Puberty Initiation in Mammals: Colocalization of NKB and Kisspeptin

Figure 1: Puberty Process ("The puberty process," 2012)

Puberty is a complexly coordinated biological process that transits an individual from childhood to adulthood1. Puberty initiation is marked by secretion of gonadotropin-releasing hormone (GnRH) from hypothalamic neurons and secreted GnRH triggers signaling cascades and gonadal activations 2. This neuro-hormonal process follows the cascade below:

Figure 2: Neurohormonal process of puberty initiation (Smith, 2008)

1. Hypothalamus release pulses of GnRH

2. Anterior pituitary cells respond by secreting Leutenizing hormone (LH) and follicle stimulating hormone (FSH) into the circulation

3. Ovaries and testis respond to increasing amounts of LH and FSH by growing and beginning to produce estradiole and testosterone

4. Increased levels of estradiol and testosterone produced by the body initiates female and male puberty

Major hormones

Neurokinin B (a tachykinin peptide) and kisspeptin (a neuropeptide): both present in the same hypothalamic neurons; critical parts of the control system that switches on the release of GnRH at the start of puberty

LH (luteinizing hormone): a larger protein hormone secreted into the general circulation by gonadotrope cells of the anterior pituitary gland; the main target cells of LH are the Leydig cells of testes and the theca cells of the ovaries

FSH (follicle stimulating hormone): another protein hormone secreted into the general circulation by the gonadotrope cells of the anterior pituitary; The main target cells of FSH are the ovarian follicles and the Sertoli cells and spermatogenic tissue of the testes

GnRH (Gonadotropin Releasing Hormone):  a peptide hormone released from the hypothalamus that stimulates gonadotrope cells of the anterior pituitary

Gonadotropin:  protein hormones secreted by gonadotrope cells of the pituitary gland of vertebrates, including the mammalian hormones follitropin (FSH)and lutropin (LH)

For more information, refer to: http://en.wikipedia.org/wiki/Puberty#Components_of_the_endocrine_reproductive_system

To see how GnRH release affect testosterone production, follow the link:

Kisspeptin and kisspeptinocentric view of GnRH release

Figure 3: Schematic diagram of kisspeptin signaling regulating GnRH release (Yeo, 2007)

Inactivation of kisspeptin signaling from kissepeptin receptor KISS1R is associated with hypogonadotropic hypogonadism and absent or delayed puberty in man3,4. As seen in study by Oakley et al., kisspeptin signaling works by signaling in the brain that mediates a negative feedback action of sex steroids on gonadotropin secretion, that generates preovulatory GnRH/LH surge, ultimately triggering the sexual maturation at puberty15.

Such discovery led to more studies on signaling pathways triggering puberty initiation in mammals and the notion of “kisspeptinocentric view”of the neural control of GnRH release!

Neurokinin B has a similar role as Kisspeptin in GnRH release

A study by Topaloglu and co-workers in 2009 reported that mutation of the genes encoding neurokininB (NKB) (TAC3) and its receptor (TAC3R) are also associated with hypogonadotropic hypogonadism as reported earlier for KISS1R mutations5,6 .

In sheep, it was found that Kisspeptin and NKB are co-expressed in the arcuate neuron, and that they are required for a pulsatile release of GnRH in humans and maintenance of adult gonadal function3,4.

Figure 4: Greater than 95% of DYN cells coexpressed NKB and Kisspeptin in sheep (Cheng 2012)

Problems Encountered

In mice experiments, deletion of KISS yielded similar results as seen in human studies; however, deletion of NKB did not lead to infertility as did in human studies. In other rodent studies NKB signaling pathway exhibited no action, or rather had an inhibitory action, on GnRH release5,6. These issues had to be reconciled with human genetics and more studies were in need to discover the NKB signaling pathway5,6.

Discovering the Effect of NKB signaling on GnRH release in Primate

In the present study by Ramaswamy et al., a series of experiments were performed to discover the action of NKB signaling on GnRH release in rhesus monkey14. Rhesus monkey was selected, especially for its attribute in having a similar post-natal pattern of pulsatile GnRH release to that in human14.

Many conditions followed in this study. Only juvenile males were selected because during stage of development, spontaneous GnRH release is more restraint in male that that of female14. Therefore, any stimulatory action of NKB on GnRH release maybe more readily detected14. Also, the subjects were castrated to eliminate any confounding effects of testicular feedback on gonadotropin secretion14.

Discussion of Role of NKB in GnRH Release

Neurokinin B, kisspeptin-10 (Kp-10), a NKB receptor (NK3R) peptide agonist senktide, and GnRH antagonist acylin and GnRH were synthesized or obtained to be injected in a dose-manner, bolus injections to observe LH discharge the rhesus monkeys.

