Kisspeptin treatment improves fetal-placental development and blocks placental oxidative damage caused by maternal hypothyroidism in an experimental rat model

Abstract

Maternal hypothyroidism is associated with fetal growth restriction, placental dysfunction, and reduced kisspeptin/Kiss1R at the maternal-fetal interface. Kisspeptin affects trophoblastic migration and has antioxidant and immunomodulatory activities. This study aimed to evaluate the therapeutic potential of kisspeptin in the fetal-placental dysfunction of hypothyroid Wistar rats. Hypothyroidism was induced by daily administration of propylthiouracil. Kisspeptin-10 (Kp-10) treatment was performed every other day or daily beginning on day 8 of gestation. Feto-placental development, placental histomorphometry, and expression levels of growth factors (VEGF, PLGF, IGF1, IGF2, and GLUT1), hormonal (Dio2) and inflammatory mediators (TNFα, IL10, and IL6), markers of hypoxia (HIF1α) and oxidative damage (8-OHdG), antioxidant enzymes (SOD1, Cat, and GPx1), and endoplasmic reticulum stress mediators (ATF4, GRP78, and CHOP) were evaluated on day 18 of gestation. Daily treatment with Kp-10 increased free T3 and T4 levels and improved fetal weight. Both treatments reestablished the glycogen cell population in the junctional zone. Daily treatment with Kp-10 increased the gene expression levels of Plgf, Igf1, and Glut1 in the placenta of hypothyroid animals, in addition to blocking the increase in 8-OHdG and increasing protein and/or mRNA expression levels of SOD1, Cat, and GPx1. Daily treatment with Kp-10 did not alter the higher protein expression levels of VEGF, HIF1α, IL10, GRP78, and CHOP caused by hypothyroidism in the junctional zone compared to control, nor the lower expression of Dio2 caused by hypothyroidism. However, in the labyrinth zone, this treatment restored the expression of VEGF and IL10 and reduced the GRP78 and CHOP immunostaining. These findings demonstrate that daily treatment with Kp-10 improves fetal development and placental morphology in hypothyroid rats, blocks placental oxidative damage, and increases the expression of growth factors and antioxidant enzymes in the placenta.

Keywords: cell stress; fetal development; inflammation; kisspeptin; rat; thyroid; trophoblast.

Figure 1

Maternal and fetal parameters of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A) Hysterectometric maternal weight gain (mean ± SEM; n = 8). (B) Free T3 (mean ± SEM; n = 4-5). (C) Free T4 (mean ± SEM; n = 4-5). (D) Representative image of fetuses and placentas from each experimental group … (E–F) Uteroplacental (E) and amniotic fluid (F) weight (mean ± SEM; n = 8). (G) Number of viable fetuses/litter (mean ± SEM; n = 8). (H) Percentage of fetal death (median, interquatile range; n = 8). (I) Fetal weight (mean ± SEM; n = 63-76). (J) Relative frequency distribution curve of fetal weight. (K) Weight of fetal organs (liver, brain, heart, lung, kidney) (mean ± SEM; n = 15-30). (L) Brain/liver ratio (mean ± SEM; n = 15-30). Significant differences were determined by ANOVA post hoc SNK except for fetal weight and fetal organs’ weight, which were determined by Generalized linear mixed-model analysis followed by the Tukey test, and fetal death which was determined by Dunn’s post hoc Kruskal-Wallis test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. KpT1, yes/no day treatment with Kp10; KpT2, daily treatment with Kp10; GD, gestational day.

Figure 2

Histomorphometry evaluation of the placenta of control, hypothyroid, and kisspeptin-10 treated-rats on the 18th GD. (A–D) Illustrative photomicrographs of the maternal-fetal interface (A) and placental layers (B), giant cells; (C), spongiotrophoblast; (D), labyrinth zone) (Hematoxylin and eosin staining; Bar = 500μm (A); 50μm (B–D). (E) Thickness of the junctional zone (giant cells+spongiotrophoblast) and labyrinth zone (mean ± SEM; n = 8). (F) Percentage of area occupied by glycogen cells, spongiotrophoblasts, and giant cells in the junctional zone (mean ± SEM; n = 8). (G) Percentage of area occupied by maternal vascular sinus, fetal capillaries, and fetal mesenchyme/trophoblast in the labyrinth zone (mean ± SEM; n = 8). Significant differences were determined by ANOVA post hoc SNK, *P<0.05, **P<0.01. MT, mesometrial triangle; BD, basal decidua; JZ, junctional zone; LZ, labyrinth zone; CV, central vessel; SP, spongiotrophoblast; Arrow, glycogen cells; Arrowhead, fetal capillaries; Asterisks, maternal vascular sinus; KpT1, yes/no day treatment with Kp-10; KpT2, daily treatment with Kp-10; GD, gestational day.

