Chronic exposures to low levels of estradiol and their effects on the ovaries and reproductive hormones: Comparison with aging

Abstract

Aging in female rats is characterized by a state called "constant estrous" in which rats are unable to ovulate, have polycystic ovaries and moderately elevated estrogen levels. We hypothesized that chronic exposure of young animals to low levels of E2 can produce reproductive changes similar to that seen in aging animals. Adult female rats were sham-implanted (control) or implanted with slow-release E2 (20 ng/day) pellets for 30, 60, or 90 days. Old constant estrous (OCE) rats were used for comparison. Estrous cyclicity was monitored periodically. At the end of treatment, animals were sacrificed, trunk blood was collected for hormone measurements and ovaries for immunohistochemistry. Young animals became acyclic with increasing duration of E2 exposure while OCE rats were in a state of acyclicity. Ovaries became increasingly more cystic with E2 exposure, and were comparable to OCE rats; however, there was a marked reduction in interstitial tissue with exogenous E2 treatment. Exogenous E2 also decreased Mullerian inhibiting substance expression, increased infiltration of macrophages without much impact on apoptosis in the ovaries. Serum testosterone levels decreased in E2-treated young animals, while it increased significantly in OCE rats. There was a marked reduction in LH but not FSH levels with E2 exposure in both young and old animals. These results indicate that even very low doses of E2 are capable of inducing aging-like changes in young animals.

Keywords: CD68; Mullerian inhibiting substance; TUNEL; estrogen; follicular cysts; ovary.

Figure 1

(A) Estrous cyclicity in control and E2-treated animals. Estrous cycles were determined by daily vaginal cytology. Graph represents percentage of animals demonstrating regular estrous cycles. (B) Serum estradiol levels in the control and treatment groups. * indicates p<0.05 when compared to control rats. “a” indicates significant difference from both control and E-30 groups.

Figure 2

(A) Representative histological sections (H&E staining) of ovaries from different groups. Control (panel 1); E30 (panel 2), E60 (panel 3), E90 (panel 4) and OCE (panel 5). X represents fresh CL and # represents old CL. (B) Total number of tertiary follicles in the different treatment groups. * indicates p<0.05 when compared to the E30 and E60 groups. (C) Total number of fresh and old CLs in the ovaries of different treatment groups. * indicates p<0.05; ** indicates p<0.0001.

Figure 3

Degenerating follicular elements with E2 treatment. Tertiary follicle in a Control ovary (A). Follicle from E90 ovary (B): Note granulosa cell (GC) nuclear pyknosis, cell dispersion with loss of polarity, antral fluid basophilia (block arrow) and mineralization with globular formation and accumulation of cellular debris, and follicular wall neutrophilic infiltration. Follicle from E90 ovary (C): Note marked accumulation of foamy macrophages. The antral fluid is degenerate and characterized by basophilia and accumulation of homogenous eosinophilic, globular debris (protein) (block arrow). (D) E90 follicle undergoing degeneration and a degenerate oocyte (block arrow) can be observed, along with fibrillar antral fluid, GC nuclear pyknosis, and GC dispersion.

Figure 4

(A) MIS immunohistochemistry in ovaries from control and E2 treated animals. Panel 1: Section from a control ovary. There was strong MIS immunoreactivity in primary, secondary, and small tertiary follicles. Panel 2 and 3: Sections from a E90 ovary and OCE ovary respectively. (B) Effect of E2 treatment on the percentage of follicles expressing MIS immunoreactivity. Total number of follicles counted ranged from 65–85 in the different groups * indicates significant decrease from control. p<0.05.

Figure 5

(A) Effect of E2 exposure in young and old (OCE) rats on CD68 expression. Arrows indicate cells in the follicles staining positive for CD68. (B) Effect of E2 exposure on follicles staining positive for CD68. The percentage of CD68 positive follicles increased significantly in the E90 and OCE groups compared to the rest of the groups (p<0.05) Total number of follicles counted ranged from 65-85 in the different groups. (C) Effect of E2 exposure on average number of CD38 positive cells per follicle. * indicates p<0.05.

Figure 6

(A) Effect of E2 exposure on serum testosterone levels. * indicates significant difference from the rest of the groups. † indicates significant decrease from control and OCE groups. p<0.05. (B) Effect of E2 exposure on serum LH levels. * indicates significant difference from control. (C) Effect of chronic E2 exposure on serum FSH levels.