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. 2022 Jun 21;20(1):91.
doi: 10.1186/s12958-022-00963-w.

Hyperandrogenism induces proportional changes in the expression of Kiss-1, Tac2, and DynA in hypothalamic KNDy neurons

Hiroe Okada  1 , Haruhiko Kanasaki  2 , Tuvshintugs Tumurbaatar  1 , Zolzaya Tumurgan  1 , Aki Oride  1 , Satoru Kyo  1
Affiliations

Hyperandrogenism induces proportional changes in the expression of Kiss-1, Tac2, and DynA in hypothalamic KNDy neurons

Hiroe Okada et al. Reprod Biol Endocrinol. .

Abstract

Background: Kisspeptin released from Kiss-1 neurons in the hypothalamus plays an essential role in the control of the hypothalamic-pituitary-gonadal axis by regulating the release of gonadotropin-releasing hormone (GnRH). In this study, we examined how androgen supplementation affects the characteristics of Kiss-1 neurons.

Methods: We used a Kiss-1-expressing mHypoA-55 cell model that originated from the arcuate nucleus (ARC) of the mouse hypothalamus. These cells are KNDy neurons that co-express neurokinin B (NKB) and dynorphin A (DynA). We stimulated these cells with androgens and examined them. We also examined the ARC region of the hypothalamus in ovary-intact female rats after supplementation with androgens.

Results: Stimulation of mHypoA-55 cells with 100 nM testosterone significantly increased Kiss-1 gene expression by 3.20 ± 0.44-fold; testosterone also increased kisspeptin protein expression. The expression of Tac3, the gene encoding NKB, was also increased by 2.69 ± 0.64-fold following stimulation of mHypoA-55 cells with 100 nM testosterone. DynA gene expression in these cells was unchanged by testosterone stimulation, but it was significantly reduced at the protein level. Dihydrotestosterone (DHT) had a similar effect to testosterone in mHypoA-55 cells; kisspeptin and NKB protein expression was significantly increased by DHT, whereas it significantly reduced DynA expression. In ovary-intact female rats, DTH administration significantly increased the gene expression of Kiss-1 and Tac3, but not DynA, in the arcuate nucleus. Exogenous NKB and DynA stimulation failed to modulate Kiss-1 gene expression in mHypoA-55 cells. Unlike androgen stimulation, prolactin stimulation did not modulate kisspeptin, NKB, or DynA protein expression in these cells.

Conclusions: Our observations imply that hyperandrogenemia affects KNDy neurons and changes their neuronal characteristics by increasing kisspeptin and NKB levels and decreasing DynA levels. These changes might cause dysfunction of the hypothalamic-pituitary-gonadal axis.

Keywords: Androgen; Hypothalamus; Kisspeptin; Prolactin; Testosterone.

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Conflict of interest statement

