3-Female_reproductive_maturation_in_the_absence[1]

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704	 VOLUME 14 | NUMBER 6 | JUNE 2011 nature neurOSCIenCe
a r t I C l e S
Puberty, in humans and mice, is a transition period of gonadal and 
behavioral maturation that occurs between juvenile stages and adult-­
hood1. Puberty onset is initiated in the CNS by an activation of GnRH 
neurons. When activated, GnRH neurons release high-­frequency 
pulses of GnRH neuropeptide into the hypophysial portal vessels, 
which leads to the stimulation of gonadotropin secretion from 
the anterior pituitary and thus activation of gonadal function1–4. 
How is the GnRH pulse generator turned on? Although it has long 
been known that different permissive signals, such as metabolic, 
environmental and even social cues, are all critical determinants 
for the initiation of reproductive maturation, little is known about 
how these inputs are integrated in the brain. Specifically, the neural 
circuits mediating coordinated control of puberty onset and sexual 
development have not been identified.
The identification of kisspeptins, neuropeptides expressed from the 
Kiss1 gene, and their receptor, GPR54, has provided an entry toward 
understanding the physiological mechanisms mediating the timing 
of puberty5–8. Inactivating mutations of the human GPR54 gene 
are linked to hypogonadotropic hypogonadism and an absence of 
puberty9,10. Knockout of the Kiss1 (refs. 11,12) or Gpr54 (refs. 12,13) 
genes in mice leads to hypogonadism and infertility. Kisspeptin 
administration triggers increased GnRH secretion and advances 
puberty onset14–17. In contrast, administration of GPR54 antago-­
nists delays puberty onset18. These data raise the possibility that 
kisspeptin neurons are needed to turn on the GnRH pulse gen-­
erator, which culminates in the awakening of the hypothalamus-­
pituitary-­gonadal (hpg) axis, marking the onset of puberty. To test 
this hypothesis, we generated mice specifically lacking kisspeptin-­
expressing and, in a complementary genetic approach, GPR54-­
expressing cells and analyzed the tempo and completeness of their 
reproductive maturation.
RESULTS
Genetic ablation of kisspeptin neurons
To ablate kisspeptin neurons in mice, we expressed the diphtheria 
toxin A (DTA) chain specifically in these cells. To do this, we bred 
kisspeptin-­IRES-­Cre (KissIC) mice to ROSA26-­DTA (R26-­DTA) 
mice. KissIC mice carry a targeted insertion of an internal ribosome 
entry site (IRES) followed by a Cre recombinase cDNA downstream 
of the kisspeptin coding sequence19. Transcription of the recombinant 
Kiss1 allele yields a bicistronic mRNA from which kisspeptin and 
Cre recombinase are independently translated. Previous experiments 
had shown that >97% of kisspeptin neurons display Cre-­mediated 
recombination in these animals19. The R26-­DTA strain carries a tran-­
scriptionally silenced DTA gene in the ROSA26 locus20. Transcription 
of the R26-DTA allele is activated by Cre-­mediated recombination 
and results in the death of the Cre-­expressing cells21 (Fig. 1a). Thus, 
Cre expression in KissIC/R26-­DTA mice should lead to the genetic 
ablation of neurons expressing kisspeptin.
KissIC/R26-­DTA mice were born with Mendelian frequencies and 
were viable. The body weight of KissIC/R26-­DTA females was slightly, 
but significantly, increased between postnatal day 31 (P31) and P46 
compared with R26-­DTA (control) littermates (KissIC/R26-­DTA 
females, 19.6 ± 0.2 g, n = 7; P40 controls, 18.3 ± 0.4 g, n = 15; P < 0.05; 
Supplementary Fig. 1a). Immunohistochemical analysis of coronal 
brain sections from control mice using antibodies to kisspeptin22 
revealed kisspeptin-­immunoreactive perikarya and fibers in the 
anteroventral periventricular nucleus (AVPV) and the arcuate nucleus 
(ARC) of the hypothalamus (Fig. 1b and Supplementary Fig. 1b), 
consistent with the described distribution of kisspeptin neurons in 
the female mouse brain23,24. In contrast, kisspeptin immunoreactivity 
was virtually absent in the ARC of P18 and adult KissIC/R26-­DTA 
mice (Fig. 1b and Supplementary Fig. 1b). No kisspeptin-­positive 
Institute for Neural Signal Transduction, Center for Molecular Neurobiology, Hamburg, Germany. Correspondence should be addressed to U.B. 
(ulrich.boehm@zmnh.uni-hamburg.de).
Received 24 January; accepted 14 March; published online 24 April 2011; doi:10.1038/nn.2818
Female reproductive maturation in the absence 
of kisspeptin/GPR54 signaling
Christian Mayer & Ulrich Boehm
Puberty onset is initiated in the brain by activation of gonadotropin-releasing hormone (GnRH) neurosecretion. Different 
permissive signals must be integrated for the initiation of reproductive maturation; however, the neural circuits controlling timely 
awakening of the reproductive axis are not understood. The identification of the neuropeptide kisspeptin as a potent activator of 
GnRH neuronal activity suggests that kisspeptin-releasing neurons might coordinate puberty onset. To test this hypothesis, we 
generated mice that specifically lack kisspeptin cells. Puberty onset in females was unaffected by kisspeptin neuron ablation. 
Furthermore, the animals were fertile, albeit with smaller ovaries. Consistent with this, female mice lacking neurons that express 
the kisspeptin receptor GPR54 were also fertile. Acute ablation of kisspeptin neurons in adult mice inhibited fertility, suggesting 
that there is compensation for the loss of kisspeptin neurons early in development. Our data indicate that the initiation and 
completion of reproductive maturation can occur in the absence of kisspeptin/GPR54 signaling.
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Maturação de reproductive feminina na ausência de kisspeptin/GPR54 sinalizar
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Onset de puberdade é iniciado no cérebro por ativação de gonadotropin-libertar hormônio (GnRH) neurosecretion. Diferente nullnulldevem ser integrados sinais permissivos para a iniciação de maturação de reproductive; porém, os circuitos de neural que pontual controlam despertando do eixo de reproductive não são ompreendidos. A identificação do kisspeptin de neuropeptide como um ativador potente de GnRH neuronal atividade sugere que kisspeptin-libertando neurônios pudessem coordenar onset de puberdade. Testar esta hipótese, nós, ratos gerados que especificamente faltam celas de kisspeptin. Onset de puberdade em fêmeas era não afetado por separação de neurônio de kisspeptin. nullnullAlém disso, os animais eram férteis, embora com ovários menores. Consistente com isto, ratos femininos neurônios necessitados que expressam o receptor de kisspeptin GPR54 também seja fértil. Separação aguda de neurônios de kisspeptin em ratos de adulto inibiu fertilidade, enquanto sugerindo que há compensação cedo para a perda de neurônios de kisspeptin em desenvolvimento. Nossos dados indicam que a iniciação e conclusão de maturação de reproductive pode acontecer na ausência de kisspeptin/GPR54 sinalizar.
Administrador
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Puberdade, nos humanos e ratos, é um período de transição de gonadal e maturação de comportamento que acontece entre fases juvenis e adulto-­ hood1. Onset de puberdade é iniciado no CNS por uma ativação de GnRH neurônios. Quando ativou, neurônios de GnRH libertam alto-­frequency pulsos de neuropeptide de GnRH nos recipientes de portal de hypophysial, que conduz à excitação de secreção de gonadotropin de o anterior pituitário e assim ativação de function1–4 de gonadal. 
Como é o GnRH pulsam gerador virado em? Embora tem muito tempo sido conhecido que sinais permissivos diferentes, como metabólico, ambiental e até mesmo sugestões sociais, é todos o determinants crítico pela iniciação de maturação de reproductive, pouco é conhecido roximadamente como estas contribuições são integradas no cérebro. Especificamente, o neural 
circuitos que medeiam controle coordenadode onset de puberdade e sexual desenvolvimento não foi identificado.
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A identificação de kisspeptins, neuropeptides expressaram do gene de Kiss1, e o receptor deles/delas, GPR54, proveu uma entrada para entender os mecanismos fisiológicos que medeiam a cronometragem nullnullde puberty5–8. são unidas mutações de Inactivating do gene de GPR54 humano a hypogonadism de hypogonadotropic e uma ausência de puberty9,10. Knockout do Kiss1 (refs. 11,12) ou Gpr54 (refs. 12,13) genes em dianteiras de ratos para hypogonadism e infertilidade. Kisspeptin administração gatilhos aumentaram secreção de GnRH e onset14–17 de puberdade de avanços. em contraste, administração de GPR54 antagonistas demoras puberdade onset18. Estes dados elevam a possibilidade que são precisados neurônios de kisspeptin virar no GnRH pulso gerador do qual culmina no despertar o hypothalamus-pituitário-gonadal (hpg) eixo, marcando o onset de puberdade. Para testar esta hipótese, nós geramos ratos que especificamente faltam kisspeptin-expressando e, em uma aproximação genética complementar, GPR54-expressando celas e analisou o tempo e perfeição do deles/delas nullnullmaturação de reproductive.
