Periventricular Kisspeptin Neurons and the GnRH / LH Surge System in Male Rats 1

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats. GPR54, Kiss1, Kiss1r, luteinizing hormone, metastin, sexual differentiation INTRODUCTION The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge is known to be sexually differentiated in rodents [1, 2]. Positive estrogen feedback to induce GnRH/LH surge is evident in female rats, whereas male rats never show LH surges, even with a preovulatory level of estradiol (E2) [3]. The LH surgegenerating system has been implicated to be disrupted by neonatal E2 converted from testosterone because a single estradiol benzoate (EB) injection during the neonatal period, such as at Day 0 or Day 5 of age, causes persistent estrus and failure of ovulation in female rats [4, 5], and because 5adihydrotestosterone, an unaromatizable androgen, did not affect estrous cyclicity in neonatal female rats [6]. Corbier [7] found that male rats castrated within 1 h after birth showed an afternoon increase in LH levels compared with the morning level, whereas neonatal castration at 24 h postnatally failed to feminize the afternoon increase in LH. Actually, circulating levels of androgen are much higher in male than female rats from prenatal Day 18 to Day 1 after birth [8]. Kisspeptin (first named metastin)-Kiss1r (previously called GPR54) signaling [9, 10] plays a critical role in regulating reproductive function via stimulation of GnRH/LH release in mammals [11–15], and loss of Kiss1r function in human and rodents causes hypogonadotropic hypogonadism [16, 17]. Several bodies of evidence suggest that kisspeptin neurons are sexually dimorphic. Our work [18] and a number of other studies [19–21] showed that cell bodies of kisspeptin neurons were distributed in the anteroventral periventricular nucleus (AVPV) and the hypothalamic arcuate nucleus (ARC) in female rats and mice. On the other hand, males show a different distribution of kisspeptin neurons, which are expressed in the ARC but are few in the AVPV. Interestingly, the AVPV has long been suggested to be a sexual dimorphic nucleus and a center for GnRH/LH surge [22, 23]. Electrolytic lesion of a small periventricular column of cells located immediately caudal to the organum vasculosum of the lamina terminalis (OVLT), which is equivalent to the AVPV, or of medial preoptic nucleus (MPO) results in persistent estrus and failure of the steroid-induced LH surge [24], whereas E2 implantation in the anterohypothalamic area, including the AVPV in the ovariectomized (OVX) rat, showed a surge-like increase in LH release [25]. Hence, AVPV kisspeptin neurons are considered to be a target of estrogen-positive feedback action to induce GnRH/LH surge. Indeed, estrogens positively regulate AVPV kisspeptin and Kiss1 mRNA expression in female rodents [18, 20]. The elegant work of Kauffman et al. [19] showed that neonatally androgenized female rats display a malelike pattern of reduced Kiss1 expression in the AVPV. Recently, Navarro et al. [26] demonstrated that neonatal EB treatment suppresses Supported by grant nos. 18658106 and 19380157 to H.T. and grant no. 18208025 to K. -I.M. from the Japan Society for the Promotion of Science’s Grants-in-Aid and by the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan. Correspondence: Hiroko Tsukamura, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. FAX: 81 52 789 4072; e-mail: htsukamura@nagoya-u.jp Received: 22 April 2009. First decision: 19 May 2009. Accepted: 24 July 2009. 2009 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod. org ISSN: 0006-3363 1216 D ow nladed rom w w w . biolrod.org. whole hypothalamic Kiss1 mRNA expressions in male and female rats. Furthermore, neonatal treatment of estrogenic substances or estrogen receptor-a (ESR1) agonist in female rats causes reduction of AVPV kisspeptin neurons [27, 28]. All of these findings provide circumstantial evidence that estrogens converted from fetal or neonatal testicular androgens cause loss of the AVPV kisspeptin expression and GnRH/LH surge mechanism in male rats. However, there has been no direct evidence for the role of neonatal testicular androgen in defeminization of AVPV kisspeptin neurons in genetically male rats. The neonatal steroidal milieu could not merely be the determinant for neuronal phenotype. Genetic factors have also been considered to be another determinant for sex difference in neurons because sex chromosome genes have been implied to cause sexual dimorphisms of behaviors and neuronal phenotypes of dopaminergic or vasopressin neurons [29, 30]. Therefore, it is of great interest to clarify the role of neonatal testicular androgen in defeminizing AVPV kisspeptin expression by removing neonatal steroids. Another issue to be addressed is whether neonatal steroids affect other AVPV neurons besides kisspeptin, because a number of neurotransmitters or neuropeptides in the AVPV, such as glutamate [31–34], gamma-aminobutyric acid (GABA) [35, 36], galanin [37, 38], neurotensin [39], and dopamine [22, 40, 41], have been suggested to be involved in the induction of the GnRH/LH surge. Ottem et al. [34] have demonstrated that ERa-immunoreactive GABA/glutamate neurons in the AVPV show sexual dimorphism and suggested that these dualphenotype neurons are the main targets of the positive estrogen feedback action on GnRH neurons. Galanin, neurotensin, and dopaminergic neurons in the AVPV are also acknowledged to be sexually dimorphic and larger in female rats than in male rats [37, 40–42], and neonatal testosterone causes masculinization of AVPV dopaminergic neurons [43]. Therefore, it is noteworthy to investigate the effects of neonatal steroidal manipulation on gene expressions of these neuronal markers in the AVPV to determine whether the manipulation specifically affects Kiss1 expression. The present study aimed to determine whether androgen released from neonatal testes defeminizes AVPV kisspeptin neurons, and then the GnRH/LH surge-generation system. To this end, we tested whether castration at birth feminizes AVPV gene expression of kisspeptin and/or the other neuropeptides, enzymes, and transporters for neurotransmitters that may result in feminization of the estrogen-positive feedback system in genetic male rats. In addition, we examined whether the attenuation of AVPV kisspeptin caused by neonatal estrogen is associated with the absence of a GnRH/LH surge system in female rats. MATERIALS AND METHODS