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Fig. 1
Fig. 1
Effect of testosterone on Kiss-1 gene and kisspeptin protein expression in mHypoA-55 cells. mHypoA-55 cells were stimulated with the indicated concentrations of testosterone for 24 h. A After stimulation, mRNA was extracted and reverse transcribed, and Kiss-1 mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over unstimulated cells and presented as the mean ± SEM of three independent experiments. **P < 0.01, *P < 0.05 vs. control. B After stimulation, lysates (20  g protein) from mHypoA-55 cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with anti-kisspeptin antibodies. The bands were visualized using an HRP-conjugated secondary antibody. C Scanning densitometry of bands was performed using ImageJ to determine differences in kisspeptin protein expression, with normalization to β-actin levels. Results are expressed as fold stimulation over the unstimulated group/control. Values are the mean ± SEM of fold stimulation from independent experiments. **P < 0.01 vs. control
Fig. 2
Fig. 2
Effect of testosterone on Tac3 gene and NKB protein expression in mHypoA-55 cells. mHypoA-55 cells were stimulated with the indicated concentrations of testosterone for 24 h. A After stimulation, mRNA was extracted and reverse transcribed. Tac3 mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over unstimulated cells and presented as the mean ± SEM of three independent experiments. *P < 0.05 vs. control. B After stimulation, lysates (20  g protein) from mHypoA-55 cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with anti-NKB antibodies. The bands were visualized using an HRP-conjugated secondary antibody. C Scanning densitometry of bands was performed using ImageJ to determine differences in kisspeptin protein expression, with normalization to β-actin levels. Results are expressed as fold stimulation over the unstimulated group/control. Values are the mean ± SEM of fold stimulation from independent experiments. **P < 0.01 vs. control
Fig. 3
Fig. 3
Effect of testosterone on DynA expression in mHypoA-55 cells. mHypoA-55 cells were stimulated with the indicated concentrations of testosterone for 24 h. A After stimulation, mRNA was extracted and reverse transcribed. DynA mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over unstimulated cells and presented as the mean ± SEM of three independent experiments. B After stimulation, lysates (20  g protein) from mHypoA-55 cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with anti-DynA antibodies. The bands were visualized using an HRP-conjugated secondary antibody. C Scanning densitometry of bands was performed using ImageJ to determine differences in DynA protein expression, with normalization to β-actin levels. Results are expressed as fold stimulation over the unstimulated group/control. Values are the mean ± SEM of fold stimulation from independent experiments. **P < 0.01 vs. control
Fig. 4
Fig. 4
Effect of DHT on the expression of kisspeptin, NKB, and DynA in mHypoA-55 cells. (A) mHypoA-55 cells were stimulated with the indicated concentrations of DHT for 24 h, and lysates (20  g protein) from the treated cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with antibodies against kisspeptin, NKB, and DynA. The bands were visualized using an HRP-conjugated secondary antibody. Scanning densitometry of bands was performed using ImageJ to determine differences in kisspeptin (A), NKB (B), and DynA (C) expression, with normalization to β-actin levels. Results are expressed as fold stimulation over the unstimulated group/control. Values are the mean ± SEM of fold stimulation from independent experiments. **P < 0.01 vs. control
Fig. 5
Fig. 5
Effect of DHT supplementation on Kiss-1, Tac3, and DynA gene expression in ovary-intact female rats. Six-week-old female rats were injected subcutaneously with DHT (5 mg/kg/day) daily for 7 days. After the rats were euthanized, hypothalamic tissues containing the ARC were removed from control (n = 4) and DHT-treated rats (n = 4), and mRNA was extracted from these tissues and reverse transcribed. Kiss-1, Tac3, and DynA mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over control and presented as the mean ± SEM. *P < 0.05 vs. control
Fig. 6
Fig. 6
Effect of NKB and DynA stimulation on Kiss-1 gene expression in mHypoA-55 cells. mHypoA-55 cells were stimulated with the indicated concentrations of NKB or DynA for 24 h. (A) After stimulation, mRNA was extracted and reverse transcribed, and Kiss-1 mRNA levels were measured by quantitative RT-PCR. Samples for each experimental group were run in duplicate and normalized to the mRNA levels of GAPDH as a housekeeping gene. The results are expressed as fold induction over unstimulated cells and presented as the mean ± SEM of three independent experiments
Fig. 7
Fig. 7
Effect of prolactin on kisspeptin, NKB, and DynA expression in mHypoA-55 cells. (A) mHypoA-55 cells were stimulated with the indicated concentrations of prolactin for 24 h. Lysates (20  g protein) from mHypoA-55 cells were analyzed by SDS-PAGE followed by immunoblotting and incubation with antibodies against kisspeptin, NKB, and DynA. The bands were visualized using an HRP-conjugated secondary antibody
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References

    1. Skorupskaite K, George JT, Anderson RA. The kisspeptin-GnRH pathway in human reproductive health and disease. Hum Reprod Update. 2014;20:485–500. doi: 10.1093/humupd/dmu009. - DOI - PMC - PubMed
    1. Clarke H, Dhillo WS, Jayasena CN. Comprehensive Review on Kisspeptin and Its Role in Reproductive Disorders. Endocrinol Metab (Seoul) 2015;30:124–141. doi: 10.3803/EnM.2015.30.2.124. - DOI - PMC - PubMed
    1. Desroziers E, Mikkelsen J, Simonneaux V, Keller M, Tillet Y, Caraty A, Franceschini I. Mapping of kisspeptin fibres in the brain of the pro-oestrous rat. J Neuroendocrinol. 2010;22:1101–1112. doi: 10.1111/j.1365-2826.2010.02053.x. - DOI - PubMed
    1. Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, et al. Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. J Reprod Dev. 2007;53:367–378. doi: 10.1262/jrd.18146. - DOI - PubMed
    1. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology. 2005;146:3686–3692. doi: 10.1210/en.2005-0488. - DOI - PubMed

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