Autophagy marker dynamics in the fetal brain and placenta of rats in hyperhomocysteinemia

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Abstract

BACKGROUND: Autophagy is essential for placenta formation and fetal brain development. Maternal hyperhomocysteinemia is a risk factor for pregnancy complications and may affect autophagy processes. However, the dynamics of autophagy markers are not studied enough so far.

AIM: The aim of this study was to assess the dynamics of key autophagy markers in the fetal brain and various parts of the placenta of rats throughout pregnancy under normal conditions and in the presence of maternal hyperhomocysteinemia.

MATERIALS AND METHODS: Pregnant Wistar rats were induced with hyperhomocysteinemia by chronic administration of L-methionine. Placental and fetal brain tissues were collected on days 14 and 20 of gestation. Levels of autophagy markers (Beclin-1; phosphatidylethanolamine conjugated form microtubule-associated protein 1A/1B light chain 3B (LC3B-II); lysosomal associated membrane protein 2 (LAMP-2)] were determined by Western blotting. Ultrastructural changes were examined using electron microscopy.

RESULTS: In the control group, by the end of pregnancy (gestational day 20) compared to gestational day 14, we observed an increase in LAMP-2 level in the maternal part of the placenta and a decrease in LC3B-II level in the fetal part of the placenta. In maternal hyperhomocysteinemia in the maternal part of the placenta, we found an increase in LAMP-2 level on gestational day 14 and in LC3B-II level from gestational day 14 to gestational day 20. In the fetal part of the placenta, under the same conditions, we observed a decrease in LC3B-II level on gestational day 14 and an increase in LAMP-2 level by the end of pregnancy. In the fetal brain, a decrease in Beclin-1 level from gestational day 14 to gestational day 20 was shown in the both study groups, while under the influence of hyperhomocysteinemia, the levels of the autophagy markers remained unchanged. Under L-methionine load, pathological ultrastructural changes were observed in the fetal part of the placenta and fetal brain at the both time points studied. Normally and under the influence of hyperhomocysteinemia, autophagosomes were found in placental cells on gestational days 14 and 20, while in brain cells, they were only present on gestational day 20.

CONCLUSIONS: The data obtained suggest that autophagy activity in the placenta and fetal brain in normal conditions and under maternal hyperhomocysteinemia depends on the gestational age. Changes in the dynamics of autophagy may be a reason for impaired placental formation and dysfunction in hyperhomocysteinemia. The absence of significant changes in autophagy markers in the fetal brain under hyperhomocysteinemia conditions may result from protective mechanisms in the placenta or/and the resilience of autophagy processes in nervous tissue.

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About the authors

Anastasiia V. Mikhel

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott; I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: anastasia.michel39@gmail.com
ORCID iD: 0000-0003-1352-9125
SPIN-code: 1064-6884

postgraduate student

Russian Federation, Saint Petersburg; Saint Petersburg

Irina V. Zalozniaia

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: irinabiolog2012@yandex.ru
ORCID iD: 0000-0002-0576-9690
SPIN-code: 2488-3790

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Anastasiia D. Shcherbitskaia

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: nastusiq@gmail.com
ORCID iD: 0000-0002-2083-629X
SPIN-code: 6913-0435

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Dmitrii S. Vasilev

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott; I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: dvasilyev@bk.ru
ORCID iD: 0000-0002-0601-2358
SPIN-code: 3752-5516

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Yuliya P. Milyutina

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: milyutina1010@mail.ru
ORCID iD: 0000-0003-1951-8312
SPIN-code: 6449-5635

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Gleb O. Kerkeshko

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: gkerkeshko@yandex.ru
ORCID iD: 0009-0005-0804-5347
SPIN-code: 3551-0320

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Alexandra V. Gorbova

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: alekss137@mail.ru
ORCID iD: 0009-0005-9774-8908
Russian Federation, Saint Petersburg

Natalia L. Tumanova

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

Email: natalia.tumanova@iephb.ru
ORCID iD: 0000-0002-9895-7892
SPIN-code: 6072-3084

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Alexander V. Arutjunyan

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Author for correspondence.
Email: alexarutiunjan@gmail.com
ORCID iD: 0000-0002-0608-9427
SPIN-code: 9938-5277

Dr. Sci. (Biology), Professor, Honored Scientist of the Russian Federation

Russian Federation, Saint Petersburg

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2. Fig. 1. Autophagy marker levels in the maternal part of the placenta on gestational days 14 (GD14) and 20 (GD20) in the control group (n = 6) and hyperhomocysteinemia group (HHС; n = 6): a, lysosomal associated membrane protein 2 (LAMP-2) levels; b, Beclin-1 levels; c, phosphatidylethanolamine conjugated form microtubule-associated protein 1A/1B light chain 3B (LC3B-II) levels; d, representative Western blot showing dynamic changes in marker expression levels relative to the control group on gestational day 14. Data are presented as median, interquartile range, minimum, and maximum values. * p < 0.05 compared to the same group on gestational day 14; ** p < 0.01 compared to control at the same gestational age

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3. Fig. 2. Autophagy marker levels in the maternal part of the placenta on gestational days 14 (GD14) and 20 (GD20) in the control group (n = 6) and hyperhomocysteinemia group (HHС; n = 6): a, lysosomal associated membrane protein 2 (LAMP-2) levels; b, Beclin-1 levels; c, phosphatidylethanolamine conjugated form microtubule-associated protein 1A/1B light chain 3B (LC3B-II) levels; d, representative Western blot showing dynamic changes in marker expression levels relative to the control group on gestational day 14. Data are presented as median, interquartile range, minimum, and maximum values. * p < 0.05 compared to the same group on gestational day 14; ** p < 0.01 compared to control at the same gestational age

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4. Fig. 3. Autophagy marker levels in the fetal brain on gestational days 14 (GD14) and 20 (GD20): a, lysosomal associated membrane protein 2 (LAMP-2) levels in the control group (n = 10) and hyperhomocysteinemia group (HHС; n = 10); b, Beclin-1 levels in the control group (n = 14) and hyperhomocysteinemia group (n = 14); c, phosphatidylethanolamine conjugated form microtubule-associated protein 1A/1B light chain 3B (LC3B-II) levels in the control group (n = 18) and hyperhomocysteinemia group (n = 18); d, representative Western blot showing dynamic changes in marker expression levels relative to the control group on gestational day 14. Data are presented as median, interquartile range, minimum, and maximum values. * p < 0.0001 compared to the same group on gestational day 14

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5. Fig. 4. Electron micrographs of the fetal (a, b, c, d) and maternal (e, f) parts of the placenta on gestational day 14 from the control group (a, b, e) and hyperhomocysteinemia group (c, d, f). Arrows indicate autophagosomes. lp, lipid granules and hyperchromatic inclusions in the cytoplasm

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6. Fig. 5. Electron micrographs of the fetal part of the placenta on gestational day 20 from the control group (a, b, e) and hyperhomocysteinemia group (c, d, f). Arrows indicate autophagosomes. lp, lipid granules and hyperchromatic inclusions in the cytoplasm; tr, trophoblastic cell nucleus; fb, accumulation of protein fibrils in the cytoplasm

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7. Fig. 6. Electron micrographs of the cortical plate tissue of rat embryos on gestational days 14 (a, b) and 20 (c, d) from the control group (a, c) and hyperhomocysteinemia group (b, d). White arrows indicate autophagosomes, the black arrow shows the area of nuclear membrane disintegration. lys, region of organelle lysis in the cytoplasm

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СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
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