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Separação genética de neurônios de kisspeptin 
A neurônios de kisspeptin de ablate em ratos, nós expressamos a difteria 
toxina UM (DTA) especificamente encadeie nestes celas. Para fazer isto, criamos nós 
kisspeptin-­IRES-­Cre (KissIC) ratos para ROSA26-­DTA (R26-­DTA) 
ratos. Ratos de KissIC levam uma inserção mirada de um ribosome interno 
local de entrada (RAIVAS) seguiu a jusante por um Cre recombinase cDNA 
do kisspeptin que codifica sequence19. Transcrição do recombinant 
Allele de Kiss1 rende um mRNA de bicistronic de qual kisspeptin e 
São traduzidos recombinase de Cre independentemente. Experiências prévias 
tinha mostrado que >97% de neurônios de kisspeptin exibem Cre-­mediated 
recombinação neste animals19. A tensão de R26-­DTA leva um tran-­ 
scriptionally silenciaram gene de DTA no locus20 de ROSA26. Transcrição 
do allele de R26-DTA é ativado através de recombinação de Cre-­mediated 
e resulta na morte do cells21 de Cre-­expressing (Figo. 1a). Assim, 
Expressão de Cre em ratos de KissIC/R26-­DTA deveria conduzir o genético separação de neurônios que expressam kisspeptin.
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Ratos de KissIC/R26-­DTA nasceram com freqüências de Mendelian e 
era viável. O peso de corpo de fêmeas de KissIC/R26-­DTA era ligeiramente, 
ut significativamente, aumentou entre dia 31 pós-natal (P31) e P46 
ompared com R26-­DTA (controle) littermates (KissIC/R26-­DTA 
EMALES, 19.6 ± 0.2 G, N = 7; P40 controla, 18.3 ± 0.4 g, n = 15; P <0.05; 
Figo de upplementary. 1a). análise de Immunohistochemical de coronal 
seções de chuva de ratos de controle que usam anticorpos a kisspeptin22 
perikarya de kisspeptin-­immunoreactive de evealed e fibras no 
núcleo de periventricular de nteroventral (AVPV) e o núcleo de arcuate 
ARCO) do hypothalamus (Figo. 1b e Figo Adicional. 1b), 
onsistent com a distribuição descrita de neurônios de kisspeptin em 
ele brain23,24 de rato feminino. em contraste, immunoreactivity de kisspeptin 
estava virtualmente ausente no ARCO de P18 e adulto KissIC/R26-­DTA 
ratos (Figo. 1b e Figo Adicional. 1b). Nenhum kisspeptin-­positive 
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neurons were detected in the AVPV of P18 KissIC/R26-­DTA (n = 4) 
females compared with 10.5 ± 4.5 kisspeptin-­positive neurons in 
 controls (n = 2). We detected 6.5 ± 3.1 kisspeptin-­positive neurons 
in the AVPV of adult KissIC/R26-­DTA (n = 3) females compared 
with 239.3 ± 10.99 in controls (n = 4) (Fig. 1c), suggesting that effi-­
cient ablation (>97%) of kisspeptin neurons occurs in these animals. 
Consistent with this, Kiss1 mRNA levels were reduced by ~95% in 
P20 and by ~98% in adult KissIC/R26-­DTA females (Fig. 1d).
Sexual maturation in the absence of kisspeptin neurons
Ovarian mass was strongly reduced in adult KissIC/R26-­DTA females 
(2.0 ± 0.1 mg, n = 12) compared with control (5.2 ± 0.4 mg, n = 14) 
animals (P < 0.001; Fig. 2a). This phenotype was already manifest at 
P20 (KissIC/R26-­DTA females, 1.0 ± 0.1 mg, n = 3; controls, 2.1 ± 
0.2 mg, n = 3; P < 0.05; Fig. 2a). Despite reduced ovarian mass, 
however, ovarian histology showed follicles at all stages of develop-­
ment and the presence of corpora lutea in KissIC/R26-­DTA females 
(Fig. 2b), raising the possibility that these animals might be fer-­
tile. Notably, all tested female KissIC/R26-­DTA mice (five of five) 
gave birth to normal-­sized litters when mated to wild-­type males. 
These data suggest that female mice can become fertile in the 
absence of kisspeptin neurons. Taken together, these data indicate 
that ablation of kisspeptin-­expressing cells leads to smaller-­sized 
ovaries that otherwise appear to be functionally normal.
Kisspeptin secretion is thought to be necessary for timely onset of 
puberty5,6,8,25. Consistent with this, kisspeptin knockout mice fail to 
undergo sexual maturation and show delayed onset of puberty11,12, 
whereas kisspeptin administration in juvenile animals can advance 
puberty onset16,17. We next asked whether ablation of kisspeptin 
neurons affects the timing of puberty onset and compared markers 
of pubertal maturation and serum hormone levels in KissIC/R26-­
DTA females with those in control mice. Unexpectedly, the day of 
vaginal opening, an external marker of puberty onset, was similar 
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Figure 1 Targeted ablation of kisspeptin neurons. (a) Genetic strategy to 
activate DTA expression specifically in kisspeptin-expressing cells. KissIC 
mice carry a recombinant Kiss1 allele from which a bicistronic mRNA is 
transcribed. R26-DTA mice carry a transcriptionally silenced DTA gene, 
which is activated on Cre-mediated recombination in KissIC/R26-DTA 
mice. (b) Immunohistochemical analysis of coronal brain sections from 
P18 (left) and adult (right) KissIC/R26-DTA (bottom) and KissIC control 
mice (top) using antibodies to kisspeptin. Kisspeptin immunoreactivity 
was found in the AVPV (top) and ARC (bottom) of the hypothalamus in 
control mice. Note the markedly reduced kisspeptin immunoreactivity in 
KissIC/R26-DTA mice, suggesting efficient ablation of kisspeptin neurons 
in both AVPV and ARC. Scale bar represents 200 µm. (c) No kisspeptin-
positive neurons were detected in the AVPV of P18 KissIC/R26-DTA 
(n = 4) females compared with controls (10.5 ± 4.5 neurons, n = 2). 
We detected 6.5 ± 3.1 kisspeptin-positive neurons in the AVPV of 
adult KissIC/R26-DTA (n = 3) females compared with 239.3 ± 10.99 
kisspeptin-positive neurons in controls (n = 4). (d) Kiss1 mRNA levels 
were reduced by ~95% in P20 KissIC/R26-DTA (n = 2) females compared 
with control (n = 8) animals and by ~98% in adult KissIC/R26-DTA (n = 8) 
females compared with controls (n = 4). Error bars represent s.e.m.
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Figure 2 Sexual maturation in female mice lacking kisspeptin neurons. 
(a) Ovarian mass of female KissIC/R26-DTA mice was strongly reduced 
at P20 and in adults. *P < 0.05, **P < 0.001. (b) Representative ovary 
section from a KissIC/R26-DTA mouse showed follicles at all stages of 
development. Scale bars represent 500 µm. (c) The day of vaginal opening 
(VO) was not significantly different in female KissIC/R26-DTA mice than in 
control animals, suggesting normal timing of puberty onset in the absence 
of kisspeptin neurons. (d) Mean serum luteinizing hormone levels were 
not substantially different in P20 and adult KissIC/R26-DTA females 
compared to controls. ns, not significant (P > 0.5). (e) Vaginal cytology of 
KissIC/R26-DTA mice (right) showed all stages of estrous cyclicity despite 
somewhat prolonged periods of vaginal cornification compared to R26-DTA 
control animals (left). C, cornified (estrous); CL, corpora lutea; L, leukocytic 
(metestrous and diestrous); N, nucleated (proestrous).
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Figure 1 separação Mirada de neurônios de kisspeptin. (um) estratégia Genética para nullnullespecificamente ative expressão de DTA kisspeptin-expressando celas. KissIC nullnullratos levam um recombinant allele de Kiss1 do qual um mRNA de bicistronic é nullnulltranscrito. Ratos de R26-DTA levam um transcriptionally silenciou gene de DTA, nullnullque é ativado em recombinação Cre-mediado em KissIC/R26-DTA nullnullratos. (b) análise de Immunohistochemical de seções de cérebro de coronal de nullnullP18 (esquerda) e adulto (direito) KissIC/R26-DTA (fundo) e controle de KissIC nullnullratos (topo) usando anticorpos a kisspeptin. Immunoreactivity de Kisspeptin nullnullfoi achado no AVPV (topo) e ARCO (fundo) do hypothalamus em nullnullcontrole ratos. Note o notadamente immunoreactivity de kisspeptin reduzido em nullnullRatos de KissIC/R26-DTA, sugestionando separação eficiente de neurônios de kisspeptin, nullnullem AVPV e ARCO. Barra de balança representa 200 µm. (c) Nenhum kisspeptin - nullnullforam descobertos neurônios positivos no AVPV de P18 KissIC/R26-DTA nullnull(n = 4) fêmeas compararam com controles (10.5 ± 4.5 neurônios, n = 2). nullnullNós descobrimos 6.5 ± 3.1 neurônios kisspeptin-positivos no AVPV de nullnulladulto KissIC/R26-DTA (n = 3) fêmeas compararam com 239.3 ± 10.99 nullnullneurônios kisspeptin-positivos em controles (n = 4). (d) Kiss1 mRNA níveis nullnullestava reduzido antes das ~95% em P20 KissIC/R26-DTA (n = 2) fêmeas compararam nullnullcom controle (n = 8) animais e antes de ~98% em adulto KissIC/R26-DTA (n = 8) nullnullfêmeas compararam com controles (n = 4). barras de Erro representam s.e.m.
