Maternal dexamethasone exposure during pregnancy in rats disrupts gonadotropin-releasing hormone neuronal development in the offspring

Abstract

The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13-20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47-52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.

Fig. 1

Gonadotropin-releasing hormone immunofluorescence (IF-GnRH) and enhanced green fluorescent protein (EGFP)-GnRH (TG-GnRH) neurons on coronal sections of the preoptic area (POA) in P0, P5 and P52 males. More IF-GnRH neurons (red) were observed compared to their respective TG-GnRH cells (green) in the POA of P0 (a) and P5 (b) males. Number of TG-GnRH neurons was comparable to the IF-GnRH neurons in P52 (c) male, as most TG-GnRH cells were colocalized with GnRH immunofluorescence (yellow) in POA. High-magnification images of TG-GnRH neurons (green) double-labeled with IF-GnRH (red) and merged images (yellow) in P0 (d–f) and P52 (g–i) males. Intensity of the IF-GnRH on TG-GnRH neurons in P0 male appeared less compared to the P52 male. Bars 100 μm (a–c) and 10 μm (d–i)

Fig. 2

Double labeling of the green fluorescent protein immunofluroscence (IF-GFP, red) in the TG-GnRH (green) neurons in the POA of P0 (a–c) and P52 (d–f) male with the merged images (yellow). The IF-GFP staining on TG-GnRH neurons appeared weak and was localized to the nuclear region in P0 male. The IF-GFP staining was distributed across the cytoplasm and along the dendrite of the TG-GnRH neurons in P52 male. Bars 10 μm (a–f)

Fig. 3

Distribution of the GnRH neurons in P0 (n = 5/sex), P5 (n = 6/sex) and young adult (P47 female, n = 3; P52 male, n = 3) transgenic rats. a Total TG-GnRH neuronal number was decreased in P0 and P5 stages compared to P47-P52 stage. b Number of IF-GnRH neurons did not differ across age and gender. c GnRH promoter activity expressed as Tg-GnRH/IF-GnRH neuron ratios was increased in the P42-P52 stage compared to the early P0 and P5 stages. Rostral to caudal distribution of TG-GnRH and IF-GnRH neurons in P0 (d–e) and P5 (f–g) male and female, plotted as the number of GnRH neurons in series of 180 μm thickness aligned by organum vasculosom of the lamina terminalis (OVLT). Increased TG-GnRH neuronal number was observed in the rostral region (−540 μm from the OVLT) and IF-GnRH neurons (−1800 μm from the OVLT) in P0 females. h–i Rostral to caudal distribution of TG-GnRH and IF-GnRH neurons in P47-52 stages, plotted as GnRH neuronal number in series of 250 μm thickness aligned by the OVLT. Gender difference was not observed in the distribution of TG-GnRH and IF-GnRH neurons in P47-P52 stages. Data are represented by the mean ± SEM for each group. *, P < 0.05; ***, P < 0.001

Fig. 4

IF-GnRH and TG-GnRH neurons on coronal sections of maternally vehicle- (VEH-P0) and dexamethasone-treated (DEX-P0) P0 male offspring. Number of TG-GnRH neurons (green) and IF-GnRH neurons (red) observed in medial septum (MS) (a–b) and POA (c–d) and OVLT (e–f) was decreased in DEX-P0 compared to VEH-P0 males. g–h IF-GnRH fibers projecting to median eminence (ME) were not different in VEH-P0 and DEX-P0 males. Bars 100 μm (a–h)

Fig. 5

Maternal DEX exposure on number and distribution of IF-GnRH and TG-GnRH neurons in P0 male offspring. a Total number of TG-GnRH and IF-GnRH neurons in VEH-P0 (n = 10) and DEX-P0 (n = 7) males. Maternal DEX exposure decreased the total number of IF-GnRH cells in P0 males but not the TG-GnRH cells. b GnRH promoter activity expressed as Tg-GnRH/IF-GnRH neuron ratio did not differ between the VEH-P0 and DEX-P0 males. Rostral to caudal distribution of TG-GnRH (c) and IF-GnRH (d) neurons in VEH-P0 and DEX-P0 males. The TG-GnRH neurons were decreased in the OVLT and rostral to the OVLT (−540 and −720 μm from OVLT) in DEX-P0 males. Number of IF-GnRH neurons were also decreased in the OVLT, rostral (−360 and −540 μm from the OVLT) and caudal (360 and 540 μm) to the OVLT in DEX-P0 males. Data are represented by the mean ± SEM for each group. *, P < 0.05; **, P < 0.01 compared to VEH-P0 group

Fig. 6

Morphological observations of GnRH neurons and fiber projections in the OVLT and ME in VEH-P0 and DEX-P0 males. Low-magnification photomontage of sagittal sections exhibited IF-GnRH neurons (red) along the caudal olfactory bulb (OB), olfactory tubercule (OT), diagonal band of Broca (DBB), MS, and OVLT/POA in VEH-P0 (a) and DEX-P0 (b) males. Few IF-GnRH neurons were observed along the forebrain distribution in DEX-P0 males. c–j GnRH fiber projections from coronal sections of OVLT and ME in VEH-P0 and DEX-P0 males. High-magnification of white insets illustrating the fiber projections in the OVLT (d, f) and ME (h, j) were shown for VEH-P0 and DEX-P0 males respectively. Thicker varicosities of IF-GnRH fibers were observed in lateral OVLT of DEX-P0 (d) compared to VEH-P0 (f) males. Bars 500 μm (a-b), 50 μm (c, e, g, i) and 20 μm (d, f, h, j)

Fig. 7

Morphological subtypes of TG-GnRH and IF-GnRH neurons in the OVLT/POA of VEH-P0 and DEX-P0 males. The TG-GnRH neurons were scored as soma, unipolar (a, c), bipolar (e, g) or branched dendritic structure (i, k), with the IF-GnRH (red) on the different morphological subtypes of TG-GnRH (green) neurons, respectively. White arrowheads indicate branching of TG-GnRH neuron dendritic structure. Labeling of IF-GnRH appeared weak in the branched GnRH dendrites, suggesting that the EGFP expression on TG-GnRH neurons is sufficient to visualize the different morphological subtypes. m Percentage of TG-GnRH neurons exhibiting branch dendritic structures was decreased in DEX-P0 males. Data are represented by the mean ± SEM for each group. **, P < 0.01 compared to VEH-P0 group. Bars 10 μm (a–l)