Hypothalamic molecular changes underlying natural reproductive senescence in the female rat

Abstract

The role of the hypothalamus in female reproductive senescence is unclear. Here we identified novel molecular neuroendocrine changes during the natural progression from regular reproductive cycles to acyclicity in middle-aged female rats, comparable with the perimenopausal progression in women. Expression of 48 neuroendocrine genes was quantified within three hypothalamic regions: the anteroventral periventricular nucleus, the site of steroid positive feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback and pulsatile GnRH release; and the median eminence (ME), the site of GnRH secretion. Surprisingly, the majority of changes occurred in the ARC and ME, with few effects in anteroventral periventricular nucleus. The overall pattern was increased mRNA levels with chronological age and decreases with reproductive cycle status in middle-aged rats. Affected genes included transcription factors (Stat5b, Arnt, Ahr), sex steroid hormone receptors (Esr1, Esr2, Pgr, Ar), steroidogenic enzymes (Sts, Hsd17b8), growth factors (Igf1, Tgfa), and neuropeptides (Kiss1, Tac2, Gnrh1). Bionetwork analysis revealed region-specific correlations between genes and hormones. Immunohistochemical analyses of kisspeptin and estrogen receptor-α in the ARC demonstrated age-related decreases in kisspeptin cell numbers as well as kisspeptin-estrogen receptor-α dual-labeled cells. Taken together, these results identify unexpectedly strong roles for the ME and ARC during reproductive decline and highlight fundamental differences between middle-aged rats with regular cycles and all other groups. Our data provide evidence of decreased excitatory stimulation and altered hormone feedback with aging and suggest novel neuroendocrine pathways that warrant future study. Furthermore, these changes may impact other neuroendocrine systems that undergo functional declines with age.

Figure 1.

Serum luteinizing hormone (A), follicle stimulating hormone (B), estradiol (C), and progesterone (D) levels during reproductive aging. Circulating serum hormone levels were measured in ovarian-intact Sprague Dawley females of the four experimental groups. Boxes show the 25th to 75th percentiles with the median value depicted as a black bar. Whiskers represent 1.5 times the interquartile range with extreme values shown as open circles. MaIrreg, middle-aged irregularly cycling; MaPE, middle-aged persistent estrus; MaReg, middle-aged regularly cycling; YgReg, young regularly cycling. *, P < .05 between YgReg and MaReg. Different letters denote significant (P < .05) differences between middle-aged groups.

Figure 2.

ARC gene expression during reproductive aging. Expression of genes with significant effects of chronological age (A) or reproductive cycle status (B) is shown. MaIrreg, middle-aged irregularly cycling; MaPE, middle-aged persistent estrus; MaReg, middle-aged regularly cycling; YgReg, young regularly cycling. +, P < .1 (trend) and *, P < .05 vs MaReg.

Figure 3.

ME gene expression during reproductive aging. Expression of genes with significant effects of chronological age (A) or reproductive cycle status (B) is shown. MaIrreg, middle-aged irregularly cycling; MaPE, middle-aged persistent estrus; MaReg, middle-aged regularly cycling; YgReg, young regularly cycling. *, P < .05 and **, P < .01 vs MaReg.

Figure 4.

Bionetwork analysis in the AVPV, ARC, and ME. Correlation networks of genes, serum hormones, and body weight of all females in the AVPV (A), the ARC (B), and the ME (C) are shown. Positive Pearson correlations are shown in red and negative in blue. Genes and hormones that displayed a significant main effect of age or cycle status are indicated by bold, italicized font (P < .05).

Figure 5.

Immunofluorescence histochemistry in the ARC during reproductive aging. The percentage of kisspeptin, ERα, and colabeled cells were quantified in the caudal ARC of middle-aged female rats during the transition to acyclicity. A, Representative micrograph from one confocal slice showing cells colabeled for kisspeptin (red) and ERα (green), marked with an asterisk, with a DAPI counterstain (blue). A higher resolution image is shown in the inset of panel A. Single-labeled cells can also be seen for both proteins. The total percentage of colabeled cells (B) and the percentage of kisspeptin cells that colabeled ERα (C) decreased at middle age in regularly cycling females. The percentage of kisspeptin cells (D) and the ratio of kisspeptin to ERα cells (E) decreased with reproductive cycle status. Kiss, kisspeptin. *, P < .05 and **, P < .01 vs MaReg. Scale bar, 30 μm (20 μm for inset).

Figure 6.

Proposed model of neuroendocrine mechanisms of reproductive aging. As regularly cycling females age from young (A) to middle aged (B), expression of many genes are increased in the ARC and ME. This up-regulation (indicated by up arrow in panel B) is proposed to compensate for a diminishing drive to GnRH neurons with age. Additionally, the percentage of kisspeptin cells in the ARC that express ERα is decreased. During aging, there are also structural changes to tanycytes in the ME, shown in young rats with a linear organization, and becoming progressively disorganized in middle-age, possibly reflecting changes in access of hypothalamic nerve terminals to the portal capillaries (58, 59). As middle-aged females transition from regular cycles (B) through acyclicity (C), they show decreased kisspeptin cell number together with decreases in neuroendocrine gene expression (indicated by down arrow in panel C). A, anterior; D, dorsal; o.c., optic chiasm; P, posterior; PIT, pituitary; V, ventral.