In the male rat, age-associated reproductive decline is thought to be due in part to diminished GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion is due to decreased GnRH gene expression by comparing GnRH mRNA and peptide content in the anterior forebrain of intact young and old male rats. Since sex steroids modulate GnRH secretion, we also determined hypothalamic-pituitary responsiveness to removal of testicular feedback by comparing GnRH mRNA and gonadotropin levels in intact and orchidectomized young and old rats. In an initial study, 10 20-mu-m coronal sections from the medial preoptic area (MPOA) were anatomically matched and compared in intact young (3-month-old) and old (24-month-old) male F344 rats (n = 5/group). In another group of young and old male rats (n = 8-12/group), animals were randomly assigned to be either orchidectomized or sham operated. Rats were killed 21 days after surgery, and comparisons were made in 12 anatomically matched sections of MPOA and 4 matched sections of diagonal band of Broca. In both studies, GnRH mRNA was quantitated by in situ hybridization, using a S-35-labeled oligodeoxynucleotide probe complementary to rat prepro-GnRH mRNA and a computerized image analysis system. In a third study, GnRH content was measured by RIA in microdissected regions of the arcuate nucleus and median eminence in intact 3- and 24-month-old male rats (n = 10 and 8, respectively). Serum LH, FSH, and testosterone (T) levels were measured by RIA in trunk blood of all animals. The number of neurons expressing the GnRH gene in the MPOA was significantly lower in sham-operated old rats (mean +/- SEM, 10.5 +/- 0.5 cells/section) than in young rats (13.7 +/- 0.7 cells/section; P < 0.01), while cellular GnRH mRNA content was unchanged with age (103 +/- 1 vs. 103 +/- 2 grains/cell). Similar results were obtained in intact old and young rats. GnRH peptide content was significantly decreased in the arcuate nucleus of intact old (0.5 +/- 0.08 ng/mg protein) compared to young animals (2.3 +/- 0.7 ng/mg protein; P < 0.05), with a trend toward a decrease in the median eminence of old (53 +/- 2 ng/mg protein) vs. young rats (69 +/- 7 ng/mg protein; P = 0.06). LH, FSH, and T levels were lower in sham-operated (intact) old compared to young rats (LH, 0.9 +/- 0.2 vs. 1.7 +/- 0.2-mu-g/liter; FSH, 2.9 +/- 0.4 vs. 7.9 +/- 0.5-mu-g/liter; T, 1.4 +/- 0.1 vs. 4.5 +/- 0.7 nmol/liter; all P < 0.05). In contrast to intact rats, orchidectomized old and young rats demonstrated similar numbers of cells expressing GnRH (11.1 +/- 0.5 vs. 11.9 +/- 0.6 cells/section) and GnRH mRNA content (105 +/- 2 vs. 104 +/- 2 grains/cell) in the MPOA. Gonadotropin and T levels were also similar in old and young orchidectomized rats (LH, 19.5 +/- 2.9 vs. 23.1 +/- 1.6-mu-g/liter; FSH, 62.0 +/- 5.1 vs. 52.6 +/- 3.6-mu-g/liter; T, 0.7 +/- 0.07 vs. 0.7 +/- 0.03 nmol/liter). In summary, the number of MPOA neurons expressing the GnRH gene, hypothalamic GnRH peptide content, and levels of gonadotropins and T were decreased in intact old compared to young male rats. In contrast, the number of neurons expressing GnRH in the MPOA and levels of gonadotropins and T were similar in orchidectomized old and young rats. We conclude that GnRH synthetic capacity is decreased in the aging male Fischer 344 rat, which may contribute to the age-associated decline in gonadotropin secretion and reproductive function. Furthermore, the age-related decreases in GnRH synthetic capacity and gonadotropin levels are dependent upon testicular feedback factors.
