Age-related alterations in hypothalamic kisspeptin, neurokinin B, and dynorphin neurons and in pulsatile LH release in female and male rats

Neurobiology of Aging 50 (2017) 30e38

Contents lists available at ScienceDirect

Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging

Age-related alterations in hypothalamic kisspeptin, neurokinin B, and dynorphin neurons and in pulsatile LH release in female and male rats Yuyu Kunimura a, Kinuyo Iwata a, Akihito Ishigami b, Hitoshi Ozawa a, * a b

Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2016 Received in revised form 6 October 2016 Accepted 12 October 2016 Available online 19 October 2016

Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) decreases during aging. Kisspeptin (encoded by Kiss1) neurons in the arcuate nucleus coexpress neurokinin B (Tac3) and dynorphin (Pdyn) and are critical for regulating the GnRH/LH pulse. We therefore examined kisspeptin neurons by histochemistry and pulsatile LH release in rats aged 2e3 (Young), 12e13 (YoungMiddle), 19e22 (Late-Middle), and 24e26 (Old) months. Total LH concentrations, sampled for 3 hours, decreased in both sexes with aging. In females, numbers of Tac3 and Pdyn neurons were significantly reduced in all aging rats, and numbers of Kiss1 neurons were significantly reduced in Late-Middle and Old rats. In males, numbers of all 3 neuron-types were significantly decreased in all aging rats. GnRH agonist induced LH release in all animals; however, the increased LH concentration in all aging rats was less than that in Young rats. These results suggest that expression of each gene in kisspeptin neurons may be controlled individually during aging, and that reduction of their expression or change in pituitary responsiveness may cause attenuated pulsatile LH secretion. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: KNDy neuron Pulse generator Aging Menopause Reproduction

1. Introduction In mammals, reproductive function is regulated by the hypothalamic-pituitary-gonadal axis. In the hypothalamus, gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile manner, and it stimulates pulsatile luteinizing hormone (LH) secretion from the pituitary. Pulsatile GnRH/LH release regulates folliculogenesis, spermatogenesis, and steroidogenesis (Filicori et al., 2002; Palermo, 2007; Ruwanpura et al., 2010). For example, pulsatile LH release acts on follicles in the ovary and stimulates the production of sex steroid hormones and formation of the corpus luteum in females. In males, LH stimulates Leydig cells to produce testosterone, which is required for sperm development. To maintain these functions, pulsatile LH release is indispensable for mammalian reproduction. It is known that aging causes reproductive decline in mammals, including hormonal abnormalities and infertility (Batista et al., 1995; Bishop, 1970; Finch, 2014). In rodents, pulsatile LH release is altered by aging, and a number of studies have suggested that the * Corresponding author at: Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Tel.: þ81-3-3822-2131 x5320; fax: þ81-3-5685-6640. E-mail address: [email protected] (H. Ozawa). 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.10.018

age-related decrease in pulsatile LH secretion is caused by alteration of the LH pulse generator. Pulsatile LH release decreases during aging in ovariectomized female rats, which have an increased LH pulse because the negative feedback effect is absent (Estes et al., 1980; Scarbrough and Wise, 1990; Wise et al., 1988). In male rats, reduced secretion of pulsatile LH was observed in intact and castrated aging animals (Karpas et al., 1983; Steiner et al., 1984). Because gonadectomized aged animals exhibit markedly reduced pulsatile LH secretion, it was suggested that the age-related reduction of LH pulses is not because of altered sensitivity of the gonadal steroid negative feedback, but rather deterioration of the LH pulse generating system. However, because the LH pulse generator was only recently discovered, the mechanism underlying alteration of the LH pulse is still unknown. Recent studies have identified neurons in the hypothalamic arcuate nucleus (ARC) that express 3 different peptides, kisspeptin, neurokinin B (NKB), and dynorphin, termed KNDy neurons. Coexpression of kisspeptin, NKB, and dynorphin have been reported in several species, including rats, mice, goats, sheep, and humans (Burke et al., 2006; Goodman et al., 2007; Lehman et al., 2010; Murakawa et al., 2016; Navarro et al., 2009; Rance, 2009; Wakabayashi et al., 2010). Several lines of evidence demonstrate ARC neurons coexpressing kisspeptin, NKB, and dynorphin to be the LH pulse generator. First, kisspeptin, a neuropeptide encoded by the

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

Kiss1 gene, is a strong activator of GnRH neurons and is required for episodic GnRH release (Goodman and Lehman, 2012; Gottsch et al., 2004; Irwig et al., 2004; Keen et al., 2008; Maeda et al., 2010; Messager et al., 2005; Oakley et al., 2009; Ohkura et al., 2009b; Roseweir et al., 2009). Furthermore, impairment of kisspeptin activity results in hypogonadotropic hypogonadism in mice and humans (d’Anglemont de Tassigny et al., 2007; de Roux et al., 2003; Lapatto et al., 2007; Seminara et al., 2003). Second, kisspeptin neurons express receptors of sex steroid hormones, and circulating estradiol/testosterone inhibits Kiss1 mRNA expression in the ARC. This means that negative feedback regulation of GnRH secretion by sex steroid hormones is mediated by ARC kisspeptin neurons (Adachi et al., 2007; Smith et al., 2005, 2006). Third, multiple-unit electrical activity (MUA) in close apposition to ARC kisspeptin neurons exhibited rhythmic volleys, and each volley was associated with an LH pulse in goats (Ohkura et al., 2009a), suggesting pulsatile kisspeptin release from ARC kisspeptin neurons. Finally, NKB showed stimulatory, whereas dynorphin showed inhibitory, effects on neuronal activity of kisspeptin neurons, and pulsatile LH release. In goats, central administration of NKB induced MUA volleys in close apposition to ARC kisspeptin neurons, whereas dynorphin inhibited MUA volleys and pulsatile LH release (Wakabayashi et al., 2010). In addition, administration of NKB or a dynorphin receptor antagonist increased LH pulse frequency, whereas administration of an NKB receptor antagonist decreased LH pulse frequency in ewes (Goodman et al., 2013). The expression of NKB and dynorphin receptors in kisspeptin neurons has also been reported in mice (Navarro et al., 2009), suggesting that the rhythmic oscillation of kisspeptin neurons is regulated by NKB and dynorphin. This evidence suggests that autosynaptic regulation of ARC kisspeptin neurons generates pulsatile kisspeptin release, which drives GnRH secretion in a pulsatile manner and hence pulsatile LH release from the pituitary (Keen et al., 2008; Navarro et al., 2009; Wakabayashi et al., 2010). These accumulating data demonstrate the indispensable roles of kisspeptin, NKB, and dynorphin in GnRH/LH pulse generation. From these findings, it can be hypothesized that the age-related decline in pulsatile LH release is caused by alterations to neurons coexpressing kisspeptin, NKB, and dynorphin; however, the involvement of these neurons in age-related pulsatile LH release remains unexamined. The present study, therefore, investigated the possible involvement of neurons expressing kisspeptin, NKB, and dynorphin in attenuated pulsatile LH release in aging rats. We determined the expression of mRNAs for kisspeptin, NKB, and dynorphin in the ARC. Female and male rats aged 2e3 (Young), 12e13 (Young-Middle), 19e22 (Late-Middle), and 24e26 (Old) months were used. We also examined GnRH-, kisspeptin-, NKB-, and dynorphin A-immunoreactive (ir) neurons, and pituitary responsiveness with aging by injecting a GnRH agonist. 2. Materials and methods 2.1. Animals and treatments Young female and male Wistar rats (2 months of age) were purchased from Tokyo Laboratory Animals Science Co (Tokyo, Japan). Middle-aged female and male Wistar rats (12e26 months of age) were obtained from Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan). They were all housed under controlled temperature (24  2  C) and lighting (14 hours light/10 hours dark, 6:00 lights on) with ad libitum access to standard rodent chow and water. We studied 4 different age groups for both females and males. To examine the GnRH/LH pulse generator without any endogenous circulating estradiol/testosterone feedback, gonadectomy (GDX) was performed on all animals. The ages of animals at

