. 1.

Transgene expression in the arcuate nucleus (ARC) of Kiss1-floxed (Kiss1^lox/lox) male mouse brain 4 or 8 weeks after the injection of adeno-associated virus (AAV) vector into the ARC. (A) Schematic representation of the site of injection (green, the ARC) adapted from the Paxinos and Franklin [25] mouse brain atlas. (B) Representative photomicrographs showing the distribution of GFP immunoreactivity (GFP-ir) in the ARC of a Kiss1-floxed male mouse injected with AAV-GFP or AAV-Cre-GFP. Insets: GFP-ir cells at a higher magnification. Scale bar, 100 μm. AAV-GFP, Kiss1-floxed male mice transduced with AAV-GFP in the ARC; AAV-Cre-GFP, Kiss1-floxed male mice transduced with AAV-Cre-GFP in the ARC; 4 wks, Kiss1-floxed male mice analyzed 4 weeks after AAV injection; 8 wks, floxed male mice analyzed in 8 weeks after AAV injection.

Fig. 2.

Inducible knockdown of Kiss1 expression in the arcuate nucleus (ARC) of castrated male Kiss1-floxed mice. (A) Representative photomicrographs showing the distribution of Kiss1 mRNA in the ARC of each group. Scale bar, 100 µm. (B) Number of Kiss1-expressing cells in the ARC, with the mean ± SEM and individual data points overlaid on the bar charts. The numbers in or on each column indicate the sample size of the group. * P < 0.05; ** P < 0.01 (vs. AAV-GFP-injected controls, Student's t-test). Abbreviations are the same as in Fig. 1.

Fig. 3.

Effect of AAV-GFP or AAV-Cre-GFP injection on pulsatile luteinizing hormone (LH) secretion in castrated male Kiss1-floxed mice. (A) Plasma LH profiles in representative animals from each group. Blood samples were collected every 3 min for 1 h a week after the castration. Arrowheads indicate the peaks of LH pulses as identified by the PULSAR computer program. (B) Mean and baseline LH concentrations, and the frequency and amplitude of LH pulses were calculated for a 1-h sampling period. Bar charts portray the mean ± SEM with individual data points overlaid. The numbers in or on each column indicate the sample size of the group. * P < 0.05 (vs. AAV-GFP-injected controls, Student's t-test). Abbreviations are the same as in Fig. 1.

Fig. 4.

Effect of AAV-GFP or AAV-Cre-GFP injection on testicular functions in male Kiss1-floxed mice. (A) Photomicrographs of testicular sections stained by hematoxylin and eosin in a representative animal from each group. Insets: Kiss1-expressing cells at a higher magnification. Scale bar, 100 µm. (B) Plasma testosterone (T) levels (left) and testicular weights (right) in each group. (C) Tubular diameters (left) and percentage of mature spermatids in total tubules (right) in each group. Bar charts portray the mean ± SEM with individual data points overlaid. Numbers in or on each column indicate the sample size of the group. Abbreviations are the same as in Fig. 1.