Maternal Vit D supplementation in AMA mice and the role of Vit D/VDR signaling in the offspring's cognition.

OBJECTIVE: To explore the molecular mechanism underlying the effect of maternal vitamin D (Vit D) supplementation before pregnancy in advanced maternal age (AMA) mice on the offspring's cognitive function. METHODS: Thirty-two-week-old female mice either received 10 IU/g body weight vitamin D3 dissolved in 200 µl corn oil (32W+VD group), or 200 µl corn oil (32W group) per day for one week. Another group of eight-week-old female mice received the same amount of corn oil as 32W group was set as normal reproductive age control (8W group). Then the three groups of female mice were mating with ten-week-old male mice at 2:1 ratio, the offspring were weaned at the age of 3 weeks and housed until the age of 6 weeks. Vit D metabolites and enzymes involved in Vit D metabolism were measured in both mothers and their offspring. Vit D receptor (VDR) and synaptic markers were determined in the offspring hippocampus. Vit D response elements in HIF-1α promoter were predicted, and VDR transcriptional target genes and related signaling molecules were also detected. RESULTS: Vit D intervention markedly improved the serum 1,25 dihydroxy vitamin D3 (1,25(OH)2D3) concentration in early pregnancy, middle pregnancy and late pregnancy stages in AMA mice. The hippocampal 1,25(OH)2D3 levels in the offspring showed the similar pattern. Subsequently, the expression of Cyp27b1, the gene encoding enzyme that converts 25(OH)D3 to 1,25(OH)2D3, in the hippocampus of the offspring from AMA mice was significantly lower than that of the offspring from normal female mice, and was restored by Vit D supplementation. VDR (Vit D receptor), which mediates the cellular actions of active 1,25(OH)2D3, was also rescued by Vit D supplementation, especially in dentate gyrus (DG) region of hippocampus. Concurrently, the synaptic markers NR1, NR2A, and PSD-93 in the hippocampus were reversed in 32W+VD group. Finally, we found that Vit D supplementation may affect PI3K-AKT, PLC-ERK1/2, and p38-MAPK signaling molecules by mediating HIF1α expression via VDR. CONCLUSION: Our findings highlight the biological significance of maternal Vit D supplementation before pregnancy on Vit D metabolism, and signaling molecules in the offspring, underlying the potential mechanism of the cognitive impairment in the offspring born to AMA mice.

Vitamin D supplementation in mice with advanced maternal age and cognitive function of the offspring.

To investigate the effects of vitamin D supplementation before pregnancy on the offspring's cognitive function in mice with advanced maternal age (AMA). Thirty-two-week-old female mice were randomly allocated into two groups: the 32 W+VD group (receiving 10 IU/g body weight vitamin D3 dissolved in 200 µl corn oil per day), and the 32 W group (receiving 200 µl corn oil per day) for one week before mating with ten-week-old male mice. Another group of eight-week-old female mice was given 200 µl corn oil for the same period of time and set as normal childbearing age controls (8 W group). The pregnancy outcomes were recorded and the offspring at the age of 6 weeks were subjected to behavioral tests. Finally, the expression level and distribution of neural cell markers in the offspring's hippocampus were detected by immunofluorescence. Mice with AMA had higher risk of adverse pregnancy outcome, smaller litter size, and offspring development. Vitamin D supplementation in mice with AMA promoted offspring development. AMA and maternal vitamin D supplementation before pregnancy did not change the anxiety and depression of young adult offspring. AMA impaired spatial learning and memory function of offspring while vitamin D supplementation before pregnancy rescued the impairment. AMA decreased NEFH (neurofilament protein) and MAP2 (microtubule binding protein) expression in offspring hippocampus, but vitamin D supplementation before pregnancy promoted NEFH and MAP2. Vitamin D supplementation before pregnancy can rescue the impaired learning and memory in offspring born to AMA mice. Our results highlight the significant impact of maternal vitamin D supplementation on the cognitive function of offspring.

Knockdown of CYP1B1 suppresses the behavior of the extravillous trophoblast cell line HTR-8/SVneo under hyperglycemic condition.

Introduction: Trophoblast plays a vital role in the embryonic implantation and function of the placenta. Exposure to a hyperglycemic environment results in the abnormal function of trophoblasts during fetoplacental development, which leads to maternal complications and poor fetal outcomes. However, the precise mechanisms of placental pathology during hyperglycemia remain elusive. We investigated the role of CYP1B1 in the functional behavior of the extravillous trophoblast (EVT) cell line HTR-8/SVneo under hyperglycemic condition.Methods: We determined the expression of CYP1B1 via real-time polymerase chain reaction and Western blot. Specific CYP1B1 inhibitors and small interfering RNA were used to knockdown CYP1B1, whereas an agonist and an adenovirus were used to overexpress CYP1B1. The proliferation, migration, and invasion of the EVT cell line (i.e. HTR-8/SVneo) were assessed via colony formation, 5-ethynyl-2-deoxyuridine, wound healing, and transwell assay.Results: CYP1B1 is highly expressed in placentas from women with gestational diabetes mellitus. The blockage of CYP1B1 inhibits EVT activities induced by hyperglycemia in vitro, including proliferation, migration, and invasion, whereas the exogenous expression of CYP1B1 exhibits the opposite effects.Discussion: Our study may offer a new method for regulating EVT motility under hyperglycemic condition via CYP1B1.

Circular RNA expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsia.