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foram descobertos neurônios no AVPV de P18 KissIC/R26-­DTA (n = 4 
fêmeas compararam com 10.5 ± 4.5 neurônios de kisspeptin-­positive dentro 
 controles (n = 2). Nós descobrimos 6.5 ± 3.1 neurônio de kisspeptin-­positive 
no AVPV de adulto KissIC/R26-­DTA (n = 3) fêmeas compararam 
com 239.3 ± 10.99 em controles (n = 4) (Figo. 1c), sugestionando aquele effi 
separação de cient (>97%) de neurônios de kisspeptin acontece nestes animais 
Consistente com isto, Kiss1 mRNA níveis estavam reduzidos antes das ~95% dentro 
P20 e antes de ~98% em adulto fêmeas de KissIC/R26-­DTA (Figo. 1d). 
Administrador
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Maturação sexual na ausência de neurônios de kisspeptin 
Massa ovariana estava fortemente reduzida em adulto fêmeas de KissIC/R26-­DTA 
(2.0 ± 0.1 mg, n = 12) comparou com controle (5.2 ± 0.4 mg, n = 14) 
animais (P <0.001; Figo. 2a). Este fenótipo já era manifesto a 
P20 (fêmeas de KissIC/R26-­DTA, 1.0 ± 0.1 mg, n = 3; controles, 2.1 ±, 
0.2 mg, n = 3; P <0.05; Figo. 2a). Apesar de massa ovariana reduzida, 
porém, histology ovariano mostraram folículos em todas as fases de desenvolver-­ 
ment e a presença de lutea de corpos em fêmeas de KissIC/R26-­DTA 
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(Figo. 2b), elevando a possibilidade que estes animais poderiam ser fer-­ 
azulejo. Notavelmente, tudo testaram ratos de KissIC/R26-­DTA femininos (cinco de cinco) 
dado à luz para normal-­sized lixos quando acasalou selvagem-­type machos. 
Estes dados sugerem que ratos femininos possam ficar férteis dentro o 
ausência de neurônios de kisspeptin. Levado junto, estes dados indicam 
aquela separação de dianteiras de celas de kisspeptin-­expressing para pequeno-­sized 
ovários que caso contrário parecem ser funcionalmente normal. 
É pensada secreção de Kisspeptin ser necessário para onset oportuno de 
puberty5,6,8,25. Consistente com isto, ratos de knockout de kisspeptin falham 
sofra maturação sexual e espetáculo demoraram onset de puberty11,12, 
considerando que administração de kisspeptin em animais juvenis pode avançar 
onset de puberdade 
16,17. nós próximo perguntou se separação de kisspeptin 
neurônios afetam a cronometragem de onset de puberdade e marcadores comparados 
de maturação de pubertal e hormônio de soro nivela em KissIC/R26-­ 
Fêmeas de DTA com esses em ratos de controle. Inesperadamente, o dia de 
abertura vaginal, um marcador externo de onset de puberdade, era semelhante 
Administrador
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Figure 2 maturação Sexual em ratos femininos neurônios de kisspeptin necessitados. nullnull(um) massa Ovariana de ratos de KissIC/R26-DTA femininos estava fortemente reduzida nullnulla P20 e em adultos. *P <0.05, * *P <0.001. (b) ovário Representativo nullnullseção de um rato de KissIC/R26-DTA mostrou folículos em todas as fases de nullnulldesenvolvimento. Barras de balança representam 500 µm. (c) O dia de abertura vaginal nullnull(A VO) não era significativamente diferente em ratos de KissIC/R26-DTA femininos que em nullnullcontrole animais, enquanto sugestionando cronometragem normal de onset de puberdade na ausência nullnullde neurônios de kisspeptin. (d) soro luteinizing hormônio níveis Ms eram nullnullnão substancialmente diferente em P20 e adulto fêmeas de KissIC/R26-DTA nullnullcomparado a controles. ns, não significante (P> 0.5). (e) citologia Vaginal de nullnullRatos de KissIC/R26-DTA (direito) mostrou todas as fases de cyclicity de estrous apesar de nullnullperíodos um pouco prolongados de cornification vaginal compararam a R26-DTA nullnullcontrole animais (esquerda). C, cornified (estrous); CL, lutea de corpos,; L, LEUKOCYTIC, nullnull(metestrous e diestrous); N, nucleated (proestrous).
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in KissIC/R26-­DTA females (P33.0 ± 1.2 d, n = 7) compared with 
control mice (P33.3 ± 0.7 d; n = 18) (Fig. 2c). This indicates that 
kisspeptin neurons are not essential for the timing of puberty onset 
in female mice. In addition, it suggests that increased GnRH neuro-­
secretion, leading to awakening of the hpg axis, can be initiated in 
the absence of kisspeptin neurons. We then compared luteinizing 
hormone levels in mutant and control animals. Serum luteinizing 
hormone levels remained low (<0.2 ng ml−1) in both KissIC/R26-­
DTA (n = 7) and control (n = 3) mice at P20 and rose to mean levels in 
adult KissIC/R26-­DTA females, which were not significantly differ-­
ent to controls (KissIC/R26-­DTA, 0.45 ± 0.09 ng ml−1, n = 16; control, 
0.33 ± 0.04 ng ml−1, n = 25; P > 0.5; Fig. 2d). Subsequent stages of 
female pubertal maturation are characterized by initiation of ovulatory 
cyclicity. Daily inspection of vaginal cytology revealed estrous cyclicity 
in KissIC/R26-­DTA females (Fig. 2e), despite somewhat prolonged 
 periods of persistent vaginal cornification. These experiments suggest 
that kisspeptin neurons are not essential for progressing through 
pubertal maturation and attaining ovulatorycyclicity.
Genetic ablation of GPR54 neurons
On the basis of these results, we reasoned that if kisspeptin neurons are 
not essential for puberty onset and fertility, then kisspeptin target cells 
might also be dispensable. We therefore performed the complementary 
genetic experiment and generated mice that lacked cells that express 
the kisspeptin receptor GPR54. We used gene targeting in embry-­
onic stem cells to insert an IRES sequence followed by a cre cDNA 
downstream of the coding region of the Gpr54 (also known as Kiss1r) 
gene (Fig. 3a–c). The resulting GPR54-­IRES-­Cre knock-­in (GPIC) 
mice were viable and showed uncompromised fertility. To monitor 
Cre expression in GPIC mice, we bred these animals to the enhanced 
ROSA26–τGFP (eR26-­τGFP) reporter line26. Hypothalamic sections 
prepared from female GPIC/eR26-­τGFP mice contained brightly fluo-­
rescent cells in the medial preoptic area (MPA; Fig. 3d) and fluo-­
rescent fiber terminals at the median eminence (data not shown). 
Immunofluorescence analysis on sections through the preoptic 
area of GPIC/eR26-­τGFP mice using antibodies to GnRH showed 
that ~99% (99 ± 1%, n = 5) of GnRH neurons of the hypothalamus 
were labeled by τGFP fluorescence and ~96% (96 ± 3%, n = 5) 
of τGFP-­positive neurons expressed GnRH (Fig. 3d), demonstrat-­
ing faithful activation of the τGFP reporter gene in GnRH/GPR54-­
expressing neurons. We also found τGFP-­positive neurons in other 
brain areas outside of the hypothalamus, such as the hippocampus of 
female GPIC/eR26-­τGFP mice (data not shown), consistent with the 
reported distribution of GPR54 neurons27.
To systemically ablate GPR54 neurons, we bred GPIC mice to R26-­
DTA mice. The number of GnRH neurons in the hypothalamus of 
female GPIC/R26-­DTA mice was reduced to 9.3% ± 0.6% (n = 4) 
of that seen in R26-­DTA control mice, coincident with a reduc-­
tion of GnRH mRNA to 6.7% in the mutant animals (Fig. 3e,f). To 
analyze whether the remaining GnRH neurons express GPR54, we 
compared coronal sections through the entire brain of triple mutant 
mice harboring one KissIC, one R26-YFP28 and one R26-DTA allele 
(KissIC/R26-­YFP/R26-­DTA mice) with sections from KissIC/R26-­
YFP animals (Supplementary Fig. 2a). YFP-­positive cells and fibers 
were completely absent in triple mutant mice (Supplementary 
Fig. 2a), demonstrating efficient ablation of all GPR54 neurons. 
Consistent with this, Gpr54 mRNA levels were reduced below the 
detection threshold in female GPIC/R26-­DTA mice (Fig. 3g).
Sexual maturation in the absence of GPR54 neurons
GPIC/R26-­DTA females had significantly reduced ovarian masses 
(GPIC/R26-­DTA, 3.9 ± 0.4 mg, n = 11; control, 5.2 ± 0.4 mg, n = 14; 
P < 0.05; Fig. 4a). This difference became even more distinct when 
ovarian masses were corrected for body weight and cycle stage 
(Supplementary Fig. 2c). These findings suggest that the absence 
of GnRH/GPR54-­expressing neurons, and thus kisspeptin/GPR54 
signaling, does affect ovarian maturation. However, all female of 
the GPIC/R26-­DTA mice that we tested produced offspring when 
mated to wild-­type males (eight of eight animals). Consistent with 
this, GPIC/R26-­DTA females displayed estrous cyclicity despite 
slightly prolonged phases of cornification (Fig. 4b). These results 
YFP
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Figure 3 Genetic ablation of GPR54 neurons. (a) Targeting strategy 
to express Cre recombinase under control of the Gpr54 promoter. The 
inserted cassette is composed of an IRES followed by the coding sequence 
for Cre recombinase and a phosphoglycerate kinase (pgk) promoter–driven 
neomycin resistance gene flanked by Flp recombinase recognition sites (FRT). 
