My research addresses the fundamental biological question of how we are transformed from children into adults and gain reproductive competence. Answering this question is important for extending human knowledge and treating pubertal and reproductive disorders that affect the lives of millions of people worldwide.

At the onset of puberty, there is an increase in coordinated, rhythmic gonadotropin-releasing hormone (GnRH) secretion from a scattered network of about 800 mainly hypothalamic GnRH-synthesizing neurons into the portal vasculature connecting the hypothalamus and pituitary. GnRH controls reproduction by binding to GnRH receptors on pituitary gonadotrophs and stimulating the secretion into the general circulation of luteinizing hormone and follicle-stimulating hormone, which are required for gonadal steroid secretion and the production of mature gametes in males and females, and hence sexual maturation and reproductive competence. The pubertal increase in GnRH secretion constitutes both a key regulatory switch in the attainment of fertility and a point at which several reproductive disorders may start. Mice and humans lacking hypothalamic GnRH neurons, or lacking GnRH because they have a deletion in the gene encoding GnRH, or administered GnRH in a continuous rather than a pulsatile manner, remain sexually immature and infertile. A pubertal increase in GnRH secretion that is either too much, too little, too early, or too late can result in disorders such as polycystic ovary syndrome (which affects between 5% and 10% of all women of reproductive age), hypogonadism, precocious puberty, or delayed puberty, respectively. However, the molecular and cellular events underlying the pubertal increase in GnRH secretion (and, for that matter, the events underlying pulsatile GnRH secretion at any age) remain a mystery.

My lab’s approach to elucidate the mechanism of the pubertal increase in GnRH secretion utilizes transgenic mice to monitor and manipulate the electrical activity, the cytoplasmic calcium levels, and the GnRH secretion of GnRH neurons. Most of the available scientific evidence supports the hypothesis that at puberty there is increased release of excitatory neurotransmitters or decreased release of inhibitory neurotransmitter(s) from presynaptic neurons onto GnRH neurons, which is controlled by a developmental clock, bodily growth, metabolic fuels such as glucose and fats, and by other as yet undiscovered factors. Increased excitation or decreased inhibition by neurotransmitters would be expected to depolarize GnRH neurons and open their voltage-gated calcium channels, or release calcium from intracellular stores such as the endoplasmic reticulum, thereby increasing cytoplasmic calcium and GnRH secretion. However, the identity of the presynaptic neurons and neurotransmitter(s), the mode of excitation of GnRH neurons, and the nature of the calcium increase in the pubertal regulation of GnRH secretion are still unknown.

A first step in testing the hypothesis is to characterize the calcium increase that underlies the increased GnRH secretion at puberty, since secretion in neurons and endocrine cells is calcium-dependent. My lab recently developed a novel mouse brain slice preparation that contains most of the 800 GnRH neurons and can be used for measuring and correlating GnRH secretion and intracellular calcium before, during, and after puberty (which occurs between one and two months of age in mice). My lab plans to express fluorescent protein-based calcium biosensors in GnRH neurons in brain slices of wild-type mice using adenoviral vectors, as well as in transgenic mice under the control of the GnRH gene promoter, and then image the calcium signals. We should then be able to obtain a detailed and accurate profile of the calcium increase within the GnRH neuronal network.

A second step in testing the hypothesis is to determine how the calcium increase in GnRH neurons occurs, by examining whether it is due to an influx of calcium through voltage-gated calcium channels in the plasma membrane, or due to a release of calcium from intracellular stores. My lab is currently measuring the activity of calcium channels and of calcium-activated potassium channels in GFP-labeled GnRH neurons in brain slices of GnRH-GFP transgenic mice before, during, and after puberty, in either the absence or the presence of selected neurotransmitters. The activity of these ion channels may also produce the calcium oscillations that likely drive rhythmic GnRH secretion.

A third step in testing the hypothesis is to determine which neurotransmitter receptors or ion channels are responsible for increasing calcium and GnRH secretion in GnRH neurons at puberty. My lab will employ gene-modified mice lacking specific neurotransmitter receptor or ion channel subunits in their GnRH neurons, and then compare their GnRH secretion to that of wild-type mice. This will involve developing methods for GnRH neuron-specific and temporally restricted (e.g., just before puberty) gene deletion.