The injection of senktide elicited a robust LH discharge. Injection of NKB also elicited a marked discharge of LH, as well as GnRH14 (Figure 5).

Figure 5: Concentrations of LH after bolus iv injections of senktide

Senktide- induced LH release was abolished when pretreated with GnRH-R antagonist, acyline (Figure 6). GnRH injection elicited LH discharge similar to that released spontaneously by endogenous pulsatile GnRH release supported by GnRH discharge from the hypothalamus of  juvenile in response senktide iv bolus being equivalent to that produced spontanesouly in pubertal and postpubertal animals14.

Figure 6: Concentrations of LH (mean ± sem) after the last GnRH (G) pulse of the priming infusion (open arrow) and sequential bolus iv injections (arrows) of senktide (S) (50 μg), NKB (N) (100 μg), and GnRH (0.3 μg) (black arrows) given to agonadal juvenile male monkeys when a GnRH-R antagonist, acyline (A) (arrowhead) 30 min after the first GnRH challenge (closed data points)

Kp-10 produced a LH discharge with amplitude greater than that observed in senktide response (Figure 7). Therefore, it is concluded that Kp-10 is the most potent releaser of GnRH14.

Figure 7: Concentrations of LH (mean ± sem) after bolus iv injections (arrows) of senktide (S) (50 μg), Kp-10 (K) (2 μg), and GnRH (G) (0.3 μg) to agonadal juvenile male monkeys

The LH response to the NK3R agonist was abolished when introduced to pretreated animal with the NK3R antagonist SB222200, but LH discharge induced by Kp-10 was not affected14, revealing that SB222200did not interfere with stimulatory action of Kp-10 (Figure 8). This proposes that NK3R and KISS1R signaling pathways are independent and that NKB pathway does not affect KP-10 induced GnRH release14.

Figure 8: Administration of NK3R antagonist (SB222200; white arrow) at −15 min abolished the LH response to an iv bolus injection of senktide (S) (50 μg; black arrow, triangle data points) given at time 0 but did not interfere with LH release when an iv bolus of KP-10 (K) (2 μg; black arrow, closed circle data points) was given at time.

Using double label fluorescence immunohistochemistry,  et al. found that NKB is expressed in arcuate kisspeptin neurons in primates (Figure 9).

Figure 9: Immunofluorescence projections showing co-localization of NKB (red fluorescence, Cy3) and kisspeptin (green fluorescence, Alexa 488) in the arcuate nucleus (ARC) and median eminence (ME), respectively. A–C, Confocal projections (magnification, ×10; 1-μm optical sections) showing immunopositive kisspeptin (A) and NKB (B) profiles and their merged image (C) in a coronal hemisection taken through the ARC. The third ventricle (3V) is seen on the right hand side of the half section. Yellow is the co-localization of NKB with kisspeptin in both ARC and ME. D and E, Higher magnification (×40, 1-μm optical sections) confocal projections taken from the ARC (D) and the ME (E) of the section shown in A–C. It is evident that NKB and kisspeptin in many of the ARC kisspeptin positive perikarya (D) and in kisspeptin axonal projections in the ME (E) are co-localized.

Ramaswamy et al. provides a description of a GnRH- releasing action of NK3R agonists in a primate14. Although repetitive activation of NK3R failed to sustain pulsatile GnRH release, a single iv bolus of NKB and senktide elicited a robust discharge of GnRH14. It also provides a clue to where NKB signaling to GnRH neurons may take place14. Because this study does not provide an answer to how NKB signaling pathway occurs and whether NKB and kisspeptin signal work in parallel or in a cascade-manner to result in pulsatile GnRH release, further studies in the future may be addressed to investigate actions of these two peptides and how they work together in driving neuro-endocrine axis, governing the initiation of puberty and reproduction14.

References

1Hwang JS, 2012 The genes associated with gonadotropin-releasing hormone-dependent precocious puberty. Korean J Pediatr.55(1): 6–10.

2Ko JM, Lee HS, Hwang JS. KISS1 gene analysis in Korean girls with central precocious puberty: a polymorphism, p.P110T, suggested to exert a protective effect. Endocr J. 2010;57:701–709

3Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley Jr WF, Apaicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627.

4de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS-1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976

5Topaloglu AK, Reimann F, Guclu M, Yalin AS, Kotan LD, Porter KM, Serin A, Mungan NO, Cook JR, Ozbek MN, Imamoglu S, Akalin NS, Yuksel B, O’Rahilly S, Semple RK 2009 TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat Genet 41:354–358.