31

the day of GDX were the following. (1) Young: 2e3 months of age (M); (2) Young-Middle: 12e13 M; (3) Late-Middle: 19e22 M; and (4) Old: 24e26 M. The estrous cycles of Young and Young-Middle females were monitored by daily vaginal smears, and animals having shown at least 2 consecutive 4-day cycles were used. Vaginal smears of Late-Middle and Old females were obtained right before the GDX and all showed diestrus stage. Bilateral GDX was performed on all animals at least 2 weeks before blood sampling. The rats were cannulated with silicon tubing (0.50-mm inner diameter; 1.00-mm outer diameter; Shin-Etsu Polymer, Tokyo, Japan) into the right atrium through the jugular vein a day before blood sampling. All surgical procedures were performed under isoflurane anesthesia. All studies were conducted according to the NIH Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Experimentation Committee, Nippon Medical School. 2.2. Blood sampling Blood samples (100 mL) were collected every 6 minutes for 3 hours from freely-moving conscious rats via a silicone cannula. An equivalent volume of red blood cells, taken from a donor rat and diluted with heparinized saline, was replaced through the cannula after each blood collection. Plasma samples (50 mL) were separated by centrifugation and stored at 25  C until LH assay. 2.3. GnRH challenge on LH release To examine the effect of aging on pituitary responsiveness, GnRH agonist (buserelin) injection and blood sampling were performed. Blood samples (100 mL) were collected every 6 minutes for 3 hours from freely-moving conscious rats via a silicone cannula. One hour after the onset of blood sampling, GnRH agonist (1 mg/ 0.1 mL in 0.05 M phosphate buffer (PB) containing 0.9% sodium chloride [PBS], Sigma-Aldrich, St Louis, MO, USA; Gajewska et al., 2002) was injected intravenously into animals through the atrial cannula. 2.4. LH assay Plasma LH concentrations in 50 mL of plasma were measured by double-antibody radioimmunoassay using a rat LH radioimmunoassay kit provided by the National Hormone and Peptide Program (Baltimore, MD, USA). LH concentrations are expressed in terms of the National Institute of Diabetes and Digestive and Kidney Diseases rat LH RP-3. The minimum and maximum levels detectable were 0.156 ng/mL and 20 ng/mL in 50-mL plasma samples, respectively. Because plasma LH concentrations in Young animals injected with GnRH agonist were higher than the maximum detectable level, samples from Young animals were divided into 2 aliquots of 25 mL, and both samples were measured. Intraassay and interassay coefficients of variation were 6.60% at 2.12 ng/mL and 10.2% at 2.12 ng/mL, respectively. 2.5. Tissue preparation After blood samples were collected, all animals were deeply anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). Animals were perfused via the left ventricle of the heart with saline solution followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4. Brains were removed, postfixed in the same fixative solution at 4  C overnight, and cryoprotected in 30% sucrose in PB at 4  C for 4 days. Brains subjected to in situ hybridization (ISH) were processed under RNase-free conditions. Coronal sections (50 mm) were cut on a sliding microtome (Leica CM3050, Heidelberg, Germany) and

32

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

stored in 0.1 M PBS at 4  C until ISH or immunohistochemistry (IHC) were conducted. 2.6. In situ hybridization for Kiss1, Tac3, and Pdyn To detect Kiss1, Tac3, and Pdyn mRNA in the ARC, nonradioactive free-floating ISH was performed with rat Kiss1-, Tac3-, and Pdynspecific digoxigenin (DIG)-labeled probes as previously described (Adachi et al., 2007). Every forth section through the ARC (from 1.72- to 3.96-mm posterior to the bregma, Paxinos and Watson, 2007) was used for each ISH. Briefly, sections were hybridized with DIG-labeled antisense complementary RNA probes for Kiss1 (position 33348; GenBank accession no. AY196983; Terao et al., 2004), Tac3 (position 180-483; GenBank accession no. NM_019162; Mostari et al., 2013), and Pdyn (position 315-731; GenBank accession no. NM_019374; Mostari et al., 2013) using a DIG RNA labeling kit (Roche Diagnostics, Mannhein, Germany). To visualize the DIG labeling, sections were incubated with an alkaline phosphatase-conjugated anti-DIG1 antibody diluted by 1:1000 (Roche Diagnostics) for 2 hours at 37  C and 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate solution for 2 hours at room temperature (RT). 2.7. Immunohistochemistry for kisspeptin, NKB, dynorphin A, and GnRH Every forth section through the ARC (from 1.72- to 3.96-mm posterior to the bregma, Paxinos and Watson, 2007) was used for each kisspeptin, NKB, and dynorphin A IHC. Free-floating sections were washed in 0.1 M PBS containing 0.3% Triton X-100 (PBST) and treated with 0.03% H2O2 in PBST to quench endogenous peroxidase activity. To detect kisspeptin, sections were incubated with PBST containing 5% normal rabbit serum at RT for 1.5 hours. Then, sections were immersed in mouse monoclonal anti-kisspeptin antibody (kindly provided by Dr Ohtaki, Takeda Pharmaceutical Co, Osaka, Japan; Kinoshita et al., 2005) diluted 1:5000 in PBST overnight at 4  C. To detect NKB, sections were incubated with 5% normal goat serum, and immersed in rabbit anti-NKB polyclonal antibody (Novus Biologicals, Littleton, CO, USA; Sawai et al., 2012) diluted 1:2500 in PBST overnight at 4  C. To detect dynorphin A, sections were incubated with 5% normal goat serum and immersed in rabbit anti-dynorphin A polyclonal antibody (Phoenix Pharmaceuticals, Burlingame, CA, USA; Yamada et al., 2008) diluted 1:5000 in PBST for 3 days at 4  C. To detect GnRH, every second section through the preoptic area (POA, from 0.48 to 0.48-mm posterior to the bregma, Paxinos and Watson, 2007) was incubated with 5% normal rabbit serum and immersed in mouse monoclonal antiGnRH antibody (LRH-13, kindly provided by Dr M.K. Park of the University of Tokyo, Japan; Park and Wakabayashi, 1986) diluted 1:1000 in PBST overnight at 4  C. Sections were incubated with biotinylated secondary antibody (HISTOFINE SAB-PO kit, Nichirei Corporation, Tokyo, Japan) diluted 1:1 in PBST for 2 hours at RT. Then, sections were incubated with streptavidin conjugated with horseradish peroxidase (HISTOFINE SAB-PO kit) diluted 1:1 in PBST for 2 hours at RT. Staining was developed using 3,30 -diaminobenzidine tetrahydrochloride (Sigma-Aldrich) with 0.009% hydrogen peroxide. Sections were washed with PBST 3 times for 10 minutes between each treatment. Sections were mounted on glass slides and completely dried overnight. Dehydration was performed through a graded ethanol series. The slides were then cleared in xylene and mounted with Permount mounting medium (Fisher Scientific, Fair Lawn, NJ, USA). The specificities of antibodies used in the experiment were previously described (Kinoshita et al., 2005; Park and Wakabayashi, 1986; Sawai et al., 2012; Yamada et al., 2008).

