Figure 1. Overexpression of Kiss1r- or Adcyap1r-expressing vectors in LβT2 cells. LβT2 cells were transfected with 2.0 μg of Kiss1r-expressing (A) or Adcyap1r-expressing (B) vectors. Cells transfected with empty vectors were used as a mock control. After 48 h, total RNA was prepared and RT-PCR was carried out for 35 cycles using specific Kiss1r (A) and Adcyap1r- (B) primers. For amplification of Gapdh, PCR was conducted for 18 cycles. Before an RT-PCR analysis was performed, the linear range of amplification was established by changing the number of PCR cycles and the amount of total RNA for reverse transcription. PCR products were resolved on a 2% agarose gel and visualized with ethidium bromide staining. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 2. Effect of kisspeptin on gonadotropin Lhb, Fshb, and Cga expressions. LβT2 cells were transfected with 2.0 μg of Kiss1r-expressing vector, together with 2.0 μg of Lhb-Luc (A) or Fshb-Luc (B) or Cga-Luc (C) and PRL-TK (0.1 μg) vectors. At 48 h after transfection, cells were treated with 100 nM kisspeptin (KP10) for 6 h. A luciferase assay was performed to examine Lhb, Fshb, and Cga promoter activity, which was normalized to PRL-TK activity and expressed as the fold difference in activation over unstimulated controls. Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). **P < 0.01 vs control. For mRNA quantification, LβT2 cells were stimulated with 100 nM kisspeptin (KP10) for 24 h, and Lhb (D), Fshb (E), and Cga (F) mRNA levels were measured by quantitative real-time PCR after mRNA extraction and reverse transcription. Samples for each experimental group were run in duplicate and normalized to Gapdh mRNA levels as a housekeeping gene. Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). Values represent the mean ± SEM of the fold stimulation from independent experiments. **P < 0.01 vs control. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 3. Effect of GnRH and kisspeptin on gonadotropin Lhb, Fshb, and Cga promoter activities. LβT2 cells were transfected with 2.0 μg of Kiss1r-expressing vector, together with 2.0 μg of Lhb-Luc (A) or Fshb-Luc (B) or Cga-Luc (C) and PRL-TK (0.1 μg) vectors. Forty-eight hours after transfection, cells were treated with 100 nM kisspeptin (KP10) or 100 nM GnRH or both for 6 h. A luciferase assay was performed to examine Lhb, Fshb, and Cga promoter activity, which was normalized to PRL-TL activity. Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). **P < 0.01 vs control. The differences between the KP10 and GnRH + KP10 for each gonadotropin subunit promoter were not statistically significant. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 4. Effect of kisspeptin and ADCYAP1 on gonadotropin Lhb, Fshb, and Cga promoter activities. LβT2 cells were transfected with 2.0 μg of Adcyap1r-expressing vector, together with 2.0 μg of Lhb-Luc (A) or Fshb-Luc (B) or Cga-Luc (C) and PRL-TK (0.1 μg) vectors. Forty-eight hours after transfection, cells were treated with 100 nM kisspeptin (KP10) or 100 nM ADCYAP1 or both for 6 h. A luciferase assay was performed to examine Lhb, Fshb, and Cga promoter activity, which was normalized to PRL-TL activity. Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). **P < 0.01 vs control. The difference between the KP10 and ADCYAP1 for Fshb-promoter was statistically significant (P < 0.01). The differences between the KP10 and KP10 + ADCYAP1 and between the ADCYAP1 and KP10 + ADCYAP1 for each gonadotropin subunit promoter were statistically significant (P < 0.01). Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 5. Effect of GnRH, kisspeptin, and ADCYAP1 on Sre and Cre promoter activities. LβT2 cells were transfected with 2.0 μg of Kiss1r- and Adcyap1r-expressing vector, together with 2.0 μg of Sre-Luc (A) or Cre-Luc (B) and PRL-TK (0.1 μg) vectors. Forty-eight hours after transfection, cells were treated with 100 nM GnRH, 100 nM kisspeptin (KP10), 100 nM ADCYAP1, or both GnRH and KP10 or KP10 and ADCYAP1 for 6 h. A luciferase assay was performed to examine Sre and Cre promoter activity, which was normalized to PRL-TL activity. Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). The differences in Sre promoter activity between GnRH and GnRH + KP10, between KP10 and KP10 + GnRH, and between ADCYAP1 and KP10 + ADCYAP1 were statistically significant (P < 0.01). The differences in Cre promoter activity between KP10 and KP10 + ADCYAP1 and between ADCYAP1 and KP10 + ADCYAP1 were statistically significant (P < 0.01). n.s., not significant. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 6. Effect of kisspeptin on Adcyap1 and Adcyap1r mRNA expression Figure 6. Effect of kisspeptin on Adcyap1 and Adcyap1r mRNA expression. LβT2 cells that were transfected with 2.0 μg of Kiss1r were treated with 100 nM kisspeptin (KP10) for 48 h. Adcyap1 (A) and Adcyap1r (B) mRNA levels were measured by quantitative real-time PCR after mRNA extraction and reverse transcription. Samples for each experimental group were run in duplicate and normalized to Gapdh mRNA levels. Results are expressed as the fold increase in stimulated vs unstimulated groups/controls. Values represent the mean ± SEM of the fold stimulation from independent experiments. **P < 0.01 vs control. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 7. Effects of ADCYAP1R overexpression on the basal levels and the kisspeptin-induced fold induction of gonadotropin Lhb, Fshb, and Cga promoter activities. LβT2 cells were transfected with 2.0 μg or 4.0 μg of Adcyap1r-expressing vector together with 2.0 μg of Lhb-Luc (A, D) or Fshb-Luc (B, E), or Cga -Luc (C, F) and PRL-TK (0.1 μg) vectors. Forty-eight hours after transfection, cells were treated with 100 nM kisspeptin (KP10) for 6 h. A luciferase assay was performed to examine Lhb, Fshb, and Cga promoter activity, which was normalized to PRLTK activity and expressed as basal (A–C) and the fold difference in activation over unstimulated controls in the mock group. The fold stimulation of kisspeptin (KP10)-stimulated cells over unstimulated cells was calculated (D–F). Data are expressed as mean ± SEM (three independent experiments performed using triplicate samples). The differences between the 2.0 μg and 4.0 μg transfection amounts of Adcyap1r in the basal activity of the Cga promoter were statistically significant (P < 0.01). Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figure 8. Effect of ADCYAP1R overexpression on Cga mRNA expression Figure 8. Effect of ADCYAP1R overexpression on Cga mRNA expression. LβT2 cells were transfected with 4.0 μg of Adcyap1r-expressing vector and cultured for 48 h. Cga mRNA levels were then measured by quantitative real-time PCR after mRNA extraction and reverse transcription. Samples for each experimental group were run in duplicate and normalized to Gapdh mRNA levels. Results are expressed as the fold increase in stimulated vs unstimulated groups/controls. Values represent the mean ± SEM of the fold stimulation from independent experiments. **P < 0.01 vs mock. Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells† Biol Reprod. 2017;96(5):1043-1051. doi:10.1093/biolre/iox030 Biol Reprod | © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com