Reports suggest that at least 25% of ovarian disorders are due to dysfunction of the brain mechanism in the hypothalamus that controls the gonadotropin-releasing hormone (GnRH), which is a molecule that governs the reproductive function in men and women by regulating the release of a range of reproductive hormones into the body, particularly gonadotropins, from the pituitary gland, which is located underneath the brain. In a healthy individual, GnRH and gonadotropins are released in pulses, while typically in individuals whose infertility is caused by hypothalmic dysfunction, the GnRH and gonadotropin pulses are supressed. Although the nature of the GnRH pulse generator has been suspected from circumstantial evidence for many years, direct evidence for it had not been found until now. The findings may provide a possible new approach for assisted reproductive technology that would be important for measures against the declining birthrate seen particularly in developed countries.
Using genetically modified female rats, researchers from Nagoya University, Japan, and the National Institute of Physiological Sciences in Okazaki City, Japan, demonstrated that a lack of properly functioning KNDy neurons specifically in the arcuate nucleus of the hypothalamus causes infertility because without a sufficient number of these neurons, not enough kisspeptin is produced to signal to the GnRH neurons to release GnRH pulses, so the pituitary gland doesn’t receive a signal to release gonadotropins. The researchers showed that kisspeptin secreted by the KNDy neurons in the arcuate nucleus is the essential factor in the healthy release of GnRH (that is, in pulses) and consequent release of gonadotropin pulses. In other words, female – and, moreover, male – mammals with a deficiency of KNDy neurons in the arcuate nucleus can never be fertile.
The researchers also demonstrated that ovarian function can be switched on by boosting the number of KNDy neurons in the arcuate nucleus: just 20% of the normal number of these is sufficient to start the GnRH and gonadotropin pulses and, moreover, maintain folliculogenesis in the ovaries, thereby enabling fertility. The number of properly functioning KNDy neurons in the arcuate nucleus was boosted from zero – the rats had been bred to have no KNDy neurons producing kisspeptin – to an adequate number by replacing a gene in the neurokinin-B neurons (the “N” in KNDy) with the Kiss1 gene, which produces kisspeptin. In this sense, the KNDy neurons were “rescued”. The research team’s gene therapy was so precise that only the KNDy neurons in the arcuate nucleus were modified, leaving those in the rest of the hypothalamus untouched.
This research work suggests at least two therapies: first, a sustained-release drug to enhance GnRH pulses in patients, based on the additional finding that neurokinin-B increases the frequency of GnRH pulses while dynorphin-A (the “Dy” in KNDy) reduces their frequency; second, gene therapy to boost the number of properly functioning KNDy neurons in the arcuate nucleus. The researchers believe that the “rescue” of KNDy neurons by gene therapy using the adeno-associated viral vector (AAV) technique to restore fertility is quite useful and applicable to animals in the near future. Indications from other published studies are that this technique is also safe for humans. The research team hopes to confirm their findings in further animal research and also hopes to eventually bring this technique to the clinic as a treatment for infertility in humans caused by abnormal GnRH secretion.
Gonadotropins are any hormones that are released from the anterior pituitary when a gonadotropin-releasing hormone (GnRH) signal arrives from the hypothalamus. They stimulate the gonads, or sex glands, to carry out their reproductive functions. The GnRH is therefore of central importance for mammalian reproduction. In fertile individuals, GnRH is secreted in a pulsatile manner, while continuous secretion of GnRH actually inhibits gonadotropin release and therefore inhibits the function of the sex glands. Reports suggest that at least 25% of ovarian disorders are due to a dysfunction of the brain mechanism in the hypothalamus controlling the release of gonadotropic hormones from the anterior pituitary gland.