2.8. Image analysis All images were captured with a light BX-51 microscope equipped with a DP73 charge-coupled device camera (Olympus, Tokyo, Japan). Numbers of Kiss1-, Tac3-, and Pdyn-expressing cells in the ARC were counted on the computer display using NIH ImageJ software (v1.49b) with cell counter plug-in. The number of GnRH-ir cells in the POA was counted under the microscope. 2.9. Statistical analysis LH pulses were identified using the PULSAR algorithm (Merriam and Wachter, 1982). Area under the curve (AUC), frequency, and amplitude of LH pulses for 3 hours sampling period were calculated individually and then, for each group. Statistical differences in body weight, gonad weight, parameters of each LH pulse, and number of GnRH-ir cells were determined by 1-way ANOVA followed by Tukey’s test. Statistical differences in numbers of neurons in the ARC were determined by 2-way ANOVA (age  mRNA, or age  protein) followed by Bonferroni’s post hoc test. The effect of buserelin injection on LH release was analyzed by 2-way ANOVA (age  sampling time point) followed by Dunnett’s post hoc test versus time point just before the buserelin injection (point 0). All data are presented as the mean  standard error of the mean. All analysis was conducted using SPSS 20 for Windows software. When the p-value was less than 0.05, differences were considered significant statistically. 3. Results 3.1. Females Female body weights of Late-Middle and Old groups were significantly increased compared with Young and Young-Middle groups. Although increased body weight was observed, weights of both ovaries of Young-Middle, Late-Middle, and Old rats were significantly decreased compared with Young rats (Table 1). The histology of ovaries examined by hematoxylin-eosin staining is shown in Supplementary Fig. 1A. The number of follicles, including primary follicles to mature follicles, and atretic follicles, was significantly reduced in Late-Middle and Old compared with Young animals (Supplementary Fig. 1B). There was no significant difference in cross-sectional area of the corpus luteum at different ages when it was corrected by ovary cross-sectional area (Supplementary Fig. 1C). Representative profiles of pulsatile LH release in females of different ages are shown in Fig. 1A. The LH AUC or total amount of LH released was significantly lower in Young-Middle, Late-Middle, and Old rats compared with that in Young rats. The frequency and amplitude of LH pulses did not significantly differ among different ages (Fig. 1B); LH pulse frequency was not detected in 1 out of 4 animals in the Old group. Representative images of Kiss1, Tac3, and Pdyn mRNA in the ARC are shown in Fig. 2A. The number of Kiss1-expressing cells did not significantly differ in Young and Young-Middle rats; however, it was significantly decreased in Late-Middle and Old rats (Fig. 2B). The numbers of Tac3-and Pdyn-expressing neurons were significantly decreased in Young-Middle, Late-Middle, and Old compared with Young rats. In Young rats, Tac3-expressing neurons were significantly more numerous than Pdyn-expressing neurons. In YoungMiddle and Late-Middle animals, the number of Pdyn-expressing neurons was significantly lower than the number of Kiss1-or Tac3-expressing neurons. In Old rats, the number of Tac3-expressing neurons was significantly higher than the number of Kiss1-or Pdyn-expressing neurons (Fig. 2B).

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

33

Table 1 Body and gonad weight in female and male rats at different ages Weight (g) Female Body weight Ovary weight/body weight Male Body weight Testis weight/body weight

Young

Young-Middle

Late-Middle

Old

214  2a 0.00040  1.2E-05a

211  3a 0.00026  1.5E-05b

280  6b 0.00024  5.9E-06b,c

299  13b 0.00021  1.2E-05c

287  17a 0.00979  2.0E-04a,b

411  7b 0.00776  1.2E-04a

388  7b,c 0.01171  7.8E-04b,c

361  13c 0.01311  1.3E-03c

Means  standard error of the mean are shown. Values with different letters are significantly different from each other (p < 0.05). Female: Young (n ¼ 15), Young-Middle (n ¼ 8), Late-Middle (n ¼ 11), Old (n ¼ 8). Male: Young (n ¼ 9), Young-Middle (n ¼ 9), Late-Middle (n ¼ 8), Old (n ¼ 7). Gonad weight is the sum of the both gonads of each animal.

