Amino Acid Transport in the Rat Placenta in Methionine-Induced Hyperhomocysteinemia

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Abstract

Maternal hyperhomocysteinemia (HHcy) is a risk factor for the development of intrauterine growth restriction, a proposed mechanism of which is a decrease in transplacental nutrient transport. In this study, we have investigated the effect of experimental HHcy caused by daily methionine administration to pregnant rats on the free amino acid content in the mother and fetal blood. The morphological and biochemical parameters on which amino acid transport through the placenta depends have also been studied. Under the influence of HHcy on the 20th day of pregnancy, an increase in the levels of most free amino acids was observed in the mother blood, while some amino acid concentrations in the fetal blood were decreased. Under conditions of HHcy in the placental labyrinth, which is an exchange site between mother and fetal circulations, the maternal sinusoids narrowed, being accompanied by blood stagnation and red blood cell aggregation. In the labyrinth zone, we also observed an increase in the protein level of neutral amino acid transporters (LAT1, SNAT2) and an activation of the downstream effector of the mTORC1 complex, 4E-BP1, which is a positive regulator of the placental transporter expression. Maternal HHcy caused an increase in the placental barrier permeability, as evidenced by an intensification in the mother to fetus transfer of the Evans blue dye. The imbalance of the free amino acid levels in the mother and fetal blood under conditions of HHcy may be due to the competition of homocysteine with other amino acids for their common transporters, as well as a decreased exchange zone area and reduced blood flow in the placental labyrinth. An increase in the amino acid transporter expression in the labyrinth zone may be a compensatory response to an insufficient intrauterine amino acid supply and a fetal growth decrease.

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Yu. P. Milyutina

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Author for correspondence.
Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

G. O. Kerkeshko

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

D. S. Vasilyev

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine; I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg; 194223, Saint Petersburg

N. L. Tumanova

I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: milyutina1010@mail.ru
Russian Federation, 194223, Saint Petersburg

I. V. Zaloznyaya

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

S. K. Bochkovsky

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

A. D. Shcherbitskaya

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

A. V. Mikhel

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

G. H. Tolibova

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

A. V. Arutyunyan

D. O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine

Email: milyutina1010@mail.ru
Russian Federation, 199034, Saint Petersburg

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2. Fig. 1. Changes under the influence of maternal hyperhomocysteinemia (HHC) in the content of free amino acids (AAs) in the serum of pregnant rats (Mother HHC) and their fetuses (Fetus HHC), as well as the ratio of the level of AAs in the blood of the fetus to that in the blood of the mother (Fetus/Mother HHC) on day 20 of gestation. a - Indispensable AAs; b - Substitutable AAs. Changes are expressed as percentage relative to the level in the control group; data are presented as Me (Q1; Q3); n = 11 females in the control group, n = 10 females in the HGC group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; t-test or Mann-Whitney U-test

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3. Fig. 2. Effect of experimental hyperhomocysteinemia on the content of amino acid transporters in the labyrinthine region of rat placenta on day 20 of gestation. Content (a) and representative immunoblot (b) of SNAT1, SNAT2, LAT1, LAT2 proteins in the fetal part of rat placenta in control and under the influence of maternal hyperhomocysteinemia (HHC); ordinate axis - intensity of bands obtained by immunoblotting expressed in conventional units; data are presented as M ± SEM; n = 4-8 females in each group; * p < 0.05, Mann-Whitney U-test; ** p < 0.01, Student t-test

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4. Fig. 3. Effect of maternal hyperhomocysteinemia (HHC) on the activity of mTOR kinase and downstream components of the mTORC1 signalling system in rat placenta at day 20 of gestation. Content and representative immunoblot of mTOR, 4E-BP1 and S6 proteins and their phosphorylated forms p-mTORSer2448, p-4E-BP1Thr37/46 and p-S6Ser235/236 in the maternal (MMP) (a and b) and fetal part (FPP) (c and d) of rat placenta in control and under the influence of maternal HGC; ordinate axis - intensity of bands obtained by immunoblotting expressed in conventional units; data are presented as M ± SEM; n = 10 females in each group; ** p < 0.05, Welch's t-test; *** p < 0.001, Student's t-test

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5. Fig. 4. Morphological changes in the rat placenta on day 20 of gestation under the influence of maternal hyperhomocysteinemia (HHC). a, b - Labyrinthine area of the placenta with trophoblast beams (BTB) and maternal sinusoids (MS) in the control (a) and experimental (b) groups. The areas of BTB and erythrocyte aggregates (AEs) whose area was quantitatively analysed are indicated in endnotes; haematoxylin-eosin staining, ×200. c - Relative area (S%) of BTB from the total area of the labyrinthine region. d-e - Electronograms of placental tissue of control (d) and experimental (e and f) groups; mb - maternal blood (maternal sinusoid), fc - fetal capillary vessel, er - erythrocytes in the vessel lumen, panels e and f show aggregates of erythrocytes filling the vessel lumen, en - endothelial cells of the fetal capillary wall. g - Area of AEs in the labyrinthine region of the placenta. h, i - Giant trophoblast cells (GTC) in the control group (h) and HGC group (i); hematoxylin-eosin staining, ×400; arrows indicate GTC nuclei; k - relative area (S%) of GTC nuclei from the total area of the GTC layer. l - Thickness of the labyrinthine region (LR) and basal area (BA) of the placenta. m - Ratio of BA thickness to LR thickness. In all graphs, data are presented as M ± SEM. For panels c and k: n = 3 placentas from 3 females in the control group, n = 14 placentas from 4 females in the HGC group; w, l and m: n = 9 placentas from 5 females in the control group, n = 18 placentas from 6 females in the HGC group; ** p < 0.01, Student's t-test

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6. Fig. 5. Effect of maternal hyperhomocysteinaemia (HHC) on the ultrastructure of the placental barrier and permeability of the rat placenta on day 20 of gestation. a-c - Electronograms of the exchange zone between maternal and fetal blood systems in the labial region; mb - maternal blood (maternal sinusoid), fc - fetal capillary vessel; layers of the placental barrier: I - cytotrophoblast layer in contact with maternal blood, II and III - two layers of syncytiotrophoblast, bm - basal membrane lining the endothelial cell layer (en) of the fetal capillary wall, er - erythrocyte in the vessel lumen, arrow - tight contact between endothelial cells, arrowhead - fenestra of the fetal capillary endothelium. d - Fluorescence intensity level of Evans blue dye in tissues of abdominal cavity organs (ACO) and fetal brain 3 h after its administration to female rats; n = 12 placentas from 2 females in the control group, n = 15 placentas from 2 females in the HGC group; *** p < 0.001, Welch's t-test

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