Intrinsic innervation of the ovary and its variations in the rat senescence process

Abstract

Ovarian functions decrease with perimenopause. The ovary has extrinsic innervation, but the neural influence on ovarian functions and dysfunction is not well-studied. The present study aimed to biochemically and morphometrically characterize the intrinsic neurons in ovaries from young adult, middle-aged, and senescent Long Evans CII-ZV rats (3, 12, and 15 months old, respectively). Ovaries were extracted from four rats of each age group (n = 12 total), cryopreserved, and processed for immunofluorescence studies with the primary NeuN/β-tubulin and NeuN/tyrosine hydroxylase (TH) antibodies. The soma area and number of intrinsic neurons in the ovarian stroma, surrounding follicles, corpus luteum, or cyst were evaluated. The intrinsic neurons were grouped in cluster-like shapes in ovarian structures. In senescent rats, the intrinsic neurons were mainly localized in the ovarian stroma and around the cysts. The number of neurons was lower in senescent rats than in young adult rats (p < 0.05), but the soma size was larger than in young adult rats. Immunoreactivity to TH indicated the presence of noradrenergic neurons in the ovary with the same characteristics as NeuN/β-tubulin, which indicates that they are part of the same neuronal group. Taken together, the findings indicate that the intrinsic neurons may be related to the loss of ovarian functions associated with aging.

Keywords: Intrinsic neurons; Ovarian function; Ovarian innervation; Senescent rat.

Fig. 1

Microphotographs show a cluster of neurons in the ovarian stroma (OS) and follicles (F) of young adult (3 M) female rats. a, e The nuclei are labeled with DAPI. b, f NeuN. c, g β-Tubulin. d, h Co-localization of NeuN and β-tubulin. The yellow head arrows show intrinsic neurons of the ovaries, in the theca layer of the follicle. The yellow double line arrow a show the average nucleus of the intrinsic neuron, twice the size of other ovarian cells (orange double line arrow). i, j, l Negative control

Fig. 2

Immunofluorescent staining was used to localize intrinsic neurons in the ovary using NeuN (green) and β-tubulin (red). a, e, i Nuclei were labeled with DAPI. b, c Distribution of the intrinsic neurons around the follicles (F) of the ovaries in a young adult rat (3 M). f, g Around a cyst (C) of the middle-aged rat (12 M) (× 60). j, k Around a cyst in a senescent rat (15 M). d, h, l Co-localization of NeuN and β-tubulin. The yellow head arrows show intrinsic neurons of the ovaries

Fig. 3

The top microphotographs are of ovaries from young adult rat (3 M), middle-aged rat (12 M), and senescent rat (15 M). Immunofluorescent staining was performed with mouse anti-NeuN (green) and rabbit anti-β-tubulin (red) antibodies, showing intrinsic neurons in the ovarian stroma (OS), corpus luteum (CL), and follicles (F). a, e, i Nuclei were labeled with DAPI. b, f, j Neu-N. c,g,k: β-Tubulin. d, h, l Co-localization of NeuN and β-tubulin. The yellow arrows show intrinsic neurons of the ovaries

Fig. 4

The top microphotographs are of ovaries from young adult rat (3 M), middle-aged rat (12 M), senescent rat (15 M). Inmunofluorescent staining was performed with mouse anti-NeuN (green) and rabbit anti-TH (red) antibodies. b, c, d show noradrenergic neurons in the theca layer of follicle. f, g, h show noradrenergic neurons around the cyst (C). j, k, l show noradrenergic neurons in ovarian stroma (OS) and around the cyst. The yellow head arrows show noradrenergic neurons around the follicle or cyst, and the yellow arrows show noradrenergic neurons on the ovarian stroma in the Senescent rat

Fig. 5

a Median number of intrinsic neurons (NeuN/β-tubulin) in ovaries from young adult rat (3 M), middle-aged rat (12 M), and senescent (15 M) CII-ZV rats. b Median number of intrinsic neurons (NeuN/TH) in ovaries c Median area of the intrinsic neurons (NeuN/β-tubulin) from ovarian follicles and cysts. d Median area of the intrinsic neurons (NeuN/β-tubulin) from ovaries in the ovarian stroma. *P < 0.05 vs. 3 M and 12 M, **P < 0.05 vs 12 M and 15 M; ANOVA followed by Tukey. Error bars indicate SEM