Gonadotropin-releasing hormone and NMDA receptor gene expression and colocalization change during puberty in female rats

Abstract

During development, an increase in gonadotropin-releasing hormone (GnRH) release occurs that is critical for the initiation of puberty. This increase is attributable, at least in part, to activation of the GnRH neurosecretory system by inputs from neurotransmitters, such as glutamate, acting via NMDA receptors. We examined changes in GnRH and NMDA-R1 gene expression by RNase protection assay of preoptic area-anterior hypothalamic (POA-AH) dissections of female rats undergoing normal puberty or in which precocious puberty was induced by treatment with the glutamate agonist NMA. GnRH mRNA levels increased significantly throughout normal development; this was accelerated by treatment with NMA. NMDA-R1 mRNA levels increased only between P10 and P20. The acceleration of the elevation in GnRH mRNA levels by NMDA suggests that a stimulation of GnRH gene expression may be a rate-limiting factor for the onset of puberty. This is attributable to a post-transcriptional mechanism because GnRH primary transcript levels, an index of proGnRH gene transcription, were not observed to change during puberty. Alterations in the colocalization of GnRH neurons with the NMDA-R1 subunit during puberty also were assessed immunocytochemically. The percentage of GnRH neurons that double-labeled with NMDA-R1 was 2% in prepubertal rats and 3% in pubertal rats; this increased to 19% in postpubertal rats. Taken together, these studies suggest that an increase in glutamatergic input to GnRH neurons plays a role in the increase in GnRH release and gene expression that occurs at the initiation of puberty.

Fig. 1.

GnRH and NMDA mRNA in representative cytoplasmic samples from rats during normal and precocious puberty. Rats were treated with NMA or saline (control) twice daily beginning on P25. Levels of GnRH, NMDA-R1, and cyclophilin were measured simultaneously in individual POA-AH dissections by RNase protection assay; a representative single cytoplasmic POA-AH fraction for both treatment groups at each postnatal age is shown. On the left is a standard curve with increasing amounts of reference RNA for the three RNA species. The specific protected bands for GnRH, NMDA-R1, and cyclophilin are indicated by arrows on the far left. GnRH mRNA levels increased during puberty; this was accelerated by treatment with NMA. No changes in NMDA-R1 or cyclophilin mRNA were observed between P25 and P41.

Fig. 2.

GnRH cytoplasmic mRNA levels increase during puberty; this is accelerated by treatment with NMA. GnRH mRNA levels (normalized to cyclophilin) were analyzed in rats from P10 to P41 treated with NMA or saline (control). The days of vaginal opening (VO) and first diestrus (D) for each group are indicated below the x-axis. GnRH mRNA levels increased significantly during puberty in control rats (p < 0.05). This was accelerated significantly by treatment with NMA (p < 0.0001). Levels of GnRH mRNA were increased significantly, as compared with those of the previous day analyzed at P20, the day of VO (P34–P46), and P41. Levels declined significantly between the day of VO (P34–P36) and first diestrus (P35–P37). GnRH mRNA levels were significantly higher in the NMA-treated group as compared with their age-matched control at VO (P29–P31) and P36. n = 6–8/group. a, p < 0.05 versus P10;b, p < 0.05 versus control P30–P32; c, p < 0.05 versus control P34–P36; d, p < 0.05 versus control P35–P37; *p < 0.05 versus corresponding control.

Fig. 3.

GnRH primary transcript in representative nuclear samples from rats during normal and precocious puberty. Levels of GnRH primary transcript were measured in individual POA-AH dissections by RNase protection assay; a representative single nuclear POA-AH fraction for both treatment groups at each postnatal age is shown. On theleft is a standard curve with increasing amounts of reference RNA for the B3C probe. The specific protected band is indicated by the arrow on the far left. GnRH primary transcript levels did not increase during puberty, nor were they affected by treatment with NMA.

Fig. 4.

GnRH nuclear primary transcript levels do not change during puberty or by treatment with NMDA. GnRH primary transcript levels were analyzed in rats from P10 to P41 treated with NMA or saline (control). See Figure 2 for description of axes and treatments. GnRH primary transcript levels did not change significantly during development and were unaffected by treatment with NMA.n = 6–8/group.

Fig. 5.

NMDA-R1 mRNA levels increase from P10 to P20 but do not change during puberty. NMA-R1 mRNA levels were analyzed in rats from P10 to P41 treated with NMA or saline (control). See Figure 2 for description of axes and treatment. NMDA-R1 mRNA levels increased significantly between P10 and P20; no further changes were observed during development. Treatment with NMA had no effect on NMDA-R1 mRNA levels in the POA-AH. *p < 0.05 versus all other days; n = 6–8/group.

Fig. 6.

GnRH neuron double-labeling with the NMDA-R1 subunit in a postpubertal rat. Double-immunolabeling of GnRH neurons with the NMDA-R1 subunit in a postpubertal rat. A, B, Images from the same section stained for GnRH and NMDA-R1 immunoreactivity, respectively. A neuron double-labeled for the two antigens is marked by the asterisk; another NMDA-R1-positive neuron that does not immunostain for GnRH is seen inB, also. C, D, Images from the same section stained for GnRH and NMDA-R1, respectively. A neuron with detectable GnRH but not NMDA-R1 immunoreactivity is indicated by thetriangle. The two GnRH-positive neurons depicted inA and C are representative of others observed in this study in that they demonstrated intense perinuclear staining. Bar in D, 10 μm.

Fig. 7.

Rostro–caudal representation of GnRH neurons double-labeling with NMDA-R1. The rostro–caudal distribution of double-labeled neurons was determined in a representative prepubertal (A21; P21; a), pubertal (A25; day of vaginal opening;b), and postpubertal (A31; P41; c) rat. A series of sections from the level of the diagonal band of Broca to the rostral hypothalamic level, illustrating GnRH neurons containing (•) or not containing (○) detectable NMDA-R1 subunit immunoreactivity, is presented. The sections represented in a–c show detectable NMDA-R1 subunit immunoreactivity in GnRH neurons in the region of the OVLT and caudal, with the highest expression in the postpubertal animal. ac, Anterior commissure;dbb, diagonal band of Broca; cc, corpus callosum; lo, lateral olfactory tubercle;poa, preoptic area; ms, medial septum;oc, optic chiasm; ovlt, organum vasculosum of the lamina terminalis; son, supraoptic nucleus.