Direct Action of Genistein on the Hypothalamic Neuronal Circuits in Female Rats

  • Lee, Woo-Cheol (Dept. of Green Life Science, Sangmyung University) ;
  • Lee, Sung-Ho (Dept. of Green Life Science, Sangmyung University)
  • Received : 2009.12.14
  • Accepted : 2010.03.09
  • Published : 2010.03.31

Abstract

Mammalian reproduction is regulated by a feedback circuit of the key reproductive hormones such as GnRH, gonadotropin and sex steroids on the hypothalamic-pituitary-gonadal axis. In particular, the onset of female puberty is triggered by gain of a pulsatile pattern and increment of GnRH secretion from hypothalamus. Previous studies including our own clearly demonstrated that genistein (GS), a phytoestrogenic isoflavone, altered the timing of puberty onset in female rats. However, the brain-specific actions of GS in female rats has not been explored yet. The present study was performed to examine the changes in the activities of GnRH neurons and their neural circuits by GS in female rats. Concerning the drug delivery route, intracerebroventricular (ICV) injection technique was employed to eliminate the unwanted actions on the extrabrain tissues which can be occurred if the testing drug is systemically administered. Adult female rats (PND 100, 210-230 g BW) were anaesthetized, treated with single dose of GS ($3.4{\mu}g$/animal), and sacrificed at 3 hrs post-injection. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). ICV infusion of GS significantly raised the transcriptional activities of enhanced at puberty1 (EAP-1, p<0.05), glutamic acid decarboxylase (GAD67, p<0.01) which are known to modulate GnRH secretion in the hypothalamus. However, GS infusion could not change the mRNA level of nitric oxide synthase 2 (NOS-2). GS administration significantly increased the mRNA levels of KiSS-1 (p<0.001), GPR54 (p<0.001), and GnRH (p<0.01) in the hypothalami, but decreased the mRNA levels of LH-$\beta$ (p<0.01) and FSH-$\beta$ (p<0.05) in the pituitaries. Taken together, the present study indicated that the acute exposure to GS could directly activate the hypothalamic GnRH modulating system, suggesting the GS's disrupting effects such as the early onset of puberty in immature female rats might be derived from premature activation of key reproduction related genes in hypothalamus-pituitary neuroendocrine circuit.

References

  1. Ahmed ML, Ong KK, Dunger DB (2009) Childhood obesity and the timing of puberty. Trends Endocrinol Metab 20:237-242. https://doi.org/10.1016/j.tem.2009.02.004
  2. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159.
  3. Cook AM, Mieure KD, Owen RD, Pesaturo AB, Hatton J (2009) Intracerebroventricular administration of drugs. Pharmacotherapy 29:832-845. https://doi.org/10.1592/phco.29.7.832
  4. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC (2009) Endocrine-disrupting chemicals: An endocrine society scientific statement. Endocr Rev 30:293-342. https://doi.org/10.1210/er.2009-0002
  5. Ebling FJ (2005) The neuroendocrine timing of puberty. Reproduction 129:675-683. https://doi.org/10.1530/rep.1.00367
  6. Fitzpatrick LA (2003) Soy isoflavones: hope or hype? Maturitas 44 Suppl 1:S21-29. https://doi.org/10.1016/S0378-5122(02)00345-6
  7. Heger S, Mastronardi C, Dissen GA, Lomniczi A, Cabrera R, Roth CL, Jung H, Galimi F, Sippell W, Ojeda SR (2007) Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis. J Clin Invest 117:2145-2154. https://doi.org/10.1172/JCI31752
  8. Kouki T, Kishitake M, Okamoto M, Oosuka I, Takebe M, Yamanouchi K (2003) Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis. Horm Behav 44:140-145. https://doi.org/10.1016/S0018-506X(03)00122-3
  9. Lee K-Y, Lee S-H (2006a) Effect of genistein on the onset of puberty in female rats. Dev Reprod 10:55-61.
  10. Lee K-Y, Lee S-H (2006b) Effects of endocrine disruptors on the expression of estrogen receptors in ovary and uterus from immature rats. Dev Reprod 10:255-261.
  11. McClain RM, Wolz E, Davidovich A, Edwards J, Bausch J (2007) Reproductive safety studies with genistein in rats. Food Chem Toxicol 45:1319-1332. https://doi.org/10.1016/j.fct.2007.01.009
  12. Massart F, Parrino R, Seppia P, Federico G, Saggese G (2006) How do environmental estrogen disruptors induce precocious puberty? Minerva Pediatr 58:247-254.
  13. Messina M, Nagata C, Wu AH (2006) Estimated Asian adult soy protein and isoflavone intakes. Nutr Cancer 55:1-12. https://doi.org/10.1207/s15327914nc5501_1
  14. Misztal T, Gorski K, Romanowicz K (2008) Differential endocrine response in rams to intracerebroventricular infusion of genistein. Acta Neurobiol Exp (Wars) 68: 43-50.
  15. Mitsushima D, Marzban F, Luchansky LL, Burich AJ, Keen KL, Durning M, Golos TG, Terasawa E (1996) Role of glutamic acid decarboxylase in the prepubertal inhibition of the luteinizing hormone releasing hormone release in female rhesus monkeys. J Neurosci 16:2563- 2573.
  16. Parent AS, Rasier G, Gerard A, Heger S, Roth C, Mastronardi C, Jung H, Ojeda SR, Bourguignon JP (2005) Early onset of puberty: tracking genetic and environmental factors. Horm Res 64 Suppl 2:41-47.
  17. Plant TM (1994) Puberty in primates. In: Knobil E & Neill JD(eds.), The Physiology of Reproduction, 2nd ed. Raven Press, New York, pp 453-485.
  18. Polkowska J, Ridderstrale Y, Wa?owska M, Romanowicz K, Misztal T, Madej A (2004) Effects of intracerebroventricular infusion of genistein on gonadotrophin subunit mRNA and immunoreactivity of gonadotrophins and oestrogen receptor-alpha in the pituitary cells of the anoestrous ewe. J Chem Neuroanat 28:217-224. https://doi.org/10.1016/j.jchemneu.2004.07.004
  19. Rasier G, Toppari J, Parent AS, Bourguignon JP (2006) Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: A review of rodent and human data. Mol Cell Endocrinol 254-255:187-201. https://doi.org/10.1016/j.mce.2006.04.002
  20. Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, McCann SM (1993) Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc Natl Acad Sci USA 90:10130-10134. https://doi.org/10.1073/pnas.90.21.10130
  21. Roa J, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M (2008) New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function. Front Neuroendocrinol 29:48-69. https://doi.org/10.1016/j.yfrne.2007.07.002
  22. Rosselli M, Keller PJ, Dubey RK (1998) Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Hum Reprod Update 4:3-24. https://doi.org/10.1093/humupd/4.1.3
  23. Wojcik-Gładysz A, Romanowicz K, Misztal T, Polkowska J, Barcikowski B (2005) Effects of intracerebroventricular infusion of genistein on the secretory activity of the GnRH/LH axis in ovariectomized ewes. Anim Reprod Sci 86:221-235. https://doi.org/10.1016/j.anireprosci.2004.08.004