6Guran T, Tolhurst G, Bereket A, Rocha N, Porter K, Turan S, Gribble FM, Kotan LD, Akcay T, Atay Z, Canan H, Serin A, O’Rahilly S, Reimann F, Semple RK, Topaloglu AK 2009 Hypogonadotropic hypogonadism due to a novel missense mutation in the first extracellular loop of the neurokinin B receptor. J Clin Endocrinol Metab 94:3633–3639.

7 Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR 1996 KiSS1, a novel human malignant melanoma metastis-suppressor gene. J Natl Cancer Inst 88:1731–1737.

8Plant TM, Witchel SF 2006 Puberty in non-human primates and humans. In: Challis JRG, de Kretser DM, Neill JD, Pfaff DW, Plant TM, Richards JS, Wassarman PM, eds. Knobil and Neill’s Physiology of Reproduction. 3rd ed. San Diego, CA: Elsevier; 2177–2230.

9Kung TT, Crawley Y, Jones H, Luo B, Gilchrest H, Greenfeder S, Anthes JC, Lira S, Wiekowski M, Cook DN, Hey JA, Egan RW, Chapman RW 2004 Tachykinin NK3-receptor deficiency does not inhibit pulmonary eosinophilia in allergic mice. Pharmacol Res 50:611–615.

10 Sandoval-Guzmán T, Rance NE 2004 Central injection of senktide, an NK3 receptor agonist, or neuropeptide Y inhibits LH secretion and induces different patterns of Fos expression in the rat hypothalamus. Brain Res 1026:307–312.

11Navarro VM, Gottsch ML, Chavkin C, Okamura H, Clifton DK, Steiner RA 2009 Regulation of gonadotropin-releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse. J Neurosci 29:11859–11866.

12Corander MP, Challis BG, Thompson EL, Jovanovic Z, Loraine Tung YC, Rimmington D, Huhtaniemi IT, Murphy KG, Topaloglu AK, Yeo GS, O’Rahilly S, Dhillo WS, Semple RK, Coll AP 2010 A study of the effects of neurokinin B upon gonadotrophin release in male rodents. J Neuroendocrinol 22:181–187.

13Goodman RL, Lehman MN, Smith JT, Coolen LM, de Oliveira CV, Jafarzadehshirazi MR, Pereira A, Iqbal J, Caraty A, Ciofi P, Clarke IJ 2007 Kisspeptin neurons in the arcuate nucleus of the ewe express both dynormphin A and neurokinin B. Endocrinology 148:5752–5760.

14 Ramaswamy S, Seminara S.B., Ali B, Ciofi P, Amin N.A., and Plant T.M. 2010 Neurokinin B Stimulates GnRH Release in the Male Monkey (Macaca mulatta) and Is Colocalized with Kisspeptin in the Arcuate Nucleus. Endocrinology 151(9): 4494–4503.

15Oakley AE, Clifton DK, and Steiner RA. 2009. Kisspeptin Signaling in Brain. Endocr Rev. 30(6):713-43.

Image References

16Smith, N. (Producer). (2008). hormone and their function during normal puberty. [Web Photo]. Retrieved from http://kallmanns.org/node/54

17Yeo, S. H. (Composer). (2007). Understanding the roles of arcuate kisspeptin neurons. [Web Photo]. Retrieved from http://physoc.org.nz/assets/images/models/Fig1.png

18(2012). The puberty process. (2012). [Web Photo]. Retrieved from https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcRw4ViQ_Ul5lJTOgY64Z-IvM0cPv_yJkRRU_uuJG8aXUJZ5tcPO

19Cheng G., Coolen L.M., Padmanabhan V., Goodman R.L., and Lehman M.N. 2010 The Kisspeptin/Neurokinin B/Dynorphin (KNDy) Cell Population of the Arcuate Nucleus: Sex Differences and Effects of Prenatal Testosterone in Sheep. Endocrinology 151 (1): 301.

In mice experiments, deletion of KISS (3)yielded similar results as seen in human studies; however, deletion of NKB (9)did not lead to infertility as did in human studies. In other rodent studies, NKB signaling pathway exhibited no action, or rather had an inhibitory action, on GnRH release(10,11). These issues had to be reconciled with human genetics and more studies were in need to discover the NKB signaling pathway.

Examination of NKB signaling on GnRH release in rhesus monkey

In the present study by Ramaswamy et al., a series of experiments were performed to discover the action of NKB signaling on GnRH release in rhesus monkey14. Rhesus monkey was selected, especially for its attribute in having a similar post-natal pattern of pulsatile GnRH release to that in human14.

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