(b) Southern blot analysis of SacI-digested embryonic stem cell DNA. The 
expected fragment sizes are indicated (wild type, 8.8 kb; mutant, 
7.5 kb). Clone 2 carries the recombinant GPIC allele (GPICneo+). 
Uncropped blot images are shown in Supplementary Figure 7. 
(c) Southern blot analysis of SacI-digested DNA from a homozygous 
mutant mouse after neomycin-selection cassette removal. The expected 
fragment size (GPICneo−) is 10.5 kb. (d) Faithful activation of a Cre-
dependent YFP reporter gene in GnRH neurons. Immunofluorescence 
analysis of sections through the MPA of the hypothalamus prepared from 
GPIC/R26-YFP mice using antibodies to GnRH. All GnRH neurons (red) 
expressed YFP (green). Scale bar represents 50 µm. (e) Efficient ablation 
of GnRH neurons in GPIC/R26-DTA mice. Immunofluorescence analysis 
of MPA sections from GPIC/R26-DTA and GPIC control mice using 
antibodies to GnRH. Scale bar represents 100 µm. (f) The number of 
GnRH neurons (top) was reduced by ~90% in GPIC/R26-DTA (n = 6), 
but not in KissIC/R26-DTA (n = 3), mice compared with R26-DTA control 
animals (n = 4). GnRH mRNA levels (bottom) were reduced by ~93% in 
GPIC/R26-DTA mice (n = 3) compared to KissIC/R26-DTA (n = 4) and 
control (n = 3) animals. (g) Gpr54 mRNA levels were below detection 
threshold (n.d.) in GPIC/R26-DTA females (n = 2) compared to controls 
(n = 5). Error bars represent s.e.m.
©
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Administrador
Note
em fêmeas de KissIC/R26-­DTA (P33.0 ± 1.2 d, n = 7) comparou com 
controle ratos (P33.3 ± 0.7 d; n = 18) (Figo. 2c). Isto indica isso 
neurônios de kisspeptin não são essenciais para a cronometragem de onset de puberdade 
em ratos femininos. Além, sugere que isso aumentasse neuro-­ de GnRH 
secreção, enquanto conduzindo a despertar do eixo de hpg, pode ser iniciado dentro 
a ausência de neurônios de kisspeptin. Nós comparamos luteinizing então 
hormônio nivela em mutante e animais de controle. Luteinizing de soro 
níveis de hormônio permaneceram baixos (<0.2 ml de ng 
-1) em ambos o KissIC/R26-­ 
DTA (n = 7) e controla (n = 3) ratos a P20 e rosa para significar níveis dentro 
adulto fêmeas de KissIC/R26-­DTA que não eram significativamente diferir-­ 
ent para controles (KissIC/R26-­DTA, 0.45 ± 0.09 ml de ng 
-1, n = 16; controle, 
0.33 ± 0.04 ML DE NG 
-1, n = 25; P> 0.5; Figo. 2d). fases Subseqüentes de 
maturação de pubertal feminina é caracterizada por iniciação de ovulatory 
cyclicity. Diariamente inspeção de citologia vaginal revelou cyclicity de estrous 
em fêmeas de KissIC/R26-­DTA (Figo. 2e), apesar de um pouco prolongado 
 períodos de cornification vaginal persistente. Estas experiências sugerem 
aqueles neurônios de kisspeptin não são essenciais por ter progredido por 
maturação de pubertal e cyclicity de ovulatory atingindo.
Administrador
Note
Separação genética de neurônios de GPR54 
Em base destes resultados, argumentamos nós que se neurônios de kisspeptin são 
não essencial para onset de puberdade e fertilidade, então kisspeptin celas designadas 
também possa ser dispensável. Nós executamos então o complementar 
experiência genética e ratos gerados que faltaram celas que expressam 
o receptor de kisspeptin GPR54. Nós usamos gene que mira emembry-­ 
onic param celas para inserir uma sucessão de RAIVAS seguida por um cDNA de cre 
a jusante da região de codificação do Gpr54 (também conhecido como Kiss1r) 
gene (Figo. 3a–c). O GPR54-­IRES-­Cre resultante bater-­in (GPIC) 
ratos eram viáveis e mostraram fertilidade de uncompromised. Monitorar 
Expressão de Cre em ratos de GPIC, nós criamos estes animais aos aumentaram 
ROSA26–tGFP (eR26-­tGFP) line26 de repórter. Seções de Hypothalamic 
preparado de ratos de GPIC/eR26-­tGFP femininos conteve fluo-­ de brightly 
celas de rescent na área de preoptic mediana (MPA; Figo. 3d) e fluo-­ 
términos de fibra de rescent à eminência mediana (dados não mostrados). 
Análise de Immunofluorescence em seções pelo preoptic 
área de ratos de GPIC/eR26-­tGFP que usam anticorpos para GnRH mostrou 
que ~99% (99 ± 1%, n = 5) de neurônios de GnRH do hypothalamus 
Administrador
Note
estava rotulado antes de fluorescência de tGFP e ~96% (96 ± 3%, n = 5) 
de neurônios de tGFP-­positive expressou GnRH (Figo. 3d), demonstrat-­ 
ing ativação fiel do gene de repórter de tGFP em GnRH/GPR54-­ 
neurônios expressando. Nós também achamos neurônios de tGFP-­positive dentro outro 
áreas de cérebro fora do hypothalamus, como o hippocampus de 
ratos de GPIC/eR26-­tGFP femininos (dados não mostrados), consistente com o 
distribuição informada de neurons27 de GPR54. 
Para ablate de systemically neurônios de GPR54, nós criamos ratos de GPIC a R26-­ 
Ratos de DTA. O número de neurônios de GnRH no hypothalamus de 
foram reduzidos ratos de GPIC/R26-­DTA femininos a 9.3% ± 0.6% (n = 4) 
disso visto em R26-­DTA controla ratos, coincidente com um reduc-­ 
tion de mRNA de GnRH para 6.7% nos animais de mutante (Figo. 3e,f). Para 
analise se os neurônios de GnRH restantes GPR54 expresso, nós, 
seções de coronal comparadas pelo cérebro inteiro de mutante triplo 
ratos que abrigam um KissIC, um R26-YFP28 e um allele de R26-DTA 
(Ratos de KissIC/R26-­YFP/R26-­DTA) com seções de KissIC/R26-­ 
Animais de YFP (Figo Adicional. 2a). celas de YFP-­positive e fibras 
estava completamente ausente em ratos de mutante triplos (Adicional 
Figo. 2a), demonstrando separação eficiente de todos os neurônios de GPR54. 
Consistente com isto, Gpr54 mRNA níveis estavam reduzidos abaixo o 
limiar de descoberta em ratos de GPIC/R26-­DTA femininos (Figo. 3g).
Administrador
Note
Maturação sexual na ausência de neurônios de GPR54 
Fêmeas de GPIC/R26-­DTA tinham reduzido massas ovarianas significativamente 
(GPIC/R26-­DTA, 3.9 ± 0.4 MG, N = 11; controle, 5.2 ± 0.4 mg, n = 14; 
P <0.05; Figo. 4a). Esta diferença ficou mais distinta até mesmo quando 
foram corrigidas massas ovarianas para peso de corpo e fase de ciclo 
(Figo adicional. 2c). Estes achados sugerem que a ausência 
de neurônios de GnRH/GPR54-­expressing, e assim kisspeptin/GPR54 
sinalizando, afete maturação ovariana. Porém, toda a fêmea de 
os ratos de GPIC/R26-­DTA que nós testamos descendência produzida quando 
acasalado selvagem-­type machos (oito de oito animais). Consistente com 
isto, fêmeas de GPIC/R26-­DTA exibiram cyclicity de estrous apesar de 
fases ligeiramente prolongadas de cornification (Figo. 4b). Estes resultados 
nature neurOSCIenCe	 VOLUME 14 | NUMBER 6 | JUNE 2011 707
a r t I C l e S
indicate that reproductive maturation can take place in the absence 
of GnRH/GPR54-­expressing neurons and suggest that kisspeptin/
GPR54 signaling is not essential for fertility. Furthermore, these data 
suggest that, in the absence of kisspeptin/GPR54 signaling, a small 
number of GnRH neurons suffices to become fertile.
Puberty onset in GPIC/R26-­DTA females (vaginal opening at P32.8 ± 
1.6 d, n = 5) was not found to be significantly different from that in 
control animals ( P > 0.05; Fig. 4c). Luteinizing hormone levels seemed 
somewhat lower in adult GPIC/R26-­DTA females (0.23 ± 0.02 ng ml−1, 
n = 15) than those in controls (0.33 ± 0.04 ng ml−1, n = 25); however, 
this difference was insignificant (P = 0.4). Taken together, these data 
indicate that, in the absence of kisspeptin/GPR54 signaling, ~10% 
of the wild-­type GnRH neuron repertoire is sufficient to develop a 
functional hpg axis.