OBJECTIVES: To identify circRNA expression profiles in the placentae of severe preeclampsia (SPE) and normal pregnant (NP) women. METHODS: Placental samples were collected from six paired SPE and NP women. CircRNA expression profiles were identified by RNA-Seq and validated in another 30 SPE and NP samples by qRT-PCR. Several bioinformatic tools were utilised to analyse the potential function of differentially expressed circRNAs (DE-circRNAs) and to predict target microRNAs (miRNAs) and proteins. Furthermore, immunohistochemistry, Western blotting and RNA binding protein immunoprecipitation (RIP) were conducted to confirm the interaction between the circRNA and protein. RESULTS: In total, 18,631 circRNAs were detected. Among them, 180 circRNAs were differentially expressed, including 94 upregulated and 86 downregulated circRNAs. Seven DE-circRNAs were selected for validation, and the results of six circRNAs (hsa_circ_0007611, hsa_circ_0011460, hsa_circ_0002888, and hsa_circ_0007445, hsa_circ_0017068, hsa_circ_0012737) were consistent with the sequencing results. Bioinformatics analyses of DE-circRNAs revealed that most of them are involved in vasodilation, regulation of blood vessel size, protein transport and localization, and pathways in cancer. In addition to the mRNA expression profile, it was interesting to find that the hsa_circ_0011460 target gene solute carrier organic anion transporter family member 2A1 (SLCO2A1, PGT) was also significantly increased in SPE placentae. Immunohistochemistry, Western blotting and qRT-PCR validated the expression and distribution of PGT. Finally, RIP of HTR-8/SVneo cells confirmed that hsa_circ_0011460 targets PGT directly. CONCLUSIONS: A total of 180 DE-circRNAs were detected. One crucial circRNA, hsa_circ_0011460, was shown to interact directly with its host gene PGT. These findings indicated that hsa_circ_0011460 may serve as a potential therapeutic target for patients with SPE.

CYP1B1 deiciency ameliorates learning and memory deficits caused by high fat diet in mice.

Our previous study showed that Cyp1b1 deficiency could protect male mice from high fat diet (HFD) obesity. In this paper, we aim to explore the role of Cyp1b1 in HFD induced learning and memory deficits in female C57BL/6J mice. METHODS: Female Cyp1b1 knock-out (KO) and wild type (WT) mice were both randomly divided into normal chew (NC) and HFD groups. All mice were fed research diet after weaning for 24 consecutive weeks. Morris Water Maze was carried out to evaluate the spatial learning and memory. Brain lipoxidation status was evaluated by malondialdehyde (MDA) level and 4-hydroxynonenal (4-HNE)-protein adducts in mice cerebral cortex. Both activity and expression of antioxidant enzymes were determined by commercial kits and realtime RT-PCR. ß-amyloid (Aß) was detected in mice brain by immunohistochemistry. Brain derived neurotrophic factor (BDNF), synaptic plasticity protein Activity-regulated cytoskeleton-associated protein (ARC), neuronal migration and positioning gene Reelin, and Nrf2, a key transcription factor in oxidative stress, and its downstream targets heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone) (NQO-1) were measured in cerebral cortex. RESULTS: Cyp1b1 deficiency mice performed better on learning and memory tests compared with WT mice after 24 weeks HFD feeding. HFD elevated brain oxidative stress, lipoxidation in mice cerebral cortex, ß-amyloid deposition in hippocampus; suppressed antioxidant genes expression in cerebral cortex, and these effects were ameliorated by Cyp1b1 deletion. BDNF expression increased in Cyp1b1 deficiency mice after HFD feeding compared with WT. HFD activated Nrf2 and its target genes and Cyp1b1 deletion reversed such impact. CONCLUSION: Cyp1b1 deficiency protects mice from HFD induced cognitive impairment. Sustained BDNF expression, reduced ß-amyloid accumulation and brain oxidative stress, and Nrf2 deactivation might be the key events in mice redox system through Cyp1b1-diet interaction.

Advanced maternal age impairs spatial learning capacity in young adult mouse offspring.

Effects of maternal aging on the offspring cognitive function remain controversial in population-based investigations, and information available in animal studies is very limited. We investigated the impact of a delayed first natural pregnancy on pregnancy outcomes in the mouse model. Spatial learning capacity in young adult mouse offspring was observed by step-down passive avoidance task and Morris water maze (MWM). Maternal serum α-klotho was measured by ELISA. Morphological characteristics of fetoplacental unit and offspring brain were identified by H&E and immunohistochemistry. Klotho, VDR and other related genes expression were quantified by real-time-RT-PCR and western blot. We found delayed pregnancy reduced fertility in female mice by three-fold (Young vs. Old: 5.0% vs. 20.7%), and increased adverse pregnant outcomes by eight-fold (Young vs. Old: 3.0% vs. 27.5%). Mice born to old mothers exhibited shorter retention trial latency in passive avoidance task and longer latency to find the platform in MWM, suggesting worse performance on the tests that measure learning and memory. Serum α-klotho level was lower in old female mice before pregnancy, whereas became comparable after pregnancy. Vitamin D receptor (VDR) expression, both in mRNA and protein, markedly decreased during the early stage of fetoplacental unit in old mice, especially in trophoblast giant cells when compared with that of young mice. Importantly, consistent with fetoplacental unit, VDR expression also declined in hippocampus from offspring born to old mice. These results suggest that young adult offspring from aged mothers exhibited worse cognitive function and the reduced VDR expression during fetoplacental development might play an important role.