A kiss from the brain to awaken the gonads

The discovery of gonadotropin-releasing hormone (GnRH) three decades ago illuminated the brain's primary role in regulating fertility, but did not resolve the mystery of how puberty and sexual maturity are postponed for at least a decade after birth in our long-lived, late-maturing species.

Professor Allan Herbison from the University of Otago told the ComBio conference in Brisbane last year that the master switch for puberty turns out to be a neuropeptide, kisspeptin, secreted by the hypothalamus. It was originally discovered in 1996, in a role unrelated to fertility.

As Herbison puts it, "fertility is not just something that happens below the belt". At 12 weeks post-conception, a human embryo's indeterminate gonads begin to differentiate into testes or ovaries, and secrete androgens and estrogens that drive male- or female-specific development of the reproductive system and central nervous system.

Not long after birth, the gonads become quiescent until childhood's end. At least a decade later, the gonads are roused to frenetic new activity, triggering puberty.

The process is triggered by specialised GnRH-secreting neurons in the hypothalamus - the seat of primal drives like hunger, thirst, sex and salt appetite - which activate and begin secreting pulses of GnRH. In response to the GnRH pulses, the pituitary gland releases two primary hormones that regulate fertility: luteinising hormone (LH) and follicle-stimulating hormone (FSH).

Half a decade ago, fertility researchers began searching for the mysterious neural signal that caused the hypothalamus to release GnRH at puberty. Although not recognised initially, it now looks as though that signal may be kisspeptin.

Box of chocolates

In 1996, Professor Danny Welch's team at the Pennsylvania College of Medicine in Hershey - home of the eponymous confectionery company -dubbed the new peptide 'kisspeptin', after the company's familiar foil-clad chocolate drop, the Hershey Kiss.

They were interested in kisspeptin and its receptor as possible targets for novel drugs for melanoma cancers. Malignant melanomas typically spawn multiple, secondary tumours in major organs, including the brain. But melanomas expressing high levels of the protein KISS-1 do not metastasise.

Amazingly, kisspeptin remained in the cancer biology field until two dramatic discoveries were reported in 2003.

In papers published only a month apart, research teams in Paris, Paradigm Therapeutics in Cambridge and Harvard Medical School, reported that humans with mutations in a G-protein coupled receptor, GCPR54, caused failure of puberty, and sterility.

Some 60 per cent of modern therapeutic drugs target GCPRs, a diverse family of cell surface protein 'switches'.

At Harvard, Professor Bill Crowley's research team was investigating families with hereditary fertility problems. They located a Saudi family in which it was customary for first cousins to marry.

The US researchers tested a number of family members who had failed to go through puberty, and found they were homozygous for a serine-leucine mutation in an external loop of the GPCR54 receptor.

Dr Nicolas De Roux in Paris reported on an Algerian family with a similar inherited disorder, in which affected individuals were homozygous for a mutation that disrupted another external loop of the receptor. They have identified several other families with similar hereditary disorders, who are also homozygous for mutations in the external, C-terminal region of the receptor protein.

In a paper the New England Journal of Medicine, Crowley's team described a GCPR54 knockout mouse, which develops tiny ovo-testes, and fails to enter puberty. It was a perfect analogue for the human disorder induced by GCPR54 C-terminal mutations.

Herbison's curiosity was piqued. Here was an orphan receptor, with a seemingly crucial role in puberty and fertility, but where was this kisspeptin-GPCR54 signaling going on?

Kiss at dawn

Certain other organs such as the placenta, pancreas and hypothalamus also express high levels of KISS-1 and GPCR54, but Herbison says the hypothalamus is the key site for expression.

His research group has shown that kisspeptin is a potent and long-lived activator of the GnRH neurons and gonadotrophin secretion.

"At the time, nobody was thinking about the entire signalling system," he says. "Five years later, everything happened in a rush. Three papers came out saying the same thing: the KISS-1 gene makes a peptide that binds to the GCPR54 receptor.

"If you dump more kisspeptin into the ventricular system of the rat brain, it's an incredibly potent activator of gonadotrophin secretion and has unusually prolonged effect. What's more, this effect is highly conserved across mammal species."

In females, a feedback loop between the hypothalamus and pituitary glands gives the system "massive plasticity", modulating both its amplitude and frequency to regulate the menstrual cycle and induce ovulation.

Herbison's team has been tracing the brain's kisspeptin-GnRH circuitry, to determine how it is activated at puberty.

They have identified three distinct populations of kisspeptin-expressing neurons in the brain - all located in the hypothalamus.

Using a synapse-hopping pseudorabies virus expressing green fluorescent protein, they were able to trace the neural connections between GnRH-secreting neurons in the pituitary, and kisspeptin-secreting neurons in the hypothalamus.

It turns out that it is the periventricular kisspeptin neuron population, lying next to fluid-filled ventricles of the hypothalamus, that modulates the activity of the GnRH neurons.

Kisspeptin's effect on the GnRH system is remarkable, Herbison says. Within two minutes of exposure to 10 picomolar kisspeptin, GnRH-secreting neurons begin firing, and continue to do so for over an hour. This is quite unlike the effects of any other regulator of GnRH neurons discovered so far.

The Pennsylvanian research team's quirky name for the molecule has proved surprisingly prescient. At puberty, it's a kiss from the brain that reawakens the slumbering gonads to vibrant life.

Jenny Clarkson, a PhD student at Herbison's laboratory, has just discovered that periventricular kisspeptin neurons only become apparent just before puberty in the mouse, around 30 days after birth (P30). At P10, there is no kisspeptin in the periventricular area and only small amounts become detectable around P25-26.

But within four or five days, at puberty, kisspeptin-secreting nerve fibres are "massively present" around GnRH-secreting neurons in the pituitary gland - the system is indistinguishable from that of the adult brain.

Interestingly, the GnRH-secreting neurons in the pituitary gland express GCPR54 receptors for kisspeptin right from birth. But they remain dormant: Herbison describes them as "lying in wait" for kisspeptin's awakening kiss at puberty.

"It looks like kisspeptin peptide levels peak right on time for puberty to occur," he says. "The developmental profile of kisspeptin inputs to GnRH neurons is compatible with a role in GnRH activation at puberty."

Clinical applications

So what is the clinical potential of kisspeptin? Herbison says environmental factors are enormously important in inducing puberty. Nutrition is one such factor and while improved food quality over the last decades may underlie the earlier onset of puberty in girls today, the flip-side is that the current obesity epidemic may have a countervailing effect.

Today, menarche typically occurs between 11 and 15. A GCPR54 antagonist - an inactive mimic of kisspeptin10 - could delay or arrest the onset of puberty and fertility, and the risk of unwanted pregnancy.

Conversely, an GPCR54 agonist could help children, typically boys, who are abnormally late to enter puberty.

"It's quite important that GCPR54 is there, waiting for kisspeptin, before puberty gets going," Herbison says. "This means that we can activate the receptor with kisspeptin.

"However, we've found something quite unexpected. If we take GnRH neurons before puberty, only about 25 per cent respond by depolarising, even though all of them are expressing GCPR54 - at least, the messenger RNA for GCPR54 is present.

"It suggests that, even though the GnRH neurons are making the receptor, somehow it's now coupling to kisspeptin-10 to activate the puberty cycle. So, we think this can be explained by a two-step mechanism.

"First, the kisspeptin-10 secreting fibres innervate the GnRH neurons at the right time, but activation requires a maturational event in which GCPR54 gets slowly coupled to the electrical output of the GnRH neuron."

The kisspeptin/GCPR54 system is also a potential target for fertility treatments, or for a unisex contraceptive, he says.