Development of a Chemical Reproductive Aging Model in Female Rats

Abstract

Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioral changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, around 45-50 years old, women are more likely to express mood disorders, anxiety, irritability and vasomotor symptoms. The current animal models of reproductive aging do not successfully replicate human perimenopause and the gradual changes that occur in this phase. While the traditional rat model of menopause involves ovariectomy or surgical menopause consisting of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones, natural or transitional menopause is achieved by the selective loss of ovarian follicles (perimenopause period). However, the natural aging rodent (around 18-24 months) model fails to reach very low estrogen concentrations and overlaps the processes of somatic and reproductive aging. The chronic exposure of young rodents to 4-vinylcyclohexene diepoxide (VCD) is a well-established experimental model for perimenopause and menopause studies. VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia (apoptosis). The VCD, ovary-intact or accelerated ovarian failure (AOF) model is the experimental model that most closely matches natural human progression to menopause mimicking both hormonal and behavioral changes typically manifested by women in perimenopause. Graphical abstract: The female reproductive system is regulated by a series of neuroendocrine events controlled by central and peripheral components. (A). The mechanisms involved in this control are extremely complex and have not yet been fully clarified. In female mammals whose ovulation (the most important event in a reproductive cycle) occurs spontaneously, reproductive success is achieved through the precise functional and temporal integration of the hypothalamus-pituitary-ovary (HPO) axis. (B). In women, loss of fertility appears to be primarily associated with exhaustion of ovarian follicles, and this process occurs progressively until complete follicular exhaustion marked by the final menstrual period (FMP). (C). While in female rodents, reproductive aging seems to begin as a neuroendocrine process, in which changes in hypothalamic/pituitary function appear independently of follicular atresia. The traditional rat model of menopause, ovariectomy or surgical menopause consists of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones. (D). The chronic exposure (15-30 days) to the chemical compound 4-vinylcyclehexene diepoxide (VCD) in young rodents accelerates gradual failure of ovarian function by progressive depletion of primordial and primary follicles, but retains residual ovarian tissue before brain alterations that occurs in women in perimenopause. Low doses of VCD cause the selective destruction of the small preantral follicles of the ovary without affecting other peripheral tissues.

Keywords: 4-vinylcyclohexene diepoxide (VCD); Accelerated ovarian failure (AOF); Chemical reproductive aging; Follicular depletion; Perimenopause.

Figure 1.. Schematic diagram showing the timeline of the experimental protocol.

Female Wistar rats (28 post-natal days) must be daily injected with VCD (160 mg/Kg) or corn oil (O; 1.25 ml/kg of body weigh) for 15 days. Approximately 55 days after the first VCD or O injection, insert implant pellets of 17β-estradiol or O s.c in the dorso-lateral region (Groups O + O; VCD + O; VCD + E). 21 days after oil or estradiol pellets implantation rats must be decapitated in the morning of diestrus.

Figure 2.. Schematic diagram showing the preparation of the accelerated ovarian failure model and control groups

Figure 3.. Schematic diagram showing the silastic capsules preparation and surgery implantation

Figure 4.. Schematic diagram showing the vaginal smear collection and estrous cycle evaluation.

Photomicrography (10×) kindly provided by Thalita de Oliveira Gonçalves and Guilherme de Souza Gagliano.

Figure 5.. Schematic diagram showing the 4-vinylcyclohexene diepoxide (VCD) dilution