Letrozole-Induced Polycystic Ovary Syndrome Attenuates Cystathionine-β Synthase mRNA and Protein Abundance in the Ovaries of Female Sprague Dawley Rats

Abstract

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 10% of reproductive-aged women and leads to hyperandrogenism, anovulation, and infertility. PCOS has been associated with elevated serum homocysteine as well as altered methylation status; however, characterization of one-carbon metabolism (OCM) in PCOS remains incomplete.

Objectives: The aim of our research was to assess OCM in a letrozole-induced Sprague Dawley rat model of PCOS.

Methods: Five-week-old female rats (n = 36) were randomly assigned to letrozole [0.9 mg/kg body weight (BW)] treatment or vehicle (carboxymethylcellulose) control that was administered via subcutaneously implanted slow-release pellets every 30 d. For both treatment groups, 12 rats were randomly assigned to be euthanized during proestrus at one of the following time points: 8, 16, or 24 wk of age. Daily BW was measured and estrous cyclicity was monitored during the last 30 d of the experimental period. Ovaries were collected to assess mRNA and protein abundance of OCM enzymes.

Results: Letrozole-induced rats exhibited 1.9-fold higher cumulative BW gain compared with control rats across all age groups (P < 0.0001). Letrozole reduced the time spent at proestrus (P = 0.0001) and increased time in metestrus (P < 0.0001) of the estrous cycle. Cystathionine β-synthase (Cbs) mRNA abundance was reduced in the letrozole-induced rats at 16 (59%; P < 0.05) and 24 (77%; P < 0.01) wk of age. In addition, CBS protein abundance was 32% lower in 8-wk-old letrozole-induced rats (P = 0.02). Interestingly, betaine-homocysteine S-methyltransferase mRNA abundance increased as a function of age in letrozole-induced rats (P = 0.03).

Conclusion: These data demonstrate that letrozole-induced PCOS Sprague Dawley rats temporally decrease the ovarian abundance of Cbs mRNA and protein in the early stages of PCOS.

Keywords: cystathionine-β synthase; letrozole; one-carbon metabolism; polycystic ovary syndrome; rat.

FIGURE 1

Cumulative body weight gain of rats on letrozole and placebo across 8, 16, and 24 wk of age. Vertical dotted lines indicate the end of the experimental period at 8 and 16 wk of age. Data are means ± SEMs; n = 6. Main effects of letrozole and age were tested in a linear mixed model of repeated measures at significance (P < 0.05). L, letrozole; P, placebo.

FIGURE 2

Change in blood glucose concentrations (A) and area under the curve (B) in rats on letrozole compared with placebo following an intraperitoneal glucose tolerance test. Data are means ± SEMs; n = 6. Main effects of letrozole and age were tested in a linear mixed model of repeated measures for change in blood glucose, whereas AUC comparisons were made at each time point using an unpaired t test at significance (P < 0.05). L, letrozole; P, placebo.

FIGURE 3

Percent frequency of days spent in each stage of the estrous cycle for a total of 30 d. Data are means ± SEMs of the percent frequency; n = 6. Main effects of letrozole and age were tested in a linear mixed model, and simple effects between letrozole and placebo groups at each age are presented at significance (**P < 0.01, and ***P < 0.001). D, diestrus; E, estrus; L, letrozole; M, metestrus; P, placebo; Pr, proestrus; U, pseudodiestrus.

FIGURE 4

Serum testosterone concentrations for letrozole-induced and placebo control Sprague Dawley rats. Data are means ± SEMs; n = 6. Significant differences were assessed by unpaired t test. *P < 0.05, **P < 0.01. L, letrozole; P, placebo.

FIGURE 5

Ovarian mRNA abundance of select enzymes in letrozole-induced and placebo control Sprague Dawley rats at 8 (A), 16 (B), and 24 (C) wk of age and cystathionine β-synthase (CBS) protein abundance (D). The abundance of mRNA was normalized to 18S ribosomal mRNA and expressed as relative to placebo rats, whereas protein abundance is expressed as arbitrary values when corrected for by α-tubulin. Data are means ± SEMs of the relative fold change of mRNA transcript abundance, protein abundance, and enzymatic activity, respectively; n = 6/group. Relative fold change, protein abundance, and enzyme activity were compared via an unpaired t test within in age group and deemed significant at P < 0.05. Bhmt, betaine-homocysteine S-methyltransferase; Cbs, cystathionine β-synthase; Cyp19A1, cytochrome P450 isoform 19a1, aromatase; Dnmt, DNA methyltransferase; Esr1, estrogen receptor 1; Gnmt, glycine N-methyltransferase; L, letrozole; Mtr, methionine synthase; P, proestrus.