However, although suspected from circumstantial evidence, the exact nature of the “GnRH pulse generator” has been a mystery ever since GnRH was discovered in 1971. In a paper published recently in the Proceedings of the National Academy of Sciences (PNAS), a research collaboration between the Graduate School of Bioagricultural Science, Nagoya University, Japan, and the National Institute of Physiological Sciences in Okazaki City, Japan, has provided the first direct evidence that KNDy neurons in the arcuate nucleus of the hypothalamus generate the GnRH pulses, and that lack of these inhibits fertility. In other words, female – and, moreover, male – mammals with a deficiency of KNDy neurons in the arcuate nucleus can never be fertile. Using a genetically-modified female rat, the study showed that the presence of just 20% of the normal number of properly functioning KNDy neurons is sufficient to start the GnRH and gonadotropin pulses and maintain folliculogenesis in the ovaries, thereby enabling fertility.
The acronym KNDy comes from the peptides (signaling molecules) that this group of neurons produces: kisspeptin, neurokinin-B and dynorphin-A. It has long been known that kisspeptin signaling is essential to the progress of puberty and normal development of sex organs, in both males and females. Adding to this, the Japanese study showed that neurokinin-B increases the frequency of GnRH pulses, while dynorphin-A reduces their frequency.
The researchers bred a female rat in which the kisspeptin gene (Kiss1) had been genetically deleted, so its kisspeptin levels were undetectable. It was infertile because hypothalamic GnRH pulses were missing and consequently pituitary gonadotropin pulses were nonexistent. The team boosted the number of properly functioning KNDy neurons precisely in the arcuate nucleus by inserting the Kiss1 gene into the neurokinin-B neurons using a viral vector technique. The researchers found that just 20% of the normal number of properly functioning KNDy neurons was enough not only to start gonadotropin pulses from the anterior pituitary, but also to maintain follicular development in the ovary in the female rats. KNDy neurons in the arcuate nucleus of the hypothalamus therefore serve as a master regulator of GnRH pulses and are indispensable for fertility in female -- and male -- mammals.
The research finding provides potential therapeutic approaches for patients with hypothalamic reproductive disorders. In particular, since the neurokinin-B activates the KNDy neurons, resulting in an increase in GnRH pulse frequency, while the dynorphin-A suppresses the KNDy neuron activity, long-term (chronic) administration of neurokinin-B or its analogues, or dynorphin-A antagonists, using a sustained-release drug could be useful in enhancing GnRH pulses in patients. This would replace pulsatile infusion of GnRH by an attached pump. This methodology could also be applied to domestic animals, because mammalian species such as cattle, sheep, goats, pigs, primates and rodents all share the same brain mechanism for regulating reproductive function; namely, the one which we now know is driven by KNDy neurons in the arcuate nucleus of the hypothalamus.
Project leader Professor Hiroko Tsukamura and her research team, together with Dr. Kei-ichiro Maeda of the University of Tokyo, who passed away during this collaboration, have been trying to solve this problem “since long before the discovery of KNDy neurons”. “At last we found direct evidence that KNDy neurons are the GnRH pulse generator,” says Professor Tsukamura. “We dedicate this study to Dr. Maeda with gratitude for his leadership, supervision, and original ideas.”
Professor Tsukamura and joint first author Professor Uenoyama believe there is a lot more work to be done to find the molecular mechanism controlling KNDy neuronal activity as the GnRH pulse generator. Nevertheless, they say, the present findings help illuminate the central mechanism underlying mammalian reproduction, and can be applied to the treatment of ovarian disorders in livestock as well as infertility in humans.
The researchers believe that the “rescue” of KNDy neurons by gene therapy using the adeno-associated viral vector (AAV) technique to restore fertility is quite useful and applicable to animals in the near future. Indications from other published studies are that this technique is also safe for humans, although more research needs to be done on this.
Citation: Nagae M, Uenoyama Y, Okamoto S, Tsuchida H, Ikegami K, Goto T, Majarune S, Nakamura S, Sanbo M, Hirabayashi M, Kobayashi K, Inoue N, Tsukamura H. (2021) “Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator,” Proc Natl Acad Sci USA.
The article may be accessed at DOI: https://doi.org/10.1073/pnas.2009156118