Fig. 2C shows the numbers of kisspeptin-, NKB-, and dynorphin A-ir neurons in the ARC. Although there was no significant interaction between age and protein, there were main effects. The total number of 3 types of neurons was significantly decreased in YoungMiddle, Late-Middle, and Old rats compared with that in Young rats (Fig. 2C). The total number of dynorphin A-ir neurons was significantly less than the number of kisspeptin- or NKB-ir neurons. There

was no significant difference in the numbers of kisspeptin- and NKB-ir neurons. Representative images of neurons showing kisspeptin-, NKB-, or dynorphin A-ir in the female ARC are shown in Supplementary Fig. 2. IHC for GnRH in the POA revealed that there was no significant difference in the number of GnRH-ir cells among different ages (Supplementary Fig. 3). Fig. 3 shows significant LH increase after i.v. buserelin (GnRH agonist) injection in all age groups. The plasma LH concentrations were significantly increased at 18, 18, 24, and 30 minutes after buserelin injection in Young, Young-Middle, Late-Middle, and Old animals, respectively. The increase in LH release was significantly higher in the Young compared with the other aged groups. Except at 12, 42, and 60 minutes before buserelin injection, the LH concentrations were significantly different between Young and YoungMiddle groups through 3 hours of sampling time. Except at 60 minutes before buserelin injection, the LH concentrations were significantly different between Young and Late-Middle groups through 3 hours of sampling time. Between Young and Old groups, the LH concentrations were significantly different over 3 hours of sampling. Between Young-Middle and Late-Middle groups, the LH concentrations were significantly different at 36 and 42 minutes before buserelin injection. The significant increases in LH levels were maintained for 2 hours after buserelin injection in all age groups.

3.2. Males

Fig. 1. Pulsatile luteinizing hormone (LH) secretion in Young, Young-Middle, LateMiddle, and Old females. (A) Plasma LH profiles in representative animals. Arrowheads indicate the peaks of LH pulses indentified with the PULSAR algorithm. (B) Area under the curve (AUC) of plasma LH concentrations, frequency, and amplitude of LH pulses. Y, Young (n ¼ 14); YM, Young-Middle (n ¼ 4); LM, Late-Middle (n ¼ 7); O, Old (n ¼ 4). Values are means  standard error of the mean. Values with different letters are significantly different (p < 0.05, 1-way ANOVA followed by Tukey’s test).

Male body weights of Young-Middle, Late-Middle, and Old rats were significantly increased compared with that of Young rats. However, the body weight of Old rats was significantly decreased compared with Young-Middle rats. Testes weight of Late-Middle animals was significantly increased compared with Young-Middle rats, and that of Old rats was significantly increased compared with Young and Young-Middle rats (Table 1). Testes histology, examined by hematoxylin-eosin staining, is shown in Supplementary Fig. 4. Seminiferous tubules were observed in Young and Young-Middle animals, and sperm production therein was detected. In contrast, the testes of Late-Middle and Old rats showed abnormal histology that was not observed in Young and Young-Middle animals; the diameter of seminiferous tubules was smaller than that of Young and Young-Middle rats, and no sperm production was observed. This abnormal histology may represent testicular tumors, which are observed in aging males (Poteracki and Walsh, 1998). Representative profiles of pulsatile LH release in different aged males are shown in Fig. 4A. The LH AUC was significantly lower in Late-Middle and Old rats compared with that in Young rats (Fig. 4B). The frequency was significantly increased in Late-Middle animals compared with that in Young animals. The amplitude of LH pulses did not significantly differ among different ages. Representative images of Kiss1, Tac3, and Pdyn mRNA in the ARC are shown in Fig. 5A. Although there was no significant interaction

34

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

Fig. 2. Expression of kisspeptin, neurokinin B (NKB), and dynorphin neurons in the arcuate nucleus (ARC) in Young, Young-Middle, Late-Middle, and Old females. (A) In situ hybridization of Kiss1, Tac3, and Pdyn mRNA in representative animals. (B) Numbers of Kiss1-, Tac3-, and Pdyn-expressing cells in the ARC. Young (n ¼ 11), Young-Middle (n ¼ 4), Late-Middle (n ¼ 7), Old (n ¼ 4). (C) Numbers of kisspeptin-, NKB-, and dynorphin A-immunoreactive (ir) cells in the ARC (n ¼ 4 each). Scale bar ¼ 200 mm. Values are means  standard error of the mean. Values with different letters and asterisks are significantly different in the number of neurons at each age and versus Young, respectively. (p < 0.05, 2way ANOVA followed by Bonferroni’s post hoc test).

between age and mRNA, there were main effects (Fig. 5B). The total number of 3 types of neurons was significantly decreased in YoungMiddle, Late-Middle, and Old rats compared with Young rats. The total number of Pdyn-expressing neurons was significantly less than the number of Kiss1-or Tac3-expressing neurons. There was no significant difference between the numbers of Kiss1-and Tac3expressing neurons. There was no significant interaction between age and protein in male animals; however, there were main effects. The total number of 3 types of neurons was significantly decreased in Late-Middle and Old rats compared with Young rats (Fig. 5C). The total number of dynorphin A-ir neurons was significantly less than the number of kisspeptin- or NKB-ir neurons. There was no significant difference between the numbers of kisspeptin- and NKB-ir neurons. Representative images of kisspeptin-, NKB-, and dynorphin A-ir neurons in the ARC are shown in Supplementary Fig. 5. There was no significant difference in the number of GnRH-ir neurons in the POA among different aged males (Supplementary Fig. 6). LH release was significantly increased by i.v. buserelin injection in all ages (Fig. 6). In Young animals, the plasma LH concentration was significantly increased from 12 minutes to 120 minutes after buserelin injection. In Young-Middle animals, the plasma LH concentration was significantly increased from 42 minutes to 120 minutes after buserelin injection, except at 102 minutes. In LateMiddle animals, the plasma LH concentration was significantly increased from 24 minutes after buserelin injection, except at 30 and 36 minutes. In Old animals, the plasma LH concentration was

significantly increased from 24 minutes to 120 minutes after buserelin injection, except at 54 minutes. The increase in LH release was significantly higher in Young rats compared with the other aged groups. Except at 0, 12, 24, and 36e54 minutes before buserelin injection, the LH concentrations were significantly different between Young and Young-Middle groups. Except at 0, 12, 48, and 54 minutes before buserelin injection, the LH concentrations were significantly different between Young and Late-Middle groups. Between Young and Old groups, the LH concentrations were significantly different at 6e120 minutes after buserelin injection. Between Young-Middle and Old groups, the LH concentrations were significantly different at 12 minutes before and 102 minutes after the buserelin injection. Between Late-Middle and Old groups, the LH concentrations were significantly different at 12, 36 to 48, and 60 minutes before and 12 minutes after buserelin injection. 4. Discussion Evidence accumulated over several decades shows that pulsatile LH release decreases with aging and, in rodents, deterioration of the GnRH/LH pulse generator has been suggested as a cause of the reduction (Estes et al., 1980; Karpas et al., 1983; Scarbrough and Wise, 1990; Steiner et al., 1984; Wise et al., 1988). However, the underlying mechanism has not been determined because the GnRH/LH pulse generator has only recently been identified to consist of neurons coexpressing kisspeptin, NKB, and dynorphin. In the present study, we revealed decreased kisspeptin, NKB, and

