The effect of steroids on GPR54 and GnRH neurons in the postpubertal male mouse [abstract]

Abstract only availableA novel neuropeptide, kisspeptin, binds to the receptor GPR54, to influence the initiation and regulation of puberty in mammals. It appears that the activation of GPR54 stimulates the release of gonadotropin releasing hormone (GnRH) from specialized neurons, resulting in the activation of the reproductive system. To further understand the role of the kisspeptin system in GnRH secretion, levels of GPR54 and GnRH mRNA were compared in hypothalamic tissue before, during, and after puberty in male mice. In addition, the expression of GPR54 and GnRH mRNA was examined after castration with or without steroid replacement in postpubertal males. Transgenic male mice that express green fluorescent protein (GFP) were used at 20, 30, and 60 days postnatal (PND). Some postnatal mice were gonadectomized (GDX), and half received testosterone (T) replacement. RNA was isolated from the septum and the basal hypothalamus (areas known to contain GnRH neurons), reverse transcribed, and subjected to real-time, quantitative PCR. Levels of mRNA were compared between GDX and GDX+T, as well as at each of the three ages. Relative amounts of mRNA from GPR54 and GnRH were compared with a housekeeping gene, RPII, using standard curves. Preliminary data suggests that the amount of GPR54 mRNA in hypothalamic tissue is unchanged at the ages studied (p<0.892), although we need to increase the number of mice studied at each age. However, the levels of GnRH mRNA in hypothalamic tissue do increase, although not significantly, with age and the pubertal transition (p<0.112). In postpubertal mice, the relative amount of GnRH mRNA is greater than GPR54 mRNA. Thus, data indicate that there is a decrease in GPR54 mRNA when compared to GnRH mRNA (p<0.05) during the pubertal transition. Studies that examine the effect of steroids on GPR54 and GnRH mRNA expression are currently in progress.Life Sciences Undergraduate Research Opportunity Progra

Kisspeptin, GPR54, and GnRH neurons: Interactions in the male mouse [abstract]

Abstract only availableFaculty Mentor: Dr. M. Cathleen Kovarik, Veterinary Biomedical SciencesA novel neuropeptide, kisspeptin, binds to the receptor GPR54, to influence the initiation and regulation of puberty in mammals. It appears that the activation of GPR54 stimulates the release of gonadotropin releasing hormone (GnRH) from specialized neurons, resulting in the activation of the reproductive system. To further understand the role of the kisspeptin system in GnRH secretion, levels of GPR54 and GnRH mRNA were compared in hypothalamic tissue before, during, and after puberty in male mice. Transgenic male mice that express green fluorescent protein (GFP) in their GnRH neurons were used at 20 (prepubertal), 30 (pubertal) and 60 (postpubertal) days postnatal. RNA was isolated from brain areas known to contain GnRH neurons. Isolated RNA was reverse transcribed and will be subjected to real-time, quantitative PCR. Relative amounts of mRNA from GPR54 and GnRH will be compared with a housekeeping gene, RPII using standard curves at each of the three ages. Currently, we have used real-time PCR to detect amplicons, of approximately 60 base pairs, for each group down to the attogram (10-18) level. We have also determined that the ABI 7000 machine and SYBR- green as a detector are optimal for our purposes. We are currently performing PCRs for each of the three genes of interest. Amounts of GPR54 and GnRH in older animals will be expressed relative to amounts from the 20 day postnatal animals. Similar techniques will be used to study the expression of GPR54 and GnRH mRNA at the level of the single GnRH neuron, as well as the effect of steroids on the system

Age Affects Spontaneous Activity and Depolarizing Afterpotentials in Isolated Gonadotropin-Releasing Hormone Neurons

Neuronal activity underlying the pulsatile secretion of GnRH remains poorly understood, as does the endogenous generation of such activity. It is clear that changes at the level of the hypothalamus are taking place during reproductive aging, yet virtually nothing is known about GnRH neuronal physiology in aging and postreproductive animals. In these studies, we performed cell-attached and whole-cell recordings in GnRH-enhanced green fluorescent protein neurons dissociated from young (3 months), middle-aged (10 months), and old (15–18 months) female mice. All mice were ovariectomized; half were estradiol replaced. Neurons from all ages fired spontaneously, most in a short-burst pattern that is characteristic of GnRH neuronal firing. Membrane characteristics were not affected by age. However, firing frequency was significantly reduced in neurons from old animals, as was spike patterning. The amplitude of the depolarizing afterpotential, evoked by a 200-msec current pulse, was significantly smaller in aged animals. In addition, inward whole-cell currents were reduced in estradiol-treated animals, although they were not significantly affected by age. Because depolarizing afterpotentials have been shown to contribute to prolonged discharges of activity after a very brief excitatory input, a decreased depolarizing afterpotential could lead to attenuated pulses in older animals. In addition, decreases in frequency and pattern generation could lead to improper information coding. Therefore, changes in the GnRH neuron during aging could lead to dysregulated activity, potentially resulting in the attenuated LH pulses observed in the transition to reproductive senescence