Modulating Morphological and Redox/Glycative Alterations in the PCOS Uterus: Effects of Carnitines in PCOS Mice

Abstract

(1) Background: Polycystic ovarian syndrome (PCOS) is a common and multifactorial disease affecting reproductive-age women. Although PCOS ovarian and metabolic features have received extensive research, uterine dysfunction has been poorly investigated. This research aims to investigate morphological and molecular alterations in the PCOS uterus and search for modulating effects of different carnitine formulations. (2) Methods: CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg L-carnitine (LC) and 0.20 mg acetyl-L-carnitine (ALC) in the presence or absence of 0.08 mg propionyl-L-carnitine (PLC). Uterine horns from the four groups were subjected to histology, immunohistochemistry and immunoblotting analyses to evaluate their morphology, collagen deposition, autophagy and steroidogenesis. Oxidative-/methylglyoxal (MG)-dependent damage was investigated along with the effects on the mitochondria, SIRT1, SOD2, RAGE and GLO1 proteins. (3) Results: The PCOS uterus suffers from tissue and oxidative alterations associated with MG-AGE accumulation. LC-ALC administration alleviated PCOS uterine tissue alterations and molecular damage. The presence of PLC prevented fibrosis and maintained mitochondria content. (4) Conclusions: The present results provide evidence for oxidative and glycative damage as the main factors contributing to PCOS uterine alterations and include the uterus in the spectrum of action of carnitines on the PCOS phenotype.

Keywords: DHEA; PCOS; SIRT1; carnitines; glycative stress; methylglyoxal; mitochondria; mouse; oxidative stress; uterus.

Figure 1

Estrous cycle in control and DHEA mice.

Figure 2

HE staining of mouse uterine horns of control (A–D), DHEA (E–H), DHEA/LC-ALC (I–L) and DHEA/LC-ALC-PLC (M–P) groups. (A,E,I,M) low-magnification LM pictures of the lumen (L), endometrium (E), myometrium (M) and perimetrium (P). (B,F,J,N) glandular epithelium (g). (C,G,K,O) luminal epithelium; (D,H,L,P) high magnification of the luminal epithelium made of columnar cells. LM, mag. 10× ((A,E,I,M) Bar: 200 µm), 20× ((B,C,F,G,J,K,N,O) Bar: 50 µm), 40× ((D,H,L,P) Bar: 20 µm).

Figure 3

Abundant vascularization (A) and presence of inflammatory cells (B) in the endometrium of the DHEA group. Black arrows in (B) indicate single inflammatory cells as eosinophils, lymphocytes and macrophages. LM, 20× (A), 40× (B).

Figure 4

Mallory Trichrome (A,B,D,E,G,H,J,K) and Col1 (C,F,I,L) staining of mouse uterine horns of control (A–C), DHEA (D–F), DHEA/LC-ALC (G–I) and DHEA/LC-ALC-PLC (J–L) groups. (A,D,G,J) low-magnification LM pictures of the lumen (L), endometrium (E), myometrium (M) and perimetrium (P). (B,E,H,K) high magnification of the luminal epithelium. (C,F,I,L) glandular epithelium (g). Inset in F shows a detail of the tunica muscularis. LM, mag. 10× ((A,D,G,J) Bar: 200 µm), 20× ((B,C,E,F,H,I,K,L) Bar: 100 µm).

Figure 5

Effects of carnitine administration on DHEA-induced autophagy in mouse uterine horns. LC3II/LC3I ratio (a) and p62 (b) Western blotting analysis and representative images (c). Three mice per experimental group were employed. Experiments were conducted in triplicate. Data obtained from n = 9 observations are presented as means ± SEM of densitometric analysis of immunoreactive bands normalized to internal reference protein (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). * p < 0.01 vs. CTRL, ** p < 0.05, # p < 0.01, t-test.

Figure 6

Immunostaining of mouse uterine horns for 17 β-HSD4 (A–D) of control (A), DHEA (B), DHEA/LC-ALC (C) and DHEA/LC-ALC-PLC (D) groups. Insets show details of the glandular epithelium. L: lumen; E: endometrium; M: myometrium; P: perimetrium. LM, mag. 10×. Insets: 20×.

Figure 7

Immunostaining of mouse uterine horns for 4-HNE (A–D) of control (A), DHEA (B), DHEA/LC-ALC (C) and DHEA/LC-ALC-PLC (D) groups. Insets show details of the glandular epithelium. L: lumen; E: endometrium; M: myometrium; P: perimetrium. LM, mag. 10×. Insets: 20×.

Figure 8

Western blot analysis of SIRT1 (A) and SOD2 (B) and representative images (C). Three mice per experimental group were employed. Experiments were conducted in triplicate. Data obtained from n = 9 observations are presented as means ± SEM of densitometric analysis of immunoreactive bands normalized to internal reference protein (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). * p < 0.01 vs. CTRL, ** p < 0.05, # p < 0.01, t-test.

Figure 9

Immunostaining of mouse uterine horns for TOMM20 (A–D) of control (A), DHEA (B), DHEA/LC-ALC (C) and DHEA/LC-ALC-PLC (D) groups. Insets show details of the glandular epithelium. L: lumen; E: endometrium; M: myometrium; P: perimetrium. LM, mag. 10×. Insets: 20×.

Figure 10

Western blot analysis of PGC1α (A) and representative images (B). Three mice per experimental group were employed. Experiments were conducted in triplicate. Data obtained from n = 9 observations are presented as means ± SEM of densitometric analysis of immunoreactive bands normalized to internal reference protein (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). * p < 0.01 vs. CTRL, t-test.

Figure 11

Immunostaining of mouse uterine horns for MG-AGE (A–D) of control (A), DHEA (B), DHEA/LC-ALC (C) and DHEA/LC-ALC-PLC (D) groups. Insets show details of the glandular epithelium. L: lumen; E: endometrium; M: myometrium; P: perimetrium. LM, mag. 10×. Insets: 20×.

Figure 12

Western blot analysis of RAGE (A) and GLO1 (B) and representative images (C). Three mice per experimental group were employed. Experiments were done in triplicate. Data obtained from n = 9 observations are presented as means ± SEM of densitometric analysis of immunoreactive bands normalized to internal reference protein (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). * p < 0.01 vs. CTRL, t-test.