Induced ablation of kisspeptin and GPR54 neurons
Finally, we asked whether uncompromised fertility and normal 
timing of puberty onset in KissIC/R26-­DTA and GPIC/R26-­DTA 
females resulted from compensatory mechanisms during develop-­
ment. To address this question, we conditionally ablated kisspeptin-­ 
and GPR54 neurons, respectively, in adult female mice. Mice were 
made susceptible to the action of diphtheria toxin by expressing a 
human diphtheria toxin receptor (DTR) transgene. We bred KissIC 
and GPIC mice to the ROSA26-­iDTR (R26-­iDTR) strain, in which a 
transcriptionally silenced DTR gene is inserted into the ROSA26 locus 
and can be activated by Cre-­mediated recombination29. KissIC/R26-­
iDTR and GPIC/R26-­iDTR develop normally and progress through 
pubertal maturation in the absence of diphtheria toxin (data not 
shown). To ablate kisspeptin and GPR54 neurons, we injected 
20-­week-­old adult female KissIC/R26-­iDTR, GPIC/R26-­iDTR (Fig. 5) 
and control mice (R26-­iDTR littermates not bearing a cre allele) twice 
intraperitoneally with 50 ng of diphtheria toxin per g body weight. 
Kisspeptin immunoreactivity was markedly reduced in KissIC/R26-­
iDTR animals 1 week after the second diphtheria toxin injection 
(Supplementary Fig. 3a). Notably, other neuropeptide-­expressing 
neuronal populations in the hypothalamus seemed to be unaffected by 
kisspeptin neuron ablation (Fig. 5c and Supplementary Fig. 4a). In 
GPIC/R26-­iDTR mice, GnRH immunoreactivity was strongly reduced 
after diphtheria toxin administration (Supplementary Fig. 3b). Taken 
together, these data suggest efficient ablation of kisspeptin and GPR54 
neurons after diphtheria toxin injection.
We then asked whether kisspeptin or GPR54 neuron abla-­
tion would have an acute effect on estrous cyclicity. In contrast 
with KissIC/R26-­DTA animals, KissIC/R26-­iDTR females did not 
exhibit normal estrous cyclicity after diphtheria toxin administra-­
tion, but vaginal lavages collected from these animals only rarely 
contained cells (Fig. 5a). These experiments suggest that adult 
females cannot compensate for the acute loss of kisspeptin neurons. 
The importance of kisspeptin neurons for ovulatory cyclicity does 
not however seem to depend on kisspeptin/GPR54 signaling itself 
because GPIC/R26-­iDTR remained cyclic after diphtheria toxin injec-­
tions (Fig. 5b). Taken together, these data indicate that kisspeptin/
GPR54 signaling, but not kisspeptin neurons, are dispensable for 
estrous cyclicity in adult mice. After monitoring vaginal cytology for 
1 month, diphtheria toxin–injected KissIC/R26-­iDTR and GPIC/R26-­
iDTR females were subsequently mated to wild-­type males. Although 
none of the KissIC/R26-­iDTR females (n = 6) produced offspring, 50% 
8
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Figure 4 Unaltered timing of puberty onset in female mice lacking 
GPR54 neurons. (a) Genetic ablation of GPR54 neurons significantly 
reduced ovarian mass (P < 0.05). (b) Vaginal cytology of GPIC/R26-DTA 
mice showed all stages of estrous cyclicity despite somewhat prolonged 
periods of vaginal cornification. (c) GPR54 neuron ablation did not affect 
the day of vaginal opening. ns, not significant (P > 0.05). (d) Mean 
serum luteinizing hormone levels in GPIC/R26-DTA females were not 
significantlydifferent from levels in controls. (e) Representative ovarian 
section showed follicles at all stages of maturation and corpora lutea in 
GPIC/R26-DTA animals. Scale bar represents 500 µm.
a
1
Day
10 20155
Exon 1 Exon 2
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GPIC/R26-iDTR
Figure 5 Acyclicity after diphtheria toxin–mediated ablation of 
kisspeptin neurons in adult KissIC/R26-iDTR mice. (a) Genetic 
strategy to conditionally ablate kisspeptin neurons via diphtheria 
toxin (DT) administration. KissIC/R26-iDTR animals did not exhibit 
normal estrous cyclicity, but showed prolonged periods of persistent 
diestrous or estrous after diphtheria toxin injection. On days without 
dots, no cells were detected in the vaginal lavage. (b) In contrast, 
vaginal cytology of GPIC/R26-iDTR mice showed all stages of estrous 
cyclicity after diphtheria toxin injection. (c) GnRH neuron numbers 
were reduced by ~97% in GPIC/R26-iDTR mice (n = 3) compared 
with R26-iDTR control mice (n = 3) after diphtheria toxin injection. 
In contrast, GnRH neuron numbers were unchanged after conditional 
ablation of kisspeptin neurons via diphtheria toxin administration in 
KissIC/R26-iDTR mice (n = 4). ***P < 0.0001. (d) Vaginal cytology of 
P40 R26-iDTR mice (left) showed all stages of estrous cyclicity after 
diphtheria toxin injection at P20. In contrast, P40 KissIC/R26-iDTR 
animals (right) did not exhibit normal estrous cyclicity, but showed 
prolonged periods of persistent diestrous after diphtheria toxin 
injection at P20. Error bars represent s.e.m.
©
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Administrador
Note
indique aquela maturação de reproductive pode acontecer na ausência 
de neurônios de GnRH/GPR54-­expressing e sugere que kisspeptin / 
GPR54 sinalizar não é essencial para fertilidade. Além disso, estes dados 
sugira que, na ausência de kisspeptin/GPR54 sinalizar, um smal 
número de neurônios de GnRH basta ficar fértil. 
Onset de puberdade em fêmeas de GPIC/R26-­DTA (abertura vaginal a P32.8 ± 
1.6 d, n = 5) não foi achado para ser significativamente diferente disso dentro 
controle animais (P> 0.05; Figo. 4c). Luteinizing hormônio níveis pareciam 
um pouco abaixe em adulto fêmeas de GPIC/R26-­DTA (0.23 ± 0.02 ml de ng 
-1 
n = 15) que esses em controles (0.33 ± 0.04 ml de ng 
-1, n = 25); porém 
esta diferença era insignificante (P = 0.4). Levado junto, estes dados 
indique que, na ausência de kisspeptin/GPR54 sinalizar, ~10% 
do selvagem-­type GnRH neurônio repertório é suficiente desenvolver um 
eixo de hpg funcional.
Administrador
Note
Separação induzida de kisspeptin e neurônios de GPR54 
Finalmente, nós perguntamos se fertilidade de uncompromised e normal 
cronometrando de onset de puberdade em KissIC/R26-­DTA e GPIC/R26-­DTA 
fêmeas foram o resultado de mecanismos compensatórios durante desenvolver-­ 
ment. Enviar esta pergunta, nós condicionalmente kisspeptin-­ de ablated 
e neurônios de GPR54, respectivamente, em adulto ratos femininos. Ratos eram 
feito suscetível à ação de toxina de difteria expressando um 
receptor de toxina de difteria humano (DTR) transgene. Nós criamos KissIC 
e ratos de GPIC para o ROSA26-­iDTR (R26-­iDTR) tensão em qual um 
transcriptionally silenciaram gene de DTR é inserido no locus de ROSA26 
e pode ser ativado através de recombination29 de Cre-­mediated. KissIC/R26-­ 
iDTR e GPIC/R26-­iDTR normalmente desenvolvem e progridem por 
maturação de pubertal na ausência de toxina de difteria (dados não 
mostrado). A kisspeptin de ablate e neurônios de GPR54, injetamos nós 
20-­week-­old adulto KissIC/R26-­iDTR feminino, GPIC/R26-­iDTR (Figo. 5) 
e controla ratos (littermates de R26-­iDTR que não agüenta um allele de cre) duas vezes 
intraperitoneally com 50 ng de toxina de difteria por g corpo peso. 
Administrador
Note
Immunoreactivity de Kisspeptin estava notadamente reduzido em KissIC/R26-­ 
animais de iDTR 1 semana depois da segunda injeção de toxina de difteria 
(Figo adicional. 3a). Notavelmente, outro neuropeptide-­expressing 
populações de neuronal no hypothalamus pareciam ser não afetado por 
separação de neurônio de kisspeptin (Figo. 5c e Figo Adicional. 4a). Em 
Ratos de GPIC/R26-­iDTR, immunoreactivity de GnRH estava fortemente reduzido 
depois de administração de toxina de difteria (Figo Adicional. 3b). Levado 
junto, estes dados sugestionam separação eficiente de kisspeptin e GPR54 
neurônios depois de injeção de toxina de difteria. 
Nós perguntamos então se kisspeptin ou GPR54 neurônio abla-­ 
tion teriam um efeito agudo em cyclicity de estrous. Em contraste 
com animais de KissIC/R26-­DTA, não fizeram fêmeas de KissIC/R26-­iDTR 
exibição cyclicity de estrous normal depois de administra-­ de toxina de difteria 
tion, mas lavages vaginal só colecionaram raramente destes animais 
celas contidas (Figo. 5a). Estas experiências sugestionam aquele adulto 
fêmeas não podem compensar para a perda aguda de neurônios de kisspeptin. 