Figure 3

Expression of VEGF, PIGF, IGF1, IGF2, GLUT1 and DIO2 in the placenta of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A–F) Photomicrographs of immunohistochemical expression of VEGF in the junctional zone (A–C) and labyrinth zone (D–F) (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (G) Immunolabeling area, in pixels, of VEGF expression in the junctional zone and labyrinth zone on the 18 GD (mean ± SEM; n = 8). (H) Relative gene expression of Vegf, Pigf, Igf1, Igf2, Glut1, and Dio2 in the placenta (mean ± SEM; n = 8). Significant differences were determined ANOVA post hoc SNK, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. KpT2, daily treatment with Kp10; GD, gestational day.

Figure 4

Expression of TNFα, IL-10, and IL-6 in the placenta of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A) Photomicrographs of immunohistochemical expression of TNFα in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (B) Immunolabeling area, in pixels, of TNFα expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (C) Photomicrographs of immunohistochemical expression of IL-10 in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (D) Immunolabeling area, in pixels, of IL-10 expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (E) Relative gene expression of Tnf, Il10, and Il6 in the placenta (mean ± SEM; n = 8). Significant differences were determined by ANOVA post hoc SNK, *P<0.05, **P<0.01. KpT2, daily treatment with Kp10; GD, gestational day.

Figure 5

Expression of HIF1α and 8-OHdG in the placenta of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A) Photomicrographs of immunohistochemical expression of HIF1α in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (B) Immunolabeling area, in pixels, of HIF1α expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (C) Immunolabeling area, in pixels, of HIF1α expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (D) Relative gene expression of Hif1α in the placenta (mean ± SEM; n = 8). Significant differences were determined by ANOVA post hoc SNK, *P<0.05, **P<0.01. KpT2, daily treatment with Kp10; GD, gestational day.

Figure 6

Expression of SOD1, catalase, and GPx1/2 in the placenta of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A) Photomicrographs of immunohistochemical expression of SOD1 in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (B) Immunolabeling area, in pixels, of SOD1 expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (C) Photomicrographs of immunohistochemical expression of catalase in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (D) Immunolabeling area, in pixels, of catalase expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (E) Photomicrographs of the immunohistochemical expression of GPx1/2 in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (F) Immunolabeling area, in pixels, of GPx1/2 expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (G) Relative gene expression of Sod1, catalase, and Gpx1 in the placenta (mean ± SEM; n = 8). Significant differences were determined by ANOVA post hoc SNK, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. KpT2, daily treatment with Kp10; GD, gestational day.

Figure 7

Expression of GRP78, CHOP, and ATF4 in the placenta of control, hypothyroid, and kisspeptin-10-treated rats on the 18th GD. (A) Photomicrographs of immunohistochemical expression of GRP78 in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (B) Immunolabeling area, in pixels, of GRP78 expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (C) Photomicrographs of immunohistochemical expression of CHOP in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (D) Immunolabeling area, in pixels, of CHOP expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (E) Photomicrographs of immunohistochemical expression of ATF4 in the junctional zone and labyrinth zone (Streptavidin-biotin-peroxidase; Harris hematoxylin; Bar = 50 µm). (F) Immunolabeling area, in pixels, of ATF4 expression in the junctional zone and labyrinth zone (mean ± SEM; n = 8). (G) Relative gene expression of Grp78 and Chop in the placenta (mean ± SEM; n = 8). Significant differences were determined by ANOVA post hoc SNK, *P<0.05, **P<0.01, ****P<0.0001. KpT2, daily treatment with Kp10; GD, gestational day.