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

35

Fig. 3. Effect of a gonadotropin-releasing hormone (GnRH) agonist on LH release in Young, Young-Middle, Late-Middle, and Old females. Buserelin (GnRH agonist, 1 mg/0.1 mL in 0.05 M PBS) was intravenously injected at 0 minutes immediately after sampling. Young (n ¼ 3), Young-Middle, Late-Middle, and Old (n ¼ 4 each). Values are means  standard error of the mean. In the 120 minutes after buserelin injection, the plasma LH concentration was significantly increased at 18, 18, 24, and 30 minutes in Young, Young-Middle, Late-Middle, and Old animals, respectively (p < 0.05 vs. just before agonist injection [time point 0], 2-way ANOVA followed by Dunnett’s post hoc test). The increase in LH release was significantly higher in Young rats compared with the other aged groups. Details for the statistical differences are described in the results.

dynorphin expressions, accompanied with a reduction of LH secretion during aging. In females, the total LH secretion was significantly decreased in Young-Middle, Late-Middle, and Old rats compared with that in Young rats, and the former groups showed significantly reduced numbers of kisspeptin-, NKB-, and dynorphin A-ir neurons (Figs. 1B and 2C). In males, both total LH secretion and the numbers of kisspeptin, NKB, and dynorphin A-ir neurons were significantly reduced in Late-Middle and Old animals compared with those in Young animals (Figs. 4B and 5C). It has been suggested that kisspeptin is a strong activator of GnRH, and NKB and dynorphin are modulators of kisspeptin release from ARC kisspeptin neurons; therefore, our results support attenuated pulsatile LH release in aging animals and suggest the involvement of neurons expressing kisspeptin, NKB, and dynorphin in age-related reduction of pulsatile LH release. In the present study, females of different ages showed no significant change in the LH pulse frequency (Fig. 1B). This seems to contradict previous reports that demonstrated decreased frequency of the LH pulse with aging in females (Sano and Kimura, 2000; Scarbrough and Wise, 1990; Wise et al., 1988). However, several evidence imply that the reproductive aging in Wistar female rat is slower compared with that in other strains. In our study, Wistar females showed regular 4 or 5-day cycles at the age of 12e13 months, whereas Long-Evans and Sprague-Dawley females started to show irregular cycles already at the age of 6 months (Ishii et al., 2012; Lapolt et al., 1986; Matt et al., 1987). In our present study, 1 out of 4 animals in Old group showed no LH pulse frequency. From these evidence, it can be suggested that a significant reduction of LH pulse frequency in Wistar females may start from around 24 months of age, which seems later compared with the other strains (Sano and Kimura, 2000; Scarbrough and Wise, 1990; Wise et al., 1988). While previous reports indicated decreased LH pulse frequency during aging in male animals (Coquelin and Desjardins, 1982; Karpas et al., 1983; Steiner et al., 1984), in our

Fig. 4. Pulsatile LH secretion in Young, Young-Middle, Late-Middle, and Old males. (A) Plasma LH profiles in representative animals. Arrowheads indicate the peaks of LH pulses indentified with the PULSAR algorithm. (B) AUC of plasma LH concentrations, frequency, and amplitude of LH pulses. Y, Young (n ¼ 9); YM, Young-Middle (n ¼ 5); LM, Late-Middle (n ¼ 4); O, Old (n ¼ 4). Values are means  standard error of the mean. Values with different letters are significantly different (p < 0.05, One-way ANOVA followed by Tukey’s test).

study, pulse frequency did not decrease in aged males (Fig. 4B). As in females, species or strain differences may explain this discrepancy in males; Sprague-Dawley rats or mice were used in previous studies (Coquelin and Desjardins, 1982; Karpas et al., 1983; Steiner et al., 1984) and Wistar rats were used in the present study. Interestingly, males of the Late-Middle group showing increased LH pulse frequency exhibited decreased total LH concentration with no change in amplitude (Fig. 4B). This may be because of the decrease in non-pulsatile LH release, which is not detected as pulses. The difference in response to GnRH in the pituitary may also be associated with the decrease in total LH levels (Fig. 6). In addition, we revealed significantly reduced LH responsiveness to GnRH in Young-Middle, Late-Middle, and Old animals compared with Young animals (Figs. 3 and 6). Our data suggest that this reduction in pituitary responsiveness might be caused by the reduced expression of kisspeptin, NKB, and dynorphin in the ARC. Normally, in young gonadectomized animals, LH gene expression in the pituitary is stimulated by an increased GnRH pulse because the negative feedback effect of gonad-derived estradiol/testosterone is absent (Dalkin et al., 1989). In this state, releasable LH is pooled in

36

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

Fig. 5. Expression of kisspeptin, NKB, and dynorphin neurons in the ARC of Young, Young-Middle, Late-Middle, and Old male animals. (A) In situ hybridization of Kiss1, Tac3, and Pdyn mRNA in representative animals. (B) Numbers of Kiss1-, Tac3-, and Pdyn-expressing cells in the ARC. Young (n ¼ 5), Young-Middle (n ¼ 5), Late-Middle (n ¼ 4), Old (n ¼ 4). (C) Numbers of kisspeptin-, NKB-, and dynorphin A-ir cells in the ARC (n ¼ 4 each). Scale bar ¼ 200 mm. Values are means  standard error of the mean. Asterisks indicates significant difference. (p < 0.05, Two-way ANOVA followed by Bonferroni’s post hoc test).

the pituitary, and GnRH stimulation results in the release of a large amount of LH from the pituitary, as shown in Young animals in our present study (Figs. 3 and 6). However, in aged animals, LH concentration after buserelin injection was significantly lower than that in Young animals. This may be caused by reduced gene and peptide expression in the ARC kisspeptin neurons because decreased kisspeptin secretion leads to less GnRH stimulation, which results in less LH gene expression in the pituitary. Smaller pools of releasable LH in the pituitary might be a reason why the pituitary responsiveness of aged animals was decreased compared with that in Young animals. It must be noted that although the buserelin-induced LH release in Young-Middle, Late-Middle, and Old animals was significantly lower than that in Young animals, it was higher than the normal pulsatile LH concentration. Although a large amount of LH, which is greater than the normal physiological pulsatile level, is pooled in the pituitary, the total LH secretion was significantly lower in aged animals (Figs. 1 and 4), indicating attenuated kisspeptin release as a cause of reduced LH secretion in aged animals. Altered GnRH receptor expression could also be a cause of reduced pituitary responsiveness in aged animals, such as reduced sensitivity of the GnRH receptor in the pituitary or kisspeptin-independent reduction of the GnRH receptor. Further studies are needed to determine the mechanism underlying reduced pituitary responsiveness in aged animals. In the present study, we also demonstrated the effects of age on the expression of Kiss1, Tac3, and Pdyn in the ARC (Figs. 2 and 5).

