Postnatal Development of Kisspeptin Neurons in Mouse Hypothalamus; Sexual Dimorphism and Projections to Gonadotropin-Releasing Hormone Neurons

Animals Male and female homozygous C57BL/6J GnRH-GFP mice ( 27 ) between postnatal day (P) 10 and P61 were used (n=4-8 for each age group and sex). All mice were housed either with their dam (for mice <P21) or in cages of 3-4 animals under conditions of 12 hour light/dark cycles (lights on 07:00h) with food and water freely available. The stage of estrous cycle in adult female mice was determined by vaginal cytology. The sex of P10 mice was confirmed with sry PCR as detailed previously ( 28 ). All procedures were approved by the University of Otago Animal Ethics Committee and carried out under project 82/05.

Two sets of sections were treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity, and then washed 3 times in TBS (10 min/wash). Sections were then incubated for 48 h at 4°C in a polyclonal rabbit anti-kisspeptin-10 antiserum (1:5,000; #566 gift from A. Caraty, Tours) in TBS containing 0.3% Triton X-100 and 0.25% BSA and 2% normal goat serum. Sections were then washed 3 times in TBS (10 min/wash) before being incubated in a biotinylated goat anti-rabbit secondary antibody (Vector Labs) at 1:400 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. After subsequent washing in TBS, the sections were incubated in Vector Elite avidin-peroxidase (Vector) at 1:100 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. The sections were again washed and immunoreactivity revealed using glucose-oxidase, nickel-enhanced diaminobenzidine hydrochloride. The sections were washed thoroughly in TBS, mounted onto gelatin-coated glass slides, air dried, dehydrated in ethanol followed by xylene and then coverslipped with DPX.

The production and characterization of the kisspeptin-10 antibody has been published ( 19 ). In brief, mouse kisspeptin-10 (YNWNSFGLRY-NH2) was coupled to BSA using glutaraldehyde and used as an immunogen in rabbits. The antiserum is highly specific to mouse kisspeptin-10 with radioimmunoassay binding not inhibited by any one of 8 different hypothalamic peptides including other RFamides such as prolactin-releasing peptide. Similarly, immunoreactivity is abolished by pre-adsorption of the antiserum with 1μM kisspeptin-10 but not 1-10μM prolactin-releasing peptide. Controls for this series of experiments included omission of the primary antibody in single and dual-label experiments and use of the kisspeptin-10 antibody preadsorbed overnight with 1μM murine kisspeptin-10 peptide (gift from A. Caraty, Tours).

Distribution of kisspeptin-10 immunoreactivity in adult female mouse hypothalamus Kisspeptin-10 immunoreactivity was examined in the hypothalamus of 4 female GnRH-GFP mice killed on the day of estrous. The Spergel GnRH-GFP mice were used as, in relation to the kisspeptin-GnRH analysis, GFP-immunostaining in this mouse line enables slightly more of the GnRH dendritic tree to be examined compared with GnRH immunocytochemistry ( 30 ). Within the medial septum and rostral preoptic area, 100% of GFP-expressing cells express GnRH ( 27 )(Herbison, unpublished observations). Three populations of kisspeptin-10-immunoreactive cell bodies were identified in the coronal sections. The first and largest population comprised a continuum of kisspeptin cell bodies lying close to the third ventricle extending from the AVPV through into the preoptic PeN ( Fig.1A-C ). A second, smaller population was observed scattered within the dorsomedial nucleus and anterior hypothalamus referred to here as the DMN group ( Fig.1E ). The third small population of cell bodies was located in the ventrolateral ARN ( Fig.2C ). In terms of kisspeptin fiber distribution, the most dramatic staining was in the ARN where a dense plexus of kisspeptin-immunoreactive fibers effectively outlined the nucleus at all levels ( Figs.1E,F & 2C ). Whereas modest staining was detected within the internal zone of the median eminence, it is noteworthy that none was found in the external zone of the median eminence ( Fig.2C ). Elsewhere in the hypothalamus, kisspeptin fiber staining was notable within the AVPV and PeN ( Fig.2G ) and extending dorsally and laterally from these nuclei into adjacent brain regions ( Figs.1A-C , 2A ), as well as within the DMN. There was a conspicuous absence of fiber staining in the VMN ( Fig.1F ). Within the preoptic area fibers were found in the median preoptic nucleus ( Fig.2A ), the medial preoptic area, and coursed through the diagonal band of Broca (DBB) to the ventral-lateral septum and the anterior portion of the bed nucleus of the stria terminalis (BNST). No fibers were detected in the MS. Scattered fibers were also found in the subfornical organ and paraventricular thalamic nucleus, as well as within the supraoptic ( Fig.2E ) and paraventricular ( Fig.2F ) nuclei. Tissue that underwent immunocytochemistry with either the omission of the primary antibody or incubation with primary antibody that had been pre-adsorbed with the kisspeptin peptide resulted in complete absence of labeling ( Figs.1D & 2B,D ).

