Shabby Chic romance is brought to life in this collection, fully embracing femininity, softness, kissing, and self-love. Layers and layers of pretty pink prints to decorate yourself, immerse yourself! Climb into silk rose gardens, lacy frills, and powder pink hues. It's love! Love is an almost indescribable feeling, but perhaps we'd compare it to climbing into a hot bath, a giant claw-foot tub, candles burning, and petals floating on the baby blue water. It's in the fit of a perfect little dress, or running across cobblestone streets in the rain, high heels clicking, and bare legs shivering as you throw on your coat. Love is getting a kiss on the cheek when you least expect it, or writing letters and bravely dropping them in someone's mailbox. It's curling up with your cat and a really good book, pulling on thick socks, and soft pyjamas. It's fresh sheets that smell like lavender soap, crisp, and neatly tucked. It's organized bedside tables decorated with small vases of roses, breakfast in bed, hot coffee with a slice of cake, love is the sound of family on the other end of the phone, or watching the stars and pointing out constellations. It's electric chills when that special someone accidentally brushes your leg with their hand. Love is pink boxes filled with chocolates and tied up with satin ribbons on a cloudy day. It's sitting snug with someone you love in a small booth at a crowded restaurant, sipping oysters and pouring way too much red wine, speaking loudly in each other's ears. It's all this and so much more... it's new babies, new romance, it's recognizing the things you love in yourself... it's the reason to live!
Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.
Kiss1 neurons produce kisspeptins, a family of neuropeptides encoded by Kiss1, that stimulate GnRH secretion acting via the G-protein coupled receptor, Gpr541. A wealth of data have conclusively documented that Kiss1 and Grp54 are essential for the acquisition and maintenance of reproductive capacity in mammals, including humans1,5; mice and humans harboring inactivating mutations of these genes suffer impuberism and hypogonadotropic hypogonadism. This essential role in the control of reproductive axis is conducted by discrete populations of Kiss1 neurons, which in rodents are mainly located in two different hypothalamic areas, namely the arcuate-nucleus (ARC) and the anteroventral periventricular nucleus (AVPV)6. While both populations produce kisspeptins, they display interesting neuro-anatomical and functional divergences. Thus, the AVPV Kiss1 neuronal population is far more prominent in females and is activated by estrogens to mediate their positive feedback for the induction of the preovulatory LH surge. In contrast, the ARC Kiss1 neuronal population is present in both sexes and is repressed by gonadal steroids to convey their negative feedback actions essential for the tonic release of GnRH7. The molecular underpinnings for these differential features remain largely unknown.
In terms of neuroendocrine responses, KiDKO and GoDKO mice presented preserved responses to effective doses of GnRH, whose primary target is the pituitary, with significant increases in serum LH levels. Admittedly, the magnitude of these responses was smaller than in controls, but this is probably due to the diminished priming activity of endogenous GnRH at the pituitary of these animals rather than a primary major pituitary defect. In contrast, the expected rise of circulating LH following removal of negative feedback of gonadal secretions by gonadectomy did not occur in any of the two genotypes (Fig. S4a, b); similarly, no post-castration rise of FSH was detected in either KiDKO or GoDKO mice. However, disparate patterns of LH responses to kisspeptin-10 (Kp-10) and the glutamate receptor agonist, NMDA, were found between KiDKO and GoDKO mice. Thus, while KiDKO mice retained their capacity to respond to Kp-10 and NMDA (Fig. S4a), albeit with modest increases in LH secretion after NMDA, GoDKO mice displayed a complete ablation of LH responses to Kp-10 and NMDA (Fig. S4b).
The neuronal basis for the reproductive phenotype of KiDKO mice during postnatal maturation was explored by expression analyses conducted in control and conditional null animals of both sexes, at 2 weeks and 4 weeks of age, and in adulthood. In situ hybridization allowed detection of numbers of Kiss1-expressing neurons in the ARC (in both males and females) and AVPV (only in females), together with relative Kiss1 expression per neuron (Fig. S6). In parallel, we took advantage of the fact that our Kiss1-Cre line express also GFP (tagged to Cre protein) under the endogenous Kiss1 promoter to detect the presence of GFP-positive neurons, as marker of viable Kiss1 neurons, in KiDKO and control mice (Fig. 3); since CreGFP is a non-secreted protein, GFP labeling is expected to be more robust than Kiss1/kisspeptin, therefore increasing the window of detection of Kiss1-expressing neurons.