A importância de neurônios de kisspeptin para cyclicity de ovulatory faz 
não porém pareça depender de kisspeptin/GPR54 que se sinaliza 
porque GPIC/R26-­iDTR permaneceu cíclico depois de injec-­ de toxina de difteria 
tions (Figo. 5b). Levado junto, estes dados indicam aquele kisspeptin / 
GPR54 sinalizando, mas não neurônios de kisspeptin, é dispensável para 
cyclicity de estrous em ratos de adulto. Depois de monitorar citologia vaginal para 
1 mês, toxin–injected de difteria KissIC/R26-­iDTR e GPIC/R26-­ 
fêmeas de iDTR foram acasaladas subseqüentemente para selvagem-­type machos. Embora 
nenhum das fêmeas de KissIC/R26-­iDTR (n = 6) produziu descendência, 50%, 
708	 VOLUME 14 | NUMBER 6 | JUNE 2011 nature neurOSCIenCe
a r t I C l e S
(three of six) of GPIC/R26-­iDTR females produced offspring after 
8 weeks of mating. After 8 weeks of mating, animals were killed and 
ablation efficiency was investigated. No kisspeptin-­positive neurons 
were detected in KissIC/R26-­iDTR animals (n = 3). In GPIC/R26-­
iDTR mice, GnRH neuron numbers were reduced by ~93% (Fig. 5c 
and Supplementary Figs. 2c and 3b). These data suggest that as little 
as ~7% of the GnRH neuron repertoire found in wild-­type mice is 
sufficient to maintain cyclic activity of the hpg axis.
To determine when female mice become sensitive to kisspeptin 
neuron ablation, we injected KissIC/R26-­iDTR animals with diphthe-­
ria toxin at P20. Because only a few neurons express kisspeptin in the 
AVPV at P20 (ref. 19), diphtheria toxin injection at this age allows us 
to mainly affect the ARC kisspeptin neuronal populations. Starting 
at P40, we monitored estrous cyclicity in these animals. In contrast 
with R26-­iDTR control littermates, KissIC/R26-­iDTR animals did 
not exhibit normal estrous cyclicity, but showed prolonged periods of 
persistent diestrous after diphtheria toxin injection at P20 (Fig. 5d). 
Consistent with this, none of the KissIC/R26-­iDTR females (n = 3) 
injected at P20 bore offspring when mated to wild-­type males for 8 
weeks after vaginal cytology inspection. These data suggest that com-­
pensation in KissIC/R26-­DTA females occurs before P20 and therefore 
mainly affects the kisspeptin neuronal population in the ARC.
DISCUSSION
To determine whether kisspeptin neurons are needed to turnon the 
GnRH pulse generator at puberty onset, we used a binary genetic 
strategy and systemically ablated kisspeptin-­expressing cells in mice. 
In a complementary genetic approach, we also generated animals 
lacking GPR54-­expressing kisspeptin target cells and analyzed their 
reproductive maturation. These experiments yield several impor-­
tant results. First, female mice lacking kisspeptin or GPR54 cells had 
significantly smaller ovaries than littermate controls, indicating an 
essential role of kisspeptin/GPR54 in gonadal maturation. Second, 
neither kisspeptin-­ nor GPR54 neuron ablation affected the timing 
of puberty onset in female mice, suggesting that the GnRH pulse 
generator can be turned on independently of kisspeptin neurons and 
kisspeptin/GPR54 signaling. Third, these animals are fertile, indi-­
cating that kisspeptin neurons and kisspeptin/GPR54 signaling are 
dispensable for coarse reproductive development. Fourth, despite 
markedly reduced numbers of GnRH neurons after GPR54 neuron 
ablation, the mice were fertile, demonstrating that ~10% of the GnRH 
neuron repertoire found in wild-­type animals is sufficient for repro-­
ductive development. Fifth, acute kisspeptin, but not GPR54, neuron 
ablation in adult animals resulted in acyclicity and infertility, indicat-­
ing that kisspeptin-­expressing neurons, but not kisspeptin/GPR54 
signaling, are essential for maintaining ovulatory cyclicity in adults. 
Finally, acute kisspeptin neuron ablation at P20 resulted in acyclicity 
and infertility, suggesting developmental compensation for ARC 
kisspeptin neurons in KissIC/R26-­DTA animals before P20.
We adopted a ‘toxin strategy’ to specifically ablate kisspeptin-­ and 
GPR54-­expressing cells in mice. With this strategy, any kisspeptin-­ 
or GPR54-­expressing cell in the body would be ablated. Along the 
hpg axis, kisspeptin and GPR54 expression has been reported in the 
anterior pituitary gland and in the ovaries30,31. Consistent with this, 
we detected some τGFP-­positive cells in the ovaries of KissIC/eR26-­
τGFP, but not of GPIC/eR26-­τGFP, animals (Supplementary Fig. 5a). 
In the ovary, Cre-­mediated recombination seemed to be restricted to a 
small subset of large luteal cells in a few corpora lutea (Supplementary 
Fig. 5a). Ablation of these cells in KissIC/R26-­DTA mice apparently 
did not preclude reproductive maturation and fertility. Cre-­mediated 
recombination was also evident in the anterior pituitary of female 
KissIC/eR26-­τGFP and GPIC/R26-­YFP mice; however, these fluores-­
cent cells did not express gonadotropins (Supplementary Fig. 5b).
Cell ablation is fundamentally different to gene knockout, which 
assesses the function of a particular gene. Instead of removing one par-­
ticular type of molecule from a cell, the toxin ablation approach elimi-­
nates the whole cell itself. In the case of neurons, a genetically defined 
type of neuron is removed from a neural circuit. Toxin-­based genetic 
cell ablation therefore assesses the role of a particular cell in a physio-­
logical process and can result in phenotypes that are different from 
those resulting from complementary gene knockout approaches32–34.
Previous studies had shown that knockout of the Kiss1 and Gpr54 
genes result in delayed puberty onset10–13. Furthermore, although 
some residual reproductive activity has been documented in these 
animals35, they are infertile. In contrast, KissIC/R26-­DTA and 
GPIC/R26-­DTA females showed normal timing of puberty onset, 
attained ovulatory cyclicity and were fertile. The knockout and abla-­
tion mouse models differ in their genetic background, which might 
contribute to some of the observed phenotypic differences. It may 
seem counterintuitive that the effects of gene deletion are more delete-­
rious than those of neuron ablation; however, one possible explanation 
for uncompromised fertility in the DTA animals could be develop-­
mental compensation. Other neurons upstream of GnRH neurons36 
might functionally take over in response to cell ablation in DTA mice, 
but not in knockout mice (Supplementary Fig. 6). Our data suggest 
that developmental compensation is occurring only when kisspeptin 
cells are ablated early in development (that is, before P20). Kisspeptin-­
expressing cell ablation in adulthood after normal development seems 
to preclude activation or formation of these alternative reproductive 
circuits. Only few neurons express kisspeptin in the AVPV before P20 
(ref. 19), suggesting that developmental compensation mainly affects 
the kisspeptin neuronal population in the ARC. Notably, develop-­
mental compensation has also been demonstrated for other neuronal 
populations in the ARC32–34.
Although we currently do not have insight into the molecular 
mechanisms underlying compensation in the DTA animals, one 
possibility might be that kisspeptin neurons coexpress some other 
neuropeptide or neurotransmitter that normally inhibits the develop-­
ment of other compensatory reproductive neural circuits. Although 
kisspeptin neuron ablation would remove this factor and lead to 
the development of the normally quiescent compensatory circuits, 
Kiss1 knockout mice would still have the inhibitory factor suppress-­
ing compensatory pathways from development. Consistent with 
this, most kisspeptin neurons express GABA37, the major inhibi-­
tory neurotransmitter in the brain. Removal of the inhibitory fac-­
tor might lead to compensatory upregulation of other neuropeptides 
that are known to modulate reproductive function. Dynorphin and 
neurokinin B are neuropeptides that are coexpressed in kisspeptin 
neurons, but are also expressed by other neurons in the hypotha-­
lamus38. However, we found that dynorphin and neurokinin B 
mRNA levels were reduced by ~80% in KissIC/R26-­DTA, but 
not in GPIC/R26-­DTA, females compared with control animals 
(Supplementary Fig. 4b), suggesting that these two neuropeptides are 
not upregulated in response to kisspeptin neuron ablation. The strong 
reduction of dynorphin mRNA levels in KissIC/R26-­DTA animals is 
surprising, as dynorphin is widely expressed in the hypothalamus.
An alternative explanation for the more deleterious phenotype 
in the knockout mice might be that dysregulation of kisspeptin-­ or 
GPR54/GnRH-­expressing neurons in a neural circuit is more det-­
rimental to reproductive function than complete removal of the 
neuron, which, in principle, allows for compensation. Potential dys-­
regulation of kisspeptin neurons by removing either kisspeptin or 
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(três de seis) de fêmeas de GPIC/R26-­iDTR descendência produziu depois 
8 semanas de acasalar. Depois de 8 semanas de acasalar, foram matados animais e 
eficiência de separação foi investigada. Nenhum neurônio de kisspeptin-­positive 
foi descoberto em animais de KissIC/R26-­iDTR (n = 3). Em GPIC/R26-­ 
ratos de iDTR, GnRH neurônio números estavam reduzidos antes das ~93% (Figo. 5c 
e Figos Adicionais. 2c e 3b). Estes dados sugerem que como pequeno 
como ~7% do GnRH neurônio repertório achados dentro selvagem-­type ratos são 
suficiente manter atividade cíclica do eixo de hpg. 