Although it is unknown whether these 3 genes are regulated in the same way during aging, it is noteworthy that the expression levels of the genes were significantly different from each other during aging in both females and males (Figs. 2B and 5B). Together with the report by Murakawa et al. (2016) demonstrating differential regulation of sorting and packaging of kisspeptin, NKB, and dynorphin within KNDy neurons, our results suggest that the expression of the 3 genes in the ARC is independently regulated during aging. In addition, it is proposed that the reduction in the number of kisspeptin, NKB, and dynorphin neurons with aging is triggered by the inhibition of gene expression, not by cell death. During aging, many changes were observed in Young-Middle animals compared with Young animals. In females, the total LH secretion over 3 hours was significantly decreased in the YoungMiddle group compared with the Young group. In NKB and dynorphin neurons, both mRNA and protein levels were significantly decreased in Young-Middleeaged rats and were maintained in later aged animals (Fig. 2B and C). In kisspeptin neurons, protein levels were decreased in the Young-Middle rats, whereas mRNA levels did not change from the level in the Young group. One possible explanation for this discrepancy between mRNA and protein levels in female Young-Middle rats is repression of Kiss1 mRNA translation. In the present study, we only examined the number of kisspeptin-ir cell bodies and not kisspeptin-ir fibers; thus, it is possible that kisspeptin peptides mostly localize in axons. Regarding males, levels of the 3 mRNAs were significantly

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

Fig. 6. Effect of a GnRH agonist on LH release in Young, Young-Middle, Late-Middle, and Old males. Buserelin (GnRH agonist, 1 mg/0.1 mL in 0.05 M PBS) was intravenously injected at 0 minutes immediately after sampling. Young (n ¼ 3), YoungMiddle, Late-Middle, and Old (n ¼ 4 each). Values are means  standard error of the mean. In the 120 minutes after buserelin injection, the plasma LH concentration was significantly increased at 12, 42, 24, and 24 minutes in Young, Young-Middle, Late-Middle, and Old animals, respectively (p < 0.05 vs. just before agonist injection [time point 0], Two-way ANOVA followed by Dunnett’s post hoc test). The increase in LH release was significantly higher in Young rats compared with the other aged groups. Details for the statistical differences are described in the results.

decreased in Young-Middle rats compared with those in Young rats (Fig. 5B), but protein levels were significantly decreased in LateMiddle and Old animals, but not in Young-Middle animals (Fig. 5C). This discrepancy between mRNA and protein levels in Young-Middle rats might be caused by a change in transportation and/or release of the peptides. A number of studies have reported dynorphin to be an inhibitor of kisspeptin neuronal activity (Goodman et al., 2013; Navarro et al., 2009; Wakabayashi et al., 2010). This evidence indicates that increased pulsatile LH release is to be expected when dynorphin activity is decreased. However, although the number of Pdyn-expressing cells was remarkably reduced in Young-Middle females, increased pulsatile LH release was not observed, and the total LH concentration was decreased (Figs. 1B and 2B). This result suggests the involvement of other regulatory factors that act instead of or together with dynorphin to inhibit kisspeptin release from ARC kisspeptin neurons. It is still unknown whether NKB and dynorphin are the only factors that regulate kisspeptin release from ARC kisspeptin neurons; therefore, age-related changes in other regulatory factors may occur when LH secretion is decreased. In addition to changes in expression of kisspeptin, NKB, and dynorphin and total LH secretion, changes in pituitary responsiveness also occurred between the ages of Young and YoungMiddle in both sexes, and the altered levels remained unchanged after the age of Young-Middle. Most surprisingly, although these changes were observed at the level of the hypothalamus and pituitary, Young-Middle females showed normal 4-day estrous cycles until the day of ovariectomy. These data suggest that 12- to 13-month-old females are able to maintain folliculogenesis and ovulation with an attenuated number of Tac3 and Pdyn neurons and inhibited LH secretion. Our results demonstrate that reproductive alterations in the hypothalamus and pituitary occur between the ages of 2e3 and 12e13 months in Wistar rats. Precise examinations

37

in this period are needed to determine when and how the alteration occurs in the GnRH/LH pulse generator. In the present study, we revealed decreased expression levels of kisspeptin, NKB, and dynorphin, which are the factors affecting LH pulse generation, but a reduction in the frequency and amplitude of pulsatile activity, which is reflected to frequency and amplitude, was not observed. However, interestingly, the total amount of LH release (AUC) was significantly reduced (Figs. 1 and 4) in aged animals expressing reduced levels of NKB and dynorphin (Figs. 2 and 5). Because kisspeptin release is dependent on NKB and dynorphin regulation (Goodman et al., 2013; Navarro et al., 2009; Wakabayashi et al., 2010), the reduction of kisspeptin release may result from decreased NKB and dynorphin expression in aged animals, resulting in a decrease in the total amount of LH release. Further studies are needed to provide evidence for pulsatile activity of kisspeptin neurons in the regulation of LH release in aged animals. In summary, our study revealed that the attenuated LH secretion in aging female and male rats was associated with reduced numbers of kisspeptin, NKB, and dynorphin neurons in the ARC. In addition, although buserelin-injected animals aged 12 months and older were capable of releasing normal physiological levels of LH pulses, the total LH secretion was decreased in these animals. This result suggests that the decreased NKB and dynorphin expression in aged animals causes the reduction of kisspeptin release from ARC kisspeptin neurons. Therefore, reduced expression of kisspeptin, NKB, and dynorphin in the ARC and/or change in pituitary responsiveness may cause attenuated LH levels in aged rats. Our study may contribute to the determination of age-related reproductive dysfunction and unexplained complaints. Disclosure statement The authors have no conflicts of interest to disclose. Acknowledgements This work was supported by grants-in-aid from JSPS (15K18979 to Iwata K., 22590230, 26460323 to Ozawa H.), and the MEXTSupported Program for the Strategic Research Foundation at Private Universities. The authors are grateful to the Takeda Pharmaceutical Company, Ltd for providing a rat Kiss1 cDNA and a monoclonal antibody for kisspeptin, Dr M.K. Park (The University of Tokyo, Japan) for providing a monoclonal anti-GnRH antibody, and Dr S. Yamada (Kyoto Prefectural University of Medicine, Japan) for providing a rat Pdyn cDNA. We are also grateful to Prof H. Tsukamura and Dr Y. Uenoyama (Nagoya University, Japan) for kindly providing 125 I-labeled rat LH for radioimmunoassays. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2016.10.018. References Adachi, S., Yamada, S., Takatsu, Y., Matsui, H., Kinoshita, M., Takase, K., Sugiura, H., Ohtaki, T., Matsumoto, H., Uenoyama, Y., Tsukamura, H., Inoue, K., Maeda, K., 2007. Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. J. Reprod. Dev. 53, 367e378. Batista, M.C., Cartledge, T.P., Zellmer, A.W., Merino, M.J., Axiotis, C., Bremner, W.J., Nieman, L.K., 1995. Effects of aging on menstrual cycle hormones and endometrial maturation. Fertil. Steril. 64, 492e499.