Female C57BL/6J mice in our colony exhibit vaginal opening at P27±1 day (SEM) and their first estrus 2 days later at P29±1 day (n=12). Male mice achieve reproductive competency (as assessed by placing juvenile males with experienced females and back-dating from litter delivery to determine the day of conception) at P45±3 (n=5). To evaluate development in relation to puberty we therefore examined 4-8 female mice at P10 (juvenile), P25 (pre-pubertal), P31 (peripubertal) and at P61 (adult). Male mice (n=5-8) were examined at P10, P25, P31, P45 (peripubertal) and adult (P61). Male and female mice showed a similar pattern of postnatal development of kisspeptin-immunoreactive cell numbers in the AVPV, rPeN and cPeN ( Figs.4 & 5 ). In male mice, no kisspeptin-immunoreactive cells were detected in the AVPV/PeN at P10 with only small numbers detected at P25. However, between P25 and P31, there was an approximately 500% increase in kisspeptin cell numbers throughout the AVPV/PeN (p<0.01; Fig.5A ). This trend continued as a smaller 30-50% increase between P31 and P45 (p<0.01) with peripubertal P45 mice not being different to adults ( Fig.5A ). In females, essentially no kisspeptin neurons were found in the AVPV/PeN at P10 but cells were clearly evident at P25 (p<0.01; Fig.4A ). There was then a doubling of kisspeptin cell numbers between P25 and P31 (p<0.01) to adult levels in the PeN ( Figs.4B,C & 5B ). In the AVPV, however, kisspeptin cell numbers did not achieve adult-like levels until after the onset of puberty with P61 numbers being approximately double that of P31 female mice (p<0.001: Fig.5B ). Although not quantified, an increase in the density of kisspeptin fibers accompanying the kisspeptin cell bodies in periventricular regions was evident ( Fig.2G,H ). The kisspeptin fiber distribution in the ARN was observed at all developmental ages in both sexes but, as kisspeptin-immunoreactive cell bodies were only occasionally visible in the ARN, no quantitative analysis was performed. The numbers of kisspeptin-immunoreactive cells in the DMN of males and females exhibited decreasing numbers of cells with postnatal development (P10 = 23.6±2.9; P25 = 12.4±1.2; P61 = 14.9±2.6; P10 vs P25 and P61, p<0.05).