Determinar quando ratos femininos ficam sensíveis a kisspeptin 
separação de neurônio, nós injetamos animais de KissIC/R26-­iDTR com diphthe-­ 
toxina de ria a P20. Porque só alguns neurônios kisspeptin expresso no 
AVPV a P20 (ref. 19), injeção de toxina de difteria a esta idade nos permite 
afetar as ARCO kisspeptin neuronal populações principalmente. Começando 
a P40, nós monitoramos cyclicity de estrous nestes animais. Em contraste 
com R26-­iDTR controlam littermates, animais de KissIC/R26-­iDTR fizeram 
não exibição cyclicity de estrous normal, mas mostrou períodos prolongados dediestrous persistente depois de injeção de toxina de difteria a P20 (Figo. 5d). 
Consistente com isto, nenhum das fêmeas de KissIC/R26-­iDTR (n = 3) 
injetado a P20 agüente descendência quando acasalou selvagem-­type machos para 8 
semanas depois de inspeção de citologia vaginal. Estes dados sugestionam aquele com-­ 
pensation em fêmeas de KissIC/R26-­DTA acontece antes de P20 e então 
principalmente afeta a população de neuronal de kisspeptin no ARCO.
Administrador
Note
Determinar se são precisados neurônios de kisspeptin virar no 
GnRH pulsam gerador a onset de puberdade, nós usamos um binário genético 
estratégia e systemically ablated kisspeptin-­expressing celas em ratos. 
Em uma aproximação genética complementar, nós geramos também animais 
kisspeptin de GPR54-­expressing necessitado celas designadas e analisou o deles/delas 
maturação de reproductive. Estas experiências rendem vários impor-­ 
tant resulta. Primeiro, ratos femininos que kisspeptin necessitado ou celas de GPR54 tiveram 
ovários significativamente menores que littermate controla, enquanto indicando um 
papel essencial de kisspeptin/GPR54 em maturação de gonadal. Segundo, 
nem kisspeptin-­ nem GPR54 neurônio separação afetaram a cronometragem 
de onset de puberdade em ratos femininos, sugerindo que o pulso de GnRH 
gerador pode ser virado independentemente em de neurônios de kisspeptin e 
kisspeptin/GPR54 sinalizando. Terço, estes animais são férteis, indi-­, 
cating que neurônios de kisspeptin e kisspeptin/GPR54 sinalizar são 
dispensável para desenvolvimento de reproductive grosso. Quarto, apesar de 
números notadamente reduzidos de neurônios de GnRH depois de neurônio de GPR54 
separação, os ratos eram férteis, enquanto demonstrando que ~10% do GnRH 
repertório de neurônio achou dentro selvagem-­type animais são suficientes para repro-­ 
desenvolvimento de ductive. Quinto, kisspeptin agudo, mas não GPR54, neurônio, 
separação em animais de adulto resultados em acyclicity e infertilidade, indicat-­, 
ing que neurônios de kisspeptin-­expressing, mas não kisspeptin/GPR54 
sinalizando, é essencial para manter cyclicity de ovulatory em adultos. 
Finalmente, separação de neurônio de kisspeptin aguda a P20 resultou em acyclicity 
e infertilidade, sugestionando compensação desenvolvente para ARCO, 
neurônios de kisspeptin em animais de KissIC/R26-­DTA antes de P20.
Administrador
Note
Nós adotamos uma ‘toxina estratégia ' para especificamente kisspeptin-­ de ablate e 
Celas de GPR54-­expressing em ratos. Com esta estratégia, qualquer kisspeptin-­ 
ou cela de GPR54-­expressing no corpo seria ablated. Ao longo do 
eixo de hpg, kisspeptin e expressão de GPR54 foi informada dentro o 
glândula pituitária anterior e no ovaries30,31. Consistente com isto, 
nós descobrimos algumas celas de tGFP-­positive nos ovários de KissIC/eR26-­ 
tGFP, mas não de GPIC/eR26-­tGFP, animais (Figo Adicional. 5a). 
No ovário, recombinação de Cre-­mediated parecia ser restringido um 
subconjunto pequeno de celas de luteal grandes em alguns lutea de corpos (Adicional 
Figo. 5a). Separação destas celas em ratos de KissIC/R26-­DTA aparentemente 
não impeça maturação de reproductive e fertilidade. Cre-­mediated 
recombinação também era evidente dentro o anterior pituitário de fêmea 
Administrador
Note
KissIC/eR26-­tGFP e ratos de GPIC/R26-­YFP; porém, este fluores-­ 
centavo celas não expressaram gonadotropins (Figo Adicional. 5b). 
Separação de cela é fundamentalmente diferente a knockout de gene que 
avalia a função de um gene particular. Em vez de remover um paridade-­ 
ticular digitam de molécula de uma cela, o toxina separação aproximação elimi-­, 
nates a própria cela inteira. No caso de neurônios, um geneticamente definiu 
tipo de neurônio é afastado de um circuito de neural. Toxina-­based genético 
separação de cela avalia o papel de uma cela particular então em um physio-­ 
processo lógico e pode resultar em fenótipos dos que são diferentes 
esse sendo o resultado de approaches32–34 de knockout de gene complementar.
Administrador
Note
Estudos prévios tinham mostrado aquele knockout do Kiss1 e Gpr54 
genes resultam em onset de puberdade atrasado 
10–13. além disso, embora 
alguma atividade de reproductive residual foi documentada nestes 
animals35, eles são estéreis. Em contraste, KissIC/R26-­DTA e 
Fêmeas de GPIC/R26-­DTA mostraram cronometragem normal de onset de puberdade, 
cyclicity de ovulatory atingido e era fértil. O knockout e abla-­ 
modelos de rato de tion diferem no fundo genético deles/delas que pode 
contribua a algumas das diferenças de phenotypic observadas. Pode 
pareça counterintuitive que os efeitos de apagamento de gene são mais apagar-­ 
rious que esses de separação de neurônio; porém, uma possível explicação 
para fertilidade de uncompromised nos animais de DTA poderia ser desenvolver-­ 
compensação mental. Outros neurônios rio acima de neurons36 de GnRH 
possa assumir funcionalmente com respeito a separação de cela em ratos de DTA, 
mas não em ratos de knockout (Figo Adicional. 6). nossos dados sugerem 
aquela compensação desenvolvente só está acontecendo quando kisspeptin 
celas são cedo ablated em desenvolvimento (quer dizer, antes de P20). Kisspeptin-­ 
separação de cela expressando em maioridade depois que desenvolvimento normal pareça 
impedir ativação ou formação deste reproductive alternativo 
circuitos. Só poucos neurônios kisspeptin expresso no AVPV antes de P20 
(ref. 19), sugerindo que compensação desenvolvente afeta principalmente 
a população de neuronal de kisspeptin no ARCO. Notavelmente, desenvolver-­ 
compensação mental também foi demonstrada para outro neuronal 
populações no ARC32–34.
Administrador
Note
Embora nós não temos perspicácia atualmente no molecular 
mecanismos compensação subjacente nos animais de DTA, um 
possibilidade poderia ser aquele coexpress de neurônios de kisspeptin algum outro 
neuropeptide ou neurotransmitter que normalmente inibem o desenvolver-­ 
ment de outros circuitos de neural de reproductive compensatórios. Embora 
separação de neurônio de kisspeptin removeria este fator e conduziria 
o desenvolvimento do normalmente circuitos compensatórios inativos, 
Kiss1 knockout ratos ainda teriam o inhibitory fatorar suprimir-­ 
ing pathways compensatório de desenvolvimento. Consistente com 
isto, a maioria dos neurônios de kisspeptin GABA37 expresso, o inhibi-­ principal, 
neurotransmitter de tory no cérebro. Remoção do fac-­ de inhibitory 
tor poderiam conduzir a upregulation compensatório de outro neuropeptides 
isso é conhecido para modular função de reproductive. Dynorphin e 
neurokinin B são neuropeptides que são coexpressed em kisspeptin 
neurônios, mas também é expressado através de outros neurônios no hypotha-­ 
lamus38. Porém, nós achamos aquele dynorphin e neurokinin B 
níveis de mRNA estavam reduzidos antes das ~80% em KissIC/R26-­DTA, mas 
não em GPIC/R26-­DTA, fêmeas compararam com animais de controle 
(Figo adicional. 4b), sugerindo que este dois neuropeptides são 
não upregulated com respeito a separação de neurônio de kisspeptin. O forte 
redução de mRNA de dynorphin nivela em animais de KissIC/R26-­DTA é 
surpreendendo, como dynorphin é expressado amplamente no hypothalamus. 
