Maternal aromatase inhibition via letrozole altered RFamide-related peptide-3 and gonadotropin-releasing hormone expression in pubertal female rats

Abstract

Objectives: Despite prevalence of polycystic ovary syndrome (PCOS) among childbearing women and development of many animal models for this syndrome, information on its etiology is still scarce. The intrauterine hyperandrogenic environment may underlie changes at the level of hypothalamus, pituitary, ovary organization in female offspring, and PCOS later in life. Letrozole has been shown to mimic reproductive and metabolic characteristics of PCOS in adult rodent models. Therefore, this research aimed to assess the condition in a prenatal letrozole-treated rat model.

Materials and methods: Twenty-eight female rats dams receiving letrozole at certain doses during late pregnancy were used in the trial. Pregnant Sprague-Dawley rats (n=21) received letrozole treatment on gestation days 16-18 at doses of 1.25, 1.0, 0.75, 0.5, and 0.25 mg/kg body weight (BW).

Results: Prenatal letrozole treatment delayed parturition time and reduced the litter size in pregnant dams (P<0.0001). Late puberty onset, irregular ovarian cyclicity, increased anogenital distance (AGD), body weight gain, serum testosterone concentration, and reduced estradiol levels (P<0.0001) were observed in the female offspring of dams receiving 1.25 and 1 mg/kg BW letrozole. Furthermore, letrozole at 1.25 and 1 mg/kg BW showed increased RFRP and decreased GnRH mRNA expression (P<0.0001). Letrozole treatment at doses of 1 mg/kg BW and lower was not fetotoxic.

Conclusion: It was concluded that 1 mg/kg BW letrozole may be suggested for prenatal PCOS induction.

Keywords: Gonadotropin-releasing – hormone; Hypothalamus; Letrozole; Polycystic ovary syndrome; Prenatal; RFamide-related peptide-3; Rat.

Figure 1

Prenatal letrozole treatment-induced effects on gestation length (A), litter size (B), and male/female offspring ratio (C) in adult rats (mean ± SEM). The lines show significant differences between groups at P<0.05

Figure 2

Prenatal letrozole treatment-induced effects on anogenital distance (AGD, A), AGD index (AGDI, B), and puberty age (C) in adult rats (mean ± SEM). The lines show statistically significant differences between groups at P<0.05

Figure 3

Prenatal letrozole treatment-induced effects on body weight gain in adult rats (mean + SEM). S (sham), C (control); The superscript letters show statistically significant differences between letrozole-treated groups with control and sham groups in each week (P<0.05)

Figure 4.

Prenatal letrozole treatment-induced effects on estrous cycle patterns during three week daily observations in adult rats. In the Y-axis, the numbers 3, 2, 1, 0 represent proestrus, estrus, metestrus, and diestrus

Figure 5

Prenatal letrozole treatment-induced effects on the percentage of each phase of the estrous cycle (A), total proestrus and estrous phases, and total metestrous and diestrous phases (B), and the number of completed and non-completed cycles (C) in letrozole-treated and control groups (mean + SEM). The mean with non-common letters has a statistically significant difference (P<0.05) in A and B. Also, the lines show statistically significant differences between groups at P<0.05 in C

Figure 6

Prenatal letrozole treatment-induced effects on the number of total follicles (A), number of atretic follicles (B), number of cystic follicles (C), and number of corpus luteum (D) (mean + SEM), and H&E staining of rat ovaries in letrozole-treated and control groups. The total follicle number in the 0.25 mg/kg BW was more than o.75 and 0.5 mg/kg BW (A). Increased number of cystic and atretic follicles in 0.25 mg/kg BW compared with 0.5 mg/kg BW was shown in (B and C). Ovarian tissue in the control group (A) that has corpus luteum (c) and antral follicles (a) ovarian tissue in the 0.75 mg group (B), which has more antral follicles and corpus luteum (c), ovarian tissue in the 0.25 mg group (C), which has a large cystic follicle with a thin granulosa layer (g) and theca layer (t), large cyst in the 0.75 mg group (D), ovarian tissue in the 0.25 mg group (E) has a large number of antral follicles (a) as well as corpus luteum (c), ovarian tissue in 1 mg group (F), which has a large number of growing follicles (f) and antral follicles. The lines show statistically significant differences between groups at P<0.05

Figure 7

Prenatal letrozole treatment-induced effects on serum concentration of testosterone (A), estradiol (B), progesterone (C), LH (D), FSH (E), and LH: FSH ratio (F) (mean + SEM) in adult rats. The lines show statistically significant differences between groups at P<0.05

Figure 8

Prenatal letrozole treatment-induced effects on relative gene expression of hypothalamic neuropeptides, Rfrp (A) and GnRH (B), (mean + SEM) in adult rats. The lines show statistically significant differences between groups at P<0.05