We report here the distribution and postnatal development of kisspeptin-10-immunoreactive neurons within the hypothalamus of the male and female mouse. We have observed that the cell bodies of kisspeptin neurons in the rostral hypothalamus exist as a continuum of cells located throughout the preoptic periventricular nuclei including the AVPV. A striking sexual dimorphism exists within this population with the numbers of kisspeptin neurons being at least 10-fold greater in adult females compared with males. This sex difference is apparent from early in postnatal development with the numbers of periventricular neurons expressing kisspeptin increasing steadily from P25 through to adulthood in females and males. In terms of kisspeptin inputs to GnRH neurons, we show that kisspeptin fibers become apparent adjacent to GnRH neurons in a topographically distinct and sexually dimorphic manner around the time of puberty in both sexes. Together, these findings support the hypothesis that periventricular kisspeptin neurons innervate GnRH neurons to help initiate their activation at puberty and, furthermore, suggest the involvement of kisspeptin in the sexually differentiated functioning of the GnRH neuronal population. The distribution of kisspeptin-10-immunoreactive cells in the rostral hypothalamus reported here with this new antibody is in excellent agreement with Kiss1 mRNA in situ hybridization studies in the mouse. In those studies, a large population of Kiss1 mRNA-expressing cells was detected within the AVPV and PeN of adult male and female mice ( 21 , 22 , 26 ). It remains unclear why other kisspeptin antibodies have failed to detect this substantial population of kisspeptin neurons ( 17 ) but the use here of an antibody generated against mouse kisspeptin-10 in mouse tissue may be significant. In situ hybridization analyses also detected a population of Kiss1 mRNA-expressing cells in the ARN ( 22 , 26 ). Abundant kisspeptin immunoreactivity was also found in the ARN, although relatively few clearly-labelled cell bodies were observed. This likely to be due to the very high density of kisspeptin fibers in the ARN that made it difficult to discern individual cell bodies, and the observation that kisspeptin biosynthesis is robustly suppressed by gonadal steroids in the ARN of intact male and female mice ( 22 , 26 ). In preliminary experiments (Clarkson & Herbison), we have examined kisspeptin immunoreactivity in ovariectomized mice and observed a large population of kisspeptin-immunoreactive cell bodies in the ARN. We also noted a third population of kisspeptin-immunoreactive cells scattered within the dorsomedial hypothalamus, as has been seen in the sheep ( 19 , 20 ) and rat ( 17 ). Interestingly, these cells have not been reported on in Kiss1 mRNA in situ hybridization experiments as yet ( 21 , 22 , 26 ). Previous studies have shown that Kiss1 mRNA levels within the whole hypothalamus of the rat and primate fluctuate over the course of postnatal development ( 9 , 23 ). We show here a clear developmental increase in kisspeptin expression within the AVPV/PeN continuum. Our earlier investigation found that the numbers of Kiss1 mRNA-expressing cells located in the AVPV increased 7-fold between P18 and adulthood in male mice ( 16 ). We now extend this result to show that (i) the same pattern of development (5-fold increase from P25 to adulthood in males) occurs for kisspeptin peptide-containing cells in the AVPV, (ii) this also occurs in the AVPV of the female mouse, and (iii) this developmental profile is also exhibited by the much larger population of kisspeptin neurons found within the PeN. Kisspeptin-immunoreactive cell numbers in the DMN exhibited a completely different developmental pattern, and fiber staining was evident in the ARN at all postnatal ages. Prior data indicate that the numbers of Kiss1 mRNA-expressing cells in this region did not change between P18 and adulthood in male mice ( 16 ). These observations demonstrate that it is only the kisspeptin neurons of the AVPV/PeN that exhibit a postnatal developmental increase in kisspeptin synthesis. Details of the ontogeny of kisspeptin signaling at a cellular level are only just emerging. Our earlier study indicated that >90% of prepubertal and adult male GnRH neurons expressed GPR54 mRNA, but that the percentage of GnRH neurons responding electrophysiologically to kisspeptin increased from 27% in prepubertal mice to nearly 100% in adult males ( 16 ). Alongside evidence for a substantial increase in the numbers of Kiss1 mRNA expressing cells in the AVPV ( 16 ), we suggested that a two-step mechanism for kisspeptin activation of GnRH neurons may exist, involving ( 1 ) a developmental change in the coupling of GPR54 to its effector pathways within GnRH neurons and ( 2 ) the development of kisspeptin inputs to GnRH neurons. Our present results provide the first direct evidence in support of the latter part of the mechanism. Appositions between kisspep