A fourth and final step in testing the hypothesis will be to determine which presynaptic neurons release the excitatory or inhibitory neurotransmitter onto GnRH neurons. For this step, my lab will identify neurons that are presynaptic to GnRH neurons using virus-infected or transgenic mice in which the presynaptic neurons are labeled with a fluorescent protein, and then measure changes in functional connectivity by stimulating the presynaptic neurons while recording the activity of GnRH neurons before, during, and after puberty in the absence and presence of blockers of selected neurotransmitter receptors.

Understanding the mechanism for the pubertal increase in GnRH secretion should give us the knowledge to intervene using pharmacological tools or gene therapy to adjust the amount or timing of the increase at the level of calcium signaling, neurotransmitter receptor activation, or presynaptic neurotransmitter release. This may result in new treatments for pubertal and other reproductive disorders, as well as in new methods for alleviating infertility, and new options for contraception. It should also enable us to better understand how network activity in other hypothalamic neuroendocrine systems and in other areas of the brain is coordinated, and undergoes normal or abnormal developmental change.

Selected Papers

Spergel DJ, Krsmanovic LZ, Stojilkovic SS, Catt KJ. (1994). Glutamate modulates [Ca2+]i and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons. Neuroendocrinology 59:309-317.

Stojilkovic SS, Krsmanovic LZ, Spergel DJ, Catt KJ. (1994). GnRH neurons: intrinsic pulsatility and receptor-mediated regulation. Trends Endocrinol Metab 5:201-209.

Spergel DJ, Krsmanovic LZ, Stojilkovic SS, Catt KJ. (1995). L-type Ca2+ channels mediate joint modulation by GABA and glutamate of [Ca2+]i and neuropeptide secretion in immortalized GnRH neurons. Neuroendocrinology 61:499-508.

Spergel DJ, Catt KJ, Rojas E. (1996). Immortalized GnRH neurons express large-conductance calcium-activated potassium channels. Neuroendocrinology 63:101-111.

Spergel DJ, Krueth U, Hanley DF, Sprengel R, Seeburg PH. (1999). GABA- and glutamate-activated channels in GFP-tagged GnRH neurons in transgenic mice. J Neurosci 19:2037-2050.

Spergel DJ, Krueth U, Shimshek DR, Sprengel R, Seeburg PH. (2001). Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies. Prog Neurobiol 63:673-686.

Shimshek DR, Kim J, Huebner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R. (2002). Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32:19-26.

Mitchell V, Loyens A, Spergel DJ, Flactif M, Poulain P, Tramu G, Beauvillain J-C. (2003). A confocal microscopic study of gonadotropin-releasing hormone (GnRH) neuron inputs to dopaminergic neurons containing estrogen receptor alpha in the arcuate nucleus of GnRH-green fluorescent protein transgenic mice. Neuroendocrinology 77:198-207.

Cronin AS, Horan TL, Spergel DJ, Brooks AN, Hastings MH, Ebling FJP. (2004). Neurotrophic effects of BDNF on embryonic gonadotropin-releasing hormone (GnRH) neurons. Eur J Neurosci 20:338-344.

Shimshek DR, Bus T, Grinevich V, Single FN, Mack V, Sprengel R, Spergel DJ*, Seeburg PH*. (2006). Impaired reproductive behavior by lack of GluR-B containing AMPA receptors but not of NMDA receptors in hypothalamic and septal neurons. Mol Endocrinol 20:219-231. *Drs. Spergel and Seeburg are the senior authors.

Spergel DJ. (2007). Calcium and small-conductance calcium-activated potassium channels in GnRH neurons before, during and after puberty. Endocrinology. In press (first published ahead of print on 02/08/2007 as doi:10.1210/en.2006-1693; http://endo.endojournals.org/cgi/content/abstract/en.2006-1693v1?papetoc).

Faculty and Research

Programs

Cancer Biology

Immunology

Microbiology

Molecular Metabolism
and Nutrition

Molecular Pathogenesis and
Molecular Medicine