Given the proven role of miRNAs in the post-transcriptional control of gene expression, kisspeptin protein levels were assessed in KiDKO mice at similar ages and at hypothalamic sites. Kiss1-specific Dicer ablation resulted in a massive drop in kisspeptin-immunoreactivity in the ARC of both males and females, which was observed already at 2 weeks of age, when protein levels were already nearly undetectable (Fig. 4a, b, g, h). In contrast, no significant differences in kisspeptin content/kisspeptin-positive cells were detected in the AVPV of KiDKO and control mice at 2- and 4-weeks of age (Fig. 4d, e). Of note, despite the massive suppression of kisspeptin content in the ARC, the infantile-pubertal transition was associated with an increase in kisspeptin-immunoreactivity at this nucleus in KiDKO mice of both sexes, which was accompanied by a significant rise in the number of kisspeptin-positive cells also in the AVPV (Fig. S7). Finally, kisspeptin-immunoreactivity was massively suppressed in adult KiDKO mice of both sexes, not only at the ARC (Fig. 4c, i) but also at the AVPV (Fig. 4f).
NKB immunoreactivity in the ARC was also affected by Dicer ablation in Kiss1-expressing cells. Yet, the profiles of protein content clearly differed from those of kisspeptin. Thus, while kisspeptin-immunoreactivity was markedly suppressed in the ARC of KiDKO mice of both sexes at the infantile period, no statistical differences in the density of NKB fibers were detected in the ARC between KiDKO and control male and female mice, at this age (2 weeks; Fig. 5a, d). In contrast, NKB protein levels were significantly diminished in the ARC of KiDKO males and females at the peripubertal period (4 weeks; Fig. 5b, e). However, the relative magnitude of such suppression was substantially milder than that of kisspeptin. Thus, while ARC kisspeptin-immunoreactivity was reduced by >160- and 10-fold in pubertal male and female KiDKO mice, respectively, the decrease in NKB protein levels was only five- and two-fold in KiDKO males and females, respectively (Fig. 5b, e). In addition, while NKB-immunoreactivity augmented during the infantile-pubertal transition in KiDKO males, this effect was not detected in conditional null females (Fig. S7). Finally, the drop in NKB immunoreactivity was maximal in adult KiDKO mice of both sexes, in which only a few NKB fibers were detected (Fig. 5c, f). This decrease in the level of NKB protein content appears to be specific to KNDy neurons, since NKB immunoreactivity was preserved in other brain regions (Fig. S8), known to harbor NKB neurons28.
To track Kiss1 neuronal survival following congenital Dicer ablation, a triple transgenic mouse line, expressing the Cre-dependent reporter (ROSA26)-YFP in Kiss1 neurons was generated on the KiDKO background. This approach permits persistent labeling of any cell ever expressing Kiss1, even if Kiss1 expression is no longer active. Analyses in this reporter mouse line were conducted at 4 weeks, 2 months, and 4 months of age (Fig. 6); namely, the developmental window in which kisspeptin (and NKB) content substantially decrease. Kiss1-YFP neurons in the ARC of KiDKO male mice were diminished already at the peripubertal period (4 weeks; Fig. 6a); yet, such a decline was substantially milder than that of kisspeptin-immunoreactivity and Kiss1 mRNA expression, which dropped to nearly negligible levels at this age (see Fig. 4b), therefore suggesting that such a decline in neuronal survival is not driven by the loss of kisspeptin per se. The decrease in the number of Kiss1-YFP cells progressed during the adult stage, with a substantial reduction in 4-month-old KiDKO males (Fig. 6b, c), which nonetheless was less pronounced than the suppression of kisspeptin content and Kiss1 expression. 2b1af7f3a8