38

Y. Kunimura et al. / Neurobiology of Aging 50 (2017) 30e38

Bishop, M.W., 1970. Ageing and reproduction in the male. J. Reprod. Fertil. Suppl. 12, 65e87. Burke, M.C., Letts, P.A., Krajewski, S.J., Rance, N.E., 2006. Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: morphologic evidence of interrelated function within the arcuate nucleus. J. Comp. Neurol. 498, 712e726. Coquelin, A., Desjardins, C., 1982. Luteinizing hormone and testosterone secretion in young and old male mice. Am. J. Physiol. 243, E257eE263. d’Anglemont de Tassigny, X., Fagg, L.A., Dixon, J.P., Day, K., Leitch, H.G., Hendrick, A.G., Zahn, D., Franceschini, I., Caraty, A., Carlton, M.B., Aparicio, S.A., Colledge, W.H., 2007. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc. Natl. Acad. Sci. U. S. A. 104, 10714e10719. Dalkin, A.C., Haisenleder, D.J., Ortolano, G.A., Ellis, T.R., Marshall, J.C., 1989. The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression. Endocrinology 125, 917e924. de Roux, N., Genin, E., Carel, J.C., Matsuda, F., Chaussain, J.L., Milgrom, E., 2003. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc. Natl. Acad. Sci. U. S. A. 100, 10972e10976. Estes, K.S., Simpkins, J.W., Chen, C.L., 1980. Alteration in pulsatile release of LH in aging female rats. Proc. Soc. Exp. Biol. Med. 163, 384e387. Filicori, M., Cognigni, G.E., Samara, A., Melappioni, S., Perri, T., Cantelli, B., Parmegiani, L., Pelusi, G., DeAloysio, D., 2002. The use of LH activity to drive folliculogenesis: exploring uncharted territories in ovulation induction. Hum. Reprod. Update 8, 543e557. Finch, C.E., 2014. The menopause and aging, a comparative perspective. J. Steroid Biochem. Mol. Biol. 142, 132e141. Gajewska, A., Siawrys, G., Bogacka, I., Przala, J., Lerrant, Y., Counis, R., Kochman, K., 2002. In vivo modulation of follicle-stimulating hormone release and beta subunit gene expression by activin A and the GnRH agonist buserelin in female rats. Brain Res. Bull. 58, 475e480. Goodman, R.L., Hileman, S.M., Nestor, C.C., Porter, K.L., Connors, J.M., Hardy, S.L., Millar, R.P., Cernea, M., Coolen, L.M., Lehman, M.N., 2013. Kisspeptin, neurokinin B, and dynorphin act in the arcuate nucleus to control activity of the GnRH pulse generator in ewes. Endocrinology 154, 4259e4269. Goodman, R.L., Lehman, M.N., 2012. Kisspeptin neurons from mice to men: similarities and differences. Endocrinology 153, 5105e5118. Goodman, R.L., Lehman, M.N., Smith, J.T., Coolen, L.M., de Oliveira, C.V., Jafarzadehshirazi, M.R., Pereira, A., Iqbal, J., Caraty, A., Ciofi, P., Clarke, I.J., 2007. Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B. Endocrinology 148, 5752e5760. Gottsch, M.L., Cunningham, M.J., Smith, J.T., Popa, S.M., Acohido, B.V., Crowley, W.F., Seminara, S., Clifton, D.K., Steiner, R.A., 2004. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145, 4073e4077. Irwig, M.S., Fraley, G.S., Smith, J.T., Acohido, B.V., Popa, S.M., Cunningham, M.J., Gottsch, M.L., Clifton, D.K., Steiner, R.A., 2004. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 80, 264e272. Ishii, M., Yamauchi, T., Matsumoto, K., Watanabe, G., Taya, K., Chatani, F., 2012. Maternal age and reproductive function in female Sprague-Dawley rats. J. Toxicol. Sci. 37, 631e638. Karpas, A.E., Bremner, W.J., Clifton, D.K., Steiner, R.A., Dorsa, D.M., 1983. Diminished luteinizing hormone pulse frequency and amplitude with aging in the male rat. Endocrinology 112, 788e792. Keen, K.L., Wegner, F.H., Bloom, S.R., Ghatei, M.A., Terasawa, E., 2008. An increase in kisspeptin-54 release occurs with the pubertal increase in luteinizing hormonereleasing hormone-1 release in the stalk-median eminence of female rhesus monkeys in vivo. Endocrinology 149, 4151e4157. Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H., Uenoyama, Y., Iwata, K., Yamada, S., Inoue, K., Ohtaki, T., Matsumoto, H., Maeda, K., 2005. Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 146, 4431e4436. Lapatto, R., Pallais, J.C., Zhang, D., Chan, Y.M., Mahan, A., Cerrato, F., Le, W.W., Hoffman, G.E., Seminara, S.B., 2007. Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice. Endocrinology 148, 4927e4936. Lapolt, P.S., Matt, D.W., Judd, H.L., Lu, J.K., 1986. The relation of ovarian steroid levels in young female rats to subsequent estrous cyclicity and reproductive function during aging. Biol. Reprod. 35, 1131e1139. Lehman, M.N., Coolen, L.M., Goodman, R.L., 2010. Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: a central node in the control of gonadotropin-releasing hormone secretion. Endocrinology 151, 3479e3489. Maeda, K., Ohkura, S., Uenoyama, Y., Wakabayashi, Y., Oka, Y., Tsukamura, H., Okamura, H., 2010. Neurobiological mechanisms underlying GnRH pulse generation by the hypothalamus. Brain Res. 1364, 103e115. Matt, D.W., Sarver, P.L., Lu, J.K., 1987. Relation of parity and estrous cyclicity to the biology of pregnancy in aging female rats. Biol. Reprod. 37, 421e430. Merriam, G.R., Wachter, K.W., 1982. Algorithms for the study of episodic hormone secretion. Am. J. Physiol. 243, E310eE318. Messager, S., Chatzidaki, E.E., Ma, D., Hendrick, A.G., Zahn, D., Dixon, J., Thresher, R.R., Malinge, I., Lomet, D., Carlton, M.B., Colledge, W.H., Caraty, A., Aparicio, S.A., 2005. Kisspeptin directly stimulates gonadotropin-releasing