Uma explicação alternativa para o fenótipo mais danoso 
nos ratos de knockout poderiam estar aquele dysregulation de kisspeptin-­ ou 
Neurônios de GPR54/GnRH-­expressing em um circuito de neural são mais det-­ 
rimental para reproductive funcionam que remoção completa do 
neurônio que, em princípio, permite compensação. Dys-­ potencial 
regulamento de neurônios de kisspeptin removendo kisspeptin ou 
nature neurOSCIenCe	 VOLUME 14 | NUMBER 6 | JUNE 2011 709
a r t I C l e S
estrogen receptor α (ERα) fromthese cells can have opposite effects 
on puberty onset in female mice. Although a knockout of ERα in 
kisspeptin neurons results in advanced puberty onset due to preco-­
cious awakening of the hpg axis19, knockout of kisspeptin leads to 
delayed onset of puberty11,12. Despite these opposite effects, dysregu-­
lation of kisspeptin neurons may preclude complete pubertal matura-­
tion and thus result in infertility in both animal models. Although 
kisspeptin neurons in the knockout mice might still release other 
neurotransmitters37, potentially disturbing downstream neurons in 
the absence of kisspeptin or ERα signaling, these would be removed 
with the kisspeptin neurons in the DTA animals. In GPR54 knock-­
out mice, the GnRH neuronal network might be dysregulated as a 
result of the absence of the GPR54 receptor on the cell surface. In 
contrast, the remaining GnRH neurons in GPIC/R26-­DTA females 
(which do not express GPR54) are not dysregulated and are appar-­
ently sufficient for reproductive development. Consistent with this 
hypothesis, previous studies have found substantial redundancy in 
the GnRH neuronal population39,40. Acute GPR54 neuron ablation 
in adults leads to prolonged phases of cornification, consistent with 
previous studies demonstrating direct effects of kisspeptin/GPR54 
signaling on GnRH/luteinizing hormone secretion14,41–43. However, 
acute ablation of GPR54 neurons in adults does not inhibit ovulatory 
cyclicity and fertility, indicating that kisspeptin/GPR54 signaling is 
not essential for reproductive function in adult females.
One potential caveat that we must consider is the following: could 
it be that, as a result of the binary genetic strategy used to ablate 
kisspeptin neurons (that is, DTA expression is indirectly coupled to 
the Kiss1 promotor via Cre-­mediated recombination), a small amount 
of kisspeptin is produced and secreted while the neurons are dying 
and that this is sufficient to activate GnRH neurosecretion? Our data 
suggest that this is probably not the explanation for the reproduc-­
tive phenotype of KissIC/R26-­DTA animals. The initial activation 
of the GnRH pulse generator is most likely mediated by the arcuate 
nucleus population of kisspeptin neurons because of the differen-­
tial onset of kisspeptin expression in the AVPV and ARC19. In the 
majority of ARC kisspeptin neurons, Cre-­mediated recombination 
is already evident at birth, that is, several weeks before vaginal open-­
ing. Virtually no kisspeptin immunoreactivity was detected in the 
ARC of P18 KissIC/R26-­DTA females, suggesting efficient ablation 
of kisspeptin neurons at this time point.
The consequences of puberty, such as the defense of territory or 
mate, pregnancy and care of young, are energetically expensive1. For 
this reason, the timing of puberty is critical. Our data indicate that 
puberty onset is precisely timed in the absence of kisspeptin/GPR54 
signaling. If the GnRH pulse generator can be activated in the absence 
of kisspeptin and GPR54 neurons, what is the physiological function 
of kisspeptin neurons and GPR54 receptors on GnRH neurons? On 
the basis of our comparison of the different mouse strains with the 
complementary knockout mice, we propose that kisspeptin neurons 
integrate various cues and relay this information to GnRH neurons 
via kisspeptin/GPR54 signaling to optimize reproductive success in 
wild-­type mice (Supplementary Fig. 6). Kisspeptin/GPR54 signaling, 
however, is neither essential for activating GnRH neurosecretion at 
puberty onset nor for becoming fertile.
METHODS
Methods and any associated references are available in the online version 
of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
AcknowledgmentS
We are indebted to O. Pongs for continuous support. A. Marquardt and 
D. Drexler provided expert technical assistance. We thank R. Kühn for providing 
the IGD3.2 embryonic stem cells and A. Waisman for providing the R26-­iDTR 
mice, I. Hermans-­Borgmeyer for help with embryonic stem cell work and 
blastocyst injections, A. Derin and U. Wolters for animal caretaking, B. Mann 
for performing the hormone assays and M. Roberson and K. Duncan for helpful 
discussions. This project was supported by Deutsche Forschungsgemeinschaft grant 
BO1743/2 to U.B.
AUtHoR contRIBUtIonS
C.M. and U.B. designed the experiments. C.M. carried out most of the experiments. 
C.M. and U.B. analyzed the data. U.B. conceived the project and wrote the paper.
comPetIng FInAncIAl InteReStS
The authors declare no competing financial interests. 
Published online at http://www.nature.com/natureneuroscience/. 
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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receptor de estrogen um (Era) destas celas podem ter efeitos opostos 
em onset de puberdade em ratos femininos. Embora um knockout de Era em 
resultados de neurônios de kisspeptin em onset de puberdade avançado devido a preco-­ 
cious que desperta do axis19 de hpg, knockout de dianteiras de kisspeptin para 
onset atrasado de puberty11,12. Apesar destes efeitos opostos, dysregu-­ 
lation de neurônios de kisspeptin podem impedir matura-­ de pubertal completo 
tion e assim resulta em infertilidade em ambos os modelos animais. Embora 
neurônios de kisspeptin nos ratos de knockout ainda poderiam libertar outro 
neurotransmitters37, perturbando a jusante potencialmente neurônios dentro, 
a ausência de kisspeptin ou Era sinalizando, estes seriam removidos 
com os neurônios de kisspeptin nos animais de DTA. Em GPR54 bater-­ 
fora ratos, a GnRH neuronal rede poderia ser dysregulated como um 
resulte da ausência do receptor de GPR54 na superfície de cela. Em 
contraste, os neurônios de GnRH restantes em fêmeas de GPIC/R26-­DTA 
(que não expressam GPR54) não é dysregulated e é appar-­ 
ently suficiente para desenvolvimento de reproductive. Consistente com isto 
hipótese, estudos prévios acharam redundância significativa dentro 
o GnRH neuronal population39,40. GPR54 neurônio separação Aguda 
em dianteiras de adultos para fases prolongadas de cornification, consistente com 
estudos prévios que demonstram efeitos diretos de kisspeptin/GPR54 
sinalizando em GnRH/luteinizing hormônio secretion14,41–43. Porém, 
separação aguda de neurônios de GPR54 em adultos não inibe ovulatory 
cyclicity e fertilidade, indicando aquele kisspeptin/GPR54 sinalizando é 
não essencial para reproductive funcione em fêmeas de adulto.
Administrador
Note
Um caveat potencial que nós temos que considerar é o seguinte: podido 
é que, como resultado da estratégia genética binária usada a ablate 
neurônios de kisspeptin (quer dizer, expressão de DTA é juntada indiretamente para 
o promotor de Kiss1 por recombinação de Cre-­mediated), uma quantia pequena 
de kisspeptin é produzido e segregou enquanto os neurônios estão morrendo 
e que isto é suficiente ativar neurosecretion de GnRH? Nossos dados 
sugira que esta provavelmente não é a explicação para o reproduc-­ 
fenótipo de tive de animais de KissIC/R26-­DTA. A ativação inicial 
do GnRH pulso gerador é mediado provável pelo arcuate 
população de núcleo de neurônios de kisspeptin por causa do differen-­ 
onset de tial de expressão de kisspeptin no AVPV e ARC19. No 
maioria de neurônios de kisspeptin de ARCO, recombinação de Cre-­mediated, 
já é evidente a nascimento, quer dizer, várias semanas antes de vaginal aberto-­ 
ing. Virtualmente nenhum immunoreactivity de kisspeptin foi descoberto dentro o 
ARCO de P18 fêmeas de KissIC/R26-­DTA, sugestionando separação eficiente, 
de neurônios de kisspeptin a este ponto de tempo.
Administrador
Note
As conseqüências de puberdade, como a defesa de território ou 
acasale, gravidez e ao cuidado de jovem, é expensive1 de energetically. Para 
esta razão, a cronometragem de puberdade é crítica. Nossos dados indicam isso 
onset de puberdade é cronometrado precisamente na ausência de kisspeptin/GPR54 
sinalizando. Se o GnRH pulsam que gerador pode ser ativado na ausência 
de kisspeptin e neurônios de GPR54, o que é a função fisiológica 
de neurônios de kisspeptin e receptores de GPR54 neurônios de GnRH acesos? Em 
a base de nossa comparação do rato diferente puxa com o 
ratos de knockout complementares, nós propomos aqueles neurônios de kisspeptin 
integre várias sugestões e retransmita esta informação a neurônios de GnRH 
por kisspeptin/GPR54 que sinaliza para aperfeiçoar sucesso de reproductive dentro 
selvagem-­type ratos (Figo Adicional. 6). Kisspeptin/GPR54 sinalizando, 
porém, nem não é essencial para ativar neurosecretion de GnRH a 
onset de puberdade nem por ficar fértil.
710	 VOLUME 14 | NUMBER 6 | JUNE 2011 nature neurOSCIenCe
a r t I C l e S
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