hormone release via G protein-coupled receptor 54. Proc. Natl. Acad. Sci. U. S. A. 102, 1761e1766. Mostari, P., Ieda, N., Deura, C., Minabe, S., Yamada, S., Uenoyama, Y., Maeda, K., Tsukamura, H., 2013. Dynorphin-kappa opioid receptor signaling partly mediates estrogen negative feedback effect on LH pulses in female rats. J. Reprod. Dev. 59, 266e272. Murakawa, H., Iwata, K., Takeshita, T., Ozawa, H., 2016. Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat. Neurosci. Lett. 612, 161e166. Navarro, V.M., Gottsch, M.L., Chavkin, C., Okamura, H., Clifton, D.K., Steiner, R.A., 2009. Regulation of gonadotropin-releasing hormone secretion by kisspeptin/ dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse. J. Neurosci. 29, 11859e11866. Oakley, A.E., Clifton, D.K., Steiner, R.A., 2009. Kisspeptin signaling in the brain. Endocr. Rev. 30, 713e743. Ohkura, S., Takase, K., Matsuyama, S., Mogi, K., Ichimaru, T., Wakabayashi, Y., Uenoyama, Y., Mori, Y., Steiner, R.A., Tsukamura, H., Maeda, K.I., Okamura, H., 2009a. Gonadotrophin-releasing hormone pulse generator activity in the hypothalamus of the goat. J. Neuroendocrinol. 21, 813e821. Ohkura, S., Uenoyama, Y., Yamada, S., Homma, T., Takase, K., Inoue, N., Maeda, K., Tsukamura, H., 2009b. Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats. Peptides 30, 49e56. Palermo, R., 2007. Differential actions of FSH and LH during folliculogenesis. Reprod. Biomed. Online 15, 326e337. Park, M.K., Wakabayashi, K., 1986. Preparation of a monoclonal antibody to common amino acid sequence of LHRH and its application. Endocrinol. Jpn. 33, 257e272. Paxinos, G., Watson, C., 2007. The Rat Brain, sixth ed. Elsevier Academic Press, London. Poteracki, J., Walsh, K.M., 1998. Spontaneous neoplasms in control Wistar rats: a comparison of reviews. Toxicol. Sci. 45, 1e8. Rance, N.E., 2009. Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback. Peptides 30, 111e122. Roseweir, A.K., Kauffman, A.S., Smith, J.T., Guerriero, K.A., Morgan, K., PieleckaFortuna, J., Pineda, R., Gottsch, M.L., Tena-Sempere, M., Moenter, S.M., Terasawa, E., Clarke, I.J., Steiner, R.A., Millar, R.P., 2009. Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation. J. Neurosci. 29, 3920e3929. Ruwanpura, S.M., McLachlan, R.I., Meachem, S.J., 2010. Hormonal regulation of male germ cell development. J. Endocrinol. 205, 117e131. Sano, A., Kimura, F., 2000. Electrical activity of the pulse generator of gonadotropinreleasing hormone in 26-month-old female rats. Neuroendocrinology 72, 199e207. Sawai, N., Iijima, N., Takumi, K., Matsumoto, K., Ozawa, H., 2012. Immunofluorescent histochemical and ultrastructural studies on the innervation of kisspeptin/ neurokinin B neurons to tuberoinfundibular dopaminergic neurons in the arcuate nucleus of rats. Neurosci. Res. 74, 10e16. Scarbrough, K., Wise, P.M., 1990. Age-related changes in pulsatile luteinizing hormone release precede the transition to estrous acyclicity and depend upon estrous cycle history. Endocrinology 126, 884e890. Seminara, S.B., Messager, S., Chatzidaki, E.E., Thresher, R.R., Acierno, J.S., Shagoury, J.K., Bo-Abbas, Y., Kuohung, W., Schwinof, K.M., Hendrick, A.G., Zahn, D., Dixon, J., Kaiser, U.B., Slaugenhaupt, S.A., Gusella, J.F., O’Rahilly, S., Carlton, M.B., Crowley, W.F., Aparicio, S.A., Colledge, W.H., 2003. The GPR54 gene as a regulator of puberty. N. Engl. J. Med. 349, 1614e1627. Smith, J.T., Cunningham, M.J., Rissman, E.F., Clifton, D.K., Steiner, R.A., 2005. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146, 3686e3692. Smith, J.T., Popa, S.M., Clifton, D.K., Hoffman, G.E., Steiner, R.A., 2006. Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J. Neurosci. 26, 6687e6694. Steiner, R.A., Bremner, W.J., Clifton, D.K., Dorsa, D.M., 1984. Reduced pulsatile luteinizing hormone and testosterone secretion with aging in the male rat. Biol. Reprod. 31, 251e258. Terao, Y., Kumano, S., Takatsu, Y., Hattori, M., Nishimura, A., Ohtaki, T., Shintani, Y., 2004. Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. Biochim. Biophys. Acta 1678, 102e110. Wakabayashi, Y., Nakada, T., Murata, K., Ohkura, S., Mogi, K., Navarro, V.M., Clifton, D.K., Mori, Y., Tsukamura, H., Maeda, K., Steiner, R.A., Okamura, H., 2010. Neurokinin B and dynorphin A in kisspeptin neurons of the arcuate nucleus participate in generation of periodic oscillation of neural activity driving pulsatile gonadotropin-releasing hormone secretion in the goat. J. Neurosci. 30, 3124e3132. Wise, P.M., Dueker, E., Wuttke, W., 1988. Age-related alterations in pulsatile luteinizing hormone release: effects of long-term ovariectomy, repeated pregnancies and naloxone. Biol. Reprod. 39, 1060e1066. Yamada, K., Park, H., Sato, S., Onozuka, M., Kubo, K., Yamamoto, T., 2008. DynorphinA immunoreactive terminals on the neuronal somata of rat mesencephalic trigeminal nucleus. Neurosci. Lett. 438, 150e154.